CTRP9 Mitigates the Progression of Arteriovenous Shunt-Induced Pulmonary Artery Hypertension in Rats

Figures

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  Figure 1. Effects on physical parameters following CTRP9 overexpression in sham and shunt rats. (a) The body weight and the weights of the lung, heart, liver, and kidney of rats. (b) mRNA expression of CTRP9 was measured by RT-qPCR in the shunt and sham groups. (c) Protein expression of CTRP9 was determined by western blotting (β-actin was loaded as the control). n = 10 of each group. Mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01 vs. the Ad-GFP-sham group. RT-qPCR: reverse transcription-quantitative PCR; CTRP9: C1q/TNF-related protein 9; Ad-GFP: adenovirus-green fluorescent protein.

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  Figure 2. PAH is prevented and attenuated by the administration of CTRP9. (a) The rats in the shunt group exhibited a significantly higher mPAP compared with that of the rats in the sham group at the end of the 12th week. (b) The rats in the shunt group exhibited a significantly higher RVSP compared with that of the rats in the sham group at the end of the 12th week. (c) Quantification of the percentage of wall thickness to vessel diameter. (d) Quantification of the lumen area to vessel total area. (e) Representative images of H&E-stained resistant pulmonary arteries from the different experimental groups. n = 10 of each group. Mean ± SEM. ∗P < 0.05 and ∗∗P < 0.01 vs. the Ad-GFP-sham group. PAH: pulmonary arterial hypertension; CTRP9: C1q/TNF-related protein 9; mPAP: mean pulmonary artery pressure; RVSP: right ventricular systolic pressure; H&E: hematoxylin and eosin; Ad-GFP: adenovirus-green fluorescent protein.

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  Figure 3. CTRP9 suppresses inflammation in vivo. (a) The interleukin 18 (IL-18), IL-10, monocyte chemoattractant protein-1 (MCP-1), IL-6, F4/80, and tumor necrosis factor alpha mRNA expression levels were analyzed by RT-qPCR. (b, d) Injection of the rats with CTRP9 in the sham and shunt groups reduced the protein expression of MCP-1, IL-6, TNF-α, and IL-18, as assessed by immunoblot analysis of the total lung tissue. (c, e) Representative western blots of the total lung tissues indicating the induction of the inflammatory markers MCP-1, IL-6, TNF-α, and IL-18 following treatment of the rats with CTRP9 in the shunt group (β-actin was used as a loading control) n = 6 for each group. In (b) and (d), the protein molecular weight of the MCP-1 (18 kDa), IL-6 (21 kDa), TNF-α (26 kDa), and IL-18 (22 kDa) was very similar, so they were all originating from the same gel. After exposure of the MCP-1 or TNF-α, we striped the primary antibody by the striping solution from the membrane and incubated the TNF-α antibody or IL-18 antibody. So, we use the same β-actin blot banding as the corresponding lading control from the same membrane. A total of 6 samples were selected from each group to perform…

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  Figure 4. CTRP9 inhibits lung apoptosis and matrix injury. Wild-type rats were injected with Ad-CTRP9 or Ad-GFP, subjected to shunt or sham operation, and samples were obtained at week 12. (a, c) Caspase-8, Bcl-2, MMP-2, and MMP-9 protein expressions were analyzed by western blotting. (b, d) Representative western blots and quantification of the changes noted in caspase-8, Bcl-2, MMP-2, and MMP-9 in the lung tissues of the sham or shunt rats treated with or without CTRP9. β-Actin was used as a loading control. The data are expressed as the mean ± SEM. n = 6 for each group. A total of 6 samples were randomly selected from each group to perform western blotting. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. the Ad-GFP-sham group. CTRP9: C1q/TNF-related protein 9; Ad-CTRP9: adenovirus-CTRP9; Ad-GFP: adenovirus GFP; Bcl-2: b-cell lymphoma; MMP: matrix metallopeptidase.

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  Figure 5. CTRP9 activates AKT signaling to relieve PAH in vivo. (a) Following the systemic delivery of Ad-CTRP9 or Ad-GFP, the wild-type rats were subjected to shunt or sham operation. CTRP9 promoted AKT, MAPK, and ERK phosphorylation during PAH. (b) The relative levels of phosphorylated AKT, p38-MAPK, and ERK were quantified using ImageJ and are expressed relative to the β-actin signal. The data are expressed as the mean ± SEM. n = 6 for each group. A total of 6 samples were randomly selected from each group to perform western blotting. ∗∗P < 0.01 and ∗∗∗P < 0.001 vs. the Ad-GFP-sham group. CTRP9: C1q/TNF-related protein 9; PAH: pulmonary arterial hypertension; Ad-CTRP9: adenovirus-CTRP9; p38-MAPK: p38-mitogen-activated protein kinase; ERK: extracellular regulated protein kinase.