Effects of Visfatin on Proliferation and Collagen Synthesis in Rat Cardiac Fibroblasts

Abstract

The proliferation of cardiac fibroblasts (CFs) and excessive extracellular matrix protein accumulation are the basic pathological processes of myocardial fibrosis. Visfatin is a novel adipokine involved in the regulation of inflammatory cytokines, however, the effects of visfatin on proliferation and collagen synthesis of CFs are unclear. The aim of this study was to determine whether visfatin has any effect on the proliferation and collagen synthesis in rat CFs. Incorporation of [³H]-thymidine and [³H]-proline were used for evaluating DNA and collagen synthesis. Flow cytometry techniques were adopted to analyze cell cycle. Enzyme-linked immunosorbent assay was used for measuring collagen type I and III production. RT-PCR and Western blot analysis were used for determining procollagen I and III mRNA expression and protein production. The inhibitors used for pathway determination were: wortmannin [phosphatiylinositol 3-kinase (PI3K)], SB203580 [p38 mitogen-activated protein kinase (MAPK)], PD98059 [extracellular signal-regulated kinase (ERK)1/2)], and JNK inhibitor II [c-Jun NH 2-terminal kinase (JNK)]. We demonstrated that visfatin significantly increased DNA and collagen synthesis in a dose- and time-dependent manner. Cell cycle analysis showed that visfatin increased S-stage percentage and proliferation index in a dose- and time-dependent manner. It was also found that visfatin upregulated collagen I and III production, procollagen I and III mRNA expression and protein production. These effects were diminished by SB203580, wortmannin, and PD98059, but not by JNK inhibitor II. These results suggest that visfatin promote CFs proliferation and collagen synthesis via p38MAPK, PI3K, and ERK 1/2 pathways rather than JNK pathways, which also indicate that visfatin might play a role in myocardial fibrosis.

