Aldehyde Dehydrogenase 2 mRNA Research Articles

LncRNA CHKB-DT Downregulation Enhances Dilated Cardiomyopathy Through ALDH2.

Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined. Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting. CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT+/- mice alleviated cardiac dilation and dysfunction. CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.

Effect of Bone Marrow Mesenchymal Stem Cells (BMSCs) on Cardiac Function and Ventricular Remodeling in Diabetic Rat Models Through Inhibiting MicroRNA (miR)-34a

Diabetes mellitus (DM) is harmful to heart tissue. We transplanted bone marrow mesenchymal stem cells (BMSCs) in this study to improve the function of diabetic heart. 45 rats were divided into healthy group, DM group (treated with high-fat diet), and BMSCs group (BMSCs transplantation into the ventricular wall) followed by analysis of miR-34a and aldehyde dehydrogenase 2 (ALDH2) levels, heart function, and ventricular remodeling in the three groups of rats. BMSCs were successfully cultured with high purity, and the myocardium in the BMSCs group was successfully labeled by DAPI. The left ventricular hypertrophy index and myocardial tissue CVF of rats with DM increased significantly, suggesting that ventricular remodeling in DM rats was stimulated. However, when BMSCs were transplanted to the ventricular wall, although the ventricular remodeling was not completely avoided, apoptosis of BMSCs group was significantly reduced. MiR-34a relative level in BMSCs group was obviously elevated, whereas ALDH2 level showed opposite trend with a negative relationship. MiR-34a can effectively bind to the 3′-UTR of ALDH2 mRNA, indicating that it can directly regulate gene expression. BMSCs can promote the synthesis of ALDH2 by inhibiting the expression of miR-34a, and improve the cardiac function and ventricular remodeling of diabetic rats.

The biological activity of bispecific trastuzumab/pertuzumab plant biosimilars may be drastically boosted by disulfiram increasing formaldehyde accumulation in cancer cells

Studies of breast cancer therapy have examined the improvement of bispecific trastuzumab/pertuzumab antibodies interacting simultaneously with two different epitopes of the human epidermal growth factor receptor 2 (HER2). Here, we describe the creation and production of plant-made bispecific antibodies based on trastuzumab and pertuzumab plant biosimilars (bi-TPB-PPB). Using surface plasmon resonance analysis of bi-TPB-PPB antibodies binding with the HER2 extracellular domain, we showed that the obtained Kd values were within the limits accepted for modified trastuzumab and pertuzumab. Despite the ability of bi-TPB-PPB antibodies to bind to Fcγ receptor IIIa and HER2 oncoprotein on the cell surface, a proliferation inhibition assay did not reveal any effect until α1,3-fucose and β1,2-xylose in the Asn297-linked glycan were removed. Another approach to activating bi-TPB-PPB may be associated with the use of disulfiram (DSF) a known aldehyde dehydrogenase 2 (ALDH2) inhibitor. We found that disulfiram is capable of killing breast cancer cells with simultaneous formaldehyde accumulation. Furthermore, we investigated the capacity of DSF to act as an adjuvant for bi-TPB-PPB antibodies. Although the content of ALDH2 mRNA was decreased after BT-474 cell treatment with antibodies, we only observed cell proliferation inhibiting activity of bi-TPB-PPB in the presence of disulfiram. We concluded that disulfiram can serve as a booster and adjuvant for anticancer immunotherapy.

Alterations in necroptosis during ALDH2‑mediated protection against high glucose‑induced H9c2 cardiac cell injury.

