The Effects Of Interleukin-8on Angiogenesis In The Ischemic Myocardium And The Underlying Mechanisms

Chapter1Construction of lentivirus vector containing IL-8geneAbstract:Objectives:Coronary atherosclerotic heart disease (CHD) is a major threat to public health in China. Study found that coronary collateral circulation (CCC) in patients was conducive to maintenance of myocardial viability, protection of heart function. Large number of animal experiments confirmed VEGF, bFGF and other angiogenic growth factor (proteins and genes) were applied to ischemic myocardium and might promote collateral vessels growth, increase the perfusion for ischemic cardiac myocytes, reduce infarct size and improve heart function. Early studies suggest that IL-8play an important role in inflammation, as IL-8and its receptor on the biological activity and mechanism of research, found that it also has mitogenic and angiogenic effect, but whether the promotion of collateral vessels growth in ischemic myocardium, the increasing of ischemic myocardial perfusion has not been reported in the literature. The study is to construct the lentivirus vector containing IL-8gene, as studies angiogenesis in ischemic myocardium lay the foundation for the role.Methods:Using PCR to angle the IL-8gene from the purchased cDNA library. Directional changed the IL-8gene into Age I enzyme cutting production (pGC-FU Vector). Then transformed bacterial competent cells. Using PCR identified the growth clones, then sequencing and comparing the PCR identified clones. The right clone is our target plasmid. The target plasmid and two auxiliary plasmids were co-transinfected into293T cells, and collect the suspension. After concentration the suspension got the concentrated solution of lentivirus and measured the titre.Results:1. By PCR amplification of human IL-8gene, and doing agarose gel electrophoresis for PCR products, obtained a343bp band which represented IL-8gene2.The directional exchange production between IL-8gene and pGC-FU Vector can transformed the bacterial competent cells. The results of PCR identification are showed in below. Positive transformants are bands of507bp, while negative band198bp. The size of the target gene is507bp-198bp=309bp. Sequencing results from Invitrogen Corporation showed that:Identities=298/298(100%), sequencing results with the target sequence (IL-8) exactly the same.3.Each of the prepared DNA solution (pGC-FU-IL8-EGFP vector20μg, pHelper1.0carrier15μg, pHelper2.0vector10μg) were cotransfected into293T cells.24-48h later, the green fluorescence could be seen.From the Western blot result, obtained a39KD band, which represented the fusion expression of IL-8and GFP.4.After virus harvesting and concentrated, Real time quantitative PCR was used as determination of titer, virus titer is2.00E+8TU/ml.Conclusions The lentivirus vectors containing IL-8gene were successfully constructed, and packaged in to293T cells. The titre after packaging is2.00E+8TU/ml. Chapter2IL-8gene expression by means of lentivirus vector mediation in rabbit models of myocardial infarction and the effects of IL-8on angiogenesis in the ischemic myocardium and its possible mechanismsAbstract:Objectives:IL-8is a multi-source cytokine, a variety of cells, such as endothelial cells, monocytes, fibroblasts, and many human tumor cells, can synthesize and secrete IL-8. IL-8not only play an important role in inflammation, but also has mitogenic and angiogenic effect. Several studies found that IL-8concentration was significantly higher than normal in the blood of patients with coronary heart disease. IL-8is engaged in the development and progression of coronary heart disease, and studies have found that some people with severe coronary atherosclerosis heart disease, with coronary collateral circulation (CCC) grading and scoring increased,serum IL-8and MMP-9concentration was correspondingly increased. However, whether IL-8can promote angiogenesis in the ischemic myocardium and the possible mechanisms are not clear. Therefore, in this study, set up rabbits models of myocardial infarction.IL-8gene carrying enhanced green fluorescent protein(EGFP) was mediated by lentiviral vector,injected to the ischemic myocardium, in order to understand whether the promotion of IL-8in the establishment of coronary collateral circulation and study the possible mechanisms.Methods:40Healthy and clean Chinese rabbits were randomly divided into4groups:sham group, myocardial infarction(MI) model group, myocardial infarction+empty lentiviral vector group, myocardial infarction+lentiviral vector containing IL-8gene group. Using traditional methods ligate proximal left anterior descending coronary artery to produce myocardial infarction. Before surgery, when modeling,30minutes after modeling, performed ECG detection, observed dynamic ECG changes.1week,6weeks after modeling did echocardiography in order to understand cardiac function. Did HE staining of myocardial tissue to observe the cardiac tissue’s morphological changes in different regions. Detected the GFP fluorescence by fluorescence microscope. Did CD34immunohisto-chemistry to meansure the density of newly formed blood vessels in the ischemic myocardium. Meansured IL-8, phosphorylated Akt, phosphorylated GSK-3βser9expression by Western Blot.Results:1.32of40modeling animals were survival to the end of the experiment (6weeks after modeling),8rabbits in each group survived.2.Modeling findings, changes of ECG, general observation of the hearts and cardiac pathology staining confirmed the model of myocardial infarction was built successfully. Visible green fluorescence was observed by microscope.That indicated IL-8labeled GFP lentiviral vector and empty lentiviral vector have been transfered to the ischemic myocardium, while myocardial infarction group and the sham control group have no green fluorescence.3. Compared with sham group,IL-8protein expression was significantly increased in ischemic myocardium of myocardial infarction model group (P<0.01).Compared with model group, IL-8expression was significantly increased in lentiviral vector containing IL-8gene group (P <0.01). That showed lentiviral vector containing