Effects Of Urotensin Ⅱ On Collagen Synthesis And Intracellular Signal Pathway In Pulmonary Arterial Smooth Muscle Cells

Background and ObjectivesPulmonary hypertension (PH) is a complicated clinical syndrome, defined as the mean pulmonary artery pressure greater than average25mmHg at rest, as measured by right heart catheterization (RHC). Congenital heart disease (CHD) is the most common pediatric heart disease. According to statistics, per1,000live births, the disease occurred about6～8babies and about150,000newborns suffer from various types of CHD each year in China. While pulmonary hypertension is a severe and frequent complication in patients after stroke, whose features are poor prognosis and high mortality. The left to right shunt CHD, such as atrial septal defect (ASD), ventricular septal defect (VSD), and patent ductus arteriosus (PDA) are common among children. With the development of disease, the blood flow of the pulmonary continues to increasing, pulmonary hypertension is formed, gradually, worse, developed into Eisenmenger syndrome, who maybe lose the opportunities of surgical operation. There is no effective treatment for pulmonary hypertension at present. The characteristic pathological changes of pulmonary hypertension are pulmonary vessels constriction response increasing and pulmonary vascular structure reconstruction. The former is functional alteration, which is reversible. The pulmonary vascular structure remodeling is pulmonary artery smooth muscle cells hypertrophy, proliferation and deposition of extracellar matrix such as collagen on the pulmonary vascular walls, result in the vascular walls thickening and luminal stenosis, that structural changes are irreversible.In recent twenty years Urotensin Ⅱ (UII) is attracting broad attention as an important regulatory factor for cardiovascular diseases (CVD). UII was originally isolated from the caudal neurosecretory of teleost fish in1985. Subsequently, the pre-proUII cDNA has been characterized in humans in1998. UⅡ is currently the most potent vasoconstrictor identified. Recent studies have indicated that UⅡ is the endogenous ligand for the orphan receptor GPR14, a G-protein-coupled receptor, now termed the "UT receptor". UⅡ and its receptor (UT receptor) display greatest expression in nervous system and cardiovascular system. UⅡ binds with UⅡ receptor UT inducing series of the biological effect in body, including vasoconstriction, cell proliferation, cell migration and so on. UⅡ plays an essential role in the development of pulmonary hypertension. Previous studies have discovered that Urantide, a UⅡ receptor antagonist, can inhibited the effect of UⅡ.This study is to investigate the UrotensinⅡ(UⅡ) and its antagonist urantide on collagen Ⅰ, collagen Ⅲ synthesis, and to study the mechanism of UⅡ-mediated mitogenic signal transduction in cultured rat pulmonary arterial smooth muscle cells(PASMCs), provide the theory basis for that Urantide may regulate the proliferation of PASMCs through ERK1/2signaling pathway and then delay the development of pulmonary hypertension.MethodsRat pulmonary arterial smooth muscle fragments were isolated and cultured with explant culture technique in vitro and the harvested cells were used in our study. Cells were divided into four groups:①UⅡ group:10-10mol/L～10-7mol/L UⅡ were added to the culture medium in order to observe the effect of UⅡ on the proliferation of cultured PASMCs.②UⅡ and Urantide group:cells were pretreated in medium for half an hour with urantide (10-7mol/L), followed by UⅡ (10"7mol/L) to explore the antagonism of urantide against the effect of UⅡ.③UⅡ and PD98059group:PD98059(10-5mol/L) was incubated30minutes prior to UⅡ treatments before to detect the effect of PD98058on the ERK1/2phosphorylation.④control group:do nothing treatment on the cells. The effect of UⅡ and Urantide on the proliferation of PASMCs was measured by cck-8method and BrdU incorporation. The protein and gene expression levels of collagen ⅠⅡ, collagenⅢ and p-ERK1/2in cultured PASMCs induced by UⅡ and urantide were evaluated by western blotting and real-time PCR, respectively. Meanwhile, detect the effect of PD98059on the phosphorylation levels of ERK1/2.Results:①UII (10"9mol/L～10-7mol/L) promoted the proliferation of PASMCs in a dose-dependent manner, with maximal effect at a concentration of10-7mol/L (P<0.05, P<0.001); it also caused a dose-dependent increased in collagen Ⅰ、Ⅲ synthesis and the levels of phosphorylated-ERK1/2, with maximal effect at a concentration of10-7mol/L (P<0.05, P<0.001).②Urantide inhibited Ull-induced cell proliferation, collagen synthesis and phosphorylation of ERK1/2(P<0.01).③PD98059significantly suppressed the UII-induced protein and gene expression levels of p-ERK1/2(P<0.05).