Role Of Aldehyde Dehydrogenase2in The Effects Of Ethanol On Endothelial Protection

BackgroundExtensive epidemiological studies links moderate alcohol consumption with reduced risk of cardiovascular morbidity and mortality. Initially, non-alcoholic components have been thought to play the primary role in the protective effects induced by drinking. However, recent studies found that ethanol may also be involved in the protection. Low-dose ethanol can rapidly activate PI3K/Akt pathway to increase endothelial nitric-oxide synthase (eNOS) activity by an adenosine receptor-dependent mechanism in human umbilical vein endothelial cells (HUVECs).In addition, ethanol, as a small exogenous liposoluble molecule, can easily pass through the cell membrane and be oxidized by metabolic enzymes systems, accompanied by reactive oxygen species (ROS) generation. ROS have detrimental or beneficial effects on eNOS activation depending on its concentrations. During ethanol exposure, ROS level is dependent on the capability of antioxidant systems to antagonize ROS generation.Mitochondrial aldehyde dehydrogenase (ALDH2), a key enzyme in ethanol metabolism, has been found to possess the antioxidant property. In vitro and in vivo studies suggest that regulation of ALDH2activity can have influence on the cellular response to oxidative stress. ALDH2activity can be affected by gene regulation or post-translational modifications. For example, ALDH2can be directly deacetylated by SIRT3resulting the inactivation of ALDH2.SIRT3, a NAD+-dependent class Ⅲ histone deacetylase, is localized in the mitochondrial matrix. It can elicit adaptive responses to metabolic stresses by regulating mitochondrial protein acetylation levels. SIRT3enzymatic activity has been found very sensitive to the NAD+/NADH ratio. Interestingly, enzymes of ethanol metabolism convert NAD+to NADH leading to the reduced NAD+/NADH ratio, which may have the potential to affect SIRT3activity.In present study, we hypothesized that ethanol can activate ALDH2to regulate ROS level, which may be involved in the protective effects of ethanol on endothelial function. SIRT3-dependent acetylation may play an important role in the potential mechanisms involved. SIRT3-dependent ALDH2acetylation may be a therapeutic target in treating cardiovascular diseases.Methods1. Cell culture and treatment:Human aortic endothelial cells (HAECs) were maintained in endothelial cell medium (ECM) in a water-saturated atmosphere of5%CO2and95%air at37℃. When cells were more that80-90%confluence, different doses of ethanol were added into cells for the designated times. Cells were treated with PI3K inhibitors and NAD for24h before incubated with20mM ethanol for another30min.2. Cell transfection:The specific siRNAs were used to inhibit ALDH2and SIRT3expression, whereas SIRT3plasmids were used to elevate SIRT3expression. Cells were transfected with the siRNA duplexes or plasmids by the Lipofectamine2000according to the manufacturer's instructions. Cells transfected with ALDH2siRNA were then incubated with or without ROS scavengers for30min before ethanol treatment for another30min.3. Real-time PCR:Total RNA were extracted from treated cells by Trizol, and then reverse-transcripted into cDNAs. Real time PCR was performed using SYBR Green detection system. The relative mRNA levels were calculated by2-ΔΔct.4. Western blotting analysis:Total cellular protein was extracted by lysis buffer. The protein expression of ALDH2,eNOS,p-eNOS,Akt,p-Akt,PI3K,SIRT3,4-HNE in HAECs were detected using Western blotting analysis.5. Immunoprecipitation:Cellular total protein was prepared by use of lysis buffer. For immunoprecipitation, the total protein was incubated with appropriate antibody precoupled to protein A/G-agarose beads at4℃overnight. Then agarose beads were removed by high-speed centrigugalization, and then the supernatant were collected to be analyzed by western blotting.6. eNOS activity assay:eNOS activity was measured by the conversion of L-arginine to NO. Then the fluorescent identity was determined by fluoro-spectrophotometer analysis at excitation495nm and emission515nm.7. ALDH2activity assay:The mitochondria ALDH2enzymatic activity was determined by monitoring the reductive reaction of NAD+to NADH at A340nm in a spectrophotometer. Enzyme activity was expressed as nmol/mg/min.8. SIRT3activity assay:The mitochondrial SIRT3activity was assayed using a deacetylase colorimetric activity assay kit according to the manufacture's instructions.9. Assessment of intracellular ROS levels:Treated cells were stained with2',7'-dichlorodihydro-fluorescein diacetate, then underwent fluorescent intensity measurement by flow cytometry.10. NAD+/NADH assay:Mitochontrial NAD+/NADH ratio were quantified using an NAD+/NADH assay kit according to the assay instructions. Total NADt and NADH were detected at450nm in a spectrophotometer. NAD+/NADH ratio is calculated as:[NADt-NADH]/NADH.Results1. Ethanol dose-dependently increased then decreased eNOS activity, with peak activity at20mM (1.67-fold, p<0.001). Additionally, time-course study showed that increase of eNOS activity began at5min after ethanol incubation and got the peak at30min (p<0.001);2. Ethanol (20mM) significantly