Prevention Of Hypoxic Pulmonary Hypertension By Inhibiting CapG

Background and objectiveAcute or sustained reduction of alveolar oxygen occurs in a variety of lung diseases eventually develop to hypoxic pulmonary hypertension (HPH), results in reconstruction of resistance vessels in the pulmonary vascular bed, and follows by right ventricular hypertrophy and heart failure. A crucial pathological feature of HPH is pulmonary vascular remodeling, including medial wall thickness of muscular vessels and muscularization of pulmonary arterioles, which implicate the involvement of pulmonary arterial smooth muscle cells (PASMCs). The imbalance between cell proliferation and apoptosis under hypoxia could be important mechanisms of pulmonary vessel remodeling associated with HPH. We previously observed that Capping protein gelsolin-like (CapG) was preferentially expressed in human PASMCs under hypoxia compared with normoxia, and reduced CapG expression was accompanied by significantly impaired migration ability in vitro, especially under hypoxia. This result demonstrated that up-regulated CapG could enhance the migration of PASMCs under hypoxia. Understanding the mechanisms responsible for PASMCs to pulmonary vascular remodeling under hypoxia could allow us to get new insights into the development of HPH. In the present study, we investigated the effects of CapG on migration, proliferation, apoptosis and cell cycle of rat PASMCs as well as in the development of HPH rat model.Methods1. Isolation of rat primary PASMCs was using the explant method. The purity of PASMCs and the distribution of CapG in PASMCs were detected by immunofluorescence. Rat PASMCs were exposed to hypoxia in a hypoxia incubator (1%O2,94%　N2, and 5%　CO2) for 0 h,6 h,12 h,24 h,48 h respectively, and then CapG expression were detected by RT-PCR and Western blot.2. To silence the expression of CapG in vitro, we transfected the PASMCs with siRNA (si-CapG), the negative control siRNA was si-NC. Cells were divided into 4 groups: Normoxia-NC, Normoxia-CapG, Hypoxia-NC, Hypoxia-CapG. To demonstrate the effect of CapG on the PASMCs, cell migration was detected by Transwell assay, cell viability was detected by CCK-8 assay, cell poliferation was detected by EdU retention assays, cell apoptosis and cell cycle was detected by flow cytometry.3. Lentiviral vector mediated shRNA (Lenti-CapG) was used to knockdown CapG expression in rat model, the negative control shRNA was Lenti-NC. Rats were divided into 3 groups:Normoxia-NC (n=8), Hypoxia-NC (n=6), Hypoxia-CapG (n=6). The HPH rat model was established by intratracheal instillation of lentiviral vector for 2 weeks and subsequent hypoxia exposure for 4 weeks. We detected CapG expression in rat lung tissues and PASMCs isolated from rats that exposed to hypoxia or normoxia and pretreated with lentiviral vector. Right ventricular systolic pressure, right ventricular hypertrophy, the percentage of medial wall thickness and vessel muscularization were measured and calcutalted to evaluate the development of HPH.Results1. Rat primary PASMCs was successfully cultured by explant method. Immunofluorescence indicated that CapG was observed in both cytoplasm and nuclei of PASMCs. CapG mRNA and protein was markedly increased in PASMCs under hypoxia.2. Hypoxia enhanced migration ability, viability and proliferation ability of PASMCs, hypoxia also induced cell apoptosis but cell cycle progression. Knock-down CapG in PASMCs resulted in impaired migration and viability and proliferation ability, increased apoptosis and induced cell cycle inhibition.3. The intratracheal administration of lentiviral vector could integrate efficiently in the pulmonary arterioles. Hypoxia induced evaluation of right ventricular systolic pressure and right ventricle hypertrophy index, promoted vascular remodeling. Down-regulation of CapG expression locally could attenuate pulmonary hypertension, pulmonary vascular remodeling and right ventricular hypertrophy in HPH rat model.ConclusionThe study shows that hypoxia can induce up-regulation of CapG in the rat primary PASMCs. Knock-down CapG in PASMCs resulted in decreased migration, viability and proliferation ability, increased apoptosis and cell cycle inhibition. Down-regulation of CapG expression locally could attenuate HPH in rat model. Our study indicated that CapG participated in the pathogenesis of pulmonary vascular remodeling in HPH rat model. We proposed CapG modulating protective effects of HPH.