Evidence for the importance of angiotensin II type 1 receptor in ischemia-induced angiogenesis

Abstract

The role of the renin-angiotensin system (RAS) in angiogenesis is little known. Here, we show that the angiotensin II (ATII) type 1 (AT1) receptor plays an important role in ischemia-induced angiogenesis. Well-developed collateral vessels and angiogenesis were observed in wild-type (WT) mice in response to hindlimb ischemia, whereas these responses were reduced in ATII type 1a receptor knockout (AT1a(-/-)) mice. Ischemia-induced angiogenesis was also impaired in WT mice treated with the AT1 receptor blocker TCV-116. These effects were not due to reduced systemic blood pressure (SBP), because hydralazine treatment preserved angiogenesis in WT mice although it reduced SBP to a level similar to that of AT1a(-/-) mice. Infiltration of inflammatory mononuclear cells (MNCs), including macrophages and T lymphocytes, was suppressed in the ischemic tissues of AT1a(-/-) mice compared with WT mice. Double immunofluorescence staining revealed that infiltrated macrophages and T lymphocytes expressed VEGF, and the expression of VEGF and monocyte chemoattractant protein-1 was also decreased in AT1a(-/-). Finally, the impaired angiogenesis in AT1a(-/-) mice was rescued by intramuscular transplantation of MNCs obtained from WT mice, further indicating the importance of MNC infiltration in ischemia-induced angiogenesis. Thus, the ATII--AT1 receptor pathway promotes early angiogenesis by supporting inflammatory cell infiltration and angiogenic cytokine expression.

Figure 1

Angiogenesis was impaired in the ischemic hindlimb of AT1a^–/– mice. (a) A low perfusion signal (dark blue) was observed in the ischemic hindlimb of an AT1a^–/– mouse, whereas a high perfusion pattern (red to orange) was detected in a control WT mouse. (b) Computer-assisted quantitative analyses of hindlimb blood perfusion demonstrated a significant reduction in the ischemic/normal hindlimb blood flow ratio in AT1a^–/– mice (n = 11) compared with WT animals (n = 13). *P < 0.05, **P < 0.01, ***P < 0.001, WT vs. AT1a^–/–. (c) Postmortem angiograms showed markedly reduced collateral vessel formation in AT1a^–/– mouse compared with WT mouse. (d) Quantitative analysis revealed a low angiographic score in AT1a^–/– mice examined on postoperative day 14 (n = 5 in each group). (e) Immunostaining of ischemic tissues with anti-CD31 mAb (brown) showed markedly reduced capillary density in an AT1a^–/– mouse compared with a WT mouse. (f) Quantitative analysis revealed a reduced capillary density in AT1a^–/– mice examined on postoperative day 14 (n = 5 in each group).

Figure 2

Effects of hydralazine and ATII receptor blockers on ischemia-induced angiogenesis. No impairment in ischemic/normal hindlimb LDBF ratio was observed in WT mice treated with hydralazine (WT + Hydralazine) (P = NS vs. WT). Administration of the AT1 receptor blocker TCV-116 (5 mg/kg/d) impaired ischemia-induced angiogenesis in WT mice (WT + TCV-116) (P < 0.01 vs. WT), so that the LDBF values were comparable to those observed in AT1a^–/– mice. Administration of the AT2 receptor blocker PD123319 (30 mg/kg/d) did not alter ischemia-induced angiogenesis in AT1a^–/– mice (AT1a^–/– + PD123319) (P = NS vs. AT1a^–/–).

Figure 3

Inflammatory responses and expression of angiogenic cytokines in ischemic tissues. (a) H&E staining revealed marked infiltration of inflammatory leukocytes in the ischemic tissues of WT mice but not in those of AT1a^–/– mice. (b) Quantitative analysis revealed reduced inflammatory cell infiltration in AT1a^–/– mice examined on days 3 and 7 after induction of limb ischemia (n = 4 in each group). (c) Double immunofluorescence staining of the ischemic tissues revealed that macrophages and T lymphocytes expressed VEGF protein. VEGF showed green fluorescence, whereas cell surface markers (F4/80 and CD3) were stained in red. Skeletal myocytes in the ischemic tissues also moderately expressed VEGF protein. (d) Quantitative analysis revealed that the number of infiltrating macrophages and T lymphocytes was significantly smaller in AT1a^–/– mice than in WT mice. The number of VEGF-positive inflammatory cells was also significantly lower in AT1a^–/– mice than in WT mice. (e) Western blot analysis revealed that the expression of VEGF and MCP-1 proteins in the ischemic tissues was weaker in AT1a^–/– mice than in WT mice on days 3 and 7 after induction of limb ischemia.

Figure 4

Impacts of the implantation of WT mouse– or AT1a^–/– mouse–derived MNCs into the ischemic hindlimbs of AT1a^–/– mice on angiogenesis. The impaired LDBF ratio in AT1a^–/– mice was partially but significantly restored after implantation of WT mouse–derived MNCs into the ischemic hindlimb of AT1a^–/– mice (P < 0.01 vs. AT1a^–/–). In contrast, implantation of AT1a^–/– mouse–derived MNCs slightly improved the LDBF ratio in AT1a^–/– mice, but the difference was not significant (vs. AT1a^–/–). Neutralizing anti-VEGF mAb treatment abolished the WT-MNC–mediated rescue of impaired angiogenesis in AT1a^–/– mice (P = NS vs. AT1a^–/–).