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Development of an Animal Model To Study the Role of ALK1 in Arteriogenesis

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Title:
Development of an Animal Model To Study the Role of ALK1 in Arteriogenesis
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YO, KYUMEE ( Author, Primary )
Copyright Date:
2008

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Blood vessels ( jstor )
Complementary DNA ( jstor )
DNA ( jstor )
Genomics ( jstor )
Hemorrhage ( jstor )
Hereditary hemorrhagic telangiectasia ( jstor )
Polymerase chain reaction ( jstor )
Receptors ( jstor )
Spleen ( jstor )
Transgenes ( jstor )

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University of Florida
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University of Florida
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Copyright Kyumee Yo. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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2/28/2007

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DEVELOPMENT OF AN ANIMAL MODEL TO STUDY THE ROLE OF ALK1 IN ARTERIOGENESIS By KYUMEE YO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Kyumee Yo

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iii TABLE OF CONTENTS page LIST OF TABLES...............................................................................................................v LIST OF FIGURES...........................................................................................................vi ABSTRACT......................................................................................................................v ii CHAPTER 1 INTRODUCTION........................................................................................................1 Clinical Manifestations.................................................................................................1 Molecular Genetics of HHT.........................................................................................2 TGFSignaling...........................................................................................................3 Endoglin....................................................................................................................... .5 Role of ALK1 in Arterializa tion and Vascular Remodeling........................................6 2 MATERIALS AND METHODS...............................................................................10 Generation of Tie2-ALK1 Transgenic Mice..............................................................10 Staining....................................................................................................................... 11 Dye Injection..............................................................................................................11 Western Blotting.........................................................................................................11 Reverse Transcription PCR........................................................................................12 Northern Analysis.......................................................................................................12 DNA Constructs..........................................................................................................13 Electroporation and ES Cell Growth..........................................................................15 Genomic Southern......................................................................................................16 3 RESULTS...................................................................................................................18 Flag-tagged Full-length Murine Alk1(mAl k1) cDNA and In Vitro Translation........18 Generation and Screening of Transgenic Founder Mice............................................20 Initial Characterization of Tg(Tie 2-Alk1-A) and Tg(Tie2-Alk1-B)..........................20 The Tg(Tie2-Alk1-B) Line Display Hemorrhages in the Spleen...............................21 Analysis of Transgene Expression..............................................................................23 Limitations of the Transgenic ALK1 Overexpression Approach...............................24 An Alternative Approach: Rosa26-caAlk1 Knockin Mice.........................................25

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iv 4 DISCUSSION.............................................................................................................27 LIST OF REFERENCES...................................................................................................32 BIOGRAPHICAL SKETCH.............................................................................................36

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v TABLE Table page 1: Mice examined at various stages before weaning age...................................................23

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vi LIST OF FIGURES Figure page 1 Schematic of the genomic structure of ENG showing location of meditations.........3 2 Schematic of the genomic structure of ALK1 showing location of meditations.......4 3 Schematic representation of the TGFsignal transduction pathway.......................5 4 Autoradiograph of In Vitro tran slated and western products. .................................18 5 Schematic diagram showing the proce dures for the generation of Tie2-Alk1 transgenic construct..................................................................................................19 6 Tie2(M)-Alk construct and PCR genotyping...........................................................21 7 Hemorrhages in the spleen of 4 week s-old Tg(Tie2-Alk1-B) mice. (A) Spleens of wild-type and Tg(B) mice....................................................................................22 8 Histological demonstration of hemorrhage s in the spleen of the Tg mice. The arrows show dark precipitants along the blood vessels. RP, red pulp; WP, white pulp; SMA, smooth muscle alpha actin...................................................................22 9 Visualized blood vessels by latex dye in the spleens. (A) wild-type and (B) Tg(Tie2-Alk1-B) having hemorrhages. No te spreading of dyes into red pulp areas (indicated by arrows) in WT but not in Tg mice.............................................23 10 RT-PCR analysis showing the transgen ic Alk1 transcripts specifically found in Tg(B) samples. Asterisk indicates a non-specific PCR band due to genomic DNA contamination.................................................................................................24 11 Schematic diagram showing Rosa 26-caAlk1 gene targeting strategy.....................26

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vii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DEVELOPMENT OF AN ANIMAL MODEL TO STUDY THE ROLE OF ALK1 IN ARTERIOGENESIS By Kyumee Yo August 2006 Chair: S. Paul Oh Major Department: Physiol ogy and Functional Genomics Hereditary Hemorrhagic Telangiectasia ( HHT) is an autosomal dominant disorder that occurs once in every 8,000 people. It is characterized by epistaxis, telangiectases and arterioveneous malformations. These symptoms are highly variable with clinical manifestations varying even among members of the same family. There are two major distinct types of HHT caused by different genes in the human genome: HHT1, which is caused by endoglin (ENG), and HHT2 which is caused by activin r eceptor-like kinase-1 (ALK1). Both are plasma membrane recep tors involved in the transduction of transforming growth factor (TGF) signals which regulate the synthesis of perivascular matrix proteins and differentiation and recruitm ent of periendothelial cells during vascular development. Restricted ex pression of ALK1 in arterial endothelium suggests that ALK1 may play a pivotal role in the maturation and stab ilization of arterial vessels. ALK1-deficiency in mice resulted in embryonic lethality, with severe dilation of large blood vessels and excessive fusion of capil laries. Impairment in the recruitment or differentiation of vascular smooth muscle ce lls was also observed in these mice. With

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viii these data we hypothesized that ALK1 is e ssential for the morphogenetic development of arterial vessels (arteriogenesis). The goal of this thesis is to investigate whether ALK1 signaling is sufficient for arte riogenesis. The approach taken was to overexpress the ALK1 gene in the entire vascular endotheliu m including veins in transgenic mice, using a pan-endothelial cell promoter, th e Tie2 regulatory sequence. Two independent transgenic lines have been generated and characterize d. Transgene-positive mice from both lines were viable and no apparent vascular malf ormations were detected, indicating that overexpression of wild-type Alk1 gene is in sufficient to cause transmorphogenesis of veins to artery-like vessels. However, this transgenic approach has a number of limitations to conclude for the role of ALK1 signaling in arteriogenesi s: first, the wildtype ALK1 may require proper stimulation by it s ligand and presence of essential Type II receptor; second, the transgene expression varies by the copy number and insertion location in the chromosome; third, the Tie2 (M ) promoter/enhancer element that we used may not be strong enough to produce a robust expression. To overcome these potential deficiencies, we modified our approaches as follows. Fo r ligand and type II receptor dependency, we decided to use a constitutive active form (always active) of Alk1 cDNA (caAlk1). For the issues of robust expression as well as dependency of the copy number transgene insertion site, we d ecided to target the Rosa26 lo cus with caAlk1. This caAlk1 expression will be activated by crossing the Rosa26-caAlk1-knockin mice with known Cre transgenic mice, such as Tie1-Cre, Flk1-Cre or Alk1-Cre. The Rosa26-caAlk1knockin targeting vector has b een successfully generated.

