Autophagy Protects Against Senescence And Apoptosis Via The RAS-mitochondria In High-glucose-induced Endothelial Cells

Endothelium plays key roles in the pathogenesis of diabetic complications. High-glucose induces endothelial damage through energy imbalance, Ca2+overload, ROS, inflammation and mitochondria-dysfunction. As the most affluent organelle of eukaryocyte, mitochondrial dysfunction contributes to cell apoptosis, senescence and autophagy. Besides digesting impaired-organelles or-proteins and maintaining cellular homeostasis, autophagy decides the final fate of cells under stresses through limiting the processes of apoptosis, necrosis and senescence. Recently reports agree that autophagy plays critical roles in cardiovascular diseases, whereas the precise evidences of autophagy in diabetes have been rarely mentioned. RAS plays a pivotal role in cardiac development and the pathogenesis of cardiovascular disease, including cellular hypertrophy, proliferation and apoptosis. But the RAS level in diabetic vascular endothelium is still unclear. The usage of RAS inhibitors, including ACEI and ARB, in diabetic patients who didn’t couple with cardiopathy is still controversial. Nevertheless, disrupting RAS has been shown to provide significant benefit for endothelial function, and this effect has been shown to rely on mitochondrial-ROS. Recently, the Ang type1receptor (AGTR1) and Ang type2receptor (AGTR2) have been reported to trigger cardiomyocyte autophagy, which may be the underlying mechanism of the negative effects caused by RAS, including the imbalance in autophagy, apoptosis and senescence. Perhaps RAS-mitochondria-autophagy also plays a critical role in maintaining vascular function. This study explores the role of RAS-mitochondria-autophagy in high-glucose-induced HUVEC, thus provides an important theoretical foundation of the early RAS intervention in diabetic patients. Part One:High-glucose induces autophagy, senescence and apoptosis in HUVECAims:In this present study, concentration-or time-dependent incubations of glucose in HUVEC are used to investigate the impacts of high glucose on autophagy, senescence and apoptosis.Methods:1. HUVECs were cultured in serum-free ECM (glucose:5.5mM) for12h before incubation with glucose at11,16.5,22,27.5or33mM for72h or at33mM for72,48,24or6h. The mannitol at33mM for72h was used to eliminate the implication of increasing osmotic pressure.2. After incubated, cell cycle analysis, SA-β-gal staining, SAHF-formation and p21expression were measured to evaluate cell senescence.3. After incubated, trigger proteins and autophagic biomarkers were detected to evaluate cell autophagy. AICAR and CC were applied to regulate the AMPK activity, a autophagic trigger.4. ANNEXIN V-FITC staining was used to evaluate cell apoptosis.Results:1. High-glucose promotes HUVEC senescence in an osmotic pressure-independent manner.2. High-glucose induces high expression of autophagic biomarkers, which would be abolished by inhibiting the phosphorylation of AMPK. 3. High-glucose does not affect HUVECs apoptosis.Conclusions:High-glucose induces HUVECs senescence and autophagy in an osmotic pressure-independent manner, whereas it does not affect HUVECs apoptosis. Part Two:High-glucose induces RAS-mitochondria dysfunction in HUVECAims:In this present study, we explore the influences of high-glucose on RAS-mitochondria, and further determine the relationship with RAS-mitochondria and senescence, autophagy, apoptosis in high-glucose-treated HUVEC.Methods:1. After incubated with glucose at33mM for72h, the ACE1、AGTR1、AGTR2mRNA and protein levels were detected.2. After incubated, JC-1staining, ROS, NADPH, cytoplasmic Ca2+levels and cytC distribution were measured to mitochondrial function.3. After incubated with Ang at1uM for72h, HUVEC mitochondrial dysfunction, senescence and autophagy were detected.4. HUVECs were pre-incutation with benazepril (1uM) or losartan (10uM) for6h before incubation with glucose at33mM for72h. Then the HUVEC mitochondrial dysfunction, senescence and autophagy were measured.5. After opening MPTP using CCCP, the HUVEC mitochondrial dysfunction, senescence, autophagy and apoptosis were measured.Results:1. High-glucose induces high expression of ACE1. AGTR1mRNA and protein, whereas the AGTR2expression is no change.2. High-glucose induces loss of ΔψPm, decreased expression of NADPH, increase of ROS, cytoplasmic Ca2+overload and cytC decoupled with mitochondria.3. Ang mimics high-glucose-induced mitochondrial damage, cell senescence and autophagy in HUVEC.4. Benazepril or losartan ameliorates high-glucose-induced mitochondrial damage, cell senescence and autophagy in HUVEC.5. CCCP induces HUVEC autophagy and apoptosis.6. CCCP abolishes beneficial effects caused by benazepril or losartanConclusions:High-glucose induces HUVECs senescence, autophagy and apoptosis through RAS-mitochondria. Part Three:Auophagy limits high-glucose-induced senescence and apoptosisAims:In this present study, we explore autophagy protects against senescence and apoptosis in high-glucose-treated HUVEC, and further discloses the underlying mechanisms.Methods:1. HUVECs were co-incubated with high glucose and3-MA, CC, CCCP or AICAR, then HUVEC senescence and apoptosis were measured.2. Deficient autophagy using LC3or beclinl siRNA for48h, we measured senescence and apoptosis in high-glucose-treated HUVEC.3. After co-incubated with3-MA and benazepril or losartan, high-glucose-induced HUVEC senescence and apoptosis were detected.4. After incubation, the NFkB expression and eNOS phosphorylation were detected.Results:1.3-MA or CC accelerates high-glucose-induced HUVEC senescence and apoptosis, meanwhile AICAR or CCCP reverses high-glucose-induced HUVEC senescence.2. LC3or beclinl knockdown promotes high-glucose-induced HUVEC senescence and induces newly-presence of increasing apoptosis.3.3-MA abolished benazepril or losartan mediated senescence decrease, and induced newly present apoptosis increase.Conclusions:Autophagy protects against senescence and apoptosis in high-glucose-treated HUVEC. Part Four:The aggregates of autophagy, senescence and apoptosis in diabetic-patient or-rat arterial endotheliumAims:In this present study, we study the expression levels of autophgy, senescence and apoptosis in diabetic-patient or-rat arterial endothelium, and investigate the protective effects of benazepril or losartan.Methods:1. STZ (70mg/kg) i.p. injections into SD rats (180-220g). The diabetic rats were randomly divided into three groups and administered with intragastric Benazepril (10mg/kg/d), Losartan (80mg/kg/d) or equal physiological saline for8weeks respectively.2. The bioassay of vasoreactivity was measured using EDR and EIR.3. The autophagic activity was detected using immunohistochemical analysis.4. The senescent level was detected using SA-β-gal activity.5. The apoptotic activity was evaluated using TUNEL staining.6. The autophagic-, senescent-, and apoptotic-biomarkers in diabetic-patient arterial endothelium were detected using western blot.Results:1. In diabetic rats, the EDR is impairment, which accompanied with autophagic biomarkers, SA-β-gal activity and TUNEL-positive cells increasing.2. Benazepril-or losartan-treatment ameliorates EDR, reduces autophagic biomarkers, SA-β-gal activity and TUNEL-positive cells in diatetic rat aortic endothelium. 3. The autophagic-, senescent-, and apoptotic-biomarkers increases in diabetic-patient arterial endothelium.Conclusions:Autophagy, senescence and apoptosis aggregate in diabetes arterial endothelium, which accompanies with EDR impairment. Moreover, interrupted RAS ameliorates EDR and decreases autophagy, senescence or apoptosis in diabetic rat aortic endothelium.