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Expression of the Receptor of Advanced Glycation Endproducts (RAGE) in Gingival Tissues of Smokers and Non-Smokers with ...

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EXPRESSION OF THE RECEPTOR OF ADVANCED GLYCATION ENDPRODUCTS (RAGE) IN GINGI VAL TISSUES OF SMOKERS AND NONSMOKERS WITH GENERALIZED PERIODONTAL DISEASE By THOMAS YOUNG HO YOON 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 Thomas Young Ho Yoon

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This document is dedicated to my fiancee and future wife Dr. Judith Lim. Without your love, support and dedication none of this could have ever been possible.

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iv ACKNOWLEDGMENTS The author would like to thank Drs. Hank Towle, Joseph Katz, Fred Brown, Gregory Horning, Arthur Vernino, Michele Beaty, Dennis Davis, Angel Santiago, Eric Shoenebeck, and Brian VanAelst from the University of Florida, Department of Periodontics, for their contri butions to this article.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................vi LIST OF FIGURES..........................................................................................................vii ABSTRACT.....................................................................................................................vi ii CHAPTER 1 INTRODUCTION........................................................................................................1 2 MATERIALS AND METHODS.................................................................................3 Periodontal Therapy......................................................................................................3 Sample Selection and Matching...................................................................................4 RNA Extraction............................................................................................................5 RNA Quantification......................................................................................................6 RT-PCR........................................................................................................................6 3 RESULTS.....................................................................................................................8 4 DISCUSSION.............................................................................................................10 Biochemistry of AGE and RAGE...............................................................................10 AGE and RAGE as a Possible Link to Periodontal Disease......................................11 Conclusion..................................................................................................................12 LIST OF REFERENCES...................................................................................................13 BIOGRAPHICAL SKETCH.............................................................................................15

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vi LIST OF TABLES Table page 1-1 Patient Data Smokers.................................................................................................5 1-2 Patient Data Non-Smokers.........................................................................................5

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vii LIST OF FIGURES Figure page 1 Apically positioned flap surgery depi cting a site where the biopsy tissue was harvested.....................................................................................................................4 2 Expression of RAGE mRNA in the gi ngiva of smokers and nonsmokers.................9

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viii 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 EXPRESSION OF THE RECEPTOR OF ADVANCED GLYCATION ENDPRODUCTS (RAGE) IN GINGI VAL TISSUES OF SMOKERS AND NONSMOKERS WITH GENERALIZED PERIODONTAL DISEASE By Thomas Young Ho Yoon May 2006 Chair: Herbert J Towle III Major Department: Periodontics Background: The association between smoking and periodontal disease is well established; however the mechanism by wh ich smoking augments the destruction of periodontal tissue is not clear. We hypothesize that there is an increased expression of the receptor of advanced glycation endproducts (R AGE) in gingival tissues of smokers which mediates the tissue toxic effect of a dvanced glycation endproducts (AGEs). Material and methods: Gingival biopsies from five smokers and five age and sex matched non-smokers were examined. RNA was extracted from the gingival tissues, reverse transcribed, amplified and run on ag arose gel. The bands on the gels were viewed using ethidium bromide and band intens ity was analyzed using Scion Image. Results: For all 10 subjects, a band was obser ved at the 326 base pairs position, which is the predicted length of the RAGE PCR product. In all pairs, the smoker biopsies expressed a greater level of RAGE as co mpared to the matched non-smoker tissues. When viewed as groups, analysis of the ba nd intensity indicated that RAGE mRNA in

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ix smokers was approximately 140 percent of the expression of that measured in nonsmokers (student’s t-test, p = 0.028). Conclusions : The presence of increased RAGE expression in human gingival epithelium of smokers with periodontal diseas e may indicate that AGEs are involved in the periodontal destruction associated with smoking.

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1 CHAPTER 1 INTRODUCTION There is ample evidence that associat e smoking with the destruction of the periodontal apparatus. The exact mechanis m by which tobacco products affects the periodontium is not well understood. Numer ous reports have documented that smokers have a higher incidence and prevalence of periodontal disease than non-smokers.1,2 Smokers have been shown to exhibit different features detrimental to gingival health. It has been reported that smokers have an increased amount of the bacteria Porphyromonas gingivalis and Bacteriodes Forsythus.3 Other proposed mechanisms for negative periodontal effects of smoking include vascular alteration, altered neutro phil function, decreased IgG production, decreased lymphocyt e proliferation, and altered fibroblast attachment and function.4 A direct link between tobacco use and th e development of advanced glycation products (AGEs) has recently been established.5,6 Nor–nicotine, a nicotine metabolite found in high concentrations in the plasma of smokers, was shown to significantly induce the formation of AGEs. AGEs have in turn been shown to induce production of cytokines such as Interleukin 1 (IL-1), IL -6 a, tumor necrosis factor(TNF) and increase oxidative stress. Recently we have shown that human gingi val cells grown in tissue culture and exposed to 1M nor-nicotine for 72 hours expressed approximately four-fold RAGE compared to non treated cells.7 We also hypothesize that AGEs and up regulation of

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2 RAGE may be involved in the pathogenesis of periodontal disease associated with smoking. The purpose of the present study was to inve stigate the hypothesis that the gingiva of smokers express elevated levels of RAGE compared to non smokers.

