Angiopoietin-2 impairs collateral artery growth associated with the suppression of the infiltration of macrophages in mouse hindlimb ischaemia

Figures

• 

  Fig. 1. r-Ang-2 impaired blood flow perfusion during ischaemia. a Representative laser-Doppler images of the plantar foot at the indicated time points after femoral artery ligation. “Pre” and “Post” are immediately before and after surgery, respectively. b The mean blood flow ratio of ischaemic to non-ischaemic hindlimb foot pads for all animals at indicated time points (n = 6/group; *P < 0.05 vs. the r-Ang-2 groupat 5 and 7 days). c Representative laser-Doppler images of the adductor region at the indicated time points after femoral artery ligation. “Pre” and “Post” are immediately before and after surgery, respectively. Outlined areas shown the regions of interest. d The mean blood flow ratio in ischaemic to non-ischaemic hindlimb footpads for all animals at the indicated time points (n = 6/group; *P < 0.05 vs. r-Ang-2 group at 5 and 7 days). e Representative images of PECAM-1+ capillaries in ischaemic gastrocnemius muscles 7 days after femoral artery ligation. Scale bars 100 μm. f Mean capillary density in ischaemic gastrocnemius muscles assessed by PECAM-1 immunostaining. g Representative images of collateral arterioles in ischaemic adductor muscles, assessed using H&E staining. h…

• 

  Fig. 2. r-Ang-2 suppresses inflammatory cell infiltration. a Recruitment of macrophages in response to ischaemia as determined by Mac-3 staining. Representative images of macrophages surrounding collateral arterioles in ischaemic adductor muscles in r-Ang-2 treated-mice on day 7 after femoral artery ligation are shown. b mRNA expression of CD11c (M1 marker), and c mRNA expression of CD206 (M2 marker) in ischemic adductor muscles as measured by real-time PCR. d Ratio of CD11c to CD206, The data were normalized to the expression level of 18S rRNA in each sample. All values represent the mean ± SEM (n = 6/group). *P < 0.05 vs. vehicle control. e Thioglycollate-elicited mouse peritoneal macrophages were used to evaluate macrophage migration. Cells were added to the upper chambers of cell culture inserts and incubated with Ang-2 (200 ng/mL) or vehicle control and then treated with MCP-1 (100 ng/mL) or vehicle control as indicated. Representative images of macrophages that migrated to lower-chamber side of transwell membrane. f The data represent the mean of triplicate experiments. *P < 0.05 vs. negative control (cells exposed neither MCP-1 nor Ang-2). **P < 0.05 vs. macrophages incubated with…

• 

  Fig. 3. r-Ang-2 modulates post-ischaemic gene regulation. mRNA expression levels of angiopoietin-1, Tie-2, PDGF-BB, VEGF-A, VEGF-C, and MCP-1 were analysed using real-time PCR in ischaemic adductor muscles. The data were normalized to the expression level of 18S rRNA in each sample. All values represent the mean ± SEM (n = 6/group). *P < 0.05 vs. vehicle control

• 

  Fig. 4. Effects of r-Ang-2 on VSMC migration in vitro. Migration of human coronary artery smooth muscle cells in the presence of r-Ang-2, followed by treatment with PDGF-BB (20 ng/mL) or vehicle control was studied. The data represent the mean of triplicate experiments. *P < 0.05 vs. negative control. **P < 0.05 vs. cells incubated with PDGF-BB only