Effects Of Icariin On Increasing And Mobilizing Endothelial Progenitor Cells To Promote Cardiovascular Angiogenesis And Its Mechanism

BackgroundMyocardial infarction is one of the common diseases of high occurrence which represents a serious threat to public health. Although a lot of progress had been made in clinical treatment, the curative effect is far from satisfaction. Promoting the growth of new vessels to increase the perfusion of ischemic tissue has become the new potential way in treating ischemic heart disease. As precursor cell of endothelial cell, endothelial progenitor cells (EPCs) can increase the flow of blood and the density of capillary, promote the formation of new vessels and play an important part in revascularization and repair of the vessels. When tissue ischemia or vessel damage occurs, EPCs are mobilized from bone marrow to peripheral blood, attach and recruit to the positions where tissue ischemia or vessel damage happens to perform their function. However, the repair by EPCs to blood vessels in the heart is limited. Instead, mobilization of the bone marrow and increase of EPCs in peripheral blood are likely to be effective strategies to treat diseases like myocardial infarction. Icariin is one of the active chemical compounds in traditional Chinese drugs, which can promote the proliferation of EPCs in human peripheral blood and protect the damage of blood vessels endothelial cells and cardiac muscle. However, the influence of icariin on biological function of EPCs proliferation is not thoroughly studied. It is unclear if icariin takes effect by helping EPCs to improve the proliferation of cardiac muscle. The mechanism of action of icariin remains unknown. As a result, study on mechanisms of action of icariin on increasing and mobilizing of EPCs to promote the angiogenesis of cardiac muscle is likely to provide theoretical basis and experimental reference to the treatment and application of icariin on MI-related diseases.AimBy establishing rat bone marrow-originated EPCs culturing model, we will observe the influence of icariin on proliferation, attachment, migration and angiogenesis of EPCs cultured in vitro. Also, we will study the influence of icariin on EPCs-related signal pathways and EPCs of peripheral blood and bone marrow of MI rats. Influence and possible mechanisms of action of icariin on angiogenesis and EPCs mobilization will be studied as well.MethodIsolate EPCs in rat bone marrow by density gradient centrifugation; identify markers of endothelial cells including VEGFR-2 and CD34 and CD133 of progenitor cells; perform identification on biological characteristics by detecting its adsorption and endocytosis of FITC-labeled lectin-1(UEA-1) and DiI labeled low density lipoprotein (Dil-ac-LDL); observe the influence of in vitro culturing conditions on the effect of icariin to the growth of EPCs; detect the influence of icariin to EPCs with MTT and EDU method; study the ability of migration of EPCs with Transwell method; observe the influence of icariin on angiogenesis with chick embryo chorioallantoic membrane assay.Establish rat MI model by ligation of left anterior descending coronary artery in rats. The rats were randomly divided into icariin administration group and control group the day after surgery. The icariin group was administrated for 28 days, and then take the abdominal aortic blood to measure myocardial enzyme spectrum, superoxide dismutase (SOD), oxidized glutathione (GSSG), glutathione (GSH) and malondialdehyde (MDA); isolate and culture the mononuclear cells from peripheral blood and bone marrow, numerate EPCs with flow cytometry, dissect the hearts and stain with nitroblue tetrazolium (NBT) to measure the infarct area, and perform pathological examination on cardiac tissue with HE staining; take ischemic myocardium immunohistochemical method to measure density of capillary and microtubule and differentiation of EPCs.ResultsMononuclear cells were isolated by gradient density centrifugation. They specifically express CD34, CD 133 and vascular endothelial growth factor receptor 2 (VEGFR-2). The proliferation of rat EPCs cultured in vitro is strong, and the cells begin to decline after 14 days. EPCs has the characteristics of DiI-ac-LDL uptake and UEA-1 binding. The proliferation of rat EPCs was detected by MTT assay, compared with the control group, icariin at all dosed tested can significantly increase the activity of cells and promote cell proliferation.5 μM and 10 μM icariin dose groups had significant increase in the number of 4h Edu-labeled cell compared with the control group. All dose groups had significant increase in the number of 24 h Edu-labeled cell compared with the control group, with the 24 h Edu-labeled cell proliferation rate at 75-80%. Cell adhesion test showed that 10μM icariin dose group can increase adsorption of EPCs compared with the control group; all dose groups of icariin had a significant increase in the number of cells across the Transwell chamber membrane compared with the control group, and the number of cells across the Transwell chamber membrane increased with the increase of the dose of icariin. The dose effect relationship was obvious. In the cell scratch assay, all dose groups of icariin had significantly narrower cell scratch gap than that of the control group. The study of influence of EPCs on vessels of chick embryo chorioallantoic membrane showed that as dose of icariin increased, the area of micro vessels of chick embryo chorioallantoic membrane increased. Quantitative real-time PCR (Real time PCR) analysis showed that the mRNA level of the chemokine receptor 4 (CXCR4) significantly increased in 5 μM and 10 μM icariin dose groups compared with the control group. In each icariin dose groups, the expression level of mRNA of phosphatidylinositol 3-kinase (PI3K) mRNA, serine/threonine protein kinase 1 (AKT1) and mRNA of endothelial nitric oxide synthase (eNOS) had significant increase compared with the control group. Moreover,10 μM icariin dose group had a obvious increase of mRNA expression level of vascular endothelial growth factor (VEGF) and SDF-1, which was significantly higher than the control group. We made the rat model of myocardial infarction, and continuously administrated the rats with icarrin. The HE staining examination of the cardiac tissue showed that rats of the control group had significant old myocardial infarction, while the icariin groups showed myocardial infarction at different degree and inflammatory infiltration. All dose groups administrated with icariin had significantly decreased infarct size compared with the control group. Icariin could reduce of the levels of glutamic oxalacetic transaminase (GOT), creatine kinase (CK), isoenzyme of creatine kinase CK-MB and alpha hydroxybutyrate dehydrogenase (alpha-HBD) of the myocardial enzyme spectrum, while it had no obvious influence on levels of lactate dehydrogenase (LDH), GSH, GSSG and SOD; icariin can reduce the content of MDA in serum. All dose groups of icariin had significant increase in the number of bone marrow EPCs in the peripheral blood, and the influence on the number of bone marrow EPCs was not significant. Immunohistochemistry staining of myocardial tissue sections showed that each icariin dose group had increase in CD 133 expression level compared with the control group; cells that expressed factor Ⅷ related antigen (vWF) increased in icariin group compared with the control group; icariin at 5 mg/kg and 10 mg/kg dose groups had significant increase in the number of CD34 positive cells of the myocardial tissue compared with the control group.Conclusion1. Icariin can promote the proliferation, adsorption, migration and angiogenesis of EPCs in vitro, and its possible mechanism of action is related to the cell signaling pathways of CXCR4/PI3K/Akt and VEGF/eNOS.2. Icariin can significantly improve the MI status of rats, and increase the number of EPCs in the peripheral blood, promote the formation of microtubule in the infarction myocardium, and this angiogenesis has the involvement of EPCs.3. Icariin can improve MI. Its mechanism of action is increasing and mobilizing EPCs from rat bone marrow and increasing the number of endothelial cell in the circulation. Furthermore, infarction foci adhere and aggregate, and promote their differentiation into vascular endothelial cells and further formation of blood vessels.