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3 CHAPTER 2 MATERIALS AND METHODS Gingival biopsy samples were obtained from 22 patients (10 smokers and 12 non smokers) with generalized chronic periodontitis All subjects were patients of the University of Florida, College of Dentis try, Department of Periodontics The study was approved by the institutional review board a nd each participant si gned a consent form. Inclusion criteria for patients involved w ith the study were 1) generalized periodontal disease consisting of loss of attachment 30 % with bleeding upon probing in the remaining dentition 2) no other significant sy stemic disease 3) current smokers and, 4) those patients who have never smoked or used tobacco products. Smokers were defined as those patients who smoked greater than 100 cigarettes over their lifetime and smoked at the time of the clinical examination.8 The exclusionary criteria were 1) lack of periodontal disease 2) previous smokers w ho had quit 3) significant systemic disease which may have an impact on periodontal stat us such as diabetes (both type 1 and 2), arthritis (both rheumatoid and osteoarthritis), or any other systemic autoimmune disease, and 4) patients on long term medication. Periodontal Therapy All subjects were provided with initial periodontal therapy including oral hygiene instructions, scaling and root planing where necessary and re-evaluation. During the reevaluation phase, patients were treatment pla nned for periodontal surgery as indicated for persistent disease. At the time of surger y, gingival tissues were obtained from the

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4 deepest probing depth and imme diately “snap frozen” at -70oC until processed for reverse transcriptase polymerase chain reaction (RT-PCR) (Figure 1). Figure 1. Apically positioned flap surgery de picting a site where the biopsy tissue was harvested Sample Selection and Matching One sample was excluded because an informed consent was not obtained. Another sample was excluded because the patient admitted to a previous smoking history after the biopsy was harvested. Five pa irs of age and sex matched pa irs, which only differed in smoking status, were subsequently chosen for analyses (table 1-1 and 1-2).

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5 Table 1-1. Patient Data Smokers Pt ID# Age Sex Smoking Status Type of Procedure Attachment Loss 1S 60 Male 2ppd x 40yrs (80 pack yrs) Multiple Extractions 4-6mm 2S 76 Male 2ppd x 55yrs (110 pack yrs) Multiple Extractions 4-6mm 3S 47 Male 1ppd x 30 yrs (30 pack yrs) APF With Osseous 4-6mm 4S 57 Female 0.5 ppd x 30yrs (15 pack yrs) APF With Osseous 4-6mm 5S 62 Female 1ppd x 40yrs (40 pack yrs) APF With Osseous 4-9mm Table 1-2. Patient Data Non-Smokers Pt ID# Age Sex Type of Procedure Attachement Loss 1S 55 Male APF With Osseous 4-6mm 2S 77 Male Single Extraction 3-6mm 3S 43 Male APF With Osseous 3-6mm 4S 58 Female APF With Osseous 4-6mm 5S 65 Female APF With Osseous 5-8mm RNA Extraction The presence of mRNA was determined by RT-PCR using a housekeeping gene as a standard. The gingival tissues were th awed and RNA was extracted using a QiaGen RNeasy Mini Kit (QiaGen RNeasy Mini Kit, Qiagen Inc., Netherlands). The DNA was digested by using the QiaGen RNase-Free DNa se Kit (QiaGen RNase-Free DNase Kit, Qiagen Inc., Netherlands). Twenty mg of sample was homogenized in 350ul of RLT Buffer with 1% b-Mercapthoethanol. The resulting solution was then centrifuged at 12000g for 3 minutes in order to produce a s upernatant. The supernatant was collected

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6 and transferred to a new tube and 350ul of 70% ethanol was added. The resultant sample was placed in an RNeasy mini column and centrifuged at 8000g for 15 seconds. The column was washed with 400ul of RW1 Bu ffer and also centrifuged at 8000g for 15 second. A solution of 10ul of DNase I and 70ul of RDD buffer was placed in each spin column and incubated at room temperature fo r 15 min. The spin column was then washed with 350ul of RW1 buffer once and with 500ul of RPE buffer twice. Finally the RNA was eluted by placing 50ul of RNase-Free wate r directly in the silica-gel membrane and centrifuged for 1 minute at 8000g. The coll ected RNA was then stored at -70oC RNA Quantification The quantity of the RNA isolated was de termined by measuring the absorbance at 260nm The purity was determined by the 260nm/280nm absorbance ratio. The minimal acceptable ratio considered was >1.75 before the RNA will be used for RT-PCR. RT-PCR The RT-PCR was performed using Promega Access RT-PCR System (Promega Access RT-PCR System, Promega Inc., Madison, WI). The RAGE primers used (forward 5'GACTCTTAGCTGGCACTTGGAT-3' and the reverse 5'GGACTTCACAGGTCAGG GTTAC-3'), yielded a 326bp product. In a 0.5ml tube 10ul AMV/Tfl 5X Reaction buffer, 1ul 0.2mM dNTP Mix, 50pmol forward primer, 50pmol reverse primer, 2ul 25 mM MgSO4, 1ul (5u/ml ) AMV Reverse Transcriptase, 1ul (5u/ml) Tfl DNA Polymerase, and 1ug of the isolated RNA were combined and brought to a volume of 50ul with RNA-Free distilled water. The first strand of cDNA synthesis was obtained by 1 cycle of 45 min at 48oC, followed by 1 cycle of 2 min at 92oC. The solution was then thermocycled at intervals of 95oC for 12 minutes, then 35 cycles of one minute at 94oC, one minute at 55oC, one minute at 72oC. The products of the RT-PCR are then

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7 separated by agarose gel el ectrophoresis and viewed with ethidium bromide and UV light. The bands were quantifie d using densitometry and statistically analyzed utilizing the student’s t-test.

