The Effect And Mechanism Of Aldehyde Dehydrogenase 2 In High Fat Diet Induced Endothelial Dysfunction

BackgroundAt present, with the increasing morbidity, cardiovascular diseases such as CAD, hypertension, and cardiovascular complications of diabetes have been the leading cause of death in the world. How to improve the levels of prevention, diagnosis and treatment of cardiovascular diseases has become a major medical and social problem which is urgent to be solved. The basic pathology of these diseases are vascular lesions which are vascular dysfunction and structural abnormalities.However, right now we are not very clear about the regulation of vascular endothelial function and structure, the molecular mechanism of pathological changes and intervention targets.The main vascular endothelial dysfunction is reducing endothelial dependent vasodilation function, inflammation and increasing vascular permeability, etc. Insulin resistance is a pathological physiological phenomenon that the effects of the organs or tissues to insulin is not sensitive. It is closely related to the occurrence and development of many metabolic and cardiovascular diseases such as coronary heart disease, diabetes, and obesity. In the early stage of these diseases, there are different kind of vascular endothelial dysfunction and insulin resistance. Usually insulin resistance and vascular endothelial dysfunction exist together and could influence each other. Usually, the insulin induced vasodilatation function is damaged in the patients having Insulin resistance. Research indicates that, the pathogenic factors such as hyperglycemia, hyperlipemia, inflammation factors and oxidative stress could damage the insulin signaling pathways and thus induce insulin resistance. The NO production in the endothelium induced by insulin through the IRS-PI3K-Akt signal pathway is decreased, leading to vascular endothelial dysfunction.The research about the inflammasome is mainly the NLRP3 inflammasome. The stimulation of NLRP3 belongs to the NOD like receptor family protein by the pathogenic factors such as hyperglycemia, hyperlipemia, inflammation factors and oxidative stress and other various types of molecules, bacteria and viruses triggers the assembly of NLRP3, ASC and caspase-1 components which subsequently oligomerize into penta or heptameric structures whis is called "inflammasome". This active inflammasome could activate caspase-1 and cleaves the precursor forms of IL-1β into mature cytokines, thus induce the inflammation reaction. Now, we are not familiar with the mechanisms of activation and regulation of NLRP3 inflammasome. The studies indicate that ROS could induce TRNIP separating with TRX and binding to NLRP3 which subsequently activate NLRP3 inflammasome. Recently, Zhou etc. Found that mitochondrial ROS is an essential mediator of NLRP3 inflammasome activation. NLRP3 inflammasome is related to many diseases. Recent research demonstrates that it plays a very important role in the development of Atherosclerosis and Diabetes.Aldehyde dehydrogenase 2 (ALDH2) which is located in the mitochondria is the main enzyme in the metabolism of ethanol. Besides its role in catalyzing the oxidation of acetaldehyde to acetic acid in ethanol metabolism, It is considered responsible for oxidation and detoxification of other reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE). So it have some anti-oxidative effects. Studies indicate that ALDH2 is closely related to cardiovascular diseases, and could ameliorate the heart function, cardiomyocyte apoptosis, mitochondrial dysfunction induced by myocardial infarction, diabetes and ethanol. We have found that ALDH2 activity was decreased in diabetic rats, the mechanism may related with oxidative stress. Several studies, including a genome-wide association study in 2012, have confirmed that ALDH2 is a susceptibility gene for coronary atherosclerotic disease.So, based on the previous studies, we make the hypothesis:1.The activation of NLRP3 inflammasome plays an important role in endothelial dysfunction induced by high fat diet.2. ALDH2 could ameliorate insulin signaling pathways which subsequently ameliorate endothelial dysfunction induced by high fat diet through the regulation of NLRP3 inflammasome.ObjectivesThe objectives of this experiment is to