The aim of the present study was to investigate whether necroptosis occurs in high glucose (HG)-induced H9c2 cardiac cell injury and whether the activation of aldehyde dehydrogenase 2 (ALDH2) can inhibit necroptosis. H9c2 cardiac cells were treated with 35 mM glucose to establish a HG-induced cell injury model. Alda-1 (20 µM), a specific activator of ALDH2 and necrostatin-1 (Nec-1, 100 µM), an inhibitor of necroptosis were used to treat H9c2 cardiac cells under HG conditions. Cell viability was measured using a Cell Counting Kit-8 assay and reactive oxygen species (ROS) generation was measured by the dihydroethidium staining method. ALDH2 activity was measured at 450 nm. The mRNA and protein expression of ALDH2, necroptosis-associated genes, receptor-interacting protein (RIP)1, RIP3 and mixed lineage kinase domain like pseudokinase (MLKL), were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. The expression of cleaved caspase-3 protein was also examined by western blotting. The results demonstrated that under HG conditions, cell viability, ALDH2 activity, mRNA and protein expression were decreased. Furthermore, ROS generation, mRNA and protein expression of RIP1, RIP3, MLKL and the protein expression of cleaved caspase-3 were increased. Treatment with Alda-1 or Nec-1 attenuated HG-induced downregulation of ALDH2 activity, mRNA and protein expression. In addition, RIP1, RIP3, MLKL mRNA, and protein expression were downregulated. Furthermore, Alda-1 but not Nec-1 decreased cleaved caspase-3 protein expression. Collectively these data indicated that activation of ALDH2 protected H9c2 cardiac cells against HG-induced injury, partly by inhibiting the occurrence of necroptosis.

Baicalin protects H9c2 cardiomyocytes against hypoxia/reoxygenation-induced apoptosis and oxidative stress through activation of mitochondrial aldehyde dehydrogenase 2.

Baicalin, a flavonoid glycoside separated from Scutellaria baicalensis, has cardioprotection against ischaemia/reperfusion (I/R) injury. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is considered as an endogenous protective mechanism against I/R injury depending on its anti-oxidant and anti-apoptotic characteristics. The present study demonstrates whether ALDH2 contributes to the cardioprotection of baicalin against hypoxia/reoxygenation (H/R)-inudced H9c2 cardiomyocytes injury. Our results observed that H/R treatment resulted in a significant decrease in cells viability and obvious increases in caspase-3 activity and apoptosis rate in H9c2 cells, while these alterations were evidently reversed by baicalin pretreatment. Simultaneously, baicalin mitigated H/R-induced the decreases in the levels of ALDH2 mRNA and protein as well as the activity of ALDH2 in H9c2 cells. However, we found that daidzin, an ALDH2 antagonist, remarkably attenuated baicalin-elicited inhibitory action on H/R-induced the downregulation of cells viability and Bcl-2 protein expression, and the upregulations of caspase-3 activity, apoptosis rate, cytochrome c and Bax proteins expressions in H9c2 cells. In addition, baicalin reversed H/R-induced oxidative stress as evidenced by the downregulation of malondialdehyde (MAD) and 4-hydroxy aldehydes (4-HNE) levels, the inhibition of endogenous reactive oxygen species (ROS) generation, and the downregulation of superoxide dismutase (SOD) activity induced by H/R treatment, while these effects were also blocked by daidzin. Furthermore, we found that Alda-1, an ALDH2 agonist, also abolished H/R-induced cytotoxicity, apoptosis, and oxidative stress, indicating that ALDH2 mediated H/R-induced H9c2 cell injury. Overall, these results suggested that baicalin prevents H/R-induced apoptosis and oxidative stress through enhancing ALDH activity and expression in H9c2 cardiomyocytes.

The Antioxidant Cofactor Alpha-Lipoic Acid May Control Endogenous Formaldehyde Metabolism in Mammals.

The healthy human body contains small amounts of metabolic formaldehyde (FA) that mainly results from methanol oxidation by pectin methylesterase, which is active in a vegetable diet and in the gastrointestinal microbiome. With age, the ability to maintain a low level of FA decreases, which increases the risk of Alzheimer's disease and dementia. It has been shown that 1,2-dithiolane-3-pentanoic acid or alpha lipoic acid (ALA), a naturally occurring dithiol and antioxidant cofactor of mitochondrial α-ketoacid dehydrogenases, increases glutathione (GSH) content and FA metabolism by mitochondrial aldehyde dehydrogenase 2 (ALDH2) thus manifests a therapeutic potential beyond its antioxidant property. We suggested that ALA can contribute to a decrease in the FA content of mammals by acting on ALDH2 expression. To test this assumption, we administered ALA in mice in order to examine the effect on FA metabolism and collected blood samples for the measurement of FA. Our data revealed that ALA efficiently eliminated FA in mice. Without affecting the specific activity of FA-metabolizing enzymes (ADH1, ALDH2, and ADH5), ALA increased the GSH content in the brain and up-regulated the expression of the FA-metabolizing ALDH2 gene in the brain, particularly in the hippocampus, but did not impact its expression in the liver in vivo or in rat liver isolated from the rest of the body. After ALA administration in mice and in accordance with the increased content of brain ALDH2 mRNA, we detected increased ALDH2 activity in brain homogenates. We hypothesized that the beneficial effects of ALA on patients with Alzheimer's disease may be associated with accelerated ALDH2-mediated FA detoxification and clearance.