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8 CHAPTER 3 RESULTS The RNA was extracted from the gingival tissues, reverse tran scribed, amplified and run on agarose gels as previously desc ribed. The bands on the gels were viewed using ethidium bromide and band intensity was analyzed using Scion Image (Scion Image, Scion Corporation, Gaithersburg, MD). For all 10 subjects a band was observed at 326 base pairs, which is the predicte d length of the RAGE PCR product. After adjusting for age and gender subjects were matched in pairs (smoker and non-smoker). In all pairs the smoker biopsy expressed a higher level of RAGE compared to the matched non-smoker. When viewed as groups, analysis of the band intensity indicated that RAGE mRNA in smokers was approximately 140 pe rcent of the expression in nonsmokers (student’s t-test, p = 0.028) (Figure 2). Thes e data suggest that smoking increases RAGE production in the oral cavity.

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9 Figure 2. Expression of RAGE mRNA in th e gingiva of smokers and nonsmokers. The top image demonstrates the RT-PCR results for RAGE mRNA for smokers and matched nonsmokers (S = smoker, N = nonsmoker). The graph is the summarized RT-PCR band intensity data for all ten subjects. The asterisk indicates p = 0.028, student’s t-test.

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10 CHAPTER 4 DISCUSSION In the present study we have demonstrated that after adjusting for age and gender, all five gingival biopsies from smokers expre ssed higher levels of RAGE compared to the non-smokers. These findings are in accordance with our previous study where we have shown in vitro that gingival fibroblasts pr eincubated with nor-ni cotine, a by product of cigarette smoking, expressed a hi gher level of RAGE compared with non treated cells. The gingival fibroblasts that were exposed to nor-nicotine were at levels that are similar to physiologic levels in smoker’s serum.7 Biochemistry of AGE and RAGE The receptors for AGE (RAGE) are cell su rface receptors which are capable of producing a pro-inflammatory re sponse. RAGEs have been shown to be expressed by a variety of cell types including endothe lial and smooth muscle cells, lymphocytes, monocytes, and neurons. It has been shown in the literature by Hofm ann et al. that once activated by AGE, RAGE can tr igger cellular activation w ith generation of key proinflammatory mediators. It has also been shown in the same study that blockade of RAGE can halt delayed-type hypersensitivity and inflammatory states by arresting the activation of central signaling pathways and expression of inflammatory gene mediators.10 The binding of AGE to RAGE stimul ates expression of RAGE itself, and generates oxidative stress, synthesi s and secretion of pro-inflammatory cytokines, and chemotaxis.9,10

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11 AGEs are the final end product of non-en zymatic glycation and oxidation of proteins found in plasma and tissues. AGEs are biologically active and may initiate a range of cellular responses including stimulation of monocyte chemotaxis, osteoclastinduced bone resorption, pro liferation of vascular endothe lial and smooth muscle cells, aggregation of platelets, and stimul ation of secretion of inflammatory cytokines, collagenase, and several growth factors.11 AGEs can be either external or internal in origin. Internal sources include certain systemic conditions such as diabetes, Alzheimer’s disease, and uraemia.12,13,14 External AGEs are produced by the combustion of nicotine in cigarette smoke. Glycation products of cigarette smoke are likely to originate during the curing of tobacco via Maillard-type reactions and have been proposed to confer aroma and flavoring to tobacco products.15 AGE and RAGE as a Possible Link to Periodontal Disease Recently it was demonstrated that AGEs can induce apoptosis in fibroblasts. In a study performed by Alikhani et al. the pr edominant AGE of skin (CML-collagen) induced fibroblast apoptosis in vivo. In vitro experiments dem onstrated that CMLcollagen induced a time and dose-dependent increase in fibroblast apoptosis when compared to control collagen.16 It is possible that a simila r pattern of fibroblast apoptosis could be found in gingival fibroblast if exposed to AGEs. AGE deposition and RAGE expression has been associated with other risk factors for periodontal disease.17 In a study performed by Lalla et al it was discovered that there is increased formation and de position of AGEs in the ging iva of diabetic mice when inoculated with either P. gingivalis or a placebo control.18 It was shown however in a later study that periodontal disease in th e same murine model was arrested by

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12 administration of soluble RAGE, probably by in hibiting the receptor fo r interacting with AGE.19 We have recently examined human diab etic patients with periodontal disease and compared them to age and sex matched cont rols. Our results showed that the gingiva of diabetic patients with periodontal diseas e exhibit a higher presence of RAGE when compared to their controls.11 These results suggested that an increased amount of RAGE may contribute to the advanced periodontal destruction commonly f ound in diabetics. Conclusion The exact mechanism of how AGEs activation of its receptor in the gingiva during smoking results in the destruction of the periodontal apparatus is unknown. Smoking promotes the production of AGEs that stimulate increased signaling through their receptors (RAGE) in the gingiva. This may tr igger the pro-inflammatory effects of AGEs by stimulating secretion of cytokines that promote destruction of the periodontal apparatus. Since AGEs induce apoptosis in fibr oblasts the possibility that apoptosis is involved in the smoking aggravated pe riodontitis, should be investigated.

