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Effects of Simulated Microgravity and Shear on Cell Behavior


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EFFECTS OF SIMULATED MICROGRAVI TY AND SHEAR ON CELL BEHAVIOR By REBECCA KATHLEEN ANDERSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA MAY 2004

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Copyright 2004 by Rebecca Kathleen Anderson

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This document is dedicated to George and Sue Anderson for their never ending support.

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iv ACKNOWLEDGMENTS I would like to acknowledge my dissertation supervisory committee for providing the guidance necessary to complete my doctora te. I would especially like to thank Dr. Tran-Son-Tay (Chair) and Dr. Don F. Cameron fo r their endless help in the pursuit of my doctorate degree in biomedical engineering. I would like to acknowledge my parents, George and Sue Anderson, for not only their patience and strength, but also their con tinuous belief in me. I want to give special thanks to my mom for being patient through al l of the times I made her listen to oral presentations over and over until I felt they were perfect. Without my mom and dad, the accomplishments that I have made over the past four years would not have been as special. I would also like to thank my brother and sister for believing in me and supporting my career goals. Special thanks go to Joelle Hushen for all of her help with cell culture using simulated microgravity. I would also like to thank Dr. Narcisse N’Dri not only for his help, guidance, and willingness to allow me to vent when I was frustrated about my project, but also for being my best frie nd. Finally, I would like to acknowledge the graduate students in the Biorheology Lab for their help regarding my project.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi LIST OF NOMENCLATURE.............................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 2 OBJECTIVES...............................................................................................................3 2.1 Rationale.................................................................................................................3 2.2 Objective.................................................................................................................4 2.3 Specific Aims..........................................................................................................5 3 BACKGROUND..........................................................................................................7 3.1 Mechanics of Simulated Microgravity...................................................................7 3.2 Cell Adhesion in Simulated Microgravity............................................................11 3.3 Endothelial Cell Behavior In Vivo vs. In Vitro....................................................18 3.4 Colon Carcinoma Behavior..................................................................................25 4 MATERIALS AND METHODS...............................................................................29 4.1 Conventional Cell Culture Technology................................................................30 4.1.1 Coating Technique......................................................................................31 4.1.2 Seeding Technique.....................................................................................32 4.2 Simulated Micrograv ity Culture Technology.......................................................32 4.3 Perfusion Flow System Technology.....................................................................33 4.4 Histological Staining Assay..................................................................................35 4.5 Morphometric Data Acquisition...........................................................................35 4.6 Quantitative Immunohistochemistry....................................................................36 4.6.1 Immunohistochemist ry Protocol................................................................37

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vi 4.6.2 Image Acquisition and Pr otein Quantification...........................................38 4.7 Statistics................................................................................................................3 9 5 RESULTS AND DISCUSSION.................................................................................40 5.1 Development of a Culture Protocol fo r a Planar Substrate in Microgravity........41 5.2 Effects of Gravity and Shear on Endothelial Cell Morphology...........................44 5.3 Effects of Gravity and Shear on Endothelial Cell Adhesion................................50 5.3.1 Cell-Substrate Adhesion.............................................................................50 5.3.2 Cell-Cell Adhesion.....................................................................................52 5.4 Effects of Gravity and Shear on Potential Cancer Marker...................................55 6 CONCLUSIONS AND FUTURE WORKS...............................................................62 APPENDICES A CHARACTERIZATION OF PERFUSION FLOW SYSTEM..................................66 B SHEAR STRESS CALCULATIONS FOR PERFUSION EXPERIMENT..............68 C QUANTITATIVE IMMUNOHISTOCHEMISTRY DATA SHEETS (HUVEC)....70 D ENDOTHELIAL CELL MORPHOLOGY DATA....................................................79 E COLON CARCINOMA C ELL MORPHOLOGY DATA.........................................86 F QUANTITATIVE IMMUNOHISTOCHEMI STRY DATA SHEETS (CACO2).....89 LIST OF REFERENCES...................................................................................................96 BIOGRAPHICAL SKETCH...........................................................................................104

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vii LIST OF TABLES Table page 1. Peristaltic Pump Calibration.........................................................................................67 2. Shear Stress Calculations for Perfusion Flow System..................................................69 3. QIHC Data for HUVEC Cultu red Conventionally (Integrin 51 Receptor).................71 4. QIHC Data for HUVEC Culture d Conventionally (E-cadherin).................................72 5. QIHC Data for HUVEC Cultu red in Microgravity (Integrin 51 Receptor)...............73 6. QIHC Data for HUVEC Cultured in Microgravity (E-cadherin)................................74 7. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2) (Integrin 51 Receptor)...........................................................................................75 8. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2 ) (Ecadherin)...................................................................................................................76 9. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2 ) (Integrin 51 Receptor)............................................................................................77 10. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2) (Ecadherin)...................................................................................................................78 11. Morphology Data for HUVEC (Perimeter)................................................................80 12. Morphology Data for HUVEC (Length 1)..................................................................82 13. Morphology Data for HUVEC (Length 2)..................................................................84 14. Morphology Data for Caco -2 (Length) (Conventional)..............................................87 15. Morphology Data forCaco-2 (Width) (Conventional)................................................87 16. Morphology Data for Caco-2 (Length) (Microgravity)...............................................88 17. Morphology Data for Caco-2 (Width) (Microgravity)................................................88 18. QIHC Data for Caco2 Cultu red Conventionally (GRP-R).........................................90

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viii 19. QIHC Data for Caco2 Cultured Conventionally (ME) (GRP-R)...............................91 20. QIHC Data for Caco2 Cultured in Microgravity (GRP-R).........................................92 21. QIHC Data for Caco2 Cultured in Microgravity (ME) (GRP-R)...............................93 22. QIHC Data for Caco-2 Cultured Conventionally (ME @ t = 6 hr)............................94 23. QIHC Data for Caco-2 Cultured in Mi crogravity (ME @ t = 6 hr) (Microgravity)...95

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ix LIST OF FIGURES Figure page 3.1 Rotary Cell Culture System (RCCS).............................................................................8 3.2 Freebody diagram of a particle....................................................................................9 4.1 Picture of planar discs..................................................................................................31 4.2 Picture of HARV co ntaining planar disc....................................................................33 4.3 Picture of perfusion flow system set-up.......................................................................34 4.4 Picture of Zeiss mo rphometric microscope................................................................36 5.1 H&E stained images of HUVEC (Matrigel)...............................................................45 5.2 HUVEC cell length (Length 1)....................................................................................46 5.3 HUVEC cell length (Length 2)....................................................................................47 5.4 HUVEC cell perimeter.................................................................................................48 5.5 Histologically stai ned HUVEC (No Matrigel)...........................................................49 5.6 Immunohistochemically staine d images of HUVEC (integrin 51 receptor).............50 5.7 Integrin receptor expression in HUVEC ( 5 subunit)..................................................51 5.8 Immunohistochemically stained im ages of HUVEC (E-cadherin)..............................53 5.9 E-cadherin expression in HUVEC...............................................................................54 5.10 Immunohistochemically stained images of Caco-2 (GRP-R)....................................56 5.11 GRP-R expression......................................................................................................57 5.12 Caco-2 cell length......................................................................................................58 5.13 Caco-2 cell width.......................................................................................................59 5.14 GRP-R expression with delayed addition of ME.......................................................60

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x LIST OF NOMENCLATURE Bn.......................................................Bombesin CACO-2.............................................Human Colon Carcinoma Cells ECM...................................................Extracellular Matrix Eu/pixel..............................................Energy Unit Per Pixel FAC....................................................Focal Adhesion Complex FAK....................................................Focal Adhesion Kinase GRP....................................................Gastrin Releasing Peptide GRP-R................................................Gastrin Releasing Peptide Receptor HARV................................................High Aspect Ratio Vessel HUVEC..............................................Human Umbilical Vein Endothelial Cells ME......................................................Methyl Ester PKC....................................................Protein Kinase C PLC....................................................Phospholipase C PVG....................................................Prosthetic Vascular Graft QIHC..................................................Quantitative Immunohistochemistry RCCS.................................................Rotary Cell Culture System STLV..................................................Slow Turning Lateral Vessel 3-D.....................................................Three-dimensional 2-D.....................................................Two-dimensional

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xi Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECTS OF SIMULATED MICROGRAVI TY AND SHEAR ON CELL BEHAVIOR By Rebecca Kathleen Anderson May 2004 Chair: Roger Tran-Son-Tay Major Department: Biomedical Engineering Based on evidence that cells cultured in si mulated microgravity exhibit in vivo characteristics similar to the parent tissue and that gravity alters metabolic activity and expression of different genes and cell signalin g in various cell lines, the present study addressed whether or not simulated microgravit y provided a better culture environment to enhance cell adhesion and to study potential markers for diseased tissue. Simulated microgravity culture promotes aggregation of anchorage dependent cells, but understanding its effect on cell structures grown on a planar surface (e.g., skin) may prove critical to the development of vasc ular grafts and our understanding of cell metastasis. Human umbilical vein endothelial cells (HUVEC) and human colon carcinoma cells (Caco-2), seeded onto planar substr ates, were investigat ed. Three culture environments (conventional cell culture, si mulated microgravity using a High Aspect Ratio Vessel, and a perfusion flow system) were used to decouple the effects of shear and gravity. Shear and gravity effects were ev aluated using histolog ical staining assays

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xii coupled with morphological measurements. Th e expressions of two endothelial adhesion proteins, integrin 51 receptor specific to fibronectin (cell-substrate) and E-cadherin (cell-cell), and a marker for colon cancer, gastrin releasing pep tide receptor (GRP-R) were determined using quantita tive immunohistochemistry (QIHC). A protocol was developed to culture anc horage dependent cells on planar discs in microgravity. Alterations in morphology and integrin 51 receptor, E-cadherin, and GRP-R expression were observed in HUVEC and Caco-2 cells cultured in microgravity. The results indicated that microgravity cult ure increased cell length, enhanced cell-cell and cell-substrate adhesion protein expressi on in HUVEC, decreased GRP-R expression in Caco-2 cells, and accelerated the kinetic s of GRP-R expression. However, in the presence of methyl ester, GRP-R expression was down-regulat ed at a slower rate in microgravity. Therefore, simulated micrograv ity culture yields results that are not obtained in conventional cell culture, and may provide a be tter culture e nvironment to study potential markers for disease.

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1 CHAPTER 1 INTRODUCTION Numerous tissues cultured under simu lated microgravity conditions have demonstrated cellular behavior similar to in vivo tissue, such as metabolic activity, cell signaling, and gene expression (Unsworth an d Lelkes 1998; Hammond et al. 2000). A microgravity environment for cell culture is de fined as a system in which the force of gravity acting on a particle is minimized (Dedolph and Dipe rt 1971; Wolf and Schwartz 1991; Gao et al. 1997) Cells cultured in simulated microgravity are able to grow and differentiate to form 3-D tissue aggregates th at structurally and functionally resemble the parent tissue (Unsworth and Lelkes 1998). Cultures grown in states of microgravity promote cell-cell interaction by up-regulating various cellcell adhesion molecules and ECM proteins (Hammond and Hammond 2001; Cowger et al. 2002). For example, kidney epithelial cells cultured under simulated microgravity show an increas e in cell-cell and cell-substrate adhesion (K aysen et al. 1999). Microgravity has been used mainly for growing 3-D structures but understanding its effect on cell-cell and/or cell-substrat e adhesion of 2-D cell structures may prove critical to the development of various biomed ical devices. In addition, studies of shear and cell adhesion on a plate are easier to pe rform and analyze than those conducted on a sphere. Microgravity studies on 2-D structures have been pe rformed in the Slow Turning Lateral Vessel (STLV) (Slentz et al. 2001). When skeletal muscle cells were cultured three-dimensionally in microgravity, large ag gregates developed a nd cells did not grow well. On the other hand, when they were att ached to 2-D silastic membrane inserts in the

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2 STLV, skeletal muscle cells reach 80% confluence within th e first 24 hours, associated with an increase in total protein level. Because the STLV requires a large volume of media and growth factors (55 ml 250 ml, depending on model), culturing in microgravity and performing chemi cal assays in this vessel can be expensive. Therefore, there is a need for a cell culture technique for growing cells on 2-D structures in the HARV, because this vessel requires 10 ml only.

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3 CHAPTER 2 OBJECTIVES The study of cell adhesion is important for understanding a wide range of fundamental biological and medical issues that include resistance change in microcirculation, cell migration, cell detachment, and cell metastasis. For example, the ability of a cell to adhere is critical for the proper functioni ng of the immune system since adhesion to vascular endothelium is a prer equisite for the circulating leukocytes to migrate into tissues. Furtherm ore, that knowledge is also es sential in the development of artificial organs. Depending on the biomedi cal application, it may be beneficial and desirable to enhance (vascular gr afts) or inhibit (art ificial implants) cell adhesion. This is also relevant to the development of cancer th erapies. In addition, the development of a method for growing cells on a planar substrat e under microgravity will allow the study of shear stress to more easily characterize cell adhesion. 2.1 Rationale Coronary heart disease, a disease caused by atherosclerosis (narrowing of vessels due to fatty build ups of plaque), is the si ngle leading cause of death in America today (American Heart Association 2004) Since prosthetic vascular grafts (PVGs) are used to replace atherosclerotic vessels or vessels weakened by diseases such as diabetes (Society for Interventional Radiology 2003) it is vital to develop techniques to enhance the adhesion of endothelial cells onto the inne r lining of PVGs if the lifetime of the implanted graft is to be optimized. Since th e isolation procedure of endothelial cells may disrupt the cell’s ability to develop normal cell-substrate and/or ce ll-cell adhesion, then a

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4 reduction in endothelial cell re tention post-implantation can be expected, greatly reducing the potential longevity of the graft. Colorectal cancer is the third most dia gnosed cancer and the second leading cause of cancer death in the United States (American Gastroenterological Association 2003). The five-year survival rate is about 90% when th e disease is detected early, compared to only 8% when it is detected in a late stage (A merican Gastroenterological Association 2003). GRP/GRP-R is aberrantly expressed in colon cancer cells, a lthough not typically expressed in normal cells (Carro ll et al. 1999a). In the co ntext of cancer, activation of GRP-R by its ligand results in the phosphoryl ation of focal adhesion kinase (FAK), an important protein tyrosine kinase that serves in the regulation of the flow of signals from the extracellular matrix (ECM ) to the actin cyto skeleton. FAK also has been shown to mediate cell growth and survival as a result of its part in cell adhesion (Parsons et al. 2000). Scientists have demonstrated that F AK is over expressed in a variety of cancers (Cance et al. 2000; Kornberg 2000). Because of its role in cell growth, survival, motility, and adhesion, it has been hypothesized that the over-expression of FAK contributes to metastatic properties of tumor cells (Kornbe rg 2000). Therefore, it was important to understand the role of GRP/GRP-R in colon cancer cells cultured in a more natural environment, such as that created using simulated microgravity culture technology. 2.2 Objective Because there is evidence that cells cu ltured in simulated microgravity exhibit in vivo characteristics sim ilar to the parent tissue (Unswo rth and Lelkes 1998; Synthecon 2000), that gravity alters metabolic activity and expression of different genes and cell signaling in various cell lines (Arase et al. 2002 ), and that exposure of seeded endothelial cells to low levels of shear stress prior to graft implantation results in increased

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5 endothelial cell retention pos t-implantation (Dardik et al. 1999), the following study intended to address whether or not simula ted microgravity provides a better culture environment to enhance cell adhe sion and to study potential ma rkers for diseased tissue. Shear stress experiments were necessary be cause shear stress and gravity have direct effects on cellular activ ities, and those effects are not decoupled in most microgravity experiments. Two cell models were used; on e model was relevant to vascular grafts (endothelial cells) and one was relevant to colon cancer (epithelial-derived colon carcinoma model). 2.3 Specific Aims The specific aims of the project were to: Develop a technique for growing cells on a 2-D substrate in microgravity. Design a cell culture protocol to decouple the effects of gravity and shear stress. Simulated microgravity was performed using a High Aspect Ratio Vessel (HARV). Shear was generated using a perfusion flow system. Conventional tissue cu lture acted as a static control. Characterize cellular morphology (as related to cellular adhesion). Cellular morphology was semi-quantified us ing a standard histological assay and morphometric microscopy. Characterize cell-cell adhesion. Cell-cell adhesion (i.e., E-ca dherin protein localization and protein level) was assessed by performing quantitat ive immunohistochemistry. Characterize cell-substrate adhesion. Cell-substrate adhesion (i.e., integrin 51 receptor locali zation and protein level) was assessed by performing quantitative immunohistochemistry. Characterize a potential marker for colon cancer.

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6 Gastrin-releasing peptide receptor expression was assessed by performing quantitative immunohistochemistry.

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7 CHAPTER 3 BACKGROUND 3.1 Mechanics of Simulated Microgravity Simulated microgravity culture technology was developed at the Johnson Space Center in Houston, Texas (Gao et al. 1997; Unsworth and Le lkes 1998). This culture technology provides a low shear, low gravit y culture environment that creates a continuous suspension due to a balance of for ces acting on a particle (i.e., a microcarrier bead or a biodegradable scaffold). Based on these properties, the microgravity bioreactor creates a culture environment that promotes 3D cell assembly. Due to the nature of this environment, cell behavior has been shown to mimic in vivo behavior, such as metabolic activity, cell signaling and ge ne expression (Unsworth an d Lelkes 1998; Hammond et al. 2000). Therefore, simulated microgravity cultu re technology may prove beneficial to the study of cellular behavior, such as pr otein expression and cell morphology. A microgravity environment for cell culture is defined as a system in which the force of gravity can be moderated, whereby the force on a particle due to gravity is minimized (Dedolph and Dipert 1971; Wolf and Schwartz 1991; Gao et al. 1997). Microgravity is simulated using the Rotary Cell Culture System (RCCS) (Figure 3.1) developed at the Johnson Space Center and produced by Synthecon, Incorporated, in Houston, TX. Two bioreactors are used to simulate micr ogravity (Freed and VunjakNovakovic 1997; Synthecon 2000). The High As pect Ratio Vessel (HARV) is a discshaped vessel that rotates a bout a horizontal axis of rota tion. A siliconized oxygenator membrane spans the diameter of the posteri or wall of the vessel. The Slow Turning

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8 Lateral Vessel (STLV) is a cy lindrical-shaped vessel in wh ich the siliconized oxygenator membrane forms the vessel core. The HARV has a “gas exchange surface per unit of cell culture volume” of roughly 5x that observed in the STLV (Freed and Vunjak-Novakovic 1997). Figure 3.1 Rotary Cell Culture System (RCCS). The RCCS was manufactured at Synthecon, Inc. in Houston, TX. The Hi gh Aspect Ratio Vessel (HARV) is a disc-shaped rotating vessel. In microgravity culture, fluid flow w ithin the vessel approximates solid body rotation with the vessel itself, eliminati ng large shear stresses (Hammond and Hammond 2001). Due to the continuous rota tion of the vessel, there is adequate mixing of nutrients and diffusion of oxygen throughout the culture envi ronment. This mixing, in part, is due to sedimentation of the particle (Hammond and Hammond 2001). An air-liquid interface is eliminated by completely filling the vessel with media. Turbulent flow is generated in the presence of air bubbles (i.e., an air-liquid interface) (Hammond and Hammond 2001). Utilization of the RCCS to generate a low shear, minimal gravity environment enables cells to develop 3D tissue constructs (Unswort h and Lelkes 1998). This BASE MOTOR HARV

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9 W = weight = mpg FL = liquid buoyant force = mlg FD = drag force Where mp = mass of particle g = gravity ml = mass of liquid = angular velocity r = radial position from center Conditions for particle movement in a circular path: Fn = mpr 2 Ft = 0 (constant speed) microgravity environment favors “high densit y” growth of anchorage dependent cells attached to microcarrier beads. A st udy by Croughan et al. (1989) demonstrated that cells cultured in a 1g culture environment requi re some agitation to maintain a continuous cell suspension, unless the microcarrier bead s are neutrally buoyant. Cell damage is minimized in a microgravity culture envir onment due to a reduction in turbulent and shear forces (Croughan 1989; Schwartz et al. 1992). Microgravity is simulated by minimizing th e force of gravity ac ting on a particle, such as a planar disc. In the inertial frame of reference, the forces acting on a particle in microgravity are shown in Figure 3.2. Figure 3.2 Freebody diagram of a particle. Th e forces acting on a particle in simulated microgravity are weight, liquid buoyant force, and fluid drag force. FL Center of Vessel W FD n t

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10 A particle moves in a circular path only when the sum of the forces is equal to mpr 2. Deviation from this condition of equality results in the particle no longer moving in a circular path. The particle may migrate towards the wall of the vessel. The shear stress acting on a microcarrier bead in simulated microgravity is approximately 0.5 dynes/cm2 (Unsworth and Lelkes 1998). Balancing the forces acting on a microcarrier bead is achieved by adjustin g the rotational speed of the vessel until sedimentation of the particle is inhibited, generating a consta nt state of free fall. When microgravity is simulated, the particle re mains in a steady state position during vessel rotation. Typically, microgravity is defined on the milli-gravity scale (10-3g) (Nechitailo and Mashinsky 1997). Theoretically, microgravity or weightlessness ac ts intracellularly by “redistributing” cellular c onstituents. Therefore, ther e is an observed relationship between chemical processes and gr avity (Nechitailo and Mashinsky 1997). Because a relative motion be tween the particle and the rotating fluid exists, the particle has the potential to migrate towa rds the vessel wall (Gao et al. 1997). For a specified g, the migration time of the particle may be affected by several parameters. The difference in the densities of the fluid and th e particle, the radius of the particle, the viscosity of the rotating flui d, the angular velocity of the rotating vessel, or the initial position of the particle within the vessel a ll affect migration time (Gao et al. 1997). Simulated microgravity culture technology provides a culture environment that promotes in vivo-like cell behavior (Hamm ond et al. 2000). Therefore, microgravity culture may provide an optimal environmen t for studying protein ex pression, alterations in cell morphology, etc.

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11 3.2 Cell Adhesion in Simulated Microgravity Gravity controls numerous cellular pr ocesses, including calcium signaling, mechanotransduction across the cytoskeletal network, cell-cell communication, various ligand-receptor interactions and possible altera tions in cellular morphology (Tairbekov 1996; Akins et al. 1997). NASA developed a rotating culture system, which creates a culture environment simulating microgravity (i.e., low gravity and low shear) (Dedolph and Dipert 1971; Wolf and Schwartz 1991). Liu et al. (2003) showed that particles cultured in microgravity only experience “micr ogravity” effects when the particle comes to a steady state position in the top half of the vessel where the level of gravity is minimized. Migration of particles in simula ted microgravity toward the center or outer wall of the vessel is dependent on the particle size, the geometry of the particle, and the density difference between the culture media an d the particle (Liu et al. 2003). Physical stimuli from the external environment play a role in cell proliferation and/or cell differentiation (Vassy et al. 2001). The question, currently, is whether or not a microgravity culture environment plays a role in the relationship between cell structure and function. Vassy et al. (2001) showed that when MCF-7 human mammary carcinoma cells were cultured in microgravity, there were changes in cellular stru cture and function. Decreased cell spreading was observed when th ese cells were cultured in microgravity. A decrease in the tensegrity of the cells (“ a looser perinuclear cy tokeratin network”) was observed in instances of decr eased cell spreading (Vassy et al. 2001). Responses of adherent cells to gravity, in vitro, can be characterized by the tensegrity model (cytoskeletal elasticity model) (Todd and Klaus 1996). Us ing this model, alterations in cell morphology and in signal tr ansduction, such as solu te transport and “buoyancy-

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12 driven flows (convection and sedimentation) ” may be observed under conditions of microgravity (Todd and Klaus 1996). Cells and tissues cultured in micrograv ity behave similarly to their in vivo counterparts in part due to the low shear a nd low gravity culture environment that is created (Unsworth and Lelkes 1998; Hammond et al. 2000). Pancreatic carcinomas cultured in simulated microgravity behaved sim ilarly to their in vivo counterparts, such as maintaining heterogeneity, cellular structur e, and biological parameters (Nakamura et al. 2002). Nakamura et al. (2002) conclude d that pancreatic carcinomas cultured under conditions of simulated microgravity exhibited proliferation rates, cellular structure, and various biological parameters, similar to in vivo tissue. Therefore, simulated microgravity culture technology provides a cultu re environment that enables the study of in vivo tissue utilizing in vitro methods (Nakamura et al. 2002). Cell behavior, such as cel l signaling, metabolic pr ocesses, and cell cycle progression may be altered in conventiona l culture due to de -differentiation of differentiated cell types culture d in 2-D (Cowger et al. 2 002). By culturing cells in simulated microgravity, a continuous cell susp ension is generated, promoting cell-cell and cell-matrix associations (Cowger et al. 2002). Varying the shear stress acting on renal epithelial cells in microgravity, such as by varying media density and by altering microcarrier bead size and/or density, the term inal or sedimentation velocity of the cells was decreased. By altering the viscosity of th e fluid media, shear and terminal velocity effects on renal cell behavior were de-coupled, demonstra ting how shear stress alters certain cellular processes (Cowger et al. 2002). Renal epithelia l cells cultured on

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13 microcarrier beads form aggregates under c onditions of microgravity resulting in cellular structure no longer resembling their in-vivo counterparts (Cowger et al. 2002). Hammond et al. (2000) demonstrated a dependence of cellular differentiation on low shear, 3-D aggregate formation and “cosp atial relation of dissimilar cell types.” Cells cultured in simulated microgravity undergo differentiation or de-differentiation based on the cell’s response to altered gravity, illustrating the benefits of microgravity culture technology (Hamm ond et al. 1999). Most differentiate d cells revert back to a dedifferentiated state when cultured using t ypical conventional culture (2-D growth) (Hammond and Hammond 2001). Suspension culture is one means to inhibit cellular dedifferentiation. When renal co rtical cells were cultured in simulated microgravity, gene expression of adhesion molecules, receptors and various intracellular signaling proteins was altered. However, when these renal cortical cells were placed in a 3g centrifuge, negligible changes in gene expression we re observed (Hammond et al. 2000). When primary human liver cells were cultured on a Matrigel coated matrix, cellular growth and de-differentiation may have been inhibite d (Yoffe 1999). These cells morphologically resembled differentiated hepatic tissue through the formation of tissue aggregates. Liver cells co-cultured with microvascular endo thelial cells demons trated angiogenic phenotypes within the tissue aggregate. Through this study, Yoffe illustrated how simulated microgravity may provide a culture environment beneficial for studying cellcell interactions and angiogenesis (Yoffe 1999) Rucci et al. (2002) showed, using a rat osteoblast-like cell line, that when 3-D aggr egates formed in microgravity, these cells produced their own extracellular matrix (Rucci et al. 2002).

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14 Currently, vascularization of organoids cultured in simulated microgravity has failed (Unsworth and Lelkes 1998). In mi crogravity, endothelial cells form solid aggregates instead of tube-like structures, demonstrating how endothe lial cells require a substratum for proper polarization and assemb lage. It may prove critical to culture endothelial cells on a planar surface in simu lated microgravity to better understand the effects of microgravity on endothelial cell be havior. A relationship between microgravity and alterations in intracellular phosphor ylation signaling pathways and in the cytoskeleton has been shown (Unsworth and Lelkes 1998). Numerous cell types cultured under microgravity conditions show up-regulati on of adhesion molecules, extracellular matrix proteins and their respective receptors Numerous studies have demonstrated the importance of cell-cell interaction for proper tissue function with respect to prostatic tissue (Cunha 1996; Thomson 1997; Margolis et al. 1999). Therefore, it may be critical to culture endothelial cells in a continuous monolayer in microgravity to properly study endothelial cell behavior. In vivo, endothelial cells t ypically are exposed to shear stresses greater than 10 dynes/cm2 (Topper and Michael A. Gimbrone 1999; Jessup et al. 2000; Resnick et al. 2003). In microgravity, endothelial cells expe rience a shear stress less than 1 dyne/cm2 (Unsworth and Lelkes 1998). Interestingly, this low level of shear stress in microgravity induces metabolic and functional changes. Using a human colorectal carcinoma cell line, Jessup et al. (2000) showed that alterations in cell proliferation and apoptosis may in part be due to shear stress, as observed in simulated micr ogravity (Jessup et al. 2000). Under microgravity conditions, endothelial cells spread, perhaps in response to reorganization of the actin cytoskeleton (Romanov et al. 2000). When confluent

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15 endothelial monolayers were e xposed to microgravity for short time intervals (1 hr for 3 days), cytoskeletal rearrangement occurred, resulting in cell spread ing and cell migration (Buravkova and Romanov 2001). A lterations in cell shape affect cell function, such as cell growth and apoptosis (Ingber 1999). Re organization of the intracellular actin cytoskeleton may result in alterations of th e forces transmitted across the cell membrane (Ingber 1999; Rucci et al. 2002). Force tran smission is greatest at cell-cell and cellsubstrate focal contacts where signaling mole cules are concentrated or clustered (i.e., integrin clustering) (Ingber 1999). Endotheli al cells, when plated in small “focal adhesion-sized islands”, formed long processes which stretched to adjacent islands of cells. The total area of cel l-substrate attachment was una ltered, however (Ingber 1999). Although integrin clustering will activate in tracellular signaling pr ocesses, integrin clustering alone will not ensure cell viability (Ingber 1999). One effect of microgravity culture on cel l morphology is cell rounding (“an actinmediated process”) (Boonstra 1999). Cytoskelet al reorganization results in altered cellmatrix binding via signal transduction across the intracellular actin cytoskeleton and the transmembrane integrin receptor network. Ther efore, it may be critical to determine the role microgravity plays in cell signaling, related to alte rations in cell morphology (Boonstra 1999). Slentz et al. (2001) devel oped a novel technique in wh ich skeletal muscle cells were cultured on a 2-D substrate placed in an STLV. When skeletal muscle cells were cultured on microcarrier beads, large cell aggr egates formed, resulting in increased shear stress acting on the cells. However, when thes e skeletal muscle cells were cultured on 2D silastic membrane inserts in microgravity, these cells reached 80% confluence within

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16 the first 24 hours and an increas e in total cellular protein was observed (Slentz et al. 2001). Sanford et al. (2002) performed a study cultu ring bovine aortic e ndothelial cells in microgravity using microcarrier beads. These cells formed tissue aggregates which tested positive for von Willebrand fact or, a typical endothelial cell marker, and a morphology similar to that of in vivo tissue. Cellcell adhesion protein ex pression, specifically proteins found in tight junctions and adhere ns junctions, was up-regulated resulting in enhanced cell-cell contact be tween cells. Although endothe lial cells form monolayers when cultured on microcarrier beads in micr ogravity, these endothelial cells formed multilayer sheets surrounding adjacent beads (Sanford et al. 2002). Jessup et al. (1994) determined that the adhesive properties of endothelial cells following culture in simulated microgravity were unaltered. However, cell manipulation, namely the subsequent digestion of dextra n microcarrier beads with collagenase and DNase occurred. To perform adhesion as says following culture in microgravity, endothelial cells must be detached from the microcarrier beads (Jessup 1994). By culturing endothelial cells on a planar substrat e, not only are the cells cultured in a 2-D monolayer as observed in vivo, but manipulatio n of the cells to study cell adhesion is unnecessary. The planar discs can be placed in a parallel plate flow system with no manipulation of the cells. Therefore, this culture protocol enable s one to directly study the effects of simulated microgr avity on epithelial cell adhesion. However, numerous cell types have dem onstrated alterations in cell adhesion when cultured in simulated microgravity (Kay sen et al. 1999; Guignandon et al. 2001; Grimm et al. 2002). Osteoblastic cells exposed to conditions of microgravity

