| BackgroundThe major pathologic feature of congenital systemic-to-pulmonary shunt disease related pulmonary arterial hypertension (PAH) is remodeling of small pulmonary arteries, during which the structural and functional changes of the vascular wall leads to increased muscularization of the muscular and part-muscular pulmonary arteries, muscularization of previously nonmuscular arteries and formation of neointima and plexiform lesions. Dysfunctions of pulmonary artery smooth muscle cells, that is excessive hypertrophy, proliferation, migration and resistance to apoptosis play important roles in pulmonary vascular remodeling.Secretory clusterin (sCLU) is a widely expressed protein in mammalians, existing in virtually all tissues and body fluids. It is generally acknowledged that sCLU is a cell protective factor with the function of promoting cell growth. Based on excessive growth of the cells in pulmonary vascular wall during PAH, we speculate that sCLU may be differently expressed in systemic-to-pulmonary shunt-related PAH and exert some effects during vascular remodeling.Recently, an original rat model of systemic-to-pulmonary shunt-related high kinetic unilateral pulmonary hypertension was established by our laboratory, which successfully simulated the hypertensive status in pulmonary circulation and the characteristic pathological features of pulmonary vascular remodeling. The present study aims to explore the expression pattern of sCLU in shunt-related PAH and the effects of sCLU in pulmonary vascular remodeling.AimsThe present study was undertaken to investigate:(1) the expression and localization of sCLU in shunt-related PAH rat lungs;(2) the effects of sCLU on pulmonary artery smooth muscle cell proliferation, migration and apoptosis resistance and the potential signaling pathways of action. Methods and Results(一) Expression of sCLU in systemic-to-pulmonary shunt-related PAH rats1. Expression of sCLU in shunt-related PAH rat lungs An original rat model of systemic-to-pulmonary shunt-related PAH was established. Real time polymerase chain reaction (real time PCR) and Western blot analysis were performed to detect the expression patterns of sCLU mRNA and protein in sham operation and systemic-to-pulmonary shunt rat lungs. Results showed that, sCLU expression was significantly elevated in systemic-to-pulmonary shunt rat lungs in a time-dependent manner.2. Expression of sCLU in shunt-related PAH rat plasma Enzyme linked immunosorbent assay (ELISA) showed that sCLU plasma levels were significantly elevated in shunt-related PAH rats in a time-dependent manner compared with sham operation rats. Spearman correlation analysis revealed that plasma sCLU levels had positive correlations with PAH index, that is, right ventricular systolic pressure, pulmonary arterial systolic pressure, mean pulmonary arterial pressure and right ventricular hypertrophy index.3. Localization of sCLU in pulmonary arteries Immunohistochemistry results showed that, there was little expression of sCLU in normal pulmonary arteries, however, it was markedly induced in the media and neointima of remodeled vessels in PAH rats and mainly localized in the cytoplasma of the medial and intimal cells and extra cellular matrix.4. Activation of ERK1/2and Akt signalling pathways in shunt-related PAH rat lungs Western blot analysis revealed that, activation of ERK1/2and Akt signalling pathways were significantly elevated in shunt-related PAH rat lungs in a time-dependent manner.(二) Effects of sCLU on pulmonary artery smooth muscle cells1. Effect of sCLU specific small interfering RNA (sCLU-siRNA) on the expression and secretion of sCLU in human pulmonary artery smooth muscle cells (HPASMCs) Real time PCR and Wstern-blot analysis revealed that, the mRNA and protein levels of sCLU were significantly decreased in sCLU-siRNA treated HPASMCs; ELISA showed that, sCLU secreted into the cell culture supernatants of sCLU-siRNA treated cells was significantly lower than that of control group.2. Cell cycle dependent expression of sCLU in HPASMCs Results from real time PCR and ELISA showed that, sCLU expression and secretion increased with the progression of cell cycle in HPASMCs, that is, the expression and secretion of sCLU were increased in proliferating (S or G2/M phase) HPASMCs compared with quisent (G0/G1phase) cells.3. Effect of sCLU on HPASMCs proliferation BrdU incorporation assay and immunofluorescence staining for Ki67demonstrated that sCLU promoted HPASMCs proliferation in concentration and time-dependent manners; Knocking down the expression and secretion of sCLU in HPASMCs significantly reduced the proliferation of the cells.4. Effect of sCLU on HPASMCs migration Transwell Boyden chamber assay showed that sCLU significantly promoted HPASMCs migration in a concentration-dependent