A Study On The Expression, Mechanism And Intervention Of Endostatin And Angiostain In Myocardial Infarction Rats

Ichemic heart disease (IHD) is one of the leading cause of death and disibility in the world. Considerable advances in drug therpy, surgical bypass and percutaneous revascularization techniques have occurred in past two decades. These interventions have improved the prognosis of most patients with IHD, but there are some patients who are not candidates because they have diffuse coronary artery disease or severe myocardial ischemia. The strategy to promote the development of coronary collaterals and myocardium angiogenesis by the administration of angiogenic growth fators have become the center of the cardiovascular therapeutics research. Using recombinant genes and growth fators for therapeutic angiogenesis in human has obtained innitial successes, but there are still some issues and problems to be resolved. Recently, coronary collaterals development and angiogenesis is thought to be regulated by the balance of growth fators and growth fator inhibitors. The latest studies indicated that endostatin expression were increased in patients with IHD.The levels of endostatin were associated with the presence or absence of collaterals in patients with IHD. We are going to investigate the expression of angiostatin, endostatin and their precursor proteins and marix metalloproteinase (plasminogen, collagen XVIII, MMP-9 mRNA) and observe the influence of Chinese medicinal herbals who have pro-angiogenetic activity on the expression of angiostatin and endostatin in ischemic myocardium of experimental myocardial infarction rats. We also observe the expression of endostatin in human umbilical vein endothelial cell between hypoxic and normoxic condition to reveale the fator that influence the expression of endostatin.Part I The expression of angiostatin and endostatin and their precursor proteins in the experimental myocardial infarction ratsSprague-Dawley(SD) rats were randomly divided into 7, 14, 21, 28 day groups after myocardial infarction.The sham-operated group acted as control. The expression of angiostatin and endostatin in ischemic myocardium were observed byImmunohistochemistry, Western blot and enzyme-linked immunosorbent assay (ELISA). The expression of VEGF and microvessels densities were also measured to observe the correlation between angiostatin or endostatin and VEGF or microvessels densities . The expression of plasminogen, collagen XVIII and marix metalloproteinase (MMP-9) mRNA in ischemic myocardium were measured by reverse transcription-polymerase chain reaction (RT-PCR).The results showed that: Immunohistochemistry demonstrated the presence of angiostatin and endostatin immunopositive zone in the edge infarct section, mainly around the blood vessels, in the experimental myocardial infarction rats. The levels of angiostatin immnopositive area were higher than the sham at 7th, 14th, 21st, 28th day after myocardial infarction (p<0.05). The level of angiostatin peaked at 7th and gradually decreased at 7th, 21st, 28th, but still higher than the sham. Western blot analysis revealed the same change trend of angiostatin with immunohistochemitry. The levels of endostatin immnopositive area were higher than the sham at 7th, 14th, 21st day after myocardial infarction (p<0.05) and peaked at 7th and gradually decreased at 14th, 21st day. The endostatin level reduced to the baseline level at 28th day (p>0.05). ELISA analysis showed the levels of endostatin were higher than the sham at 7th, 14th, 21st day(p<0.05). RT-PCR revealed the presence of plasminogen mRNA, collagen XVIII mRNA and MMP-9 mRNA in the edge of infarct section in the experimental myocardial infarction rats. The expression of plasminogen mRNA, collagen XVIII mRNA and MMP-9 mRNA were increased in the edge of infarct section in myocardial infarction and higher than those of the sham (p<0.05). The levels of plasminogen mRNA, collagen XVIII mRNA peaked at 7th day and gradually decreased at 14th, 21st, 28th day. MMP-9 mRNA expression did not change among the infarct groups.Part II The effects of HONG jingtian and JIANG xiang on the expression of angiostatin and endostatin in the experimental myocardial infarction ratsThe experimental myocardial infarction rats were randomly divided into the control group, HONG jingtian group and JIANG xiang group. The sham-operated rats were the sham group. HONG jingtian group and JIANG xiang group were seprately given the aqueous extracts of HONG jingtian or JIANG xiang by oral. The sham andcontrol group were given normal saline by oral. The levels of angiostatin and endostatin were measured after 2 weeks by Wetern blot and ELISA. The numbers of CD34 immunopositive microvessel in the edge of infarct section of myocardial infarction rats were measured by Immunohistochemistry.The results showed: The numbers of CD34 immunopositive microvessel in the edge of infarct section of myocardial infarction rats were increased in the HONG jigtian and JIANG xiang group. Meanwhile, Western blot showed that the level of angiostatin in the HONG jigtian and JIANG xiang group were lower than the control (p<0.05). ELISA also revealed the expression of endostatin decreased compared with the control (p<0.05). No significant difference were found between the HONG jiangtian and the JIANG xiang group (p<0.05).Part m The effects of hypoxia on the expression of endostatin and collagen XVIII mRNA in human umbilical vein endothelial cellsHuman umbilical vein endothelial cells (HUVECs) were cultured in vitro in normoxic and hypoxic condition. The levels of endostatin in the culture media of HUVECs were measured by ELISA. The expression of collagenXVIII mRNA in HUVECs were measured by RT-PCR.The results showed: RT-PCR demonstrated the presence of collagenXVIII mRNA in HUVECs. The expression of collagen XVIII mRNA were up-regulated by hypoxia (p<0.05). ELISA revealed the presence of endostatin in the culture midia of HUVECs. The level of endostatin increased by hypoxia compared with the normoxic group (p<0.05). The expression of endostatin and collagen XVIII mRNA decreased in normoxic condition after hypoxia (p<0.05).Conclusions1. The level of angiostatin and endostatin increased in the edge of infarct section in experimental myocardial infarction rats and gradually decreased during the later period.2. The expression of plasminogen and collagen XVIII mRNA were up-regulated...