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17 demonstrated a down-regulation in adhesion prot eins such as vincul in and extracellular matrix proteins such as fibronectin (Gui gnandon et al. 2001). Because exposure to a microgravity environment may reduce “cytoskel eton-generated tensi ons” acting on a cell, subsequent signaling pathways involving the phosphorylation of specific adhesion proteins such as focal adhesion kinase may be affected (Guignandon et al. 2001). Studies using various osteoblastic cell lines demons trated that microgravity alters cellular morphology and gene expression of such proteins as matrix proteins and various growth factors (Carmeliet and Bouillon 1999; H ughes-Fulford and Gilbertson 1999). Fibronectin is continuously produced by osteoblasts during exposure to microgravity. Therefore, changes in cell morphology are not due to a down-regulation of fibronectin in the extracellular matrix (Hughes-Fulford and Gilbertson 1999). Kaysen et al. (1999) showed that when renal epithelial cells were cultured in micrograv ity, changes in gene expression of various shear stress response el ement dependent genes such as ICAM and VCAM were observed. These results correlate to those observed when endothelial cells are exposed to flow-induced shear stress. Th erefore, they concluded that shear stress plays a definitive role in genetic alterations due to exposure to microgravity (Kaysen et al. 1999). When human follicular thyroid carci noma cells were exposed to microgravity conditions, up-regulation of extr acellular matrix proteins, su ch as collagen I and III, fibronectin and laminin was observed (Grimm et al. 2002). Both cell-cell and cell-substrate adhesion have been shown to be altered under microgravity conditions. Increased levels of E-cadherin were observed in 3-D tumor constructs cultured in simulated microgravity (Ingram et al. 1997). Because microgravity

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18 promotes the formation of 3-D tissue aggreg ates, it makes sense that cell-cell adhesion and its respective adhesion proteins would be up-regulated (Ingram et al. 1997). A study by Felix et al. (2000) looked at the effects of simu lated microgravity culture on Ca2+ dependent signaling pathways, such as cell-cell adhesion via E-cadherin. This Ca2+ dependence is through activation of the protein kinase C (PKC) second messenger system. Tracheal epithelial cells cultured in simulated microgravity retain their mechanically activated Ca2+ signaling pathway activit y. They concluded that tracheal epithelial cells cultured in simulated microgravity were more PKC sensitive than cells cultured conventionally. Th is translates to the epithelia l cells’ increased ability to activate this second messenger system. They also observed a morphological change in tracheal epithelial cells culture d in microgravity. The cells appeared taller and more cuboidal than those cells cultured conventionally (Fe lix et al. 2000). 3.3 Endothelial Cell Behavior In Vivo vs. In Vitro Mechanical forces alter cell behaviors, such as cell shape, cell growth, cell proliferation, extracellular matrix remodeling, and signal tr ansduction (Chicurel et al. 1998). Mechanical forces may not distribu te evenly across the cell membrane, but instead may localize at sites of attachment such as at cell-cell and cell-matrix junctions. The binding of integrin receptors to extracel lular matrix proteins, forming focal adhesion complexes (FAC), is a strong bond perhaps due to receptor clustering and the number of bound receptors (Chicurel et al. 1998). This is known as mechanical coupling. FAC links the extracellular matr ix to the intrac ellular actin cytoskeleton, providing a continuous network through which a mechanical stimulus can traverse across the cell from one pole to the opposite pole. Other FAC associated protei ns include vinculin, paxillin, -actinin and tyrosine kinases. Th is signal transduction may explain why

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19 endothelial cells and fibroblasts orient in the direction of flow when a shear is applied. When the balance of forces acting on a cell is altered, gene expr ession as well as extracellular matrix remodeling can occur (Chicurel et al. 1 998). The greatest alteration in cell behavior that occurs when there is a shift in the balance of forces acting on a cell is changes in cell morphology. If th e strength of the cell-matrix attachment is greater than the cytoskeletal arrangement, then the cell wi ll flatten and spread. Altered cell shape may result in inhibited cell growth and enhanced cell differentiation (C hicurel et al. 1998). Mechanotransduction is one process that activates phospholipase C (PLC), which in turn activates a signaling cascade, resu lting in the release of intracellular Ca2+ (Felix et al. 1998). However, when epithelial cells are cultured in microgravity, mechanical forces acting on the cells are reduce d, and therefore, activation of this second messenger system may be decreased. This reduced activation ma y be due to alterations in the cytoskeleton and arrangement of actin stress fibers when cells are cultured in microgravity. Felix et al. (1998) concluded from their study that although the mechanical forces which typically activate this particular signaling cascade are reduced in simulated microgravity, the release of intercellular Ca2+ is not altered in states of mi crogravity (Felix et al. 1998). Cell-substrate adhesion is created by binding the intracellular cytoskeletal network composed of actin filaments to the underlying extracellular matrix via the integrin receptor family, forming a focal a dhesion (Parsons et al. 2000). It has been shown that signals are transmitted acro ss the focal adhesions through the actin cytoskeleton, regulating various cellular processes. Pars ons et al. (2000) conducted a study to determine what role focal adhesion kinase plays in the regulation of signal transmission from the extrace llular matrix to th e intracellular actin cytoskeleton. The

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20 adhesion complex is composed intracellu larly of actin, vinculin, talin, and -actinin. It has been shown that tyrosine kinase phosphor ylation plays a role in the assembly of adhesion proteins to form FAC. Focal adhe sion kinase (FAK) is a non-receptor protein tyrosine kinase recruited to the focal a dhesion complex upon binding of the integrin receptor to the underlying extrace llular matrix (Parsons et al. 2000). When endothelial cells were grown on Matrigel coated substr ates, enhanced tyrosine phosphorylation of several adhesion proteins was observed (Williams et al. 1996). It is thought that FAK plays a role in cell morphology and migr ation. When FAK activation occurred, morphological and migrational alterations oc curred, thereby producing, “capillary-like structures” in which cells formed cord-like structures (Williams et al. 1996). Kano et al. (2000) showed that when a fluid shear stress was applied to endothelial cells, increased focal adhesion kinase expression near the basal su rface of the cells occurred. It was concluded that when the endot helial cells experience a shea r stress, they increase the strength of their cell-substrate focal adhesi ons by recruiting a greater concentration of focal adhesion kinase, therefore increasing the size of the focal adhesions. They have also been able to conclude that the apic al surface of the cell, in which the plasma membrane is attached to actin stress fibe rs, may be involved in mechanotransduction directly or that the shear force is transmitte d via the cytoskeletal network to the focal adhesions or to the site of ce ll-cell adhesion (Kan o et al. 2000). When endothelial cells are exposed to a fl uid shear, several major cellular events occur (Ballermann et al. 1998; Dardik et al. 1999; Topper and Michael A. Gimbrone 1999). First, the endothelial cells reorganize their actin cytosk eleton in order to orient the cells in the direction of flow The cells also demonstrate alterations in their metabolic

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21 processes, cell cycle activity, and expression of various cell adhesion proteins in the presence of shear stress. Cell surface receptors and expression of various signal transduction proteins are altered in the presen ce of shear stress (Bal lermann et al. 1998; Dardik et al. 1999; Topper a nd Michael A. Gimbrone 1999). Epithelial cells, when adhered to a surf ace, are polarized (Blaschuk and Rowlands 2000; Braga 2000). The basal surface of the ce ll attaches to the underlying basement membrane and the apical surface faces the lume n of the vessel or duct. Epithelial cells are able to maintain a specific cell shape, such as squamous with respect to endothelial cells, due to the interaction of the intrac ellular cytoskeleton and cell-cell and cellsubstrate adhesion. Adherens j unctions, such as tight junctions form cell-cell contacts. Cadherins are calcium dependent adhesion proteins which make up the adherens junctions (Blaschuk and Rowlands 2000; Br aga 2000). When ca dherin function is altered, typical epithelial features are inhib ited. Cadherins are composed of parallel dimers, which bind adjacent cells extracellu larly. The intracellular cadherin domains bind to the intracellular actin cytoskeleton via such proteins as -catenin, plakoglobin and -catenin. When cell-cell adhesion occurs, cadherin molecules cluster at the site of adhesion, similar to integrin receptor coupling for cell-substrate adhesion. Once the cadherins associate with the in tracellular actin cyto skeleton, mechanical forces act on the cell at these adhesion contac ts, therefore mediating a sp ecific polarized morphology (Braga 2000). Vascular endothelial cadheri n (VE-cadherin) is a single pass transmembrane protein expressed by endothelial cells. In or der for endothelial cells to form a functional confluent monolayer, both cell-substrate and cell-cell adhesion

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22 complexes must form. Cadherins, therefore, pl ay a pivotal role in the maintenance of a functional endothelial monolayer (Blaschuk and Rowlands 2000). The morphology of vascular endothelial cells is that of a spindle shape, aligning along the “longitudinal axis” of the vessel (Yamada et al. 2000). However, when endothelial cells are cultured conventiona lly, they have a cobblestone morphology and have no specific organizational pattern. Th e morphology of endothelia l cells in vivo is believed to be regulated by blood flow induced shear stress and wall pressure due to stretch (Yamada et al. 2000). Damage to the endothelial lining of blood ve ssels due to atherosclerosis or various medical procedures, such as angioplasty, results in the development of intimal hyperplasia (i.e., the migrati on of smooth muscle cells in to the endothelial region) (Underwood et al. 2002). Development of intimal hyperplasia results in the occlusion of the vessel or even occlusion of a vascular gr aft. Underwood et al. (2002) showed that mature cell-cell junctions via cadherin bindi ng are formed following 24 hours of seeding endothelial cells, possibly i nhibiting cell migration and proliferation. When exogenous vascular endothelial growth f actor (VEGF) is present, cad herin junctions are disrupted due to phosphorylation of the cadherin protein. Therefore, in the presence of VEGF, cell proliferation and migration (spread ing) occurs (Underwood et al. 2002). Fisher et al. (2001) conducte d studies on how endothelial cells sense alterations in levels of shear stress acting on them. Cell membrane and integrin receptors on the basal surface of the cell membrane can sense alterations in shear and then transmit these changes in stimuli to the intracellular actin cytoskeleton, where reorganization of the

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23 actin cytoskeleton results in alterations in endoth elial cell morphology (Fisher et al. 2001). For cell detachment to occur, a shear stre ss great enough to disrupt cell-cell and/or cell-substrate adhesions must be imposed (Pee l and DiMilla 1999). Cell detachment is dependent on the morphology and orientation of cells with respect to fluid flow. In a study by Peel et al. (1999), th e critical shear stress for det achment was determined for stromal cells. Strength of adhesion was gr eatest in moderately confluent cultures, whereas 100% cell detachment was observe d when only cell-cell interactions were present. When the cells were subconfluen t, cell detachment was dependent on cellsubstrate interactions (Peel and DiMilla 1999). Cell cycle progression is dependent on cell-substrate adhesion (Schwartz and Assoian 2001). It has been shown that the integrin 51 receptor associates with other proteins, namely caveolin and Shc, to initiate the cell cycle. Therefore, cell-substrate adhesion may be necessary for epithelial (e ndothelial) cells to grow (Schwartz and Assoian 2001). Levenberg et al. (1998) showed that ther e is an autoregulatory pathway that is activated by the presence of cell-cell or cel l-substrate adhesion si tes. Two distinct pathways have been identified. When ce ll-cell adhesion is enhanced, cell-matrix adhesion is decreased and when cell-matrix adhesion is enhanced, cell-cell adhesion is decreased. Both processes ar e dependent on tyrosine phosphor ylation but appear to be distinct from one another (Levenberg et al. 1998). A study by Ko et al. (2001) addressed what role Ca2+ signaling plays in cell-cell adhesion, perhaps through activation of PLC. It is known that in order for cell-cell

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24 adhesion to occur by way of ca dherin binding, extracellular Ca2+ must be present. They showed that cell-cell co ntacts induced the releas e of intracellular calcium, facilitating the binding of cadherins and -catenin to the actin filament s comprising the cytoskeleton, which resulted in increased strength of cell-cell contacts (Ko et al. 2001). Three major types of cell-cell junctions are present in endothelial cells, tight junctions, adherens junctions, a nd gap junctions. The adherens junction is formed by the binding of cadherin to its respec tive intracellular catenin. Sc hnittler et al. (2001) showed that when minor actin depolymerization o ccurred, changes in cell morphology occurred, namely a decrease in cell height due to a de creased stress acting on the cell. When a large degree of actin depolymerization occurr ed, cell-cell adhesion was interrupted. They showed that actin dynamics must be in balance in order to maintain cell-cell adhesion and that decreased cell-cell adhesion wa s due to decreased binding of -actinin to the cell-cell junction (Schnittler et al. 2001). It has been determined that the apical surface of the endothelium experiences the shear stress initially (Lang ille 2001). The major morphologi c response endothelial cells have to shear stress is the re-orientation of the cells in the direc tion of flow. A minor morphologic response is cell elongation due to the shear force. When endothelial cells are exposed to large levels of shear, the actin cytoskel eton undergoes reorganization, therefore resulting in the reduction of cell-cell contacts and an increase in focal adhesions at the cell-matrix region. It has been dete rmined that the greatest alterations in cell morphology appear to be due to assembly and disassembly of adherens junctions (Langille 2001).

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25 A study by Urbich et al. (2000) concluded that shear st ress does result in an upregulation of integrin recept ors. However, the productio n of nitric oxide by the endothelium in response to shea r stress also resulted in the up-regulation of integrin receptors. Because cells experience low levels of laminar shear stress during microgravity culture, some up-regulation of the integrin receptor prot ein in comparison to a static conventional culture environmen t is expected (Urbich et al. 2000). 3.4 Colon Carcinoma Behavior Activation of GRP/GRP-R may play a cri tical role in altered cell adhesion, resulting in tumor progression (Schumann et al. 2003). GRP, a bombesin-like peptide, acts as either an autocrine hormone (secrete d by and acts on same cell) or a paracrine hormone (secreted by one cell and acts on a local cell) within the gastrointestinal tract. Bombesin-like receptors are thought to act lik e mitogens, acting as an autocrine growth factor that plays a role in tumor progressi on. When bombesin-like peptides bind to the GRP-R, a second messenger signaling cascade is initiated through the activation of phopholipase C (PLC) (Schuma nn et al. 2003). Although GRP-R mRNA is often expresse d in colon cancer cell lines, the message may become mutated resulting in non -functional GRP-R (Carroll et al. 2000a). From a previous study, it was shown that th at a down-regulation of GRP-R resulted in dedifferentiation of tumor cells (Carroll et al. 2000). Hist ologically, well-differentiated tumors are similar to the tissue of origin whereas poorly differentiated tumors show a disorganized histology. A study by Carroll et al. (1999) showed that GRP/GRP-R is aberrantly expressed in well-differentiate d tumors, but a decrea sed expression of GRP/GRP-R is observed in tumors undergoi ng dedifferentiation. Therefore, GRP/GRPR acts both as a mitogen (stimulates mitosi s) and a morphogen (stimulates differentiation

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26 of cells/tissue; involved in embryogenesis) (Carroll et al. 1999; Glover et al. 2003). Although GRP may cause cell pro liferation, it does not act as an “oncogenic growth factor” (Carroll et al. 1999a). Carroll et al. (1999) demonstrat ed that aberra nt expression of both GRP and GRP-R is observed in adeno carcinomas of the colon, but do not appear to up-regulate as the tumor stages. Becau se it was shown that GRP/GRP-R expression plays a direct role in tumor cell differentiation, it was concluded that GRP/GRP-R may act in an autocrine manner with respect to cell differentiation. GRP/GRP-R has been shown to play a role in the alteration of cell-cell attachment, therefore acting as a morphogen (Carroll et al. 1999). Because GRP/GRP-R plays a role in the differentiation of colon cancers, it was concluded that GRP/ GRP-R expression may re-capitulate its role in normal intestinal developm ent (Carroll et al. 2002). Bombesin plays a role in alterations in cell morphology, alterations in the actin cytoskeleton in “nontransformed” cells, an d enhancing cell motility of cancer cells (Saurin et al. 1999). The process of meta stasis requires many steps including cell locomotion, cell-substrate adhesion, and cel l proliferation. Sa urin et al. (1999) demonstrated that bombesin stimulated ce ll spreading, formation of lamellipodia, and adhesion to the extracellular matrix (collagen type I). From this study, it was concluded that GRP-R may play a role in the “invasive properties” of colonic cancer cells. The formation of lamellipodia has been shown to be related to the process of tumor cell metastasis (Saurin et al. 1999). Epithelial cells lining a normal GI tract do not typically express FAK. FAK is shown to be present in well-differentiated tu mors. FAK expression was up-regulated as the tumors became better differentiated (Ca rroll et al. 2000). Focal adhesion kinase

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27 (FAK) is a cytoplasmic adhesion protein lo calized at focal contacts. Although FAK expression is up-regulated in colon cancers, th e degree of FAK activat ion due to tyrosine phosphorylation is critical (The ocharis et al. 2003; Matkowskyj et al. 2003a). Human colon cancers are typically heterogeneously diffe rentiated. Differentiation with respect to tumors defines how much tumor cells rese mble the normal tissue of origin. Tumor differentiation plays a role in tumor metast asis. Although GRP/GRPR is not typically expressed by the epithelial lining of the gastroin testinal tract, an increased expression of GRP/GRP-R was observed in “post-neopla stic transformation, resulting in betterdifferentiated tumors” (Theocharis et al. 2003 ; Matkowskyj et al. 2003a). A relationship between better differentiated tumors and the expression of focal adhesion kinase (FAK) via the GRP/GRP-R signaling pathway has been determined. It was shown that FAK phosphorylation at tyrosine 397 and tyrosi ne 407 is a function of tumor cell differentiation and the expression of GRP/GR P-R. Therefore, GRP/GRP-R plays a role in cancer cell differentiation via FAK phosphorylat ion at specific sites (Theocharis et al. 2003; Matkowskyj et al. 2003a). One study demonstrated that the presence of GRP-R in colonocytes resulted in a constitutive activation of the receptor, resulti ng in enhanced cell grow th in the absence of agonist binding, growth factors, and serum (Ferris et al. 1997). This stud y identified the constitutive activation of GRP-R in the abse nce of mutations, therefore concluding that GRP-R may act as an oncogene with respect to colon cancer. Transition of cells from an epithelial to a mesenchymal state (epithelialmesenchymal transition) plays a role in morphogenesis and in the development of

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28 carcinomas (Thiery 2003b). Carcinoma progres sion has been linked to de-differentiation of tumoragic epithelial cells which furt her leads to invasion and metastasis. The process of cell adhesion plays a role in other cellular processes including proliferation, differentiation, migration, and death (Thiery 2003a ). Tissue types comprised of epithelial cells are more likely to develop into cancer, typically carcinomas (~ 90% if tumor types), than other cell types. Due to alterations in adhesive properties of epithelial cells, tumor progression occurs originally from an adenoma towards development of invasive carcinomas. Du ring this progression, polarization of the epithelial cells regresses and a disorganized arrangement of the cells within the tumor occurs (Thiery 2003a; Thiery 2003b). Alterations in gravity, such as hypogravity or hypergrav ity, result in the activation of the protein kinase C (PKC) signaling cascad e (Han et al. 1999). Activated PKC plays a role in cell prolifer ation. With respect to human tu mor cells, alterations in gravity resulted in mutations in “microsatellite sequences” (“short repeats of 1-4 nucleotide units”). These minor mutations result in “mismatch repair-deficient human tumor cells” (Han et al. 1999). A study by Rhee et al. (2001) demonstrated that when prostatic cancer cells are cultured three-dimensionally in simulated microgravity, the epithelial derived cells demonstrated potential metastatic markers no t typically observed in conventional culture. They concluded that transiti oning of carcinogenic prosta tic tissue from an androgendependent to an androgen-independent pathway is mechanistically related to the presence of tumorigenic and metastatic markers (Rhee et al. 2001).

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29 CHAPTER 4 MATERIALS AND METHODS The present study investigated the effect s of shear stress and gravity on cell behavior, such as cell morphology and membra ne bound receptor levels when anchorage dependent cells were cultured on planar discs in simulated microgravity. A protocol for culturing cells seeded onto planar discs in simulated microgravity, in a perfusion flow chamber or in conventional culture, and to st udy the effects of shear stress and gravity on cell behavior was developed. It was proposed that simu lated microgravity culture technology would enhance cell-ce ll and/or cell-substrate a dhesion protein levels in human umbilical vein endothelial cells (HUVE C) and might provide a better environment to study potential markers for diseased tissue. Typical 3-D substrat es commonly used for seeding and culturing cells in simulated mi crogravity include microcarrier beads and PLA/PGA scaffolds. However, the evaluation of the strength of e ndothelial cell adhesion cannot be easily performed on these spherical b eads. Therefore, 2-D planar discs were used to culture adherent cells in micrograv ity. Microgravity was simulated using the HARV and the perfusion flow sy stem was composed of a parallel plate flow chamber (both plates fixed) driven by a peristaltic pump. Histologic al assays were performed using a standard histological staining assay (hematoxylin and eosin staining) in order to analyze alterations in cell morphology (cell width, cell length and cell perimeter) due to changes in the levels of shear and gravity placed on the cells. Quantitative immunohistochemistry was employed to id entify the presence, localization and concentration of three membrane bound receptors, integrin 51 receptor (cell-substrate

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30 receptor specific to fibronectin), E-cadherin (cell-cell integral pr otein), and gastrinreleasing peptide r eceptor (GRP-R). 4.1 Conventional Cell Culture Technology The protocols for cell culture were perf ormed as described previously in the Clonetics catalog (2000-1). HUVEC (Cambre x, Inc., Walkersville MD) cells were initially plated onto non-coated T-75 flasks (Fisher Scientific, USA) and grown to roughly 80% confluence prior to harvesting. The cells were cultured in a 37 C and 5% CO2 – 95% air environment. Upon reaching 80% confluence (5-9 days), the cells were washed with Hanks Balanced Salt Solution (Fis her Scientific, USA) to remove any serum supplemented media remaining on the cells. The cells were then trypsinized using a 0.25% trypsin/EDTA (Fisher Scientific, USA) so lution until the cells began to round up and detach from the flask surface. The cell solution was placed in a 15 ml conical tube and centrifuged at 1000g for a pproximately 3 min. The supern atant was aspirated and the cells were further washed 2x in unsuppl emented endothelial basal media (EBM) (Cambrex, Inc., Walkersville, MD). Cell viab ility was determined by staining a sample of the cell suspension with trypan blue. Cells which took up the stain, were not viable. Caco-2 cells were initially plated onto non-coated T-25 flasks (Fisher Scientific, USA) and grown to roughly 80% confluence prior to harvesti ng. The cells were cultured in a 37 C and 5% CO2 – 95% air environment. Upon reaching 80% c onfluence (2-3 days), the cells were washed with Hanks Bala nced Salt Solution (Fisher Scientific, USA) to remove any serum supplemented media rema ining on the cells. The cells were then trypsinized using a 0.25% trypsin /EDTA (Fisher Scientific, US A) solution until the cells began to round up and detach from the flask surface. The cells were counted using a hemacytometer to determine a specified cell concentration.

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31 4.1.1 Coating Technique. Circular planar discs were prepared from plastic cover slips using a ” hole punch (Figure 4.1). For the endothelial study, ci rcular planar discs were prepared from 22 mm x 22 mm unbreakable plas tic cover slips (Fisher Scien tific, USA) using a ” hole puncher. The discs were sterilized by placi ng them in a Petri dish containing a 70% ethanol solution for approximately 5 min. Th e ethanol was aspirated off and the discs were allowed to dry under UV light in a ti ssue culture hood. The planar discs were coated by dipping the discs in a 20% solution of Matrigel (Fisher Scientific, USA) in unsupplemented EBM (Cambrex, Inc., Walkersville, MD). The discs were then placed in a 24 well-plate, three discs per well. While harvesting the cells, the 24 well-plate was placed in the incubator to allow the Matrig el coating to polymerize on the discs. Figure 4.1 Picture of planar discs. Planar discs were constructed from plastic cover slips. Planar discs for the endothelial st udy were constructed from unbreakable plastic cover slips. Planar discs for the Caco-2 study were constructed from Thermanox plastic cover slips. For the Caco-2 study, circular planar disc s were prepared from Thermanox plastic cover slips (Fisher Scie ntific, USA) using a ” hole punche r. The discs were sterilized via autoclaving. These discs were not coated with Matrigel. Matrigel interrupts the role

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32 of methyl ester as an antagonist for GRP-R. The discs were then placed in a 24 wellplate, three discs per well. 4.1.2 Seeding Technique HUVEC were trypsinized, as stated in th e conventional cell cu lture section, and the cell suspension was placed in a 15 ml cen trifuge tube. The cell suspension was centrifuged at 1000 rpm for 3-5 min and the s upernatant was aspirated off. The cells were washed in unsupplemented EBM at least 2 times. The cells were resuspended in 5 ml of media supplemented w ith antibiotics, growth fact ors and 10% FBS. The cell suspension was then evenly distributed amongst the wells. Th e cells seeded in conventional culture until roughly 80% confluen t (approximately 4-6 days). Following the seeding period, the discs were either placed in simulate d microgravity culture for an additional 48 hrs, placed in a perfusion flow chamber set at a specified flow rate for an additional 48 hrs, or left in conventi onal culture for an additional 48 hrs. Caco-2 cells were trypsinize d, as stated in the conven tional cell culture section, and the cell suspension was placed in a 15 ml centrifuge tube. The cell suspension was diluted in approximately 10 ml of medi a and the cells were counted using a hemacytometer. A cell concentr ation of approximately 60,000 cells/cm2 was determined for seeding. The cells seeded for approxima tely 12 hrs. Following the seeding period, the discs were either placed in simulated mi crogravity culture for tim e periods of 2, 6, 12, 24, and 48 hrs or left in conventional culture for time periods of 2, 6, 12, 24, and 48 hrs. 4.2 Simulated Micrograv ity Culture Technology To culture anchorage dependent cells on a planar substrate in the HARV, modification of the conventional culture prot ocol was necessary. Following seeding, a single disc was placed in each HARV via st erile forceps (Figure 4.2). The disc was

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33 placed on the membrane attached to the poste rior wall of the HARV. The anterior and posterior portions of the HAR V were screwed together and 10 ml of media supplemented with 2 g of 2000 kDa MW dextran in 50 ml of EBM were used to fill the chamber. The dextran was used to increase the viscosity of the media. The HARVs were then placed on the rotating base which was placed inside th e incubator. The ro tational speed of the HARVs was adjusted until the discs came to a steady state position in the vessel. Figure 4.2 Picture of HARV contai ning planar disc. A single pl anar disc is placed within the interior of the HARV using sterile forceps. The anterior and posterior portions of the HARV are screwed togeth er and the vessel is filled with media. 4.3 Perfusion Flow System Technology For the perfusion flow system, due to an increase in the fluid viscosity, the flow rate was decreased in order to maintain a shear stress similar to that observed in simulated microgravity. For the perfusion flow chamber, the discs were placed on top of a glass slide, which fit directly into the flow channel (Figure 4.3). The discs were approximately 0.2 mm in height. Therefore the chamber height was reduced from 1.5 mm to 1.3 mm. The disc s were prepared as previously stated. The discs were then placed back into the original punched out spac es in the cover slips and the cover slips region of HARV filled with media (10 ml)

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34 were placed in the chamber. Via calculation of the entrance length and the length of the flow channel, approximately 10 substrates could be placed in the flow channel per experiment (approximately 2 cover slips). Figure 4.3 Picture of perfusion flow system setup. A parallel plate flow chamber driven by a peristaltic pump comprised the perf usion flow system. The pump was set to run at flow rates (Q) correspondi ng to 2 mL/min, 4mL/min and 8 mL/min. The shear stress applied to seeded HUVEC ranged from 0.25-1.01 dynes/cm2 using the following formula, = 6 Q/wh2, calculated from the flow between two parallel plates where Q is the flow rate, is the fluid viscosity an d w and h are the width and height of the chamber, respectively. A Ra inin Dynamax peristaltic pump was used to drive the media into the flow chamber. Th e pump generated a multi, unidirectional pass flow. The Dynamax peristaltic pump was co nnected to the perfusion flow chamber via 3/16” silastic tubing (Fisher Scientific, USA). The perfusion flow chamber was placed in the incubator at 37 C and 5% CO2 – 95% air for 48 hrs. planar discs placed in perfusion flow system parallel plate flow chamber peristaltic pump

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35 4.4 Histological Staining Assay Following the specified culture period, HUVEC were fixed with 4% paraformaldehyde/PBS while still remaining attached to the plan ar discs. The cells were subsequently stained with hematoxylin and eo sin. Harris’ hematoxylin (Fisher Scientific, USA) is a basic stain that will stain the nucleus and nucleolus of cells blue. Eosin (Fisher Scientific, USA) is an acidic stain that wi ll stain the protein por tion of a cell pink. Morphological measurements (length, width, peri meter, and aspect ratio) were collected via digital photomicroscopy and the imag ing program, Axiovi sion 3.1 (Carl Zeiss Microimaging, Inc., Thornwood, NY). The protocol for typi cal histological staini ng is described belo w. The cells were fixed in 4% paraformaldehyde for 20 min. Th e cells were then ri nsed 3x (5 min each wash) with phosphate buffered saline (PBS). Hematoxylin was placed on the cells for roughly 2 min. Following the 2 min of hema toxylin stain, the cells were washed with tap water (acts as a bluing solution). Eosi n was placed on the cells for roughly 1 min. Following the 1 min of eosin stain, the cel ls were dipped approximately 15x in 95% ethanol to prevent the eosin from washing out in subseque nt washes. The discs were mounted cell side down onto a glass side usi ng a water based glycer ol/gelatin mounting agent. 4.5 Morphometric Data Acquisition Specific morphological parameters were measured. These parameters included perimeter, length, width, and aspect ratio (L/W ). Images of the cells were taken at a 10x magnification for the endothelial cell study and at a 40x magnification for the Caco-2 study using a Zeiss microscope with an Axioplan camera (Figure 4.4).

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36 Figure 4.4 Picture of Zeiss morphometric micr oscope. A Zeiss microscope connected to an Axioplan camera was used to collect images. Morphological measurements were taken using the image acquisition program, Axiovision 3.1 (Carl Zeiss Microimaging, Inc., Thornwood, NY). Length and width were measured using the Length icon. Peri meter was measured using the Curved Spline icon. Each morphological parameter was measur ed three times for a ccuracy. An average of each measurement was recorded. Three cells were selected per three clusters of cells containing 5-7 cells on each disc. From each cluster, 3 cells were analyzed. Three discs were used for analysis for each culture environment. 4.6 Quantitative Immunohistochemistry Immunohistochemistry is a technique used to identify the presen ce of a particular protein using an antibody against that prot ein. Immunohistochemistry was performed on cells cultured in simulated microgravity, in a perfusion flow syst em generating a shear stress of 0.51 dynes/cm2 and 1.01 dynes/cm2, and in conventional culture, using antibodies against the integrin 51 receptor (cell-substrate) a nd E-cadherin (cell-cell) for

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37 the endothelial study and GRP-R for the Caco -2 study. All three of these receptor proteins are integral membrane proteins. 4.6.1 Immunohistochemistry Protocol. Following the specified culture time period for the endothe lial cells or the Caco-2 cells, the discs were placed in a 24 well plate and washed with tris buffered saline (TBS) to remove excess dextran from the discs. Any debris, such as dextran, remaining on the cells prior to fixation resulted in dark staining in that region. Theref ore, washing the cells prior to fixation was critical The cells were then fixe d in a 3.7% formaldehyde/TBS solution at 37 C and 5% CO2 – 95% air for 30 min. The fixative was aspirated off and the cells were washed with TBST (tris bu ffered saline and Tween 20). TBST was used for cytosolic proteins (to perforate the membra ne). The three protei ns of interest were integral membrane proteins, containing an extracellular domain, a membrane-spanning domain, and a cytosolic domai n. Therefore, the cell membra nes required perforation in order to expose the entire protein. The cell membranes were permeabilized for 5 min at room temperature with TBST. Endogena se peroxidase activ ity was blocked by incubating the cells in 0.03% hydrogen peroxide for 5 min. The cells were rinsed with distilled water and then further washed with TBST. The endothelial cells were incubated with primary antibodies against the integrin 51 receptor and E-cadherin. The Caco-2 cells incubated with a primary antibody agains t GRP-R. The antibodi es, diluted in Dako antibody diluent, were placed on the cells fo r 60 min at room temperature. The cells were washed for 5 min (x2) with TBST to remove excess primary antibody. The cells were incubated with anti-mouse secondary anti body for 15 min at room temperature. The cells were then washed for 5 min (x2) with TBST. A Streptavidin link was placed on the cells for 15 min at room temperature. The ce lls were washed with TBST. The cells were

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38 incubated with DAB for 5-10 min at room temper ature. The cells were then washed with tap water (color changed from purple to brown with water wash). The cells then incubated for 2 min with Autostainer he matoxylin (Gill’s Hematoxylin) at room temperature. The cells were then rinsed well with TBST. TBST works as a bluing solution (color changed from purple to blue with TBST wash). The discs were then mounted onto glass slides cell side down usi ng a water soluble glycerol/gelatin mounting agent. The mounted cells were then dried overni ght prior to image acquisition and data analysis. 4.6.2 Image Acquisition and Protein Quantification Digitized images of the immunohistochemica lly stained cells were collected using the image acquisition program, Axiovision 3.1 (Carl Zeiss Microimaging, Inc., Thornwood, NY). The images were prepared for protein analysis using Adobe PhotoShop as previously described (Matkowsk yj et al. 2003b). Th e digitized images were saved as *.tif files and were prepar ed in Adobe Photoshop for analysis. The background of each image was set to white using the Level command. A single cell was then cropped from the image file. Usi ng the magic wand tool, the brown color (indicating the presence/locali zation of the protein of choice) was isolated and copied/pasted into a new *.tif file. This image file was then opened in the program, TIFFALYZER. From this program, the energy unit per pixel value wa s quantified. This value was subtracted from the value obtained for negative samples (those samples not incubated with primary antibody ) to obtain the energy unit pe r pixel value (i.e., protein level) for each specified protein. Thirty cel ls were randomly chosen for each adhesion protein analysis for the endothe lial cell study. Fourteen cell s were randomly chosen for

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39 protein analysis of GRP-R for the Caco-2 st udy. The control tissue fo r the endothelial cell study was human dermal fibroblasts (C ambrex, Inc., Walkersville, MD). 4.7 Statistics. Analysis of Variance (ANOVA) was perf ormed to determine significance between the three culture environments. Statistical analysis was performed using the online statistical software program, Graphpad Quic k Calcs (www.graphpad.com). Comparisons were made for morphology and protein expre ssion. Significance was taken at p<0.05 or p<0.01.