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1 CHAPTER 1 INTRODUCTION Hereditary Hemorrhagic Telangiectasia (HHT) or Rendu-Osler-Weber syndrome is an autosomal dominant diso rder characterized primarily by epistaxis (spontaneous and recurrent nosebleeds), telangiectases (dilati on of blood vessels creating small red spots) and arterioveneous malformations (AVMs) . Telangiectasis is a vascular lesion caused by direct connections of arteri oles and venuoles without inte rvening capillaries, and often found in the mucosal layer of skin of HHT patients. Large AVMs occur in the internal organs such as the lung, live r, brain and gut of the body. A minimal prevalence rate of HHT is estimated to be one in 8,000 (1). Some of the most devasta ting consequences of this disease result from cerebral vascular malformations. These become evident as either arteriovenous fistulae (AVF), small nidus-t ype AVMs (places where disease lodges and multiplies) or micro-AVMs with a nidus less than 1 cm in size (1). Clinical Manifestations People with HHT have a wide range of symptoms and there exists great variability of clinical manifestations be tween families and even among members of the same family. A spontaneous recurrent nosebleed from telangiectasis in the nasal mucosa is the most common sign in more than 90% of HHT patients. The severity and frequency of nosebleeds generally increase with age and can lead to chronic anemia and ultimately require blood transfusion. Telangiectases can al so develop in the gastrointestinal tract, particularly in the stomach and small bowel of older patients, usually in their fifth or sixth decades of life. Liver involvement is now more widely recognized and reported in up to

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2 40% of HHT patients. It is usually asymptomatic in up to 50% of the affected individuals and is due to the presence of multiple intrahepat ic telangiectases that lead to formation of shunts between the major vessels of the liver (2). A significant num ber of HHT patients also have AVMs in the pulmonary and cer ebral/spinal circulation. Pulmonary AVMs (PAVMs) have been reported in up to 50% of patients and are due to a direct connection between pulmonary artery and vein. This le ft-to-right shunting of blood by a PAVM can lead to hypoxemia, stroke and brain abs cess. Cerebral AVMs (CAVMs), aneurysms or cavernous angiomas can lead to seizures and life threatening or disabling hemorrhagic stroke (3). Molecular Genetics of HHT The pathogenic mechanisms involved in the development of the malformed vessels in HHT are of interest to scientists and clinicians alike. There are two distinct locations for HHT genes within the human genome, HHT1, which maps to chromosome 9, is shown to encode the type III TGFcoreceptor endoglin (ENG). The HHT2 locus, which maps to chromosome 12, is shown to be a gene that encodes a further member of the TGFsuperfamily of cytokines, known as ac tivin receptor-like kinase-1 (ALK1) (1). It is a mutation in the normal expression of either gene that is the cause of the abnormal vasculature leading to HHT. The majo rity of ENG and ALK1 mutations appear to lead to a loss of function. As an au tosomal dominant condition, the presence of a heterozygous mutant allele appears sufficien t to predispose to th e development of the classic vascular anomalies seen with HHT. Ho wever, these are site specific rather than generalized, suggesting that othe r events, either genetic or environmental, are necessary to switch the normal equilibrium of vascular cells from a maintenance state. To date, no

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3 specific evidence for a second genetic hit of eith er gene has been brought forward but it is a suspected mechanism. Expression studies in human umbilical vein endothelial cells and peripheral blood monocytes have confirmed hapl oinsufficiency as the mechanism in both main forms of HHT. The identification and characterization of mutations in HHT patients revealed extensive molecular heterogeneity (5). As a result of different selection criteria, populations and detection methods for the mu tation analysis, diffe rent groups report different mutation detection rates. ENG has 14 exons and occupies 30 kb and to date, 114 mutations have been reported and the distribu tion of these mutations in the gene is shown in figure 1 (6). ALK1 is made up of 10 e xons and spans more than 15 kb of genomic DNA. ALK1 mutations are dist ributed over the entire coding region with exons 3, 7, and 8 being most frequently affected with a tota l of 80 mutations (6). (Figure 2) When the possible founder effects are excluded, the percentage of ENG (53%) and ALK1 (47%) mutations causing HHT is almost the same (5). Figure 1 : Schematic of the genomic structure of ENG showing locati on of meditations TGFSignaling Endoglin and activin receptor-like kinase-1 are plasma membrane-bound receptors involved in the transducti on of transforming growth factor (TGF) signals

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4 Figure 2 : Schematic of the genomic structure of ALK1 showing loca tion of meditations which regulate the synthesis of perivascular matrix proteins and differentiation and recruitment of periendothelial cells during vascular development. This pathway also regulates many other biological processes as we ll as inhibiting pro liferation, stimulation of extracellular matrix depos ition, and modulation of the i mmune response, therefore TGFsignaling has been linked to various diseas es such as cancer, pulmonary and liver fibrosis, autoimmune diseases and vascular disorders (6). Although ENG and ALK1 can potentially be expressed in many different cells, transcripts fro m both ENG and ALK1 are expressed predominantly in vascular endo thelial cells. The gene ral and most studied model of TGFsignaling requires ligand binding to its cell surface type II receptor which will self phosphorylate the intracellula r kinase domain at serine and threonine residues, located in the cytoplasmic GS-domai n. This process enables recruitment of the type I receptor into the activated comple x, and once activated, the type I receptor phosphorylates members of the Smad family of intracellular mediator s, which act as cell type-specific transcription factors. Specifici ty of the response is determined by the components of the cascade that are activated. (Figure 3) For example, interaction of the

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5 type I receptor ALK1 leads to phosphorylation of receptor Smad 1, 5, and 8. Less well characterized Smad independent pathways, t ogether with Smad dependent processes, may be further regulated by the expression of type III receptors such as ENG (8). Figure 3: Schematic representation of the TGFsignal trans duction pathway. Endoglin ENG is a type III (nonsignaling) TGFcoreceptor (9). ENG binds different members of the TGFsuperfamily in the presence of th e signaling receptors types I and II (10). Human ENG is a homodimeric transmembrane glycoprotein constitutively expressed at high levels on endot helial cells of capillaries, ve ins and arteries. It has been postulated that endoglin might also have the ability to recruit other Smads or downstream second messengers into the signaling comple x. Because ENG facilitates signaling by growth factors it is plausible that a mutation in ENG could alter the constraints placed on growth of endothelial cells and smooth mu scle cells, therefore stimulating vessel expansion as seen in HHT (11). Studies have shown that targeted inactivation of the TGFSignaling Pathway II I p p T R-II ActRIIA ActRIIB BMPRII MIS-II ALK1-7 Smads Gene Regulation

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6 endoglin gene results in vascular and cardi ovascular defects in early mouse embryos. From embryonic day 9.0, the primitive vascular plexus of the yolk sac fails to form mature structures leading to vessel dilation, rupture and he morrhage. The vasculature of the ENG null embryos is also very fragile a nd bleeds, demonstrating a crucial role for ENG in remodeling and sprouting required for angiogenesis. Angiogene sis refers to the process of neovascularization by sprouting from preexisting blood ve ssels. Heart defects are also observed in ENG null mice. Cushion tissue formation, essential for valve development and heart septation, does not occur and pericardial edema is observed. These vascular developmental anomalies lead to death at embryonic day 10–10.5 (12). Activin Receptor-like Kinase-1 In the TGFpathway, there are currently seven type I receptors identified and designated as activin receptor-like kinase (A LK) 1 to 7. The ligand specificity of these ALKs has been determined primarily by thei r ability to bind to a given ligand and to activate specific downstream genes in the presence of corresponding type II receptors. ALK1 is able to bind to TGF-ß1 or activins in the presence of either T R-II or activin type II receptors, respective ly (13). Restricted expression of ALK1 in the arterial endothelium suggests that ALK1 may play a pivotal role in the maturation and stabilization of arterial vessels. It has been shown by Oh et al that ALK1-deficiency in mice results in embryonic lethality, with se vere dilation of large blood vessels and excessive fusion of capillaries. Impairment in the recruitment or differentiation of vascular smooth muscle cells also being observed in these mice (14). Role of ALK1 in Arterialization and Vascular Remodeling The structure of arteries and veins is designed to fulfill the pressure requirements of directional blood flow. The disruption of this network can have devastating

