The Effects RPL22on ET-1Induced Proliferation Of Human Pulmonary Arterial Smooth Muscle Cells

Pulmonary hypertension (PH) is a clinically insidious and complex disease involving both the cardiovascular and pulmonary systems. The hemodynamic characteristic of PH is progressive elevation of pulmonary vascular resistance. According to the updated guidelines of ESC/ERS2009, PH has been defined as an increase in mean pulmonary arterial pressure≥25mmHg at rest as assessed by right heart catheterization (RHC). PH is a disease involved variety of etiological factors and can be developed from deferent diseases. Due to the increased pulmonary artery pressure, PH patients are suffered from right heart overload and often complicated by right ventricular hypertrophy, right heart failure and serious arrhythmia. Sudden death is the most dangerous complication. PH has high morbidity and mortality, the patient’s prognosis is very poor. Although there is already several decades of research history, the mechanisms of this disease are still not very clear. Clinically, there lack of ideal therapeutic methods.The pulmonary circulation is a low-pressure, high-flow system with a great capacity for recruitment of normally unperfused vessels. As a consequence, the walls of pulmonary arteries are thin, in keeping with their low transmural pressure. Studies showed that abnormal pulmonary vascular contraction, pulmonary vascular wall remodeling, thrombosis in situ are the most important pathophysiological changes of PH. The pathological lesions of PH mainly affect the distal pulmonary arterials (<500μm of diameter), characterized by vascular narrowing leading to a progressive increase in pulmonary vascular resistance. Pulmonary vascular wall has a three-tier structure, the inner are endothelial cells (ECs), the middle cells are pulmonary arterial smooth muscle cells (PASMCs) and the outside are fibroblasts. Under pathological conditions, cells structure of the pulmonary vascular occur different degrees of proliferation. The pathological lesions are medial hypertrophy, intimal proliferation and fibrotic changes, adventitial thickening with moderate perivascular inflammatory infiltrates, complex lesions and thrombotic lesions, pulmonary capillary bed damage and neovascularization. These pathological lesions lead to increase resistance of the pulmonary cavity, resulting in pulmonary hypertension. In the pathophysiology of these vascular changes, medial hypertrophy of pulmonary artery wall is particularly significant. It is believed that proliferation of PASMCs is the major pathophysiological basis for the development of PH. The development of PH involves a complex and heterogeneous constellation of multiple genetic, molecular, and humoral abnormalities, presenting a final manifestation of vascular remodeling in which fibroblasts, smooth muscle and endothelial cells all play the roles. Several mechanisms have been proved involving in the PASMCs abnormal proliferation in PH, though the mechanism is not clearly understood.Ribosome is a cellular organelle where mRNAs are translated into proteins in all eucaryon cells. Ribosome has about80different proteins and ribosomal dysfunction can affect protein synthesis and protein function. Ribosome proteins (RPs) are important constituent of ribosome that act as an essential role in the formation of a fully function of ribosome. It can be predicted that the defects in ribosomal proteins can cause the ribosome dysfunction. Although ribosomal proteins are essential for cell growth, the effects of their mutations and their roles in human diseases have been ignored. With the development of cell biology and molecular genetics, further researches into the mechanisms of ribosomal proteins responsible for pulmonary hypertension have been possible. More and more functions of ribosomal proteins have been discovered. Studies showed that the regulation of ribosomal protein gene can produce a wide range of physiological functions of the body cells. The gene mutations and abnormal expression of ribosomal proteins will cause functional changes of the ribosome expression or inappropriate protein modification resulting in serious consequences, for example, disorder of cell membrane structure and function, abnormal cell metabolism, cell surface antibody decreased, abnormal cell growth and apoptosis. Therefore, it is very necessary to explore the function of ribosomal proteins in PH.Ribosome protein L22(RPL22), a component of the60S eukaryotic ribosomal subunit, is one of the important proteins in ribosomal protein family. Study showed that RPL22deficiency selectively upregulated p53in αβ-lineage thymocytes by increasing p53synthesis. Moreover, RPL22deficiency selectively arrested development of T cells at the (3-selection checkpoint by inducing their death. Our preceding research showed that Iptakalim, a selective KATP channel opener, suppressed ET-1-induced PASMC proliferation and significantly inhibited RPL22expression in PASMCs. It was presumed that RPL22might involve in the proliferation of PASMCs. In the present study, siRNA against RPL22and RPL22plasmid were used to investigate the effects of RPL22on HPASMC proliferation and the potential mechanisms.In the present study, we established human pulmonary artery smooth muscle cells (HPASMCs) model. Cells were cultured by tissue-piece inoculation method, purified by differential attachment method and identified by positive staining with a smooth muscle α-actin antibody by immunocytochemistry. HPASMCs were transfected by siRNA-RPL22and RPL22plasmid, respectively. ET-1was selected as the trigger of cells proliferation actor. Realtime-PCR and Western blot were used to measure the expression of RPL22mRNA and RPL22protein; PCNA, CCK-8immunohistochemistry and flow cytometry analysis were used to evaluate HPASMCs proliferation. Cyclin D1(CCND1), an important cyclin protein involving cell growth, was also assayed. The results suggest that by siRNA-RPL22transfection, RPL22were effectively inhibited. HPASMCs proliferation was inhibited after RPL22gene was knocked-out. CCND1was inhibited when RPL22deficiency. By transfected with RPL22plasmid, HPASMCs proliferation was significantly enhanced and CCND1expression upregurated accordingly. In conclusion, the data suggest that RPL22is a key factor involving the HPASMCs proliferation. The effects of RPL22on HPASMCs proliferation are considered to be mediated through interfering the expression of CCND1expression.