investigate:I.The role and mechanism of NLRP3 inflammasome in endothelial dysfunction induced by high fat diet.2. The role and mechanism of ALDH2 in endothelial dysfunction induced by high fat diet.3. The regulation and mechanism of ALDH2 to NLRP3 inflammasome.Methods1.Cell culture:Human umbilical vein endothelial cells (HUVECs) purchased from American ATCC were cultured in endothelial cell medium (ECM) containing 5% FBS. Using Palmitic acid (PA) and LPS to stimulate HUVECs to induce endothelial dysfunction.2. Animal model:Male C57BL/6J mice (n=40),7 weeks old(20g) were divided into 4 groups:Control Diet+GFP Virus, High Fat Diet+GFP Virus, Control Diet+ALDH2 Virus and High Fat Diet+ALDH2 Virus. The High Fat Diet (HFD) contains 60% energy.10 weeks later the mice were injected with GFP or ALDH2 overexpression LentiVirus from the tail vein. At the end of 12 weeks, the mice were killed.3. LentiVirus transfection:HUVECs were stimulated with PA and LPS after transfected with GFP or ALDH2 overexpression LentiVirus for 48h.4. Small Interfering RNA (siRNA) transfection:HUVECs were stimulated with PA and LPS after transfected with control, NLRP3, ASC or caspase-1 siRNA for 48h.5. RT-PCR:Total RNA was extracted from grouped HUVECs. The mRNA levels of ALDH2 was analyzed through RT-PCR.6. Measurement of ALDH2 Activity:HUVECs mitochondria were extracted. Using the mitochondria to detect ALDH2 activity.7. Western blot (WB):Total proteins with the same concentration were extracted from HUVECs. The protein expression of p-eNOS(Ser1177)、 t-eNOS、p-Akt(Ser473、t-Akt、p-AMPKa(Thr172)、t-AMPKα、NLRP3、ASC、 Pro-Caspase-1、cleaved Caspase-1 (p10)、Pro-IL-1、IL-1β、ALDH2 and β-actin were detected.8. Statistical Analysis:Data were expressed as mean ±SD. SPSS was used for statistical analysis. p< 0.05 was considered statistically significant.Results1. PA significantly inhibit IRS-1-Akt-eNOS signaling pathway.Compared with the control and LPS group, different concentrations of PA alone could inhibit p-Akt(Ser473) and p-eNOS(Ser1177) without changing Akt or eNOS expression. But giving LPS pre-stimulation, PA could significantly inhibit the phosphorylation of Akt and eNOS.2. NLRP3 inflammasome mediated the impairment of the insulin signaling pathway induced by PA.Compared with the control and LPS group, different concentrations of PA alone could not change the expression of NLRP3 inflammasome, cleaved Caspase-1(p10) or IL-1β. But giving LPS pre-stimulation, PA could significantly increase the expression of cleaved Caspase-1(p10) and 1L-1β without changing the expression of NLRP3 inflammasome. Different concentrations of IL-1β could inhibit p-Akt(Ser473) and p-eNOS(Ser1177) without changing Akt or eNOS expression. Inhibiting NLRP3 inflammasome expression using siRNA transfection decrease IL-1(3 expression and increase the expression of p-Akt(Ser473) and p-eNOS(Ser1177).3. AMPKa negatively regulate PA induced activation of NLRP3 inflammasome.Compared with the control and LPS group, different concentrations of PA alone could inhibit p-AMPKa(Thr172) without changing AMPKa expression. But giving LPS pre-stimulation, PA could significantly inhibit the phosphorylation of AMPKa. AMPK activator AICAR could inhibit cleaved Caspase-1(p10) and IL-1(3 expression and increase the expression of p-Akt(Ser473) and p-eNOS(Ser1177).4. Increasing ALDH2 protein expression or activity ameliorate the activity of the insulin signaling pathway.Using ALDH2 overexpression LentiVirus to increase ALDH2 expression, the expression of p-Akt(Ser473) and p-eNOS(Ser1177) were increased.5. Increasing ALDH2 protein expression or activity could activate AMPKa and subsequently inhibit the activation of NLRP3 inflammasome.The WB showed that ALDH2 overexpression LentiVirus could increase the phosphorylation of AMPKa and inhibit the expression of cleaved Caspase-1(p10) and IL-1β.6. ALDH2 ameliorate aortic endothelial function and the activity of the insulin signaling pathway in vivo.Compared with the Control Diet group, ALDH2 overexpression alone did not change endothelial function or the phosphorylation of Akt and eNOS. HFD could significantly induce endothelial dysfunction and inhibit p-Akt(Ser473) and p-eNOS(Ser1177) expression. Compared with the HFD group, ALDH2 overexpression could ameliorate HFD induced endothelial dysfunction and