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7 consequences (15). Recent identification of genes that are differentially expressed in arteries over veins reveals information about the mechanisms determining arterial and venous vessel formation. These genes includ e notch ligand Dll4, basic helix-loop-helix transcription factor gridlock, and membrane -bound ligand ephrin-B2. It was shown that gridlock favors the differentiation of comm on angioblasts to prear terial angioblasts (16,17). Notch signaling, which is associated with cell fate determination through lateral specification, may also be involved in the differentiation of arterial versus venous endothelium (18). Membrane-bound ephrineB2 and its receptor EphB4 (putative receptor for ephrine-B2) show restricted expr ession in arteries and veins, respectively, and their bidirectional interactions ar e required for proper angiogenesis (12). Various physiological factors, such as blood flow (shear stress) or pressure (cyclic strain), have been shown to trigger the remodeling of bl ood vessels, which involves the synthesis of extracellular matr ix proteins and the prolifera tion of vascular smooth muscle cells. Induced ALK1 expression in preexisting feed arteries distant from the wound and tumor during wound healing and tumorigenesis, as well as in the small mesenteric arteries corresponding to the increase of blood flow, suggest that AL K1 signaling is also involved in the vascular remodeling of arteries . Findings by Oh et al are consistent with the findings that TGFand ALK1 expression was elevat ed in the pulmonary vessels with increased blood flow, and that increased TGF1 mRNA and protein from endothelial cells were stimulat ed by laminar shear stress (14). The exact revealing event or tr igger for the formation of va scular abnormalities in HHT patients is not yet identified. One theory is that the expression of familial diseases such as the development of an arteriovenous malf ormation in HHT requires a second hit to

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8 inactivate the normal copy of a gene. The fact that nosebleeds and mucocutaneous telangiectasis in the eyes and lips are the most common symptoms among variable disease manifestations of HHT is suggestive of this second hit theory (1). However, a more recent hypothesis, hypoxia, has come into light and its basis is on new data on the regulation of ENG gene expr ession. Hypoxia causes an increa se in expression of ENG mRNA and protein in human endothelial a nd monocytic cell lines. In addition, when these same cell lines are treated with TGFand cultured under hypoxic conditions, a synergistic effect on ENG expression is observed. Since hypoxia and TGFhave a similar effect on VEGF expression in human endothelial cells and because VEGF plays an important role in the promotion of angi ogenesis, hypoxia appears to be a valid reason for the trigger of vascular remodeling that oc curs in HHT patients. Although this theory is appealing, it still leaves some questions open, such as, why hypoxia would lead to a different expression depending on the time of the revealing trigger, why no de-novo lesions appear, and why only the ve nous site is affected (3). The purpose of this study has been to investigate whether Alk1 signaling is sufficient for arteriogenesis. It has been show n by Oh et al that deletion of ALK1 shows embryonic lethality, hyperdilation of large bl ood vessels, excessive fusion of capillaries and impairment in recruitment of vascular smooth muscle cells to which they could conclude that ALK1 plays an important role in vascular development. More specifically my hypothesis is that ALK1 may play an important role in arteriogenesis and the remodeling of arteries. My approach involved overexpression of the ALK1 gene in the entire vascular endothelium including veins in transgenic mice, using a pan-endothelial cell promoter, the Tie2 regulatory sequence. However, this transgenic approach had a

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9 number of limitations to conclude for the ro le of ALK1 signaling in arteriogenesis: including ligand and type II receptor dependency by wild -type ALK1, the transgene expression variation by the copy number and in sertion location in the chromosome and, the Tie2 (M) promoter/enhancer element not being strong enough to produce a robust expression. To overcome these potential defi ciencies, the approach was modified as follows. For ligand and type II receptor depe ndency, a constitutive active form (always active) of Alk1 cDNA (caAlk1). For the issues of robust expression as well as dependency of the copy number transgene inser tion site, we decided to target the Rosa26 locus with caAlk1. This caAlk1 expressi on will be activated by crossing the Rosa26caAlk1-knockin mice with known Cre transgenic mice, such as Tie1-Cre, Flk1-Cre or Alk1-Cre.

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10 CHAPTER 2 MATERIALS AND METHODS Generation of Tie2-ALK1 Transgenic Mice The construct was done by Jihye Yun at the University of Florida College of Medicine. The full length murine ALK1 cD NA was obtained by join ing the previously isolated partial mALK1 cDNA and an EST clone. DNA sequence analysis confirmed the full length mALK1 cDNA. The Flag tag was added at the C-terminal end using PCR based modification. The 3Â’ Primer containe d the coding sequence of the last 5 amino acids of ALK1, a flag tag and a stop codon. An in vitro translation experiment using TNT Quick Coupled Transcription/ Translation Sy stem (Promega) was done to test for the Alk1 protein product from the mAlk1-Fl ag sequence. For uniform endothelium expression of ALK1, the Tie2 promoter and e nhancer element from Dr. Thomas Sato at the University of Texas Southwestern Medi cal School was used. Two transgene vectors were engineered and designated Tie2 (M)-A LK1 and Tie2 (F)-ALK1, both contained the Tie2 promoter and mALK1-Flag followed by SV40 polyA signal. The Tie2 (F) enhancer has more consistent and uniform expressi on during postnatal st ages but since the immediate goal is to test whether ALK1 is sufficient for arteriogenesis either would suffice. The transgenic mice were generate d using standard methods by the transgenic core with Tie2(M)-ALK1 at the University of Florida College of Medicine. The offsprings were screened by genomic PCR anal ysis using primers from a primer set from two exons that would distinguish from th e transgene Alk1 and genomic ALK1 and 2 mice were identified as positive for the transgene.

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11 Staining Adult mice were dissected with remova l of the lungs, spleen and kidney which were fixed in 4% formaldehyde. The spleen samples were serially dehydrated and then paraffin wax embedded. Samples were secti oned at 7 µm thickness and placed on glass slides. For H&E staining, secti ons were rehydrated and then stained in hematoxylin for 6 minutes and eosin for 2 minutes. For immunohistochemistry, sections were deparaffinized and hydrated and treated with 3% hydrogen peroxide fo r 10 minutes. The Antibodies used to identify specific prot eins were monoclonal anti-smooth muscle actin (Sigma) and anti-flag (Sigma) using ve ctastain immunohistoc hemical staining kits. (Vector Laboratory, CA, USA) Dye Injection Adult mice were injected with 0.5 ml of av ertin, and then their chest cavities were opened with an incision in the right atrium. Two units of he parin in PBS were perfused through the left ventricle followed by 1 mL of blue latex dye using steady pressure. The spleens were removed for microscopic examin ation and cleared with organic solvent. Western Blotting Tissues of interest were homogenized on ice in 500 L of sample buffer (62.5 mM Tris-Cl (pH 6.8), 2.5% SDS, 0.1% bromophenol blue, 1% 2 Beta-mercaptoethanol, 10% glycerol) followed by the sonic dismembraner . Lysates were centrifuged at 14,000 x g for 5 minutes and the pellet discarded. Cleared cel l lysates were boiled at 100 degrees for 10 minutes. Analysis by electrophoresis was done in 12% SDS-PAGE gel and then the proteins were electr otransferred to nitrocellulose me mbranes and soaked in blocking solution (PBT, 5% skim milk, 2% BSA) incubating in 4 degrees overnight.