Increased expression of microRNA-378a-5p in acute ethanol exposure of rat cardiomyocytes.

Alcohol abuse is a risk factor for a distinct form of congestive heart failure, known as alcoholic cardiomyopathy (ACM). Here, we investigate how microRNAs may participate in the induction of cardiomyocyte apoptosis associated with ethanol exposure in vitro. Increasing the concentrations of ethanol to primary rat cardiomyocytes resulted in elevated apoptosis assessed by annexin V and propidium iodide staining, and reduced expression of an enzyme for alcohol detoxification aldehyde dehydrogenase 2 (ALDH2). These ethanol effects were accompanied by a substantial elevation of miR-378a-5p. Driving miR-378a-5p overexpression in cardiomyocytes decreased ALDH2. The specific interaction of miR-378a-5p with the 3'UTR of ALDH2 was examined by luciferase reporter assays, and we found that miR-378a-5p activity depends on a complementary base pairing at the 3'-UTR region of ALDH2 mRNA. Finally, ethanol-induced apoptosis in cardiomyocytes was attenuated in the presence of anti-miR378a-5p. Collectively, these data implicate a likely involvement of miR-378a-5p in the stimulation of cardiomyocyte apoptosis through ALDH2 gene suppression, which might play a potential role in the pathogenesis of ACM.

Abstract 12949: Elamipretide ( Bendavia TM ) Restores 4-Hydroxy-2-Nonenal Protein Adducts and Aldehyde Dehydrogenase-2 Activity and mRNA Expression in Left Ventricular Myocardium of Dogs With Advanced Heart Failure

Background: 4-hydroxy-2-nonenal (4HNE), a reactive aldehyde, is generated in the failing heart and contributes to cardiomyocyte injury and death. Aldehyde dehydrogenase-2 (ALDH2) plays a key role in detoxifying mitochondrial (MITO) reactive aldehydes. We showed that chronic therapy with elamipretide (ELA), a novel MITO targeting peptide, improves LV systolic function, normalizes MITO function and reduces the burden of reactive oxygen species (ROS) in LV of dogs with microembolization-induced heart failure (HF). Hypothesis: This study tested the hypothesis that chronic therapy with ELA normalizes protein levels of 4-HNE-adducts and ALDH2 activity and expression in LV myocardium of HF dogs (LV ejection fraction ~30%). Methods: LV tissue from 14 HF dogs randomized to 3 months therapy with subcutaneous injections of ELA (0.5 mg/kg once daily, n=7) or subcutaneous injections of saline (control, CTR, n=7) and tissue from 6 normal (NL) dogs was used in the study. Level of 4-HNE-protein adducts was quantified using an Elisa kit. ALDH2 activity was assayed spectrophotometrically in LV MITO fractions by monitoring NADH formation from NAD to NADH at 340 nm. Protein levels of ALDH2 and porin (an internal loading control) were determined by Western blotting and band intensity quantified in densitometric units (du). mRNA expression of ALDH2 and GAPDH was measured in using real-time PCR. Results: Data are shown in the table. Levels of 4HNE-protein adducts were significantly increased and ALDH2 activity, mRNA expression and protein levels significantly decreased in CTR compared to NL dogs. ELA normalized 4-HNE levels, ALDH2 activity and expression. There were no differences in the level of porin or GAPDH among the 3 study groups. Conclusions: Chronic therapy with ELA normalizes ALDH2 and 4-HNE levels in LV myocardium of HF dogs. By limiting myocardial ROS burden, ELA contributes to cardiac end-organ protection and hence, to overall improvement in global LV function.

