The Study On Tissue-repair Of Acute Myocardial Infarction In Rat Model By Rosuvastatin Treatment In Vivo

With the progress of urbanization in modern society, myocardial infarction (MI) and post-MI heart failure become the major threat against public health and cost huge expenditure of health care funding. Post-MI heart failure is one of the major causes of death and disability in the developed world, with a dismal five-year mortality rate of50%-70%in symptomatic patients. Stem cell therapy is a potentially new therapeutic option for post-MI heart failure. Although preclinical studies have shown encouraging results suggesting potential benefits of stem cell-based cell therapy for post-MI cardiac repair and regeneration, the overall clinical benefits are marginal and sometimes transient, showing a lack of durability1.Recent studies have shown that the injury and death of cardiomyocytes could trigger the mechanism of tissue self-repair including the modification of microenvironment and the promotion of cell renewing and angiogenesis in the impaired tissue. The ischemic myocardium can express several cytokines in autocrine and paracrine pattern2. Gene expression analysis after MI revealed increased levels of several genes including stromal-cell derived factor-1(SDF-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9(MMP-9), intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1)3. These molecules act as chemotactic signals to facilitate mobilization and incorporation of stem cells and progenitor cells into the ischemic region. Meanwhile, these cytokines also participate in the inflammation and scar-formation in the injured myocardium. It can be implied that the microenvironment of ischemic tissue is critical to tissue renewing and heart remodeling after MI, which may decide the prognosis of post-MI heart function. The present study is designed to investigate the way and the timing of optimizing the microenvironment, by which can we keep balance between the promotion of tissue renewing and the amelioration of scar formation and heart remodeling.Statins are3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, extensively used to lower plasma cholesterol levels. Additionally, statins can protect the heart against ischemic injury and preserve the post-MI function through mechanisms independent from the lipid-lowering potent. Rosuvastatin is proved to possess remarkable ability in reducing low-density lipoprotein cholesterol levels. It is known that statin can promote the mobilization and incorporation of bone marrow progenitor cells, improve endothelial progenitor cells associated neovascularization in mice with acute MI. However, the mechanisms and the targets of the statins, especially rosuvastatin, in initiating the tissue-repair still remain unknown. We also know little about the modification of ischemic microenvironment by rosuvastin treatment in vivo after acute MI.The present study was designed to address the relationship between oral rosuvastatin treatment and the modulation of cytokines, like SDF-1/CXCR-4axis, HIF-1α, VEGF, ICAM and etc, expressed in circulation and different sites of heart in rat model of acute MI. We also investigated the time course of cytokines expression modulated by rosuvastatin treatment, which may help to optimize the timing of cellular cardiomyoplasty after MI.The study is composed of3parts:Part Ⅰ Rosuvastatin Attenuates Left Ventricular Remodeling and Improves Post-infarction Systolic FunctionMale SD rats (250g) at about6weeks old were randomized to receive either rosuvastatin (5mg/Kg QD) or vehicle (5%CMC-Na) by daily gavage from7days before open-chest surgery for permanent ligation of the left anterior descending coronary artery to establish the acute MI model of left anterior wall. Rats of two groups were on the same dose of either rosuvastatin or vehicle after MI surgery until the day before sacrifice.Echocardiographic measurements of cardiac morphology and heart function were performed on the10th and28th day after MI. Rat hearts were harvested on the28th day after MI for hematoxylin-eosin (HE) and Masson staining.Two-dimensional and M-mode ultrasound cardiographic measurements demonstrated that the volume of left ventricle was decreased in the rosuvastatin group. Compared with the untreated animals, the rosuvastatin-treated ones retained the post-infarct left ventricle function, defined by improved left ventricle ejection fraction (LVEF) and left ventricle short axis fractional shortening (LVFS) on the28th day after MI. We confirmed that the thickness of left anterior wall was increased, the range of infarction was decreased, and the collagen fibrosis was attenuated in the rosuvastatin-treated group through the microscope. These histological changes by rosuvastatin-treatment