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12 Immunodetection was done by anti-flag an tibody (1:1000, Sigma) and membranes were developed by chemiluminescence for 5 minutes. (Supersignal, Pierce) Reverse Transcription PCR RNA was extracted using Trizol reagent (Invitrogen) and Dnase I treated. First strand cDNA was synthesized using Superscr ipt III First-Strand synthesis kit for RTPCR. (Invitrogen) Forty cycles of PCR were ran with 2 primer sets: forward 1: 5’ AAG AGT CGC AAT GTG CTG GTCAAG 3’, reve rse: 5’ GTC GTC ATC GTC TTT GTA GTC GTG 3’, and forward 2: 5’ TTG T GG AGG ATT ACA GGC CAC CTT 3’ from mALK1-flag DNA. The PCR products were separated on 1.5% agarose gel and visualized with ethidium bromide. Northern Analysis RNA was extracted using Trizol reagent (Invitrogen). Formamide was used to dissolve the final RNA and total RNA was qua ntified by measuring the absorbance at 260nm. All samples were stored at -80°C and incubated in a 65 C water bath for 5 minutes prior to Northern blot analysis with immediate loadi ng afterwards of 10 g. Total RNAs together with size markers were electrophoresed on a 1% agarose gel containing 10mM Sodium Phosphate buffer at pH 6.8 at 3 to 7 volts V/cm. Because the buffering capacity of the electrophoretic buffer is relatively weak, constant recirculation of the buffer is maintained. At the completion of the electrophoresis the gels were examined on a UV light box. The gel was washed with shaking for 10 minutes in 7% (v/v) formaldehyde and then washed twice with 50 C water for 15 minute each. The agarose gel was then blotted with a sheet of nylon membrane (Hybond – N). The RNA was fixed to the membrane by using an op timized UV cross-linking procedure and the

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13 position of the bands of the RNA ladder were marked on the filter. The blot was then wrapped and stored at -20° C prior to prehybridization. DNA Constructs The constitutively active form of human AL K1 (caALK1) with a Ha tag added at the C-terminal end was given to us by Eiic hi Oeda from Yamaguc hi Medical School and inserted into the pcDNA3 vector (Invitrog en). The pcDNA3 vector with caALK1 was double digested the enzymes Spe I and Sal I, and at the same time the pBigT vector was double digested by Nhe I and XhoI. The caALK1 DNA was then run on 0.8% gel and the 1.5kb band was cut out while the vector st ill incubated. Both the caALK1 DNA and vector were purified with Stra ta Prep DNA gel extraction kit. (Stratagene, LaJolla, CA) Once the elution of DNA was verified, both the caALK1 DNA and pBigT vector were ligated with T4 DNA ligase overnight in a 16 C water bath. Competent cells were then transformed with 10µl of ligated pBigT-caA LK1 DNA with the plating of 100 µl on a LB amp plate left overnight in 37 C. Next, microcentrifuge tube s and plates were inoculated with the colonies that grew overnight and left to cultu re overnight at 37 C. The tubes were then minipreped by the boiling met hod and then checked for correct ligation by digestion with EcoRI which would give bands at 2.7 and 4.8 kb if there was ligation in the correct orientation. Af ter positive clones were identified, one was picked for verification by digestion with Bgl II ( 5.6, 1.5, 200kb), Sac I (6.3, 1.3), Sph I (3.2, 2.5, 1.0, 0.8kb), EroRV/Not I (5, 2.5) produci ng bands at the noted sizes. Then 8mL of LB amp in a 50ml conical tube was inoculated with th e positive colony and incubated overnight at 37 C. The DNA was then minipreped and isol ated using Fast Plasmid Mini (Eppendorf) and sequenced using the primers pBig T-R (5-CACCTA CTCAGACAATGCGATG-3),

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14 pBigT-F2 (5-GTCTGGATCCCCATCAAGC TG-3), hA1seq (5-TGGACAGTGACTGC ACACAG-3), hA1seq2 (5-CAAATGACCCCAGCTTTGAGGAC-3), and hA1seq3 (5GCTCTCACACGTGCAGGTCAC-3) using Big Dye Terminator 3.1 at the sequencing core at the University of Florida. For ubiquitous endothelium expr ession of ALK1, the Rosa26 promoter was used. The final linear ization needed a rare cutting restriction enzyme that needed to be modified by a Fse I adaptor oli go (5-phosGGCCGGTAC-3) because the proposed Kpn I site is also in caALK1 DNA. The adaptor oligo (100pM) was prepared by boiling for 5 minutes and then cooled for 30 minutes at room temperature before using. The Rosa 26 vector was digested with Kpn I and then purified using StrataPrep DNA gel extraction kit, eluti ng 10µl. The digested and purified Rosa 26 vector DNA was ligated for 1 hour with the adaptor oligo at 37 C. Prior to transformation with 5 µl of the ligated DNA, it was digested with Kpn I to cut up any Rosa 26 vectors that may have ligated back together. Next, 4ml of LB-amp was inoculated and left to culture overnight at 37 C with DNA isolation using Fast Plasmid Mini. Eluted DNA was verified for FseI modification by digestion by Fse I, and a positive clone was picked for sequencing using M13 reverse primer (5GGAAACAGCTATGACCATG-3). The positiv e clone for caALK1 in pBigT being inserted into the modified Rosa26 vector was done the same way as mentioned for caALK1 ligation with the pBigT vector. PBigT-caALK1 DNA and Rosa26 were double digested with Asc I and Pac I, the pBigT-ALK1 DNA produced bands at 4.7 and 2.8, with the 4.7 band containing the caALK1 DNA insert. Verification for ligation was done by using the same enzymes and positive cl ones generating bands at 11 and 4.7kb and sequenced using M13 F (5-GTAAAACGAC GGCC AG-3). There was a problem

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15 encountered with culturing the positive final clones at 37 C as well as poor DNA quality. Therefore, the positive clone was inoculated in 10mL of LB amp and also streaked on a plate and grown overnight at 30 C. Five milliliters was then inoculated into 240mL of LB amp and grown overnight at 30 C in preparation for a large prep using Qiagen Maxi kit. Electroporation and ES Cell Growth Digestion of the final DNA was done to prepare for electroporation. This was done with 60 µ g of DNA in 100 µ l with 5 µ l AsiSI incubating for 2 hours at 37 C with another 5 µ l of enzyme added and incubated ag ain for another 2 hours. Two hundred microliters of deionized water was adde d followed by phenol/chloroform extraction. Two hundred and seventy microliters was transf erred to a new tube with ethanol added and kept at -20 C until used for electroporation. ES cells were cultured in a culture plate for 2-3 days followed by centrifuging with et hanol. DNA was prepared by dissolving it in deionized water and adding it into a freezing tube . ES cells were then washed with Hepes followed by trypsin and incubated at 37 C. Next ES + LIF media was added and then centrifuged followed by washing with electropor ation buffer. The ES cells were then added to the tube containing DNA and transf erred into a cuvette. Electroporation was done under the parameters of 0.25 kV, 0.25 µ F x 1000. These ES cells were incubated at room temperature while the feeder cells were quickly prepared by suspending them in an ES + LIF and EF + LIF media mix. Both cells were then plated and incubated overnight. The plates were then washed with He pes and the ES+LIF+G418 media was changed daily. Once the ES cell colonies looked to be of good size, they were picked. A 96 well plate was prepared with 20 µ l of trypsin. The media in the ES cell plate was changed out

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16 to Hepes and each picked and placed in a different well. The 96 well plate was then incubated at 37 C for 8 minutes, and at its conclusion ES media was added into each well and pipetted 15 times. The ES cells were then transferred to feeder cells with the ES media being changed daily for 4 days. Genomic Southern Twenty four well plates were prepared with 0.5ml feeder ce lls per well. Media was then removed from the 96 well plate with ES cells and washed with Hepes followed by adding trypsin. After a 10 minute incubation, 70 µ l of ES media was added and mixed 20 times by pipetting. Forty microliter s of freezing media along with 40 µ l of ES cells are transferred to a freezing plate and sealed with parafilm and stored at -80 C. The remaining ES cells were transferred into th e 24 well plate. The media was changed with 1/5 ES media daily for a week. At its c onclusion, the DNA was extracted from the ES cells by adding 250 µ l of lysis buffer containing p k20. The plates were wrapped thoroughly and incubated at 55 C over night with shaking at about 210 rpm. The next day the plates were allowed to cool to room temperature and then 250 µ l of isopropanol was added and mixed. The DNA was carefully picked up by forceps; excess isopropanol was removed and then resuspended in 25 µ l of deionized water. The tubes with the DNA mixture was then incubated at 55 C overnight for complete melting. Afterwards the DNA was stored at 4 C until southern blot analysis. Th e DNA was loaded onto a 0.8% gel and run at 20 mV overnight. After the gel was run, a picture was taken with a ruler next to the marker. Next the gel was washed in 0.25 N HCl with light shaking for 30 minutes in order to chop the DNA into small pieces. The gel was then rinsed several times with deionized water. The gel was then washed in 0.4N NaOH with shaking for 30 minutes

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17 twice with washing with water in betw een. Next the DNA was transferred to a membrane. The membrane was hybridized with 250 kb probe from the 5 prime end of the Rosa26 vector.