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40 CHAPTER 5 RESULTS AND DISCUSSION The goal of the present study was to determine whether or not simulated microgravity culture technology provides a bett er culture environment to enhance cell adhesion and to study potential markers for diseased tissues. Through the development of a culture protocol enabli ng anchorage dependent cells to be cultured on a planar substrate in simulated microgravity, the direct effects of gravity and shear stress on cell adhesion using an endothelial ce ll model and the role of a potential colon cancer marker, GRP-R using a colon cancer cell model were determined. Since endothelial cells naturally form a confluent monolayer, it was im portant to simulate th is growth pattern in order to study the direct effect s of gravity and shear stress on endothelial cell behavior. It has previously been shown by Sanford et al (2002) that endothelial cells do form a monolayer around a single microcarrier bead, but form multilayer sheets of endothelial cells when the microcarrier beads aggregate to gether. Therefore, the native state of the endothelial cells is compromised during the culture period. By cu lturing the endothelial cells on a planar substrate, monolayer forma tion is maintained during the entire culture period. A second advantage to cu lturing on a planar substrate in microgravity is that no cell manipulation must occur in order to st udy specific cell behaviors, such as adhesion protein expression and cell morphology. The s ubstrate does not interf ere with analysis, such as morphological analysis or determina tion of protein levels. Also, the planar

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41 substrates can further be placed in a perfusion flow system to study cell detachment in the presence of shear. 5.1 Development of a Culture Protocol for a Planar Substrate in Microgravity A method for growing cells on a 2-D stru cture in simulated microgravity was developed. A brief description of the me thod is provided here but more detailed information can be found elsewhere (Anderson 2 001). Cells were cult ured on ” circular discs punched from a 22 x 22 mm2 square plastic cover slip (Fisher Scientific, USA). The discs were coated with a 1:5 Matrigel solution to promote cell binding to the cover slips. Matrigel was selected as the coat ing material because it is a non-synthetic extracellular matrix solution comprised of almost every ECM protein. Unbreakable plastic cover slips were utilized to mini mize the weight of the substrate in the microgravity vessel. The substrates were suspended in a highly viscous fluid in the HARV, so that during rotation, the substr ate was suspended. Media viscosity was increased approximately 12 fold by adding high molecular wei ght dextran. As previously shown, cells cultured in media supplemented with dextran and cells cultured in media containing no dextran do not de monstrate differences in ce ll growth or morphology (Anderson 2001). To isolate gravity and shear effects, 3 culture environments were selected. Conventional tissue culture provi ded a static, 1g environment. Simulated microgravity provided a low shear, low gravity environment. A perfusion flow system of variable shear (0.25, 0.5, and 1.0 dynes/cm2) provided a link between a static and a low shear culture environment. This link enabled th e effects of gravity and/or shear on cell behavior to be decoupled. The first shear stress of 0.25 dynes/cm2 provided a link between a static culture environment (0 dynes/cm2) and the shear acti ng on a microcarrier

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42 r rU r r U r U 1bead in a simulated microgr avity environment (0.5 dynes/cm2). A shear stress of 0.5 dynes/cm2 was selected because this shear was co mparable to the shear stress acting on a microcarrier bead in simulated microgravity (Gao et al. 1997). The third shear stress of 1.0 dynes/cm2 provided a shear stress comparable to the shear acting on a plate in microgravity. Generally, a simulated microgravity environment will create a shear stress of less than 0.5 dynes/cm2. The media (fluid) in the HARV rota tes in a circular path as a solid body with the vessel due to the elimination of an air-liquid inte rface at low angular velocities ( ) (Gao et al. 1997). The velocity of the rotating fluid is defined in the direction only and the shear rate is locally defined. Shear stress is equal to zero with respect to solid body rotation ( r = 0). The shear stress in the r plane (Gao et al. 1997) is where r = shear stress, = fluid viscosity, U = velocity in respective plane (r ), r = location of particle in vessel. Relative moti on between the particle (cell/tissue construct) and the fluid, allowing for mixing of me dia, generates small shear stresses. The shear stress generated due to the re lative motion between the rotating fluid and the particle is dependent on the relative speed ( rel) between the fluid and the particle (Gao et al. 1997). Therefore, the maximum shear stress ( max) acting on a particle of radius, a, within a simulated microgravity environment (Gao et al. 1997) can be defined as (5.1) a vrel2 3max v v vP rel(5.2)

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43 r V Vdl p ReThe proposed study has demonstrated that microgravity can be simulated using a planar disc. The drag force (FD) on a planar disc in si mulated microgravity was calculated from the following equation where Ap = particle area l = fluid density V = V0 = velocity CD = drag coefficient on a planar disc. CD was determined using a CD vs. Re chart (Roberson and Crowe 1997). The Re was calculated from the following equation where dp = particle diameter r = radius of vessel = angular velocity = viscosity. For the case where = 12.24 cP; dp = 0.006 m; l = 1.02 g/mL; V = 0.25 m/s; r = 0.03 m; = 8.2 rpm, the Re was 13.6 (Anderson et al. 2004a). Using this Re, the drag force on a planar disc in microgravity was approximately 4.97 10-4 N, where Ap = 126.6 mm2; l = 1.02 g/mL; V = V0 = 0.25 m/s (Gao et al. 1997) (Anderson et al. 2004a). It should be noted that the drag force on a planar disc in simulated mi crogravity was on the same order as the drag force acting on a micr ocarrier bead in simulated microgravity. Using equation 5.2, the shear stress acting on a planar disc in microgravity was approximately 1.0 dynes/cm2, where a = 0.02 m; = 12.24 cP; V = 0.017 m/s V A C Fl p D D 22 0(5.3) (5.4) (5.5)

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44 (Anderson et al. 2004a). Therefore, the shear stress acting on a planar disc in microgravity is approximately 2x the shear stress acting on a microcarrier bead in microgravity (Unsworth and Lelkes 1998). 5.2 Effects of Gravity and Shear on Endothelial Cell Morphology HUVEC cultured on a planar substrate in simulated microgravity demonstrated a morphologically distinct shape when compared to HUVEC cultured in a static or variable shear 1g environment (Figur e 5.1). HUVEC appeared el ongated and developed fine, cytoplasmic projections when cultured in simu lated microgravity (Anderson et al. 2004a). A “cobblestone” morphology was observed in HUVEC cultured in a static or variable shear 1g environment. Previous studies indica te that endothelial ce lls exposed to shear stress elongate and orient in the directio n of flow (Thoumine et al. 1995; Topper and Michael A. Gimbrone 1999). Al terations in the actin cytoskel eton result in alterations of the focal adhesion complexes comprised of the integrin 51 receptors (Chicurel et al. 1998). Because simulated microgravity cultur e technology creates a culture environment that enables cells to behave similarly to their in vivo count erparts (Unsworth and Lelkes 1998; Hammond and Hammond 2001), the initiati on of angiogenesis may be occurring in the absence of exogenous vascular endothelial growth factor (VEGF) which is typically required for initiation of angiogenesis in conventional culture. These cytoplasmic extensions observed in HUVEC cultured in mi crogravity may represent the initiation of capillary formation (Anderson et al. 2004a).

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45 Figure 5.1 H&E stained images of HUVEC (Matrigel). a) Ce lls cultured using conventional tissue culture technique. Note the cobblestone appearance of cells cultured conventionally. b) Cells cultured using simulated microgravity. Note the long, hair-like projectio ns extending from the cells. Length 1 represents the total length of the cell; length 2 represents the cell length without the hair-like projections. c) Cells cultured using simulated microgravity. Note the tube-like clustering of cells. All of these images were taken at a 10x magnification. From this study, we found that cells cu ltured in simulated microgravity were significantly longer than those cells cultured in the perfusion flow system set-up to generate three distinct shear stresses and those cells cultured conventionally (Figure 5.2 and Figure 5.3) (Anderson et al. 2004a). Tw o length measurements were determined. a) b) c)

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46 Morphologic Measurement (Length 1)0 20 40 60 80 100 120 140 160 180 200microns Microgravity Conventional 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 *Length 1 represented the entire length of the cell, including the long hair-like projections as observed in cells cultured using simulate d microgravity. Length 2 represented the cell length excluding the long, hair-like projections. Figure 5.2 HUVEC cell length (Length 1). Lengt h 1 includes the cytoplasmic projections observed in simulated microgravity cultu re. Length 1 of HUVEC cultured in simulated microgravity was significantly greater than the length of HUVEC cultured conventionally or in a perfusion flow system. Morphologic measurements were obtained using Ax iovision 3.1 (Carl Ze iss Microimaging, Thornwood, NY). The asterisk denote s significance at a p<0.01.

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47 Morphologic Measurement (Length 2)0 20 40 60 80 100 120microns *Microgravity Conventional 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Figure 5.3 HUVEC cell length (Length 2). Morphometric measurement (Length 2) acquired using the image acquisition program, Axiovision 3.1. Length 2 represents the cell body only. Lengt h 2 of HUVEC cultured in simulated microgravity was significantly greate r than the length of HUVEC cultured conventionally or in a perfusion fl ow system. The asterisk denotes significance at a p<0.01. HUVEC cultured using simulated microgravit y had a significantly larger perimeter when compared to cells cultured using conve ntional tissue culture technique or in the perfusion flow system (Figure 5.3) (Anderson et al. 2004a). This observed increase in cell perimeter is proportional to the increase in cell length for those cells cultured in simulated microgravity. Although the width of the HUVEC cultured in simulated microgravity was greater than the width of HUVEC cultured in a static environment, no significant difference was

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48 Morphologic Measurement (Perimeter)0 50 100 150 200 250 300 350 400 450 500microns Microgravity Conventional 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 *observed. Because cell height was not meas ured, it was difficult to speculate how cell width is altered with changes in cell leng th. However, it is clear that microgravity produces larger cells as give n by cell length and cell peri meter (Figure 5.4). However, HUVEC cultured under increasing shear did demonstrate an increasing trend in perimeter. Figure 5.4 HUVEC cell perimeter. Morphomet ric measurement (perimeter) acquired using the image acquisition program, Axiovision 3.1. The measured perimeter of the cells includes the hair-l ike projections extending from the cell bodies. The perimeter of HUVEC cultu red in simulated microgravity was significantly greater than the perimete r of HUVEC cultured conventionally or in a perfusion flow system. The aste risk denotes signifi cance at a p<0.05. Additionally, HUVEC were s eeded onto non-coated planar discs and cultured in microgravity for 48 hrs (Figure 5.5) (Ande rson et al. 2004a). Morphologically, HUVEC developed long, cytoplasmic projections exte nding from each pole of the cell and/or aggregated together to form tube-like stru ctures similar to HUVEC cultured on Matrigel

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49 coated planar discs in microgravity. Th erefore, morphologic alterations in HUVEC cultured in microgravity do not appear to be due to the Matrigel coating (Anderson et al. 2004a). HUVEC undergo morphologic alterati ons in response to the microgravity environment. Figure 5.5 Histologically stained HUVEC (No Matrigel ). HUVEC cultured on noncoated planar discs demonstrated morphology similar to those HUVEC cultured on Matrigel coated substrates. In summary, it can be seen that both gravity and shear play a role in morphological differences in HUVEC. Tradit ionally, shear stress has been shown to result in the re-organization of the actin cy toskeleton, therefore resulting in reorientation of the cells in the direction of flow (Topper and Michael A. Gimbrone 1999). It is known that endothelial cells in vivo typically are exposed to shear stresses greater than 10 dynes/cm2, but the low shear that endothelial ce lls experience in simulated microgravity is great enough to induce meta bolic and functional changes (Jessup et al. 2000). Our results confirm those results, and suggest that gravity, or a minimization of it, may also contribute to the reorganization of the actin cy toskeleton, therefore resu lting in alterations in cell length.

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50 5.3 Effects of Gravity and Shear on Endothelial Cell Adhesion The expression of 2 major adhesion protei ns was studied; an integrin receptor specific to fibronectin for characterizing cell-surface adhesi on and E-cadherin for characterizing cell-cell adhesion. 5.3.1 Cell-Substrate Adhesion The level of expression of the integrin r eceptor is altered in the absence/presence of shear and gravity. Figure 5.6 shows i mmunohistochemically stained images of HUVEC cultured in the presence/absence of shear and altered gravity. Figure 5.6 Immunohistochemically stai ned images of HUVEC (integrin 51 receptor). ac) human dermal fibroblasts cultured c onventionally, in microgravity, or in a perfusion flow system, respectively. d-f) HUVEC cultured conventionally, in microgravity, or in a perfusion flow system, respectively. The expression of the 5 subunit of the integrin 51 receptor was different when HUVEC were cultured conventionally, in a pe rfusion flow system generating a shear stress of 0.5 dynes/cm2 or a shear stre ss of 1.0 dynes/cm2, and in simulated microgravity. a) b) c) d) e) f)

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51 Integrin Receptor (Alpha 5) Expression0 25 50 75 100 125 150 175 200Eu/pixelConventional Microgravity 0.5 dynes/cm2 1.0 dynes/cm2 *The control tissue used for immunohistochemi stry was human dermal fibroblasts. This control tissue was incubated with an tibodies specific to the integrin 51 receptor and to E-cadherin. By performing immunohistochemi stry on the control tissue, the presence and location of the proteins of interest were identified. Figure 5.7 shows the important role of shear in the up-regulati on of the integrin 51 receptor, specifically the 5 subunit specific to fibronectin. Figure 5.7 Integrin recept or expression in HUVEC ( 5 subunit). Expression was determined by QIHC. Integrin re ceptor expression was significantly upregulated in HUVEC cultured in micrograv ity and cultured in a perfusion flow system generating shear stresses of 0.5 and 1.0 dynes/cm2 compared to HUVEC cultured conventionally. No si gnificant difference in integrin receptor expression was observed be tween HUVEC cultured in microgravity or a perfusion flow system generatin g shear stresses of 0.5 and 1.0 dynes/cm2. The asterisk denotes significance at a p<0.05.

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52 The integrin concentration is about 160 Eu/pixel when HUVEC are cultured in simulated microgravity or in the perfusi on flow system generating a shear of 0.5 dynes/cm2 or a shear of 1.0 dynes/cm2 (the shear acting on a plan ar disc in microgravity) (Anderson et al. 2004a), and a lit tle less than 140 Eu/pixel when they cultured in a static environment. The concentration unit Eu/pixel represents the mathematical energy of the data image file calculated for a single pixel (Matkowskyj et al. 2003b). Perinuclear localization of the 1 subunit of the integrin receptor was observed in osteoblasts cultured in simulated microgravity (S arkar et al. 2000). They determined that the perinuclear localization was in part due to a disruption of the actin cytoskeleton. A trend of perinuclear localization of the 5 subunit of the integrin 51 receptor, similar to the Sarkar et al. study, was observed when HU VEC were cultured in microgravity. This specific localization of the protein may in pa rt be due to re-orientation of the actin cytoskeleton due to the shear for ces acting on the cells (Ingber 1999). Typically, the integrin r eceptors will couple in the area of greatest stress. Therefore, because the receptors tend to loca lize perinuclearly in simulated microgravity, the area of greatest stress acting on the endot helial cells is most likely located in the region of the nucleus. On the other hand, because the integrin receptors do not appear to localize in a specific area th roughout cells culture d conventionally, the stress is in all likelihood distributed evenly across the cell membrane. 5.3.2 Cell-Cell Adhesion The level of expression of E-cadherin is al tered in the absence/presence of shear and gravity. Figure 5.8 shows immunohist ochemically stained images of HUVEC cultured in the presence/absence of shear and altered gravity.

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53 Figure 5.8 Immunohistochemically stained imag es of HUVEC (E-ca dherin). a-c) human dermal fibroblasts cultured conventiona lly, in microgravity, or in a perfusion flow system, respectively. d-f) HUVEC cultured co nventionally, in microgravity, or in a perfusion flow system, respectively. The expression of E-cadherin was di fferent when HUVEC were cultured conventionally, in a perfusion flow system generating a shear stress of 0.5 and 1.0 dynes/cm2, and in simulated microgravity. Figure 5.9 shows that HUVEC cultured in simulated microgravity demonstrated signifi cantly greater levels of E-cadherin when compared to those cells culture d in a perfusion flow system generating a shear stress of 0.5 dynes/cm2 or a shear stress of 1.0 dynes/cm2 (Anderson et al. 2004a). a) b) c) d) e) f)

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54 E-cadherin Levels0 20 40 60 80 100 120 140 160 180 200Eu/pixel *Conventional Microgravity 0.5 dynes/cm2 1.0 dynes/cm2 Figure 5.9 E-cadherin expression in HUVEC. E-cadherin expr ession was determined by QIHC. The expression of E-cadherin was signifi cantly up-regulated in HUVEC cultured in simulated microgravity and in HUVEC cultured conventionally compared to HUVEC cultu red in a perfusion flow system generating a shear stress of 0.5 and 1.0 dynes/cm2. The asterisk denotes significance at a p<0.05. A small difference in E-cadherin leve ls was observed between HUVEC cultured conventionally and in simulated microgravit y. An increased level of E-cadherin in HUVEC cultured in simulated microgravity was expected beca use this culture environment promotes 3-D cell aggregate fo rmation. The main staple of cell-cell aggregation is an up-regulation of cell-cell adhe sion molecules. A statistically significant

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55 difference in E-cadherin levels was also obser ved when cells were cultured in a perfusion flow system generating a shear of 0.5 dynes/cm2 when compared to cells cultured conventionally. It is clear that if shear stress down regul ates E-cadherin expression, then microgravity significantly up regulates it. When comparing the data obtained for cell-substrate adhesion and the data obtained for cell-cell adhesion, it was shown that cell-cell adhesion and cell-substrate adhesion was shown to be greater in simu lated microgravity than in the other two environments, namely static and perfusion fl ow (Anderson et al. 2004a). While shear can inhibit or promote cell adhesion by upor dow nregulating the expr ession of adhesion proteins, an environment where the force of gravity is minimized appears to oftentimes promote adhesion, resulting in the up-regula ted expression of adhesion proteins. Changes in adhesion protein expression are due both to gravity and shear. However, other factors, such as diffusion of oxygen and mixing of media during rotation, most likely play a role in the present cell behavior. 5.4 Effects of Gravity and Shear on Potential Cancer Marker Gastrin-releasing peptide receptor (GRP -R) is not normally expressed in the epithelial lining of the gastrointestinal tract. However, this receptor is expressed in colon carcinoma, an epithelial-derived tumor type (C arroll et al. 1999). Th e goal of this study was to determine whether or not simulated micr ogravity culture technology could be used to enhance the response of a potential ma rker for colon cancer. Through quantitative immunohistochemistry, it was shown that GR P-R expression in C aco-2 cells (colon carcinoma cell line) cultured conventionally does not down-regulate within the first 48 hrs of culture (Figure 5.10 and Figure 5.11). However, GRP-R expression in Caco-2 cells cultured on a planar substrate in si mulated microgravity rapidly down-regulates

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56 within the first 48 hrs. (Figure 5.10 and Figure 5.11) (Anderson et al. 2004b) This down-regulation may in part be due to the in ternalization of the receptor. GRP-R is expressed in well-differentiated tumors, but is minimally expressed in poorly differentiated tumors (Carroll et al. 1999). Figure 5.10 Immunohistochemically stained imag es of Caco-2 (GRP-R). Over time, GRP-R expression in Caco-2 cells is significantly down-re gulated, as shown by minimal brown staining in the above images. Perhaps, Caco-2 cells on planar substr ates in simulated microgravity dedifferentiate over time, resulting in the dow n-regulation of GRP-R expression. Because GRP/GRP-R activation results in the production of fo cal adhesion kinase (FAK), a constituent of the intracellular adhesion network, GRP/GRP-R expr ession and activation play a role in cell adhesion. Adhesion plays a critical role in cancer metastasis with respect to FAK. From our study, we concl uded that GRP-R expression is down-regulated Microgravity (+ME) Conventional (+ME) Microgravity Conventional 6 hr 12 hr 24 hr 48 hr

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57 GRP-R Expression0 50 100 150 200 250 300 061218243036424854 hoursEu/pixel Conv no ME Conv ME HARV ME HARV no MEin microgravity, both in the absence and pres ence of a GRP-R antagonist, methyl ester (ME) (Figure 5.11). As an antagonist, ME shoul d not have an effect on the expression of GRP-R. GRP-R expression in Caco-2 cells cultured conventionally remains constant in the absence or presence of ME over a 48 hr time period (Figure 5.11) (Anderson et al. 2004b). In contrast, GRP-R expression in Caco-2 cells cultured in microgravity on a planar substrate down-regulates rapidly over the first 48 hrs in the absence of ME. Figure 5.11 GRP-R expression. GRP-R expre ssion does not change in the absence or presence of ME when Caco-2 cells are cultured conventionall y within the first 48 hrs of culture. GRP-R expression down regulates immediately when Caco2 cells are cultured in the absence of ME in simulated microgravity. GRP-R expression down-regulates at a slower ra te when Caco-2 ce lls are cultured in the presence of ME in simulated microgravity.

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58 Caco2 Length0 5 10 15 20 25 30 35 40 45 50 01020304050607080 hoursmicrons Microgravity Microgravity ME Conventional ME ConventionalThis down-regulation of GRP-R is also observed when the Caco-2 cells are treated with ME in micrograv ity, although the down-regulation is milder and appears to level off at approximately 24 hrs (Anderson et al. 2004b). If the activation time of ME (typically 18 hrs) is altered in simulated microgravity, then GRP-R may be internalized at a slower rate in microgravity when ME is present. ME should not ha ve an effect on GRP-R expre ssion because ME acts as an antagonist, binding to and ultimately inactiva ting GRP-R. Inactiva tion of GRP-R results in inactivation of the production of FAK. Th erefore, alterations in cell adhesion may be observed. This data indicates a direct link be tween Caco-2 cell morphology and GRP-R expression. Caco-2 cells cultured in simula ted microgravity were significantly longer than Caco-2 cells cultured conventionally (Figure 5.12) (Anders on et al. 2004b). Figure 5.12 Caco-2 cell length. The length of Caco-2 cells cu ltured in simulated microgravity were significantly longer than those cultured conventionally in the presence or absence of ME. Howe ver, in the presence of ME, Caco-2 length did not demonstrate an increasing trend over time.

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59 Caco-2 Width0 5 10 15 20 25 30 35 06121824303642485460667278hoursmicrons Microgravity Microgravity ME Conventional Conventional MEInterestingly, this increase in cell le ngth correlates with a decrease in GRP-R expression. In the presence of ME, Caco-2 cell length was not signi ficantly different for those cells cultured conventionally or in simulated microgravity. Caco-2 cell width (Figure 5.13) also appeared to be greater for those cells cultured in simulated microgravity, although the diffe rence was not significant. Next, the dependence of morphological alterations on GR P-R expression and how these alterations were affected by a change in GRPR expression were determined. Figure 5.13 Caco-2 cell width. The width of Caco-2 cells cultured in simulated microgravity was greater than the width of Caco-2 cells cultured conventionally in the presence or absen ce of ME. However, in the presence of ME, Caco-2 width did not show as gr eat of an increase over time as Caco-2 cells cultured in the absence of ME. To study the role of GRP-R expression in morphological alterations, ME was added to Caco-2 cells cultured conventionally or in simulated microgravity after the first

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60 GRP-R Expression (delayed ME addition)0 20 40 60 80 100 120 140 160 180 200 0612182430hoursEu/pixel Conventional Microgravity6 hrs of the experiment (Figure 5.14). ME was added at this time point, because GRP-R down-regulation in microgravity is initiated wi thin the first 6 hours of culture. When ME was added following the first 6 hours of culture in microgravity, GRP-R expression rapidly down-regulated and was almost gone w ithin the first 24 hours. Therefore, some aspect of the microgravity culture environm ent, independent of GRP-R expression, is causing morphological alterations in Caco -2 cells (Anderson et al. 2004b). Figure 5.14 GRP-R expression with delayed a ddition of ME. GRP-R expression does not change when ME is added 6 hrs following the start of the experiment for Caco-2 cells cultured conventionally or in simulated microgravity within the first 24 hrs of culture. GRP-R expre ssion also down-regulated significantly by the time the ME was added to Caco-2 cells cultured in simulated microgravity. A study by Glover et al. (2004) utilized a non-malignant epithelial cell line (293 cells) which expressed GRP/GR P-R normally to show the role of GRP/GRP-R in cell

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61 detachment and cell deformability (Delaney 2003; Glover et al. 2004). From this study, it was shown that when GRP/GRP-R is activ ated and therefore, FAK was produced, cell detachment was inhibited. When 293 cells were exposed to ME during culture, GRP/GRP-R activation was blocked, inhib iting FAK production, and cell detachment was increased. Therefore, they concluded that GRP/GRP-R does play a role in cellsubstrate adhesion and may help to regulate cancer metastasis. From this study, it was also concluded that cell deformability was enhanced when FAK production was inhibited. This data correlates with th e morphological data showing that when GRP/GRP-R activation was inhibited in the presence of ME, cell length and cell width were minimally altered (Anderson et al. 2004b). However, when GRP/GRP-R activation occurred, enhanced alterations in cell lengt h and cell width were observed, the more extreme case observed in simulated micrograv ity (Delaney 2003; Glover et al. 2004). This data correlates with the idea that 293 cells are less deformable when FAK activation is uninhibited.

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62 CHAPTER 6 CONCLUSIONS AND FUTURE WORKS Culture in simulated microgravity is not limited to 3-D cell association. Endothelial cells seeded onto planar substr ates and cultured in simulated microgravity behave in a similar fashion to endothelial cells cultured on micr ocarrier beads in simulated microgravity. Culturing cells on a 2-D substrate in microgravity enables the direct testing of various cell parameters w ithout subsequent cell manipulation, such as digestion of the microcarrier bead or centr ifugation (Anderson et al 2004a; Anderson et al. 2004b). Future studies in the area of a ngiogenesis and cell detachment will further determine endothelial cell behavior when cultured in 2-D in microgravity. With respect to morphology, endothelial ce lls cultured in simulated microgravity demonstrated a distinct cell shape not observed when cells were cultured using conventional tissue culture tech nique or in a perfusion flow system. Endothelial cells cultured in simulated microgravity exhibite d a distinct morphology because microgravity culture technology may provide a culture environment where cell behavior mimics native tissue. Therefore, the cells cultured in si mulated microgravity exhibited characteristics similar to those observed in vivo, such as cel l elongation and flatteni ng of the cell. The endothelial cells that were cultured in si mulated microgravity exhi bited fine, hair-like projections extending from the cell body. These fine projecti ons were not observed when the cells were cultured in the other two culture environments. Because the cells cultured in simulated microgravity exhibited the gr eatest difference with respect to cell

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63 morphology when compared to cells cultured using conventional tissue culture technique or in a perfusion flow system, it was conclude d that gravity has a gr eater effect on cell morphology than shear stress. Cells cultured using conventional tissue culture technique or in a perfusion flow system did not appear to show great differences with respect to cell morphology. Interestingly, the cells that we re cultured in simulated microgravity exhibited a distinct cell aggregate formation not observed when cells were cultured in the other two culture environments. A future study involves looking at the induction of angiogenesis when endothelial cells are cu ltured in simulated microgravity without exogenous stimulation of vascular en dothelial growth factor. With respect to adhesion protein expr ession, HUVEC cultured in simulated microgravity did demonstrate an up-regulation in both cell-cell and cell-substrate adhesion protein. The increased expression of these adhesion proteins is not only due to the effects of shear but it is also due to the effects of gravity and other factors like oxygenation. These results confirm the con cept that simulated microgravity culture promotes cell-cell and cell-substrate associatio ns. We, therefore, have developed a new method for culturing cells on a pl anar substrate in simulated microgravity that provides a better way for studying adhesion, gravity and shear effects than on 3D microcarrier beads. Because the microgravity environment is comprised of numerous parameters which facilitate in vivo-like cell behavior, it is difficult to isolate which parameter has the greatest effect on Caco-2 cell behavior. Down-regulation of GRP-R in microgravity occurred at a much earlier time point than in conventional culture, indicating a shift in the kinetics of GRP-R expression. The advantage of accelerated kinetics of GRP-R

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64 down-regulation is that a grea ter concentration of experime nts can be conducted at much earlier time points in microgravity as opposed to 4 days in conven tional culture. More importantly, because microgravity culture has been shown to induce in vivo-like cell behavior, perhaps, on a developmental note, no rmal intestinal development is favored in microgravity culture, illustrati ng a hypothesis for this extr eme and rapid down-regulation of GRP-R. A second hypothe sis, which the data may s upport, is alte rations in differentiation states of tumors (i.e., welldifferentiated to poorly differentiated) may provide an explanation for this extrem e and rapid down-regul ation of GRP-R in microgravity. By developing a culture protocol which enables a direct comparison to be made between in vitro (conventional) cell behavior and in vivo-like cell behavior (simulated microgravity), better culture techniques can be developed that enable researchers to better understand the role of specific parameters, such as GRP-R expression, in morphological changes a nd cell adhesion in diseased cells. Future studies for the endothelial study include looking at the process of angiogenesis in simulated microgravity. One study would involve looking at the expression of vascular endothe lial growth factor and its subsequent receptors when HUVEC are cultured in simulated microgravity. Based on the morphology (e.g., the tube-like clustering of HUVEC in micrograv ity), it appears as though angiogenesis is taking place in the absence of exogenous vasc ular endothelial growth factor. A second study would be to address the adhesive stre ngth of HUVEC cultured in microgravity. Following culture in simulated microgravit y, HUVEC seeded on planar discs may be placed in a perfusion system generating high shear stresses. From this study, the shear stress required to detach s eeded HUVEC could be determined.

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65 Future studies for the Caco-2 study involve looking at the differentiation state of Caco-2 cells cultured in micr ogravity. One study would be to isolate well differentiated, moderately differentiated, and poorly differen tiated cells within th e carcinoma and grow them up separately to form homogeneous tu mors to study parameters for metastasis. A second study would be to determine the ro le phopholipase C signaling pathway plays in metastasis (i.e., cell adhesion, FAK, and GRPR expression). This pathway is altered in simulated microgravity and is affiliated with the process of cell adhesion. Several studies could be designed to develop a culture protocol which will involve development of a microchamber which will facilitate the grow th of a colon carcinoma (or any tumor type) in microgravity, using minimal cell density, minimal media, etc. A second microdevice could be developed that will mimic an epithelial lining, prov iding an attachment surface similar to the epithelial lining of the gastrointestinal tract. By attaching the 3-D tumor to this surface, in vivo like cell behavior during metastasis may more easily be studied. Definitely, surface topography will play a role in cell adhesion and metastasis. Finally, using specific time points, determine as a colon carcinoma (developed in 3-D in microgravity) stages (and begins the process of metastasis), how the differentiation of the tumor changes. During this analysis, we c ould identify alte rations in vari ous markers, like GRPR expression. Ultimately, this study would lead to the development of a 3-D model for colon carcinoma study. It may be beneficial to study colon carcinoma models to determine how well an in vivo-like model could be designed for in vitro experimentation.

