Role Of LOX-1in Pulmonary Vascular Remodeling Of Hypoxia Induced Pulmonary Arterial Hypertension And The Underlying Mechanisms

[Background] Pulmonary arterial hypertension (PAH) is present when mean pulmonary artery pressure exceeds25mm Hg at rest or30mm Hg with exercise, or pulmonary vascular resistance higher than3Wood units.PAH is a syndrome resulting from restricted flow through the pulmonary arterial circulation resulting in increased pulmonary vascular resistance and ultimately in right heart failure. The predominant cause of increased pulmonary vascular resistance is loss of vascular luminal cross section due to vascular remodeling. Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a key role in vascular remodeling.Lectin-like oxidized low density lipoprotein recepter-1(LOX-1) plays an important role in vascular remodeling of various cardiovascular dieases such as atherosis and hypertension. LOX-1mediates vascular endothelial cell injury and proliferation of cadiac cell and vascular smooth muscle cells via oxidase stress and proinflammatory pathway, which results in remodeling of heart, aorta and vessel of kidney. It has recently been shown that the expression of LOX-1is increased in PASMCs from patients with chronic thromboembolic pulmonary hypertension and mediates the proliferation of PASMCs induced by C-reative protein. The role of LOX-1in vascular remodeling in PAH remains unclear. We therefore explored the role of LOX-1in vascular remodeling in PAH.[Method] Sprague-Dawley (SD) rats (180～220g) were kept under hypoxia (10%O2) for3weeks to induce PAH. At the end of experiments, RVSP and mPAP were monitored by the method of right external jugular vein cannula. The right, left ventricle (RV, LV) and interventricular septum (IS) were dissected from the heart and weighed for calculating the ratio of RV to (LV+IS). Blood was collected from carotid artery to determine the plasma levels of sLOX-1by ELISA. The freshly isolated pulmonary arterial or lung tissues samples were used for determination of LOX-1, Ki-67, PCNA expression by real-time PCR or Western blot. Excised left lung was fixed in4%paraformaldehyde for evaluation of morphology.Primary rat PASMCs were prepared from the pulmonary artery of male10-week-old SD rats using explant method. In all the experiments, passage3to6cells were used. Immunocytochemistry was performed to identify the differentiated cells.3%O2was used to induce cell proliferation.PASMCs were pre-treated with LOX-1-neutralizing antibody (5,10,20ng/ml) for1h, then subjected to hypoxia (3%O2). Cell proliferation was measured by MTS, flow cytometry and the expression of proliferation marker, Ki-67and PCNA. The expression of LOX-1was analysed by real-time PCR or Western blot.After PASMCs grown to60%to70%confluence, LOX-1siRNA were transfected with TurboFect transfection reagent into cells. The transfection efficiency was evaluated by LOX-1mRNA and protein expression using real-time PCR and Western blot analysis, respectively. Twenty-four hours after transfection, PASMCs were subjected to hypoxia. Cell proliferation was measured by MTS, flow cytometry and the expression of proliferation marker, Ki-67and PCNA.[Results] In rats with hypoxia induced PAH, RVSP and mPAP were higher than that of normoxic rats. The thick of pulmonary arterial wall was also increased in PAH rats. The mRNA expression of Ki-67and PCNA was up-regulated in pulmonary arteries of PAH rats. The level of sLOX-1in plasma and the expression of LOX-1was increased in pulmonary arteries of PAH rats.Hypoxia significantly induced proliferation of PASMCs, and dramatically up-regulated expression of LOX-1. Both LOX-1-neutralizing antibody (TS-20)(5,10,20ng/ml) and LOX-1siRNA inhibited hypoxia-induced proliferation of PASMCs, down-regulated the expression of Ki-67and PCNA.