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18 111 73 47 mAlk1-Flag +111 73 47 mAlk1-Flag +-CHAPTER 3 RESULTS Flag-tagged Full-length Murine Alk1(m Alk1) cDNA and In Vitro Translation The full-length murine Alk1 cDNA was obtained by joining the previously isolated partial mAlk1 cDNA and an EST clone (IMAGE clone: ID 2939277). DNA sequence analysis confirmed that the combined cDNAs represented the full-le ngth mAlk1 cDNA. To identify the Alk1 transgene, we added th e Flag tag at the C-te rminal end of mAlk1 using a PCR based modification (mAlk1-Flag). The 3Â’ PCR primer contained the coding sequences of the last 5 amino acids of Alk1, a Flag tag, and a stop codon. To test whether mAlk1-Flag produces Alk1 protei n product, we performed an in vitro translation experiment using TNT Quick Coupled Transcri ption/Translation System (Promega). The expected size of in vitro translated mAlk1-Flag protein is 57.6 kDa. As shown in Figure 4, the main product of in vitro translated protein wa s about 60 kDa. Western blot analysis using anti-FLAG mAb (Sigma Chemical Co.) recognized a major band at around 60 kDa (figure 4). Figure 4 . Autoradiograph of In Vitro translated and western products. (A) Autoradiogram of In vitro translated mAlk1-Flag. (B) Westen blot of in vitro translated product with anti-Flag an tibody. Expected size of mAlk1-flag is about 60 kDa. The size markers are indicated in kDa.

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19 Construction of Tie2-Alk1 Transgene Vector Figure 5. Schematic diagram showing the procedur es for the generation of Tie2-Alk1 transgenic construct For uniform endothelium expression of Alk1, we propose to use Tie2 promoter and enhancer element, which are well characterized in transgenic mice (18-20). Plasmid vectors containing Tie2 promoter and Tie2 mi nimal enhancer elements were kindly given to us from Dr. Thomas N. Sato (University of Texas Southwestern Medical School) (18). With multiple subcloning steps, we successf ully engineered two transgene vectors, designated Tie2(M)-Alk1, which contains Tie2 promoter and mAlk1-Flag followed by SV40 poly A signal and the 2kb Tie2 minimal e nhancer. Transgenic studies have shown that the 2kb Tie2 minimal enhancer is iden tical in terms of sufficient expression in embryonic vascular endothelium in comparis on to the 10kb Tie2 full-length enhancer Tie2 enhancer pA Tie2 Promoter pA mAlk1-Flag Tie2 (M) enhancer SalI NotIPmeI/NaeISalI Tie2 Promoter pA mAlk1-Flag SalI NotIPmeISalI Tie2 (M) enhancer NaeISalI SalI Tie2 Promoter pA mAlk1-Flag SalI NotIPmeISalI HindIIINotI HindIII Tie2 Promoter SalI NotIXhoI HindIII KpnI Replacement Tie2 minimum enhancer with PmeIlinler pSPTg.T2FpAXK pSPTg.T2FpAPmeI Tie2-Alk1 pgSo-2.11 HindIII pSPTg.T2FAlk1pAPmeI XhoI Step 1 Step 2 Step 3 Tie2 enhancer pA Tie2 Promoter pA mAlk1-Flag Tie2 (M) enhancer SalI NotIPmeI/NaeISalI Tie2 Promoter pA mAlk1-Flag SalI NotIPmeISalI Tie2 (M) enhancer NaeISalI SalI Tie2 Promoter pA mAlk1-Flag SalI NotIPmeISalI HindIIINotI HindIII Tie2 Promoter SalI NotIXhoI HindIII KpnI Replacement Tie2 minimum enhancer with PmeIlinler pSPTg.T2FpAXK pSPTg.T2FpAPmeI Tie2-Alk1 pgSo-2.11 HindIII pSPTg.T2FAlk1pAPmeI XhoI Step 1 Step 2 Step 3

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20 (Tie2(F)). It was reported that Tie2(F) enhancer has more consistent and uniform expression during postnatal stages than Tie2(M ) enhancer (18). Since Alk1 expression is predominant in embryonic and early postnatal stages and our immediate goal is to test whether Alk1 is sufficient for arteriogenesis, either Tie2(M) or Tie2(F) would equally suffice for our goal. With this reason we d ecided to use the Tie2(M )-Alk1 construct. Generation and Screening of Transgenic Founder Mice Transgenic founder mice were genera ted using standard methods by the transgenic core at the University of Florida, College of Medicine. Briefly, SalI-SalI fragment of the Tie2(M)-Alk1 transgene inse rt was microinjected into the pronuclei of fertilized eggs from FVB female mice. Su ccessfully grown two-cell stage embryos were transferred into the oviduct of pseudopregnant females (21). Offsprings were screened by genomic PCR analysis using primer se ts from two exons, which allow us to distinguish transgene Alk1 cDNA from Alk1 genomic DNA containing introns. We have obtained a total of 23 offspring. PCR screening has identified 2 mice positive for the transgene (figure 6). These founder mice were designated as Tg(Tie2-Alk1A ) and Tg(Tie2-Alk1B ). Initial Characterization of Tg(T ie2-Alk1-A) and Tg(Tie2-Alk1-B) The two founder mice were initially cro ssed with wild-type FVB females. We obtained transgene-positive viable F1 offspri ng from each line. At least three F1 males from each line were further crossed w ith wild-type FVB or 129Sv/C57BL6 hybrid females, and observed transgene-positive F2 mice in an expected Mendelian ratio at weaning age, indicating that the Tie2-Alk1 tr ansgene insertion in these two lines do not affect normal embryonic and postnatal devel opment. Morphological and histological

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21 Tie2 Promoter mAlk1-Flag pA Tie2 (M) SalI SalI 450 bp 1340bp 450bp 8910111213 14 15161718 19 20212223 Tie2 Promoter mAlk1-Flag pA Tie2 (M) SalI SalI 450 bp 1340bp 450bp 8910111213 14 15161718 19 20212223 Figure 6: Tie2(M)-Alk construct and PCR genotyping of 23 offspring from pronuclei injections. A primer set was genera ted from two exons separated by 1.34kb. The Tie2(M)-Alk1 construct contains Al k1 cDNA, and thus the same primer set amplifies 450bp band from transgen e positive founder mice (#14 and #19). analysis of transgene-positive embryos show ed no discernible defects in arteries and veins (data not shown). The Tg(Tie2-Alk1-B) Line Disp lay Hemorrhages in the Spleen We performed autopsies of Tg mice at the weaning age and found that Tg-positive mice from the Tg(Tie2-Alk1-B) line have si gns of hemorrhages in the spleen. The hemorrhage spots were localized to the lower dorsal side of the spleen (figure 7). This splenic hemorrhage phenotype had been obser ved on a consistent basis with a high penetrance (5/6) in the initial phase of character ization. Histological analysis revealed that the red pulp areas appeared to be irregular and dark precip itants were found along the blood vessels indicating chronic hemorrhages (figure 8) . To visualize blood vessels in the spleen, latex dye was injected into the left ventricle of the heart, and the spleen was taken out for microscopic examinations after clearing with organic solvent. Sple ens having no sign of hemorrhages (either wild-type or Tg-positive mice) showed more rigorous spreading of the dye into the red pulp areas in comparison with hemorrhagic spleens (figure 9). In order to

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22 determine the initial stage when hemorrhage begi ns to occur, offspring from Tg mice were examined at various embryonic (E) and postn atal (PN) stages, in cluding PN5, PN7, PN14, and PN21. The earliest age that the hemorrh agic spleens were observed was at PN14. (table 1) As we analyze the splenic hemo rrhage phenotype further, penetrance of the phenotype diminished. Out of a a total of 69 Tg-positive adult mice examined, 11 mice exhibited the splenic hemorrhag e phenotype. We also found one out of 20 wild-type adult mice which displayed the similar phenotype. Figure 7: Hemorrhages in the spleen of 4 weeksold Tg(Tie2-Alk1-B) mice. (A) Spleens of wild-type and Tg(B) mice, showing hemorrhage (indicated by asterisk). The arrow indicates abnormal shape of the spleen often associated with hemorrhagic spleen. (B) A higher magnified view of the Tg(B) spleen shown in (A). (C) Hemorrhagic spleen found in another Tg(B) line. Figure 8: Histological demonstration of hemorrhages in the spleen of the Tg mice. The arrows show dark precipitants along the blood vessels. RP, red pulp; WP, white pulp; SMA, smooth muscle alpha actin.

