Research Of Farnesyl Pyrophosphate Synthase In Ang Ⅱ-induced Cardiac Fibrosis

Part1Inhibition of farnesyl pyrophosphate synthase reverses in vitro cardiac fibrosis induced by Ang IIBackground:Cardiac fibroblast (CF) is the main cell of the heart that plays an important role in the process of cardiac fibrosis. Various stimuli can induce cardiac fibrosis, such as angiotensin II (AngⅡ), ET-1and so on. A variety of cellular signaling transduction pathways were involved in AngⅡ-induced in vitro cardiac fibrosis, including RhoA activation pathway.Previous studies have shown bisphosphonates, such as alendronate and zoledronate, inhibit a key enzyme--farnesyl pyrophosphate synthase (FPPS) in the mevalonate pathway with subsequently decrease of farnesylation pyrophosphate (FPP) and geranyl geranyl pyrophosphate (GGPP), thereby reducing the synthesis of isoprene. Actually, GGPP is very important for RhoA geranyl geranyl activation and pyrophosphatation.Purpose:We take this experiment to study whether FPPS inhibition by zoledronate is relevant with in vitro cardiac fibrosis induced by Ang II, and whether it involves with RhoA pathway. Method:(1). CFs were incubated in serum-free medium (SFM) for24hours before incubated with10-30μM zoledronate for30minutes and then co-incubated with0.1μM Ang II for48hours. Cardiac fibrosis marker genes such as collagen I, collagen III, transforming growth factor β1(TGFβ1) and FPPS protein levels were assayed.(2). CFs were incubated in SFM for24hours before incubated with10μM zoledronate and10μM GGOH or10μM FOH for30minutes and then co-incubated with0.1μM Ang II for48hours. Cardiac fibrosis marker genes such as collagen I, collagen III, TGFβ1and FPPS protein and RNA levels were assayed.(3). CFs were incubated in SFM for24hours before incubated with10μM zoledronate and10μM GGOH for48hours and then co-incubated with1μM Ang II for15minutes. Active and total RhoA were detected by pull-down assay kits and western blot.(4). CFs were incubated with50μM siRhoA or control siRNA for24hours and incubated in SFM for24hours, then incubated with0.1μM Ang II for48hours. Cardiac fibrosis marker genes such as collagen I, collagen III, TGF β1and FPPS protein and RNA levels were assayed.Results:(1). FPPS inhibition by zoledronate dose-dependently down-regulated in vitro cardiac fibrosis marker genes such as collagen I, collagen III, TGFβ1and FPPS protein expression.(2). The inhibitory effect of zoledronate on the expression of in vitro cardiac fibrosis marker genes such as collagen I, collagen III, TGFβ1and FPPS protein was reversed partially by GGOH but not FOH.(3). In vitro cardiac fibrosis induced by Ang II is relevant with RhoA pathway.(4). siRhoA down-regulated Ang II-induced in vitro cardiac fibrosis marker genes such as collagen I, collagen III, TGF β1and FPPS protein and RNA expression.Conclusion:The results showed that FPPS inhibition by zoledronate can effectively reverse Ang Ⅱ-mediated in vitro cardiac fibrosis, which involved with RhoA geranyl geranyl pyrophosphatation. Meanwhile, RhoA-silencing down-regulated in vitro cardiac fibrosis and FPPS expression. Part2Silencing of farnesyl pyrophosphate synthase reverses in vitro cardiac fibrosis induced by Ang IIBackground:The first part of the experiment showed that the inhibition of FPPS can reverse in vitro cardiac fibrosis induced by Ang II, possibly suggesting that FPPS was an important target in Ang II-mediated in vitro cardiac fibrosis. Previous studies showed that FPPS expression was significantly up-regulated both in in vivo and in vitro models of cardiac hypertrophy, meanwhile, active RhoA was increased significantly in Ang II-mediated cardiac fibrosis. The above evidence possibly showed the important role of FPPS in Ang Ⅱ-mediated in vitro cardiac fibrosis with the activation of RhoA.Purpose:The experiment explores whether FPPS expression was increased in Ang II-mediated in vitro cardiac fibrosis, and whether FPPS silencing modulated in vitro cardiac fibrosis, and furthermore explore FPPS-relative RhoA activity. Method:(1). FPPS transcriptional and translational expression in Ang II-mediated in vitro cardiac fibrosis was assayed by western blot and real-time PCR.(2). FPPS recombination lentivirus transfected neonatal CFs. Cardiac fibrosis marker genes such as collagen Ⅰ, collagen III, TGF β1and FPPS were assayed.(3). The involvement of FPPS in in vitro cardiac fibrosis was furthered by investigation of downstream, to find alteration of active and total RhoA by pull-down assay and western blot. Results:(1). FPPS expression in transcriptional and translational levels was up-regulated both in in vivo and in vitro models of cardiac fibrosis.(2). Silencing of FPPS significantly down-regulated FPPS expression and cardiac fibrosis marker genes such as collagen I, collagen III, TGF β1.(3). Silencing of FPPS effectively down-regulated active RhoA in in vitro cardiac fibrosis induced by Ang II.Conclusion:FPPS was up-regulated both in in vivo and in vitro models of Ang II-mediated cardiac fibrosis, showing the important role of FPPS in Ang II-mediated cardiac fibrosis. Meanwhile, RhoA activation was relevant with FPPS in Ang II-mediated in vitro cardiac fibrosis.