Role Of Angiopoietin-2(Ang-2) In Neovascularization In A Mouse Hindlimb Ischemia Model

Objective: to explore the role of angiopoietin-2(Ang-2) in neovascularization in a mouse hindlimb ischemia model. Methods:1. The murine hindlimb ischemia model and intraperitoneal administration: After anesthesia, briefly, the left femoral artery was exposed and ligated distally to the origin of the arteria profunda femoris, then cut the femoral artery between two ligations and sutured the skin. After rewarming, relative blood flow to the foot was measured under standardized conditions by laser Doppler imaging. Measurements were performed pre- and post-artery ligation, additionally on postoperative days 3, 5, and 8. The left-to-right ratio(occluded-to-nonoccluded leg) was calculated for each animal. Six mice were equably seperated into two groups. Each mouse in the first group was intraperitoneal injected normal saline with the same dose of recombination-human-Angiopoietin-2 in the second group. On the days of 1, 3 and 5, we did the injection. On the eighth day, mice were killed. The blood was drawn, and the adductor and gastrocnemius were harvested. 2. Detect r-Ang-2 level in serum with enzyme-linked immunosorbent assay technique: Follow the ELISA protocol, test the r-Ang-2 level in serum. 3. Analyse target gene expression in muscle sample with real time PCR technique: grind mice muscle tissue with liquid nitrogen, lyse the mice muscle tissue with trizol reagent, extract RNA with traditional method, examine the quality of RNA by agarose gel electrophoresis, reverse transcribe m RNA into c DNA, analyse target genes(including Ang-1, Ang-2, Tie-2, PDGF-B, PDGFR-beta, VEGF-A, VEGF-C) with real time PCR technique. 4. Test neovascularization in ischemia tissue with immumofluorescence method: the tissue was fixed with 4% paraformaldehyde and embedded with paraffin and cut into slices. We use α‐SMA to quantify pericyte coverage. quantification was performed by analyzing at least 3 sections and 3 fields per tissue. 5. Study the influence of Ang-2 to PDGF-BB signaling pathway: human umbilical smooth muscle cells were cultured in dishes. We set up 4 groups, containing blank-group, Ang-2-group, PDGF-BB-group, Ang-2-PDGF-BB-group. We did two assays(cell scratch assay and westernblotting). 6. Statistical analysis: Data are presented as the mean ± SEM and were analyzed by ANOVA and by unpaired two‐tailed Student’s t test. P values of <0.05 were regarded as statistically significant. Results: 1. After the day of ligation, blood flow in control group gradually increases in adductor but not in Ang-2 group. And there were no apparent changes in gastrocnemius in two groups. 2. From the ELISA results, r-Ang-2 can be detected in Ang-2 group but not in control group. 3. Ang-1, Ang-2, PDGF-BB, VEGF-A, VEGF-C were down-regulated in adductor in Ang-2 group in real time PCR results. 4. Compared with control group, the diameter of vessel in adductor in Ang-2 group is smaller in immunofluorescence. 5. In cell scratch assay, PDGF-BB-group restores fastest, Ang-2-PDGF-BB group faster, Ang-2-group fast, compared with control group; in western blot, Ang-2 blocks the PDGF-BB-induced PDGFR-beta phosphorylation. Conclusions: 1. Ang-2 inhibits vessels’ formation and maturity in adductor, but has no obvious influence in gastrocnemius. 2. R-Ang-2 exists in serum of mice and influences vascularization. 3. Ang-2 down-regulated vascular factors(including Ang-1, Ang-2, PDGF-BB, VEGF-A, VEGF-C) in vessel formation in ischemia tissue. 4. Ang-2 can inhibit the growth of vessel in adductor after ischemia. 5. The PDGF-BB signaling pathway can be weaken or inhibited by Ang-2.