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23 Figure 9: Visualized blood vessels by latex dye in the spleens. (A) wild-type and (B) Tg(Tie2-Alk1-B) having hemorrhages. No te spreading of dyes into red pulp areas (indicated by arrows) in WT but not in Tg mice. Table 1: Mice examined at various stages before weaning age # of Wt # of Tg PN5 12 (0) 4 (0) PN7 5 (0) 5 (0) PN 14 12 (2) 5(1) PN 21 2 (0) 5 (1) These are total numbers of nice examined at each stage with the numbers in parenthesis representing the number of mice w ith hemorrhaging in the spleen. Analysis of Transgene Expression In order to investigate whether transgene is expressed in the Tg-positive mice, we performed RT-PCR as well as Western blot analysis. To specifically amplify the transgenic Alk1 transcripts, we generated a 3Â’ primer from the Flag se quence inserted at the 3Â’ end of mAlk1 cDNA. We also generated tw o different 5Â’ primers, with which we would be able to detect the transgenic Alk1 transc ripts by RT-PCR in two different sizes, 537 and 281 base pairs, when they are combined with the 3Â’ primer. Total RNA was isolated from the lung of 4 week-old wild-type and Tg(B )-positive mice. PCR amplification was performed using the two primer sets on th e first strand cDNA templates which were

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24 generated with the reverse tr anscriptase. As shown infF igure 10, PCR band with the anticipated sizes were detected only in Tgpositive samples. Non-specific PCR band that was found in the wild-type sample was appear ed to be due to genomic DNA contamination as such bands disappeared when RNA sample was treated with DNAse (data not shown). To detect transgenic ALK1 in Tg samples, we performed the Western blot analysis using both anti-Alk1 antibody (purch ased from R&D and Santa Cruise) as well as anti-Flag antibodies. We were unable to detect the tr ansgenic ALK1 protein from either ALK1 or Flag antibodies, even after much struggle, ex cept for a faint band at the expected size. Immunostaining was also unsuccessful. These results indicate a low level of transgenic ALK1 expression. Figure 10: RT-PCR analysis showing the transgenic Alk1 transcripts specifically found in Tg(B) samples. Asterisk indicates a non-specific PCR band due to genomic DNA contamination. Limitations of the Transgenic ALK1 Overexpression Approach Due to uncontrolled insertion sites and copy number of transgene, it requires multiple independent transgenic lines to conclu de that a phenotype observed in a transgenic line is resulted from the transgene overe xpression. Although we observed no apparent vascular phenotype from two st able and independent transgenic lines, it is premature to

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25 conclude that increased ALK1 signaling in ve ins is insufficient for transmorphogenesis of veins to artery-like vessels, becau se it could be owing to low level of transgene expression. This may indicate that the Tie2 (M) promoter/e nhancer element that we used is not strong enough to produce a robust expression of the tran sgene. In addition, the wild-type ALK1 may require proper stimulation by its ligand and presence of essential Type II receptor. An Alternative Approach: Rosa26-caAlk1 Knockin Mice We sought to overcome these limitations, and decided to modify our approaches as follows. For lignad and type II receptor dependency, we decided to use a constitutive active form (always active) of Alk1 cDNA (caAlk1), which is independent from availability of its ligand and t ype II receptor. For the issues of robust expression as well as dependency of the copy number and transgene insertion site, we decided to target the Rosa26 locus with caAlk1. Rosa26 is one of the most well characterized gene locus, which drives robust and ubiqu itous expression (23). Lastly, to control tissue specific caAlk1 overexpression, the caAlk1 cDNA is designed to be inserted into th e 3Â’ region of a translational stop codon flanked by the loxP sequences in a Ro sa26 construct (14). Thus, the inserted caALK1 would be silenced in all tissues, but constitutively overexpressed in the tissue only where active Cre recombinase is present. Well characterized Cre transgenic mice, such as Tie1-Cre, Tie2-Cre, or Flk1-Cre will be used for the pan-endothelium specific caALK1 overexpression. To disti nguish from the endogenous mAlk1, we use human Alk1 and attach the Flag sequence at the carboxyl-terminal of the ca-hAlk1 cDNA. The Rosa26-caAlk1 gene targeting construct has been successfully generated as detailed in Materials and Method section. The linearized targe ting vector was electroporated into embryonic stem (ES) cells. We picked 96 G418resistant ES clones, and homologous recombination is currently being screened by genomic Southern blot analysis. Once

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26 available, the targeted ES cells will be inje cted into blastocysts to generate germline chimeras. Figure 11: Schematic diagram showing Rosa26-caAlk1 gene targeting strategy

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27 CHAPTER 4 DISCUSSION The long-term goal of this project is eluc idate the role of ALK1, an endotheliumspecific TGFtype I receptor, in the developmen t and remodeling of blood vessels. Since heterozygous ALK1 mutation causes HHT, understanding in vivo role of ALK1 in vascular biology would help us to illuminate underlyi ng pathogenetic mechanism for HHT as well as to develop a novel therapeutic scheme for su ch a malady. Based on data using various genetically altered mouse mode ls, Dr. OhÂ’s group hypothesized that ALK1 signaling is essential for arte riogenesis (13). This hypothesi s can be tested by either deletion of Alk1 in arteries (knockout or conditional KO) for its necessity, or ectopic expression of Alk1 in veins (trans genic) for its sufficiency. Dr. OhÂ’s and Dr. Dean LiÂ’s groups have previously generated three independent constitutive knockout mice for the Alk1 gene, and demonstrated that Alk1-defici ency results in seve re dilation of blood vessels and in formation of arteriovenous ma lformation (7). In addition, our laboratory has been characterizing a condi tional Alk1-knockout allele. Preliminary data show that Alk1-deficiency leads to hyperd ilation arteries, similar to the morphology of veins (Park et al., unpublished). These data supports the necessity issue of ALK1 signaling in arteriogenesis. The current transgenic approach deals with the sufficiency issue, which is complementary to the ongoing conditional knockout studies. In this thesis, we showed generation and characterization of two independent transgenic mouse lines, in which wild-typ e murine Alk1 is expressed under a pan-

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28 endothelium promoter, Tie2. We confirmed st able integrations of the transgene in each line as the transgene was detected in mice through multiple generations. In one of the transgenic lines, the Tg(Tie2-Alk1-B) line, there was an interesting phenotype exhibiting hemorrhages in the dorsal part of the splee n. Histological sections also showed chronic hemorrhagic lesions along blood vessels. Although only 16% of Tg-B adult mice exhibited such a phenotype, this result i ndicates that Alk1 overexpression in entire vascular beds may impact development or ma intenance of some selected vessels. Other than this minor vascular phenotype, however , Tg-positive mice from both line did not exhibited any apparent mor phological or functional de fects in blood vessels. Nonetheless, we believe it is premature to conclude that elevated ALK1 signaling in veins may not be sufficient to change the mor phologies of veins to those of arteries for the following reasons. First, we were able to detect transgenic Alk1 transcripts, yet failed to demonstrate protein expression or localiza tion in the vascular endotheli um, indicating a low level of transgene expression. We used both anti-A lk1 and anti-Flag antibodies. Although we have been unsuccessful to detect ALK1 usi ng the anti-Alk1 antibodies even in wild-type tissues, the anti-Flag antibodies have been successfully used to detect the in vitro translated Alk1-Flag proteins and other Flag -tagged proteins in our hands, and thus the failure of detecting transgenic ALK1 prot ein should not be attributed to the poor condition of the antibodies or Western blot. Since each transgenic line has independent insertion site and copy number of the tran sgene, it is uncommon to observe a poor transgene expression in a few transgenic lines. Unfortunately we were unable to examine tissues of the Tg(Tie2-Alk1-A) mice becau se we have lost the colonies.