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APPENDIX A CHARACTERIZATION OF PERFUSION FLOW SYSTEM

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67 Dynamx Peristaltic Pump Calibration 0 5 10 15 20 25 30 0102030405060 Pump Speed (rpm)Volumetric Flow Rate (mL/min) Flow Rate Calibration Linear (Flow Rate Calibration)The Dynamax peristaltic pump was calibra ted using 3/16” silastic tubing. The predetermined calibration equations for specifi ed tubing size were not compatible with the tubing of choice. Th erefore, the pump was calibra ted by hand for the before mentioned tubing size. Table 1. Peristaltic Pump Calibration Pump Speed (rpm) Volumetric Flow Rate (ml/min) 1 0.8 2.7 1.9 6.25 4 10 6.3 14.8 8.8 25 15 48 27.4

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APPENDIX B SHEAR STRESS CALCULATIONS FOR PERFUSION EXPERIMENT

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69Table 2. Shear Stress Calculations for Perfusion Flow System. Tau Mu (g/cms) h (cm) w (cm) Q (ml/s)Q (ml/min) A (cm2) V (cm/s) Rho (kg/cm3) D (cm) Re Le (mm) 0.128 0.122 0.2002.400 0.017 1.000 19.350 0.001 0.001 0.2000.001 0.000 0.255 0.122 0.2002.400 0.033 2.000 19.350 0.002 0.001 0.2000.003 0.000 0.383 0.122 0.2002.400 0.050 3.000 19.350 0.003 0.001 0.2000.004 0.001 0.510 0.122 0.2002.400 0.067 4.000 19.350 0.003 0.001 0.2000.006 0.001 0.638 0.122 0.2002.400 0.083 5.000 19.350 0.004 0.001 0.2000.007 0.001 0.765 0.122 0.2002.400 0.100 6.000 19.350 0.005 0.001 0.2000.009 0.001 0.893 0.122 0.2002.400 0.117 7.000 19.350 0.006 0.001 0.2000.010 0.001 1.020 0.122 0.2002.400 0.133 8.000 19.350 0.007 0.001 0.2000.011 0.001 1.148 0.122 0.2002.400 0.150 9.000 19.350 0.008 0.001 0.2000.013 0.002 1.275 0.122 0.2002.400 0.167 10.000 19.350 0.009 0.001 0.2000.014 0.002 1.403 0.122 0.2002.400 0.183 11.000 19.350 0.009 0.001 0.2000.016 0.002 1.530 0.122 0.2002.400 0.200 12.000 19.350 0.010 0.001 0.2000.017 0.002 1.658 0.122 0.2002.400 0.217 13.000 19.350 0.011 0.001 0.2000.019 0.002 1.785 0.122 0.2002.400 0.233 14.000 19.350 0.012 0.001 0.2000.020 0.002 1.913 0.122 0.2002.400 0.250 15.000 19.350 0.013 0.001 0.2000.021 0.003 2.040 0.122 0.2002.400 0.267 16.000 19.350 0.014 0.001 0.2000.023 0.003 2.168 0.122 0.2002.400 0.283 17.000 19.350 0.015 0.001 0.2000.024 0.003 2.295 0.122 0.2002.400 0.300 18.000 19.350 0.016 0.001 0.2000.026 0.003 2.423 0.122 0.2002.400 0.317 19.000 19.350 0.016 0.001 0.2000.027 0.003 2.550 0.122 0.2002.400 0.333 20.000 19.350 0.017 0.001 0.2000.029 0.003

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APPENDIX C QUANTITATIVE IMMUNOHISTOCHEMISTRY DATA SHEETS (HUVEC)

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71 Table 3. QIHC Data for HUVEC Cu ltured Conventionally (Integrin 51 Receptor) Negative Integrin 51 Receptor Total Protein (Eu/Pixel) 600.1 465 135.1 600.1 474.8 125.3 600.1 485.5 114.6 600.1 447.5 152.6 600.1 485.6 114.5 600.1 448.3 151.8 600.1 477.7 122.4 600.1 466.7 133.4 600.1 480.5 119.6 600.1 486.9 113.2 600.1 465.7 134.4 600.1 481.7 118.4 600.1 489.7 110.4 600.1 457.7 142.4 600.1 451.4 148.7 600.1 470.9 129.2 600.1 474.1 126 600.1 469.6 130.5 600.1 451.9 148.2 600.1 476.1 124 600.1 449.9 150.2 600.1 449.3 150.8 600.1 437.4 162.7 600.1 437.3 162.8 600.1 451.4 148.7 600.1 434.2 165.9 600.1 445.3 154.8 600.1 435.9 164.2 600.1 457 143.1 600.1 469.4 130.7

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72 Table 4. QIHC Data for HUVEC Cultu red Conventionally (E-cadherin) Negative E-Cadherin Total Protein (Eu/Pixel) 600.1 473.4 126.7 600.1 513.3 86 600.1 483.6 116.5 600.1 493 107.1 600.1 494 106.1 600.1 491.7 108.4 600.1 488.3 111.8 600.1 471.3 128.8 600.1 467.9 132.2 600.1 470.9 129.2 600.1 456.7 143.4 600.1 478.1 122 600.1 464 136.1 600.1 461.8 138.3 600.1 485.7 114.4 600.1 484.8 115.3 600.1 479.3 120.8 600.1 473.8 126.3 600.1 477.3 122.8 600.1 485.3 114.8 600.1 465 135.1 600.1 462.6 137.5 600.1 487.3 112.8 600.1 470.9 129.2 600.1 490.7 109.4 600.1 473 127.1 600.1 481.8 118.3 600.1 468.3 131.8 600.1 461 139.1 600.1 464.6 135.5

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73 Table 5. QIHC Data for HUVEC Cu ltured in Microgravity (Integrin 51 Receptor) Negative Integrin 51 Receptor Total Protein (Eu/Pixel) 605.3 415 499.3 605.3 452.5 459.6 605.3 486 493.6 605.3 435.5 500.9 605.3 429.7 468.5 605.3 474.1 503 605.3 432.2 468.4 605.3 438.8 472.9 605.3 423 460.6 605.3 457.5 481.4 605.3 435.9 458 605.3 459.1 458.1 605.3 419.5 439.1 605.3 417.4 462.5 605.3 420.5 495.4 605.3 430 469.2 605.3 428.1 494.5 605.3 469.8 492.2 605.3 428.9 492.6 605.3 481.4 488.7 605.3 422 436.6 605.3 477.7 436.8 605.3 482.3 471.3 605.3 470 483.2 605.3 459.3 460 605.3 457.6 485.3 605.3 430 490.1 605.3 441.8 447.1 605.3 437.2 450.6 605.3 428.2 463.4

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74 Table 6. QIHC Data for HUVEC Culture d in Microgravity (E-cadherin) Negative E-Cadherin Total Protein (Eu/Pixel) 605.3 190.3 106 605.3 152.8 145.7 605.3 119.3 111.7 605.3 169.8 104.4 605.3 175.6 136.8 605.3 131.2 102.3 605.3 173.1 136.9 605.3 166.5 132.4 605.3 182.3 144.7 605.3 147.8 123.9 605.3 169.4 147.3 605.3 146.2 147.2 605.3 185.8 166.2 605.3 187.9 142.8 605.3 184.8 109.9 605.3 175.3 136.1 605.3 177.2 110.8 605.3 135.5 113.1 605.3 176.4 112.7 605.3 123.9 116.6 605.3 183.3 168.7 605.3 127.6 168.5 605.3 123 134 605.3 135.3 122.1 605.3 146 145.3 605.3 147.7 120 605.3 175.3 115.2 605.3 163.5 158.2 605.3 168.1 154.7 605.3 177.1 141.9

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75 Table 7. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2) (Integrin 51 Receptor) Negative Integrin 5 1 Receptor Total Protein (Eu/Pixel) 595.6 411.6 489.2 595.6 461.2 479.3 595.6 444.9 478.6 595.6 441.7 480.4 595.6 438.6 497.5 595.6 446 456.7 595.6 429.2 449.7 595.6 419.9 492.5 595.6 449.8 500.2 595.6 460.5 502.2 595.6 433 487.4 595.6 430.7 494.4 595.6 435.1 492.1 595.6 465.1 485.8 595.6 441.4 485.8 595.6 433.3 490.3 595.6 414.3 502 595.6 413.6 497.4 595.6 464.6 472.7 595.6 445.6 468.6 595.6 453.8 459.4 595.6 428.6 445.9 595.6 442.7 504.9 595.6 436.9 486.1 595.6 441.9 467 595.6 431.4 472 595.6 419.7 446.8 595.6 436.6 504.8 595.6 422.4 460.2 595.6 426.4 461.5

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76 Table 8. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2 ) (E-cadherin) Negative E-Cadherin Total Protein (Eu/Pixel) 595.6 184 106.4 595.6 134.4 116.3 595.6 150.7 117 595.6 153.9 115.2 595.6 157 98.1 595.6 149.6 138.9 595.6 166.4 145.9 595.6 175.7 103.1 595.6 145.8 95.4 595.6 135.1 93.4 595.6 162.6 108.2 595.6 164.9 101.2 595.6 160.5 103.5 595.6 130.5 109.8 595.6 154.2 109.8 595.6 162.3 105.3 595.6 181.3 93.6 595.6 182 98.2 595.6 131 122.9 595.6 150 127 595.6 141.8 136.2 595.6 167 149.7 595.6 152.9 90.7 595.6 158.7 109.5 595.6 153.7 128.6 595.6 164.2 123.6 595.6 175.9 148.8 595.6 159 90.8 595.6 173.2 135.4 595.6 169.2 134.1

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77 Table 9. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2 ) (Integrin 51 Receptor) Negative Integrin 51 Receptor Total Protein (Eu/Pixel) 595.6 422.6 173 595.6 444.5 151.1 595.6 419.9 175.7 595.6 426.6 169 595.6 418.3 177.3 595.6 434.5 161.1 595.6 428.3 167.3 595.6 422.7 172.9 595.6 420.1 175.5 595.6 421.8 173.8 595.6 422.1 173.5 595.6 434.3 161.3 595.6 430.9 164.7 595.6 451.2 144.4 595.6 451.5 144.1 595.6 447.7 147.9 595.6 425.1 170.5 595.6 473.8 121.8 595.6 420.5 175.1 595.6 439.8 155.8 595.6 421.7 173.9 595.6 435.4 160.2 595.6 432.8 162.8 595.6 421.3 174.3 595.6 430.5 165.1 595.6 448.6 147 595.6 444.3 151.3 595.6 421.3 174.3 595.6 427.4 168.2 595.6 427.1 168.5

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78 Table 10. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2) (E-cadherin) Negative E-Cadherin Total Protein (Eu/Pixel) 595.6 449 146.6 595.6 455.5 140.1 595.6 484 111.6 595.6 444.8 150.8 595.6 461.6 134 595.6 456.5 139.1 595.6 493.9 101.7 595.6 462.6 133 595.6 472.7 122.9 595.6 436 159.6 595.6 494.4 101.2 595.6 440.5 155.1 595.6 492.5 103.1 595.6 481.4 114.2 595.6 477.5 118.1 595.6 470.7 124.9 595.6 492.6 103 595.6 484.9 110.7 595.6 481.7 113.9 595.6 480 115.6 595.6 496.7 98.9 595.6 465.1 130.5 595.6 478.7 116.9 595.6 473.1 122.5 595.6 484.9 110.7 595.6 482.4 113.2 595.6 480.7 114.9 595.6 505.1 90.5 595.6 488.2 107.4 595.6 505.7 89.9

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APPENDIX D ENDOTHELIAL CELL MORPHOLOGY DATA

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80 Table 11. Morphology Data for HUVEC (Perimeter) Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Perimeter Perimeter Perimeter Perimeter Perimeter 183.2 466.5 197.3 180.2 172.3 167.3 574.0 231.4 177.3 128.7 190.9 551.2 271.9 162.2 170.2 165.6 573.0 150.7 151.3 222.6 152.3 563.3 198.3 144.1 212.3 182.3 1174.8 134.2 158.8 164.3 184.2 486.2 178.9 214.2 191.6 175.8 668.2 151.9 182.2 167.4 197.4 484.6 190.9 176.8 171.6 172.7 247.2 171.9 178.7 236.7 182.6 192.9 194.6 231.4 181.0 186.0 240.1 178.4 206.7 226.9 166.5 189.4 143.6 215.9 216.3 153.5 297.0 151.1 146.6 195.3 296.6 196.1 176.2 155.8 201.7 206.5 392.2 219.2 153.3 229.7 144.8 243.3 245.4 186.7 200.8 126.2 298.3 200.7 144.9 201.3 156.9 677.9 126.3 178.4 162.3 168.1 717.9 168.8 145.5 181.3 158.0 292.9 179.6 178.0 166.2 173.7 760.9 237.1 223.8 180.5 178.5 309.6 196.0 232.6 154.2 151.0 220.5 194.4 207.1 194.1 187.7 480.6 176.2 217.9 196.7 192.5 271.9 166.6 214.5 210.7 185.1 405.9 162.2 196.4 191.9 196.9 348.6 152.9 227.4 210.1 342.2 388.8 135.5 274.9 223.7 154.3 341.9 136.8 261.6 234.4 208.5 407.1 137.9 317.9 229.3 149.6 324.5 139.9 227.6 185.9 156.9 316.9 141.7 206.0 149.1 262.3 508.1 155.5 193.7 242.1 246.8 421.3 146.2 153.5 193.7 189.7 265.6 167.6 195.8 175.3

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81 Table 11 cont. Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Perimeter Perimeter Perimeter Perimeter Perimeter 251.9 343.2 240.1 213.2 233.0 229.0 630.3 249.2 166.0 203.4 199.8 419.0 257.0 233.4 188.1 172.9 549.8 204.7 171.2 175.8 203.1 298.3 185.3 224.9 171.4 206.2 331.5 155.9 272.5 237.2 198.0 427.6 176.0 251.1 183.7 205.9 244.4 164.3 202.7 225.7 156.3 271.5 166.9 165.0 189.3 131.6 594.0 170.8 181.8 195.1 136.2 495.0 157.9 240.9 222.3 123.5 365.6 164.3 168.8 187.9 143.5 585.5 190.8 234.7 186.4 115.9 357.3 164.4 186.8 172.4 136.7 449.6 155.4 185.1 183.2 138.4 381.0 215.5 166.9 186.6 139.0 307.2 207.9 157.9 195.6 136.4 435.7 223.6 223.9 179.9

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82 Table 12. Morphology Data for HUVEC (Length 1) Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Length Length 1 Length Length Length 50.02 194.8 40 44.6 55.4 40.69 208 58.2 64.1 40.8 58.41 208.8 87.2 44.4 57.8 49.71 211 32.2 37.6 73.4 48.88 204.1 50.4 41 73.6 52.62 473.8 51.3 35 45.3 53.45 199.5 49.1 71.1 60.3 50.53 266.3 44.5 58.8 52.6 47.3 171.5 58.9 56.4 52.3 55.1 110.4 56.7 59.7 74 66 68 58.5 83.3 68.4 51.9 88.5 55.4 68.8 79.6 59.6 60.6 47.6 66.3 71.2 46.8 89.4 36.4 47.3 66.7 99.5 70.7 59.8 46.9 76.8 65.7 135.2 69.3 44.5 73.7 37.7 79.3 62.6 65.3 77.7 46.9 102.1 58 35.3 69.2 44.97 222.2 36.2 54.6 60.7 48.73 243.9 53.6 42.5 60.8 46.08 99.9 50.1 64.3 49.2 46.59 289.6 69.5 69.1 63.5 42.35 127.9 40.1 66.4 51 55.72 83.7 59.9 55.8 68 67.32 176.5 52.5 72.3 77 66.33 97.4 43.6 79.1 80.2 54.48 139.6 46.9 59.1 68.7 60.6 127.6 49.9 68.7 77 132.1 146.1 41.1 100.1 79.2 45.9 126.8 41.7 89.5 77.3 62.8 164.4 41 96.2 70.4 42.7 134 32.8 67.6 62.3 46.9 114.2 45.9 67.3 44.5 82.8 196.8 53 55.7 68.9 76.4 158.2 45.5 50.3 63.4 55.9 97.5 50.9 60.2 52.2

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83 Table 12 cont. Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Length Length 1 Length Length Length 78.3 106.7 70.3 66.4 85.1 81.3 258.4 82.3 49.8 68.6 62.9 160.8 65.2 76.6 62.3 62.3 191.6 51.3 56.9 53.5 59.6 99.4 54.5 88.4 57.9 73.8 117.6 44.9 89.8 83.2 65.4 142.2 43.5 84.3 58.3 61.9 79 34.3 64.4 85.9 39.5 93 52.8 50.4 62.5 42.56 196 45.3 60.7 71.9 39.62 195.5 47.5 76.9 83.3 38.64 125.8 50.4 45.6 69.6 46.49 200.4 48.6 74.5 63.6 37.35 117.8 53.3 67.1 56.1 45.56 137 54.2 49.7 58.2 46.53 139.4 58.9 48.6 55.6 49.39 104 59.6 45.3 64.7 47.17 147.3 49.5 77.5 66.8

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84 Table 13. Morphology Data for HUVEC (Length 2) Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Length Length 2 Length Length Length 50.02 99.1 40 44.6 55.4 40.69 54.4 58.2 64.1 40.8 58.41 67.7 87.2 44.4 57.8 49.71 79.8 32.2 37.6 73.4 48.88 101 50.4 41 73.6 52.62 98.8 51.3 35 45.3 53.45 50.5 49.1 71.1 60.3 50.53 56.4 44.5 58.8 52.6 47.3 75 58.9 56.4 52.3 55.1 110.4 56.7 59.7 74 66 68 58.5 83.3 68.4 51.9 88.5 55.4 68.8 79.6 59.6 60.6 47.6 66.3 71.2 46.8 89.4 36.4 47.3 66.7 99.5 70.7 59.8 46.9 76.8 65.7 76.2 69.3 44.5 73.7 37.7 79.3 62.6 65.3 77.7 46.9 102.1 58 35.3 69.2 44.97 95.9 36.2 54.6 60.7 48.73 74.2 53.6 42.5 60.8 46.08 99.9 50.1 64.3 49.2 46.59 127.6 69.5 69.1 63.5 42.35 127.9 40.1 66.4 51 55.72 83.7 59.9 55.8 68 67.32 176.5 52.5 72.3 77 66.33 97.4 43.6 79.1 80.2 54.48 73 46.9 59.1 68.7 60.6 127.6 49.9 68.7 77 132.1 84.6 41.1 100.1 79.2 45.9 71.3 41.7 89.5 77.3 62.8 65.9 41 96.2 70.4 42.7 134 32.8 67.6 62.3 46.9 114.2 45.9 67.3 44.5 82.8 67.8 53 55.7 68.9 76.4 82.1 45.5 50.3 63.4 55.9 97.5 50.9 60.2 52.2

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85 Table 13 cont. Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2 Length Length 2 Length Length Length 78.3 106.7 70.3 66.4 85.1 81.3 63.7 82.3 49.8 68.6 62.9 55.5 65.2 76.6 62.3 62.3 50.2 51.3 56.9 53.5 59.6 99.4 54.5 88.4 57.9 73.8 117.6 44.9 89.8 83.2 65.4 142.2 43.5 84.3 58.3 61.9 79 34.3 64.4 85.9 39.5 93 52.8 50.4 62.5 42.56 93.4 45.3 60.7 71.9 39.62 75 47.5 76.9 83.3 38.64 125.8 50.4 45.6 69.6 46.49 73.2 48.6 74.5 63.6 37.35 117.8 53.3 67.1 56.1 45.56 72.9 54.2 49.7 58.2 46.53 139.4 58.9 48.6 55.6 49.39 104 59.6 45.3 64.7 47.17 73.1 49.5 77.5 66.8

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APPENDIX E COLON CARCINOMA C ELL MORPHOLOGY DATA

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87 Table 14. Morphology Data for C aco-2 (Length) (Conventional) 6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME 28.7 32.9 33.9 34.7 33.3 31.8 36.4 30.1 38.8 31.6 30.7 35.5 31.6 37.5 36.1 36.7 33.4 42.3 31.5 33.8 29.0 30.6 37.3 32.0 38.4 39.1 39.5 33.2 28.3 34.8 32.7 38.3 32.7 36.3 37.4 42.9 40.2 30.4 36.6 35.6 40.7 34.3 37.1 37.2 37.7 29.1 33.9 28.5 30.1 38.7 30.7 37.0 40.6 38.1 28.0 33.2 34.8 32.3 31.0 33.3 35.8 37.0 39.4 33.0 35.1 33.8 34.4 36.8 35.5 34.6 34.2 38.2 31.0 33.8 36.8 32.6 45.8 34.1 36.1 35.1 35.0 33.8 29.8 29.8 38.1 36.3 32.2 34.9 46.7 33.0 31.5 34.0 34.4 30.5 33.2 34.0 33.1 37.8 37.4 30.2 33.6 28.6 34.0 38.5 35.6 34.9 41.1 41.1 31.3 32.9 32.6 37.0 36.7 36.3 38.9 37.6 35.4 34.9 33.5 29.3 39.2 32.3 34.2 39.8 48.6 40.6 Table 15. Morphology Data forCaco-2 (Width) (Conventional) 6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME 21.1 19.6 21.8 24.8 29.5 27.5 24.0 24.7 28.9 23.8 19.5 25.8 24.2 33.8 20.7 23.7 21.7 22.6 17.9 20.7 2.3 25.8 24.2 27.2 26.3 27.2 26.0 15.0 18.9 24.0 24.1 26.7 28.7 24.2 25.2 32.3 17.3 21.2 26.1 21.7 25.9 21.6 26.6 24.2 29.7 19.9 18.1 20.4 19.7 27.9 27.1 26.7 25.8 30.5 20.6 17.9 25.4 22.1 31.6 28.0 23.7 24.0 32.7 21.7 19.3 19.3 25.1 22.3 25.8 19.3 26.1 22.4 21.2 16.5 20.4 27.7 24.8 30.8 26.9 24.8 20.6 20.0 21.5 18.2 25.6 28.7 29.0 27.3 25.9 21.6 23.5 18.6 18.9 25.3 29.4 26.3 23.8 23.1 25.4 18.7 18.4 18.9 23.0 29.6 2.7 23.9 23.0 31.9 21.2 20.5 18.2 22.5 28.2 30.1 25.5 23.8 24.8 24.0 21.4 20.9 21.7 23.6 25.1 27.7 26.8 26.7

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88 Table 16. Morphology Data for Caco -2 (Length) (Microgravity) 6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME 40.4 37.7 42.1 39.4 46.8 46.0 33.7 40.0 42.8 36.8 35.4 40.2 38.5 44.4 41.4 36.4 40.8 40.6 37.9 43.1 41.8 47.0 40.2 40.7 35.7 43.7 37.8 35.8 41.4 40.7 39.6 42.2 39.1 41.1 39.1 44.6 37.9 43.3 42.9 48.7 45.1 40.0 44.1 43.7 38.1 42.1 44.9 40.5 47.4 40.0 38.5 40.2 45.4 41.5 44.7 42.0 42.2 41.8 58.2 43.2 41.4 47.2 44.1 39.8 43.4 41.8 49.8 46.7 36.6 38.5 46.6 47.7 40.8 36.8 44.7 43.5 43.7 34.0 45.2 39.9 40.1 43.4 40.4 38.4 41.4 42.0 33.3 38.1 39.1 35.8 35.2 43.8 37.9 35.4 48.1 36.1 41.6 37.2 38.8 39.2 38.4 37.9 36.8 41.2 34.2 46.2 39.6 38.8 43.1 40.6 37.8 44.4 52.5 43.3 44.2 37.0 47.3 47.7 43.8 36.7 39.0 47.3 39.2 44.1 42.3 39.8 Table 17. Morphology Data for Caco-2 (Width) (Microgravity) 6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME 23.0 19.1 25.4 37.4 31.2 27.0 25.5 28.3 23.4 21.5 17.7 26.7 26.2 29.9 27.4 25.5 24.8 32.1 22.0 24.0 22.9 26.3 28.8 24.2 28.2 32.9 25.6 24.0 23.3 21.2 27.6 37.2 25.0 29.7 26.8 26.0 26.5 19.7 25.1 23.7 29.7 28.7 28.4 30.1 26.4 22.1 16.8 22.2 28.4 33.3 27.6 28.4 25.5 24.1 25.2 26.2 24.6 21.1 29.2 27.9 27.4 22.9 26.0 21.6 28.0 22.5 32.6 34.0 28.7 27.4 31.3 33.9 25.7 29.0 25.9 32.2 30.5 28.7 26.8 24.1 31.3 27.4 24.7 21.3 32.7 30.6 27.0 27.8 28.4 25.9 24.7 21.0 15.9 28.4 32.3 27.1 29.8 29.7 28.6 20.0 20.6 23.5 33.2 26.2 27.8 24.3 25.4 27.4 19.6 22.1 21.5 30.1 24.7 28.0 27.1 24.2 31.9 15.3 25.0 23.0 25.0 36.3 27.3 23.2 29.8 26.2

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APPENDIX E QUANTITATIVE IMMUNOHISTOCHEMI STRY DATA SHEETS (CACO2)

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90Table 18. QIHC Data for Caco2 Cultured Conventionally (GRP-R) Negative 6 hr Total Protein (Eu/Pixel) 12 hr Total Protein (Eu/Pixel) 24 hr Total Protein (Eu/Pixel) 48 hr Total Protein (Eu/Pixel) 623.2 422.4 200.8 424.8198.4 439.8183.4 385.8237.4 623.2 408.3 214.9 424.7198.5 406.9216.3 381.1242.1 623.2 409.0 214.2 421.2202.0 427.3195.9 414.4208.8 623.2 423.8 199.4 417.9205.3 423.4199.8 384.5238.7 623.2 399.9 223.3 420.3202.9 414.9208.3 394.2229.0 623.2 416.3 206.9 416.8206.4 409.2214.0 381.7241.5 623.2 422.6 200.6 433.3189.9 409.6213.6 396.4226.8 623.2 400.0 223.2 412.0211.2 422.7200.5 403.7219.5 623.2 440.9 182.3 413.1210.1 427.5195.7 402.9220.3 623.2 447.8 175.4 440.5182.7 443.2180.0 419.7203.5 623.2 421.1 202.1 415.0208.2 406.3216.9 443.4179.8 623.2 442.2 181.0 424.4198.8 429.3193.9 443.4179.8 623.2 446.1 177.1 436.2187.0 410.6212.6 485.9137.3 623.2 442.7 180.5 412.9210.3 409.6213.6 468.9154.3 623.2 439.6 183.6 410.6212.6 415.6207.6 462.0161.2 623.2 443.4 179.8 420.2203.0 424.3198.9 465.0158.2 623.2 424.0 199.2 434.0189.2 413.4209.8 430.1193.1 623.2 421.0 202.2 415.7207.5 419.0204.2 441.1182.1 623.2 396.5 226.7 414.7208.5 425.9197.3 471.2152.0 623.2 453.2 170.0 449.5173.7 420.2203.0 436.2187.0

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91Table 19. QIHC Data for Caco2 Cult ured Conventionally (ME) (GRP-R) Negative 6 hr Total Protein (Eu/pixel) 12 hr Total Protein (Eu/pixel) 24 hr Total Protein (Eu/pixel) 48 hr Total Protein (Eu/pixel) 623.2 428.5 194.7 417.7 205.5 436.2 187.0 440.8 182.4 623.2 413.7 209.5 417.9 205.3 451.5 171.7 413.2 210.0 623.2 426.1 197.1 408.3 214.9 421.7 201.5 407.1 216.1 623.2 431.8 191.4 414.6 208.6 445.6 177.6 401.3 221.9 623.2 434.6 188.6 410.4 212.8 438.9 184.3 427.9 195.3 623.2 424.5 198.7 401.9 221.3 447.3 175.9 416.4 206.8 623.2 446.2 177.0 405.5 217.7 420.9 202.3 445.8 177.4 623.2 432.4 190.8 426.7 196.5 390.9 232.3 444.5 178.7 623.2 436.7 186.5 422.6 200.6 426.1 197.1 438.5 184.7 623.2 431.5 191.7 440.2 183.0 417.6 205.6 449.2 174.0 623.2 447.9 175.3 443.3 179.9 437.8 185.4 422.7 200.5 623.2 444.7 178.5 440.5 182.7 430.8 192.4 413.8 209.4 623.2 444.1 179.1 426.1 197.1 452.5 170.7 402.6 220.6 623.2 423.7 199.5 422.4 200.8 433.0 190.2 421.5 201.7 623.2 444.1 179.1 454.9 168.3 431.9 191.3 410.9 212.3 623.2 424.4 198.8 421.0 202.2 452.2 171.0 408.1 215.1 623.2 430.3 192.9 416.9 206.3 416.8 206.4 435.3 187.9 623.2 418.7 204.5 412.8 210.4 405.1 218.1 422.9 200.3 623.2 441.0 182.2 454.4 168.8 410.2 213.0 395.8 227.4 623.2 429.8 193.4 416.8 206.4 434.3 188.9 445.1 178.1

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92Table 20. QIHC Data for Caco2 Cu ltured in Microgravity (GRP-R) Negative 6 hr Total Protein (Eu/Pixel) 12 hr Total Protein (Eu/Pixel) 24 hr Total Protein (Eu/Pixel) 48 hr Total Protein (Eu/Pixel) 613.2 432.5 180.7 465.9 147.3 520.9 92.3 601.6 11.6 613.2 419.6 193.6 459.5 153.7 524.8 88.4 590.5 22.7 613.2 428.1 185.1 475.3 137.9 580.6 32.6 595.9 17.3 613.2 415.2 198 466.6 146.6 510 103.2 573.8 39.4 613.2 431.8 181.4 470.7 142.5 551 62.2 592.9 20.3 613.2 423.9 189.3 482.8 130.4 557.8 55.4 601.6 11.6 613.2 468.2 145 482.4 130.8 536.2 77 584.1 29.1 613.2 424.9 188.3 466.2 147 534.4 78.8 575.1 38.1 613.2 438.4 174.8 459.3 153.9 524.5 88.7 591.6 21.6 613.2 452.5 160.7 455.6 157.6 539.1 74.1 548 65.2 613.2 410.9 202.3 436.4 176.8 563.8 49.4 572.5 40.7 613.2 449.3 163.9 451.1 162.1 558.9 54.3 570.6 42.6 613.2 440.3 172.9 413.1 200.1 521.9 91.3 560.7 52.5 613.2 401.7 211.5 457.1 156.1 558.9 54.3 560.1 53.1 613.2 423.8 189.4 441.7 171.5 560.9 52.3 582.7 30.5 613.2 457.6 155.6 462.8 150.4 520.9 92.3 580.5 32.7 613.2 427.7 185.5 427.4 185.8 524.8 88.4 586.9 26.3 613.2 406.3 206.9 416.6 196.6 558.9 54.3 560.7 52.5 613.2 426.7 186.5 434.6 178.6 531.7 81.5 568 45.2 613.2 413.3 199.9 459.4 153.8 536.8 76.4 574.2 39

