The Critical Role Of Aldehyde Dehydrogenase2(ALDH2) In Angiogenesis Of Chronic Ischemic Tissue

Ischemic heart disease is one of the major killers of human health. According to statistics, total prevalence rate of ischemic heart disease is more than7.0%in adult more than20-year-old. In China, with economic growth and improvement of living standards, ischemic heart disease morbidity and mortality is increasing every year. Despite drug therapy, interventional therapy, surgical treatment has made a lot of progress, approximately20%of patients could not be treated with these types of treatment or could be treated with these but ineffective. Clinical studies had demonstrated that good collateral circulation could protect the ischemic myocardium, and improve the survival rate of patients with ischemic heart disease. How to promote the formation of collateral circulation has become a hot topic in the prevention and treatment of ischemic heart disease. In patients with severe ischemic heart disease, vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-la (HIF-la) had been shown to promote the formation of collateral circulation effectively.Aldehyde dehydrogenase (ALDH2) is an important aldehyde oxidase in mitochondria. There are some common mutations of ALDH2gene. Especially Glu504Lys mutation has dramatically high frequency in East Asian populations, for example, in the Chinese Han population the frequency is about34%, and is about46%in the Japanese population. Pets heterozygous for the mutation will cause the self-reduction in enzyme activity, only one-sixteenth of the wild-type ALDH2activity. While the mutant homozygotes have almost no enzyme activity in vivo. Traditionally ALDH2was taken as a key enzyme in the metabolism of alcohol, catalyzing toxic acetaldehyde metabolism to non-toxic acetic acid, which may continue to participate in the reaction for energy supply. Science reported in2003that ALDH2played a significant protective effect in ischemia-reperfusion injury model by activation of protein kinase C ε (PKCs) pathway. In recent years, ALDH2gradually had been proven to be an important endogenous cardio-protective factor, playing a very critical role in protection such as heart failure caused by myocardial infarction, cardiac ischemia-reperfusion injury, alcoholic cardiomyopathy and other aspects. Our previous study found that:ALDH2might play a protective role for chronic ischemic myocardium after myocardial infarction, by detoxing aldehydes such as4-hydroxynonenal (4-HNE) which generated by lipid peroxidation, thereby inhibiting JNK/p53pathway. In2007Nature had reported that p53inhibited angiogenesis further deterioration of cardiac function by down-regulating HIF-1a exacerbating the relatively ischemia of myocardial cells in transverse aortic constriction (TAC) model which mimicked cardiac pressure overload and cardiomyocyte hypertrophy. Conversely, inhibition of p53could promote myocardial cells surrounding angiogenesis and myocardial blood supply effectively, thereby enhancing the mouse heart ejection fraction. Our team members had participated in this research. Therefore, dose ALDH2involved in the regulation of angiogenesis? On the other hand, ALDH2participated in the generation of Reactive oxygen species (ROS), which could also regulate HIF-1α/VEGF pathway. Therefore, this study is amid at, as an endogenous ischemic protection factor, whether ALDH2could regulate angiogenesis in the course of chronic ischemic by regulating of HIF-1α/VEGF pathway, thus improving the blood supply compensatory to tissues and promote ischemic tissue functions. No such study has been reported yet.Chronic coronary occlusion (CTO) lesion was considered as the "last frontier" of coronary interventional medical specialties because of its severity and complexity. Formation of coronary collateral circulation was essential for patients with CTO to maintain their heart function. Considering that there were nearly half of Asians were ALDH2mutation carrier, impaction of ALDH2genotypes on formation of collateral circulation in CTO patients would has significant influence in clinical prevention of CTO in people at high risk, the choice of treatment strategy and prognosis. This study was to illustrate the relation between ALDH2genotype and the grade of coronary collateral circulation classified by Rentrop stander in CTO patients.In summary, this study aimed to study the role of ALDH2in regulating angiogenesis of chronic ischemic tissue, and further elucidate the molecular mechanism. Also this study was to verify the relation between ALDH2genotypes and grade of coronary collateral circulation through the clinical research in CTO patients, especially in East Asian populations. It would clarify the cardio-protective role of ALDH2for chronic ischemic myocardium from the perspective of angiogenesis, and illustrate the influence of ALDH2genotypes on CCVs formation of CTO patients providing evidence to individual treatment for such patients, such as ALDH2-deficient patients might get more benefits by taking a more active revascularization strategy. The text was divided into the following three parts:Part Ⅰ:ALDH2Regulates Angiogenesis in Chronic Ischemic TissuesObjective Angiogenesis is essential for the development and prognosis of ischemic heart disease. myocardial protection role of aldehyde dehydrogenase2(ALDH2) had been concerned in recent years. The purpose of this research is to study the role of ALDH2in regulating angiogenesis of chronic ischemic tissues. Methods8weeks male ALDH2-knockout mice (KO) and wild-type C57BL/6mice (WT) mice were taken to produce