PAGE 22

13 LIST OF REFERENCES 1. Ismail AI, Burt BA, Eklund SA. Epidemiologic patterns of smoking and periodontal disease in the United States. J Am Dent Assoc 1983;106:617-21. 2. Horning GM, Hatch CL, Cohen ME. Risk indicators for periodontitis in a military treatment population. J Periodontol 1992;63:297-302. 3. Zambon JJ, Grossi SG, Machtei EE, Ho AW, Dunford R, Genco RJ. Cigarette smoking increases the risk for subgingiva l infection with pe riodontal pathogens. J Periodontol 1996;67:1050-4. 4. Johnson GK, Hill M. Cigarette smoking and the periodontal patient. J Periodontol 2004;75:196-209. Review. 5. Dickerson TJ, Janda KD. A previously undescribed chemical link between smoking and metabolic disease. Proc Natl Acad Sci 2002;99:15084 – 15088. 6. Hasegawa G, Nakano K, Sawada M, Uno K, Shibayama Y, Ienaga K, Kondo M. Possible role of tumor necrosis factor and interleukin –1 in the development of diabetic nephropathy. Kidney Int 1991;40:1007-12. 7. Katz J, Caudle RM, Bhattacharyya I, Stewart CM, Cohen, DM. Receptor for advanced glycation endproducts (R AGE) upregulation in human gingival fibroblasts incubated with nor-nicotine. J Periodontol 2005;76: 130-3. 8. Tomar SL, Asma S. Smoking-attributable periodontitis in the United States: findings from NHANES III. National Health and Nutrition Examination Survey. J Periodontol 2000;71:743-51. 9. Takane M, Sugano N, Ezawa T, Uchiyama T, Ito K. A marker of oxidative stress in saliva: association with periodontally -involved teeth of a hopeless prognosis. J Oral Sci 2005;47:53-7. 10. Hofmann, MA, Drury S, Fu C, Qu W, Ta guchi A, Lu Y, Avila C, Kambham N, Bierhaus A, Nawroth P, Neurath MF, Slat tery T, Beach D, McClary J, Nagashima M, Morser J, Stern D, Schmidt AM. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 1999;97:889–901.

PAGE 23

14 11. Katz J, Bhattacharyya I, Farkhondeh-Kish F, Perez FM, Caudle RM, Heft M. Expression of the receptor of advanced glycation end products in the gingival tissues of type 2 diabetic patients wi th chronic periodontal disease: A study utilizing immunohistochemistry and RT-PCR. J Clin Periodontol 2005;32:40-44. 12. Dawnay A, Millar DJ. The pathogenesis and consequences of AGE formation in uraemia and its treatment. Cell Mol Biol 1998;44:1081-94. Review. 13. Yonekura H, Yamamoto Y, Sakurai S, Wata nabe T, Yamamoto H. Roles of the receptor for advanced glycation endproducts in diabetes-induced vascular injury. J Pharmacol Sci 2005;97:305-11. 14. Richter T, Munch G, Luth HJ, Arendt T, Kientsch-Engel R, Stahl P, Fengler D, Kuhla B. Immunochemical crossreactivity of antibodies specific for "advanced glycation endproducts" with "advan ced lipoxidation endproducts." Neurobiol Aging 2005;26:465-74. 15. Cerami C, Founds H, Nicholl I, Mitsuhashi T, Giordano D, Vanpatten S, Lee A, Al-Abed Y, Vlassara H, Bucala R, Cerami A. Tobacco smoking is a source of toxin reactive glycation products. Proc Natl Acad Sci 1997;94: 12915 – 13920. 16. Alikhani Z, Alikhani M, Boyd CM, Naga o K, Trackman PC, Graves DT. Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytopl asmic and mitochondrial pathways. J Biol Chem 2005;280:12087-95. 17. Lalla E, Lamster IB, Schmidt AM. Enhanced interaction of advanced glycation end products with their cellular receptor RAGE: implications for the pathogenesis of accelerated periodontal disease in diabetes. Ann Periodontol 1998;3:13-9. Review. 18. Lalla E, Lamster IB, Feit M, Huang L, Schmidt AM. A murine model of accelerated periodontal disease in diabetes. J Periodontal Res 1998;33:387-99. 19. Lalla E, Lamster IB, Feit M, Huang L, Spe ssot A, Qu W, Kislinger T, Lu Y, Stern DM, Schmidt AM. Blockade of RAGE suppresses periodontitis-associated bone loss in diabetic mice. J Clin Invest 2000;105:1117-1124.

PAGE 24

15 BIOGRAPHICAL SKETCH Dr. Thomas Young Ho Yoon obtained his BA and DDS from the University of Missouri at Kansas City six year advanced BA/DDS program. Af ter graduation in 2001 he successfully completed a General Practice Residency at Barnes Jewish Hospital in Saint Louis, Missouri. Wanting to furthe r pursue the surgical arts, Dr. Yoon then attended and completed an internship in oral and maxillofacial surgery at the University of Florida. He finished his surgical tr aining with the Depart ment of Periodontology where he served as their chief resident during his final year. Currently, Dr. Yoon practices periodontics in Winter Park, Florida, and maintains a part-time facu lty associate position at the University of Flor ida, Department of Periodontology.


Permanent Link: http://ufdc.ufl.edu/UFE0014383/00001

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Title: Expression of the Receptor of Advanced Glycation Endproducts (RAGE) in Gingival Tissues of Smokers and Non-Smokers with Generalized Periodontal Disease
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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Material Information

Title: Expression of the Receptor of Advanced Glycation Endproducts (RAGE) in Gingival Tissues of Smokers and Non-Smokers with Generalized Periodontal Disease
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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EXPRESSION OF THE RECEPTOR OF ADVANCED GLYCATION
ENDPRODUCTS (RAGE) IN GINGIVAL TISSUES OF SMOKERS AND NON-
SMOKERS WITH GENERALIZED PERIODONTAL DISEASE











By

THOMAS YOUNG HO YOON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006

































Copyright 2006

by

Thomas Young Ho Yoon

































This document is dedicated to my fiancee and future wife Dr. Judith Lim. Without your
love, support and dedication none of this could have ever been possible.















