| Cardiovascular disease is the global leading cause of death in the whole world. Currently available approaches for treating patients with ischemic heart disease include medical therapy or coronary revascularization by percutaneous coronary angioplasty (PCA) or coronary artery by pass grafting (CABG). However, a significant number of these patients are not candidates for coronary revascularization procedures or achieve incomplete revascularization with these procedures. Consequently, many of these patients have persistent symptoms of myocardial ischemia despite intensive medical therapy. Preliminary clinical experiences suggest that therapeutic angiogenesis may provide additional blood flow to incompletely revascularized areas. More recently, several studies suggest that implanted bone marrow cells may induce angiogenesis in ischemic myocardium.Currently there are 3 major approches for therupetic angiogenesis, that is gene therapy, protein therapy and cellbased transplation. Howerve, theses methods are not only expessive but also on the stduy phase, and can not be used in clinical.Endothelial dysfunction has been implicated in the pathogenesis of many diseases affecting the cardiovascular system. Experimental and clinical studies have shown that endothelial dysfunction may play a cusial role in the development and progression of vascular diseases, such as hypertension ro restenosis, as well as their complications, including myocardial infarction. Mature endothelial cells (ECs), however, have limited regenerative capacity. A growing body of evidence indicates that endothelial progenitor cells (EPCs) promote endothelial repair and contribute to ischemia-induced neovascularization. Increasing evidence suggests that circulating progenitor cells contribute to postnatal neovascularization except in embro. These cells are derived form bone-marrow, in some physiological and pathological status, they are released from bone-marrow into circulation, home to sites of ischemia, adopt an endothelial phenotype, and contribute to new blood vessel formation. Hence, the discovery of candidate molecules able to augment EC funtion to stimulate myocardial angiogenesis has stirred a growing interest in using these molecules for therapeutic application.Shuangshentongguan (SSTG) is made up of ginsenosides, salvianolic acids and corydalines. Our previous pharmacological studies showed that SSTG could relieve myocardial damage of dog and/or acute myocardial ischemia of rat, show protective effects on myocardium infartion. But, there is little information about its'effect on theraputic angiogenesis and its matierial basis.So, we designed the experiments to test SSTG effects on angiogenesis, it is realised by 3 models, they are as follows:chick embryo chorioallantoic membrane, endothelial progenitor cell and myocardial ischemia in rats induced by ligation of the left anterior descending (LAD) coronary artery.Chapter 1:Effects of SSTG on angiogenesis of the chick embryo chorioallantoic membranePart 1:Effects of SSTG on angiogenesis of the chick embryo chorioallantoic membraneObjective:To investigate the effect of SSTG on angiogenesis of the chick embryo chorioallantoic membrane (CAM). Methods:Fifty 8-day-old fertilized eggs were obtained from a commercial source (Beijing Merial Vital Laboratory Animal Technology Co., Ltd). On day 9 of the incubation, the fertile eggs were randomly allocated into five groups. Fertilized eggs are first disinfected with 75%alcohol, a window opening is punctured at the blunt end of the egg facing upwards using sterilized forceps. The eggshell above the air cell was removed and the shell membrane attached to it, was cut off for further examination. Normal development was verified and embryos with malformations or dead embryos were excluded. CAMs (10 eggs per group) were treated as described as follows:1) overlaying them with 20-mm2 sterilized filter discs that had been loaded with 10μ1 of serum without any test materials (negative control),10μl of serum containing SSTG, ginsenosides, salvianolic acids and corydalines. The window was sealed with tape and the eggs were returned to the incubator. After incubation for an additional 72 hours, all samples were immediately fixed in methanol and acetone (1:1) for at least 15min, then they were photographed using a stereomicroscope equipped with a camera and image analysis system. The angiogenic response was evaluated by counting the vessel density converging toward the filter discs..Result:The angiogenesis area in SSTG, ginsenosides, salvianolic acids groups was obviously increased compared with control group (P<0.05, P<0.05, P<0.05). Conclusion:The result showed that SSTG, ginsenosides, salvianolic acids can obviously stimulate angiogenesis of the chick embryo CAM.Part 2:Effects of salvianolic acids, salvianolic acid A and salvianolic acid B on angiogenesis of the chick embryo chorioallantoic membraneObjective:To investigate the effect of salvianolic acids, salvianolic acid A and salvianolic acid B on angiogenesis of the chick embryo chorioallantoic membrane (CAM). Methods:the same as above-mentioned. Result:The angiogenesis area in salvianolic acids 100 mg/L (2.97±0.39),300 mg/L (3.07±0.36) groups was obviously increased compared with control group (2.47±0.35) (P<0.05, P<0.05); the angiogenesis area in salvianolic acid A 5.56 mg/L (4.42±1.16),16.67 mg/L (5.51±1.15),50 mg/L (4.60±0.71) was obviously increased compared with control group (3.07±0.85) (P<0.05, P<0.001, P<0.01); the angiogenesis area in salvianolic acid B 50mg/L (2.18±0.31),150 mg/L (2.43±0.32) was obviously increased compared with control group (1.86±0.30) (P<0.05, P<0.01). Conclusion:The result showed that salvianolic acids, salvianolic acid A and salvianolic acid B can obviously