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29 Second, it is not certain wh ether overexpression of ALK1 alone would have been sufficient to elicit the signaling cascade. The chice of the transgenic approach is based on the assumption that TGFand other signaling partners such as TGFtype II receptor and relevant Smads are present in venous e ndothelium. It has been shown that TGFis expressed in the developing blood vessels, a nd physiologically sufficient quantity of TGFis present in the serum. TGFtype II receptor (TGFBR2 ) expression in blood vessels has not been clearly shown. Howe ver, Alk1 expression in blood vessels and similar vascular phenotype of Tgfbr2-null embryos with TGF1-null embryos suggest that a low level of TGFBR2 is present in blood vessels indi scriminately. Therefore, it was not unreasonable to assume that the TGFin the serum can interact and elicit signaling via receptors expressed in the endothelial cells. When we deliberated how to pursue this pr oject further, we had to make a choice between the Rosa26-Alk1 knockin approach and generating more transgenic lines using the same Tie2(M) or other pan-endothelial pr omoters such as Tie1 and Tie2(F). The conventional transgenic approach requires less time for the production of transgenic mice, but a huge effort for careful analysis of multiple lines. In this regard, the Rosa26Alk1 knockin approach has a clear advantag e since Alk1 overexpres sion can be induced by well-characterized pan-endot helium-Cre mice. We also considered the “tet-on” system system so that we can turn on and o ff the transgenic Alk1 expression. However, this system requires two independent transg enic strains, and demands more time and money compare to the knockin approach. In the new approach, we decided to use the constitutively active form of Alk1 instead of the wild-type. Certainly, the us e of caAlk1 would allow us to overcome the

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30 ligand and/or type II recept or-dependent limitations. Since caALK1 activates Smads 1, 5, and 8, regardless of ligand, however, this may result in mimicking the activation of BMP signals. Robust, constitutive over expression of caALK1 under the Rosa26 promoter could be another pitfall of the revised approach in terms of physiological relevance. Therefore, the in terpretation of the result woul d require careful attention. Although the revised approach still has potential limitations, it appears to be the best choice to directly address whether elevated ALK1 signaling in the vein could alter the vessel morphology. With the revisions in the new approach some additional invivo and invitro studies that can be employed to examine possible ph enotypes are studies examining the retina, apoptosis, and capillary endothelial cell culture s. First some invivo studies include the retina and apoptotic studies. The retina, ha ving a very sensitive ve ssel capillary network, makes it a prime location to study any malformati ons in vasculature. Al so due to the ease of examining the retina without invasive procedures in vi sual examination makes is a more alluring site to study. As ALK1 is a receptor in the TGFsignaling pathway, a possible approach lies in the biological processes it takes part in. Among the roles that the TGFsignaling pathway takes part in is proa poptosis. Therefore w ith the increase in ALK1 signaling there may be an increase in apoptosis and its detection can be done by utilizing the Tunnel method and intranucleos omal DNA fragment detection by insitu end labeling. Aside from these invivo technique s ALK1 overexpression may also be tested by studying capillary endotheli al cell cultures and ex amining if there are any malformations in the visible long fibrous cap illary networks. These methods along with

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31 the previous studies done in th e transgenic mice should shed more light on the role of AlK1 in angiogenesis

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32 LIST OF REFERENCES 1. Krings T., Ozanne A., Chng S.M., Alva rez H., Rodesch G., Lasjaunias P.L. Neurovascular Phenotypes In Hereditary Haemorrhagic Telangiectasia Patients According To Age Review of 50 Consecutive Patients Aged 1 day – 60 Years. Neuroradiology 2005; 47(10):711-720. 2. Argyrion L., Pfitzmann R., Wehner L., Twel kemeyer S., Neuhaus P., Nayernia K., Engel W. Alk-1 Mutations in Liver Transplanted Patients With Hereditary Hemorrhagic Telangiectasia. Liver Transplantation 2005; 11:1132-1135. 3. Abdalla S.A., Letarte M. Hereditary Haemorrhagic Telangiectasia: Current Views On Genetics and Mechanisms of Diseas e. J. Med. Genet. 2006; 43(2):97-110. 4. Cole S., Begbie M., Wallace G., Shovlin C. A New Locus For Hereditary Haemorrhagic Telangiectasia (HHT3) Ma ps to Chromosome 5. J. Med. Genet. 2005; 42(7):577-582. 5. Schlaeger T.M., Bartunkova S., Lawitts J.A., Teichmann G., Risau W., Deutsch U., Sato T.N. Uniform Vascular-endothelialcell-surface Gene Expression In Both Embryonic and Adult Transgenic Mice. Proc. Natl. Acad. Sci. 1997;94:3058-3063. 6. Bertolino P., Deckers M., Lebrin F., ten Dijke P. Transforming Growth FactorSignal Transduction In Angiogenesis and Vascular Disorders. Chest. 2005;128:585S-590S. 7. Kisanuki Y.Y., Hammer R.E., Miyazaki J., Williams S.C., Richardson J.A., Yanagisawa M. Tie2-cre Transgenic Mice: A New Model For Endothelial Celllineage Analysis In Vivo. Dev. Biol. 2001; 230:230-242. 8. Lodish H., Berk A., Matsudaira P., Kaiser C., Krieger M., Scott M., Zipursky S., Darnell J. Molecular Cell Biology. New York: W.H. Freeman and Co. 2004. 9. Jacobson B. Hereditary Hemorrhagic Telangiectasia A Model For Blood Vessel Growth and Enlargement. Am. J. Path. 2000; 156(3):737-741. 10. Toydemir P., Mao R., Lewin S., McDonald J. Hereditary Hemorrhagic Telangiectasia: An Overview of Diagnosis and Management In the Molecular Era For Clinicians. Genetics in Medicine 2004; 6(4): 175-191.