ACKNOWLEDGMENTS

The author would like to thank Drs. Hank Towle, Joseph Katz, Fred Brown,

Gregory Horning, Arthur Vernino, Michele Beaty, Dennis Davis, Angel Santiago, Eric

Shoenebeck, and Brian VanAelst from the University of Florida, Department of

Periodontics, for their contributions to this article.
















TABLE OF CONTENTS

page

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

L IST O F T A B L E S ................................................................... .............. vi

L IST O F FIG U R E S .... ....... ................................................ .... ..... .. ............. vii

A B S T R A C T ......... .................................. ................................................... v iii

CHAPTER

1 IN TR O D U C TIO N ......................................................................... .... .. ........

2 M A TERIALS AND M ETH OD S ............................................. .......................... 3

Periodontal Therapy................ .. ... .................... ........3
Sam ple Selection and M watching ...........................................................................4
R N A E x traction ....................................................... ................ 5
R N A Q uantification ........ ....... ................................................................ ......... . ..
R T -P C R ........................................................................... . 6

3 R E SU L T S ................................................................ .8

4 D ISC U SSIO N ..................................................... 10

Biochemistry of AGE and RAGE............................................................................10
AGE and RAGE as a Possible Link to Periodontal Disease .............. .... ............... 11
C conclusion ....................................................................................................... ........ 12

L IST O F R EFER EN CE S ..................................... .............................................................. 13

B IO G R A PH IC A L SK E T C H ........................................................................................ 15











v
















LIST OF TABLES

Table pge

1-1 P patient D ata Sm okers ................................................................. ....................... 5

1-2 Patient Data Non-Smokers .......................... ........... .. ...... ............... 5
















LIST OF FIGURES


Figure


1 Apically positioned flap surgery depicting a site where the biopsy tissue was
h a rv e ste d ....................................................... ................ 4

2 Expression of RAGE mRNA in the gingiva of smokers and nonsmokers .............9


page















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EXPRESSION OF THE RECEPTOR OF ADVANCED GLYCATION
ENDPRODUCTS (RAGE) IN GINGIVAL TISSUES OF SMOKERS AND NON-
SMOKERS WITH GENERALIZED PERIODONTAL DISEASE

By

Thomas Young Ho Yoon

May 2006

Chair: Herbert J Towle III
Major Department: Periodontics

Background: The association between smoking and periodontal disease is well

established; however the mechanism by which smoking augments the destruction of

periodontal tissue is not clear. We hypothesize that there is an increased expression of the

receptor of advanced glycation endproducts (RAGE) in gingival tissues of smokers which

mediates the tissue toxic effect of advanced glycation endproducts (AGEs).

Material and methods: Gingival biopsies from five smokers and five age and sex

matched non-smokers were examined. RNA was extracted from the gingival tissues,

reverse transcribed, amplified and run on agarose gel. The bands on the gels were

viewed using ethidium bromide and band intensity was analyzed using Scion Image.

Results: For all 10 subjects, a band was observed at the 326 base pairs position,

which is the predicted length of the RAGE PCR product. In all pairs, the smoker biopsies

expressed a greater level of RAGE as compared to the matched non-smoker tissues.

When viewed as groups, analysis of the band intensity indicated that RAGE mRNA in









smokers was approximately 140 percent of the expression of that measured in

nonsmokers (student's t-test, p = 0.028).

Conclusions: The presence of increased RAGE expression in human gingival

epithelium of smokers with periodontal disease may indicate that AGEs are involved in

the periodontal destruction associated with smoking.














CHAPTER 1
INTRODUCTION

There is ample evidence that associate smoking with the destruction of the

periodontal apparatus. The exact mechanism by which tobacco products affects the

periodontium is not well understood. Numerous reports have documented that smokers

have a higher incidence and prevalence of periodontal disease than non-smokers.1,2

Smokers have been shown to exhibit different features detrimental to gingival health. It

has been reported that smokers have an increased amount of the bacteria Porphyromonas

gingivalis and Bacteriodes Foi \yilnt\ 3 Other proposed mechanisms for negative

periodontal effects of smoking include vascular alteration, altered neutrophil function,

decreased IgG production, decreased lymphocyte proliferation, and altered fibroblast

attachment and function.4

A direct link between tobacco use and the development of advanced glycation

products (AGEs) has recently been established.5'6 Nor-nicotine, a nicotine metabolite

found in high concentrations in the plasma of smokers, was shown to significantly induce

the formation of AGEs. AGEs have in turn been shown to induce production of cytokines

such as Interleukin 1 (IL-1), IL-6 a, tumor necrosis factor- a (TNF- a) and increase

oxidative stress.

Recently we have shown that human gingival cells grown in tissue culture and

exposed to 1 [M nor-nicotine for 72 hours expressed approximately four-fold RAGE

compared to non treated cells.7 We also hypothesize that AGEs and up regulation of






2


RAGE may be involved in the pathogenesis of periodontal disease associated with

smoking.

The purpose of the present study was to investigate the hypothesis that the gingiva

of smokers express elevated levels of RAGE compared to non smokers.














