The Protective Effects And Mechanisms Of Angiopoietin-1 On Myocardial Ischemia In Diabetic Mouse

【Hyperglycemia Altered Angiopoietin /Tie-2 Expression and Aggravated Myocardial Injury induced by Ischemia】Cardiovascular disease increases the risk of morbidity and mortality in patients who are diabetic verses patients who are not.This increase in risk may be due to the impairment of myocardial angiogenesis that occurs in diabetic conditions.It has been shown that the Angiopoietin(Ang) /Tie-2 system plays a critical role in controlling angiogenesis in diabetes. There has been a positive and significant correlation between ratios of Ang-1 and Ang-2 and coronary artery disease.The relationship between the Ang/Tie-2 system and cardiovascular disease in diabetes,however, has not yet been defined.The first part of our study was to analyze the Ang/Tie-2 expression in streptozotocin(STZ)-induced diabetic mice that had undergone myocardial ischemia.Ligation of the left anterior descending coronary artery of mice was performed to create the model for ischemia.Our western blot analysis showed that wild-type hearts subjected to ischemia for 24 hours resulted in a significant increase in Ang-2,Tie-2 and phospho-AKT protein expression.In STZ diabetic mice,however, ischemia failed to induce Tie-2 and phospho-AKT protein expression. There was little affect on Ang-1 expression.The relative ratio of Ang-2 /Ang-1 in response to ischemia was significantly increased in diabetic-mice hearts compared to results found in control wild-type mice. The second part of our study was to analyze myocardial injury induced by ischemia in STZ diabetic mice.After ischemia,we measured the infarct area by triphenyltetrazolium chloride(TTC)staining and analyzed myocardial apoptosis by Tunel and caspase3 immuno-staining.STZ mice showed a significant increase in infarct area and myocardial apoptosis.Angiopoietins represent a new family of angiogenic factors.In order to find the angiogenesis that are influenced by hyperglycemia via the Ang /Tie-2 system,we measured myocardial angiogenesis by Fluorescence Staining.The results showed that the density of capillaries and micro-arteries were significantly increased in wild-type hearts two weeks after induced myocardial ischemia but were decreased in diabetic mice. At the same time,angiogenesis paralleled compensatory hypertrophy. These data suggest that hyperglycemia exacerbated myocardial injury induced by ischemia and impaired angiogenesis by disrupting the Ang /Tie-2 system.【The Protective Effects of Ang-1 on Myocardial Ischemia in Diabetic Mouse】In order to determine whether a shift in the Ang-2 /Ang-1 ratio favoring Ang-1 could minimize the hyperglycemia-induced exacerbation of myocardial injury,we pretreated STZ mice with adenovirus vectors expressing Ang-1(Ad-Ang-1)or B-galactosidase by intravenous tail injection two days before surgery.STZ mice treated with Ad-Ang-1 showed a significant increase in activation of protective AKT protein and a remarkable decrease in apoptosis and the infarct area after ischemia compared with STZ mice receiving the control vector.Moreover,STZ mice treated with Ad-Ang-1 showed a significant increase in capillary and arteriole densities.These data indicate that Ang-1 can protect ischemic heart exacerbated by hyperglycemia via restoration of the Ang-2 /Ang-1 balance.【The Protective Mechanisms of Angiopoietin-1 on Myocardial Ischemia in Diabetic Mouse】Diabetic aggravated injury of myocardial ischemia is strongly associated with hyperglycemia-induced endothelial dysfunction.Cardiac microvascular endothelial dysfunction plays an important role in the initiation and perpetuation of diabetic cardiomyopathy.So our study emphasized the effect of Ang-1 on cardiac microvascular endothelium that was cultured in hyperglycemic media.Our Immunoprecipiation(IP)and western blot data showed that exposure of mouse heart myocardial endothelial cells(MHMEC)treated with high glucose(HG)resulted in a dramatic up-regulation of Ang-2 expression and a decrease in phosphorylation of Tie-2/AKT/eNOS signaling pathways compared to that of normal glucose(LG)conditions. Ang-1 significantly increased the Tie-2 and AKT posphorylation in HG condition,though Ang-1 increased slightly phosphorylation of eNOS. Ang-1/Tie-2/AKT signaling is a probable contributor to protect MHMEC under hyperglycemic conditions.We used MTT to determine cellular viability,Tunel and caspase3 enzyme linked immunosorbent assay (ELISA)for cellular apoptosis,and western blot to analyze survivin protein expression.Treatment of MHMEC with Ang-1 increased cellular viability and survivin protein expression after serum starvation for two days in HG conditions.Similarly,Ang-1 suppressed serum starvation-induced caspase3 activation and endothelial cell apoptosis in HG conditions,but Ang-2 impaired the effects of Ang-1.These data suggested that Ang-1 can protect ischemic heart exacerbated by hyperglycemia via inhibition of microvascular endothelial apoptosis.Angiogensis,which is impaired in diabetes,is another important function in cardiac microvascular endothelia.Endothelial sprouting is the primary method of angiogenesis.Since endothelial sprouting depends on endothelial migration,proliferation and capillary tube formation,we checked the effect of Ang-1 on angiogenesis in cardiac microvascular endothelia by using tubular formation and aortic ring assay.Ang-1 promoted tubular formation that was previously impaired due to hyperglycemia.Similarly,Ang-1 increased the length of sprouts in aorta sections.These data suggested that Ang-1 can protect ischemic heart exacerbated by hyperglycemia via increase of angiogenesis.Angiogenesis and vasculogenesis are two consecutive processes during blood vessel development.Endothelial progenitor cells(EPC)play a crucial role in postnatal vasculogenesis.We used endothelial mark CD31 to determine the number of EPCs in cultured bone marrow cells. We found Ang-1 increase the number of EPC impaired by hyperglycemia.Taken together,Ang-1 can restore the ang-2/Ang-1 balance that was disrupted by hyperglycemia and activate the Ang-1/Tie-2/AKT signaling that was weaken by hyperglycemia.Ang-1 can protect ischemic heart exacerbated by hyperglycemia via inhibition of microvascular endothelial apoptosis and increases of angiogenesis and vasculogenesis.