Activation of ALDH2 with Low Concentration of Ethanol Attenuates Myocardial Ischemia/Reperfusion Injury in Diabetes Rat Model.

The aim of this paper is to observe the change of mitochondrial aldehyde dehydrogenase 2 (ALDH2) when diabetes mellitus (DM) rat heart was subjected to ischemia/reperfusion (I/R) intervention and analyze its underlying mechanisms. DM rat hearts were subjected to 30 min regional ischemia and 120 min reperfusion in vitro and pretreated with ALDH2 activator ethanol (EtOH); cardiomyocyte in high glucose (HG) condition was pretreated with ALDH2 activator Alda-1. In control I/R group, myocardial tissue structure collapse appeared. Compared with control I/R group, left ventricular parameters, SOD activity, the level of Bcl-2/Bax mRNA, ALDH2 mRNA, and protein expressions were decreased and LDH and MDA contents were increased, meanwhile the aggravation of myocardial structure injury in DM I/R group. When DM I/R rats were pretreated with EtOH, left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 expression were increased; LDH, MDA, and myocardial structure injury were attenuated. Compared with DM + EtOH I/R group, cyanamide (ALDH2 nonspecific blocker), atractyloside (mitoPTP opener), and wortmannin (PI3K inhibitor) groups all decreased left ventricular parameters, SOD, Bcl-2/Bax, and ALDH2 and increased LDH, MDA, and myocardial injury. When cardiomyocyte was under HG condition, CCK-8 activity and ALDH2 protein expression were decreased. Alda-1 increased CCK-8 and ALDH2. Our findings suggested enhanced ALDH2 expression in diabetic I/R rats played the cardioprotective role, maybe through activating PI3K and inhibiting mitoPTP opening.

Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

Aldehyde dehydrogenase-independent bioactivation of nitroglycerin in porcine and bovine blood vessels

The vascular bioactivation of the antianginal drug nitroglycerin (GTN), yielding 1,2-glycerol dinitrate and nitric oxide or a related activator of soluble guanylate cyclase, is catalyzed by aldehyde dehydrogenase-2 (ALDH2) in rodent and human blood vessels. The essential role of ALDH2 has been confirmed in many studies and is considered as general principle of GTN-induced vasodilation in mammals. However, this view is challenged by an early report showing that diphenyleneiodonium, which we recently characterized as potent ALDH2 inhibitor, has no effect on GTN-induced relaxation of bovine coronary arteries (De La Lande et al., 1996). We investigated this issue and found that inhibition of ALDH2 attenuates GTN-induced coronary vasodilation in isolated perfused rat hearts but has no effect on relaxation to GTN of bovine and porcine coronary arteries. This observation is explained by low levels of ALDH2 protein expression in bovine coronary arteries and several types of porcine blood vessels. ALDH2 mRNA expression and the rates of GTN denitration were similarly low, excluding a significant contribution of ALDH2 to the bioactivation of GTN in these vessels. Attempts to identify the responsible pathway with enzyme inhibitors did not provide conclusive evidence for the involvement of ALDH3A1, cytochrome P450, or GSH-S-transferase. Thus, the present manuscript describes a hitherto unrecognized pathway of GTN bioactivation in bovine and porcine blood vessels. If present in the human vasculature, this pathway might contribute to the therapeutic effects of organic nitrates that are not metabolized by ALDH2.

Aberrant hypermethylation of aldehyde dehydrogenase 2 promoter upstream sequence in rats with experimental myocardial infarction.

