The modulation of fibrosis in scleroderma by 3-deoxyglucosone PRINCIPAL INVESTIGATOR:

s have been included. Decreased E xpression of P er tinent E xtracellular M atr ix Molecules i n Systemic S clerosis Utilizing a Glucose Metabolite that Modulates p44/p42 MAP Kinase pathway. Carol M . A r tlett, Sihem S assi-Gaha, Danielle T L oughlin. Drexel U niversity C ollege of Medicine. Purpose: Scleroderma ( SSc) i s a f ibrotic di sease of unknow n or igin. W hat i s a pparent i s t he uncontrolled f ibrosis i n t he de rmis a nd i nternal or gans t hat affects m orbidity a nd l eads t o mortality in these patients. Understanding the mechanisms whereby fibroblasts interact with the extracellular matrix (ECM), are central to the understanding of fibrosis in SSc. TGF-beta signals through the p44/p42 MAP kinase pathway inducing the fibrogenic pathology observed in SSc. Glucose metabolites have been implicated in nu merous pa thologies but more importantly t hey have be en implicated in collagen cross-linking. Therefore, w e und ertook a nalyses t hat investigated t he r ole of one of t hese m etabolites, D yn18, i n t he m odulation of ECM g ene expression in SSc dermal cell lines. Methods: Primary dermal cell lines from the active lesions from patients with diffuse SSc were obtained f rom C arol F eghali-Bostwick at P ittsburgh U niversity a nd r e-established i n c ulture. Fibroblasts w ere t reated with D yn18 and c ultured 24 h be fore h arvesting f or RNA. cDNA transcripts of COL1A1, COL3A1, elastin, fibrillin-1, CTGF, and TGF-beta genes were measured in SSc and compared t o untreated SSc f ibroblast cel l l ines. We also investigated the phosphorylation of p44/ p42 M APK i n S Sc fibroblasts t reated w ith D yn18 a nd compared i t t o untreated fibroblasts. Results: With only 24 h incubation, we found that the glucose metabolite, Dyn18, significantly decreased the expression of ECM molecules in the dermal fibroblast lines from the SSc patients: COL1A1 decreased by 24%, COL3A1 by 23%, TGF-beta by 70%, fibrillin-1 by 53%, elastin by 6%, and CTGF by 35%. We also found that we reduced the phosphorylation of p44/p42 MAPK by 40%. Conclusions: We de monstrate f or the f irst time , that a g lucose me tabolite c an be ut ilized to decrease t he ex pression of pe rtinent E CM pr oteins t hat a re ove r expressed in SSc de rmal fibroblasts. W e a lso f ound t hat w e decreased the phos phorylation of p44/p42 M AP kinase confirming that t his pa thway i s i ntegrally i nvolved in t he over expression of ECM proteins in SSc. Taken t ogether t his da ta i s pr ovocative a nd s uggests a ne wly di scovered therapeutic t hat may be effective in controlling fibrosis in patients with SSc. 3-Deoxyglucosone Alters Integrin Expression and Collagen Specific Transcription Factors in Scleroderma Fibroblasts Sihem Sassi-Gaha and Carol M. Artlett Systemic sclerosis (SSc) is a fibrotic disease of unknown origin. We previously demonstrated that collagen was decreased by SSc dermal fibroblasts with the advance glycation end-product, 3-deoxyglucosone (3DG) and this led us to speculate that this was mediated through collagen receptors on the cell surface. Integrins bind collagen and sense the environment external to the cell and allow the cells to respond to that environment. Therefore, we investigated the expression of integrins on SSc fibroblasts isolated from the fibrotic active lesions cultured on collagen that had been modified by 3DG. 3DG decreased the expression of integrin alpha2 and beta1, but induced the expression of alpha1, alpha5, alphav, and beta3, while decreasing type 1 collagen and TGF-beta. When we investigated the transcription factors that are known the modulate collagen, we found c-myc, c-fos and Sp1 protein were decreased. We also found that elastin, fibrillin and alpha-smooth muscle actin mRNA transcripts were depressed, suggesting that the modification of lysine and arginine by 3DG is important and can affect the expression of multiple extracellular matrix proteins. Indeed, culturing fibroblasts on non-modified collagen matrices and supplementing with 3DG-modified arginine depressed collagen expression. Further studies are underway to understand the effect of 3DG on the fibroblast and to determine if this can be utilized as a potential therapeutic for SSc. 3-Deoxyglucosone Modifies Fibroblast Focal Adhesions and Induces GADD153 Leading to Altered Wound Healing Danielle T Loughlin and Carol M. Artlett The interaction of fibroblasts with the extracellular matrix (ECM) is critical for wound healing and for the functional integrity of the tissue. Advanced end glycation products (AGEs) occur as a result of fructose and glucose metabolism resulting in the glycation of long lived proteins such as collagens. This alteration leads to abnormal pathophysiology of the ECM which may complicate chronic wounds. One of the precursors to AGE is 3deoxyglucosone (3DG). 3DG has been found to be elevated in patients with diabetes and accumulates on collagen with increasing chronological age. Since the process of wound repair is dependent on fibroblast migration, proliferation, and expression of ECM proteins at the wound site, we examined the role of 3DGmodified collagens and the subsequent response of fibroblasts to this modification. We demonstrate that fibroblasts adhere more strongly to 3DG-modified collagen, express less collagen, and are unable to migrate efficiently into the wound site, when compared to unmodified collagen. This suggested an impaired organization of the actin cytoskeleton. Focal adhesion kinase and paxillin are important proteins that make up focal adhesions (FA), which are involved in cell migration. We further show that cells treated with 3DG exhibit unorganized actin cytoskeleton and different localization of key FA proteins. Additionally, these cells express higher levels of the misfolded indicator protein GADD153. This data suggests that fibroblast/matrix interactions alter as AGEs accumulate and affect focal adhesion formation. These findings also suggest that 3DG may be an factor mediating chronic wounds observed in patients with diabetes, and in the elderly by altering the signaling within the fibroblast and inducing the misfolding of proteins. INVESTIGATOR'S RESPONSIBILITIES Drexel University College ofMedicine operates in compliance with all applicable laws and regulations and good clinical practice. Drexel University relies upon Drexel University College ofMedicine's IRBs for the review and approval ofall protocols. Investigators are responsible for the implementation ofprotocols as approved by the IRB. Fonnal guidance to investigators, standard operating procedures ofIRB and fonns to be used are posted on the Office ofRegulatory Research Compliance website www.research.drexe1.edu. Additional educational infonnation can be obtained at www.irbforum.research.drexe1.edu. Consent Forms Only approved and stamped consent forms must be used to enroll subjects. 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