Potential role of cartilage oligomeric matrix protein in the modulation of pulmonary arterial smooth muscle superoxide by hypoxia

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.

Keywords: NADPH oxidases; extracellular matrix; pulmonary hypertension; smooth muscle phenotype; superoxide dismutase.

Fig. 1.

Effects of hypoxia on the level of cartilage oligomeric matrix protein (COMP) in the lungs of mice and organoid-cultured bovine arteries. Representative Western blot bands and quantitative analysis of COMP protein in the lungs from 3 wk chronic hypoxia (10% O₂)-exposed male mice with pulmonary hypertension (PH) and control normoxic (21% O₂) mice (**P < 0.01, PH mice versus control mice, Student’s t test, n = 5) (A); and in bovine pulmonary artery (BPA) organoid cultured under 3% or 21% O₂ for 48 h (*P < 0 0.05, 3% versus 21% O₂, paired t test, n = 5 (B); and in left anterior descending (paired t test, n = 5) (C); and left circumflex coronary arteries (n = 7) organoid cultured under 3% vs. 21% O₂ (paired t test, n = 7) (D).

Fig. 2.

Effect of hypoxia and cartilage oligomeric matrix protein (COMP) on the levels of mitochondrial matrix and extra-mitochondrial matrix superoxide detected by MitoSOX and dihydroethidium (DHE), respectively. A: level of superoxide-specific metabolites of MitoSOX and DHE detected by HPLC in bovine pulmonary artery (BPA) cultured under 21 or 3% O₂ for 48 h in the absence or presence of COMP. **P < 0.01 (two-way ANOVA with Sidak correction, n = 10; A). B: COMP protein level deceased by COMP siRNA (*P < 0.05, paired t test, n = 5; B). C: level of MitoSOX (n = 14) and DHE (n = 8) superoxide detected by HPLC in BPA treated with COMP siRNA or scramble siRNA (*P < 0.05, **P < 0.01 for COMP siRNA versus scramble siRNA, paired t test).

Fig. 3.

Effects of hypoxia and cartilage oligomeric matrix protein (COMP) on the expression of the different forms of SOD present in bovine pulmonary artery (BPA). The level of individual forms of SOD measured by Western blotting in BPA cultured under 21% (CTL) or 3% O₂ for 48 h (H48) in the absence and presence of COMP (C, 0.5 μM) is shown. A–C: representative Western blot bands and quantitative analysis of Cu,Zn-SOD1 (A) (n = 8), mitochondrial SOD2 (B) (n = 6), and extracellular SOD3 (C) (n = 6). *P < 0.05, two-way ANOVA with Sidak correction. D: representative Western blot bands and quantitative analysis showing the effect of knockdown of COMP with siRNA decreasing protein levels of SOD2 (n = 9) and SOD3 (n = 7). *P < 0.05, COMP-siRNA vs. scramble siRNA, paired t test. (H48, 3% O₂ for 48 h; H48 + C, 3% O₂ for 48 h and treated with 0.5 μM COMP at the same time; COMPi, siRNA-COMP.) B and C were edited between the 2nd and 3rd channels that are shown separated by a line to remove two additional channel bands from the original imaging capture of the same membrane.

Fig. 4.

Effects of hypoxia and cartilage oligomeric matrix protein (COMP) on the expression of NADPH oxidases NOX2 and NOX4 detected by Western blotting. Bovine pulmonary artery (BPA) were cultured under 21% (CTL) or 3% O₂ (H48) for 48 h in the absence and presence of COMP (0.5 μM). A and B: representative Western blot bands and quantitative analysis of changes in NOX2 (A) and NOX4 (B). *P < 0.05, two-way ANOVA with Sidak correction, n = 7. C: effects of siRNA knockdown of COMP on detection of NOX2 (n = 8) and NOX4 (n = 6) protein levels. Representative Western blot bands and quantitative analysis of NOX2 and NOX4. **P < 0.01, ***P < 0.001, COMP siRNA vs. scramble siRNA, paired t test. (H48 + C, 3% O₂ for 48 h and treated with 0.5 μM COMP at the same time; COMPi, COMP siRNA.)

Fig. 5.

Effects of scavengers of mitochondrial (mitoTEMPO) or extra-mitochondrial (TEMPO) superoxide on the hypoxia-elicited decreases in the levels of SM22α and calponin. Representative Western blot bands and quantitative analysis of cartilage oligomeric matrix protein (COMP) level under 1% and 3% O₂ (*P < 0.05, one-way ANOVA with Newman-Keuls correction, n = 5) (A) and calponin (n = 8) and SM22α (n = 9) under 1% O₂ (*P < 0.05, **P < 0.01, one-way ANOVA with Newman Keuls correction) (B). CTL, normoxia; H48, hypoxia for 48 h; T, 0.5 μM TEMPO under normoxia; H48 + T, 1% O₂ for 48 h in the presence of 0.5 μM TEMPO; MT, 0.5 μM mitoTEMPO under normoxia; H48 + MT, 1% O₂ for 48 h in the presence of 0.5 μM mitoTEMPO.

Fig. 6.

