Angiotensin AT(1) receptor signalling modulates reparative angiogenesis induced by limb ischaemia

Abstract

1. The concept that angiotensin II exerts pro-angiogenic activity is not universally accepted. We evaluated whether inhibition of the renin-angiotensin system (RAS) would influence reparative angiogenesis in a murine model of limb ischaemia. 2. Perfusion recovery following surgical removal of the left femoral artery was analysed by laser Doppler flowmetry in mice given the ACE inhibitor ramipril (1 mg kg(-1) per day), the AT(1) antagonist losartan (15 mg kg(-1) per day), or vehicle. Muscular capillarity was examined at necroscopy. Ramipril-induced effects were also studied under combined blockade of kinin B(1) and B(2) receptors. Furthermore, the effects of ischaemia on AT(1) gene expression and ACE activity were determined. 3. In untreated mice, muscular AT(1a) gene expression was transiently decreased early after induction of limb ischaemia, whereas AT(1b) mRNA was up-regulated. ACE activity was reduced in ischaemic muscles at 1 and 3 days. Gene expression of AT(1) isoforms as well as ACE activity returned to basal values by day 14. Spontaneous neovascularization allowed for complete perfusion recovery of the ischaemic limb after 21 days. 4. Reparative angiogenesis was negatively influenced by either ramipril (P<0.02) or losartan (P<0.01), leading to delayed and impaired post-ischaemic recovery (50 - 70% less compared with controls). Ramipril-induced effects remained unaltered under kinin receptor blockade. 5. The present study indicates that (a) expression of angiotensin II AT(1) receptors and ACE activity are modulated by ischaemia, (b) ACE-inhibition or AT(1) antagonism impairs reparative angiogenesis, and (c) intact AT(1) receptor signalling is essential for post-ischaemic recovery. These results provide new insights into the role of the RAS in vascular biology and suggest cautionary use of ACE inhibitors and AT(1) antagonists in patients at risk for developing peripheral ischaemia.

Figure 1

Expression of angiotensin II AT_1a and AT_1b receptors in muscles of mice at 0, 1, 3, 7, and 14 days following induction of limb ischaemia. Levels of GAPDH mRNAs are shown for comparisons.

Figure 2

Bar graph shows the ACE activity in limb muscles of mice at 0, 1, 3, 7, and 14 days from induction of ischaemia (n=4-6 mice per each time points). Values are the mean±s.e.mean. ^*P<0.05 vs basal; +P<0.05 vs day 1.

Figure 3

Line graph shows the time-course of perfusion recovery after induction of ischaemia evaluated by laser Doppler flowmetry. Pharmacological treatment started 1 day prior to surgery. Mice were given tap water to drink (vehicle, hatched squares) or water containing losartan (15 mg kg⁻¹ per day, open triangles), or ramipril (1 mg kg⁻¹ per day, open circles). Ramipril was also given in combination with IP bradykinin (BK) B₁ and B₂ receptor antagonists (des-Arg⁹-Leu⁸-BK at 50 nmol kg⁻¹ and Arg,[Hyp³,Thi⁵D-Tic⁷,Oic⁸]-BK at 1 μmol kg⁻¹ per day, respectively, full circles). Values are the mean±s.e.mean and represent ischaemic to non-ischaemic perfusion ratio at the level of distal hindlimb. The perfusion ratio prior to induction of ischaemia (a full square) is shown as a reference. +P<0.01 vs basal (day 0); ^*P<0.05 vs vehicle.

Figure 4

Bar graph shows the effect of chronic treatment with losartan (Los), ramipril (Ram), ramipril in combination with DALBK and Icatibant (Ram+anta), or vehicle (V) on the spontaneous angiogenic response to limb ischaemia. Capillary density per mm² of adductor (A) and the capillary to myofiber ratio (B) were evaluated in ischaemic adductors at day 21 after femoral artery removal. The capillary density of normoperfused adductors of untreated mice (C) was also counted. Values are mean±s.e.mean. ^*P<0.001 vs normoperfused controls. +P<0.01 vs ischaemic adductors of vehicle-treated mice.