Effect Of Methionine On Angiogenesis And Key Gene Expression In Porcine Endothelial Cell

The placenta is located at the maternal-fetal interface and modulates the in utero environment to promote optimal fetal development.The dense networks of blood vessels within the placenta are responsible for proper fetal growth throughout pregnancy.It has been found that increasing the methionine level in the diet during pregnancy can increase the litter weight and the birth weight of the piglets.However,the regulatory role of methionine on placental vascular development and its regulatory mechanisms are unclear.In this study,porcine endothelial cells were used as the research object,a method of co-culturing porcine endothelial cells and porcine trophoblast cells was established.The regulation of methionine on angiogenesis in porcine endothelial cells was studied.Further studies were conducted from the perspective of key gene expression regulation.The key mechanism of methionine regulation of angiogenesis key genes in endothelial cells.The main research contents and results are as follows:1.Effect of increasing methionine concentration on angiogenesis in porcine endothelial cells.By establishing an in vitro co-culture model of porcine endothelial cells and porcine trophoblast cells,The effect of increase the physiological concentration(0.05 mmol/L)of methionine to 0.2 and 0.5 mmol/L on angiogenesis of of porcine endothelial cells in cultured porcine endothelial cells alone and under co-culture conditions were investigated.The results showed that increasing the methionine levels significantly promoted endothelial cells tube formation regardless of culture or co-culture conditions(P<0.05),but there was no interaction between methionine and culture conditions,suggesting that methionine promoting endothelial cell angiogenesis doesn't rely on trophoblast cells.2.Increased methionine levels affect the primary event of angiogenesis in porcine endothelial cells.After increasing methionine levels,the number of tube fornation,the total branch length,and the total number of nodes in endothelial cells increased significantly(P<0.05).By MTT assay and flow cytometry,the effects of different concentrations of methionine treatment on cell proliferation were examined.The results showed that with the increase of methionine levels,cell viability increased significantly(P<0.05),but there was no effect on cell cycle.Using wound-healing assy and trans-well assy assays to determine the effect of increasing methionine levels on cell migration,it was found that as the concentration of methionine increased,cell migration was significantly increased compared to physiological concentrations of methionine(P<0.05).It was demonstrated that increasing the methionine level mainly affects the cell migration events in endothelial cell angiogenesis.3.Methionine regulates the key genes of angiogenesis in endothelial cells.The expression of mRNA genes related to cell migration and angiogenesis gene in endothelial cells were detected.The results showed that increased methionine levels(0.05 mmol/L Vs 0.5 mmol/L)had no effect on mRNA expression of MMP2,but significantly increased mRNA levels of MMP9,VEGF-A,VEGF120,VEGF164,VEGFR-1(P<0.05),and the negative angiogenic gene s VEGFR-1 were also increased(P<0.05).Under the condition of 0.5 mmol/L methionine,the mRNA expression of VEGF-A in porcine endothelial cells was detected by different treatment time of methionine.The results showed that with the increase of methionine treatment time,the mRNA expression of VEGF-A gradually increased.When the treatment time reached 24 h,the expression of VEGF-A mRNA was significantly promoted(P<0.05).In addition,methionine different treatment time or different concentrations of methionine had no effect on VEGF-A mRNA expression in porcine placental trophoblast cells.4.VEGF-A plays a role in mediating methionine-promoting angiogenesis in endothelial cells.In this study,cells were treated with the VEGFA inhibitor Bevacizumab,and the role of VEGF-A in mediating methionine levels to promote endothelial cell angiogenesis was investigated.The results showed that compared with the high methionine treatment group,the cell migration ability of the high methionine plus Bevacizumab group was significantly reduced(P<0.05),but there was no significant difference compared with the physiological concentration of methionine.In the angiogenesis assay,the number of tube formation,total branch length and total number of nodes formed by the high methionine plus Bevacizumab treatment group was significantly lower than that of the high concentration methionine treatment group(P<0.05),and the total branch length was still significant increased compared with the physiological concentration of methionine(P<0.05),there is no significant difference in the total number of nodes.It was demonstrated that VEGF-A plays a key role in mediating methionine-promoting angiogenesis in endothelial cells.5.Study on the mechanism of methionine to regulate the expression of VEGF-A.Increased mRNA levels of VEGF-A may involve the regulation of transcriptional and post-transcriptional levels.This study examined the effect of methionine on the ability of RNA polymerase II to bind to the CDS region of the VEGF-A gene by chromatin immunoprecipitation.The results showed that there was no significant change in the binding of RNA polymerase II to VEGF-A at high methionine levels.Treatment with actinomycin D showed that the mRNA stability of VEGF-A in the high methionine treatment group was significantly higher than that in the low methionine treatment group when the treatment time was increased to 30 and 60min(P<0.05).It was demonstrated that increasing methionine had no effect on the level of VEGF-A transcription,but significantly affected the post-transcriptional stability of VEGF-A mRNA.In summary,the conclusion of this study is:1.Increasing methionine levels promotes migration and angiogenesis of porcine endothelial cells.However,this effect does not depend on trophoblast cells.2.Methionine promotes endothelial cell migration and angiogenesis by increasing VEGF-A levels.3.Methionine increases VEGF-A mRNA levels by affecting post-transcriptional stability of VEGF-A mRNA.