Background. Aldehyde dehydrogenase 2 (ALDH2) plays a crucial role in myocardial protection against ischemia. Downregulation of ALDH2 was evidenced after myocardial infarction and the underlying mechanism is not fully understood. DNA methylation can regulate gene transcription in epigenetic level. We thus hypothesized that DNA methylation may affect ALDH2 expression in myocardial infarction (MI). Methods. MI was induced in Sprague-Dawley rats. MI border zone tissues were harvested at 1st week, 2nd week, and 3rd week after MI. Bisulfite sequencing PCR (BSP) was performed to detect the methylation levels of ALDH2 core promoter. Sequenom MassARRAY platform (MassARRAY) was used to examine the methylation levels of ALDH2 promoter upstream sequence. ALDH2 protein and mRNA expression were assayed by Western blot and real-time PCR, respectively. Results. Compared with Sham group, ALDH2 protein and mRNA expression of MI groups was significantly downregulated. Compared with Sham group, DNA methylation level of CpG sites in ALDH2 promoter upstream sequence was significantly higher in MI groups in a time-dependent manner (CpG1, CpG2, and CpG7, P < 0.01). DNA methylation did not affect ALDH2 core promoter sequence during the progress of MI. No significant difference was detected in DNA methylation level of ALDH2 promoter upstream sequence among MI groups. Conclusion. Aberrant hypermethylation of CpG sites in ALDH2 promoter upstream sequence is associated with myocardial ischemia injury and may partly result in ALDH2 downregulation after MI.

ASSA13-03-28 Effect of ALDH2 on Myocardial Ischemia/Reperfusion Injury in Diabetic Rats and Related Mechanism Research

Objective To observe the role of activation of aldehyde dehydrogenase 2 (ALDH2) on myocardial ischemia/reperfusion injury in diabetic rats, and study the relationships of ALDH2, PI3K-Akt and mitochondrial permeability transition pore in myocardial injury. Methods Diabetic rat model was simulated by intraperitoneal injection 55 mg/kg streptozotocin (STZ) and divided into diabetes and ethanol + diabetes groups. After 8 weeks, myocardial ischemia/reperfusion model was mimicked in vitro, and subjected to drug intervention, divided into: diabetic I/R + ethanol +cyanamide (DM +EtOH I/R +CYA); diabetic I/R + ethanol +atractyloside (DM+EtOH I/R+Atr), diabetic I/R + ethanol +wortmannin (DM+EtOH I/R+Wor). The ventricular hemodynamic parameters and lactate dehydrogenase (LDH) content in coronary flow were determined. SOD activity and MDA content were evaluated, the activities of caspase-3 and ALDH2 were evaluated, and the expressions of Bcl-2, Bax and ALDH2 mRNA were detected by RT-PCR; ALDH2 protein expression was evaluated by western blot. Results In contrast to I/R in control rat, left ventricular development pressure (LVDP), maximal rise/fall rate of left ventricular pressure (± dp/dtmax), left ventricular work (RPP), SOD and ALDH2 activities were decreased in diabetic rat, left ventricular end diastolic pressure (LVEDP), LDH release, caspase-3 activity and MDA content were increased, and the expression of Bcl-2/Bax mRNA, ALDH2 mRNA and protein expressions were decreased. Compared with DM I/R group, in DM+EtOH I/R group, LVDP, ± dp/dtmax, RPP, SOD and ALDH2 activities were increased, LVEDP, LDH release, caspase-3 activity and MDA content were increased, and the expression Bcl-2/Bax mRNA, ALDH2 mRNA and protein expressions were increased. Compared with DM+EtOH I/R group, in DM+EtOH+CYA I/R group, DM+EtOH+Atr I/R group and DM+EtOH+ Wor I/R group, LVDP, ±dp/dtmax, RPP, SOD and ALDH2 activities were further decreased, and LVEDP, caspase-3 activity and MDA content were increased, and the expression Bcl-2/Bax mRNA, ALDH2 mRNA and protein expressions were decreased. Conclusions Enhanced mitochondrial ALDH2 expression in diabetic rats can play cardiac protective role, and activation of ALDH2 plays the myocardial protective effect through inhibiting mitoPTP opening and activation of PI3K-Akt signalling pathway.