Key words

visfatin - proliferation - collagen synthesis - cardiac fibroblasts

References

• 1 Brilla CG, Reams GP, Maisch B, Weber KT. Renin-angiotensin system and myocardial fibrosis in hypertension: regulation of the myocardial collagen matrix.  Eur Heart J. 1993;  14 ((Suppl J)) 57-61
• 2 Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium: fibrosis and renin-angiotensin-aldosterone system.  Circulation. 1991;  83 1849-1865
• 3 Weber KT, Brilla CG, Janicki JS. Myocardial fibrosis: functional significance and regulatory factors.  Cardiovasc Res. 1993;  27 341-348
• 4 Hammarstedt A, Pihlajamäki J, Rotter Sopasakis V, Gogg S, Jansson PA, Laakso M, Smith U. Visfatin is an adipokine, but it is not regulated by thiazolidinediones.  J Clin Endocrinol Metab. 2006;  91 1181-1184
• 5 Moschen AR, Kaser A, Enrich B, Mosheimer B, Theurl M, Niederegger H, Tilg H. Visfatin, an adipocytokine with proinflammatory and immunomodulating properties.  J Immunol. 2007;  178 1748-1758
• 6 Luk T, Malam Z, Marshall JC. Pre-B cell colony-enhancing factor (PBEF)/ visfatin: a novel mediator of innate immunity.  J Leukoc Biol. 2008;  83 804-816
• 7 Wang P, Xu TY, Guan YF, Su DF, Fan GR, Miao CY. Perivascular adipose tissue-derived visfatin is a vascular smooth muscle cell growth factor: role of nicotinamide mononucleotide.  Cardiovasc Res. 2009;  81 370-380
• 8 Adya R, Tan BK, Punn A, Chen J, Randeva HS. Visfatin induces human endothelial VEGF and MMP-2/9 production via MAPK and PI3K/Akt signalling pathways: novel insights into visfatin-induced angiogenesis.  Cardiovasc Res. 2008;  78 356-365
• 9 Kim SR, Bae SK, Choi KS, Park SY, Jun HO, Lee JY, Jang HO, Yun I, Yoon KH, Kim YJ, Yoo MA, Kim KW, Bae MK. Visfatin promotes angiogenesis by activation of extracellular signal-regulated kinase 1/2.  Biochem Biophys Res Commun. 2007;  357 150-156
• 10 Song HK, Lee MH, Kim BK, Park YG, Ko GJ, Kang YS, Han JY, Han SY, Han KH, Kim HK, Cha DR. Visfatin: a new player in mesangial cell physiology and diabetic nephropathy.  Am J Physiol Renal Physiol. 2008;  295 F1485-F494
• 11 Xie H, Tang SY, Luo XH, Huang J, Cui RR, Yuan LQ, Zhou HD, Wu XP, Liao EY. Insulin-like effects of visfatin on human osteoblasts.  Calcif Tissue Int. 2007;  80 201-210
• 12 Liu SW, Qiao SB, Yuan JS, Liu DQ. Visfatin Stimulates Production of Monocyte Chemotactic Protein-1 and Interleukin-6 in Human Vein Umbilical Endothelial Cells.  Horm Metab Res. 2009;  41 281-286
• 13 Liu SW, Qiao SB, Yuan JS, Liu DQ. Association of plasma visfatin levels with inflammation, atherosclerosis and acute coronary syndromes (ACS) in humans.  Clinical Endocrinology. 2009;  71 202-207
• 14 Kim NN, Villarreal FJ, Printz MP, Lee AA, Dillmann WH. Trophic effects of angiotensin II on neonatal rat cardiac myocytes are mediated by cardiac fibroblasts.  Am J Physiol. 1995;  269 E426-E437
• 15 Peterkofsky B, Diegelmann R. Use of a mixture of proteinase-free collagenases for the specific assay of radioactive collagen in the presence of other proteins.  Biochemistry. 1971;  10 988-994
• 16 Takahashi S, Lee MJ. Quantitative study of tissue collagen metabolism.  Anal Biochem. 1987;  162 553-561
• 17 Callera GE, Touyz RM, Tostes RC, Yogi A, He Y, Malkinson S, Schiffrin EL. Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src.  Hypertension. 2005;  45 773-779
• 18 Dubey RK, Gillespie DG, Jackson EK. Adenosine inhibits collagen and protein synthesis in cardiac fibroblasts. Role of A2b receptors.  Hypertension. 1998;  31 943-948
• 19 Tsiotra PC, Tsigos C, Yfanti E, Anastasiou E, Vikentiou M, Psarra K, Papasteriades C, Raptis SA. Visfatin, TNF-alpha and IL-6 mRNA expression is increased inmononuclear cells from type 2 diabetic women.  Horm Metab Res. 2007;  39 758-763
• 20 Fukuhara A, Matsuda M, Nishizawa M, Segawa K, Tanaka M, Kishimoto K, Matsuki Y, Murakami M, Ichisaka T, Murakami H, Watanabe E, Takagi T, Akiyoshi M, Ohtsubo T, Kihara S, Yamashita S, Makishima M, Funahashi T, Yamanaka S, Hiramatsu R, Matsuzawa Y, Shimomura I. Visfatin: a protein secreted by visceral fat that mimics the effects of insulin.  Science. 2005;  307 426-430
• 21 Zhu J, Schott M, Liu R, Liu C, Shen B, Wang Q, Mao X, Xu K, Wu X, Schinner S, Papewalis C, Scherbaum WA, Liu C. Intensive glycemic control lowers plasma visfatin levels in patients with type 2 diabetes.  Horm Metab Res. 2008;  40 801-805
• 22 Weber KT, Brilla CG. Pathological hypertrophy and cardiac interstitium: fibrosis and rennin-angiotensin-aldosterone system.  Circulation. 1991;  83 1849-1865
• 23 Chapman D, Weber KT, Eghabali M. Regulation of fibrillar collagen typeI, III and basement membrane type IV collagen gene expression in pressure overloaded rat myocardium.  Circ Res. 1990;  67 784-789
• 24 Porter KE, Turner NA. Cardiac fibroblasts: At the heart of myocardial remodeling.  Pharmacology & Therapeutics. 2009;  123 255-278
• 25 Olson ER, Shamhart PE, Naugle JE, Meszaros JG. Angiotensin II-induced extracellular signal-regulated kinase 1/2 activation is mediated by protein kinase C delta and intracellular calcium in adult rat cardiac fibroblasts.  Hypertension. 2008;  51 704-711
• 26 Jiang XY, Gao GD, Du XJ, Zhou J, Wang XF, Lin YX. The signaling of AT₂ and the influence on the collagen metabolism of AT2 receptor in adult rat cardiac fibroblasts.  Acta Cardiol. 2007;  62 429-438
• 27 Villarreal F, Epperson SA, Ramirez-Sanchez I, Yamazaki KG, Brunton LL. Regulation of cardiac fibroblast collagen synthesis by adenosine: roles for Epac and PI3K.  Am J Physiol Cell Physiol. 2009;  296 C1178-C1184
• 28 Gao X, He X, Luo B, Peng L, Lin J, Zuo Z. Angiotensin II increases collagen I expression via transforming growth factor-beta1 and extracellular signal-regulated kinase in cardiac fibroblasts.  Eur J Pharmacol. 2009;  606 115-120
• 29 Kannel WB, Hjortland M, Castelli WP. Role of diabetes in congestive heart failure: the Framingham study.  Am J Cardiol. 1974;  34 29-34
• 30 Gottdiener JS, Arnold AM, Aurigemma GP, Polak JF, Tracy RP, Kitzman DW, Gardin JM, Rutledge JE, Boineau RC. Predictors of congestive heart failure in the elderly: the Cardiovascular Health Study.  J Am Coll Cardiol. 2000;  35 1628-1637
• 31 Nichols GA, Hillier TA, Erbey JR, Brown JB. Congestive heart failure in type 2 diabetes: prevalence, incidence, and risk factors.  Diabetes Care. 2001;  24 1614-1619

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