The Effects Of Hypoxia On Adult Rat Cardiac Fibroblast Dna Synthesis And The Major Matrix Protein Expression And Mechanism

Effects of hypoxia on the expression of several majorextracellular matrix proteins and DNA synthesis in adultrat cardiac fibroblasts and its mechanismsAbstractObjective:The goal of this study was to determine the effects of hypoxia on the expression of several major extracellular matrix proteins and DNA synthesis in cultured adult rat cardiac fibroblasts and its mechanisms.Methods:1. We cultured cardiac fibroblasts(CFs) from adult Wistar rat ventricule. Studies were conducted on the second passage of CFs and they were randomized into 4 groups: 1). normoxia group; 2).normoxia with captopril group; 3).hypoxia(1%02) group;4).hypoxia(1%02) with captopril group. 2. The changes of DNA synthesis was determined by measuring the incorporation of 3H-TdR into DNA when CFs was exposed to hypoxic environment for 6h, 12h and 24h. 3. The changes of expression of pro-cd(I) collagen, pro-al(III), matrix metalloproteinase I, fibronectin, TGF-131 and HIF-la mRNA were measured by in situ hybridization when CFs was exposed to hypoxic environment for lh, 2h, 4h, 8h, 12h and 24h.Results:1.The 3H-TdR incorporation of CFs was increased by 34% (P0.05 )and 36%( P < 0.01) after 6h, 12h hypoxic exposure respectively. As compared with normoxia, the 3H-TdR incorporation was not significant different from under hypoxa for 24h.2.The expression of fibronectin mRNA increased when the cells were cultured under hypoxia for lh, then decreased and were notdifferent from normoxia.3. The expression of pro-al(III) and matrix metalloproteinase ImRNA increased when cells were cultured under hypoixa for 2h. Butthere was not detectable difference in the level of pro-a1(III) mRNAand matrix metalloproteinase I mRNA between the normoxia andhypoxia fOr 4h, 8h, l2h, 24h.4. The level of pro-al(I) collagen mRNA was significantlyelevated in the cells under hypoxia for 4h, 8h, and l2h. Theexpression of pro-al (I) collagen mRNA in cells exposured to hypoxiafor 24h also decreased and was not different from under normoxia.5. The expression of TGF-61 mRNA increased when the cellswere cultured under hypoxia fOr lh, 2h, 4h, then decreased and wasnot different from normoxia.6. Hypoxia induced the expression of HIF-l Q mRNA in thecultured CFs at the early phase(l~4h). There was not detectablechanges of HIF-l Q mRNA expression in CFs exposured to hypoxiafOr 8-- 24h compared with the cells under normoxia.7. The stimulative effects of hypoxia on incorporation ofsH-TdR and expression of fibronectin, pro-a1(I) collagen mRNAwere attenutated by the presence of captopril. Captopril alsoabolished the stimulative effective of hypoxia on the expression ofTGF-61 mRNA.C o n clu si o n:1. Hypoxia alone can reguate DNA synthesis and expression ofextracelluar matrix(ECM) proteins in adult rat cardiac fibroblsts.2. The alteration of ECM proteins mRNA expression in adult ratcardiac fibroblasts during hypoxia shows time-dependece. At theearly phase(lh) of hypoxia, the expression of fibronectin mRNA isIIincreased transiently . A much later, sustained(4挆 12h) increase in expression of pro-ctl(III) mRNA follow the increase of pro-ctl(III) and matrix metalloproteinase I mRNA expression.3.Hypoxia can upregulate the expression of TGF-pl in dult rat cardiac fibroblsts via the effects of angiotensin II.4.ACE inhibitor can inhibit the increases induced by hypoxia in3H-TdR incorporation and expression of fibronectin and pro-x1(III) mRNA. These results suggest that local angiotensin II may play a important role in the regulation of cardiac fibroblasts function during hypoxia.5.Hypoxia can induce the expression of HJF-hx in adult rat cardiac fibroblasts.