Tolerance to nitroglycerin through proteasomal down‐regulation of aldehyde dehydrogenase‐2 in a genetic mouse model of ascorbate deficiency

Background and PurposeL-gulonolactone oxidase-deficient (Gulo(-/-)) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation.Experimental ApproachAscorbate deficiency was induced in Gulo(-/-) mice by ascorbate deprivation for 4 weeks. Some of the animals were concomitantly treated with the proteasome inhibitor bortezomib and effects compared with ascorbate-supplemented Gulo(-/-), untreated or nitrate-tolerant wild-type mice. Aortic relaxation of the experimental groups to GTN, ACh and a NO donor was studied. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined.Key ResultsLike GTN treatment, ascorbate deprivation induced vascular tolerance to GTN that was associated with markedly decreased rates of GTN denitration. Ascorbate deficiency did not affect ALDH2 mRNA levels, but reduced ALDH2 protein expression and the total amount of ubiquitinated proteins to about 40% of wild-type controls. These effects were largely prevented by ascorbate supplementation or treating Gulo(-/-) mice with the 26S proteasome inhibitor bortezomib.Conclusions and ImplicationsOur data indicate that ascorbate deficiency results in vascular tolerance to GTN via proteasomal degradation of ALDH2. The results support the view that impaired ALDH2-catalysed metabolism of GTN contributes significantly to the development of vascular nitrate tolerance and reveal a hitherto unrecognized protective effect of ascorbate in the vasculature.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase2 (ALDH2) and glutathioneS-transferase pi1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatinA increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

Activation of mitochondrial aldehyde dehydrogenase 2 and inhibition of mitochondrial permeability transition pore involved in cardioprotection of ethanol postconditioning

To investigate whether activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) and inhibition of mitochondrial permeability transition pore (mitoPTP) were involved in the cardioprotection of ethanol postconditioning in isolated rat heart. Hearts isolated from male Sprague-Dawley rats were perfused on a langendorff apparatus and subjected to 30 min of regional ischemia (occlusion of left anterior descending artery) followed by 120 min of reperfusion. The ventricular hemodynamic parameters and lactate dehydrogenase (LDH) release during reperfusion were measured. Infarct size was measured by TTC staining method and the expression of ALDH2 at mRNA level of left anterior myocardium was detected by RT-PCR. In contrast to ischemia and reperfusion, ethanol postconditioning improved the recovery of left ventricular developed pressure, maximal rise/fall rate of left ventricular pressure during reperfusion, reduced LDH release and infarct size. The expression of ALDH2 mRNA level was increased. Administration of mitoPTP activator atractyloside attenuated the effect of ethanol postconditioning, LDH release and infarct size were increased, and the recovery of hemodynamic parameters was inhibited. The expression of ALDH2 mRNA was decreased. Ethanol postconditioning has cardioprotection effect, which may be associated with upregulating mitochondrial ALDH2 mRNA expression and inhibiting the opening of mitochondrial permeability transition pore.

A Promoter Polymorphism in the ALDH2 Gene Affects Its Basal and Acetaldehyde/Ethanol-Induced Gene Expression in Human Peripheral Blood Leukocytes and HepG2 Cells

To assess the effect of the -360G/A polymorphism in the promoter region of the human aldehyde dehydrogenase-2 (ALDH2) gene on its transcription, basal and acetaldehyde/ethanol-induced gene expression was examined by in vivo and in vitro experiments. Human peripheral blood leukocytes were collected before and after alcohol ingestion (0.4 g/kg body weight) in 21 healthy young Japanese volunteers with a deficient phenotype of ALDH2 ((487)Glu/Lys), and the levels of ALDH2 mRNA were quantified by real-time RT-PCR. The transcriptional activity of the ALDH2 promoter was investigated by a reporter assay using HepG2 cells in the presence or absence of acetaldehyde/ethanol. The basal level of ALDH2 mRNA was significantly higher in -360A heterozygous subjects than in -360G homozygous subjects. In all subjects, regardless of the genotype, ALDH2 mRNA increased following ethanol ingestion. The promoter activity of a reporter plasmid for -360G was significantly lower than that of a reporter plasmid for -360A. Exposure to acetaldehyde induced a significant increase in the transcriptional activity of the -360G reporter, but not the -360A reporter. In vivo and in vitro experiments showed that the -360G allele has lower basal transcriptional activity than the -360A allele, whereas acetaldehyde/ethanol-induced gene expression, in general, seems to be more enhanced in individuals homozygous for the -360G allele than in those with the -360A allele. Thus, the promoter polymorphism may be involved in individual differences in acetaldehyde elimination.