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93Table 21. QIHC Data for Caco2 Cult ured in Microgravity (ME) (GRP-R) Negative 6 hr Total Protein (Eu/Pixel) 12 hr Total Protein (Eu/Pixel) 24 hr Total Protein (Eu/Pixel) 48 hr Total Protein (Eu/Pixel) 613.2 422.2 191 455.7 157.5 536.8 76.4 512.9 100.3 613.2 434.5 178.7 442.6 170.6 533 80.2 500.4 112.8 613.2 433.6 179.6 431.7 181.5 538.6 74.6 500.8 112.4 613.2 446.7 166.5 432.9 180.3 521.8 91.4 511.8 101.4 613.2 437.7 175.5 427.3 185.9 528.5 84.7 490.8 122.4 613.2 445.4 167.8 442.1 171.1 514.3 98.9 511.7 101.5 613.2 431.2 182 434.1 179.1 515.3 97.9 502.2 111 613.2 449.8 163.4 430.4 182.8 493.9 119.3 505.4 107.8 613.2 445.5 167.7 448.6 164.6 502.5 110.7 503.6 109.6 613.2 453.4 159.8 427 186.2 513.3 99.9 512.1 101.1 613.2 444.6 168.6 440.5 172.7 532.6 80.6 525.2 88 613.2 451.1 162.1 422.2 191 534.2 79 525 88.2 613.2 441.9 171.3 428.8 184.4 531.4 81.8 538.6 74.6 613.2 433.7 179.5 460.3 152.9 524.8 88.4 542 71.2 613.2 446.7 166.5 442.8 170.4 521.3 91.9 521.5 91.7 613.2 443.9 169.3 435 178.2 528.7 84.5 539.1 74.1 613.2 433 180.2 418 195.2 515.6 97.6 512.2 101 613.2 435.5 177.7 425.9 187.3 522.6 90.6 531 82.2 613.2 445.3 167.9 430.3 182.9 500.9 112.3 531.6 81.6 613.2 439.1 174.1 436.2 177 503.7 109.5 522.2 91

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94Table 22. QIHC Data for Caco-2 Cultu red Conventionally (ME @ t = 6 hr) Negative 12 hr ME (t = 6 hr) Total Protein (Eu/Pixe l) 24 hr ME (t = 6 hr) Total Protein (Eu/Pixel) 623.2 460.5 162.7 474.4 148.8 623.2 464.9 158.3 490.5 132.7 623.2 438.6 184.6 454.8 168.4 623.2 457.9 165.3 476.4 146.8 623.2 477.6 145.6 456.0 167.2 623.2 412.8 210.4 469.6 153.6 623.2 434.5 188.7 491.9 131.3 623.2 439.1 184.1 485.3 137.9 623.2 418.4 204.8 459.5 163.7 623.2 475.0 148.2 454.8 168.4 623.2 462.2 161.0 435.4 187.8 623.2 483.6 139.6 444.8 178.4 623.2 471.0 152.2 455.7 167.5 623.2 485.8 137.4 450.7 172.5 623.2 497.7 125.5 409.6 213.6 623.2 439.7 183.5 409.2 214.0 623.2 458.3 164.9 468.4 154.8 623.2 477.7 145.5 440.4 182.8 623.2 446.3 176.9 421.3 201.9 623.2 469.5 153.7 419.8 203.4

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95Table 23. QIHC Data for Caco-2 Cultured in Microgravity (ME @ t = 6 hr) (Microgravity) Negative 12 hr ME (t = 6 hr) Total Protein (Eu/Pixe l) 24 hr ME (t=6 hr) Total Protein (Eu/Pixel) 613.2 550.8 62.4 577.9 35.3 613.2 519.6 93.6 546.6 66.6 613.2 559.7 53.5 525.6 87.6 613.2 569.5 43.7 531.9 81.3 613.2 563.5 49.7 521.3 91.9 613.2 575 38.2 499 114.2 613.2 510.5 102.7 517.1 96.1 613.2 578 35.2 515.9 97.3 613.2 527.2 86 502.3 110.9 613.2 583.7 29.5 571.7 41.5 613.2 519.2 94 593.3 19.9 613.2 583.8 29.4 571.5 41.7 613.2 591.4 21.8 572.3 40.9 613.2 514.6 98.6 566.2 47 613.2 549.1 64.1 563.9 49.3 613.2 510.7 102.5 539.8 73.4 613.2 518.2 95 522.4 90.8 613.2 509.9 103.3 540.9 72.3 613.2 534.1 79.1 555.1 58.1 613.2 561.3 51.9 520.3 92.9

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103 Topper, J. N. and J. Michael A. Gimbrone (1999). "Blood flow and the vascular gene expression: fluid shear stress as a modul ator of endothelial phenotype." Molecular Medicine Today 5 (1): 40-46. Underwood, P. A., P. A. Bean and J. R. Gamb le (2002). "Rate of endothelial expansion is controlled by cell:cell adhesion." The Internat ional Journal of Bioc hemistry and Cell Biology 34 : 55-69. Unsworth, B. R. and P. I. Lelkes (1998). "Growing tissues in microgravity." Nature Medicine 4 (8): 901-907. Urbich, C., D. H. Walter, A. M. Zeiher a nd S. Dimmeler (2000). "Laminar shear stress upregulates integrin expression: role in endothelial cell adhesion and apoptosis." Circulation Research 87 : 683-689. Vassy, J., S. Portet, M. Beil, G. Millot, F. Fauvel-Lafeve, A. Karniguian, G. Gasset, T. Irinopoulou, F. Calvo, J. P. Rigaut a nd D. Schoevaert (2001). "The effect of weightlessness on cytoskeleton architecture and proliferation of huma n breast cancer cell line MCF-7." FASEB Journal 15 : 1104-1106. Williams, G. M., S. J. G. Kemp and N. P. J. Brindle (1996). "Involvement of protein tyrosine kinases in regulation of endotheli al cell organization by basement membrane proteins." Biochemical and Ci ophysical Research Communications 229 : 375-380. Wolf, D. A. and R. P. Schwar tz (1991). "Analysis of grav ity-induced particle motion and fluid perfusion flow in the NASA-designe d rotating zero-head-space tissue culture vessel." NASA Technical Paper 3143 Yamada, T., K. Naruse and M. Sokabe (2000) "Stretch-induced mo rphological changes of human endothelial cells depend on the intracellular level of Ca2+ rather than a cAMP." Life Sciences 67 : 2605-2613. Yoffe, B., G.J. Darlington, H.E. Soriano, B. Krishnan, D. Risin, N.R. Pellis, and V.I. Khaoustov (1999). "Cultures of human liv er cells in simu lated microgravity environment." Adv. Space Res. 24 (6): 829-836.

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104 BIOGRAPHICAL SKETCH Rebecca Kathleen Anderson obtained Bachel or of Science degrees in chemical engineering and biology from the Universi ty of South Florida in August 1998. She matriculated in August 1998 into the Medical Sciences Ph.D. program with a concentration in anatomy in the College of Medi cine at the University of South Florida. In January 1999, she began working with Dr. Alway, conducting research in the area of muscle physiology and aging. During her time with Dr. Alway, she published four abstracts and two papers with a postdoctoral fellow, Hans Degens, Ph.D. In January 2000, she transferred into the Depa rtment of Biomedical Engin eering at the University of Florida, specializing in cell and tissue engi neering. In 2001, Ms. Anderson established a collaboration between the University of South Florida and the University of Florida in the area of simulated microgravity research. She received a Master of Science in biomedical engineering in December 2001. In August 2002, she was awarded a Florida Space Grant Fellowship through the Kennedy Space Center. During her time with Dr. Tran-Son-Tay, she published three abstracts and submitte d two papers for peer review.


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EFFECTS OF SIMULATED MICROGRAVITY AND SHEAR ON CELL BEHAVIOR


By

REBECCA KATHLEEN ANDERSON


















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


MAY 2004


































Copyright 2004

by

Rebecca Kathleen Anderson





















This document is dedicated to George and Sue Anderson for their never ending support.















ACKNOWLEDGMENTS

I would like to acknowledge my dissertation supervisory committee for providing

the guidance necessary to complete my doctorate. I would especially like to thank Dr.

Tran-Son-Tay (Chair) and Dr. Don F. Cameron for their endless help in the pursuit of my

doctorate degree in biomedical engineering.

I would like to acknowledge my parents, George and Sue Anderson, for not only

their patience and strength, but also their continuous belief in me. I want to give special

thanks to my mom for being patient through all of the times I made her listen to oral

presentations over and over until I felt they were perfect. Without my mom and dad, the

accomplishments that I have made over the past four years would not have been as

special. I would also like to thank my brother and sister for believing in me and

supporting my career goals.

Special thanks go to Joelle Hushen for all of her help with cell culture using

simulated microgravity. I would also like to thank Dr. Narcisse N'Dri not only for his

help, guidance, and willingness to allow me to vent when I was frustrated about my

project, but also for being my best friend. Finally, I would like to acknowledge the

graduate students in the Biorheology Lab for their help regarding my project.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TA BLE S ....................................................... .. ........... ............ .. vii

LIST OF FIGURES ......... ......................... ...... ........ ............ ix

ABSTRACT .............. .......................................... xi

LIST OF N OM EN CLA TURE .............................................. ....... ..............................

CHAPTER

1 INTRODUCTION ............... ................. ........... ................. ... .... 1

2 O B JEC TIV E S ................................................................. ..... .......... 3

2 .1 R atio n ale .......................................................................................... . 3
2 .2 O bjectiv e .............................................. 4
2.3 Specific A im s............................................................ 5

3 B A C K G R O U N D .................... .... .............................. .......... ........ .......... .. ....

3.1 M echanics of Simulated M icrogravity ....................................... ............... 7
3.2 Cell Adhesion in Simulated Microgravity .........................................................11
3.3 Endothelial Cell Behavior In Vivo vs. In Vitro..................................................18
3.4 Colon Carcinom a Behavior ........................................................................... 25

4 M ATERIALS AND M ETHODS ........................................ ......................... 29

4.1 Conventional Cell Culture Technology ....................................................30
4 1.1 C eating T echniqu e ........................................................... .......... .. .... 3 1
4.1.2 Seeding T technique ............................................. ............................ 32
4.2 Simulated Microgravity Culture Technology.....................................................32
4.3 Perfusion Flow System Technology ............................................ ...............33
4.4 H istological Staining A ssay............................................ ........... ............... 35
4.5 M orphom etric D ata A cquisition................................................ ...... ......... 35
4.6 Quantitative Immunohistochemistry ....................................... ............... 36
4.6.1 Immunohistochemistry Protocol. .................................... .................37


v









4.6.2 Image Acquisition and Protein Quantification........................... ........38
4 .7 S statistic s ........................................................................ 3 9

5 RESULTS AND DISCU SSION ........................................... .......................... 40

5.1 Development of a Culture Protocol for a Planar Substrate in Microgravity ........41
5.2 Effects of Gravity and Shear on Endothelial Cell Morphology .........................44
5.3 Effects of Gravity and Shear on Endothelial Cell Adhesion .............................50
5.3.1 C ell-Substrate A dhesion....................................... .......................... 50
5.3.2 C ell-C ell A dhesion ......................... .. ................................. .. ..... .... 52
5.4 Effects of Gravity and Shear on Potential Cancer Marker .................................55

6 CONCLUSIONS AND FUTURE WORKS................................... ...............62

APPENDICES

A CHARACTERIZATION OF PERFUSION FLOW SYSTEM .................................66

B SHEAR STRESS CALCULATIONS FOR PERFUSION EXPERIMENT ..............68

C QUANTITATIVE IMMUNOHISTOCHEMISTRY DATA SHEETS (HUVEC) ....70

D ENDOTHELIAL CELL MORPHOLOGY DATA ..............................................79

E COLON CARCINOMA CELL MORPHOLOGY DATA.......................................86

F QUANTITATIVE IMMUNOHISTOCHEMISTRY DATA SHEETS (CACO2).....89

L IST O F R E F E R E N C E S ........................................................................ .....................96

BIOGRAPHICAL SKETCH ............................................................. ............... 104















LIST OF TABLES


Table page

1. Peristaltic Pum p C alibration ............................................... .............................. 67

2. Shear Stress Calculations for Perfusion Flow System ............................................69

3. QIHC Data for HUVEC Cultured Conventionally (Integrin a531 Receptor)................71

4. QIHC Data for HUVEC Cultured Conventionally (E-cadherin)..............................72

5. QIHC Data for HUVEC Cultured in Microgravity (Integrin 5a31 Receptor) ..............73

6. QIHC Data for HUVEC Cultured in Microgravity (E-cadherin) .............................74

7. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2)
(Integrin as31 R eceptor) ................................................ ............................... 75

8. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2 ) (E-
c a d h e rin ) ...................................... ................................................. 7 6

9. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2 )
(Integrin a5P 1 R eceptor)........................................................................... 77

10. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2) (E-
cadherin) ..................................... .................................. ........... 78

11. Morphology Data for HUVEC (Perimeter) ..................................... .................80

12. Morphology Data for HUVEC (Length 1)...................................... ............... 82

13. Morphology Data for HUVEC (Length 2)...................................... ............... 84

14. Morphology Data for Caco-2 (Length) (Conventional)......................................87

15. Morphology Data forCaco-2 (Width) (Conventional)..............................................87

16. Morphology Data for Caco-2 (Length) (Microgravity)..............................................88

17. Morphology Data for Caco-2 (Width) (Microgravity) .............................................88

18. QIHC Data for Caco2 Cultured Conventionally (GRP-R) ........................................90









19. QIHC Data for Caco2 Cultured Conventionally (ME) (GRP-R)............. ...............91

20. QIHC Data for Caco2 Cultured in Microgravity (GRP-R) .......................................92

21. QIHC Data for Caco2 Cultured in Microgravity (ME) (GRP-R) ............................93

22. QIHC Data for Caco-2 Cultured Conventionally (ME @ t = 6 hr) ..........................94

23. QIHC Data for Caco-2 Cultured in Microgravity (ME @ t = 6 hr) (Microgravity)...95
















LIST OF FIGURES

Figure page


3.1 Rotary Cell Culture System (RCCS) ........................................ ......................... 8

3.2 Free- body diagram of a particle......... ................. ............................... ...............

4.1 Picture of planar discs ....... ...... ......... .. .... ................ .. ..... 31

4.2 Picture of HARV containing planar disc ........................................ ............... 33

4.3 Picture of perfusion flow system set-up............... ................... .....................34

4.4 Picture of Zeiss morphom etric microscope ..................................... .................36

5.1 H&E stained images of HUVEC (M atrigel)......................................................... 45

5.2 H U V EC cell length (Length 1) ......................................................... ............... 46

5.3 HUVEC cell length (Length 2) ........... .............................. 47

5.4 H U V E C cell perim eter........................................................................ ................... 48

5.5 Histologically stained HUVEC (No Matrigel). .................................. .................49

5.6 Immunohistochemically stained images of HUVEC (integrin aS 1 receptor).............50

5.7 Integrin receptor expression in HUVEC (la subunit).........................................51

5.8 Immunohistochemically stained images of HUVEC (E-cadherin)..............................53

5.9 E-cadherin expression in H U VEC.................................................... ...............54

5.10 Immunohistochemically stained images of Caco-2 (GRP-R).............................. 56

5.11 G R P -R expression........... .................................................................. ........ ... .... 57

5.12 C aco-2 cell length ...................... .................... .. .. ........... .... .. .....58

5.13 Caco-2 cell w idth ............... ................. ........... ....................... 59

5.14 GRP-R expression with delayed addition of ME..................................................60

ix
















LIST OF NOMENCLATURE


Bn ........ .... ..... .. ..............Bombesin

CACO-2 ..............................Human Colon Carcinoma Cells

ECM ................... .... ...............Extracellular M atrix

Eu/pixel ....... ........................Energy Unit Per Pixel

FAC ...................................................Focal Adhesion Com plex

FAK .................. ........ ...............Focal A dhesion K inase

GRP.............. .... ...............Gastrin Releasing Peptide

GRP-R...............................Gastrin Releasing Peptide Receptor

HARV ........................................... High Aspect Ratio Vessel

HUVEC.................. ..................Human Umbilical Vein Endothelial Cells

M E.................................... ........ ....... M ethyl Ester

PK C ...... .... .................................... Protein K inase C

PLC ...... .... ....... ..... ...............Phospholipase C

PVG............................Prosthetic Vascular Graft

QIHC.............................................Quantitative Immunohistochemistry

RCCS ................................................Rotary Cell Culture System

STLV ................. ..............................Slow Turning Lateral Vessel

3-D .................................. ...............Three-dim ensional

2-D ....................................................Tw o-dim ensional



x















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

EFFECTS OF SIMULATED MICROGRAVITY AND SHEAR ON CELL BEHAVIOR

By

Rebecca Kathleen Anderson

May 2004

Chair: Roger Tran-Son-Tay
Major Department: Biomedical Engineering

Based on evidence that cells cultured in simulated microgravity exhibit in vivo

characteristics similar to the parent tissue and that gravity alters metabolic activity and

expression of different genes and cell signaling in various cell lines, the present study

addressed whether or not simulated microgravity provided a better culture environment to

enhance cell adhesion and to study potential markers for diseased tissue. Simulated

microgravity culture promotes aggregation of anchorage dependent cells, but

understanding its effect on cell structures grown on a planar surface (e.g., skin) may

prove critical to the development of vascular grafts and our understanding of cell

metastasis.

Human umbilical vein endothelial cells (HUVEC) and human colon carcinoma

cells (Caco-2), seeded onto planar substrates, were investigated. Three culture

environments (conventional cell culture, simulated microgravity using a High Aspect

Ratio Vessel, and a perfusion flow system) were used to decouple the effects of shear and

gravity. Shear and gravity effects were evaluated using histological staining assays









coupled with morphological measurements. The expressions of two endothelial adhesion

proteins, integrin as51 receptor specific to fibronectin (cell-substrate) and E-cadherin

(cell-cell), and a marker for colon cancer, gastrin releasing peptide receptor (GRP-R)

were determined using quantitative immunohistochemistry (QIHC).

A protocol was developed to culture anchorage dependent cells on planar discs in

microgravity. Alterations in morphology and integrin a531 receptor, E-cadherin, and

GRP-R expression were observed in HUVEC and Caco-2 cells cultured in microgravity.

The results indicated that microgravity culture increased cell length, enhanced cell-cell

and cell-substrate adhesion protein expression in HUVEC, decreased GRP-R expression

in Caco-2 cells, and accelerated the kinetics of GRP-R expression. However, in the

presence of methyl ester, GRP-R expression was down-regulated at a slower rate in

microgravity. Therefore, simulated microgravity culture yields results that are not

obtained in conventional cell culture, and may provide a better culture environment to

study potential markers for disease.














CHAPTER 1
INTRODUCTION

Numerous tissues cultured under simulated microgravity conditions have

demonstrated cellular behavior similar to in vivo tissue, such as metabolic activity, cell

signaling, and gene expression (Unsworth and Lelkes 1998; Hammond et al. 2000). A

microgravity environment for cell culture is defined as a system in which the force of

gravity acting on a particle is minimized (Dedolph and Dipert 1971; Wolf and Schwartz

1991; Gao et al. 1997). Cells cultured in simulated microgravity are able to grow and

differentiate to form 3-D tissue aggregates that structurally and functionally resemble the

parent tissue (Unsworth and Lelkes 1998). Cultures grown in states of microgravity

promote cell-cell interaction by up-regulating various cell-cell adhesion molecules and

ECM proteins (Hammond and Hammond 2001; Cowger et al. 2002). For example,

kidney epithelial cells cultured under simulated microgravity show an increase in cell-cell

and cell-substrate adhesion (Kaysen et al. 1999).

Microgravity has been used mainly for growing 3-D structures but understanding

its effect on cell-cell and/or cell-substrate adhesion of 2-D cell structures may prove

critical to the development of various biomedical devices. In addition, studies of shear

and cell adhesion on a plate are easier to perform and analyze than those conducted on a

sphere. Microgravity studies on 2-D structures have been performed in the Slow Turning

Lateral Vessel (STLV) (Slentz et al. 2001). When skeletal muscle cells were cultured

three-dimensionally in microgravity, large aggregates developed and cells did not grow

well. On the other hand, when they were attached to 2-D silastic membrane inserts in the






2


STLV, skeletal muscle cells reach 80% confluence within the first 24 hours, associated

with an increase in total protein level. Because the STLV requires a large volume of

media and growth factors (55 ml 250 ml, depending on model), culturing in

microgravity and performing chemical assays in this vessel can be expensive. Therefore,

there is a need for a cell culture technique for growing cells on 2-D structures in the

HARV, because this vessel requires 10 ml only.














CHAPTER 2
OBJECTIVES

The study of cell adhesion is important for understanding a wide range of

fundamental biological and medical issues that include resistance change in

microcirculation, cell migration, cell detachment, and cell metastasis. For example, the

ability of a cell to adhere is critical for the proper functioning of the immune system since

adhesion to vascular endothelium is a prerequisite for the circulating leukocytes to

migrate into tissues. Furthermore, that knowledge is also essential in the development of

artificial organs. Depending on the biomedical application, it may be beneficial and

desirable to enhance (vascular grafts) or inhibit (artificial implants) cell adhesion. This is

also relevant to the development of cancer therapies. In addition, the development of a

method for growing cells on a planar substrate under microgravity will allow the study of

shear stress to more easily characterize cell adhesion.

2.1 Rationale

Coronary heart disease, a disease caused by atherosclerosis (narrowing of vessels

due to fatty build ups of plaque), is the single leading cause of death in America today

(American Heart Association 2004). Since prosthetic vascular grafts (PVGs) are used to

replace atherosclerotic vessels or vessels weakened by diseases such as diabetes (Society

for Interventional Radiology 2003), it is vital to develop techniques to enhance the

adhesion of endothelial cells onto the inner lining of PVGs if the lifetime of the

implanted graft is to be optimized. Since the isolation procedure of endothelial cells may

disrupt the cell's ability to develop normal cell-substrate and/or cell-cell adhesion, then a

3









reduction in endothelial cell retention post-implantation can be expected, greatly reducing

the potential longevity of the graft.

Colorectal cancer is the third most diagnosed cancer and the second leading cause of

cancer death in the United States (American Gastroenterological Association 2003). The

five-year survival rate is about 90% when the disease is detected early, compared to only

8% when it is detected in a late stage (American Gastroenterological Association 2003).

GRP/GRP-R is aberrantly expressed in colon cancer cells, although not typically

expressed in normal cells (Carroll et al. 1999a). In the context of cancer, activation of

GRP-R by its ligand results in the phosphorylation of focal adhesion kinase (FAK), an

important protein tyrosine kinase that serves in the regulation of the flow of signals from

the extracellular matrix (ECM) to the actin cytoskeleton. FAK also has been shown to

mediate cell growth and survival as a result of its part in cell adhesion (Parsons et al.

2000). Scientists have demonstrated that FAK is over expressed in a variety of cancers

(Cance et al. 2000; Kornberg 2000). Because of its role in cell growth, survival, motility,

and adhesion, it has been hypothesized that the over-expression of FAK contributes to

metastatic properties of tumor cells (Kornberg 2000). Therefore, it was important to

understand the role of GRP/GRP-R in colon cancer cells cultured in a more natural

environment, such as that created using simulated microgravity culture technology.

2.2 Objective

Because there is evidence that cells cultured in simulated microgravity exhibit in

vivo characteristics similar to the parent tissue (Unsworth and Lelkes 1998; Synthecon

2000), that gravity alters metabolic activity and expression of different genes and cell

signaling in various cell lines (Arase et al. 2002), and that exposure of seeded endothelial

cells to low levels of shear stress prior to graft implantation results in increased









endothelial cell retention post-implantation (Dardik et al. 1999), the following study

intended to address whether or not simulated microgravity provides a better culture

environment to enhance cell adhesion and to study potential markers for diseased tissue.

Shear stress experiments were necessary because shear stress and gravity have direct

effects on cellular activities, and those effects are not decoupled in most microgravity

experiments. Two cell models were used; one model was relevant to vascular grafts

(endothelial cells) and one was relevant to colon cancer (epithelial-derived colon

carcinoma model).

2.3 Specific Aims

The specific aims of the project were to:

Develop a technique for growing cells on a 2-D substrate in microgravity.

Design a cell culture protocol to decouple the effects of gravity and shear stress.

Simulated microgravity was performed using a High Aspect Ratio Vessel
(HARV).
Shear was generated using a perfusion flow system.
Conventional tissue culture acted as a static control.

Characterize cellular morphology (as related to cellular adhesion).

Cellular morphology was semi-quantified using a standard histological assay
and morphometric microscopy.

Characterize cell-cell adhesion.

Cell-cell adhesion (i.e., E-cadherin protein localization and protein level) was
assessed by performing quantitative immunohistochemistry.

Characterize cell-substrate adhesion.

Cell-substrate adhesion (i.e., integrin 1531 receptor localization and protein
level) was assessed by performing quantitative immunohistochemistry.

Characterize a potential marker for colon cancer.






6


* Gastrin-releasing peptide receptor expression was assessed by performing
quantitative immunohistochemistry.














CHAPTER 3
BACKGROUND

3.1 Mechanics of Simulated Microgravity

Simulated microgravity culture technology was developed at the Johnson Space

Center in Houston, Texas (Gao et al. 1997; Unsworth and Lelkes 1998). This culture

technology provides a low shear, low gravity culture environment that creates a

continuous suspension due to a balance of forces acting on a particle (i.e., a microcarrier

bead or a biodegradable scaffold). Based on these properties, the microgravity bioreactor

creates a culture environment that promotes 3-D cell assembly. Due to the nature of this

environment, cell behavior has been shown to mimic in vivo behavior, such as metabolic

activity, cell signaling and gene expression (Unsworth and Lelkes 1998; Hammond et al.

2000). Therefore, simulated microgravity culture technology may prove beneficial to the

study of cellular behavior, such as protein expression and cell morphology.

A microgravity environment for cell culture is defined as a system in which the

force of gravity can be moderated, whereby the force on a particle due to gravity is

minimized (Dedolph and Dipert 1971; Wolf and Schwartz 1991; Gao et al. 1997).

Microgravity is simulated using the Rotary Cell Culture System (RCCS) (Figure 3.1)

developed at the Johnson Space Center and produced by Synthecon, Incorporated, in

Houston, TX. Two bioreactors are used to simulate microgravity (Freed and Vunjak-

Novakovic 1997; Synthecon 2000). The High Aspect Ratio Vessel (HARV) is a disc-

shaped vessel that rotates about a horizontal axis of rotation. A siliconized oxygenator

membrane spans the diameter of the posterior wall of the vessel. The Slow Turning

7









Lateral Vessel (STLV) is a cylindrical-shaped vessel in which the siliconized oxygenator

membrane forms the vessel core. The HARV has a "gas exchange surface per unit of cell

culture volume" of roughly 5x that observed in the STLV (Freed and Vunjak-Novakovic

1997).

HARV



MIOTOR









BASE



Figure 3.1 Rotary Cell Culture System (RCCS). The RCCS was manufactured at
Synthecon, Inc. in Houston, TX. The High Aspect Ratio Vessel (HARV) is a
disc-shaped rotating vessel.

In microgravity culture, fluid flow within the vessel approximates solid body

rotation with the vessel itself, eliminating large shear stresses (Hammond and Hammond

2001). Due to the continuous rotation of the vessel, there is adequate mixing of nutrients

and diffusion of oxygen throughout the culture environment. This mixing, in part, is due

to sedimentation of the particle (Hammond and Hammond 2001). An air-liquid interface

is eliminated by completely filling the vessel with media. Turbulent flow is generated in

the presence of air bubbles (i.e., an air-liquid interface) (Hammond and Hammond 2001).

Utilization of the RCCS to generate a low shear, minimal gravity environment

enables cells to develop 3-D tissue constructs (Unsworth and Lelkes 1998). This










microgravity environment favors "high density" growth of anchorage dependent cells

attached to microcarrier beads. A study by Croughan et al. (1989) demonstrated that

cells cultured in a Ig culture environment require some agitation to maintain a continuous

cell suspension, unless the microcarrier beads are neutrally buoyant. Cell damage is

minimized in a microgravity culture environment due to a reduction in turbulent and

shear forces (Croughan 1989; Schwartz et al. 1992).

Microgravity is simulated by minimizing the force of gravity acting on a particle,

such as a planar disc. In the inertial frame of reference, the forces acting on a particle in

microgravity are shown in Figure 3.2.


(I


FL


FD




n


W


W = weight = mpg Center of Vessel
FL = liquid buoyant force = mig
FD = drag force
Where mp = mass of particle
g = gravity
mi = mass of liquid
o = angular velocity
r = radial position from center

Conditions for particle movement in a circular path:
YFn = mpro2
YFt = 0 (constant speed)



Figure 3.2 Free- body diagram of a particle. The forces acting on a particle in simulated
microgravity are weight, liquid buoyant force, and fluid drag force.









A particle moves in a circular path only when the sum of the forces is equal to mprco2.

Deviation from this condition of equality results in the particle no longer moving in a circular

path. The particle may migrate towards the wall of the vessel.

The shear stress acting on a microcarrier bead in simulated microgravity is

approximately 0.5 dynes/cm2 (Unsworth and Lelkes 1998). Balancing the forces acting

on a microcarrier bead is achieved by adjusting the rotational speed of the vessel until

sedimentation of the particle is inhibited, generating a constant state of free fall. When

microgravity is simulated, the particle remains in a steady state position during vessel

rotation. Typically, microgravity is defined on the milli-gravity scale (10-3g) (Nechitailo

and Mashinsky 1997). Theoretically, microgravity or weightlessness acts intracellularly

by "redistributing" cellular constituents. Therefore, there is an observed relationship

between chemical processes and gravity (Nechitailo and Mashinsky 1997).

Because a relative motion between the particle and the rotating fluid exists, the

particle has the potential to migrate towards the vessel wall (Gao et al. 1997). For a

specified g, the migration time of the particle may be affected by several parameters. The

difference in the densities of the fluid and the particle, the radius of the particle, the

viscosity of the rotating fluid, the angular velocity of the rotating vessel, or the initial

position of the particle within the vessel all affect migration time (Gao et al. 1997).

Simulated microgravity culture technology provides a culture environment that

promotes in vivo-like cell behavior (Hammond et al. 2000). Therefore, microgravity

culture may provide an optimal environment for studying protein expression, alterations

in cell morphology, etc.









3.2 Cell Adhesion in Simulated Microgravity

Gravity controls numerous cellular processes, including calcium signaling,

mechanotransduction across the cytoskeletal network, cell-cell communication, various

ligand-receptor interactions, and possible alterations in cellular morphology (Tairbekov

1996; Akins et al. 1997). NASA developed a rotating culture system, which creates a

culture environment simulating microgravity (i.e., low gravity and low shear) (Dedolph

and Dipert 1971; Wolf and Schwartz 1991). Liu et al. (2003) showed that particles

cultured in microgravity only experience "microgravity" effects when the particle comes

to a steady state position in the top half of the vessel where the level of gravity is

minimized. Migration of particles in simulated microgravity toward the center or outer

wall of the vessel is dependent on the particle size, the geometry of the particle, and the

density difference between the culture media and the particle (Liu et al. 2003). Physical

stimuli from the external environment play a role in cell proliferation and/or cell

differentiation (Vassy et al. 2001). The question, currently, is whether or not a

microgravity culture environment plays a role in the relationship between cell structure

and function. Vassy et al. (2001) showed that when MCF-7 human mammary carcinoma

cells were cultured in microgravity, there were changes in cellular structure and function.

Decreased cell spreading was observed when these cells were cultured in microgravity.

A decrease in the tensegrity of the cells ("a looser perinuclear cytokeratin network") was

observed in instances of decreased cell spreading (Vassy et al. 2001). Responses of

adherent cells to gravity, in vitro, can be characterized by the tensegrity model

(cytoskeletal elasticity model) (Todd and Klaus 1996). Using this model, alterations in

cell morphology and in signal transduction, such as solute transport and "buoyancy-









driven flows (convection and sedimentation)" may be observed under conditions of

microgravity (Todd and Klaus 1996).

Cells and tissues cultured in microgravity behave similarly to their in vivo

counterparts in part due to the low shear and low gravity culture environment that is

created (Unsworth and Lelkes 1998; Hammond et al. 2000). Pancreatic carcinomas

cultured in simulated microgravity behaved similarly to their in vivo counterparts, such

as maintaining heterogeneity, cellular structure, and biological parameters (Nakamura et

al. 2002). Nakamura et al. (2002) concluded that pancreatic carcinomas cultured under

conditions of simulated microgravity exhibited proliferation rates, cellular structure, and

various biological parameters, similar to in vivo tissue. Therefore, simulated

microgravity culture technology provides a culture environment that enables the study of

in vivo tissue utilizing in vitro methods (Nakamura et al. 2002).