upregulated p-Akt protein expression at30min, which protective effects of ethanol could be blocked by PI3K inhibitors, confirming that ethanol can activate PI3K/Akt pathway to increase eNOS activity;3. ALDH2activity increased significantly after15min of ethanol incubation, and got the peak at30min (1.65-fold,p<0.001), and then decreased rapidly. However, ALDH2mRNA and protein expression were not affected; 4. ALDH2siRNA could inhibit the protective effects of ethanol on activation of Akt and eNOS;5. ROS level had no change after incubation with ethanol. However, ROS concentration was markedly increased by approximately60%in cells expressing ALDH2siRNA after ethanol treatment. Administration of ROS scavengers into cells expressing ALDH2siRNA inhibited ethanol-induced ROS accumulation and increased activation of Akt and eNOS;6. After ethanol treatment, ALDH2acetylation showed time-dependent increase and then decrease, with peak at30min (2.2-fold, p<0.001), consistently with the changes of ALDH2activity;7. After incubation with ethanol, SIRT3activity decreased with time, and got the minimum value at30min. Moreover, SIRT3overexpression could reverse ethanol-elevated ALDH2acetylation and activity;8. ROS level increased significantly in cells overexpressing SIRT3after ethanol incubation. Subsequently, SIRT3overexpression reversed ethanol-induced Akt/eNOS activaton;9. Mitochondrial NAD+/NADH radio decreased by65%at30min after ethanol treatment. NAD treatment reversed the effects of ethanol on SIRT3, ALDH2, Akt and eNOS.Conclusion1. Ethanol can upregulate eNOS activity through activating PI3K-Akt pathway;2. Ethanol can increase ALDH2activity;3. ALDH2is an endogenous anti-oxidant factor, which activation mediates the protective effects of ethanol on eNOS activation through preventing ROS accumulation;4. Ethanol-induced ALDH2activation is positively relevant with acetylation of ALDH2dependent on reduced SIRT3activity;5. Ethanol can inhibit SIRT3activity through reducing NAD+/NADH ratio. BackgroundEndothelial senescence-induced endothelial dysfunction plays an important role in the initiation and the progression of atherosclerosis (AS). Endothelial senescence, as a key characteristic of vascular aging, is companied by reduced telomere length and telomerase activity, which is also called replicative senescence contributing to the increased risk for AS mortality in old-aged population. Moderate alcohol consumption has been found prevent the AS, delay aging and extend lifespan in old persons. Thus detecting the molecular mechanisms involved the inhibitory effects of ethanol on aging may help explore the novel targets for prevention and therapies of AS.Some researchers found that low-dose ethanol can increase eNOS activity and NO release in silent endothelial cells. We presumed that this protective effect of ethanol may also be detected in the senescent endothelial cells. Moreover, a previous study suggests that moderate ethanol ingestion can resist aging-related cognitive dysfunction. Thus, ethanol may prevent aging-related AS through improving endothelial function.SIRT1, a NAD+-dependent class III histone deacetylase, plays an important role in cell survival and replicative senescence. It plays an important role in a wide variety of processes, including apoptosis, senescence, differentiation and aging by interactions with a variety of chromatin-associated proteins and non-histone targes. SIRT1activity can be affected by gene regulation, NAD+/NADH ratio, oxidative stress, post-translational modifications and nucleocytoplasmic shuttling. Our previous studies have found that ethanol can rapidly upregulate NAD+/NADH ratio to affect SIRT3activity. In view of the high degree of homology between SIRT1and SIRT3, SIRT3biological function may also be affected by ethanol.Our previous findings suggest that the protective effects of ethanol on eNOS activity can be mediated by aldehyde dehydrogenase2(ALDH2), which functions as the molecular swich. However, whether ethanol can regulate ALDH2to affect endothelial senescence has no related reports until now. Mitochondrial ALDH2, known as an important enzyme in ethanol metabolism, exists abundantly in cardiovascular system. Epidemiological studies report that ALDH2with high enzymatic activity may prevent the development of AS. Interestingly, ALDH2has been found to regulate aging processes in vitro and in vivo. Furthermore, ALDH2possesses the property to inhibit oxidative stress, which is known as an important contributor for aging especially through affecting SIRT1function. Thus, ALDH2may have an association with SIRT1function to regulate senescence-related diseases, which means ALDH2may be the new target for preventing AS.In present study, we hypothesized that ethanol has the protective effects against endothelial senescence in HAECs, in which progression SIRT1and ALDH2may both be involved. Low-dose ethanol may upregulate ALDH2activity to induce the inhibitory effects of SIRT1on endothelial senescence.Methods1. Cell culture:Human aortic endothelial cells (HAECs) were maintained in endothelial cell medium (ECM; Sciencell, USA) in a water-saturated atmosphere of5%CO2and95%air at37℃.2. Endothelial replicative senescence model:The replicative