The Role Of Platelet Derived Growth Factor-BB In Cultured Cardiomyocytes Exposed To Hypoxia

BackgroudCardiomyocytes are sensitive to hypoxia.Hypoxia can not only lead to the abnormality of cardiac electrical activity,but also the necrosis and apoptosis of cardiomyocytes. Many kinds of ischemic heart diseases including coronary atherosclerosis heart diseases can lead to hypoxic damage.In clinical practice, drug treatment is a common strategy in treating cardiac ischemia. The effect of treatment on heart failure is not ideal. Therefore, carrying out the mechanism of hypoxic damage to cardiomyocytes and finding a new method to repair heart failure are scientific problems that need to solve.Platelet derived growth factor is a kind of important growth factor which including PDGF -AA,PDGF -AB,PDGF-BB,PDGF -CC and PDGF -DD.In physiological state,PDGF exists in platelets.PDGF existed in damaged endothelial cells,macrophages,smooth muscle cells and fibroblasts would release when the tissue was demaged.The main effect of PDGF is promoting cell divison,chemotaxis and vasoconstriction.In PDGF family,PDGF-BB most closely relates to cardiovascular diseases.PDGF can induce the proliferation and migration of endothelial cells, vascular smooth muscle cell, which is a growth fator to promoting angiogenesis growth factors and regeneration.Platelet derived growth factor mainly promoted the proliferation of vascular endothelial cells in previous reports and also played an important role in some specific cell survival in subsequent research,also,it take part in regeneration after the apex of heart. PDGF-BB can effectively promote formation of new blood-vessels in the infarcted myocardium and further decrease the area of the infarct. Therefore, PDGF-BB may tend to protect the cardiomyocytes. We propose that PDGF-BB may be a protective factor for myocardial cells based on these reports. Previous study showed that PDGF can stimulate cell differentiation and proliferation. Meanwhile, hypoxia can increase the expression of PDGF-BB in vascular endothelial cell. These results all study indicate that PDGF may be a crucial molecular in myocardial cells under the condition of hypoxia. Apoptosis is an improtant reason of heart failure, cardiac arrhythmias and hemodynamic dysfunction.Ischemia and hypoxia can induce damage of myocardial cells through many ways to lead to the necrosis and apoptosis of cells.Thus,we need to know the changes of endogenic PDGF-BB after the hypoxia of cardiomyocytes.Then, the protective role of exogenous PDGF-BB on culturing myocardial cells under the condition of hypoxia in vitro and the related potential molecular mechanism need further research.At present, exogenous PDGF-BB overexpression in cells is induced by PDGF-BB transduction that has been reported, but the expression efficiency need to improve. Gene transduction technology means that purified exogenous gene was transducted into cells to research the function. Gene transfection technology means that purified exogenous gene was transferred into cells to research the function. Viral vectors, such as retrovirus vectors, adenovirus, adeno-associated virus, lentivirus and herpes simplex virus (HSV), are the most effective ones. Adenovirus could contain large gene information and avoid transgenic gene inserting into genome. But there is a strong immunogenicity and long period in this adenovirus combination. Lentivirus vectors, come from HIV-1, have an advantage in infection ability. But the gene inserted site is uncontrolled, which restricts its application. HSV-1, a double DNA virus about 152 kb, half of total 84 genes in HSV genome is unessential.Large gene information, as more as 30 kb, could be inserted into after these genes are deleted. Moreover, expression of HSV-target gene could be restricted in some certain cells after some unessential gens were deleted. HSV has a strong infection ability,therefore, application of HSV-1 has a bright future in vectors for transgene.The study includes two parts.The study includes two parts.Part One:Changes of PDGF-BB of cardiomyocytes exposed to hypoxia.Objective:To observe the degree of injury of cardiomyocytes of neonatal rats sufferting hypoxia and to investigate the expression of PDGF-BB both in gene level and protein level in order to explore changes of PDGF-BB in the cardiomyocytes after cultured in hypoxia conditions.Methods1.The culture of cardiomyocytes and the establishment of hypoxia model of cardiomyocytesHearts from neonatal SD rats at 2-3 days were isolated to the culture of cardiomyocytes using 0.1% trypsin and 0.16% Ⅱ collagenase, which were divided into normal group and hypoxia group randomly. And cells in hypoxia group were placed in the incubator for anoxia handling with the setting conditions of 37℃, 95%N2 and 5%CO2 for 3h,6h and 12h, respectively. Thus, Cardiomyocytes were devided into NC group, H3h Group, H6h Group and H12h Group randomly.2.Detection of damage to the cardiomyocytes in this hypoxia modleObserve cardiomyocytes in hypoxic and normoxic state.LDH kit was used to detect the content of lactate dehydrogenase to know the damage to the cardiomyocytes in this hypoxia modle.TUNEL kit was used to detect the apotosis of cardiomyocytes after cultured in hypoxia condition for 3h,6h and 12h.3. Detection of expression of PDGF-BB in cardiomyocytes in hypoxia conditionImmunofluorescence technique was used to identify cardiomyocytes. In order to determine the gene expression and protein expression of PDGF-BB in hypoxia condition, both quantitative polymerase chain reaction(Q-PCR) and western blotting were performed.4.Statistical analysisAll data were presented as the mean±SEM. Raw data were