Cell behavior, such as cell signaling, metabolic processes, and cell cycle

progression may be altered in conventional culture due to de-differentiation of

differentiated cell types cultured in 2-D (Cowger et al. 2002). By culturing cells in

simulated microgravity, a continuous cell suspension is generated, promoting cell-cell

and cell-matrix associations (Cowger et al. 2002). Varying the shear stress acting on

renal epithelial cells in microgravity, such as by varying media density and by altering

microcarrier bead size and/or density, the terminal or sedimentation velocity of the cells

was decreased. By altering the viscosity of the fluid media, shear and terminal velocity

effects on renal cell behavior were de-coupled, demonstrating how shear stress alters

certain cellular processes (Cowger et al. 2002). Renal epithelial cells cultured on









microcarrier beads form aggregates under conditions of microgravity resulting in cellular

structure no longer resembling their in-vivo counterparts (Cowger et al. 2002).

Hammond et al. (2000) demonstrated a dependence of cellular differentiation on

low shear, 3-D aggregate formation and "cospatial relation of dissimilar cell types."

Cells cultured in simulated microgravity undergo differentiation or de-differentiation

based on the cell's response to altered gravity, illustrating the benefits of microgravity

culture technology (Hammond et al. 1999). Most differentiated cells revert back to a de-

differentiated state when cultured using typical conventional culture (2-D growth)

(Hammond and Hammond 2001). Suspension culture is one means to inhibit cellular de-

differentiation. When renal cortical cells were cultured in simulated microgravity, gene

expression of adhesion molecules, receptors and various intracellular signaling proteins

was altered. However, when these renal cortical cells were placed in a 3g centrifuge,

negligible changes in gene expression were observed (Hammond et al. 2000). When

primary human liver cells were cultured on a Matrigel coated matrix, cellular growth and

de-differentiation may have been inhibited (Yoffe 1999). These cells morphologically

resembled differentiated hepatic tissue through the formation of tissue aggregates. Liver

cells co-cultured with microvascular endothelial cells demonstrated angiogenic

phenotypes within the tissue aggregate. Through this study, Yoffe illustrated how

simulated microgravity may provide a culture environment beneficial for studying cell-

cell interactions and angiogenesis (Yoffe 1999). Rucci et al. (2002) showed, using a rat

osteoblast-like cell line, that when 3-D aggregates formed in microgravity, these cells

produced their own extracellular matrix (Rucci et al. 2002).









Currently, vascularization of organoids cultured in simulated microgravity has

failed (Unsworth and Lelkes 1998). In microgravity, endothelial cells form solid

aggregates instead of tube-like structures, demonstrating how endothelial cells require a

substratum for proper polarization and assemblage. It may prove critical to culture

endothelial cells on a planar surface in simulated microgravity to better understand the

effects of microgravity on endothelial cell behavior. A relationship between microgravity

and alterations in intracellular phosphorylation signaling pathways and in the

cytoskeleton has been shown (Unsworth and Lelkes 1998). Numerous cell types cultured

under microgravity conditions show up-regulation of adhesion molecules, extracellular

matrix proteins and their respective receptors. Numerous studies have demonstrated the

importance of cell-cell interaction for proper tissue function with respect to prostatic

tissue (Cunha 1996; Thomson 1997; Margolis et al. 1999). Therefore, it may be critical

to culture endothelial cells in a continuous monolayer in microgravity to properly study

endothelial cell behavior.

In vivo, endothelial cells typically are exposed to shear stresses greater than 10

dynes/cm2 (Topper and Michael A. Gimbrone 1999; Jessup et al. 2000; Resnick et al.

2003). In microgravity, endothelial cells experience a shear stress less than 1 dyne/cm2

(Unsworth and Lelkes 1998). Interestingly, this low level of shear stress in microgravity

induces metabolic and functional changes. Using a human colorectal carcinoma cell line,

Jessup et al. (2000) showed that alterations in cell proliferation and apoptosis may in part

be due to shear stress, as observed in simulated microgravity (Jessup et al. 2000).

Under microgravity conditions, endothelial cells spread, perhaps in response to

reorganization of the actin cytoskeleton (Romanov et al. 2000). When confluent









endothelial monolayers were exposed to microgravity for short time intervals (1 hr for 3

days), cytoskeletal rearrangement occurred, resulting in cell spreading and cell migration

(Buravkova and Romanov 2001). Alterations in cell shape affect cell function, such as

cell growth and apoptosis (Ingber 1999). Reorganization of the intracellular actin

cytoskeleton may result in alterations of the forces transmitted across the cell membrane

(Ingber 1999; Rucci et al. 2002). Force transmission is greatest at cell-cell and cell-

substrate focal contacts where signaling molecules are concentrated or clustered (i.e.,

integrin clustering) (Ingber 1999). Endothelial cells, when plated in small "focal

adhesion-sized islands", formed long processes which stretched to adjacent islands of

cells. The total area of cell-substrate attachment was unaltered, however (Ingber 1999).

Although integrin clustering will activate intracellular signaling processes, integrin

clustering alone will not ensure cell viability (Ingber 1999).

One effect of microgravity culture on cell morphology is cell rounding ("an actin-

mediated process") (Boonstra 1999). Cytoskeletal reorganization results in altered cell-

matrix binding via signal transduction across the intracellular actin cytoskeleton and the

transmembrane integrin receptor network. Therefore, it may be critical to determine the

role microgravity plays in cell signaling, related to alterations in cell morphology

(Boonstra 1999).

Slentz et al. (2001) developed a novel technique in which skeletal muscle cells

were cultured on a 2-D substrate placed in an STLV. When skeletal muscle cells were

cultured on microcarrier beads, large cell aggregates formed, resulting in increased shear

stress acting on the cells. However, when these skeletal muscle cells were cultured on 2-

D silastic membrane inserts in microgravity, these cells reached 80% confluence within









the first 24 hours and an increase in total cellular protein was observed (Slentz et al.

2001).

Sanford et al. (2002) performed a study culturing bovine aortic endothelial cells in

microgravity using microcarrier beads. These cells formed tissue aggregates which tested

positive for von Willebrand factor, a typical endothelial cell marker, and a morphology

similar to that of in vivo tissue. Cell-cell adhesion protein expression, specifically

proteins found in tight junctions and adherens junctions, was up-regulated resulting in

enhanced cell-cell contact between cells. Although endothelial cells form monolayers

when cultured on microcarrier beads in microgravity, these endothelial cells formed

multilayer sheets surrounding adjacent beads (Sanford et al. 2002).

Jessup et al. (1994) determined that the adhesive properties of endothelial cells

following culture in simulated microgravity were unaltered. However, cell manipulation,

namely the subsequent digestion of dextran microcarrier beads with collagenase and

DNase occurred. To perform adhesion assays following culture in microgravity,

endothelial cells must be detached from the microcarrier beads (Jessup 1994). By

culturing endothelial cells on a planar substrate, not only are the cells cultured in a 2-D

monolayer as observed in vivo, but manipulation of the cells to study cell adhesion is

unnecessary. The planar discs can be placed in a parallel plate flow system with no

manipulation of the cells. Therefore, this culture protocol enables one to directly study

the effects of simulated microgravity on epithelial cell adhesion.

However, numerous cell types have demonstrated alterations in cell adhesion

when cultured in simulated microgravity (Kaysen et al. 1999; Guignandon et al. 2001;

Grimm et al. 2002). Osteoblastic cells exposed to conditions of microgravity









demonstrated a down-regulation in adhesion proteins such as vinculin and extracellular

matrix proteins such as fibronectin (Guignandon et al. 2001). Because exposure to a

microgravity environment may reduce "cytoskeleton-generated tensions" acting on a cell,

subsequent signaling pathways involving the phosphorylation of specific adhesion

proteins such as focal adhesion kinase may be affected (Guignandon et al. 2001). Studies

using various osteoblastic cell lines demonstrated that microgravity alters cellular

morphology and gene expression of such proteins as matrix proteins and various growth

factors (Carmeliet and Bouillon 1999; Hughes-Fulford and Gilbertson 1999).

Fibronectin is continuously produced by osteoblasts during exposure to microgravity.

Therefore, changes in cell morphology are not due to a down-regulation of fibronectin in

the extracellular matrix (Hughes-Fulford and Gilbertson 1999). Kaysen et al. (1999)

showed that when renal epithelial cells were cultured in microgravity, changes in gene

expression of various shear stress response element dependent genes such as ICAM and

VCAM were observed. These results correlate to those observed when endothelial cells

are exposed to flow-induced shear stress. Therefore, they concluded that shear stress

plays a definitive role in genetic alterations due to exposure to microgravity (Kaysen et

al. 1999). When human follicular thyroid carcinoma cells were exposed to microgravity

conditions, up-regulation of extracellular matrix proteins, such as collagen I and III,

fibronectin and laminin was observed (Grimm et al. 2002).

Both cell-cell and cell-substrate adhesion have been shown to be altered under

microgravity conditions. Increased levels of E-cadherin were observed in 3-D tumor

constructs cultured in simulated microgravity (Ingram et al. 1997). Because microgravity









promotes the formation of 3-D tissue aggregates, it makes sense that cell-cell adhesion

and its respective adhesion proteins would be up-regulated (Ingram et al. 1997).

A study by Felix et al. (2000) looked at the effects of simulated microgravity

culture on Ca2+ dependent signaling pathways, such as cell-cell adhesion via E-cadherin.

This Ca2+ dependence is through activation of the protein kinase C (PKC) second

messenger system. Tracheal epithelial cells cultured in simulated microgravity retain

their mechanically activated Ca2+ signaling pathway activity. They concluded that

tracheal epithelial cells cultured in simulated microgravity were more PKC sensitive than

cells cultured conventionally. This translates to the epithelial cells' increased ability to

activate this second messenger system. They also observed a morphological change in

tracheal epithelial cells cultured in microgravity. The cells appeared taller and more

cuboidal than those cells cultured conventionally (Felix et al. 2000).

3.3 Endothelial Cell Behavior In Vivo vs. In Vitro

Mechanical forces alter cell behaviors, such as cell shape, cell growth, cell

proliferation, extracellular matrix remodeling, and signal transduction (Chicurel et al.

1998). Mechanical forces may not distribute evenly across the cell membrane, but

instead may localize at sites of attachment such as at cell-cell and cell-matrix junctions.

The binding of integrin receptors to extracellular matrix proteins, forming focal adhesion

complexes (FAC), is a strong bond perhaps due to receptor clustering and the number of

bound receptors (Chicurel et al. 1998). This is known as mechanical coupling. FAC

links the extracellular matrix to the intracellular actin cytoskeleton, providing a

continuous network through which a mechanical stimulus can traverse across the cell

from one pole to the opposite pole. Other FAC associated proteins include vinculin,

paxillin, a-actinin and tyrosine kinases. This signal transduction may explain why









endothelial cells and fibroblasts orient in the direction of flow when a shear is applied.

When the balance of forces acting on a cell is altered, gene expression as well as

extracellular matrix remodeling can occur (Chicurel et al. 1998). The greatest alteration

in cell behavior that occurs when there is a shift in the balance of forces acting on a cell is

changes in cell morphology. If the strength of the cell-matrix attachment is greater than

the cytoskeletal arrangement, then the cell will flatten and spread. Altered cell shape may

result in inhibited cell growth and enhanced cell differentiation (Chicurel et al. 1998).

Mechanotransduction is one process that activates phospholipase C (PLC), which

in turn activates a signaling cascade, resulting in the release of intracellular Ca2+ (Felix et

al. 1998). However, when epithelial cells are cultured in microgravity, mechanical forces

acting on the cells are reduced, and therefore, activation of this second messenger system

may be decreased. This reduced activation may be due to alterations in the cytoskeleton

and arrangement of actin stress fibers when cells are cultured in microgravity. Felix et al.

(1998) concluded from their study that although the mechanical forces which typically

activate this particular signaling cascade are reduced in simulated microgravity, the

release of intercellular Ca2+ is not altered in states of microgravity (Felix et al. 1998).

Cell-substrate adhesion is created by binding the intracellular cytoskeletal

network composed of actin filaments to the underlying extracellular matrix via the

integrin receptor family, forming a focal adhesion (Parsons et al. 2000). It has been

shown that signals are transmitted across the focal adhesions through the actin

cytoskeleton, regulating various cellular processes. Parsons et al. (2000) conducted a

study to determine what role focal adhesion kinase plays in the regulation of signal

transmission from the extracellular matrix to the intracellular actin cytoskeleton. The









adhesion complex is composed intracellularly of actin, vinculin, talin, and a-actinin. It

has been shown that tyrosine kinase phosphorylation plays a role in the assembly of

adhesion proteins to form FAC. Focal adhesion kinase (FAK) is a non-receptor protein

tyrosine kinase recruited to the focal adhesion complex upon binding of the integrin

receptor to the underlying extracellular matrix (Parsons et al. 2000). When endothelial

cells were grown on Matrigel coated substrates, enhanced tyrosine phosphorylation of

several adhesion proteins was observed (Williams et al. 1996). It is thought that FAK

plays a role in cell morphology and migration. When FAK activation occurred,

morphological and migrational alterations occurred, thereby producing, "capillary-like

structures" in which cells formed cord-like structures (Williams et al. 1996). Kano et al.

(2000) showed that when a fluid shear stress was applied to endothelial cells, increased

focal adhesion kinase expression near the basal surface of the cells occurred. It was

concluded that when the endothelial cells experience a shear stress, they increase the

strength of their cell-substrate focal adhesions by recruiting a greater concentration of

focal adhesion kinase, therefore increasing the size of the focal adhesions. They have

also been able to conclude that the apical surface of the cell, in which the plasma

membrane is attached to actin stress fibers, may be involved in mechanotransduction

directly or that the shear force is transmitted via the cytoskeletal network to the focal

adhesions or to the site of cell-cell adhesion (Kano et al. 2000).

When endothelial cells are exposed to a fluid shear, several major cellular events

occur (Ballermann et al. 1998; Dardik et al. 1999; Topper and Michael A. Gimbrone

1999). First, the endothelial cells reorganize their actin cytoskeleton in order to orient the

cells in the direction of flow. The cells also demonstrate alterations in their metabolic









processes, cell cycle activity, and expression of various cell adhesion proteins in the

presence of shear stress. Cell surface receptors and expression of various signal

transduction proteins are altered in the presence of shear stress (Ballermann et al. 1998;

Dardik et al. 1999; Topper and Michael A. Gimbrone 1999).

Epithelial cells, when adhered to a surface, are polarized (Blaschuk and Rowlands

2000; Braga 2000). The basal surface of the cell attaches to the underlying basement

membrane and the apical surface faces the lumen of the vessel or duct. Epithelial cells

are able to maintain a specific cell shape, such as squamous with respect to endothelial

cells, due to the interaction of the intracellular cytoskeleton and cell-cell and cell-

substrate adhesion. Adherens junctions, such as tight junctions, form cell-cell contacts.

Cadherins are calcium dependent adhesion proteins which make up the adherens

junctions (Blaschuk and Rowlands 2000; Braga 2000). When cadherin function is

altered, typical epithelial features are inhibited. Cadherins are composed of parallel

dimers, which bind adjacent cells extracellularly. The intracellular cadherin domains

bind to the intracellular actin cytoskeleton via such proteins as p-catenin, plakoglobin and

a-catenin. When cell-cell adhesion occurs, cadherin molecules cluster at the site of

adhesion, similar to integrin receptor coupling for cell-substrate adhesion. Once the

cadherins associate with the intracellular actin cytoskeleton, mechanical forces act on the

cell at these adhesion contacts, therefore mediating a specific polarized morphology

(Braga 2000). Vascular endothelial cadherin (VE-cadherin) is a single pass

transmembrane protein expressed by endothelial cells. In order for endothelial cells to

form a functional confluent monolayer, both cell-substrate and cell-cell adhesion









complexes must form. Cadherins, therefore, play a pivotal role in the maintenance of a

functional endothelial monolayer (Blaschuk and Rowlands 2000).

The morphology of vascular endothelial cells is that of a spindle shape, aligning

along the "longitudinal axis" of the vessel (Yamada et al. 2000). However, when

endothelial cells are cultured conventionally, they have a cobblestone morphology and

have no specific organizational pattern. The morphology of endothelial cells in vivo is

believed to be regulated by blood flow induced shear stress and wall pressure due to

stretch (Yamada et al. 2000).

Damage to the endothelial lining of blood vessels due to atherosclerosis or various

medical procedures, such as angioplasty, results in the development of intimal

hyperplasia (i.e., the migration of smooth muscle cells into the endothelial region)

(Underwood et al. 2002). Development of intimal hyperplasia results in the occlusion of

the vessel or even occlusion of a vascular graft. Underwood et al. (2002) showed that

mature cell-cell junctions via cadherin binding are formed following 24 hours of seeding

endothelial cells, possibly inhibiting cell migration and proliferation. When exogenous

vascular endothelial growth factor (VEGF) is present, cadherin junctions are disrupted

due to phosphorylation of the cadherin protein. Therefore, in the presence of VEGF, cell

proliferation and migration (spreading) occurs (Underwood et al. 2002).

Fisher et al. (2001) conducted studies on how endothelial cells sense alterations in

levels of shear stress acting on them. Cell membrane and integrin receptors on the basal

surface of the cell membrane can sense alterations in shear and then transmit these

changes in stimuli to the intracellular actin cytoskeleton, where reorganization of the









actin cytoskeleton results in alterations in endothelial cell morphology (Fisher et al.

2001).

For cell detachment to occur, a shear stress great enough to disrupt cell-cell and/or

cell-substrate adhesions must be imposed (Peel and DiMilla 1999). Cell detachment is

dependent on the morphology and orientation of cells with respect to fluid flow. In a

study by Peel et al. (1999), the critical shear stress for detachment was determined for

stromal cells. Strength of adhesion was greatest in moderately confluent cultures,

whereas 100% cell detachment was observed when only cell-cell interactions were

present. When the cells were subconfluent, cell detachment was dependent on cell-

substrate interactions (Peel and DiMilla 1999).

Cell cycle progression is dependent on cell-substrate adhesion (Schwartz and

Assoian 2001). It has been shown that the integrin a531 receptor associates with other

proteins, namely caveolin and Shc, to initiate the cell cycle. Therefore, cell-substrate

adhesion may be necessary for epithelial (endothelial) cells to grow (Schwartz and

Assoian 2001).

Levenberg et al. (1998) showed that there is an autoregulatory pathway that is

activated by the presence of cell-cell or cell-substrate adhesion sites. Two distinct

pathways have been identified. When cell-cell adhesion is enhanced, cell-matrix

adhesion is decreased and when cell-matrix adhesion is enhanced, cell-cell adhesion is

decreased. Both processes are dependent on tyrosine phosphorylation but appear to be

distinct from one another (Levenberg et al. 1998).

A study by Ko et al. (2001) addressed what role Ca2+ signaling plays in cell-cell

adhesion, perhaps through activation of PLC. It is known that in order for cell-cell









adhesion to occur by way of cadherin binding, extracellular Ca2+ must be present. They

showed that cell-cell contacts induced the release of intracellular calcium, facilitating the

binding of cadherins and P-catenin to the actin filaments comprising the cytoskeleton,

which resulted in increased strength of cell-cell contacts (Ko et al. 2001).

Three major types of cell-cell junctions are present in endothelial cells, tight

junctions, adherens junctions, and gap junctions. The adherens junction is formed by the

binding of cadherin to its respective intracellular catenin. Schnittler et al. (2001) showed

that when minor actin depolymerization occurred, changes in cell morphology occurred,

namely a decrease in cell height due to a decreased stress acting on the cell. When a

large degree of actin depolymerization occurred, cell-cell adhesion was interrupted. They

showed that actin dynamics must be in balance in order to maintain cell-cell adhesion and

that decreased cell-cell adhesion was due to decreased binding of a-actinin to the cell-cell

junction (Schnittler et al. 2001).

It has been determined that the apical surface of the endothelium experiences the

shear stress initially (Langille 2001). The major morphologic response endothelial cells

have to shear stress is the re-orientation of the cells in the direction of flow. A minor

morphologic response is cell elongation due to the shear force. When endothelial cells

are exposed to large levels of shear, the actin cytoskeleton undergoes reorganization,

therefore resulting in the reduction of cell-cell contacts and an increase in focal adhesions

at the cell-matrix region. It has been determined that the greatest alterations in cell

morphology appear to be due to assembly and disassembly of adherens junctions

(Langille 2001).









A study by Urbich et al. (2000) concluded that shear stress does result in an up-

regulation of integrin receptors. However, the production of nitric oxide by the

endothelium in response to shear stress also resulted in the up-regulation of integrin

receptors. Because cells experience low levels of laminar shear stress during

microgravity culture, some up-regulation of the integrin receptor protein in comparison to

a static conventional culture environment is expected (Urbich et al. 2000).

3.4 Colon Carcinoma Behavior

Activation of GRP/GRP-R may play a critical role in altered cell adhesion,

resulting in tumor progression (Schumann et al. 2003). GRP, a bombesin-like peptide,

acts as either an autocrine hormone (secreted by and acts on same cell) or a paracrine

hormone (secreted by one cell and acts on a local cell) within the gastrointestinal tract.

Bombesin-like receptors are thought to act like mitogens, acting as an autocrine growth

factor that plays a role in tumor progression. When bombesin-like peptides bind to the

GRP-R, a second messenger signaling cascade is initiated through the activation of

phopholipase C (PLC) (Schumann et al. 2003).

Although GRP-R mRNA is often expressed in colon cancer cell lines, the

message may become mutated resulting in non-functional GRP-R (Carroll et al. 2000a).

From a previous study, it was shown that that a down-regulation of GRP-R resulted in

dedifferentiation of tumor cells (Carroll et al. 2000). Histologically, well-differentiated

tumors are similar to the tissue of origin whereas poorly differentiated tumors show a

disorganized histology. A study by Carroll et al. (1999) showed that GRP/GRP-R is

aberrantly expressed in well-differentiated tumors, but a decreased expression of

GRP/GRP-R is observed in tumors undergoing dedifferentiation. Therefore, GRP/GRP-

R acts both as a mitogen (stimulates mitosis) and a morphogen (stimulates differentiation









of cells/tissue; involved in embryogenesis) (Carroll et al. 1999; Glover et al. 2003).

Although GRP may cause cell proliferation, it does not act as an "oncogenic growth

factor" (Carroll et al. 1999a). Carroll et al. (1999) demonstrated that aberrant expression

of both GRP and GRP-R is observed in adenocarcinomas of the colon, but do not appear

to up-regulate as the tumor stages. Because it was shown that GRP/GRP-R expression

plays a direct role in tumor cell differentiation, it was concluded that GRP/GRP-R may

act in an autocrine manner with respect to cell differentiation. GRP/GRP-R has been

shown to play a role in the alteration of cell-cell attachment, therefore acting as a

morphogen (Carroll et al. 1999). Because GRP/GRP-R plays a role in the differentiation

of colon cancers, it was concluded that GRP/GRP-R expression may re-capitulate its role

in normal intestinal development (Carroll et al. 2002).

Bombesin plays a role in alterations in cell morphology, alterations in the actin

cytoskeleton in "nontransformed" cells, and enhancing cell motility of cancer cells

(Saurin et al. 1999). The process of metastasis requires many steps including cell

locomotion, cell-substrate adhesion, and cell proliferation. Saurin et al. (1999)

demonstrated that bombesin stimulated cell spreading, formation of lamellipodia, and

adhesion to the extracellular matrix (collagen type I). From this study, it was concluded

that GRP-R may play a role in the "invasive properties" of colonic cancer cells. The

formation of lamellipodia has been shown to be related to the process of tumor cell

metastasis (Saurin et al. 1999).

Epithelial cells lining a normal GI tract do not typically express FAK. FAK is

shown to be present in well-differentiated tumors. FAK expression was up-regulated as

the tumors became better differentiated (Carroll et al. 2000). Focal adhesion kinase









(FAK) is a cytoplasmic adhesion protein localized at focal contacts. Although FAK

expression is up-regulated in colon cancers, the degree of FAK activation due to tyrosine

phosphorylation is critical (Theocharis et al. 2003; Matkowskyj et al. 2003a). Human

colon cancers are typically heterogeneously differentiated. Differentiation with respect to

tumors defines how much tumor cells resemble the normal tissue of origin. Tumor

differentiation plays a role in tumor metastasis. Although GRP/GRP-R is not typically

expressed by the epithelial lining of the gastrointestinal tract, an increased expression of

GRP/GRP-R was observed in "post-neoplastic transformation, resulting in better-

differentiated tumors" (Theocharis et al. 2003; Matkowskyj et al. 2003a). A relationship

between better differentiated tumors and the expression of focal adhesion kinase (FAK)

via the GRP/GRP-R signaling pathway has been determined. It was shown that FAK

phosphorylation at tyrosine 397 and tyrosine 407 is a function of tumor cell

differentiation and the expression of GRP/GRP-R. Therefore, GRP/GRP-R plays a role

in cancer cell differentiation via FAK phosphorylation at specific sites (Theocharis et al.

2003; Matkowskyj et al. 2003a).

One study demonstrated that the presence of GRP-R in colonocytes resulted in a

constitutive activation of the receptor, resulting in enhanced cell growth in the absence of

agonist binding, growth factors, and serum (Ferris et al. 1997). This study identified the

constitutive activation of GRP-R in the absence of mutations, therefore concluding that

GRP-R may act as an oncogene with respect to colon cancer.

Transition of cells from an epithelial to a mesenchymal state (epithelial-

mesenchymal transition) plays a role in morphogenesis and in the development of









carcinomas (Thiery 2003b). Carcinoma progression has been linked to de-differentiation

of tumoragic epithelial cells which further leads to invasion and metastasis.

The process of cell adhesion plays a role in other cellular processes including

proliferation, differentiation, migration, and death (Thiery 2003a). Tissue types

comprised of epithelial cells are more likely to develop into cancer, typically carcinomas

(- 90% if tumor types), than other cell types. Due to alterations in adhesive properties of

epithelial cells, tumor progression occurs originally from an adenoma towards

development of invasive carcinomas. During this progression, polarization of the

epithelial cells regresses and a disorganized arrangement of the cells within the tumor

occurs (Thiery 2003a; Thiery 2003b).

Alterations in gravity, such as hypogravity or hypergravity, result in the activation

of the protein kinase C (PKC) signaling cascade (Han et al. 1999). Activated PKC plays

a role in cell proliferation. With respect to human tumor cells, alterations in gravity

resulted in mutations in "microsatellite sequences" ("short repeats of 1-4 nucleotide

units"). These minor mutations result in "mismatch repair-deficient human tumor cells"

(Han et al. 1999).

A study by Rhee et al. (2001) demonstrated that when prostatic cancer cells are

cultured three-dimensionally in simulated microgravity, the epithelial derived cells

demonstrated potential metastatic markers not typically observed in conventional culture.

They concluded that transitioning of carcinogenic prostatic tissue from an androgen-

dependent to an androgen-independent pathway is mechanistically related to the presence

of tumorigenic and metastatic markers (Rhee et al. 2001).














CHAPTER 4
MATERIALS AND METHODS

The present study investigated the effects of shear stress and gravity on cell

behavior, such as cell morphology and membrane bound receptor levels when anchorage

dependent cells were cultured on planar discs in simulated microgravity. A protocol for

culturing cells seeded onto planar discs in simulated microgravity, in a perfusion flow

chamber or in conventional culture, and to study the effects of shear stress and gravity on

cell behavior was developed. It was proposed that simulated microgravity culture

technology would enhance cell-cell and/or cell-substrate adhesion protein levels in

human umbilical vein endothelial cells (HUVEC) and might provide a better environment

to study potential markers for diseased tissue. Typical 3-D substrates commonly used for

seeding and culturing cells in simulated microgravity include microcarrier beads and

PLA/PGA scaffolds. However, the evaluation of the strength of endothelial cell adhesion

cannot be easily performed on these spherical beads. Therefore, 2-D planar discs were

used to culture adherent cells in microgravity. Microgravity was simulated using the

HARV and the perfusion flow system was composed of a parallel plate flow chamber

(both plates fixed) driven by a peristaltic pump. Histological assays were performed

using a standard histological staining assay (hematoxylin and eosin staining) in order to

analyze alterations in cell morphology (cell width, cell length and cell perimeter) due to

changes in the levels of shear and gravity placed on the cells. Quantitative

immunohistochemistry was employed to identify the presence, localization and

concentration of three membrane bound receptors, integrin a531 receptor (cell-substrate









receptor specific to fibronectin), E-cadherin (cell-cell integral protein), and gastrin-

releasing peptide receptor (GRP-R).

4.1 Conventional Cell Culture Technology

The protocols for cell culture were performed as described previously in the

Clonetics catalog (2000-1). HUVEC (Cambrex, Inc., Walkersville, MD) cells were

initially plated onto non-coated T-75 flasks (Fisher Scientific, USA) and grown to

roughly 80% confluence prior to harvesting. The cells were cultured in a 37 C and 5%

CO2 95% air environment. Upon reaching 80% confluence (5-9 days), the cells were

washed with Hanks Balanced Salt Solution (Fisher Scientific, USA) to remove any serum

supplemented media remaining on the cells. The cells were then trypsinized using a

0.25% trypsin/EDTA (Fisher Scientific, USA) solution until the cells began to round up

and detach from the flask surface. The cell solution was placed in a 15 ml conical tube

and centrifuged at 1000g for approximately 3 min. The supernatant was aspirated and the

cells were further washed 2x in unsupplemented endothelial basal media (EBM)

(Cambrex, Inc., Walkersville, MD). Cell viability was determined by staining a sample

of the cell suspension with trypan blue. Cells, which took up the stain, were not viable.

Caco-2 cells were initially plated onto non-coated T-25 flasks (Fisher Scientific,

USA) and grown to roughly 80% confluence prior to harvesting. The cells were cultured

in a 37 C and 5% CO2 95% air environment. Upon reaching 80% confluence (2-3

days), the cells were washed with Hanks Balanced Salt Solution (Fisher Scientific, USA)

to remove any serum supplemented media remaining on the cells. The cells were then

trypsinized using a 0.25% trypsin/EDTA (Fisher Scientific, USA) solution until the cells

began to round up and detach from the flask surface. The cells were counted using a

hemacytometer to determine a specified cell concentration.









4.1.1 Coating Technique.

Circular planar discs were prepared from plastic cover slips using a 14" hole

punch (Figure 4.1). For the endothelial study, circular planar discs were prepared from

22 mm x 22 mm unbreakable plastic cover slips (Fisher Scientific, USA) using a 14" hole

puncher. The discs were sterilized by placing them in a Petri dish containing a 70%

ethanol solution for approximately 5 min. The ethanol was aspirated off and the discs

were allowed to dry under UV light in a tissue culture hood. The planar discs were

coated by dipping the discs in a 20% solution of Matrigel (Fisher Scientific, USA) in

unsupplemented EBM (Cambrex, Inc., Walkersville, MD). The discs were then placed in

a 24 well-plate, three discs per well. While harvesting the cells, the 24 well-plate was

placed in the incubator to allow the Matrigel coating to polymerize on the discs.
















Figure 4.1 Picture of planar discs. Planar discs were constructed from plastic cover slips.
Planar discs for the endothelial study were constructed from unbreakable
plastic cover slips. Planar discs for the Caco-2 study were constructed from
Thermanox plastic cover slips.

For the Caco-2 study, circular planar discs were prepared from Thermanox plastic

cover slips (Fisher Scientific, USA) using a 4" hole puncher. The discs were sterilized

via autoclaving. These discs were not coated with Matrigel. Matrigel interrupts the role









of methyl ester as an antagonist for GRP-R. The discs were then placed in a 24 well-

plate, three discs per well.

4.1.2 Seeding Technique

HUVEC were trypsinized, as stated in the conventional cell culture section, and

the cell suspension was placed in a 15 ml centrifuge tube. The cell suspension was

centrifuged at 1000 rpm for 3-5 min and the supernatant was aspirated off. The cells

were washed in unsupplemented EBM at least 2 times. The cells were resuspended in 5

ml of media supplemented with antibiotics, growth factors and 10% FBS. The cell

suspension was then evenly distributed amongst the wells. The cells seeded in

conventional culture until roughly 80% confluent (approximately 4-6 days). Following

the seeding period, the discs were either placed in simulated microgravity culture for an

additional 48 hrs, placed in a perfusion flow chamber set at a specified flow rate for an

additional 48 hrs, or left in conventional culture for an additional 48 hrs.

