C1q/TNF-Related Protein 9 Promotes Revascularization in Response to Ischemia via an eNOS-Dependent Manner

Figure 1. CTRP9-KO mice show reduction of perfusion recovery and capillary density in ischemic limbs. (A) Representative laser Doppler blood flow (LDBF) images in ischemic limb of CTRP9-KO or WT mice. A low perfusion signal (dark blue) was observed in the ischemic hindlimb of CTRP9-KO mice, whereas a high perfusion signal (red) was detected in WT mice at postoperative days 14 and 28. (B) Quantitative analysis of the ischemic to nonischemic LDBF ratio in the WT and CTRP9-KO mice before surgery and at different time points after surgery. Results are shown as the mean ± SD (n = 7-8 mice for each group). *p < .05, **p < .01 vs CTRP9-KO mice. (C) Fluorescence staining of ischemic tissues with anti-CD31 monoclonal antibody (red) at postoperative day 28. (D) Quantitative analysis of capillary density in WT and CTRP9-KO mice on postoperative day 28. Capillary density was expressed as the number of capillaries per muscle fiber. Results are presented as mean ± SD (n = 3 mice for each group).

Figure 2. CTRP9 supplementation enhances perfusion of ischemic limbs in WT and CTRP9-KO mice. (A) CTRP9 expression in plasma in WT and CTRP9-KO mice after treatment with Ad-CTRP9 or Ad-β-gal (control). Ad-CTRP9 or Ad-β-gal was injected into the jugular vein of WT and CTRP9-KO mice (7 × 108 ifu in each group) at day 5 before surgery. Plasma CTRP9 levels were assessed at day 5 after adenoviral vector injection by Western blot analysis. (B) Quantitative analysis of ischemic to nonischemic laser Doppler blood flow (LDBF) ratio in WT and CTRP9-KO mice receiving Ad-β-gal or Ad-CTRP9 on postoperative day 14 (n = 4–9 mice for each group). (C) Quantitative analysis of capillary density in WT and CTRP9-KO mice receiving Ad-β-gal or Ad-CTRP9. Capillary density was expressed as the number of capillaries per muscle fiber. Results are presented as mean ± SD (n = 3 mice for each group).

Figure 3. CTRP9 promotes network formation and migration of cultured endothelial cells. (A) Endothelial cell network formation after stimulation with CTRP9 or vehicle. After 24 h of serum deprivation, human umbilical vein endothelial cells (HUVECs) were treated with recombinant CTRP9 (10 μg/ml), or PBS (control) followed by culture in Matrigel-coated dishes. Representative photomicrographs of HUVEC network formation is shown (upper panel). Quantitative analyses of the network area of tubes are shown (lower panel) (n = 3 independent culture experiments). (B) Endothelial cell migration after stimulation with CTRP9 or vehicle. A modified Boyden chamber assay was performed using HUVECs. HUVECs were treated with CTRP9 (10 µg/ml), or PBS (control). Results are presented as mean ± SD. Results are expressed relative to the values compared with control (n = 8 independent assays).

Figure 4. Effect of CTRP9 on the phosphorylation of AMP-activated kinase (AMPK), Akt, and endothelial nitric oxide synthase (eNOS) in endothelial cells and ischemic muscle. (A) Time-dependent changes in the phosphorylation of AMPK, Akt, and eNOS signaling in endothelial cells following stimulation with CTRP9. Changes in the phosphorylation of AMPK (p-AMPK), Akt (p-Akt), and eNOS (p-eNOS) after CTRP9 treatment were determined by Western blot analysis. Representative blots are shown. (B) Quantitative analysis of relative changes in phosphorylated AMPK, Akt, and eNOS in endothelial cells following stimulation with CTRP9 (n = 3–4 independent culture experiments). (C) The phosphorylation of AMPK, Akt, and eNOS in the ischemic muscle of WT and CTRP9-KO mice at day 7 after surgery. Phosphorylation of AMPK, Akt, and eNOS was determined by Western blot analysis. (D) Quantitative analysis of relative changes in the phosphoylation of AMPK, Akt, and eNOS in the ischemic muscle of WT and CTRP9-KO mice at day 7 after surgery (n = 3–4 mice for each group). Relative phosphorylation levels were normalized to total protein signal. Relative phosphorylation levels of AMPK, Akt, and eNOS were quantified using…

Figure 5. Endothelial nitric oxide synthase (eNOS) signaling is required for angiogenic response to CTRP9. (A) Effect of Compound C and LY294002 on CTRP9-stimulated eNOS phosphorylation. Human umbilical vein endothelial cells (HUVECs) were pretreated with Compound C (10 µmol/L), LY294002 (10 µmol/L), or vehicle (DMSO) for 30–60 min and treated with CTRP9 (10 μg/ml) or PBS (control) for 15 min. Phosphorylated eNOS (p-eNOS) was determined by Western blot analysis. (B) Quantitative analysis of relative changes in p-eNOS is shown. Relative phosphorylation levels of eNOS were quantified by using ImageJ software. Relative phosphorylation levels were normalized to total protein signal. Results are presented as mean ± SD (n = 5 independent culture experiments). (C) Contribution of eNOS to CTRP9-induced network formation of HUVECs. HUVECs were pretreated with L-NAME (1 mmol/L) or vehicle (PBS) and treated with CTRP9 (10 μg/ml), or PBS (control) for 17 h. Representative photomicrographs of HUVEC network formation are shown (upper panel). Quantitative analyses of the network area of tubes are shown. Results are presented as mean ± SD (lower panel) (n = 5–9 independent culture experiments). (D)…

Figure 6. Proposed scheme for the angiogenic actions of CTRP9.