Expression And Regulation Of HIF-1 In Hypoxia Myocardium Of Diabetic Rats

IntroductionIschemic disease,including mainly ischemic heart disease,stroke and peripheral vascular disease,is a major cause of morbidity and mortality in the world.The initial treatments include modifying risk factors and reducing oxygen demand with medical therapy.When these treatments fail,revascularization to restore blood supply by percutaneous coronary intervention(PCI)or coronary artery bypass grafting(CABG)is often necessary.However,a significant number of patients have diffuse coronary artery disease,absent conduits after previous bypass surgery,small distal vessels,and comorbidities that preclude either procedure.This patient population has been estimated to comprise up to 12%of individuals presenting to interventional referral centers.With the widespread use of revascularization in the future will increase significantly.Since there is an inverse correlation between infarct size and collateral blood flow,therapeutic angiogenesis presents an exciting new method of improving blood supply to the ischemic myocardium and therefore providing symptomatic relief to a large and growing population of patients.Vascular endothelial cell growth factor(VEGF)and fibroblast growth factor (FGF)have shown to play key roles in therapeutic angiogenesis and are the most extensively investigated and utilized in clinical and preclinical studies.It has been demonstrated that they promote angiogenesis both in animal models and in clinical trials either by gene transfer or by protein therapy.However,the resulting capillaries are often leaky and accompanied by edema,inflammation,and spontaneous hemorrhagic ulcers.Administration of VEGF in combination with other angiogenic factors alleviates some of the vascular defects.Hypoxia inducible factor-1(HIF-1)is a transcriptional factor playing a key role in angiogenesis by activating gene transcription of VEGF and other proangiogenic factors.It has been shown that the resulting blood vessels from HIF-1 were structurally indistinguishable from normal capillaries in both morphology and function.Importantly,as a transcriptional factor,HIF-1 can not only promote angiogenesis,but also mediate cellular and systemic homeostatic responses to reduced O2 availability by promoting the transcription of dozens of its target genes, thus contributing to the survival of ischemic/hypoxia cells.Therefore,increasing HIF-1 activity may provide a new promising therapeutic strategy for ischemic disease.HIF-1 is a major regulator of the hypoxic response after myocardial infarction. This transcription factor consists of two basic-helix-loop-helix-PAS transcription factors,HIF-1αand HIF-1β.Both HIF-1αprotein expression and transactivation activity are regulated by changes in cellular oxygen level.Under normoxic conditions, HIF-1αis degraded by 26S proteasome,which is mediated by von Hippel-Lindau tumor suppressor gene product(VHL),that together with elongin B and C and a RING finger protein,form a complex harboring E3 ubiquitin ligase activity.The binding of pVHL to HIF-1αis dependent on the hydroxylation of proling 563 and 402 of HIF-1αby prolyl hydroxylases.VHL can also form a ternary complex with HIF-1αand the co-repressor FIH-1(factor inhibiting HIF-1).Both pVHL and FIH-1 recruit histone deacetylases that may contribute to the loss of HIF-1αtranscriptional activity under non-hypoxic conditions.HIF-1αis sensitive to hypoxia and is rapidly degraded by the ubiquitin proteasomal pathway under normoxic conditions.Decreased tissue oxygen causes altered availability of HIF-1αto the von Hippel-Lindau protein and ubiquitination,blocking its degradation.This results in nuclear accumulation of HIF-1αprotein.HIF-1 binds to hypoxia response element(HRE,5'-RCGTG-3')of its target genes and enhance the expression of target genes.Therefore it may become a new therapeutics that regulating the expression and activity of HIF-1α. The morbidity and mortality of ischemic heart disease are high,whose major pathological factor is oxygen deficiency of cardiomyocytes resulting from ischemia.