lower limb ischemia model, to detect the level of lower limb perfusion with a laser Doppler measurement after1days,7days and21days post-surgery. Rat cardiac micro-vascular endothelial cells (MECs) were cultured and the ALDH2low expression MECs was achieved by transfecting ALDH2-siRNA. These two kind of MECs were cultured in normoxia and hypoxia6hours respectively on Metrigel to observe the ability of micro-vascular endothelial cells to formation tubes. Results It was found by laser Doppler detection that the blood flow recovery of ALDH2-KO mice lower extremity is worse than the wild-type mice, and there was a statistically significant difference at day21(p<0.05). The blood flow was represented by the ratio of the reperfusion of ischemic limb and the non-ischemic limb of each mouse. In cellular experiments, it was observed that given ALDH2-siRNA interference on rat myocardial MECs, compared with the control group, had significantly weakened tube formation capacity both under normoxia and6hours of hypoxia cultured on Metrigel. Conclusion These results indicate that the the ALDH2defects can significantly weakened angiogenesis capacity of chronic ischemic tissue, which in turn hinder the self-revascularization and functional reconstruction. This explained the myocardial protection role of ALDH2on chronic ischemic from the point of view of angiogenesis. It would provide potential targets of new drug for the clinical treatment of ischemic disease. Part Ⅱ:ALDH2Promote Angiogenesis Through Regulating HIF-la/VEGF PathwayObjective In recent years, ALDH2had been proven to be an important endogenous cardio-protective factor, protecting heart failure after myocardial infarction, cardiac ischemia-reperfusion injury, alcoholic cardiomyopathy and other aspects. The first part of the study found that the ALDH2defects can significantly weakened angiogenesis of chronic ischemic tissue, therefore, this study aimed to clarify its intrinsic molecular mechanisms. Methods Bilateral quadriceps tissue of each mouse were taken to extract proteins after1day,7days and21days post-surgery, with the same animal grouping and modeling of the first part. Human Umbilical Vein Endothelial Cells (HUVECs) were cultured, and ALDH2low expression and high expression HUVECs cell lines were established by transfection of corresponding virus. Each line set normoxia group and six hours hypoxia group. Western Blot was employed to detect ALDH2, hypoxia-inducible factor-1alpha (HIF-1α), vascular endothelial growth factor (VEGF) and other relative proteins. Results HIF-1α and VEGF protein levels detected by Western Blot were significantly lower in ALDH2-KO mice ischemic side quadriceps tissues than that of wild type mice7days after surgery (p<0.05). Western Blot detection of cultured HUVECs cells found that cells transfected with adenovirus ALDH2which mimicked high expression model showed significantly increased HIF-1α and VEGF level; conversely, cells transfected with the plasmid to inhibit the expression of ALDH2showed significantly reduced HIF-1α and VEGF level. Conclusion These results indicate that ALDH2played a key regulatory role of promoting chronic ischemic tissue angiogenesis by regulating Hif-1α/VEGF pathway, thus improved ischemic tissue blood supply and promoted ischemic tissue function. This study explains ALDH2intrinsic molecular mechanism of regulating angiogenesis. Part Ⅲ:The Clinical Impact of ALDH2Genotypes on the Formation of Coronary Collateral Vessels (CCVs) in Coronary Total Occlusion (CTO) Patients Objective Coronary chronic total occlusion (CTO) is considered to be the "last frontier" of coronary interventional medical specialties. The formation of coronary collateral circulation (CCVs) in patients with CTO is essential to maintain the cardiac function. Nearly half of Asian populations carry the ALDH2mutation Glu504Lys. Therefore, this study aimed to clarify the role of ALDH2genotype on chronic ischemic myocardial angiogenesis from a clinical point of view. Methods With approval of ethical review of Zhongshan Hospital and obtain the consent of the patients, CTO patients were extracted by the inclusion criteria of1) at least one of the right coronary artery, left main coronary artery, left anterior descending artery or left circumflex artery flow0, and2)duration of ischemic manifestations of more than3months; by the exclusion criteria of1) undergoing acute myocardial infarction,2) previous percutaneous coronary intervention or coronary artery bypass grafting, or3) congenital heart diseases or valvular heart diseases. According to the coronary angiography image CCVs was graded by Rentrop classification, and in this study the grade0or I were taken as poor CCVs and grade Ⅱ or Ⅲ as rich CCVs. Using ALDH2gene sequencing to get the genotype. SAS software and logistic regression methods were used for statistical analysis. Results A total of154cases were included. It was observed that the percentage of individuals with poor CCVs in ALDH2*1/1group (n=83) was38.5%; the percentage of ALDH2*1/2individuals (n=63) with poor CCVs was44.4%; and it was71.4%in ALDH2*2/2group (n=7) possessing of poor CCVs. Therefore homozygous had significant higher percentage of poor CCVS than the combined group (p<0.05). Conclusions These data suggest that ALDH2contributes to the angiogenesis in chronic ischemic tissue, like formation of CCVs in CTO. Notably, ALDH2*2homozygous (6-10%of East Asian people) showed much lower ability to formatting CCVs than non-ALDH2*2homozygous. Therefore, it might be beneficial for ALDH2*2homozygous to undergo CTO lesions recanalization at early stage.