manner and sCLU-siRNA treatment markedly attenuated the migratory capability of HPASMCs.5. Effect of sCLU on HPASMCs apoptosis Flow cytometry analysis revealed that, sCLU inhibited H2O2induced HPASMCs apoptosis concentration-dependently and that sCLU depletion by RNA interference resulted in spontaneous apoptosis of HPASMCs.6. Effect of sCLU on on ERK1/2and Akt activation Western blot analysis demonstrated that sCLU promoted phosphorylation of ERK1/2and Ak both in a time-dependent pattern, while treatment of HPASMCs with sCLU-siRNA resulted in significant decrease of ERK1/2and AKT activation.ConclusionIn conclusion, sCLU was significantly elevated with the progression of PAH in systemic-to-pulmonary shunt rats; sCLU promoted HPASMCs proliferation, migration and apoptosis resistance, at least in part, through ERK1/2and Akt signailing pathways, contributing to the remodeling of pulmonary arteries, which suggest that disrupting sCLU may be an attractive strategy for shunt-related PAH therapy; In addition, plasma sCLU may be a potential biomarker for congenital intracardiac shunt related PAH. BackgroundVentricular remodeling is one of the most important mechanisms of heart failure after myocardial infarction. Cardiac fibroblasts (CFs), the most abundant cells in the heart, migrate into the ischemic area, prolifetate, secrete various cytokines and growth factors and alter extracellular matrix (ECM) turn over, playing important roles in ventricular remodeling after myocardial infarction.Dermatopontin (DPT) is a secretory protein, playing important roles in interaction of cells with ECM and ECM assembly. Previous reports showed that, the mRNA level of DPT was significantly elevated in the infarct zone of experimental myocardial infarction rats, however, the effect of sCLU on cardiac fibroblasts and the exact role that DPT exerts in ventricular remodeling after myocardial infarction remains unclear.AimsThe present study was undertaken to investigate:(1) The expression and secretion of DPT in CMs and CFs under conditions of ischemia and oxygen deficient;(2) The effects of DPT on CFs adhesion, spreading, migration and proliferation, which are important aspects of ventricular remodeling.Methods and Results1. Hypoxia and serum deprivation (Hypoxia/SD) induced expression and secretion of DPT in CMs and CFs Hypoxia/SD conditions were used to mimic the in vivo conditions of ischemic myocardium. Real time PCR and Western blot analysis showed that, Hypoxia/SD induced DPT mRNA and protein expression both in CMs and CFs in a time-dependent manner. Moreover, ELISA showed that, under conditions of Hypoxia/SD the secretion of DPT in CMs and CFs was also significantly elevated.2. DPT promotes CFs adhesion via integrin α3β1 Using a solid-phase cell adhesion assay, we found that DPT promoted the adhesion of CFs in a concentration-related mode, furthermore, integrin α3and/or integrin β1neutralizing antibodies inhibited the adhesion of CFs to DPT and the inhibition was most effective when they were simultaneously used, suggesting that, integrin α3β1was at least one receptor for CFs adhesion to DPT.3. DPT promotes CFs spreading Immunofluorescent staining with phalloidin and an anti-vinculin antibody showed that CFs adhered to DPT formed well organized actin flaments and focal adhesions containing vinculin.4. DPT promotes CFs migration via integrin α3β1Using transwell cell culture chamber assays, we found that DPT stimulated CFs migration when it was added into the lower compartments of the chambers and inhibited CFs migration towards2%FBS or10μg/ml Fn when it was added into the upper compartments of the chambers, both in a concentration-dependent manner. Moreover, these effects of DPT were all inhibited by functional blocking antibodies specific for integrin α3and integrin β1, which interestingly expounded that DPT served as a chemotaxin for CFs migration via integrin α3β1.5. DPT has no effect on CFs proliferation MTS and BrdU incorporaion assay were performed to investigate the impact of DPT on CFs proliferation, however, no effect with statistical significance of DPT on CFs proliferation was finally detected. These results achieved here confirmed that the pro-migratory effect of DPT on CFs was not attributed to its proliferative effects.ConclusionThe expression and secretion of DPT was elevated in CMs and CFs under conditions of ischemia and oxygen deficient; Elevated DPT in myocardial infarction/ischemia region promoted CFs adhesion, spreasding and migration via integrin α3β1. Therefore, DPT may contribute to the progression of ventricular remodeling through the above-mentioned mechanism and it may be a potential theraputic target for excessive ventricular remodeling after myocardial infarction. |
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