â…¡-type diabetes is one of the most common complications in ischemic heart disease, and the reduction of compensatory circulation,aggravation of myocardial damage and the worse prognosis in patients with coronary heart disease combined withâ…¡-type diabetes perhaps are correlated with the decrease of HIF-1αexpression.The aim of this study was to investigate expression of HIF-1αin GK diabetic rats with myocardial infarction and explored the effect of chronic cobalt chloride treatment on blood glucose,myocardial infarction size,HIF-1αexpression in GK diabetic rats through myocardial infarction models of diabetic rats and non-diabetic rats,which may provide a new therapeutics of coronary heart disease combined with diabetes through regulating HIF-1 signal transduction system.Partâ… Effect and Mechanism of High Glucose on Expression of Hypoxia-Indueible Factor 1αin Cultured Neonatal Rat Cardiomyocytes under Hypoxia and Non-hypoxia ConditionsAimsTo investigate the effect and mechanism of different high glucose(Glu) concentrations on the expression of hypoxia-inducible factor 1α(HIF-1α)in cultured neonatal rat cardiomyocytes under hypoxia and non-hypoxia conditions.Materials and MethodsNeonatal rat cardiomyocytes were cultured for 6 hours under different conditions and were divided into 6 groups:â‘ Negative control group(5.5mMGlu);â‘¡Cobalt chloride(Cocl₂)mimicking hypoxia group(5.5mMGlu+400uM Cocl₂);â‘¢Different high Glu concentrations groups:(11.1mM,22.2mM,33.3mMGlu); â‘£Cocl₂+different high Glu concentrations groups(11.1mMGlu+400uM Cocl₂, 22.2mMGlu+400uM Cocl₂,33.3mMGlu+400uM Cocl₂);⑤High Glu concentrations+antioxidantα-tocopherol group(33.3mM Glu +100uMα-tocopherol);â‘¥High Glu concentrations+antioxidantα-tocopherol+Cocl2 group(33.3mMGlu +400uM Cocl₂+100uMα-tocopherol).To observe the effect of high Glu concentrations,Cocl₂ and high Glu concentrations plus Cocl₂ on the expression of HIF-1αmRNA and protein in cultured neonatal rat cardiomyocytes,as well as the effect of high Glu concentrations plus antioxidantα-tocopherol,high Glu concentrations plus Cocl₂ and antioxidantα-tocopherol on the expression of HIF-1αmRNA and protein.Results1,Compared with negative control group(5.5mMGlu),the expression of HIF-1αincreased under Cocl₂ mimicking hypoxia(5.5mMGlu+400uM Cocl₂,11.1mMGlu +400uM Cocl₂,22.2mMGlu+400uM Cocl₂,33.3mMGlu +400uM Cocl₂);2,The expression of HIF-1αincreased gradually followed by the increasing of Glu concentrations(5.5mM,11.1mM,22.2mM and 33.3mMGlu);3,The expression of HIF-1αdecreased gradually followed by the increasing of Glu concentrations under certain Cocl₂ plus different high Glu concentrations (5.5mMGlu+400uM Cocl₂,11.1 mMGlu+400uM Cocl₂,22.2mMGlu+400uM Cocl₂,33.3mMGlu+400uM Cocl₂);4,Under high Glu concentration plus antioxidantα-tocopherol(33.3mM Glu +100Umα-tocopherol),the expression of HIF-1αincreased less than that of the same high Glu concentration(33.3mM Glu);5,Under high Glu concentration plus Cocl₂(33.3mMGlu+400uM Cocl₂),the expression of HIF-1αincreased less than that of the same high Glu concentration plus antioxidantα-tocopherol and Cocl₂(33.3mMGlu+400uM Cocl₂+100uMα-tocopherol). ConclusionsHigh glucose concentrations increase the expression of HIF-1αunder non-hypoxia,but blunt the expression of HIF-1αunder hypoxia(mimicking)in cultured neonatal rat cardiomyocytes.Some mechanisms such as ROS(reactive oxygen species)signal transduction system and oxidative stress may be involved in it.Partâ…¡Effect of Chronic Cobalt Chloride Treatment on Hypoxia-Inducible Factor 1 Expression and Myocardial Infarction Size in GK Diabetic RatsAimsTo investigate whether chronic cobalt chloride treatment decreases blood glucose and myocardial infarct size in GK diabetic rats,as well as increases hypoxia-inducible factor 1α(HIF-1α)expression in ischemic zone.Materials and MethodsExperiments were carded out in male GK diabetic rats and male Wistar rats(both weighing 200g-250g).Left anterior descending coronary artery(LADCA)was ligated with silk ligature in anesthetized rats.Rats were divided into six groups(Wistar sham-operated group,Wistar infarct group,Wistar infarct+cobalt chloride treatment group,GK sham-operated group,GK infarct group,GK infarct+cobalt chloride treatment group)and raised with different treatments for 3 weeks.Before operation, blood glucose of each rat was measured.3 weeks after operation,blood glucose of each rat was also measured before hearts of all survived rats were excised,then myocardial infarct size and HIF-1αexpression were detected in all groups. Results1.Compared with that of GK sham-operated group and GK infarct group,blood glucose of GK infarct+cobalt chloride treatment group decreased;2.Compared with that of GK infarct group,myocardial infarct size of GK infarct +cobalt chloride treatment group decreased;3.Compared with that of Wistar infarct group,myocardial infarct size of GK infarct group increased;4.HIF-1αexpression of GK infarct+cobalt chloride treatment group increased in ischemic zone than that of GK infarct group;5.HIF-1αexpression of GK infarct group decreased than that of Wistar infarct group.ConclusionsChronic cobalt chloride treatment can decrease blood glucose and myocardial infarct size in GK diabetic rats,also increase HIF-1αexpression in ischemic zone, which may provide a new insight and therapeutics on treatment of coronary heart disease combined diabetes through regulating HIF-1 signal transduction system.