C1q/TNF-related protein-9 ameliorates hypoxia-induced pulmonary hypertension by regulating secretion of endothelin-1 and nitric oxide mediated by AMPK in rats

Figures

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  Figure 1. Hypoxia induced cardiopulmonary remodeling and reduction of serum CTRP9 content in rats. (A–D) Quantitative data of cardiopulmonary hemodynamics, including right ventricular systolic pressure (RVSP, A), mean carotid artery pressure (mCAP, B), and right ventricular hypertrophy index (C, D), are presented. € Paraffin-embedded sections of rat lungs were assessed for pulmonary arterial remodeling by staining (hematoxylin & eosin; × 200 magnification). Scale bar = 20 μm. (F, G) Blinded quantitative analyses of MT% and MA% of peripheral pulmonary arteries (30 vessels/3 sections from one animal, 30–100 μm in diameter) were done using Image-Pro Plus. (H) CTRP9 concentrations in the serum of rats suffering hypoxia or not (n = 6). Data are the mean ± SEM. ※P < 0.05, ※※P < 0.01 vs. normoxia group. N normoxia for 4 weeks, H2 hypoxia for 2 weeks, H4 hypoxia for 4 weeks.

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  Figure 2. CTRP9 overexpression ameliorated HPH. (A) CTRP9 concentrations in rat serum are shown (nN+AAV-Control = 4, nN+AAV-CTRP9 = 5, nH+AAV-Control = 5, nH+AAV-CTRP9 = 6). (B, C) Quantitative data of cardiopulmonary hemodynamics, including right ventricular systolic pressure (RVSP; nN+AAV-Control = 4, nN+AAV-CTRP9 = 7, nH+AAV-Control = 6, nH+AAV-CTRP9 = 6) and mean carotid artery pressure (mCAP; n = 4), are presented. (D) Paraffin sections of rat lungs were assessed for pulmonary-arterial remodeling by staining (hematoxylin and eosin, × 400 magnification). Scale bar = 40 μm. (E, F) Indices of right ventricular remodeling are shown (E:n = 3; F: nN+AAV-Control = 6, nN+AAV-CTRP9 = 7, nH+AAV-Control = 6, nH+AAV-CTRP9 = 7). Data are the mean ± SEM. (G, H) Blinded quantitative analyses of MT% (n = 4) and MA% (nN+AAV-Control = 4, nN+AAV-CTRP9 = 4, nH+AAV-Control = 4, nH+AAV-CTRP9 = 5) of peripheral pulmonary arteries (30 vessels/3 sections per animal, 30–100 μm in diameter) were done using Image-Pro Plus. ※P < 0.05 vs. group N + AAV-Control; #P < 0.05 vs. group H + AAV-Control. N + AAV-Control transfection of AAV-Control vectors maintained at normoxia for 4 weeks, N + AAV-CTRP9 transfection of…

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  Figure 3. CTRP9 overexpression improved endothelial function in pulmonary arteries from HPH rats. (A) Dose–response curves for ACh-induced relaxation were obtained from pulmonary arteries of different groups (n = 6). Data are expressed as the percentage of the contraction to phenylephrine (PE). (B) Dose–response curves for SNP-induced relaxation were obtained from pulmonary arteries of different groups (n = 6). Data are expressed as the percentage of the contraction to PE. Data are the mean ± SEM. ※※P < 0.01 vs. group N; #P < 0.05 vs. group H + AAV-Control. N normoxia for 4 weeks, H4 hypoxia for 4 weeks, H + AAV-Control transfection of AAV-Control vectors maintained at hypoxia for 4 weeks, H + AAV-CTRP9 transfection of AAV-CTRP9 vectors maintained at hypoxia for 4 weeks.

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  Figure 4. CTRP9 overexpression promoted phosphorylation of AMPK and eNOS and decreased ERK1/2 phosphorylation in isolated endothelial cells from the lungs of HPH rats. (A–C) Western blotting of expression of AMPK and p-AMPK (A, n = 3; blots are cropped from different gels), eNOS and p-eNOS (B, n = 3; blots are cropped from different gels), ERK1/2 and p-ERK1/2 (C, n = 3; blots are cropped from different gels) in endothelial cells from lungs of HPH rats using GAPDH as an internal control. The ratio of p-AMPK to AMPK, p-eNOS to eNOS, and p-ERK1/2 to ERK1/2 was calculated. (D, E) Levels of NO (nN+AAV-Control = 3, nN+AAV-CTRP9 = 3, nH+AAV-Control = 4, nH+AAV-CTRP9 = 3) and ET-1 (nN+AAV-Control = 5, nN+AAV-CTRP9 = 5, nH+AAV-Control = 4, nH+AAV-CTRP9 = 5) in rat serum were measured. Data are the mean ± SEM. ※P < 0.05, ※※P < 0.01 vs. group N + AAV-Control; #P < 0.05 vs. group H + AAV-Control. N + AAV-Control transfection of AAV-Control vectors maintained at normoxia for 4 weeks, N + AAV-CTRP9 transfection of AAV-CTRP9 vectors maintained at normoxia for 4 weeks, H + AAV-Control transfection of AAV-Control vectors maintained at hypoxia for 4 weeks, H + AAV-CTRP9 transfection of AAV-CTRP9 vectors…

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  Figure 5. AMPK mediated CTRP9-induced NO production and reduced ET-1 production by activating eNOS and inactivating ERK1/2. (A–C) Western blotting of expression of AMPK and p-AMPK (A, n = 3; blots were cropped from different parts of the same gel), eNOS and p-eNOS (B, n = 3; blots are cropped from different gels), ERK1/2 and p-ERK1/2 (C, n = 3; blots cropped from different gels) in rat PMVECs. (D, E) Levels of NO (nN+AAV-Control = 3, nN+AAV-CTRP9 = 3, nH+AAV-Control = 5, nH+AAV-CTRP9 = 4) and ET-1 (nN+AAV-Control = 5, nN+AAV-CTRP9 = 5, nH+AAV-Control = 5, nH+AAV-CTRP9 = 6) in PMVECs culture medium. Data are the mean ± SEM. ※P < 0.05 vs. group N; #P < 0.05 vs. group H; &P < 0.05 vs. group H + CTRP9. H hypoxia, N normoxia, C.C compound C.

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  Figure 6. Knockdown of AMPK expression inhibited CTRP9-induced alterations in expression of p-AMPK, p-eNOS, p-ERK1/2, NO and ET-1. (A–C) Western blotting of expression of AMPK and p-AMPK (A, n = 3; blots were cropped from different parts of the same gel), eNOS and p-eNOS (B, n = 3; blots were cropped from different gels), ERK1/2 and p-ERK1/2 (C, n = 3; blots were cropped from different gels) in rat PMVECs. The protein ratio of p-AMPK to AMPK, p-eNOS to eNOS, and p-ERK1/2 to ERK1/2 was calculated. (D, E) Levels of NO (n = 3) and ET-1 (n = 4) in PMVECs culture medium were examined. Data are the mean ± SEM. ※P < 0.05 vs. H + LV-Control group; #P < 0.05 vs. H + LV-Control + CTRP9 group. H hypoxia, LV-Control lentivirus control, LV-shAMPK lentivirus with AMPK interruption.