The Wnt Signaling Pathway Involved In Myocardial Infarction Myocardial Tissue Of The Healing Process Control,

Myocardial infarction (MI) is one of the leading causes of morbidity and mortality in the whole world. Restoration of the blood supply to the infarcted area and prevention of cardiac remodeling to improve the cardiac function have been the subjects of study for many years. Recently, studies in animals and humans pointed out the importance of the infarct itself as a potential target for intervention, so more attention has been paid to the wound healing process after MI.Although, the mechanisms that govern the architectural control of the processes involved in infarction healing remain to be elucidated, it has been suggested that the migration and proliferation of cells, including myofibroblast and endothelial cells may contribute to these processes. Myofibroblasts not only take responsibility for the deposition of the extracellular matrix in the granulation tissue but also possess contractile properties that prevent the dilatation of the infarction area. Endothelial cells are thought to be involved in neovascularization process of the infarction area.The Wnt signaling pathway is thought to be functionally conserved in vertebrates and invertebrates and plays an important role in cell proliferation, differentiation, organogenesis and oncogenesis. And several members of the Wnt-frizzled signal transduction pathway were found to be expressed during cardiac development in vertebrates. In cardiovascular pathology, re-expression of a fetal gene expression pattern is a generally observed phenomenon.To verify the assumption that Wnt-frizzled pathway may be involved in the repair process after MI, we designed both in vivo and in vitro experiments. First, we established the disease model, the left descending coronary arteries of rats were ligated to induce MI. Immunohistochemistry SABC method and in situ hybridization were performed to detect the expression of two of the most important signaling molecules β -catenin and Dishevelled-1.The results indicated that on day 4 after MI, p-catenin positive cells appeared in granulation tissue and the border zone of the infarct area; the expression of p-catenin increased significantly at 7 days after MI and returned to levels comparable to the sham samples 28 days later. After MI the expression of Dvl-1 gene precede that of P-catenin and 1 day after MI the Dvl-1 mRNA could be easily detected at the border zone, although the protein was still rather hard to detect at that time. 4 days after MI the expression of Dishevelled-1 increased exclusively; 7 days after MI, it seems that the expression reached its peak, the positive staining evenspread into the endothelial and smooth muscle cells of the newly formed and pre-existing blood vessels in the infarction area; after that the Dishevelled-1 expression decreased abruptly and could hardly be detected 28 days after MI.It seems that the time-dependent expression of Dvl-1 and the formation of granulation tissue matched well after MI in rats. These results make a clue that Wnt signaling pathway maybe involved in the proliferation and migration of myofibroblasts and endothelial cells during the process of wound healing after MI in vivo.To clarify the molecular mechanism that Wnt signaling pathway may control the growth and several phonotypes of fibrolasts and endothelial cells related to the repair process after MI in vitro, we need to establish a Wnt-signaling-pathway-constantly-activating cell model. Rat-1, a kind of Rat fibroblasts was chosen as experimental material. Full length Wnt-3a cDNA was inserted into myc tagged pcDNA 3.1 vector to construct the Wnt-3a mammalian expression vector, this vector was stably transfected into Rat-1 cells, then we detected the expression of myc tagged Wnt-3a protein in the culture medium of the Rat-1 AVnt-3a cells by Western blot.Further, we verified that overexpression of Wnt-3a protein make cytoplasmic P-catenin accumulaltion. Thus, we are certain that we established Wnt-signaling-pathway -constantly-activating cell model successfully.Then, we investigated the regulation of Wnt signaling pathway to the growth and several phenotypes of Rat-1 cells and the expression level of several genes related to those phenotypes.The results showed that when Wnt signaling pathway was activated constantly, Rat-1 cells exhibited morphological changes: grew more densely as a monolayer, adopted an elongated and refractile appearance, forming cord-like bundles lined up in a uniform direction. The results of MTT assay and FCM analysis indicated that more Rat-l/Wnt-3a cells enter into G2-phase and the proliferation rate of the Rat-1/ Wnt-3a cells increased significantly compared to the non-transfected cells. Though the migration of Rat-1/Wnt-3a cells increased slightly by the method of Transwell migration assay, there was no statistic significance compared to the non-transfected cells. The result of in vitro scrape wound healing assay showed that for Rat-l/Wnt-3a cells the time course of wound healing decreased significantly.And for the Rat-l/Wnt-3a cells, the expression level of proliferation related gene CyclinDl increased significantly, and the expression level of ECM degradation related gene MMP2 decreased significantly, and the expression level of ECM deposition related gene Fibronectin increased exclusively.For this...