Caco-2 cells were trypsinized, as stated in the conventional cell culture section,

and the cell suspension was placed in a 15 ml centrifuge tube. The cell suspension was

diluted in approximately 10 ml of media and the cells were counted using a

hemacytometer. A cell concentration of approximately 60,000 cells/cm2 was determined

for seeding. The cells seeded for approximately 12 hrs. Following the seeding period,

the discs were either placed in simulated microgravity culture for time periods of 2, 6, 12,

24, and 48 hrs or left in conventional culture for time periods of 2, 6, 12, 24, and 48 hrs.

4.2 Simulated Microgravity Culture Technology

To culture anchorage dependent cells on a planar substrate in the HARV,

modification of the conventional culture protocol was necessary. Following seeding, a

single disc was placed in each HARV via sterile forceps (Figure 4.2). The disc was









placed on the membrane attached to the posterior wall of the HARV. The anterior and

posterior portions of the HARV were screwed together and 10 ml of media supplemented

with 2 g of 2000 kDa MW dextran in 50 ml of EBM were used to fill the chamber. The

dextran was used to increase the viscosity of the media. The HARVs were then placed

on the rotating base which was placed inside the incubator. The rotational speed of the

HARVs was adjusted until the discs came to a steady state position in the vessel.





? O



region of HARV filled
Switch media (10 ml)






Figure 4.2 Picture of HARV containing planar disc. A single planar disc is placed within
the interior of the HARV using sterile forceps. The anterior and posterior
portions of the HARV are screwed together and the vessel is filled with
media.

4.3 Perfusion Flow System Technology

For the perfusion flow system, due to an increase in the fluid viscosity, the flow

rate was decreased in order to maintain a shear stress similar to that observed in

simulated microgravity. For the perfusion flow chamber, the discs were placed on top of

a glass slide, which fit directly into the flow channel (Figure 4.3). The discs were

approximately 0.2 mm in height. Therefore the chamber height was reduced from 1.5

mm to 1.3 mm. The discs were prepared as previously stated. The discs were then

placed back into the original punched out spaces in the cover slips and the cover slips








were placed in the chamber. Via calculation of the entrance length and the length of the

flow channel, approximately 10 substrates could be placed in the flow channel per

experiment (approximately 2 cover slips).






planar discs
S'...--- placed in perfusion
S. ,, flow system peristaltic
\ p mp




parallel plate flow chamber




-r- 5

Figure 4.3 Picture of perfusion flow system set-up. A parallel plate flow chamber driven
by a peristaltic pump comprised the perfusion flow system. The pump was set
to run at flow rates (Q) corresponding to 2 mL/min, 4mL/min and 8 mL/min.

The shear stress applied to seeded HUVEC ranged from 0.25-1.01 dynes/cm2

using the following formula, c = 6LQ/wh2, calculated from the flow between two parallel

plates where Q is the flow rate, [t is the fluid viscosity and w and h are the width and

height of the chamber, respectively. A Rainin Dynamax peristaltic pump was used to

drive the media into the flow chamber. The pump generated a multi, unidirectional pass

flow. The Dynamax peristaltic pump was connected to the perfusion flow chamber via

3/16" silastic tubing (Fisher Scientific, USA). The perfusion flow chamber was placed in

the incubator at 37 C and 5% CO2 95% air for 48 hrs.









4.4 Histological Staining Assay

Following the specified culture period, HUVEC were fixed with 4%

paraformaldehyde/PBS while still remaining attached to the planar discs. The cells were

subsequently stained with hematoxylin and eosin. Harris' hematoxylin (Fisher Scientific,

USA) is a basic stain that will stain the nucleus and nucleolus of cells blue. Eosin (Fisher

Scientific, USA) is an acidic stain that will stain the protein portion of a cell pink.

Morphological measurements (length, width, perimeter, and aspect ratio) were collected

via digital photomicroscopy and the imaging program, Axiovision 3.1 (Carl Zeiss

Microimaging, Inc., Thornwood, NY).

The protocol for typical histological staining is described below. The cells were

fixed in 4% paraformaldehyde for 20 min. The cells were then rinsed 3x (5 min each

wash) with phosphate buffered saline (PBS). Hematoxylin was placed on the cells for

roughly 2 min. Following the 2 min of hematoxylin stain, the cells were washed with

tap water (acts as a bluing solution). Eosin was placed on the cells for roughly 1 min.

Following the 1 min of eosin stain, the cells were dipped approximately 15x in 95%

ethanol to prevent the eosin from washing out in subsequent washes. The discs were

mounted cell side down onto a glass side using a water based glycerol/gelatin mounting

agent.

4.5 Morphometric Data Acquisition

Specific morphological parameters were measured. These parameters included

perimeter, length, width, and aspect ratio (L/W). Images of the cells were taken at a 10x

magnification for the endothelial cell study and at a 40x magnification for the Caco-2

study using a Zeiss microscope with an Axioplan camera (Figure 4.4).

























--V



Figure 4.4 Picture of Zeiss morphometric microscope. A Zeiss microscope connected to
an Axioplan camera was used to collect images.

Morphological measurements were taken using the image acquisition program,

Axiovision 3.1 (Carl Zeiss Microimaging, Inc., Thornwood, NY). Length and width

were measured using the Length icon. Perimeter was measured using the Curved Spline

icon. Each morphological parameter was measured three times for accuracy. An average

of each measurement was recorded. Three cells were selected per three clusters of cells

containing 5-7 cells on each disc. From each cluster, 3 cells were analyzed. Three discs

were used for analysis for each culture environment.

4.6 Quantitative Immunohistochemistry

Immunohistochemistry is a technique used to identify the presence of a particular

protein using an antibody against that protein. Immunohistochemistry was performed on

cells cultured in simulated microgravity, in a perfusion flow system generating a shear

stress of 0.51 dynes/cm2 and 1.01 dynes/cm2, and in conventional culture, using

antibodies against the integrin a531 receptor (cell-substrate) and E-cadherin (cell-cell) for









the endothelial study and GRP-R for the Caco-2 study. All three of these receptor

proteins are integral membrane proteins.

4.6.1 Immunohistochemistry Protocol.

Following the specified culture time period for the endothelial cells or the Caco-2

cells, the discs were placed in a 24 well plate and washed with tris buffered saline (TBS)

to remove excess dextran from the discs. Any debris, such as dextran, remaining on the

cells prior to fixation resulted in dark staining in that region. Therefore, washing the cells

prior to fixation was critical. The cells were then fixed in a 3.7% formaldehyde/TBS

solution at 37 C and 5% CO2 95% air for 30 min. The fixative was aspirated off and

the cells were washed with TBST (tris buffered saline and Tween 20). TBST was used

for cytosolic proteins (to perforate the membrane). The three proteins of interest were

integral membrane proteins, containing an extracellular domain, a membrane-spanning

domain, and a cytosolic domain. Therefore, the cell membranes required perforation in

order to expose the entire protein. The cell membranes were permeabilized for 5 min at

room temperature with TBST. Endogenase peroxidase activity was blocked by

incubating the cells in 0.03% hydrogen peroxide for 5 min. The cells were rinsed with

distilled water and then further washed with TBST. The endothelial cells were incubated

with primary antibodies against the integrin a531 receptor and E-cadherin. The Caco-2

cells incubated with a primary antibody against GRP-R. The antibodies, diluted in Dako

antibody diluent, were placed on the cells for 60 min at room temperature. The cells

were washed for 5 min (x2) with TBST to remove excess primary antibody. The cells

were incubated with anti-mouse secondary antibody for 15 min at room temperature. The

cells were then washed for 5 min (x2) with TBST. A Streptavidin link was placed on the

cells for 15 min at room temperature. The cells were washed with TBST. The cells were









incubated with DAB for 5-10 min at room temperature. The cells were then washed with

tap water (color changed from purple to brown with water wash). The cells then

incubated for 2 min with Autostainer hematoxylin (Gill's Hematoxylin) at room

temperature. The cells were then rinsed well with TBST. TBST works as a bluing

solution (color changed from purple to blue with TBST wash). The discs were then

mounted onto glass slides cell side down using a water soluble glycerol/gelatin mounting

agent. The mounted cells were then dried overnight prior to image acquisition and data

analysis.

4.6.2 Image Acquisition and Protein Quantification

Digitized images of the immunohistochemically stained cells were collected using

the image acquisition program, Axiovision 3.1 (Carl Zeiss Microimaging, Inc.,

Thornwood, NY). The images were prepared for protein analysis using Adobe

PhotoShop as previously described (Matkowskyj et al. 2003b). The digitized images

were saved as *.tif files and were prepared in Adobe Photoshop for analysis. The

background of each image was set to white using the Level command. A single cell was

then cropped from the image file. Using the magic wand tool, the brown color

(indicating the presence/localization of the protein of choice) was isolated and

copied/pasted into a new *.tif file. This image file was then opened in the program,

TIFFALYZER. From this program, the energy unit per pixel value was quantified. This

value was subtracted from the value obtained for negative samples (those samples not

incubated with primary antibody) to obtain the energy unit per pixel value (i.e., protein

level) for each specified protein. Thirty cells were randomly chosen for each adhesion

protein analysis for the endothelial cell study. Fourteen cells were randomly chosen for






39


protein analysis of GRP-R for the Caco-2 study. The control tissue for the endothelial cell

study was human dermal fibroblasts (Cambrex, Inc., Walkersville, MD).

4.7 Statistics.

Analysis of Variance (ANOVA) was performed to determine significance between

the three culture environments. Statistical analysis was performed using the online

statistical software program, Graphpad Quick Calcs (www.graphpad.com). Comparisons

were made for morphology and protein expression. Significance was taken at p<0.05 or

p<0.01.














CHAPTER 5
RESULTS AND DISCUSSION


The goal of the present study was to determine whether or not simulated

microgravity culture technology provides a better culture environment to enhance cell

adhesion and to study potential markers for diseased tissues. Through the development

of a culture protocol enabling anchorage dependent cells to be cultured on a planar

substrate in simulated microgravity, the direct effects of gravity and shear stress on cell

adhesion using an endothelial cell model and the role of a potential colon cancer marker,

GRP-R using a colon cancer cell model were determined. Since endothelial cells

naturally form a confluent monolayer, it was important to simulate this growth pattern in

order to study the direct effects of gravity and shear stress on endothelial cell behavior. It

has previously been shown by Sanford et al. (2002) that endothelial cells do form a

monolayer around a single microcarrier bead, but form multilayer sheets of endothelial

cells when the microcarrier beads aggregate together. Therefore, the native state of the

endothelial cells is compromised during the culture period. By culturing the endothelial

cells on a planar substrate, monolayer formation is maintained during the entire culture

period. A second advantage to culturing on a planar substrate in microgravity is that no

cell manipulation must occur in order to study specific cell behaviors, such as adhesion

protein expression and cell morphology. The substrate does not interfere with analysis,

such as morphological analysis or determination of protein levels. Also, the planar









substrates can further be placed in a perfusion flow system to study cell detachment in the

presence of shear.

5.1 Development of a Culture Protocol for a Planar Substrate in Microgravity

A method for growing cells on a 2-D structure in simulated microgravity was

developed. A brief description of the method is provided here but more detailed

information can be found elsewhere (Anderson 2001). Cells were cultured on 14" circular

discs punched from a 22 x 22 mm2 square plastic cover slip (Fisher Scientific, USA).

The discs were coated with a 1:5 Matrigel solution to promote cell binding to the cover

slips. Matrigel was selected as the coating material because it is a non-synthetic

extracellular matrix solution comprised of almost every ECM protein. Unbreakable

plastic cover slips were utilized to minimize the weight of the substrate in the

microgravity vessel. The substrates were suspended in a highly viscous fluid in the

HARV, so that during rotation, the substrate was suspended. Media viscosity was

increased approximately 12 fold by adding high molecular weight dextran. As previously

shown, cells cultured in media supplemented with dextran and cells cultured in media

containing no dextran do not demonstrate differences in cell growth or morphology

(Anderson 2001).

To isolate gravity and shear effects, 3 culture environments were selected.

Conventional tissue culture provided a static, Ig environment. Simulated microgravity

provided a low shear, low gravity environment. A perfusion flow system of variable

shear (0.25, 0.5, and 1.0 dynes/cm2) provided a link between a static and a low shear

culture environment. This link enabled the effects of gravity and/or shear on cell

behavior to be decoupled. The first shear stress of 0.25 dynes/cm2 provided a link

between a static culture environment (0 dynes/cm2) and the shear acting on a microcarrier









bead in a simulated microgravity environment (0.5 dynes/cm2). A shear stress of 0.5

dynes/cm2 was selected because this shear was comparable to the shear stress acting on a

microcarrier bead in simulated microgravity (Gao et al. 1997). The third shear stress of

1.0 dynes/cm2 provided a shear stress comparable to the shear acting on a plate in

microgravity.

Generally, a simulated microgravity environment will create a shear stress of less

than 0.5 dynes/cm2. The media (fluid) in the HARV rotates in a circular path as a solid

body with the vessel due to the elimination of an air-liquid interface at low angular

velocities (co) (Gao et al. 1997). The velocity of the rotating fluid is defined in the 0

direction only and the shear rate is locally defined. Shear stress is equal to zero with

respect to solid body rotation (Tre = 0). The shear stress in the rO plane (Gao et al. 1997)

is U U 1 U r (5.1)

Sr r r 80

where Tre = shear stress, t = fluid viscosity, U = velocity in respective plane (rO), r =

location of particle in vessel. Relative motion between the particle (cell/tissue construct)

and the fluid, allowing for mixing of media, generates small shear stresses.

The shear stress generated due to the relative motion between the rotating fluid

and the particle is dependent on the relative speed (Vrei) between the fluid and the particle

(Gao et al. 1997). Therefore, the maximum shear stress (Tmax) acting on a particle of

radius, a, within a simulated microgravity environment (Gao et al. 1997) can be defined

as (5.2)
3 P vreI Vrel = VP V_ o
max 2
2a









The proposed study has demonstrated that microgravity can be simulated using a

planar disc. The drag force (FD) on a planar disc in simulated microgravity was

calculated from the following equation

A 2 (5.3)
D C 2

where Ap = particle area
pi = fluid density
V = Vo = velocity
CD = drag coefficient on a planar disc.
CD was determined using a CD vs. Re chart (Roberson and Crowe 1997). The Re

was calculated from the following equation


Re = VdPI (5.4)

V = cr (5.5)

where d = particle diameter
r = radius of vessel
co = angular velocity
[ = viscosity.
For the case where t = 12.24 cP; dp = 0.006 m; pi = 1.02 g/mL; V = 0.25 m/s; r

= 0.03 m; co = 8.2 rpm, the Re was 13.6 (Anderson et al. 2004a). Using this Re, the drag

force on a planar disc in microgravity was approximately 4.97 10-4 N, where Ap = 126.6

mm2; pi = 1.02 g/mL; V = Vo = 0.25 m/s (Gao et al. 1997) (Anderson et al. 2004a). It

should be noted that the drag force on a planar disc in simulated microgravity was on the

same order as the drag force acting on a microcarrier bead in simulated microgravity.

Using equation 5.2, the shear stress acting on a planar disc in microgravity was

approximately 1.0 dynes/cm2, where a = 0.02 m; t = 12.24 cP; V = 0.017 m/s









(Anderson et al. 2004a). Therefore, the shear stress acting on a planar disc in

microgravity is approximately 2x the shear stress acting on a microcarrier bead in

microgravity (Unsworth and Lelkes 1998).

5.2 Effects of Gravity and Shear on Endothelial Cell Morphology

HUVEC cultured on a planar substrate in simulated microgravity demonstrated a

morphologically distinct shape when compared to HUVEC cultured in a static or variable

shear Ig environment (Figure 5.1). HUVEC appeared elongated and developed fine,

cytoplasmic projections when cultured in simulated microgravity (Anderson et al. 2004a).

A "cobblestone" morphology was observed in HUVEC cultured in a static or variable

shear Ig environment. Previous studies indicate that endothelial cells exposed to shear

stress elongate and orient in the direction of flow (Thoumine et al. 1995; Topper and

Michael A. Gimbrone 1999). Alterations in the actin cytoskeleton result in alterations of

the focal adhesion complexes comprised of the integrin a5P1 receptors (Chicurel et al.

1998). Because simulated microgravity culture technology creates a culture environment

that enables cells to behave similarly to their in vivo counterparts (Unsworth and Lelkes

1998; Hammond and Hammond 2001), the initiation of angiogenesis may be occurring in

the absence of exogenous vascular endothelial growth factor (VEGF), which is typically

required for initiation of angiogenesis in conventional culture. These cytoplasmic

extensions observed in HUVEC cultured in microgravity may represent the initiation of

capillary formation (Anderson et al. 2004a).













B'
U

V


rry


Figure 5.1 H&E stained images of HUVEC (Matrigel). a) Cells cultured using
conventional tissue culture technique. Note the cobblestone appearance of
cells cultured conventionally. b) Cells cultured using simulated microgravity.
Note the long, hair-like projections extending from the cells. Length 1
represents the total length of the cell; length 2 represents the cell length
without the hair-like projections. c) Cells cultured using simulated
microgravity. Note the tube-like clustering of cells. All of these images were
taken at a 10x magnification.

From this study, we found that cells cultured in simulated microgravity were

significantly longer than those cells cultured in the perfusion flow system set-up to

generate three distinct shear stresses and those cells cultured conventionally (Figure 5.2

and Figure 5.3) (Anderson et al. 2004a). Two length measurements were determined.










Length 1 represented the entire length of the cell, including the long hair-like projections

as observed in cells cultured using simulated microgravity. Length 2 represented the cell

length excluding the long, hair-like projections.

Morphologic Measurement (Length 1)
200


180


160-


140


120
)
C
100
E
80


60


40


U
Microgravity Conventional 0.25 dyneslcm2 0.5 dyneslcm2 1.0 dyneslcm2


Figure 5.2 HUVEC cell length (Length 1). Length 1 includes the cytoplasmic projections
observed in simulated microgravity culture. Length 1 of HUVEC cultured in
simulated microgravity was significantly greater than the length of HUVEC
cultured conventionally or in a perfusion flow system. Morphologic
measurements were obtained using Axiovision 3.1 (Carl Zeiss Microimaging,
Thornwood, NY). The asterisk denotes significance at a p<0.01.






47


Morphologic Measurement (Length 2)
120

*

100



80



0 60
E


40 -



20 -



0
Microgravity Conventional 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2

Figure 5.3 HUVEC cell length (Length 2). Morphometric measurement (Length 2)
acquired using the image acquisition program, Axiovision 3.1. Length 2
represents the cell body only. Length 2 of HUVEC cultured in simulated
microgravity was significantly greater than the length of HUVEC cultured
conventionally or in a perfusion flow system. The asterisk denotes
significance at a p<0.01.

HUVEC cultured using simulated microgravity had a significantly larger perimeter

when compared to cells cultured using conventional tissue culture technique or in the

perfusion flow system (Figure 5.3) (Anderson et al. 2004a). This observed increase in

cell perimeter is proportional to the increase in cell length for those cells cultured in

simulated microgravity.

Although the width of the HUVEC cultured in simulated microgravity was greater

than the width of HUVEC cultured in a static environment, no significant difference was










observed. Because cell height was not measured, it was difficult to speculate how cell

width is altered with changes in cell length. However, it is clear that microgravity

produces larger cells as given by cell length and cell perimeter (Figure 5.4). However,

HUVEC cultured under increasing shear did demonstrate an increasing trend in

perimeter.

Morphologic Measurement (Perimeter)
500

450 -

400

350

300

0 250
2
E
200

150

100

50 -

0
Microgravity Conventional 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2


Figure 5.4 HUVEC cell perimeter. Morphometric measurement (perimeter) acquired
using the image acquisition program, Axiovision 3.1. The measured
perimeter of the cells includes the hair-like projections extending from the cell
bodies. The perimeter of HUVEC cultured in simulated microgravity was
significantly greater than the perimeter of HUVEC cultured conventionally or
in a perfusion flow system. The asterisk denotes significance at a p<0.05.

Additionally, HUVEC were seeded onto non-coated planar discs and cultured in

microgravity for 48 hrs (Figure 5.5) (Anderson et al. 2004a). Morphologically, HUVEC

developed long, cytoplasmic projections extending from each pole of the cell and/or

aggregated together to form tube-like structures similar to HUVEC cultured on Matrigel






49


coated planar discs in microgravity. Therefore, morphologic alterations in HUVEC

cultured in microgravity do not appear to be due to the Matrigel coating (Anderson et al.

2004a). HUVEC undergo morphologic alterations in response to the microgravity

environment.


4*


t A .(















cultured on Matrigel coated substrates.

In summary, it can be seen that both gravity and shear play a role in

morphological differences in HUVEC. Traditionally, shear stress has been shown to

result in the re-organization of the actin cytoskeleton, therefore resulting in reorientation

of the cells in the direction of flow (Topper and Michael A. Gimbrone 1999). It is known

that endothelial cells in vivo typically are exposed to shear stresses greater than 10

dynes/cm2, but the low shear that endothelial cells experience in simulated microgravity

is great enough to induce metabolic and functional changes (Jessup et al. 2000). Our

results confirm those results, and suggest that gravity, or a minimization of it, may also

contribute to the reorganization of the actin cytoskeleton, therefore resulting in alterations

in cell length.
resuts cnfir thoe reult andsugs thtgaiy r iiiain fimya
contribu-e to the reraizto of *h *'i yokltn hrfrersligi lea

in cel l:e ^ ^









5.3 Effects of Gravity and Shear on Endothelial Cell Adhesion

The expression of 2 major adhesion proteins was studied; an integrin receptor

specific to fibronectin for characterizing cell-surface adhesion and E-cadherin for

characterizing cell-cell adhesion.

5.3.1 Cell-Substrate Adhesion

The level of expression of the integrin receptor is altered in the absence/presence

of shear and gravity. Figure 5.6 shows immunohistochemically stained images of

HUVEC cultured in the presence/absence of shear and altered gravity.













c) human. .l .,ibrblse -l my. or i--
,....4r 7.7 -. -









"7 .l '. -.- ... ..
"i ., ,-i,,
r.--






Figure 5.6 Immunohistochemically stained images of HUVEC (integrin a531 receptor), a-
c) human dermal fibroblasts cultured conventionally, in microgravity, or in a
perfusion flow system, respectively. d-f) HUVEC cultured conventionally, in
microgravity, or in a perfusion flow system, respectively.

The expression of the ac subunit of the integrin a 53i receptor was different when

HUVEC were cultured conventionally, in a perfusion flow system generating a shear

stress of 0.5 dynes/cm2 or a shear stress of 1.0 dynes/cm2, and in simulated microgravity.






51


The control tissue used for immunohistochemistry was human dermal fibroblasts. This

control tissue was incubated with antibodies specific to the integrin asP5 receptor and to

E-cadherin. By performing immunohistochemistry on the control tissue, the presence

and location of the proteins of interest were identified.

Figure 5.7 shows the important role of shear in the up-regulation of the integrin

as51 receptor, specifically the a5 subunit specific to fibronectin.

Integrin Receptor (Alpha 5) Expression


200

175
T
150

S 125
X
0C
100

75

50

25

0 -
Conventional Microgravity 0.5 dynes/cm2 1.0 dynes/cm2


Figure 5.7 Integrin receptor expression in HUVEC (a5 subunit). Expression was
determined by QIHC. Integrin receptor expression was significantly up-
regulated in HUVEC cultured in microgravity and cultured in a perfusion flow
system generating shear stresses of 0.5 and 1.0 dynes/cm2 compared to
HUVEC cultured conventionally. No significant difference in integrin
receptor expression was observed between HUVEC cultured in microgravity
or a perfusion flow system generating shear stresses of 0.5 and 1.0 dynes/cm2.
The asterisk denotes significance at a p<0.05.









The integrin concentration is about 160 Eu/pixel when HUVEC are cultured in

simulated microgravity or in the perfusion flow system generating a shear of 0.5

dynes/cm2 or a shear of 1.0 dynes/cm2 (the shear acting on a planar disc in microgravity)

(Anderson et al. 2004a), and a little less than 140 Eu/pixel when they cultured in a static

environment. The concentration unit Eu/pixel represents the mathematical energy of the

data image file calculated for a single pixel (Matkowskyj et al. 2003b).

Perinuclear localization of the P1 subunit of the integrin receptor was observed in

osteoblasts cultured in simulated microgravity (Sarkar et al. 2000). They determined that

the perinuclear localization was in part due to a disruption of the actin cytoskeleton. A

trend of perinuclear localization of the a5 subunit of the integrin a531 receptor, similar to

the Sarkar et al. study, was observed when HUVEC were cultured in microgravity. This

specific localization of the protein may in part be due to re-orientation of the actin

cytoskeleton due to the shear forces acting on the cells (Ingber 1999).

Typically, the integrin receptors will couple in the area of greatest stress.

Therefore, because the receptors tend to localize perinuclearly in simulated microgravity,

the area of greatest stress acting on the endothelial cells is most likely located in the

region of the nucleus. On the other hand, because the integrin receptors do not appear to

localize in a specific area throughout cells cultured conventionally, the stress is in all

likelihood distributed evenly across the cell membrane.

5.3.2 Cell-Cell Adhesion

The level of expression of E-cadherin is altered in the absence/presence of shear

and gravity. Figure 5.8 shows immunohistochemically stained images of HUVEC

cultured in the presence/absence of shear and altered gravity.






53







Figre.8 c n im of H ( a- h
S '..

... ....


mi b" i .e.










T 'E i' w e : HV









dermal fibroblasts cultured conventionally, in microgravity, or in a perfusion
flow system, respectively. d-f) HUVEC cultured conventionally, in
microgravity or in a perfusion flow system, respectively.














dynes/cm2, and in simulated microgravity. Figure 5.9 shows that HUVEC cultured in

simulated microgravity demonstrated significantly greater levels of E-cadherin when


compared to those cells cultured in a perfusion flow system generating a shear stress of

0.5 dynes/cm2 or a shear stress of 1.0 dynes/cm2 (Anderson et al. 2004a).









E-cadherin Levels

200

180

160

140


















Conventional Microgravity 0.5 dynes/cm2 1.0 dynes/cm2

Figure 5.9 E-cadherin expression in HUVEC. E-cadherin expression was determined by
QIHC. The expression of E-cadherin was significantly up-regulated in
HUVEC cultured in simulated microgravity and in HUVEC cultured
conventionally compared to HUVEC cultured in a perfusion flow system
generating a shear stress of 0.5 and 1.0 dynes/cm2. The asterisk denotes
0_100
LuI
80

60

40

20



Conventional Microgravity 0.5 dynes/cm2 1.0 dynes/cm2






significance at a p<0.05.
Figure 5.9 E-cadherence in E-cadherin levels was obserinved betweession HUVEC culturdetermined
conventionally and in simulated microgravity. An increased level of E-cadherin in
H~TUVEC cultured in simulated microgravity was expected because this culture
conventionally compared to HUVEC cultured in a perfusion flow system
generating a shear stress of 0.5 and 1.0 dynes/cm2. The asterisk denotes
significance at a p<0.05.

A small difference in E-cadherin levels was observed between HUVEC cultured

conventionally and in simulated microgravity. An increased level of E-cadherin in

HUVEC cultured in simulated microgravity was expected because this culture

environment promotes 3-D cell aggregate formation. The main staple of cell-cell

aggregation is an up-regulation of cell-cell adhesion molecules. A statistically significant









difference in E-cadherin levels was also observed when cells were cultured in a perfusion

flow system generating a shear of 0.5 dynes/cm2 when compared to cells cultured

conventionally. It is clear that if shear stress down regulates E-cadherin expression, then

microgravity significantly up regulates it.

When comparing the data obtained for cell-substrate adhesion and the data

obtained for cell-cell adhesion, it was shown that cell-cell adhesion and cell-substrate

adhesion was shown to be greater in simulated microgravity than in the other two

environments, namely static and perfusion flow (Anderson et al. 2004a). While shear can

inhibit or promote cell adhesion by up- or down- regulating the expression of adhesion

proteins, an environment where the force of gravity is minimized appears to oftentimes

promote adhesion, resulting in the up-regulated expression of adhesion proteins.

Changes in adhesion protein expression are due both to gravity and shear. However,

other factors, such as diffusion of oxygen and mixing of media during rotation, most

likely play a role in the present cell behavior.

5.4 Effects of Gravity and Shear on Potential Cancer Marker

Gastrin-releasing peptide receptor (GRP-R) is not normally expressed in the

epithelial lining of the gastrointestinal tract. However, this receptor is expressed in colon

carcinoma, an epithelial-derived tumor type (Carroll et al. 1999). The goal of this study

was to determine whether or not simulated microgravity culture technology could be used

to enhance the response of a potential marker for colon cancer. Through quantitative

immunohistochemistry, it was shown that GRP-R expression in Caco-2 cells (colon

carcinoma cell line) cultured conventionally does not down-regulate within the first 48

hrs of culture (Figure 5.10 and Figure 5.11). However, GRP-R expression in Caco-2

cells cultured on a planar substrate in simulated microgravity rapidly down-regulates









within the first 48 hrs. (Figure 5.10 and Figure 5.11) (Anderson et al. 2004b) This

down-regulation may in part be due to the internalization of the receptor. GRP-R is

expressed in well-differentiated tumors, but is minimally expressed in poorly

differentiated tumors (Carroll et al. 1999).


6 hr










24 hr




48 hr




Microgravity Conventional Microgravity (+ME) Conventional (+ME)

Figure 5.10 Immunohistochemically stained images of Caco-2 (GRP-R). Over time,
GRP-R expression in Caco-2 cells is significantly down-regulated, as shown
by minimal brown staining in the above images.

Perhaps, Caco-2 cells on planar substrates in simulated microgravity de-

differentiate over time, resulting in the down-regulation of GRP-R expression. Because

GRP/GRP-R activation results in the production of focal adhesion kinase (FAK), a

constituent of the intracellular adhesion network, GRP/GRP-R expression and activation

play a role in cell adhesion. Adhesion plays a critical role in cancer metastasis with

respect to FAK. From our study, we concluded that GRP-R expression is down-regulated







57


in microgravity, both in the absence and presence of a GRP-R antagonist, methyl ester

(ME) (Figure 5.11). As an antagonist, ME should not have an effect on the expression of

GRP-R.

GRP-R expression in Caco-2 cells cultured conventionally remains constant in the

absence or presence of ME over a 48 hr time period (Figure 5.11) (Anderson et al.

2004b). In contrast, GRP-R expression in Caco-2 cells cultured in microgravity on a

planar substrate down-regulates rapidly over the first 48 hrs in the absence of ME.

GRP-R Expression

300
-*- Conv no ME
--Conv ME
--HARVME
250 -A-HARV no ME



200



'150



100



50




0 6 12 18 24 30 36 42 48 54
hours

Figure 5.11 GRP-R expression. GRP-R expression does not change in the absence or
presence of ME when Caco-2 cells are cultured conventionally within the first
48 hrs of culture. GRP-R expression down regulates immediately when Caco-
2 cells are cultured in the absence of ME in simulated microgravity. GRP-R
expression down-regulates at a slower rate when Caco-2 cells are cultured in
the presence of ME in simulated microgravity.











This down-regulation of GRP-R is also observed when the Caco-2 cells are

treated with ME in microgravity, although the down-regulation is milder and appears to

level off at approximately 24 hrs (Anderson et al. 2004b).

If the activation time of ME (typically 18 hrs) is altered in simulated

microgravity, then GRP-R may be internalized at a slower rate in microgravity when ME

is present. ME should not have an effect on GRP-R expression because ME acts as an

antagonist, binding to and ultimately inactivating GRP-R. Inactivation of GRP-R results

in inactivation of the production of FAK. Therefore, alterations in cell adhesion may be

observed.

This data indicates a direct link between Caco-2 cell morphology and GRP-R

expression. Caco-2 cells cultured in simulated microgravity were significantly longer

than Caco-2 cells cultured conventionally (Figure 5.12) (Anderson et al. 2004b).

Caco2 Length
50
45 -- -- Microgravity
...... ..- Microgravity ME
40 -A-Conventional ME
35 --------- Conventional

30
t--------- ^ ^

0 25
E
20

15

10

5

0
0 10 20 30 40 50 60 70 80
hours

Figure 5.12 Caco-2 cell length. The length of Caco-2 cells cultured in simulated
microgravity were significantly longer than those cultured conventionally in
the presence or absence of ME. However, in the presence of ME, Caco-2
length did not demonstrate an increasing trend over time.