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33 11. Letteboer T., Mager H., Snijd er R., Koeleman B., Lindhout D., Ploos van Amstel H., Westermann K. Genotype-phenotype Rela tionship In Hereditary Hemorrhagic Telangiectasia. J. Med. Genet. 2006; 43(4):371-377. 12. Blanco F., Sanitbanez J., Guerrero-Esteo M., Langa C., Vary C., Bernabeu C. Interaction and Functio nal Interplay Between Endoglin and ALK-1, Two Components of the Endothelial Transforming Growth FactorReceptor Complex. J. Cell. Path. 2005; 204:574-584. 13. Seki T., Yun J., Oh P. Arterial Endothe lium-specific Activin Receptor-like Kinase 1 Expression Suggests Its Role In Arteri alization and Vascular Remodeling. Circ. Res. 2003; 93:682-689. 14. Srinivas S., Watanabe T., Lin C., William C ., Tanabe Y., Jessell T., Constantini F. Cre Reporter Strains Produced By Targeted Insertion of EYFP and ECFP Into the ROSA26 Locus. BMC Dev. Bio. 2001; 1:4. 15. Zhong T., Rosenberg M., Mohideen M., We instein B., Fishman M. Gridlock, An HLH Gene Required For Assembly of the Aorta In Zebrafish. Science. 2000; 287:1820 –1824. 16. Shutter J., Scully S., Fan W., Richards W ., Kitajewski J., Deblandre G., Kintner C., Stark K. Dll4, A Novel Notch Ligand Expr essed In Arterial Endothelium. Genes. Dev. 2000; 14:1313–1318. 17. Wang H., Chen Z., Anderson D. Molecular Distinction and Angiogenic Interaction Between Embryonic Arteries and Veins Re vealed By ephrin-B2 and Its Receptor Eph-B4. Cell. 1998; 93:741–753. 18. Sorensen L., Brooke B., Li D., Urness L. Loss of Distinct Arterial and Venous Boundaries In Mice Lacking Endogl in, A Vascular-specific TGF Coreceptor. Dev. Bio. 2003; 261:235-250. 19. Zhong T., Childs S., Leu J., Fishman M. Gridlock Signaling Pathway Fashions the First Embryonic Artery. Nature. 2001; 414:216 –220. 20. Isermann B., Hendrickson S.B., Zogg M., Wing M., Cummiskey M., Kisanuki Y.Y., Yanagisawa M., Weiler H. Endotheliumspecific Loss of Murine Thrombomodulin Disrupts the Protei n C Anticoagulant Pathway and Causes Juvenile-onset Thrombosis. J. Clin. Invest. 2001; 108:537-546. 21. Oh S.P., Warman M.L., Seldin M.F., Ch eng S.D., Knoll J.H., Timmons S., Olsen B.R. Cloning of cDNA and Genomic DNA Encoding Human Type XVIII Collagen and Localization of the Alpha 1(XVIII) Collagen Gene To Mouse Chromosome 10 and Human Chromosome 21. Genomics 1994; 19:494-499.

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34 22. Zambrowicz B.P., Imamoto A., Fiering S., Herzenberg L.A., Kerr W.G., Soriano P. Disruption of Overlapping Transcripts In the ROSA beta geo 26 Gene Trap Strain Leads to Widespread Expression of beta -galactosidase In Mouse Embryos and Hematopoietic Cells. Proc. Natl. Acad. Sci. 1997; 94:3789-3794. 23. Parera M., van Dooren M., van Kempen M ., de Krijger R., Grosveld F., Tibboel D., Rottier R. Distal Angiogenesis: A New Concept For Lung Vascular Morphogenesis. Am. J. Physiol. Lung Cell. Mol. Physiol. 2005; 288:L141-L149. 24. Nakao A., Imamura T., Souchelnytskyi S ., Kawabata M., Ishisaki A., Oeda E., Tamaki K., Hanai J., Heldin C ., Miyaazono K., ten Dijke P. TGFReceptormediated Signaling Through Smad2, Smad3 and Smad4. EMBO journal. 1997; 16(17): 5353-5362. 25. Barbara N., Wrana J., Letarte M. Endoglin Is An Accessory Protein That Interacts With the Signaling Receptor Complex of Multiple Members of the Transforming Growth FactorSuperfamily. J. Bio. Chem. 1999; 274(2):584-594. 26. Pece-Barbara N., Cymerman U., Vera S ., Marchuk D., Letarte M. Expression Analysis of Four Endoglin Missense Muta tions Suggests That Haploinsufficiency is the Predominant Mechanism for Hereditary Hemorrhagic Telangiectasia Type 1. Human Molecular Genetics. 1999; 8(12): 2171-2181. 27. Gougos A., Letarte M. Primary Structur e of Endoglin, An RGDcontaining Glycoprotein of Human E ndothelial Cells. J. Bio. Chem. 1990; 265(15):8361-8364. 28. Abdalla S., Pece-Barbara N., Vera S., Tapi a E., Paez E., Bernabeu C., Letarte M. Analysis of Alk-1 and Endoglin In Newborns From Families With Hereditary Hemorrhagic Telangiectasia Type 2. Hu man Molecular Genetics. 2000; 9(8):12271237. 29. Bourdeau A., Faughnan M., Letarte M. E ndoglin-deficient Mice, a Unique Model To Study Hereditary Hemorrhagic Tela ngiectasia. Trends Cardiovasc. Med. 2000;10:279-285. 30. Oh S., Seki T., Goss K., Imamura T., Yi Y., Donahoe P., Li L., Miyaazono K., ten Dijke P., Kim S., Li E. Activin Receptor –like Kinase 1 Modulates Transforming Growth Factor1 Signaling in the Regulation of Angiogenesis. PNAS. 2000; 97(6):2626-2631. 31. Letarte M., McDonald M., Li C., Kathirka mathamby K., Vera S., Pece-Barbara N., Kumar S. Reduced Endothelial Secretion and Plasma Levels of Transforming Growth Factor1 in Patients With Hereditary Hemorrhagic Telangiectasia Type 1. Cardiovascular Research. 2005; 68:155-164. 32. Seki T., Hong K., Oh S. Nonoverlapping E xpression Patterns of ALK1 and ALK5 Reveal Distinct Roles of Each Recepto r In Vascular Development. Laboratory Investigation. 2006; 86:116-129.

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35 33. Seki T., Hong K., Yun J., Kim S., Oh S. Isolation of a Regulatory Region of Activin Receptor-like Kinase1 Gene Suffi cient for Arterial Endothelium-specific Expression. Circ. Res. 2004; 94:e72-e77. 34. Albe E., Escalona E., Rajagopal R., Javi er J., Chang J., Azar D. Proteomic Identification of Activin Receptor-like Ki nase-1 as a Differentially Expressed Protein During Hyaloid Vascular Sy stem Regression. FEBS. 2005; 579:5481-5486. 35. Fernandez A., Sanz-Rodriquez F., Zarrabe itia R., Perez-Molino A., Hebbel R., Nguyen J., Bernabeu C., Botella L. Blood Outgrowth Endothelial Cells From Hereditary Haemorrhagic Telangietasia Patients Reveal Abnormalities Compatable With Vascular Lesions. Cardiova scular Research. 2005; 68(2):235-248. 36. Srinivasan S., Hanes M., Dickens T., Porteous M., Oh S., Hale L., Marchuk D. A Mouse Model For Hereditary Hemorrhagi c Telangiectasia (HHT) Type 2. Human Molecular Genetics . 2003; 12(5):473-482. 37. Gu Y., Jin P., Zhang L., Zhao X., Gao X., Ning Y., Meng A., Chen Y. Functional Analysis of Mutations In the Kinase Domain of the TGFReceptor ALK1 Reveals Different Mechanisms For Induction of Hereditary Hemorrhagic Telangiectasia. Blood First Edition. 2005; 107(5):1951-1954. 38. Bauer S., Bauer R., Liu Z., Chen H., Goldstein L., Velazquez O. Vascular Endothelial Growth Factor-C Promotes Vasculogenesis, Angiogenesis, and Collagen Constriction In Three-dimensi onal Collagen Gels. J. Vasc. Surg. 2005; 41:699-707. 39. Schepper A., Vanoenacker F., de Beeck B., Gielen J., Parizel P. Vascular Pathology of the Spleen, Part I. Abdom. Imaging. 2005; 30:96-104. 40. Ignjatovic D., Stimec B., Zivanoic V. The Basis For Splenic Segmental Dearterialization: A Post-mortem St udy. Surg. Radiol. Anat. 2005; 27:15-18. 41. Harvey N., Oliver G. Choose Your Fate: Artery, Vein Or Lymphatic Vessel? Curr. Opin. in Genetics & Dev. 2004; 14:499-505.

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36 BIOGRAPHICAL SKETCH Kyumee went to high school at Atlan tic Community High in Delray Beach, Florida, successfully completing the Intern ational Baccalaureate pr ogram and received her I.B. diploma in 1999. She continued her educ ation with a Bachelor of Science degree in nutrition at the University of Florid a, graduating in 2003. In 2004, she joined the Department of Physiology and Functional Ge nomics at the Univers ity of Florida to continue to her masterÂ’s e ducation under the mentorship of Dr. S Paul Oh. After her graduation she will continue researching at the University of Florida in the Department of Oral & Maxillofacial Surgery and Diagnostic Sciences