CTRP7 as a molecular biomarker associating with responsiveness to pulmonary vasodilators: insights from human and animal studies in pulmonary arterial hypertension

Figures

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  Figure 1. Up-regulation of C1q/TNF-related protein 7 in patients with PAH and PH animal models. (A) Volcano plot of RNA sequencing using cultured PASMCs from patients with PAH and non-PH individuals, identifying variations in 3017 genes. (B) Representative western blot and quantification of PTGIR expression in PASMCs from patients with PAH and intracellular cAMP accumulation in PASMCs from patients with PAH or from non-PH individuals treated with MRE-269 (0 and 1000 nM for 30 min); volcano plot of RNA sequencing utilizing cultured PASMCs from patients with PAH divided by PTGIR expression levels, identifying variations in 704 genes. (C) Elevated CTRP7 plasma levels can be observed in patients with PAH (n = 114) compared with those in non-PH individuals (n = 70), analysed using Welch's t-test. Schematic diagram of the selection of CTRP7 as a candidate gene based on multiple RNA sequencing and comprehensive plasma assessment. (D) Single-cell RNA sequencing of mouse lung tissues and expression of actin alpha 2 (ACTA2) and CTRP7. Data were obtained from the Tabula Muris project. Expression in corresponding tissue of origin of all cells (upper graph). The cells were clustered using the…

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  Figure 2. CTRP7 down-regulates prostacyclin receptors in patients with PAH and rodent animal models. (A) Representative western blots and quantification of CTRP7, PTGIR, sGCβ1, ENDRA, and GAPDH expression in cultured human PASMCs transfected with si-CTRP7 or si-Scramble (n = 3), analysed using Welch's t-test. (B) Representative western blots and quantification of PTGIR expression in PASMCs from patients with PAH and from non-PH individuals transfected with si-CTRP7 or si-Scramble, analysed using a two-way ANOVA. (C–E) Representative images of immunostaining of αSMA and PTGIR in the distal PAs of patients with PAH, hypoxic mice (10% O2 for 4 weeks) Sugen/hypoxia rats, and corresponding controls, analysed using Welch's t-test. Scale bars = 100 μm. (F) Representative western blots and quantification of PTGIR and GAPDH expression in lung homogenates from hypoxic mice (each n = 5) and Sugen/hypoxia rats and their corresponding controls (each n = 5), analysed using Welch's t-test. PTGIR, prostacyclin analogue receptor; sGCβ1, soluble guanylate cyclase β1 subunit; ENDRA, endothelin receptor type A; PASMCs, pulmonary artery smooth muscle cells; CTRP7, C1q/TNF-related protein 7; PAH, pulmonary…

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  Figure 3. IL-6 increases CTRP7 expression via transcriptional mechanisms in PASMCs. (A) Plasma levels of CTRP7 and IL-6 in patients with non-CTD-PH (n = 30–50), those with CTD-PAH (n = 30–64), and in non-PH individuals (n = 20–70), analysed using a one-way ANOVA. (B) Correlation between CTRP7 expression and the plasma levels of inflammatory factors in patients with PAH and non-PH individuals (n = 123), analysed using Pearson product-moment correlation. (C) mRNA expression of CTRP7 in human PASMCs treated with IL-6 (20 ng/mL) or vehicle for 24 h, analysed using Welch's t-test. (D) Chromatin immunoprecipitation targeting H3K27 acetylation and H3K4 methylation of CTRP7 promoter in PASMCs treated with IL-6 (n = 8), analysed using a two-way ANOVA, followed by Tukey’s honestly significant difference test for multiple comparisons. (E) Protein expression of CTPR 7 and PTGIR in human PASMCs treated with IL-6 (20 ng/mL) for 24 h (n = 6), analysed using Welch's t-test. CTRP7, C1q/TNF-related protein 7; IL-6, interleukin-6; non-CTD-PH, non-connective tissue disease-associated pulmonary arterial hypertension; PASMCs, pulmonary artery smooth muscle cells; H3K27, histones 3 lysine 27; H3K4, histone 3…

