Atorvastatin Inhibits The Apoptosis Of Human Umbilical Vein Endothelial Cells Induced By Angiotensin Ii Via The Lysosomal-Mitochondrial Axis

Objective:The renin-angiotensin-aldosterone system(RAAS)plays an important role in the pathogenesis of cardiovascular diseases,including hypertension and atherosclerosis.Angiotensin II(Ang ?)is the main active peptide hormone of the RAAS and plays a major role in endothelial dysfunction,vascular remodeling and vascular inflammation,which are closely related with hypertension and atherosclerosis.Statins have exerted a variety of protective effects including upregulation of endothelial nitric oxide(NO)expression and antioxidant effects,which is independent of lowering cholesterol concentrations.Apoptosis is an evolutionary conserved mechanism of elimination of the unwanted cells,which is mediated by extrinsic and intrinsic pathways.The intrinsic pathway,known as "mitochondria-dependent pathway",is triggered by various intracellular and extracellular stimuli in which the signals are finally transmitted to mitochondria.On the basis of former researches,in 2006,Terman A et al.put forward a new concept named as "lysosomal-mitochondrial axis" theory.This study was aimed to evaluate lysosomal-mitochondrial cross-signaling in Angiotensin ?(Ang ?)-induced apoptosis of human umbilical vein endothelial cells(HUVECs)and whether atorvastatin played a protective role via lysosomal-mitochondrial axis.Methods: 1.HUVECs were cultured with 1 ?M Ang ? or 10 ?M atorvastatin for 24 h.2.The green fluorescent protein(GFP)-expressing lentiviral plasmids containing short interference(si)RNA against human CTSD or control siRNA were used in the knockdown experiments 3.The GFP-expressing lentiviral plasmids containing wild-type or catalytically inactive mutant(D295 N)CTSD cDNA were used in the overexpression experiments.4.MTT Colorimetric analysis,Hoechst 33342 staining,AO/EB staining and flow cytometry were used to detect effects of Ang ? or atorvastatin on the cell viability and apoptosis of HUVECs.5.TMRE staining was used to detect effects of Ang ? or atorvastatin on mitochondrial membrane potential of HUVECs 6.DCFH-DA staining was used to detect effects of Ang ? or atorvastatin on reactive oxygen species of HUVECs.7.AO staining and Lysotracker staining were used to detect effects of Ang ? or atorvastatin on lysosomal functions of HUVECs.8.Western blot were used to detect the protein expression changes of caspase 3,caspases 9,Bad,Bax,Bcl-2,cathepsin D and cytochrome c.9.Cathepsin D activity was detected by Cathepsin D Activity Fluorometric Assay Kit.Results: 1.Atorvastatin attenuated Ang ?-induced apoptosis in HUVECs.2.Atorvastatin showed a protective effect on mitochondrial dysfunction induced by Ang ?.3.Atorvastatin showed a protective effect on lysosomal dysfunction induced by Ang ?.4.Ang ?-induced mitochondrial dysfunction was downstream of the lysosomal permeabilization.5.Pharmacological inhibition of CTSD protected mitochondrial dysfunction and apoptosis.6.Genetic inhibition of CTSD protected mitochondrial dysfunction and apoptosis.7.Overexpression of CTSD showed no affection on mitochondrial dysfunction and apoptosis.Conclusion: 1.Ang ?-induced apoptosis of HUVECs was associated with release of cytochrome c into the cytosol,activation of proapoptotic proteins through lysosomal-mitochondrial axis.2.Atorvastatin could inhibit apoptosis induced by Ang ? through stabilizing lysosomal membrane.