CHAPTER 2
MATERIALS AND METHODS

Gingival biopsy samples were obtained from 22 patients (10 smokers and 12 non

smokers) with generalized chronic periodontitis. All subjects were patients of the

University of Florida, College of Dentistry, Department of Periodontics The study was

approved by the institutional review board and each participant signed a consent form.

Inclusion criteria for patients involved with the study were 1) generalized periodontal

disease consisting of loss of attachment > 30 % with bleeding upon probing in the

remaining dentition 2) no other significant systemic disease 3) current smokers and, 4)

those patients who have never smoked or used tobacco products. Smokers were defined

as those patients who smoked greater than 100 cigarettes over their lifetime and smoked

at the time of the clinical examination.8 The exclusionary criteria were 1) lack of

periodontal disease 2) previous smokers who had quit 3) significant systemic disease

which may have an impact on periodontal status such as diabetes (both type 1 and 2),

arthritis (both rheumatoid and osteoarthritis), or any other systemic autoimmune disease,

and 4) patients on long term medication.

Periodontal Therapy

All subjects were provided with initial periodontal therapy including oral hygiene

instructions, scaling and root planing where necessary and re-evaluation. During the re-

evaluation phase, patients were treatment planned for periodontal surgery as indicated for

persistent disease. At the time of surgery, gingival tissues were obtained from the









deepest probing depth and immediately "snap frozen" at -70C until processed for reverse

transcriptase polymerase chain reaction (RT-PCR) (Figure 1).
























Figure 1. Apically positioned flap surgery depicting a site where the biopsy tissue was
harvested

Sample Selection and Matching

One sample was excluded because an informed consent was not obtained. Another

sample was excluded because the patient admitted to a previous smoking history after the

biopsy was harvested. Five pairs of age and sex matched pairs, which only differed in

smoking status, were subsequently chosen for analyses (table 1-1 and 1-2).










Table 1-1. Patient Data Smokers
Pt ID# Age Sex Smoking Type of Attachment
Status Procedure Loss
1S 60 Male 2ppd x 4 i-s Multiple 4-6mm
(80 pack yrs) Extractions
2S 76 Male 2ppd x 55yrs Multiple 4-6mm
(110 pack yrs) Extractions
3S 47 Male Ippd x 30 yrs APF With 4-6mm
(30 pack yrs) Osseous
4S 57 Female 0.5 ppd x 30yrs APF With 4-6mm
(15 pack yrs) Osseous
5S 62 Female 1ppd x 41- is APF With 4-9mm
(40 pack yrs) Osseous



Table 1-2. Patient Data Non-Smokers
Pt ID# Age Sex Type of Attachement
Procedure Loss
1S 55 Male APF With 4-6mm
SOsseous
2S 77 Male Single 3-6mm
SExtraction
3S 43 Male APF With 3-6mm
Osseous
4S 58 Female APF With 4-6mm
Osseous
5S 65 Female APF With 5-8mm
Osseous


RNA Extraction

The presence of mRNA was determined by RT-PCR using a housekeeping gene

as a standard. The gingival tissues were thawed and RNA was extracted using a QiaGen

RNeasy Mini Kit (QiaGen RNeasy Mini Kit, Qiagen Inc., Netherlands). The DNA was

digested by using the QiaGen RNase-Free DNase Kit (QiaGen RNase-Free DNase Kit,

Qiagen Inc., Netherlands). Twenty mg of sample was homogenized in 350ul of RLT

Buffer with 1% b-Mercapthoethanol. The resulting solution was then centrifuged at

12000g for 3 minutes in order to produce a supernatant. The supernatant was collected









and transferred to a new tube and 350ul of 70% ethanol was added. The resultant sample

was placed in an RNeasy mini column and centrifuged at 8000g for 15 seconds. The

column was washed with 400ul of RW1 Buffer and also centrifuged at 8000g for 15

second. A solution of 10ul of DNase I and 70ul of RDD buffer was placed in each spin

column and incubated at room temperature for 15 min. The spin column was then washed

with 350ul of RW1 buffer once and with 500ul of RPE buffer twice. Finally the RNA

was eluted by placing 50ul of RNase-Free water directly in the silica-gel membrane and

centrifuged for 1 minute at 8000g. The collected RNA was then stored at -700C .

RNA Quantification

The quantity of the RNA isolated was determined by measuring the absorbance at

260nm The purity was determined by the 260nm/280nm absorbance ratio. The minimal

acceptable ratio considered was >1.75 before the RNA will be used for RT-PCR.

RT-PCR

The RT-PCR was performed using Promega Access RT-PCR System (Promega

Access RT-PCR System, Promega Inc., Madison, WI). The RAGE primers used (forward

5'- GACTCTTAGCTGGCACTTGGAT-3' and the reverse 5'-

GGACTTCACAGGTCAGGGTTAC-3'), yielded a 326bp product. In a 0.5ml tube 10ul

AMV/Tfl 5X Reaction buffer, lul 0.2mM dNTP Mix, 50pmol forward primer, 50pmol

reverse primer, 2ul 25 mM MgSO4, lul (5u/ml) AMV Reverse Transcriptase, lul (5u/ml)

Tfl DNA Polymerase, and lug of the isolated RNA were combined and brought to a

volume of 50ul with RNA-Free distilled water. The first strand of cDNA synthesis was

obtained by 1 cycle of 45 min at 480C, followed by 1 cycle of 2 min at 92C. The solution

was then thermocycled at intervals of 95C for 12 minutes, then 35 cycles of one minute

at 94C, one minute at 550C, one minute at 720C. The products of the RT-PCR are then






7


separated by agarose gel electrophoresis and viewed with ethidium bromide and UV

light. The bands were quantified using densitometry and statistically analyzed utilizing

the student's t-test.