stimulate angiogenesis of the chick embryo CAM. Chapter 2:Effects of Salvianolic acids (SAs) and Salvianolic acid A on the numbers and function of endothelial progenitor cellsPart 1:cultivation and identification of endothelial progenitor cellsObjective:To investigate the methods of isolation, cultivation and identification of endothelial progenitor cells (EPCs) from rat spleen, as well as their ability of differentiating into endothelial cells. Methods:The mononuclear cells were isolated f rom SD rat spleen and were cultured in vitro via adhesion selection methods. The expression of CD34 and VEGFR-2 were assessed by immunocytochemistry after 9d cultivation. And the adherent cells were stained with DiI complexed acetylated low-density lipoprotein (DiI-acLDL) and fluorescein Ulex Europaeus agglutinin-1 (FITC-UEA-1), and then viewed by laser scanning confocal microscope (LSCM) to confirm EPCs lineage. EPCs phenotype was also tested by ultrastructural analyses. And EPCs were assessed by lightmicroscopy for their capacity of independent tubulogenesis. Results:The adherent cells stretched and exhibited the clone-like morphology in about 20d of cultivation. Immunocytochemistry stain showed that the adherent cells were positive for CD34 and VEGFR-2 from 9d after culture. The cells could take up DiI-acLDL, and bind to FITC-UEA-1, showed double positive fluorescence under LSCM. EPCs phenotype was also confirmed by the presence of Weibel-Palade body in cytoplasm by ultrastructural analyses. Branched interconnecting EC-specific tubules formed with EPCs. Conclusion:EPCs are enriched in rat spleen and may exhibited some of the characteristics of endothelial cells (ECs) after 9d inducing culture in vitro.Part 2:Effects of Salvianolic acids (SAs) on the numbers and function of endothelial progenitor cells in normal and H2O2 injured stationObjective:Salvia miltiorrhiza (SM, also known as DanShen) is one of the well-known widely used Chinese herbal medicines in clinical. In this study we aimed to demonstrate the effects of Salvianolic acids (SAs), the main active components of aqueous extract of SM on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station. Methods:To do this, new-born rat spleen mononuclear cells were isolated by density gradient and EPCs were expanded. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, transwell chamber assay, and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Results:Incubation of EPCs with SAs increased the numbers of EPCs and promoted EPCs migratory, adhesive, and in vitro vasculogenesis capacity, could also augment function of EPCs injured by H2O2. Conclusion:The results of the present study suggest that SAs may be developed as a new therapeutic remedy for various ischemic diseases such as coronary artery disease.Part 3:Effects of Salvianolic acid A on the numbers and function of endothelial progenitor cells in normal and H2O2 injured stationObjective:Salvianolic acid A (SA)is one of the well-known widely used Chinese herbal medicines in clinical. In this study we aimed to demonstrate the effects of SA on the numbers and function of endothelial progenitor cells in normal and H2O2 injured station. Methods:same as above-mentioned. Results:Incubation of EPCs with SA increased the numbers of EPCs and promoted EPCs migratory, adhesive, and in vitro vasculogenesis capacity. Besides, SA could augment function of EPCs injured by H2O2 Conclusion:The results of the present study suggest that SA may be developed as a new therapeutic remedy for various ischemic diseases such as coronary artery disease.Chapter 3:Effects of Salvianolic acid A on angiogenesis after myocardial ischemia in rats and underlying mechanism.Objective:To investigate the effects of SA on angiogenesis after myocardial ischemia in rats and underlying mechanism. Methods:myocardial ischemia was induced by occluding the left anterior descending (LAD) coronary artery of the hearts of wistar rats. After operation, they were randomly departed into 6 groups: vehicle-treated, SA 2.5,5,10mg/kg groups, positive group and sham-operated group. On 2 day of operation, ischemiac rats received tail intravenous injection once a day. At 7 day post ischemia, these experiments were done:1) to test of area of ischemiac myocardium (NBT staining); 2) to observe pathological chang in myocardium by HE staining; 3) to evaluate the localization and expression of VEGF and VEFGR-2 and CD34-labeled microvascular density (MVD) of the ischemic boundary zone by immunohistochemistry methods; 4) to test SDF-1 and MMP-9 expression in serum by ELISA. Results:compared to sham-operated rats CD34 positive cells(P<0.05), the expression of VEGF (P<0.001) and VEGFR-2 (P<0.001) significantly augmented, exposure to SA with 10,5mg/kg markedly promoted the expression of VEGF (P<0.001, P<0.05) and VEGFR-2 (P<0.001, P<0.001) and CD34-labeled MVD (P <0.001,P<0.001,P<0.001) of the ischemic boundary zone compared to vehicle-treated rats; compared to sham-operated rats, the expression of SDF-1 (P<0.05) and MMP-9 (P<0.05) significantly augmented, exposure to SA with 10,5,2.5mg/kg markedly promoted the expression of SDF-1 (P<0.01, P<0.05) and MMP-9 (P<0.001, P<0.001, P<0.05). Conclusion:the results demonstrated that SA could decrease size of infarciton in myocardial ischemic rats, the underlying mechanisms might be to promote angiogenesis mediated by upregulating expression of VEGF and VEGFR-2, and increasing of SDF-1 and MMP-9.Taken all together, Salvianolic acid A is one of the effectvie material basis of the promotion of angiogenesis by SSTG the underlying mechanisms might be to promote angiogenesis mediated by upregulating expression of VEGF and VEGFR-2, and increasing of SDF-1 and MMP-9.
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