Influence Of Hirudin On Thrombin-Induced Proliferation Of Vascular Smooth Muscle Cells And Protection Of Endothelial Cells

BackgrandTissue lesion, thrombosis, and endangium proliferation are involving the physiology and pathobiology process of atherosclerosis and restonosis.Thrombin is a serine protease that potently activates platelets and catalyzes the conversion of fibrinogen to fibrin. Thrombin also exerts direct effects on vascular cells, via interactions with members of the protease-activated receptor family. Evidence in several animal models and clinical trials implicates thrombin-mediated signaling events in the response to injury that typifies vascular lesion formation in atherosclerosis and restenosis. The prototypical direct thrombin inhibitor hirudin was originally isolated from the salivary glands of the medicinal leech and produced by recombinant DNA technology nowdays. Composed of 65 or 66 amino acids, hirudin inhibits thrombin directly,both in the free and in the thrombus-bound state,and does not requeir any endogenoud cofactors. Owing to its high affinity and specificity for thrombin, hirudin has been intensively investigated for research and theraeutic purposes. These unique properties of hirudin launched a series of in vivo studies and clinical trials that explored not merely its potential as a novel antithrombotic agent but also—on the basis of the distinct differences in whichhirudin inhibits thrombin induced other biological responses,such as reducing platelet deposition and thrombosis after catheter-induced vascular injury.The aim of this study was to compare the effects of recombinant hirudin and heparin on thrombin induced vascular smooth muscle cells (VSMC) development and human umbilical vein endothelial cells (HUVEC) activity of matrix metalloproteinases-2, which is a class of zinc-utilizing proteolytic enzymes that function to degrade extracellular matrix components and regulate of cell proliferation, migration and matrix invation. And to undersand how during those process, nuclear factor kappaB(NF-B), one of the most critical transcription factors responsible for both the initiation and expansion of lesions in atherosclerosis and restonosis to play its role. Understanding the different physiologic and pathologic processes of thrombin's multiple effects on endothelium and smooth muscle cells and how hirudin plays its role offers the hope of intervening to delay or prevent atherosclerosis and restonosis progression and complications. Part 1. Influence of Hirudin on Thrombin-induced Proliferation of Vascular Smooth Muscle CellsAim To study the influence of hirudin on thrombin-induced proliferation of vascular smooth muscle cellls(VSMC).And to investigate its the possible signaling pathways involved through analysis of cell growth,cycle distribution, platelet-drived growth factor(PDGF) and proliferating cell nuclear antigen(PCNA) in VSMCs. Methods Vesscular smooth muscle cells were isolated from thoracic aorta of rabbits by plant method and cultured in in medium Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum. Passages between 2 and 5 were used. After the cells were incubated in cluture flask or well plates up to 70~80% of confluence, their growth was arrested by culturing overnight in serum-free medium. Then the tests compared with four main groups ,which were thrombin(4.0 ku/L); thrombin(4.0 ku/L)+hirudin ( 6.0 ku/L ) ; thrombin(4.0 ku/L)+ heparin (6.0k u/L) ; and serum-free DMEM ascontrol group.Cell proliferation assays were performed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-dimethyltetrazolium bromide (MTT, Sigma) reagent . Cell cycle distribution was analysesed with flow cytometry. And expression of PDGF and PCNA in VSMCs were evaluated by streptavidin biotin complex(SABC) immunocytochemistry technique and computer image analysis system. Results are shown as mean±SEM. Statistical analyses were performed with ANOVA followed by Fisher's protected least significant difference test to compare 2 treatments. A value of P<0.05 was considered statistically significant.Results The thrombin promoted HUVEC growth, which remarked by ascending 12.7%> 29.7% and 16.4%of optical density at 12h> 24h and 48h respectively (p<0.05~ p<0.01) . Hirudin, a potent and specific thrombin inhibitor, completely inhibited thrombin-induced the proliferation of VSMC. Hirudin also blacked the role of thrombin involving in facilitating cell-cycle transition from Gi to S phase and also promoting from G2- to M-phase transition. The result of streptavidin biotin complex(SABC) immunocytochemistry technique and computer image analysis system showed that, under the treated with hirudin, the reduced numbers of postive cells in expression of PDGF and PCNA was 14.67±2.81 and 23.71±2.50 , 0.201±0.07 respectively (p<0.05~p<0.01 )<,Part 2.Influence of Hirudin on the Production and Activation of MMP-2 in Human Umbilical Vein Endothelial CellsAim Based on the central role of thrombin in diverse aspects of endothelial cell function and the potential importance of matrix metalloproteinases (MMPs) in angiogenesis, to investigate the possibility that thrombin may be involved in disfunction of HUVEC and upregulation of HUVEC to express MMP-2 and membrane-type matrix metalloproteinase(MT-MMP), the key enzymes for degradation of extracellular matrix composing.And whether during the process of MMP-2 express, there were also following the activation of signalingpathway-neuclear factor-/cB(NF-kB). Methods Human umbilical vein endothelial cells (HUVECs) were isolated from primary human umbilical vein by digestion with collagenase type II and cultured in medium Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum. Passages between 2 and 5 were used. After the cells were incubated in cluture flask or well plates up to 70~80% of confluence, their growth was arrested by culturing overnight in serum-free medium. Then the tests compared with four main groups ,which were thrombin(4.0 ku/L); thrombin(4.0 ku/L)+hirudin (6.0 ku/L) ; thrombin(4.0 ku/L)+ heparin (6.0k u/L) ; and serum-free DMEM as control group. The roles of thrombin and hirudin to the living activity of HUVECs were quentified with colormetric assay by using MTT reagent. The expression level of MMP-2 and MT1-MMP of HUVEC was assessed with reverse transcriptase-polymerase chain reactin(RT-PCR). The location and distribution of nuclear factor kappaB(NF- k B) protein expression was detected by SABC immunocytochemistry technique and Northern Blot Analyses. Results are shown as mean + SEM. Statistical analyses were performedwith ANOVA followed by Fisher ' s protected least significant difference test to compare 2 treatments. A value of P<0.05 was considered statistically significant.Result : The thrombin interfered with HUVEC growth, which remarked by descending in optical density of MTT. Thrombin treatment of endothelial cells within 24h resulted in a dose- and time-dependent manner generation of MMP-2 and MT-MMP with a start at 1 hour, followed by a high peak at 24 hours. Hirudin, a potent and specific thrombin inhibitor, completely inhibited thrombin-induced MMP-2 activation, demonstrating that the proteolytic activity of thrombin is essential. However, activation was also dependent upon membrane-type-MMP (MT-MMP) action, which rapidly (within lh) increased cellular MT-MMP activity, and at longer time points (24h). Immunocytochemistry and Western blot analyses showed thatthrombin could induce the transfer of NF-kB form cytoplasma to nucleus. Compared with thrombin , hirudin treatment HUVEC resulted in much less activation of NF-kB at 15~3Omin, but there was no consistent decrease at 24 h.Part 3. Hirudin decreases expression of adhesive molecules on polymorphonuclear cells and human umbilical vein endothelial cell Aim: To observe whether thrombin enhanced expression of CDllb on polymorphonuclear leukocyte (PMN) in whole blood and ICAM-1 on human umbilical vein endothelial cells(HUVEC).And whether hirudin blocked the role of thrombin.The PMN was isolated in whole blood by density gradient centrifugation method and its altered expression in CDllb was assayed by immunofluorence flow cytometery. The HUVEC was isolated from primary human umbilical vein by digestion with collagenase type II and its altered expression in ICAM-1 was assayed by enzymelinkedimmunosorbentassay.Result The result showed that both CDllb expressions on the PMN and ICAM-1 on HUVEC gradually increased with the passage of time from 6 h after incubated with thrombin, and was the highest at 24h, on both of which hirudin was an effective inhibitor. The finding suggested The hirudin blocked the expression of the adhesive molecule induced by thrombin, which might related to leukocyte adhesion and migration, even influence the pathogenesis of atherosclerosis.Conclusion and Significance Firstly the stimulatory effect of conditioned medium from thrombin on VSMC was significantly reduced by hirudin, which might be carried out though inhibition of PDGF and PCNA expression and involvement of cell-cycle regulation. Secendly the stimulatory effect of conditioned medium from thrombin on HUVEC was significantly reduced by hirudin, which might be carried out though inhibition NF-kB signal transduction pathways. Although the exact mechanisms that lead to atherosclerosis and restonosis remain largely unknown, It is no doubt the contribution of early migration and proliferation of medial SMCs and disfunction Of endothelial EC ,shch as increased MMP-2 expression and activity as well...