IL-8gene successfully transfected into the ischemic myocardium. There was no difference between the model group and empty lentiviral vector group.4.Compared with the sham group, cardiac function of model group was significantly decreased1or6weeks after MI(P<0.01). There was no difference of cardiac function between the model group and lentiviral vector containing IL-8gene group1week after MI, but after6weeks, the LVEDd, LVESd reduced, LVFS, LVEF increased, showing improvement in cardiac function, but the difference was not significant (P>0.05).5. Compared with the sham group, micro vessel density of the ischemic myocardium was significantly increased in model group (P <0.01).Compared with model group, microvessel density was significantly increased in lentiviral vector containing IL-8gene group (P <0.01). There was no difference between the model group and empty lentiviral vector group.6. Compared with sham group, phosphorylation Akt, phosphorylation GSK-3βser9expression were significantly increased in ischemic myocardium of model group (P<0.01).Compared with model group, phosphorylation Akt, phosphorylation GSK-3βser9expression were significantly increased in lentiviral vector containing IL-8gene group (P <0.01). There was no difference between the model group and empty lentiviral vector group.Conclusions:1. Rabbit models of acute myocardial infarction were successfully constructed,and lentivirus-mediated IL-8gene successful transfected into the ischemic myocardium.2. IL-8can promote the formation of micro vessels in the ischemic myocardium,which is related to increase phosphorylation Akt and phosphorylation GSK-3βser9expression. Chapter3The effects of IL-8on proliferation and apoptosis of hypoxic human umbilical vein endothelial cells and its possible mechanismsAbstract:Objectives Endothelial cells are single flat cells which cover in blood and lymph vessels with a variety of physiological functions. It acts as the first barrier between the blood and tissue. And it also be one of the first cell to feel the hypoxia. Endothelial cells play a major role in angiogenesis. In changing conditions and environment, the endothelial cells go from the resting period into division, it causes angiogenesis. Studies found that inducing apoptosis of endothelial cells would offset proliferation,thereby inhibiting angiogenesis and leading to the decline of blood vessels. And inhibition of endothelial cells’ apoptosis can promote angiogenesis. In this part,prepare hypoxic human umbilical vein endothelial cells and explore the effect of IL-8on proliferation and apoptosis of hypoxic human umbilical vein endothelial cells (HUVECs) and its possible mechanisms.Methods:Culture HUVECs12,24,48h with different concentrations of CoCl2(50,100,200,400μmol/L). Use MTT assay for cell survival rate detection, and select the appropriate concentration of CoCl2to produce hypoxic HUVECs; With different concentrations of recombinant human IL-8(1,10,50,100ng/ml) cultured hypoxic HUVECs, use MTT assay for cell survival rate detection and select the appropriate concentration of IL-8and time for the next step mechanism researchment. Choose normal HUVECs and hypoxic HUVECs, and add recombinant human IL-8(lOng/ml), Anti-IL-8(10μg/ml) or LY294002(20umol/L) to incubate cells for48h. Use MTT colorimetric analysis for cell survival rate detection. Annexin V/PI was engaged in the assayment of apoptosis for human umbilical vein endothelial cells. The expression level of phosphorylated AKT, phosphorylated GSK-3βser9, Caspase3protein were measured by Western blot.Results:1. Incubation HUVECs with CoCl2(50,100uM) for12h can promote the proliferation of HUVECs. Incubation for24h or48h, compared with the control group, the high concentrations of CoCl2 (200,400uM) can inhibite the proliferation, which shows significant effects of hypoxia. The most significant effect time is48h (P<0.01).2.Incubation hypoxic HUVECs with IL-8(1,10,50,100ng/ml) for24h can improve the anoxia-induced proliferation inhibition of HUVECs. But there is no statistical difference. Incubation for48h or72h, compared with hypoxic HUVECs,10,50,100ng/ml groups can significantly improve the anoxia-induced proliferation inhibition of HUVECs, which shows the promoting ability for cell proliferation (P<0.01). Incubation hypoxic HUVECs with1ng/ml IL-8for24h,48h,72h can improve the anoxia-induced proliferation inhibition of HUVECs,but that has no statistical difference.3. In normal groups,compared with the normal control group, IL-8(lOng/ml) could promote proliferation of HUVECs after48h, and can inhibit apoptosis of HUVECs. And LY294002(20uM) or IL-8antibody (10μg/ml) inhibited those effection of IL-8significantly (P<0.01). In hypoxia groups, Compared with normal control group,the proliferation decreased and apoptosis increased significantly in hypoxia groups(P<0.01). compared with the hypoxic group, IL-8(10ng/ml) can stimulate proliferation and inhibit apoptosis of hypoxic HUVECs after48h. LY294002(20uM) or IL-8antibody (10μg/ml) inhibited those effection of IL-8significantly (P<0.01).4. In normal groups,compared with the normal control group, IL-8(lOng/ml) significantly increased the protein expression of phosphorylation Akt, phosphorylation GSK-3βser9after48h,and also reduced the protein expression of Caspase3. LY294002(20uM) or IL-8antibody (10μg/ml) significantly inhibited the effection of IL-8(P<0.01). In hypoxia groups, compared with normal group, the protein expression of phosphorylation Akt, phosphorylation GSK-3βser9in hypoxia group were significantly decreased, while Caspase3was significantly increased (P<0.01). compared with hypoxia group, incubation hypoxia HUVECs with IL-8(lOng/ml) for48h can significantly increase the protein expression of phosphorylation Akt, phosphorylation GSK-3βser9,reduce the protein expression of Caspase3. LY294002(20uM) or IL-8antibody (10μg/ml) inhibited those effects of IL-8significantly(P<0.01). Conclusions:IL-8can improve proliferation and inhibit apoptosis of hypoxic HUVECs induced by CoCl2, which is related to increase the protein expression of phosphorylation Akt, phosphorylation GSK-3βser9and decrease the protein expression of caspase3.