[Conclusion] LOX-1mediates proliferation of PASMCs induced by hypoxia, which contributes to vascular remodeling in rats with hypoxia induced pulmonary arterial hypertension. Part2The upstream signaling of LOX-1up-regulation in hypoxia-treated pulmonary artery smooth muscle cells[Background] LOX-1is a multiligand receptor, whose repertoires of ligands include ox-LDL and AGEs. The LOX-1promoter is constitutively active only at a low level though its expression can be induced by its ligands and other stimulates such as Ang Ⅱ, fluid shear stress, proinflammatory cytokines. Our preliminary study demonstrated that LOX-1is up-regulated in PASMCs in hypoxia induced PAH, but the upstream signaling of LOX-1is unclear.Based on the recent study that microRNA let-7g targets the LOX-13’UTR and inhibits LOX-1expression, we proposed that hypoxia up-regulates LOX-1expression by inhibiting let-7g gene expression. Interestingly, several studies have reported that LOX-1activation could enhance downstream gene regulation including miRNAs. LOX-1affects the binding of transcriptional repressor OCT-1to the let-7g promoter. We therefore speculate that OCT-1is involved in the feedback regulation between LOX-1and let-7g.Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular fuctions such as cytoskeletal remodeling, cell cycle progression, gene expression and apoptotic cell death. It is reported that the calpain inhibitor MDL28170prevents the progression of established PAH by monocrotaline. It is known that LOX-1activates OCT-1by Ca2+-PKC signalling pathway. PKC, one of the substract of calpain, could be activated by limited proteolytic of calpain. We therefore tested the hypothesis that the signal transduction that causes a feedback regulation between LOX-1and let-7g may be via LOX-1-calpains-PKC-OCT-1-let-7g.[Method] Sprague-Dawley (SD) rats (180～220g) were kept under hypoxia (10%O2) for3weeks to induce PAH. At the end of experiments, RVSP and mPAP were monitored by the method of right external jugular vein cannula. The right, left ventricle (RV, LV) and interventricular septum (IS) were dissected from the heart and weighed for calculating the ratio of RV to (LV+IS). The freshly isolated pulmonary arterial or lung tissues samples were used for determination of LOX-1, calpain-1, calpain-2, calpain-4, OCT-1, let-7g expression by real-time PCR or Western blot.Primary rat PASMCs were prepared from the pulmonary artery of male10-week-old SD rats using explant method. In all the experiments, passage3to6cells were used. Immunocytochemistry was performed to identify the differentiated cells.3%O2was used to induce cell proliferation. After PASMCs grown to70%to80%confluence, let-7g mimics were transfected with TurboFect transfection reagent into cells. The transfection efficiency was evaluated by let-7g mRNA expression using real-time PCR. Twenty-four hours after transfection, PASMCs were subjected to hypoxia. The expression of LOX-1were analysed by real-time PCR or Western blot. Cell proliferation was measured by MTS, flow cytometry and the expression of proliferation marker, Ki-67and PCNA.PASMCs were pre-treated with LOX-1-neutralizing antibody (5,10,20ng/ml) or chelerythrine (1μM) for1h, then subjected to hypoxia (3%O2). The expression of LOX-1, calpain-1, salpain-2, calpain-4, OCT-1and let-7g were analysed by real-time PCR or Western blot.After PASMCs grown to60%to70%confluence, LOX-1, calpain-1, calpain-2, calpain-4and OCT-1siRNA were transfected with TurboFect transfection reagent into cells, respectively. The transfection efficiency was evaluated by its mRNA and protein expression using real-time PCR and Western blot analysis, respectively. Twenty-four hours after transfection, PASMCs were subjected to hypoxia. The expression of LOX-1, calpain-1, calpain-2, calpain-4, OCT-1and let-7g were analysed by real-time PCR or Western blot.[Results] Calpain-1, calpain-2, calpain-4and OCT-1are increased while let-7g is decreased in hypoxia induced PAH. Let-7g mimic inhibited hypoxia induced upregulation of LOX-1expresion and proliferation of PASMCs. At the same time, inhibition of LOX-1expression also reversed the decreased let-7g expression in PASMCs induced by hypoxia.PKC inhibitor, calpain-1siRNA, calpain-2siRNA, calpain-4siRNA and OCT-1siRNA inhibited upregulation of LOX-1expression and reversed the decreased let-7g expression in PASMCs induced by hypoxia. At the same time, inhibition of LOX-1expression reversed the incresed calpain-1, calpain-2, calpain-4, OCT-1expression in PASMCs induced by hypoxia.