Interestingly, this increase in cell length correlates with a decrease in GRP-R

expression. In the presence of ME, Caco-2 cell length was not significantly different for

those cells cultured conventionally or in simulated microgravity.

Caco-2 cell width (Figure 5.13) also appeared to be greater for those cells cultured

in simulated microgravity, although the difference was not significant. Next, the

dependence of morphological alterations on GRP-R expression and how these alterations

were affected by a change in GRP-R expression were determined.

Caco-2 Width
35
*- Microgravity
3. -- Microgravity ME
-A- Conventional
25 ... --" -4- Conventional ME
25 ------


u) 20-
0

E 15


10


5


0
0 6 12 18 24 30 36 42 48 54 60 66 72 78
hours


Figure 5.13 Caco-2 cell width. The width of Caco-2 cells cultured in simulated
microgravity was greater than the width of Caco-2 cells cultured
conventionally in the presence or absence of ME. However, in the presence
of ME, Caco-2 width did not show as great of an increase over time as Caco-2
cells cultured in the absence of ME.

To study the role of GRP-R expression in morphological alterations, ME was

added to Caco-2 cells cultured conventionally or in simulated microgravity after the first







60


6 hrs of the experiment (Figure 5.14). ME was added at this time point, because GRP-R

down-regulation in microgravity is initiated within the first 6 hours of culture. When ME

was added following the first 6 hours of culture in microgravity, GRP-R expression

rapidly down-regulated and was almost gone within the first 24 hours. Therefore, some

aspect of the microgravity culture environment, independent of GRP-R expression, is

causing morphological alterations in Caco-2 cells (Anderson et al. 2004b).

GRP-R Expression (delayed ME addition)
200
Conventional
180 -4- Microgravity

160

140

120
X
100
uL.
80-

60

40

20

0
0 6 12 18 24 30
hours


Figure 5.14 GRP-R expression with delayed addition of ME. GRP-R expression does not
change when ME is added 6 hrs following the start of the experiment for
Caco-2 cells cultured conventionally or in simulated microgravity within the
first 24 hrs of culture. GRP-R expression also down-regulated significantly
by the time the ME was added to Caco-2 cells cultured in simulated
microgravity.

A study by Glover et al. (2004) utilized a non-malignant epithelial cell line (293

cells) which expressed GRP/GRP-R normally to show the role of GRP/GRP-R in cell









detachment and cell deformability (Delaney 2003; Glover et al. 2004). From this study,

it was shown that when GRP/GRP-R is activated and therefore, FAK was produced, cell

detachment was inhibited. When 293 cells were exposed to ME during culture,

GRP/GRP-R activation was blocked, inhibiting FAK production, and cell detachment

was increased. Therefore, they concluded that GRP/GRP-R does play a role in cell-

substrate adhesion and may help to regulate cancer metastasis. From this study, it was

also concluded that cell deformability was enhanced when FAK production was

inhibited. This data correlates with the morphological data showing that when

GRP/GRP-R activation was inhibited in the presence of ME, cell length and cell width

were minimally altered (Anderson et al. 2004b). However, when GRP/GRP-R activation

occurred, enhanced alterations in cell length and cell width were observed, the more

extreme case observed in simulated microgravity (Delaney 2003; Glover et al. 2004).

This data correlates with the idea that 293 cells are less deformable when FAK activation

is uninhibited.














CHAPTER 6
CONCLUSIONS AND FUTURE WORKS

Culture in simulated microgravity is not limited to 3-D cell association.

Endothelial cells seeded onto planar substrates and cultured in simulated microgravity

behave in a similar fashion to endothelial cells cultured on microcarrier beads in

simulated microgravity. Culturing cells on a 2-D substrate in microgravity enables the

direct testing of various cell parameters without subsequent cell manipulation, such as

digestion of the microcarrier bead or centrifugation (Anderson et al. 2004a; Anderson et

al. 2004b). Future studies in the area of angiogenesis and cell detachment will further

determine endothelial cell behavior when cultured in 2-D in microgravity.

With respect to morphology, endothelial cells cultured in simulated microgravity

demonstrated a distinct cell shape not observed when cells were cultured using

conventional tissue culture technique or in a perfusion flow system. Endothelial cells

cultured in simulated microgravity exhibited a distinct morphology because microgravity

culture technology may provide a culture environment where cell behavior mimics native

tissue. Therefore, the cells cultured in simulated microgravity exhibited characteristics

similar to those observed in vivo, such as cell elongation and flattening of the cell. The

endothelial cells that were cultured in simulated microgravity exhibited fine, hair-like

projections extending from the cell body. These fine projections were not observed when

the cells were cultured in the other two culture environments. Because the cells cultured

in simulated microgravity exhibited the greatest difference with respect to cell









morphology when compared to cells cultured using conventional tissue culture technique

or in a perfusion flow system, it was concluded that gravity has a greater effect on cell

morphology than shear stress. Cells cultured using conventional tissue culture technique

or in a perfusion flow system did not appear to show great differences with respect to cell

morphology. Interestingly, the cells that were cultured in simulated microgravity

exhibited a distinct cell aggregate formation not observed when cells were cultured in the

other two culture environments. A future study involves looking at the induction of

angiogenesis when endothelial cells are cultured in simulated microgravity without

exogenous stimulation of vascular endothelial growth factor.

With respect to adhesion protein expression, HUVEC cultured in simulated

microgravity did demonstrate an up-regulation in both cell-cell and cell-substrate

adhesion protein. The increased expression of these adhesion proteins is not only due to

the effects of shear but it is also due to the effects of gravity and other factors like

oxygenation. These results confirm the concept that simulated microgravity culture

promotes cell-cell and cell-substrate associations. We, therefore, have developed a new

method for culturing cells on a planar substrate in simulated microgravity that provides a

better way for studying adhesion, gravity and shear effects than on 3D microcarrier

beads.

Because the microgravity environment is comprised of numerous parameters

which facilitate in vivo-like cell behavior, it is difficult to isolate which parameter has the

greatest effect on Caco-2 cell behavior. Down-regulation of GRP-R in microgravity

occurred at a much earlier time point than in conventional culture, indicating a shift in

the kinetics of GRP-R expression. The advantage of accelerated kinetics of GRP-R









down-regulation is that a greater concentration of experiments can be conducted at much

earlier time points in microgravity as opposed to 4 days in conventional culture. More

importantly, because microgravity culture has been shown to induce in vivo-like cell

behavior, perhaps, on a developmental note, normal intestinal development is favored in

microgravity culture, illustrating a hypothesis for this extreme and rapid down-regulation

of GRP-R. A second hypothesis, which the data may support, is alterations in

differentiation states of tumors (i.e., well-differentiated to poorly differentiated) may

provide an explanation for this extreme and rapid down-regulation of GRP-R in

microgravity. By developing a culture protocol which enables a direct comparison to be

made between in vitro (conventional) cell behavior and in vivo-like cell behavior

(simulated microgravity), better culture techniques can be developed that enable

researchers to better understand the role of specific parameters, such as GRP-R

expression, in morphological changes and cell adhesion in diseased cells.

Future studies for the endothelial study include looking at the process of

angiogenesis in simulated microgravity. One study would involve looking at the

expression of vascular endothelial growth factor and its subsequent receptors when

HUVEC are cultured in simulated microgravity. Based on the morphology (e.g., the

tube-like clustering of HUVEC in microgravity), it appears as though angiogenesis is

taking place in the absence of exogenous vascular endothelial growth factor. A second

study would be to address the adhesive strength of HUVEC cultured in microgravity.

Following culture in simulated microgravity, HUVEC seeded on planar discs may be

placed in a perfusion system generating high shear stresses. From this study, the shear

stress required to detach seeded HUVEC could be determined.









Future studies for the Caco-2 study involve looking at the differentiation state of

Caco-2 cells cultured in microgravity. One study would be to isolate well differentiated,

moderately differentiated, and poorly differentiated cells within the carcinoma and grow

them up separately to form homogeneous tumors to study parameters for metastasis. A

second study would be to determine the role phopholipase C signaling pathway plays in

metastasis (i.e., cell adhesion, FAK, and GRPR expression). This pathway is altered in

simulated microgravity and is affiliated with the process of cell adhesion. Several studies

could be designed to develop a culture protocol which will involve development of a

microchamber which will facilitate the growth of a colon carcinoma (or any tumor type)

in microgravity, using minimal cell density, minimal media, etc. A second microdevice

could be developed that will mimic an epithelial lining, providing an attachment surface

similar to the epithelial lining of the gastrointestinal tract. By attaching the 3-D tumor to

this surface, in vivo like cell behavior during metastasis may more easily be studied.

Definitely, surface topography will play a role in cell adhesion and metastasis. Finally,

using specific time points, determine as a colon carcinoma (developed in 3-D in

microgravity) stages (and begins the process of metastasis), how the differentiation of the

tumor changes. During this analysis, we could identify alterations in various markers,

like GRPR expression. Ultimately, this study would lead to the development of a 3-D

model for colon carcinoma study. It may be beneficial to study colon carcinoma models

to determine how well an in vivo-like model could be designed for in vitro

experimentation.















APPENDIX A
CHARACTERIZATION OF PERFUSION FLOW SYSTEM







67


The Dynamax peristaltic pump was calibrated using 3/16" silastic tubing. The

predetermined calibration equations for specified tubing size were not compatible with

the tubing of choice. Therefore, the pump was calibrated by hand for the before

mentioned tubing size.

Dynamx Peristaltic Pump Calibration


0 10 20 30
Pump Speed (rpm)

Table 1. Peristaltic Pump Calibration


40 50 60


Pump Speed (rpm) Volumetric Flow Rate (ml/min)
1 0.8
2.7 1.9
6.25 4
10 6.3
14.8 8.8
25 15
48 27.4















APPENDIX B
SHEAR STRESS CALCULATIONS FOR PERFUSION EXPERIMENT





















CDCD -N N N N N m m m
E ooooooo~fil~~l~"mmCD

't 11 r al t 1C r- al 7 r- l
C) C) (= (= 000 00
0000 01= 1= 1=000






In~ ~ 3b~~3b~
- - - - - -J



7t all all 7t 11C r-







5 5 5 5 5 5 5 5 500
5 5 5 5 5 5 5 5 -







7t t t 7 7 7t 7t 7t I I I-I-I- t 7 7 7






- - - - -




N t7 1 l
000000 0 N 7t N 7t0














APPENDIX C
QUANTITATIVE IMMUNOHISTOCHEMISTRY DATA SHEETS (HUVEC)










Table 3. QIHC Data for HUVEC Cultured Conventionally (Integrin at531 Receptor)

Negative Integrin a531 Receptor Total Protein (Eu/Pixel)


600.1 465 135.1
600.1 474.8 125.3
600.1 485.5 114.6
600.1 447.5 152.6
600.1 485.6 114.5
600.1 448.3 151.8
600.1 477.7 122.4
600.1 466.7 133.4
600.1 480.5 119.6
600.1 486.9 113.2
600.1 465.7 134.4
600.1 481.7 118.4
600.1 489.7 110.4
600.1 457.7 142.4
600.1 451.4 148.7
600.1 470.9 129.2
600.1 474.1 126
600.1 469.6 130.5
600.1 451.9 148.2
600.1 476.1 124
600.1 449.9 150.2
600.1 449.3 150.8
600.1 437.4 162.7
600.1 437.3 162.8
600.1 451.4 148.7
600.1 434.2 165.9
600.1 445.3 154.8
600.1 435.9 164.2
600.1 457 143.1
600.1 469.4 130.7










Table 4. QIHC Data for HUVEC Cultured Conventionally (E-cadherin)

Negative E-Cadherin Total Protein (Eu/Pixel)


600.1 473.4 126.7
600.1 513.3 86
600.1 483.6 116.5
600.1 493 107.1
600.1 494 106.1
600.1 491.7 108.4
600.1 488.3 111.8
600.1 471.3 128.8
600.1 467.9 132.2
600.1 470.9 129.2
600.1 456.7 143.4
600.1 478.1 122
600.1 464 136.1
600.1 461.8 138.3
600.1 485.7 114.4
600.1 484.8 115.3
600.1 479.3 120.8
600.1 473.8 126.3
600.1 477.3 122.8
600.1 485.3 114.8
600.1 465 135.1
600.1 462.6 137.5
600.1 487.3 112.8
600.1 470.9 129.2
600.1 490.7 109.4
600.1 473 127.1
600.1 481.8 118.3
600.1 468.3 131.8
600.1 461 139.1
600.1 464.6 135.5










Table 5. QIHC Data for HUVEC Cultured in Microgravity (Integrin asP5 Receptor)

Negative Integrin asP5 Receptor Total Protein (Eu/Pixel)


605.3 415 499.3
605.3 452.5 459.6
605.3 486 493.6
605.3 435.5 500.9
605.3 429.7 468.5
605.3 474.1 503
605.3 432.2 468.4
605.3 438.8 472.9
605.3 423 460.6
605.3 457.5 481.4
605.3 435.9 458
605.3 459.1 458.1
605.3 419.5 439.1
605.3 417.4 462.5
605.3 420.5 495.4
605.3 430 469.2
605.3 428.1 494.5
605.3 469.8 492.2
605.3 428.9 492.6
605.3 481.4 488.7
605.3 422 436.6
605.3 477.7 436.8
605.3 482.3 471.3
605.3 470 483.2
605.3 459.3 460
605.3 457.6 485.3
605.3 430 490.1
605.3 441.8 447.1
605.3 437.2 450.6
605.3 428.2 463.4










Table 6. QIHC Data for HUVEC Cultured in Microgravity (E-cadherin)


Negative E-Cadherin Total Protein (Eu/Pixel)


605.3 190.3 106
605.3 152.8 145.7
605.3 119.3 111.7
605.3 169.8 104.4
605.3 175.6 136.8
605.3 131.2 102.3
605.3 173.1 136.9
605.3 166.5 132.4
605.3 182.3 144.7
605.3 147.8 123.9
605.3 169.4 147.3
605.3 146.2 147.2
605.3 185.8 166.2
605.3 187.9 142.8
605.3 184.8 109.9
605.3 175.3 136.1
605.3 177.2 110.8
605.3 135.5 113.1
605.3 176.4 112.7
605.3 123.9 116.6
605.3 183.3 168.7
605.3 127.6 168.5
605.3 123 134
605.3 135.3 122.1
605.3 146 145.3
605.3 147.7 120
605.3 175.3 115.2
605.3 163.5 158.2
605.3 168.1 154.7
605.3 177.1 141.9










Table 7. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2)
(Integrin a531 Receptor)


Negative Integrin a531 Receptor Total Protein (Eu/Pixel)


595.6 411.6 489.2
595.6 461.2 479.3
595.6 444.9 478.6
595.6 441.7 480.4
595.6 438.6 497.5
595.6 446 456.7
595.6 429.2 449.7
595.6 419.9 492.5
595.6 449.8 500.2
595.6 460.5 502.2
595.6 433 487.4
595.6 430.7 494.4
595.6 435.1 492.1
595.6 465.1 485.8
595.6 441.4 485.8
595.6 433.3 490.3
595.6 414.3 502
595.6 413.6 497.4
595.6 464.6 472.7
595.6 445.6 468.6
595.6 453.8 459.4
595.6 428.6 445.9
595.6 442.7 504.9
595.6 436.9 486.1
595.6 441.9 467
595.6 431.4 472
595.6 419.7 446.8
595.6 436.6 504.8
595.6 422.4 460.2
595.6 426.4 461.5










Table 8. QIHC Data for HUVEC Cultured in a Perfusion Flow System (0.5 dynes/cm2)
(E-cadherin)

Negative E-Cadherin Total Protein (Eu/Pixel)


595.6 184 106.4
595.6 134.4 116.3
595.6 150.7 117
595.6 153.9 115.2
595.6 157 98.1
595.6 149.6 138.9
595.6 166.4 145.9
595.6 175.7 103.1
595.6 145.8 95.4
595.6 135.1 93.4
595.6 162.6 108.2
595.6 164.9 101.2
595.6 160.5 103.5
595.6 130.5 109.8
595.6 154.2 109.8
595.6 162.3 105.3
595.6 181.3 93.6
595.6 182 98.2
595.6 131 122.9
595.6 150 127
595.6 141.8 136.2
595.6 167 149.7
595.6 152.9 90.7
595.6 158.7 109.5
595.6 153.7 128.6
595.6 164.2 123.6
595.6 175.9 148.8
595.6 159 90.8
595.6 173.2 135.4
595.6 169.2 134.1










Table 9. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2)
(Integrin a5P1 Receptor)


Negative Integrin a531 Receptor Total Protein (Eu/Pixel)


595.6 422.6 173
595.6 444.5 151.1
595.6 419.9 175.7
595.6 426.6 169
595.6 418.3 177.3
595.6 434.5 161.1
595.6 428.3 167.3
595.6 422.7 172.9
595.6 420.1 175.5
595.6 421.8 173.8
595.6 422.1 173.5
595.6 434.3 161.3
595.6 430.9 164.7
595.6 451.2 144.4
595.6 451.5 144.1
595.6 447.7 147.9
595.6 425.1 170.5
595.6 473.8 121.8
595.6 420.5 175.1
595.6 439.8 155.8
595.6 421.7 173.9
595.6 435.4 160.2
595.6 432.8 162.8
595.6 421.3 174.3
595.6 430.5 165.1
595.6 448.6 147
595.6 444.3 151.3
595.6 421.3 174.3
595.6 427.4 168.2
595.6 427.1 168.5










Table 10. QIHC Data for HUVEC Cultured in a Perfusion Flow System (1.0 dynes/cm2)
(E-cadherin)


Negative E-Cadherin Total Protein (Eu/Pixel)


595.6 449 146.6
595.6 455.5 140.1
595.6 484 111.6
595.6 444.8 150.8
595.6 461.6 134
595.6 456.5 139.1
595.6 493.9 101.7
595.6 462.6 133
595.6 472.7 122.9
595.6 436 159.6
595.6 494.4 101.2
595.6 440.5 155.1
595.6 492.5 103.1
595.6 481.4 114.2
595.6 477.5 118.1
595.6 470.7 124.9
595.6 492.6 103
595.6 484.9 110.7
595.6 481.7 113.9
595.6 480 115.6
595.6 496.7 98.9
595.6 465.1 130.5
595.6 478.7 116.9
595.6 473.1 122.5
595.6 484.9 110.7
595.6 482.4 113.2
595.6 480.7 114.9
595.6 505.1 90.5
595.6 488.2 107.4
595.6 505.7 89.9
















APPENDIX D
ENDOTHELIAL CELL MORPHOLOGY DATA










Table 11. Morphology Data for HUVEC (Perimeter)


Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2
Perimeter Perimeter Perimeter Perimeter Perimeter

183.2 466.5 197.3 180.2 172.3
167.3 574.0 231.4 177.3 128.7
190.9 551.2 271.9 162.2 170.2
165.6 573.0 150.7 151.3 222.6
152.3 563.3 198.3 144.1 212.3
182.3 1174.8 134.2 158.8 164.3
184.2 486.2 178.9 214.2 191.6
175.8 668.2 151.9 182.2 167.4
197.4 484.6 190.9 176.8 171.6
172.7 247.2 171.9 178.7 236.7
182.6 192.9 194.6 231.4 181.0
186.0 240.1 178.4 206.7 226.9
166.5 189.4 143.6 215.9 216.3
153.5 297.0 151.1 146.6 195.3
296.6 196.1 176.2 155.8 201.7
206.5 392.2 219.2 153.3 229.7
144.8 243.3 245.4 186.7 200.8
126.2 298.3 200.7 144.9 201.3
156.9 677.9 126.3 178.4 162.3
168.1 717.9 168.8 145.5 181.3
158.0 292.9 179.6 178.0 166.2
173.7 760.9 237.1 223.8 180.5
178.5 309.6 196.0 232.6 154.2
151.0 220.5 194.4 207.1 194.1
187.7 480.6 176.2 217.9 196.7
192.5 271.9 166.6 214.5 210.7
185.1 405.9 162.2 196.4 191.9
196.9 348.6 152.9 227.4 210.1
342.2 388.8 135.5 274.9 223.7
154.3 341.9 136.8 261.6 234.4
208.5 407.1 137.9 317.9 229.3
149.6 324.5 139.9 227.6 185.9
156.9 316.9 141.7 206.0 149.1
262.3 508.1 155.5 193.7 242.1
246.8 421.3 146.2 153.5 193.7
189.7 265.6 167.6 195.8 175.3










Table 11 cont.


Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2
Perimeter Perimeter Perimeter Perimeter Perimeter

251.9 343.2 240.1 213.2 233.0
229.0 630.3 249.2 166.0 203.4
199.8 419.0 257.0 233.4 188.1
172.9 549.8 204.7 171.2 175.8
203.1 298.3 185.3 224.9 171.4
206.2 331.5 155.9 272.5 237.2
198.0 427.6 176.0 251.1 183.7
205.9 244.4 164.3 202.7 225.7
156.3 271.5 166.9 165.0 189.3
131.6 594.0 170.8 181.8 195.1
136.2 495.0 157.9 240.9 222.3
123.5 365.6 164.3 168.8 187.9
143.5 585.5 190.8 234.7 186.4
115.9 357.3 164.4 186.8 172.4
136.7 449.6 155.4 185.1 183.2
138.4 381.0 215.5 166.9 186.6
139.0 307.2 207.9 157.9 195.6
136.4 435.7 223.6 223.9 179.9










Table 12. Morphology Data for HUVEC (Length 1)


Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2
Length Length 1 Length Length Length

50.02 194.8 40 44.6 55.4
40.69 208 58.2 64.1 40.8
58.41 208.8 87.2 44.4 57.8
49.71 211 32.2 37.6 73.4
48.88 204.1 50.4 41 73.6
52.62 473.8 51.3 35 45.3
53.45 199.5 49.1 71.1 60.3
50.53 266.3 44.5 58.8 52.6
47.3 171.5 58.9 56.4 52.3
55.1 110.4 56.7 59.7 74
66 68 58.5 83.3 68.4
51.9 88.5 55.4 68.8 79.6
59.6 60.6 47.6 66.3 71.2
46.8 89.4 36.4 47.3 66.7
99.5 70.7 59.8 46.9 76.8
65.7 135.2 69.3 44.5 73.7
37.7 79.3 62.6 65.3 77.7
46.9 102.1 58 35.3 69.2
44.97 222.2 36.2 54.6 60.7
48.73 243.9 53.6 42.5 60.8
46.08 99.9 50.1 64.3 49.2
46.59 289.6 69.5 69.1 63.5
42.35 127.9 40.1 66.4 51
55.72 83.7 59.9 55.8 68
67.32 176.5 52.5 72.3 77
66.33 97.4 43.6 79.1 80.2
54.48 139.6 46.9 59.1 68.7
60.6 127.6 49.9 68.7 77
132.1 146.1 41.1 100.1 79.2
45.9 126.8 41.7 89.5 77.3
62.8 164.4 41 96.2 70.4
42.7 134 32.8 67.6 62.3
46.9 114.2 45.9 67.3 44.5
82.8 196.8 53 55.7 68.9
76.4 158.2 45.5 50.3 63.4
55.9 97.5 50.9 60.2 52.2










Table 12 cont.


Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2
Length Length 1 Length Length Length


78.3 106.7 70.3 66.4 85.1
81.3 258.4 82.3 49.8 68.6
62.9 160.8 65.2 76.6 62.3
62.3 191.6 51.3 56.9 53.5
59.6 99.4 54.5 88.4 57.9
73.8 117.6 44.9 89.8 83.2
65.4 142.2 43.5 84.3 58.3
61.9 79 34.3 64.4 85.9
39.5 93 52.8 50.4 62.5
42.56 196 45.3 60.7 71.9
39.62 195.5 47.5 76.9 83.3
38.64 125.8 50.4 45.6 69.6
46.49 200.4 48.6 74.5 63.6
37.35 117.8 53.3 67.1 56.1
45.56 137 54.2 49.7 58.2
46.53 139.4 58.9 48.6 55.6
49.39 104 59.6 45.3 64.7
47.17 147.3 49.5 77.5 66.8










Table 13. Morphology Data for HUVEC (Length 2)


Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2
Length Length 2 Length Length Length

50.02 99.1 40 44.6 55.4
40.69 54.4 58.2 64.1 40.8
58.41 67.7 87.2 44.4 57.8
49.71 79.8 32.2 37.6 73.4
48.88 101 50.4 41 73.6
52.62 98.8 51.3 35 45.3
53.45 50.5 49.1 71.1 60.3
50.53 56.4 44.5 58.8 52.6
47.3 75 58.9 56.4 52.3
55.1 110.4 56.7 59.7 74
66 68 58.5 83.3 68.4
51.9 88.5 55.4 68.8 79.6
59.6 60.6 47.6 66.3 71.2
46.8 89.4 36.4 47.3 66.7
99.5 70.7 59.8 46.9 76.8
65.7 76.2 69.3 44.5 73.7
37.7 79.3 62.6 65.3 77.7
46.9 102.1 58 35.3 69.2
44.97 95.9 36.2 54.6 60.7
48.73 74.2 53.6 42.5 60.8
46.08 99.9 50.1 64.3 49.2
46.59 127.6 69.5 69.1 63.5
42.35 127.9 40.1 66.4 51
55.72 83.7 59.9 55.8 68
67.32 176.5 52.5 72.3 77
66.33 97.4 43.6 79.1 80.2
54.48 73 46.9 59.1 68.7
60.6 127.6 49.9 68.7 77
132.1 84.6 41.1 100.1 79.2
45.9 71.3 41.7 89.5 77.3
62.8 65.9 41 96.2 70.4
42.7 134 32.8 67.6 62.3
46.9 114.2 45.9 67.3 44.5
82.8 67.8 53 55.7 68.9
76.4 82.1 45.5 50.3 63.4
55.9 97.5 50.9 60.2 52.2










Table 13 cont.


Conventional HARV 0.25 dynes/cm2 0.5 dynes/cm2 1.0 dynes/cm2
Length Length 2 Length Length Length

78.3 106.7 70.3 66.4 85.1
81.3 63.7 82.3 49.8 68.6
62.9 55.5 65.2 76.6 62.3
62.3 50.2 51.3 56.9 53.5
59.6 99.4 54.5 88.4 57.9
73.8 117.6 44.9 89.8 83.2
65.4 142.2 43.5 84.3 58.3
61.9 79 34.3 64.4 85.9
39.5 93 52.8 50.4 62.5
42.56 93.4 45.3 60.7 71.9
39.62 75 47.5 76.9 83.3
38.64 125.8 50.4 45.6 69.6
46.49 73.2 48.6 74.5 63.6
37.35 117.8 53.3 67.1 56.1
45.56 72.9 54.2 49.7 58.2
46.53 139.4 58.9 48.6 55.6
49.39 104 59.6 45.3 64.7
47.17 73.1 49.5 77.5 66.8















APPENDIX E
COLON CARCINOMA CELL MORPHOLOGY DATA










Table 14. Morphology Data for Caco-2 (Length) (Conventional)


6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME

28.7 32.9 33.9 34.7 33.3 31.8 36.4 30.1 38.8
31.6 30.7 35.5 31.6 37.5 36.1 36.7 33.4 42.3
31.5 33.8 29.0 30.6 37.3 32.0 38.4 39.1 39.5
33.2 28.3 34.8 32.7 38.3 32.7 36.3 37.4 42.9
40.2 30.4 36.6 35.6 40.7 34.3 37.1 37.2 37.7
29.1 33.9 28.5 30.1 38.7 30.7 37.0 40.6 38.1
28.0 33.2 34.8 32.3 31.0 33.3 35.8 37.0 39.4
33.0 35.1 33.8 34.4 36.8 35.5 34.6 34.2 38.2
31.0 33.8 36.8 32.6 45.8 34.1 36.1 35.1 35.0
33.8 29.8 29.8 38.1 36.3 32.2 34.9 46.7 33.0
31.5 34.0 34.4 30.5 33.2 34.0 33.1 37.8 37.4
30.2 33.6 28.6 34.0 38.5 35.6 34.9 41.1 41.1
31.3 32.9 32.6 37.0 36.7 36.3 38.9 37.6 35.4
34.9 33.5 29.3 39.2 32.3 34.2 39.8 48.6 40.6




Table 15. Morphology Data forCaco-2 (Width) (Conventional)

6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME

21.1 19.6 21.8 24.8 29.5 27.5 24.0 24.7 28.9
23.8 19.5 25.8 24.2 33.8 20.7 23.7 21.7 22.6
17.9 20.7 2.3 25.8 24.2 27.2 26.3 27.2 26.0
15.0 18.9 24.0 24.1 26.7 28.7 24.2 25.2 32.3
17.3 21.2 26.1 21.7 25.9 21.6 26.6 24.2 29.7
19.9 18.1 20.4 19.7 27.9 27.1 26.7 25.8 30.5
20.6 17.9 25.4 22.1 31.6 28.0 23.7 24.0 32.7
21.7 19.3 19.3 25.1 22.3 25.8 19.3 26.1 22.4
21.2 16.5 20.4 27.7 24.8 30.8 26.9 24.8 20.6
20.0 21.5 18.2 25.6 28.7 29.0 27.3 25.9 21.6
23.5 18.6 18.9 25.3 29.4 26.3 23.8 23.1 25.4
18.7 18.4 18.9 23.0 29.6 2.7 23.9 23.0 31.9
21.2 20.5 18.2 22.5 28.2 30.1 25.5 23.8 24.8
24.0 21.4 20.9 21.7 23.6 25.1 27.7 26.8 26.7










Table 16. Morphology Data for Caco-2 (Length) (Microgravity)


6hr 12 hr 24 hr 48 hr 72 hr 6hrME 12hrME 24hrME 48hrME

40.4 37.7 42.1 39.4 46.8 46.0 33.7 40.0 42.8
36.8 35.4 40.2 38.5 44.4 41.4 36.4 40.8 40.6
37.9 43.1 41.8 47.0 40.2 40.7 35.7 43.7 37.8
35.8 41.4 40.7 39.6 42.2 39.1 41.1 39.1 44.6
37.9 43.3 42.9 48.7 45.1 40.0 44.1 43.7 38.1
42.1 44.9 40.5 47.4 40.0 38.5 40.2 45.4 41.5
44.7 42.0 42.2 41.8 58.2 43.2 41.4 47.2 44.1
39.8 43.4 41.8 49.8 46.7 36.6 38.5 46.6 47.7
40.8 36.8 44.7 43.5 43.7 34.0 45.2 39.9 40.1
43.4 40.4 38.4 41.4 42.0 33.3 38.1 39.1 35.8
35.2 43.8 37.9 35.4 48.1 36.1 41.6 37.2 38.8
39.2 38.4 37.9 36.8 41.2 34.2 46.2 39.6 38.8
43.1 40.6 37.8 44.4 52.5 43.3 44.2 37.0 47.3
47.7 43.8 36.7 39.0 47.3 39.2 44.1 42.3 39.8


Table 17. Morphology Data for Caco-2 (Width) (Microgravity)


6 hr 12 hr 24 hr 48 hr 72 hr 6 hr ME 12 hr ME 24 hr ME 48 hr ME

23.0 19.1 25.4 37.4 31.2 27.0 25.5 28.3 23.4
21.5 17.7 26.7 26.2 29.9 27.4 25.5 24.8 32.1
22.0 24.0 22.9 26.3 28.8 24.2 28.2 32.9 25.6
24.0 23.3 21.2 27.6 37.2 25.0 29.7 26.8 26.0
26.5 19.7 25.1 23.7 29.7 28.7 28.4 30.1 26.4
22.1 16.8 22.2 28.4 33.3 27.6 28.4 25.5 24.1
25.2 26.2 24.6 21.1 29.2 27.9 27.4 22.9 26.0
21.6 28.0 22.5 32.6 34.0 28.7 27.4 31.3 33.9
25.7 29.0 25.9 32.2 30.5 28.7 26.8 24.1 31.3
27.4 24.7 21.3 32.7 30.6 27.0 27.8 28.4 25.9
24.7 21.0 15.9 28.4 32.3 27.1 29.8 29.7 28.6
20.0 20.6 23.5 33.2 26.2 27.8 24.3 25.4 27.4
19.6 22.1 21.5 30.1 24.7 28.0 27.1 24.2 31.9
15.3 25.0 23.0 25.0 36.3 27.3 23.2 29.8 26.2