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  Figure 4. IL-6 regulates the expression of CTRP 7 and prostacyclin receptors in the PASMCs and lungs of PH animal models. (A) Representative western blots and quantification of CTRP7 and PTGIR expression in PASMCs transfected with si-CTRP7 or si-Scramble, treated with IL-6 (20 ng/mL) or vehicle, analysed using a two-way ANOVA, followed by Tukey’s honestly significant difference test for multiple comparisons. (B) Representative western blots and quantification of CTRP7, GAPDH, and transferrin expression in the lung homogenates and plasma harvested from hypoxic mice (n = 6), analysed using a one-way ANOVA. (C) mRNA expression in the lungs of hypoxic mice (n = 6) analysed using a one-way ANOVA. (D) Representative immunofluorescent images and quantification of αSMA and PTGIR expression in the pulmonary arteries of hypoxic mice (n = 6), analysed using a one-way ANOVA. (E) UMAP plot of mouse pulmonary arteries analysed at the single-cell level. Correlation between IL-6 and CTRP7 expression in the isolated pulmonary arteries of control mice and hypoxic mice analysed at the single-cell level. Data were obtained from the NCBI Gene Expression Omnibus (GEO) database (GSE228644). CTRP7,…

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  Figure 5. CTRP7 down-regulates prostacyclin receptor via Rab5a in PASMCs. (A) Western blot and quantification of Rab5a expression in cultured PASMCs from patients with PAH and non-PAH individuals (n = 6 each), analysed using Welch's t-test. (B) Western blot and quantification of CTRP7, Rab5a, and GAPDH expression in cultured human PASMCs transfected with si-CTRP7 or si-Scramble (n = 6 each), analysed using Welch's t-test. (C) Analysis of representative western blots and quantification of CTRP7, Rab5a, and GAPDH expression in PASMCs treated with recombinant CTRP7 (1000 ng/mL), lysosome inhibitor chloroquine (100 μM), or vehicle (n = 6 each), analysed using a one-way ANOVA. (D) Representative immunofluorescent images of PTGIR (green), Rab5a (red), and DAPI (blue) in human PASMCs treated with IL-6 or vehicle, along with transfection of si-CTRP7 or si-Scramble for 24 h, analysed using a two-way ANOVA, followed by Tukey’s honestly significant difference test for multiple comparisons. Scale bar = 100 μm. The yellow arrow indicates the co-localization of PTGIR and Rab5a. (E) Flow cytometric analyses and quantification of cell surface expression of PTGIR in human cultured PASMCs transfected with…

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  Figure 6. Down-regulation of CTRP7 improves the treatment response to selexipag in hypoxic mice. (A) Experimental timeline for AAV6-shCTRP7 and treatment with selexipag (1 mg/kg/day gavage) in a hypoxia-induced PH mouse model. (B) Western blots and quantification of CTRP7, GAPDH, and transferrin expression in the lungs and plasma samples collected from hypoxic mice treated with AAV6-shCTRP7 or AAV6-scramble via intratracheal instillation (n = 6–7), analysed using Welch's t-test. (C) Representative immunofluorescence images of αSMA, PTGIR, Rab5a, and DAPI of the distal PAs in hypoxic mice (10% O2 for 4 weeks) with down-regulation of CTRP7 using AAV6-shCTRP7; quantification was analysed using a one-way ANOVA. Scale bar = 50 μm. (D) RVSP was assessed using a catheter. CO was assessed using ultrasonography. TPR was calculated as RVSP per CO. (E) RVH was assessed from the weight ratio of the right ventricle to the left ventricle plus the septum, (F) Exercise capacity was assessed using the workload of the treadmill exercise. Quantification (D–F) was analysed a using two-way ANOVA, followed by Tukey’s honestly significant difference test for multiple comparisons. (G) Concentration–relaxation…

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  Figure 7. Inhibition of IL-6 signalling improves the treatment response to selexipag in hypoxic mice. (A) Experimental timeline for inhibiting IL-6 signalling and treatment with selexipag (1 mg/kg/day gavage) in a hypoxic model of PH in mice. (B) mRNA expression in the lungs of hypoxic mice (n = 7), analysed using Welch's t-test. (C)Western blots and quantification of CTRP7, GAPDH, and transferrin expression in the lungs and plasma samples collected from hypoxic mice intraperitoneal injected an IL-6-R neutralizing antibody or control IgG (n = 7), analysed using Welch's t-test. (D) Representative immunofluorescence images of αSMA, PTGIR, and DAPI in the distal PAs in hypoxic mice (10% O2 for 4 weeks) with inhibition of IL-6 signalling; quantification was analysed using Welch's t-test. Scale bar = 50 μm. (E) RVSP was assessed using a catheter. Ultrasonography was performed to assess CO. TPR was calculated as RVSP per CO. (F) RVH was assessed from the weight ratio of the right ventricle to the left ventricle plus the septum. (G) Exercise capacity was assessed using the workload of the treadmill exercise. Quantification (E–G) was analysed using a two-way ANOVA, followed by Tukey’s honestly…