MAPK Pathway Mediates The Effects Of Fibrin(Ogen) On The Proliferation And Migration Of Vascular Smooth Muscle Cells

Background and Objective Evidence from epidemiological and perspective clinical studies suggests that fibrinogen is an independent risk factor for atherosclerosis. Its transformation into fibrin is confirmed to be closely correlated with atherosclerosis progression, thrombogenesis and restenosis after percutaneous coronary intervention. Whether fibrinogen is a pathologic factor to atherosclerosis, however, is still controversial and it is not clear whether fibrinogen itself, instead of fibrin, contribute to the development of atherosclerosis. A major step in the pathogenesis of atherosclerosis is the vectorial migration of vascular smooth muscle cells (VSMC) from the arterial media through the internal elastic lamina into the intima and their subsequent proliferation in the intima. In this experiment, we investigate the effect of fibrinogen and fibrin on the proliferation, chemotaxis and migration of VSMC to explore the possible function and mechanism of fibrinogen itself in the formation of atherosclerosis and to make a comparison between fibrinogen and fibrin on their role in the formation of atherosclerosis. Mitogen activated protein kinase is the common and central pathway between extracellular signal and nuclear reaction, but it has never been reported about the role of MAPK in mediating the function of fibrin(ogen) on VSMC. Therefore, we explore the role of MAPK in regulating fibrin(ogen)-induced VSMC proliferation and migration. Methods (1) Rat aorta smooth muscle cells (RASMC) were primarily cultured. RASMC identity was confirmed morphologically and by positive immunostaining for SM α-actin. Growth curve was drawn to assess the proliferation ability and cell growth kinetics. (2) The preparation of fibrin was performed using thrombin and fibrinogen. (3) Effects of fibrinogen and fibrin on the proliferation of RASMC were observed by means of cell counting and BrdU incorporation test and cell cycle analysis performed by flow cytometry. (4) RASMC random migration was measured with scrape assay and time-lapse microcinematography and their chemotaxis done with Boyden's chemotaxis assay and by observation of RASMC upward movement in the fibrin gel. Gelatinase A expression and activity were assessed by Western blotting and zymography techniques. (5) Western blot was used to assess the effect of fibrin on the activation of ERK, p38 and JNK with the result scanned and analyzed densitometrically with Bio-Rad Geldoc 2000 image analyzer. Selective inhibitor of ERK, p38 or JNK was used to assess their specific role in the proliferation and chemotaxis of RASMC induced by fibrinogen or fibrin. Results (1) RASMC were characterized by positive SM α-actin immunofluorescence staining and by specific hill-and-valley appearance. They had strong viability with doubling generation time 64 h. Cells of 3-10 passages were used for the present study. (2) Both fibrinogen and fibrin could stimulate the proliferation of SMC dose-dependently in the range between 0.2 g/L and 3.2 g/L with the effect of fibrin more significant. (3) Fibrinogen had no apparent effect on random migration of RASMC but both fibrinogen and fibrin could induce chamotaxis of RASMC in a dose-dependent manner with stronger effect of fibrin than fibrinogen of the same concentration. Both can induce the up-regulation of expression and activity of gelatinase A which made it possible to speculate that the increase of gelatinase secretion stimulated by fibrinogen and fibrin may help RASMC move from media to intima. (4) Fibrinogen induced activation of ERK and p38 time-dependently and so did fibrin on ERK, p38 and JNK. (5) Fibrin induced RASMC proliferation partly by activation of ERK and JNK, instead of p38, but activation of p38, instead of ERK and JNK may partly attribute to fibrin-induced RASMC chemotaxis. (6) Phosphorylation and nuclear translocation of ERK were closely correlated with RASMC's survivability of serum withdrawal and the heterogeneity of VSMC. The time-dependent changes of ERK activity and subcellular location were closely correlated with fibrinogen...