Effects Of Adrenomedullin On The Metabolism Of Collagens And The Interaction Between Adrenomedullin And TGF-β1 In Hepatic Stellate Cell

Liver fibrosis is the common pathological consequence of clinical liver diseases, which can be induced by many factors. Hepatic stellate cell (HSC) accounts for 15 percent of all the hepatic cells and plays an important role in hepatic fibrogenesis. HSC has been widely regarded as the target for anti-fibrosis therapy.Adrenomedullin (AM) is a new hypotensive peptide recently identified in human pheochromocytoma arising from adrenal medulla, belongs to the family of calcitonin gene related peptide. Besides hypotensive activity, AM acts as a circulating hormone as well as a hind of cytokine which elicits multiple biological activities in a paracrine or autocrine manner . Several reports have demonstrated that AM inhibited proliferation of glomerular mesangial cells and cardiac fibroblasts, antagonized the secretion of collagens and tapered off fibrosis. AM can be produced and secreted by various cells including hepatic stellate cell. AM secretion can be regulated by many cytokines. There were relatively less reports about the effect of TGF-β1 on AM secretion and it was determined from previous reports that the regulation of TGF-1β on AM synthesis seems to be paradoxical dependent on cell types.Hepatic fibrogenesis and development is owed to the roles of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor (PDGF). TGF-β1 regulates the expression levels and activities of MMPs/TIMPs as well as inducing production of ECM in HSC; PDGF acting as the most efficient mitogen of HSC exerts multiple activities such as upregulating TGF-β1 production and MMP-2 expression in HSC. It was recently reported that MMP-2 had the capability of degrading AM and AM could be regarded as the new substrate of MMP-2, some research in rat aortic adventitial fibroblasts showed that AM induced expression and activity of MMP-2. However, in hepatic stellate cell, no any reports is about theinteraction between AM and TGF- 3 1 or PDGF.The aim of the present study was to investigate, in cultured HSC, the effects of AM gene overexpression on cell growth and metabolism of collagens; the interaction of TGF-pi and AM at transcription and protein expression level as well as the effect of AM on TGF-pi or PDGF-induced MMP-2 expression and the possible mechanism. Solution of above questions is no doubt beneficial to further understanding the pathogenesis of hepatic fibrosis and provides a clue to develop a new therapeutic strategy for anti-hepatic fibrosis.Part One Effect of AM gene overexpression on cell growth, procollagens production and MMPs/TIMPs system in HSCObjective: To investigate the effect of AM gene overexpression on cell growth, procollagens production and MMPs/TIMPs system in HSC by stable transfection with AM gene.Methods: HSC which expresses low basal levels of AM was stably transfected with an expression construct containing rat AM gene or with empty expression vector. Expression of AM in HSC was determined by Reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA); Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry as well as using the cell counting kit; Hoechst staining and flowcytometer analysis were used to evalue apoptosis of HSC; RT-PCR and western blot were used to test the expressions of procollagen type I and III, MMP-1, MMP-2 and TIMP-2.Results: Two cell clones (A-2,A-8) transfected with the AM gene expressed higher levels of AM mRNA and protein (P<0.05). AM gene overexpression had an inhibitory effects on cell proliferation of HSC (P<0.05) and induced the apoptosis of HSC (P<0.05); the expressions of procollagen type I and III were suppressed by AM gene overexpression (P<0.05). Compared with cells non-transfected and transfected with empty vector, these two clones had lower expression levels of TIMP-2 and MMP-2, but displayed a higher expression levels of MMP-1 (PO.05).Conclusions: AM exerts negative influence in some extent on liver fibrosis by inhibiting HSC growth and production of procollagen in addition to inducing ECM degradation.Part Two The interactive regulation between AM and TGF-01 at gene transcript and protein expression levels in HSCObjective: To examine whether interactive regulation between AM and TGF-pi at gene transcript and protein expression levels exists in hepatic stellate cell.Methods: Realtime RT-PCR, radioimmunoassay and western blot were used to test gene transcript levels and protein expression levels of AM and TGF-pi in HSC.Results: Cells were stimulated for 8 h with the indicated concentrations of TGF-pi (Ong/ml, O.lng/ml, lng/ml and 5ng/ml) or stimulated with TGF-pi (lng/ml) for indicated incubation time (Oh, 4h, 8h and 14h), realtime RT-PCR results showed that AM gene transcript levels relative to GAPDH (xlO"7) were 4.37±0.3, 2.95±0.27, 1.28±0.08, 1.4±0.08 and 4.17±0.6, 3.84±0.2, 2.04±0.4, 3.3U0.17, respectively (PO.05) . TGF-pl was of no effect on AM secretion level from HSC (P>0.05) . Cells were stimulated for 24 h with the indicated concentrations of AM (OM, 10"9M, 10"8M and 10"7M), TGF-pi gene transcript levels relative to GAPDH (xlO'7) showed by realtime RT-PCR were 4.71±0.03, 4.56±0.22, 4.37±0.05 and 4.45±0.04, respectively (P<0.05) and TGF-pl protein expression levels were 1.97±0.1, 1.16±0.06, 0.29±0.06 and 0.58±0.07, respectively (P<0. 05). When cells were treated with AM (10'8M) for indicated time (Oh, 12h, 24h and 36h), TGF-pl gene transcript levels relative to GAPDH (xlO~7) were 3.8±0.2, 4.16±0.1, 2.41±0.08, 1.9±0.06 and TGF-pi protein expression levels were 12.27±0.29, 8.04±0.17, 1.53±0.08, 0.83±0.13, respectively (P<0. 05).Conclusions: In hepatic stellate cell, TGF-pi dose-dependently and time-dependently inhibits AM gene transcription, but has no effect on AM secretion. AM also has negative effect on TGF-pi gene transcription and protein expression in dose-dependent and time-dependent manners. AM may elicit certain anti-hepatic fibrosis activity by downregulating the expression of TGF-pi.