phosphorylation of Akt and eNOS.7. ALDH2 activate AMPKa and inhibit the activation of NLRP3 inflammasome in mice aortic endothelium.Western Blots of the mice aortic endothelium showed that the expression of NLRP3 inflammasome did not change in each group. Compared with the Control Diet group, ALDH2 overexpression alone did not change the expression of p-AMPKa(Thr172), cleaved Caspase-1(p10) and IL-1(3. HFD could significantly inhibit AMPKa phosphorylation and increase cleaved Caspase-1(p10) and IL-1β expression. Compared with the HFD group, ALDH2 overexpression could increase AMPKa phosphorylation and inhibit cleaved Caspase-1(p10) and IL-1β expression.Conclusions1.HFD could impair the insulin signaling pathway through the activation of NLRP3 inflammasome and subsequently induce endothelial dysfunction.2.AMPKa mediated the activation of NLRP3 inflammasome induced by HFD.3.ALDH2 could increase the activity of the insulin signaling pathway and ameliorate FHD induced endothelial dysfunction, may through activating AMPKa and inhibiting the activation of NLRP3 inflammasomeBackgroundSirtuin-1 (SIRT1), an NAD+-dependent type III histone deacetylase, mainly located in the nucleus. It is widely distributed in tissues. Except histone, it has some other substrates such as p53, FOXO, eNOS, iNOS, NF-κB, PARP-1. It has been implicated in the regulation of DNA repairing, oxidative stress, inflammation, cellular aging, metabolism and other physiopatholocial processes. It plays an important role in several cardiovascular diseases such as atherosclerosis and diabetes.Healthy vascular endothelial function depends on the activity of endothelial nitric oxide synthase (eNOS) and production of NO. NO has a vasodilator activity, inhibits the accumulation of atherosclerotic plaques and promotes angiogenesis. eNOS activity is induced by SIRT1 deacetylation. SIRT1 also protects the vascular endothelial function from increased inflammation and apoptosis.Understanding the mechanism is important, as it will provide insights into the modulation of this molecule as a potential treatment for these diseases. However, the regulation of SIRT1 protein turnover is poorly defined. Emerging evidence suggests that eNOS positively regulates SIRT1 protein expression. Also, the p38 kinase-mediated proteasomal degradation of SIRT1 contributed to the cellular senescence. Recent studies identified eNOS derived NO as an endogenous inhibitor of 26S proteasome functionality.So, based on the previous studies, we make the hypothesis:NO could increase SIRT1 protein expression by inhibiting 26S proteasome functionality.ObjectivesThe objectives of this experiment is to investigate:The mechanisms of SIRT1 protein expression induced by NO.Methods1. Cell culture:Human umbilical vein endothelial cells (HUVECs), human embryonic kidney cell line 293T (HEK 293T) and GFPu-1 cells were obtained from ATCC (Manassas, VA). U20S cells expressing the GFP-LC3B reporter were procured from EMD Millipore (Billerica, MA). The immortalized mouse embryonic fibroblasts (MEF) were made from Ulk1-/-, Ulk2-/-,Ulk1-/-2-/- and wild-type mice. HUVECs were cultured in EBM containing 5% fetal bovine serum (FBS). Mouse embryonic fibroblasts (MEF), HEK 293T, U20S and GFPu-1 cells were grown in DMEM with 10% FBS.2. Animal experiments:Male eNOS-/- mice (10 weeks), db/db mice (25 weeks) and the age-matched C57BL/6J WT mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME) at younger ages.3. AdenoVirus, Plasmid and siRNA transfection:AdenoVirus transfection:HUVECs and MEF cells were transfected with adenovirus encoding eNOS and GFP (as adenoviral infection control) for 48h.Plasmid transfection:HEK 293T cells were transfected with plasmid encoding UbG76V-GFP for 48h.SiRNA transfection:HUVECs were transfected with control or target siRNA (ULK1, β-TrCP1) based on the protocols provided by Santa Cruz Biotechnology (Santa Cruz, CA) for 48h.4. RT-PCR:Total cellular RNA was isolated from HUVECs and HEK 293T cells using the Total RNA Kit. The mRNA levels of SIRT1 and 18s was analyzed through RT-PCR.5. Determination of 26S proteasome activity:26S proteasome activity was determined by the chymotrypsin-like activity which was measured using a fluorogenic proteasome substrate SucLLVY-7-amido-4-methylcoumarin (AMC). ATP-dependent cleavage activity was