CHAPTER 3
RESULTS

The RNA was extracted from the gingival tissues, reverse transcribed, amplified

and run on agarose gels as previously described. The bands on the gels were viewed

using ethidium bromide and band intensity was analyzed using Scion Image (Scion

Image, Scion Corporation, Gaithersburg, MD). For all 10 subjects a band was observed

at 326 base pairs, which is the predicted length of the RAGE PCR product. After

adjusting for age and gender subjects were matched in pairs (smoker and non-smoker). In

all pairs the smoker biopsy expressed a higher level of RAGE compared to the matched

non-smoker. When viewed as groups, analysis of the band intensity indicated that RAGE

mRNA in smokers was approximately 140 percent of the expression in nonsmokers

(student's t-test, p = 0.028) (Figure 2). These data suggest that smoking increases RAGE

production in the oral cavity.










S N S N S N S N S N


326 bp -


30 -

25
ES
S20


S -15



U 5


Smokers Nonsmokers


Figure 2. Expression of RAGE mRNA in the gingiva of smokers and nonsmokers. The
top image demonstrates the RT-PCR results for RAGE mRNA for smokers
and matched nonsmokers (S = smoker, N = nonsmoker). The graph is the
summarized RT-PCR band intensity data for all ten subjects. The asterisk
indicates p = 0.028, student's t-test.














CHAPTER 4
DISCUSSION

In the present study we have demonstrated that after adjusting for age and gender,

all five gingival biopsies from smokers expressed higher levels of RAGE compared to the

non-smokers. These findings are in accordance with our previous study where we have

shown in vitro that gingival fibroblasts preincubated with nor-nicotine, a by product of

cigarette smoking, expressed a higher level of RAGE compared with non treated cells.

The gingival fibroblasts that were exposed to nor-nicotine were at levels that are similar

to physiologic levels in smoker's serum.

Biochemistry of AGE and RAGE

The receptors for AGE (RAGE) are cell surface receptors which are capable of

producing a pro-inflammatory response. RAGEs have been shown to be expressed by a

variety of cell types including endothelial and smooth muscle cells, lymphocytes,

monocytes, and neurons. It has been shown in the literature by Hofmann et al. that once

activated by AGE, RAGE can trigger cellular activation with generation of key pro-

inflammatory mediators. It has also been shown in the same study that blockade of

RAGE can halt delayed-type hypersensitivity and inflammatory states by arresting the

activation of central signaling pathways and expression of inflammatory gene

mediators.10 The binding of AGE to RAGE stimulates expression of RAGE itself, and

generates oxidative stress, synthesis and secretion of pro-inflammatory cytokines, and

chemotaxis.9,10









AGEs are the final end product of non-enzymatic glycation and oxidation of

proteins found in plasma and tissues. AGEs are biologically active and may initiate a

range of cellular responses including stimulation of monocyte chemotaxis, osteoclast-

induced bone resorption, proliferation of vascular endothelial and smooth muscle cells,

aggregation of platelets, and stimulation of secretion of inflammatory cytokines,

collagenase, and several growth factors.11

AGEs can be either external or internal in origin. Internal sources include certain

systemic conditions such as diabetes, Alzheimer's disease, and uraemia.12'13'14 External

AGEs are produced by the combustion of nicotine in cigarette smoke. Glycation

products of cigarette smoke are likely to originate during the curing of tobacco via

Maillard-type reactions and have been proposed to confer aroma and flavoring to tobacco

products.15

AGE and RAGE as a Possible Link to Periodontal Disease

Recently it was demonstrated that AGEs can induce apoptosis in fibroblasts. In a

study performed by Alikhani et al. the predominant AGE of skin (CML-collagen)

induced fibroblast apoptosis in vivo. In vitro experiments demonstrated that CML-

collagen induced a time and dose-dependent increase in fibroblast apoptosis when

compared to control collagen.16 It is possible that a similar pattern of fibroblast apoptosis

could be found in gingival fibroblast if exposed to AGEs.

AGE deposition and RAGE expression has been associated with other risk factors

for periodontal disease.17 In a study performed by Lalla et al. it was discovered that there

is increased formation and deposition of AGEs in the gingiva of diabetic mice when

inoculated with either P. gingivalis or a placebo control.18 It was shown however in a

later study that periodontal disease in the same murine model was arrested by









administration of soluble RAGE, probably by inhibiting the receptor for interacting with

AGE.19 We have recently examined human diabetic patients with periodontal disease

and compared them to age and sex matched controls. Our results showed that the gingiva

of diabetic patients with periodontal disease exhibit a higher presence of RAGE when

compared to their controls.1 These results suggested that an increased amount of RAGE

may contribute to the advanced periodontal destruction commonly found in diabetics.

Conclusion

The exact mechanism of how AGEs activation of its receptor in the gingiva during

smoking results in the destruction of the periodontal apparatus is unknown. Smoking

promotes the production of AGEs that stimulate increased signaling through their

receptors (RAGE) in the gingiva. This may trigger the pro-inflammatory effects of AGEs

by stimulating secretion of cytokines that promote destruction of the periodontal

apparatus. Since AGEs induce apoptosis in fibroblasts the possibility that apoptosis is

involved in the smoking aggravated periodontitis, should be investigated.















