The ADMA/DDAH Pathway Regulates CT-1-Induced Angiogensis

Background and Objective:Angiogenesis is one of the modes of vasculogene-sis,and is the formation of new blood vessels from pre-existing vessels,which is complex and involves several discrete steps such as extracellular matrix degradation,proliferation and migration of endothelial cells,and morphological differentiation of endothelial cells to form tubes.Recently,a series of studies demonstrated that a certain degree of angiogenesis is accompany with the pathological and physiological procedure of myocardial hypertrophy.Cardiotrophin-1(CT-1)is a member of the interleukin-6 super family of cytokines.CT-1 has been shown to mainly associate with myocardial hypertrophy.But it was not clear whether CT-1 could induce angiogenesis in the process.A series of signal transduction pathway have been shown to involve in the regulation of angiogenesis.ADMA/DDA-H pathway play an important role in angiogenesis.We hypothesized that CT-1 may activate ADMA/DDAH pathway and exert the function of angiogenesis.In this study,an eukaryotic expression vector for CT-1 was constructed successfully and HUVECs were transfected with it.We will investigate whether CT-1 could induce endothelial cells proliferation,migration and formation of blood vessels.In addition,we will further explore whether CT-1 could regulate angiogenesis through the ADMA/DDAH pathway from molecular level.lt will be a new target for the therapy of ischemic heart disease.Methods:(1)pEGFP-N1-CTF1-GFP and pEGFP-N1 were constructed,and were transiently transfected to HUVECs using LipofectamineTM 2000.(2)HUVECs were divided into four groups:?HUVECs group:normal control,? GFP group:transfected with pEGFP-N1,? CT-1 group:transfected with pEGFP-N1-CTF1-GFP,? CT-1+ADMA group:24 hours post transfecting with pEGFP-N1-CTF1-GFP,HUVECs were cultured with 100?mol/L ADMA.(3)48 hours post transfecting,endothelial cells proliferation assay was evaluated using MTT method,migration assay was performed using transwell,tube formation test was examined on Matrigel.Simultaneously,the cells were collected to prepare total RNA and protein lysates.The mRNA and protein levels of CT-1 and other related genes were mesured by qRT-PCR and western blot,respectively.Then,the level of ADMA and the activity of DDAH were measured by High Performance Liquid Chromatography,while NOS activity and the concentration of NO were assayed by L-[3H]citrulline production from L-[3H]arginine.(4)We used statistical analysis software SPSS 14.0 to calculate standard deviation(SD)and statistically significant differences between samples.Data were presented as means±SD.The statistically significant changes with P values calculated by Student T Test,P values<0.05 were considered statistically significant.Results:(1)PCR identification and DNA sequencing showed that the eukaryotic expression vector for CT-1 was constructed successfully.The transfection efficiency was 40%-60%at 48h after transfetion.Accordingly,we selected the time point of 48h post-transfection for further study.(2)The MTT assay,transwell assay and tube formation study indicated that comparing with control,up-regulated of CT-1 significantly incressed the proliferation,migration and number of tube formation of endothelial cells.Whereas transfected with CT-1 and cultured with ADMA decreased the proliferation,migration and number of tube formation of endothelial cells.(3)Overexpression of CT-1 decreased the concentration of ADMA,while improv-ed the protein expression levels of VEGF,DDAH I and DDAHII and elevated the activity of DDAH.Also,we found that the mRNA levels of eNOS,the activity of NOS and the concertration of NO were increased accompanied with CT-1 overexpression.However,transfected with CT-1 and cultured with ADMA could partly attenuate these results induced by up-regulated CT-1.Couclusions:(1)The eukaryotic expression vector for CT-1 was constructed successfully.The expression of CT-1 was up-regulation obviously in HUVECs.(2)Overexpression of CT-1 significantly increased the HUVECs proliferation,migration and formation of blood vessels.CT-1 may induce angiogenesis.(3)CT-1 may regulate angiogenesis through ADMA/DDAH pathway.It will be a new target for the therapy of ischemic heart disease.