analyzed with SPSS 17.0 software. Each experimentwas repeated three times.Statistical analysis for multiple group comparisons was performed by one-way ANOVA, followed by post hoc least significant difference (LSD) tests. All P values were two sided, and p<0.05 was considered statistically different.ResultsOn the phenomenon level, we have gotten these results as follows:1.The immunofluorescence results showed the purity of cardiomyocytes was over 98%. In normal group, the cardiomyocytes grew well and the cell kept intact. While in hypoxia group, the cell menbrane shrinkaged, cytoplasmic granules increased, foot processes reduced or even disappeared. Moreover, cell beating was weak and irregular.2.The results of LDH level showed, compared with normal group, the level of cardiomyocytes in H12h was significantly higher (p<0.05). The results of TUNEL staining showed that the rate of apoptosis was remarkedly raised after hypoxia. Compared with normal group, the apoptosis of cardiomyocytes in H12h group increased markedly(p<0.05).3. The results of Q-PCR:Compared with normal group, the level of PDGF-BB mRNA of cardiomyocytes in H12h group was significantly upregulated (p<0.05).4.The results of Western Blot:While compared with normal group, the expression of PDGF-BB in cardiomyocytes was greatly increased in H12h group (p<0.05).Part Two:Mechanism of the effect of PDGF-BB in cardiomyocytes exposed to hypoxia.ObjectiveTo construct the recombinant vector NX01-U6H1-rPDGFi and to confirm the transduction rate.To transduct the cardiomyocytes and to observe whether the lower expression of PDGF-BB would increase hypoxia-induced apoptosis to explore the possible role of endogenous PDGF-BB in cardiomyocytes.To construct the recombinant vector HSV-CMV-PDGF so as to transduct the cardiomyocytes and to observe whether the higer expression of PDGF-BB would decrease hypoxia-induced apoptosis to explore the protective role of exogenous PDGF-BB in hypoxia-induced apoptosis in cardiomyocytes. To study the possible associated signal pathway under hypoxia by detecting the expression of PI3K, AKT and ERK in these cardiomyocytes.Methods1. Construction of HSV-PDGF-BB recombinant vector.The recombinant of HSV-PDGF-BB was estabilished in order to explore the role of PDGF-BB in cardiocytes exposed to hypoxia. PDGF-BB coding sequence and interference RNA was cloned into plasmids, and enveloped by using HSV-1. Cardiomyocytes were conducted by HSV--PDGF-BB recombinant vectors and cultured in hypoxia condition.Then we detected the expression of PDGF-BB by western blot.2.Changes of apotosis in cultured cardiomyocytes after over expression and lower expresion of PDGF-BBCardiomyocytes were divided into five groups randomly:nomorxia group(NC group),,hypoxia group(H group), empty vector group under hypoxia(H+vector group), PDGF-BB overexpression group (H+PDGF-BB group)and PDGF-BB lower exprssion group (H+SiPDGF-BB group) under hypoxia condition randomly.LDH and TUNEL kits were used respectively to detect LDH level and the apotosis of cardiomyocytes after transduction with the recombinant vectors to observe the change of apotosis after over expression and lower expresion of PDGF-BB.3.Mechanism of the Anti-apoptotic effect of PDGF-BBWe detected the level of PI3K、AKT、pAKT、ERK、pERK before and after hypoxia and observe the difference between these groups to study the possible associated signal pathway under hypoxia4.Statistical analysisAll data were presented as the mean±SEM. Raw data were analyzed with SPSS software. Each experimentwas repeated three times.Statistical analysis for multiple group comparisons was performed by one-way ANOVA, followed by post hoc least significant difference (LSD) tests. All P values were two sided, and P<0.05 was considered statistically different.ResultsOn the functional level, we have gotten these results as follows:1.HSV-1 recombinant vector HSV-CMV-PDGF and NX01-U6H1-rPDGFi have been successfully constructed. The transduction efficiency of HSV-1 is 80%.PDGF-BB level was upregulated in cardiomyocytes constructed with HSV-CMV-PDGF compared with empty vector group under hypoxia condition.(p<0.05). PDGF-BB level was decreased in cardiomyocytes constructed with NX01-U6H1-rPDGFi compared with empty vector group under hypoxia condition (p<0.05).2.1n order to know the role of endogenous PDGF-BB, we performed PDGF-BB knockdown in cultured cardiomyocytes. There was a marked increase on the apoptotic number of cells and the level of LDH in H+SiPDGF-BB group compared with H group and H+vector group(p<0.05).3.In order to know the role of exogenous PDGF-BB,the cardiomyocytes were transducted with empty vector and HSV-1 carried PDGF-BB overexpression.There was a marked decrease on the apoptotic number of cells and the level of LDH in H+PDGF-BB group compared with H group and H+vector group(p<0.05).. Overexpression of PDGF-BB in cardiomyocytes help to repair damages induced by hypoxia.4.The expression of PI3K and pAKT were higher in H+PDGF-BB group than in H group and H+vector group (p<0.05). The expression of pERK have no difference in these groups. PI3K/AKT may be involved in the protective effect of PDGF-BB on cardiomyocytes suffering hypoxia.Conclusions1.Our results showed apoptosis of cardiomyocytes could be induced by hypoxia, companied by the upregulated expression of PDGF-BB.2.Our findings suggested PDGF-BB could alleviate apoptosis of cardiomyocytes induced by hypoxia.3.Our findings suggested PDGF-BB may regulate PI3K-AKT signaling pathway to affect apoptosis of cardiomyocytes cultured in vitro. Our results may contribute to the new therapeutic strategies involving myocardial ischemia by using PDGF-BB interventional methods.