may contribute to the improvement of the heart systolic function.Part Ⅱ The Modulation of Cytokines by Rosuvastatin after Myocardial InfarctionPlasma levels of SDF-1, VEGF, HIF-1α, VCAM-1and ICAM-1of the animals in two groups were evaluated by ELISA on the3rd,7th, and28th day respectively after MI.Rat hearts were harvested on the4th,7th, and14th day after MI. Real-time RT-PCR and Western blot were performed to assess the expression of SDF-1, CXCR-4, HIF-1α, VEGF, and ICAM-1mRNAs and proteins in peri-infarct region (left anterior wall) and non-infarct region (left posterior wall), repectively.Rats were sacrificed on the28th day after MI and myocardial samples were fixed in4%formaldehyde. Immunohistochemical measurements were performed to detect the expression of SDF-1, CXCR-4, HIF-la, VEGF, and ICAM-1in the ischemic region.The study of Part II demonstrated the following results:The plasma level of SDF-1and VCAM-1were increased after acute MI. Oral rosuvastatin treatment can promote the elevation of plasma SDF-1level within the first week after MI.The expressions of SDF-1, CXCR-4, HIF-la, ICAM-1, and VEGF in myocardium were increased within the first week after MI, and went down in the second week. Compared to the non-infarct region, the cytokine response was more significant in the ischemic region. Rosuvastatin treatment can optimize the local microenvironment of post-MI myocardium by further up-regulating the SDF-1expression and down-regulating the CXCR-4, HIF-la, ICAM-1and VEGF expression.The intensity of ischemic microenvironment modulation by rosuvastatin is of regional and timing difference. The impact of rosuvastatin is more significant in the peri-infarct region in the first week after acute MI.Part Ⅲ Rosuvastatin Increases the Number of Progenitor Cells and Promote Angiogenesis in the Peri-infarct MyocardiumRats were sacrificed on the28th day after MI and myocardial samples of ischemic region were fixed in4%formaldehyde. To evaluate the progenitor cells which may contribute to cardiomyogenesis and angiogenesis in the ischemic region, immunohistochemical measurements were performed to detect the expression of CD90(+)19-20,c-kit (+)21-22and CD133(+)23-26cells. The results demonstrated that rosuvastatin treatment can increase the number of CD90+, c-kit+, and CD133+progenitor cells and promote angiogenesis in the peri-infarct region. It can be implied that the local microenvironment of peri-infarct region plays an important role in recruiting and supporting these progenitor cellsConclusionOur study demonstrated that oral administration of rosuvastatin (5mg/kg QD) from1week pre-infarction to4weeks post-infarction can significantly promote the tissue repair of infarcted territory. The underlying mechanisms of the rosuvastatin treatment are probably as follows:1. Rosuvastatin significantly improves heart systolic function by suppressing the collagen fibrosis, decreasing the size of infarction and ameliorating the heart remodeling after MI.2. Rosuvastatin can optimize the individual internal environment and regional ischemic microenvironment after acute MI by modulating the circulating and local expression of cytokines. Rosuvastatin keeps the balance between tissue renewing and the over response of cytokines by up-regulating the SDF-1expression and down-regulating the CXCR-4, HIF-la, ICAM-1and VEGF expressions in the post-infarct tissue.3. The effect of Rosuvastatin is of timing and regional difference, more significant in the peri-infarct region in the first week after acute MI.4. Rosuvastatin increases the number of CD90+, c-kit+, and CD133+progenitor cells and promote angiogenesis in the peri-infarct region, which may contribute to tissue repair and function preservation.The present study described the expression of SDF-1/CXCR-4pathway, HIF-la, VEGF, ICAM-1, etc in circulation and local myocardium after acute MI in rat. The time course and the regional difference of the cytokine response after acute MI were reported in this research. The study investigated the modification of individual internal environment and ischemic regional microenvironment through modulating the expression of SDF-1/CXCR-4pathway, HIF-1α, VEGF, ICAM-1, etc by rosuvastatin treatment in vivo. To my knowledge, it is the first time to confirm the multiple mechanisms of ischemic injury protection and function preservation after myocardial infarction by rosuvastatin intervention in vivo, including up-regulation of SDF-1level, mobilization of stem cells, suppression of cytokines over-responses, attenuation of fibrosis and scar-formation, and amelioration of heart remodeling. The results of the study provide the evidence for therapeutic potent of rosuvastatin in cytokine modulation in cellular cardioplasty for ischemic heart disease.