monitored continuously by detection of free AMC with a Fluorescence Microplate Reader at 380/460 nm at 37℃6. Detection of O-GlcNAc-modified proteins:Cell lysates were prepared in lysis buffer. Agarose bound Wheat Germ Agglutinin Kit (WGA) was used to pull down proteins modified by O-linked GlcNAc.7. Western blot (WB):Total proteins with the same concentration were extracted from the cells. The protein expression of ULK1, OGT, eNOS, SIRT1, O-GlcNAc, LC3B, Beclin-1, p62, β-TrCP1, GFP, Rpt2,β7 and β-actin were detected.8. Statistical Analysis:Data were expressed as mean ± SD. SPSS was used for statistical analysis. p< 0.05 was considered statistically significant.Results1.NO stabilizes and increases SIRT1 protein expression in vascular endothelial cells.Increased eNOS protein expression increase SIRT1 protein. The upregulation was recapitulated in a time-dependent fashion, when HUVECs were pretreated with A23187, an eNOS activator or DETA-NONOate (Diethylenetriamine NONOate), an NO donor. However, NONOate did not significantly increase SIRT1 mRNA. In chase experiments, cycloheximide (CHX) alone reduced SIRT1 protein expression in a time-dependent fashion. In the presence of NONOate, SIRT1 protein stability was significantly increased. In addition, MG132, a potent 26S proteasome inhibitor, upregulated SIRT1 expression.2. NO stabilizes and increases ULK1 protein expression in vascular endothelial cells.Overexpression of eNOS in HUVECs upregulated ULK1 protein level. This effect was reproduced by administration of A23187 or NONOate in a time-dependent manner. In chase experiments, both NONOate and A23187 markedly prolonged SIRT1 protein stability in the presence of CHX.3. ULK1 mediated the regulation of NO induced SIRT1 protein expression.Overexpression of eNOS upregulated SIRT1 protein expression in WT and Ulk2-/- MEF, but this did not occur in Ulk1-/- and Ulk1-/-2-/- MEF. Administration of NONOate produced similar results. A23187 and NONOate increased the SIRT1 protein levels in control siRNA-treated cells, but failed to do so in the ULK1-siRNA treated cells. Transient expression of ULK1 plasmid in HEK 293T cells increased SIRT1 protein levels without altering SIRT1 mRNA levels.4. Modulation of autophagy does not mimic the regulation of SIRT1 by NO or ULK1Rapamycin induced autophagy marker LC3B and autophagic flux, but did not increase SIRT1 protein expression. The effects of NONOate on autophagic flux were minimal. The impact of NONOate on common autophagy markers such as p62, Beclin-1 and LC3B was not significant, similar to the effects of eNOS and A23187. Baf A1, an autophagy inhibitor, did not restore SIRT1 protein expression in MEF cells.5. ULK1 regulates 26S proteasome functionality.Upregulation of ULK1 in GFPu-1 cells increased both GFP protein levels and GFP fluorescence, indicating 26S proteasome suppression. These changes were accompanied by reduced 26S proteasome activity. We treated the MEF cells with plasmids expressing UbG76V -GFP, a 26S proteasome reporter, the UIk1-/- MEF presented significantly less GFP protein compared with WT MEF. Ulk1-/- MEF and ULK1-siRNA treated HUVECs was associated with increased proteasome activity.6. ULK1 regulates 26S proteasome functionality via OGT.Overexpression of ULK1 significantly upregulated OGT protein expression accompanied by elevated levels of O-GlcNAc-modified protein. We used WGA to enrich O-GlcNAc modified proteins and detected the increased O-GlcNAcylation of Rpt2 by ULK1 Overexpression. In contrast, downregulation of ULK1 by siRNA treatment decreased OGT protein expression and O-GlcNAc-modified protein levels. NONOate increased the levels of OGT and O-GlcNAc modified proteins in HUVECs. However, these effects were compromised in ULK1-siRNA treated cells.7. The NO-ULK1-SIRT1 axis seems operative in eNOS-knockout mice and db/db mice.In the lungs of eNOS-KO mice, the absence of eNOS was associated with the downregulation of ULK1 and Sirti proteins. In type 2 diabetic db/db mice, eNOS protein expression was reduced in the lungs and hearts. This reduction was correlated with a significant reduction of ULK1 and SIRT1 protein expression.ConclusionsNO could inhibit 26S proteasome activity through increasing ULK1 and OGT expression independent of autophagy which subsequently enhance the expression of SIRT1.