ABSTRACT
Changes in reactive oxygen species and extracellular matrix seem to participate in pulmonary hypertension development. Because we recently reported evidence for chronic hypoxia decreasing expression of cartilage oligomeric matrix protein (COMP) and evidence for this controlling loss of pulmonary arterial smooth muscle bone morphogenetic protein receptor-2 (BMPR2) and contractile phenotype proteins, we examined if changes in superoxide metabolism could be an important factor in a bovine pulmonary artery (BPA), organoid cultured under hypoxia for 48 h model. Hypoxia (3% O2) caused a depletion of COMP in BPA, but not in bovine coronary arteries. Knockdown of COMP by small-interfering RNA (siRNA) increased BPA levels of mitochondrial and extra-mitochondrial superoxide detected by MitoSOX and dihydroethidium (DHE) HPLC products. COMP siRNA-treated BPA showed reduced levels of SOD2 and SOD3 and increased levels of NADPH oxidases NOX2 and NOX4. Hypoxia increased BPA levels of MitoSOX-detected superoxide and caused changes in NOX2 and SOD2 expression similar to COMP siRNA, and exogenous COMP (0.5 µM) prevented the effects of hypoxia. In the presence of COMP, BMPR2 siRNA-treated BPA showed increases in superoxide detected by MitoSOX and depletion of SOD2. Superoxide scavengers (0.5 µM TEMPO or mitoTEMPO) maintained the expression of contractile phenotype proteins calponin and SM22α decreased by 48 h hypoxia (1% O2). Adenoviral delivery of BMPR2 to rat pulmonary artery smooth muscle cells prevented the depletion of calponin and SM22α by COMP siRNA. Thus, COMP regulation of BMPR2 appears to have an important role in controlling hypoxia-elicited changes in BPA superoxide and its potential regulation of contractile phenotype proteins.
Subject(s)
Bone Morphogenetic Protein Receptors, Type II/genetics , Cartilage Oligomeric Matrix Protein/genetics , Hypoxia/genetics , Myocytes, Smooth Muscle/drug effects , Oxygen/pharmacology , Superoxides/metabolism , Animals , Bone Morphogenetic Protein Receptors, Type II/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cartilage Oligomeric Matrix Protein/antagonists & inhibitors , Cartilage Oligomeric Matrix Protein/metabolism , Cattle , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Gene Expression Regulation , Heart/drug effects , Hypoxia/metabolism , Lung/drug effects , Lung/metabolism , Male , Mice , Mice, Inbred C57BL , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/metabolism , NADPH Oxidase 2/genetics , NADPH Oxidase 2/metabolism , NADPH Oxidase 4/genetics , NADPH Oxidase 4/metabolism , Primary Cell Culture , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Tissue Culture Techniques , CalponinsABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5-3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-ß1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-ß1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-ß1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-ß1 signaling should be persuaded.