[Conclusion] The signaling of the upstream of LOX-1in proliferation of PASMCs induced by hypoxia may be LOX-1-calpains-PKC-OCT-1-Let-7g feedback pathway. Part3The downtream singnaling of LOX-1induced proliferation of pulmonary artery smooth muscle cells in hypoxia induced pulmonary arterial hypertension[Background] VSMCs are highly plastic and their phenotypes can be modulated between proliferative and differentiated states in response to extracellular cues. Abnormalities in differentiation of VSMCs are responsible for a variety of disorders, including atherosclerosis and hypertension. Most smooth muscle contractile protein genes are controlled by SRF, which binds to a sequence known as a CArG box and recruits MRTFs, a coactivator family that is necessary and sufficient for smooth muscle gene expression. In addition, CArG boxes are originally identified in transcriptional regulatory elements controlling expression of a set of growth-or serum-responsive genes including c-fos and egr-1. SRF also regulates genes involved in cell proliferation, which opposes the smooth muscle differentiation program.It is reported that stimulation of SRF activity via two independent pathways, one dependent on phosphorylation of ternary complex factors,(TCFs) by a MAPK cascade, the other dependent on Rho signaling and actin dynamics. Suppression of differentiation of VSMCs by growth factor signalling is mediated, at least in part, by displacement of myocardin from SRF by growth-dependent TCFs. Myocardin and Elk-1compete for interaction with a common docking site on SRF and Elk-1acts as a signal-responsive repressor of smooth muscle gene expression by displacing myocardin from SRF within the context of native chromatin. Growth factor and/or vascular injury stimulate proliferation of VSMCs via two pathways, one dependent on phosphorylation of TCFs by a MAPK cascade, the other dependent on depressed MRTFs pathway.Our pilot study demonstrated that LOX-1is upregulated in PASMCs in hypoxia induced PAH leading to proliferation of PASMCs. The downstream of LOX-1is unclear. Several studies have reported that LOX-1activation could enhance downstream gene regulation including NADPH oxidase, MAPKs, PKC. We proposed that LOX-1could induce phosphorylation of Elk by a MAPK signalling resulting in the expression of c-fos. At the same time, Elk-1acts as a signal-responsive repressor of smooth muscle gene expression by displacing MRTF-A from SRF. All of these results in PASMCs phenotypic modulation and proliferation. We speculate that LOX-1induces proliferation of PASMCs via LOX-1-ERK1/2-pElk/MRTF-A-SRF-c-fos pathway in hypoxia induced PAH.[Method] Sprague-Dawley (SD) rats (180～220g) were kept under hypoxia (10%O2) for3weeks to induce PAH. At the end of experiments, RVSP and mPAP were monitored by the method of right external jugular vein cannula. The right, left ventricle (RV, LV) and interventricular septum (IS) were dissected from the heart and weighed for calculating the ratio of RV to (LV+IS). The freshly isolated pulmonary arterial or lung tissues samples were used for determination of pERK1/2, pElk, MRTF-A, SRF, c-fos, a-SMA expression by real-time PCR or Western blot.Primary rat PASMCs were prepared from the pulmonary artery of male10-week-old SD rats using explant method. In all the experiments, passage3to6cells were used. Immunocytochemistry was performed to identify the differentiated cells.3%O2was used to induce cell proliferation.PASMCs were pre-treated with LOX-1-neutralizing antibody (5,10,20ng/ml) for1h, then subjected to hypoxia (3%O2). The expression of pERK1/2,pElk, MRTF-A, SRF, c-fos were analysed by real-time PCR or Western blot.After PASMCs grown to60%to70%confluence, LOX-1siRNA was transfected with TurboFect transfection reagent into cells. The transfection efficiency was evaluated by LOX-1mRNA and protein expression using Real-Time PCR and Western blot analysis, respectively. Twenty-four hours after transfection, PASMCs were subjected to hypoxia. The expression of pERK1/2, pElk, MRTF-A, SRF, c-fos, a-SMA were analysed by real-time PCR or Western blot.[Results] The expression of pERK1/2, pElk, SRF, c-fos in pulmonary artery was increased accompanied by decreased expression of MRTF-A, but not changed in α-SMA, in hypoxia induced PAH. Inhibition of LOX-1reversed the activation of pERK1/2, pElk and the increased expression of SRF and c-fos as well as the decreased MRTF-A expression in PASMCs induced by hypoxia. Hypoxia markedly decreased a-SMA expression in cultured PASMCs, which was not reversed by inhibition of LOX-1.[Conclusion] The signaling of the downstream of LOX-1in mediating proliferation of PASMCs induced by hypoxia may be the LOX-1-ERK1/2-pElk/MRTF-A-SRF-c-fos pathway.