senescence model of HAECs was constructed by consecutive passage assay. Before each passage, the total cell number were obtained to calculate the population doubling level (PDL). We found that At PDL22, cells exhibited senescence-associated phenotype.3. Cell treatment:When senescent cells were more that80-90%confluence, different doses of ethanol (OmM,5mM,20mM,50mM,100mM)was added into cells for the designated times.4. Cell viability assay:HAECs were seeded in the96-well plates, and the cell viability was detected using the CCK-8assays according to the manufacturer's instructions. The absorption at450nm was measured spectroscopically.5. Senescence-associated β-galactosidase (SA-β-gal) assay:Cells were stained with β-galactosidase, and the blue-stained cells and the total number of cells were counted under light microscopy.6. BrdU incorporation assay:BrdU incorporates into cellular DNA during cell proliferation, and is detected by immunofluorescent staining. Then the fluorescent identity was determined by fluoro-spectrophotometer analysis at450nm.7. Cell transfection:The specific siRNAs were used to inhibit ALDH2and SIRT1expression, whereas ALDH2and SIRT1plasmids were used to elevate their expression. Cells were transfected with the siRNA duplexes or plasmid by the Lipofectamine2000according to the manufacturer's instructions. Cells transfected with siRNAs or plasmids were then incubated with ethanol.8. Real-time PCR:Total RNA were extracted from treated cells by Trizol, and then reverse-transcripted into cDNAs. Real time PCR was performed using SYBR Green detection system. The relative mRNA levels were calculated by2-ΔΔct.9. Western blotting analysis:Total cellular protein was extracted by lysis buffer. The protein expression of ALDH2,eNOS,SIRT1in HAECs were detected using Western blotting analysis.10. eNOS activity assay:eNOS activity was measured by the conversion of L-arginine to NO by use of a nitricoxide synthase assay kit. Then the fluorescent identity was determined by fluoro-spectrophotometer analysis at excitation495nm and emission515nm.11. ALDH2activity assay:The mitochondria ALDH2enzymatic activity was determined by monitoring the reductive reaction of NAD+to NADH at A340nm in a spectrophotometer. Enzyme activity was expressed as nmol/mg/min. 12. Immunofluorescence Microscopy:Cells were grown on sterilized glass coverslips and processed for immunofluorescence microscopy by staining eNOS, ALDH2and Sirt1.Results1. After purifying HAECs by consecutive passage, we identified PDL8cells as young cells and PDL22cells as senescent cells according to the cellular changes in morphology, SA-β-gal activity and BrdU incorporation.2. Senescent cells (PDL22) exhibited decreased eNOS protein and mRNA expression, accompanied by reduced activity, indicating that eNOS inactivation can be observed in senescent cells.3.20mM ethanol may delay endothelial cell replicative senescence. However, other doses of ethanol (5,50,100mM) had no beneficial effects on senescent cells.4. Ethanol treatment dose-dependently increased then decreased eNOS protein and mRNA expression, with peak value at20mM. As well, eNOS activity was also increased by20mM ethanol.5. Compared to young cells, in senescent cells, the nuclear expression of SIRT1decreased whereas the cytoplasmic expression increased at the same time. However, the total protein expression of SIRT1had no change in senescent cells, indicating SIRT1translocation from nucleus into cytoplasm in senescent cells.6. In order to determine the association of this alteration in SIRT1subcellular localization with the cellular senescence, SIRT1-EGFP (nuclear SIRT1) and mtNLS-EGFP (cytoplasmic SIRT1) were used in this study. The results suggest that the nuclear but not cytoplasmic SIRT1has impact against replicative senescence.7.20mM ethanol decreased the cytoplasmic SIRT1protein expression and increased the nuclear SIRT1protein expression, indicating20mM ethanol can inhibit SIRT1nuclear export in senescent cells.8. In senescent HAECs, ALDH2activity decreased. Further study found that ALDH2protein and mRNA expression were also decreased in senescent cells.9. ALDH2overexpression could delay endothelial senescence. 10.20mM ethanol could increase ALDH2protein and mRNA expression, accompanied by the increased ALDH2activity in senescent cells. After transfection with ALDH2siRNA to inhibit its expression, the protective effects of ethanol against senescence were attenuated significantly.11. In the presence of ALDH2siRNA, the inhibitory effects of ethanol on SIRT1nuclear export were attenuated.Conclusion1. The replicative senescent model of HAECs can be constructed by consecutive passage assay;2. Ethanol can delay endothelial senescence and improve endothelial function in HAECs;3. SIRT1translocates from nucleus into cytoplasm in senescent HAECs, which can be blocked by ethanol to delay endothelial senescence;4. The expression and activity of ALDH2decrease in senescent HAECs, which can be inhibited by ethanol to delay endothelial senescence;5. Ethanol can upregulate ALDH2expression and activity to inhibit SIRT1nuclear export, which may be the important molecular mechanisms in the protective effects of ethanol against endothelial senescence.