Effects of bone morphogenetic protein receptor-2 (BMPR2) on the expression of contractile phenotype proteins associated with the actions of cartilage oligomeric matrix protein (COMP) under normoxia. Representative Western blot bands and quantitative analysis of BMPR2 in bovine pulmonary artery (BPA) cultured under 3% O₂ for 48 h (*P < 0.05, **P < 0.01, two-way ANOVA with Sidak correction, n = 6) (A); BMPR2 and calponin in BPA treated with scramble siRNA, BMPR2 siRNA, or BMPR2 siRNA plus COMP (*P < 0.05, one-way ANOVA with Newman Keuls correction, n = 6) (B); and the levels of BMPR2, SM22α, and calponin in cultured pulmonary artery smooth muscle cells (PASMCs) derived from male rats treated with adenovirus-vector (Ad-V or V) or adenovirus-BMPR2 (Ad-B or B) without or with scramble siRNA (S) or COMP siRNA (Ci) [*P < 0.05, **P < 0.01, ***P < 0.001, BMPR2: (n = 3) one-way ANOVA with Newman Keuls correction and SM22α (n = 5) or calponin (n = 5): two-way ANOVA with Sidak correction] (C). CTL, normoxia; H48, hypoxia for 48 h; H48 + C, 1% O₂ for 48 h in the presence of 0.5 μM COMP; Bi, BMPR2 siRNA; Bi + C, COMP was added to the culture media for 24 h after BMPR2 siRNA treatment for 24 h; Ad-V, treatment with 1.6 × 10⁻⁵ plaque-forming units (PFU) of adenovirus-vector for 48 h; Ad-B, treatment with 1.2 × 10⁻⁵ PFU of adenovirus-BMPR2 for 48 h; S + Ad-V/Ad-B, treatment with scramble siRNA for 24 h and then adding Ad-V/Ad-B for another 48 h; Ci + Ad-V/Ad-B, treatment with COMP siRNA for 24 h and then adding Ad-V/Ad-B for another 48 h).

Fig. 7.

Effects of bone morphogenetic protein receptor-2 (BMPR2) on the detection of superoxide and levels of SOD and NADPH oxidase NOX2. A: levels of MitoSOX (n = 10) and dihydroethidium (DHE, n = 7)-detected superoxide by HPLC analysis in bovine pulmonary artery (BPA) treated with scramble siRNA, BMPR2 siRNA, or BMPR2 siRNA plus cartilage oligomeric matrix protein (COMP). *P < 0.05, **P < 0.01, analysis by one-way ANOVA with Newman-Keuls correction. B: representative Western blot bands and quantitative analysis of SOD1, SOD2, SOD3, and NOX2 in BPA treated with scramble siRNA, BMPR2 siRNA, or BMPR2 siRNA plus COMP. *P < 0.05, analysis of one-way ANOVA with Newman-Keuls correction, n = 6. C: representative Western bands and quantitative analysis of BMPR2 in BPA cultured under 3% O₂ for 48 h treated with or without catalase, SOD, and gp91 ds-tat. *P < 0.05, analysis of one-way ANOVA with Newman-Keuls correction, n = 8. Bi, BMPR2 siRNA; Bi + C, COMP added to the culture media for 24 h after BMPR2 siRNA treatment for 24 h; CTL, normoxia; H48, hypoxia for 48 h; H-cat, 3% O₂ for 48 h in the presence of 1 μM catalase; H-SOD, 3% O₂ for 48 h in the presence of 1 μM SOD; H-gp91, 3% O₂ for 48 h in the presence of 50 μM gp91 ds-tat. Note that the box-and-whisker format of the SOD data in B shows the median, upper, and lower quartile box and extreme data points.

Fig. 8.

Model showing some of the hypothesized actions resulting from a depletion of cartilage oligomeric matrix protein (COMP) from bovine pulmonary smooth muscle resulting from organoid culture under hypoxia for 48 h that are potentially contributing to the observed increases in mitochondrial and extra-mitochondrial matrix in superoxide that seem to participate in a depletion of contractile phenotype proteins based on the actions of siRNA depletion of COMP and attenuation of the actions of hypoxia by treatment with COMP. COMP depletion promotes decreases in SOD2 and increases in NADPH oxidase NOX2, associated with increases in mitochondrial superoxide (detected by MitoSOX) and extra-mitochondrial superoxide [detected by dihydroethidium (DHE)]. The actions of superoxide scavengers TEMPO and mitoTEMPO on inhibiting the effects of hypoxia suggest roles for increases in the different sources of superoxide contribute to depleting contractile phenotype protein levels. Depletion of bone morphogenetic protein receptor-2 (BMPR2) by siRNA promoting decreased levels of calponin and adenovirus-BMPR2 reversal of reduced calponin and SM22α caused by COMP siRNA indicate COMP regulation of BMPR2 expression is potentially a key process maintaining the contractile phenotype. Moreover, BMPR2 siRNA increases superoxide (as supported by detection of MitoSOX and DHE) and promotes contractile protein depletion in bovine pulmonary artery, which cannot be prevented by exogenous COMP. Thus, protective effects of COMP on the contractile phenotype of pulmonary artery smooth muscle cells (PASMCs) appear to be via maintenance of BMPR2 signaling. SOD3 and NOX4 are shown in parentheses because changes in the expression of these proteins in pulmonary arteries observed with siRNA depletion of COMP or by more prolonged in vivo exposure of animals to hypoxia reported in the literature were not observed in the 48-h organoid culture hypoxia model used in the present study.