LIST OF REFERENCES


1. Ismail AI, Burt BA, Eklund SA. Epidemiologic patterns of smoking and
periodontal disease in the United States. JAm Dent Assoc 1983;106:617-21.

2. Horning GM, Hatch CL, Cohen ME. Risk indicators for periodontitis in a military
treatment population. JPeriodontol 1992;63:297-302.

3. Zambon JJ, Grossi SG, Machtei EE, Ho AW, Dunford R, Genco RJ. Cigarette
smoking increases the risk for subgingival infection with periodontal pathogens.
JPeriodontol 1996;67:1050-4.

4. Johnson GK, Hill M. Cigarette smoking and the periodontal patient.
JPeriodontol. 2004;75:196-209. Review.

5. Dickerson TJ, Janda KD. A previously undescribed chemical link between smoking
and metabolic disease. Proc NatlAcad Sci 2002;99:15084 15088.

6. Hasegawa G, Nakano K, Sawada M, Uno K, Shibayama Y, lenaga K, Kondo M.
Possible role of tumor necrosis factor and interleukin -1 in the development of
diabetic nephropathy. Kidney Int 1991;40:1007-12.

7. Katz J, Caudle RM, Bhattacharyya I, Stewart CM, Cohen, DM. Receptor for
advanced glycation endproducts (RAGE) upregulation in human gingival
fibroblasts incubated with nor-nicotine. JPeriodontol 2005;76: 130-3.

8. Tomar SL, Asma S. Smoking-attributable periodontitis in the United States:
findings from NHANES III. National Health and Nutrition Examination Survey. J
Periodontol 2000;71:743-51.

9. Takane M, Sugano N, Ezawa T, Uchiyama T, Ito K. A marker of oxidative stress
in saliva: association with periodontally-involved teeth of a hopeless prognosis.J
Oral Sci 2005;47:53-7.

10. Hofmann, MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila C, Kambham N,
Bierhaus A, Nawroth P, Neurath MF, Slattery T, Beach D, McClary J, Nagashima
M, Morser J, Stem D, Schmidt AM. RAGE mediates a novel proinflammatory axis:
a central cell surface receptor for S100/calgranulin polypeptides. Cell
1999;97:889-901.









11. Katz J, Bhattacharyya I, Farkhondeh-Kish F, Perez FM, Caudle RM, Heft M.
Expression of the receptor of advanced glycation end products in the gingival
tissues of type 2 diabetic patients with chronic periodontal disease: A study
utilizing immunohistochemistry and RT-PCR. J Clin Periodontol 2005;32:40-44.

12. Dawnay A, Millar DJ. The pathogenesis and consequences of AGE formation in
uraemia and its treatment. CellMolBiol 1998;44:1081-94. Review.

13. Yonekura H, Yamamoto Y, Sakurai S, Watanabe T, Yamamoto H. Roles of the
receptor for advanced glycation endproducts in diabetes-induced vascular injury. J
Pharmacol Sci 2005;97:305-11.

14. Richter T, Munch G, Luth HJ, Arendt T, Kientsch-Engel R, Stahl P, Fengler D,
Kuhla B. Immunochemical crossreactivity of antibodies specific for "advanced
glycation endproducts" with "advanced lipoxidation endproducts." Neurobiol
Aging 2005;26:465-74.

15. Cerami C, Founds H, Nicholl I, Mitsuhashi T, Giordano D, Vanpatten S, Lee A,
Al-Abed Y, Vlassara H, Bucala R, Cerami A. Tobacco smoking is a source of
toxin reactive glycation products. Proc NatlAcad Sci 1997;94: 12915 13920.

16. Alikhani Z, Alikhani M, Boyd CM, Nagao K, Trackman PC, Graves DT. Advanced
glycation end products enhance expression of pro-apoptotic genes and stimulate
fibroblast apoptosis through cytoplasmic and mitochondrial pathways. JBiol Chem
2005;280:12087-95.

17. Lalla E, Lamster IB, Schmidt AM. Enhanced interaction of advanced glycation end
products with their cellular receptor RAGE: implications for the pathogenesis of
accelerated periodontal disease in diabetes. Ann Periodontol 1998;3:13-9. Review.

18. Lalla E, Lamster IB, Feit M, Huang L, Schmidt AM. A murine model of
accelerated periodontal disease in diabetes. JPeriodontalRes 1998;33:387-99.

19. Lalla E, Lamster IB, Feit M, Huang L, Spessot A, Qu W, Kislinger T, Lu Y, Stern
DM, Schmidt AM. Blockade of RAGE suppresses periodontitis-associated bone
loss in diabetic mice. J Clin Invest 2000; 105:1117-1124.















BIOGRAPHICAL SKETCH

Dr. Thomas Young Ho Yoon obtained his BA and DDS from the University of

Missouri at Kansas City six year advanced BA/DDS program. After graduation in 2001

he successfully completed a General Practice Residency at Barnes Jewish Hospital in

Saint Louis, Missouri. Wanting to further pursue the surgical arts, Dr. Yoon then

attended and completed an internship in oral and maxillofacial surgery at the University

of Florida. He finished his surgical training with the Department of Periodontology

where he served as their chief resident during his final year. Currently, Dr. Yoon

practices periodontics in Winter Park, Florida, and maintains a part-time faculty associate

position at the University of Florida, Department of Periodontology.