Blockade of advanced glycation end-product formation restores ischemia-induced angiogenesis in diabetic mice

Abstract

We hypothesized that formation of advanced glycation end products (AGEs) associated with diabetes reduces matrix degradation by metalloproteinases (MMPs) and contributes to the impairment of ischemia-induced angiogenesis. Mice were treated or not with streptozotocin (40 mg/kg) and streptozotocin plus aminoguanidine (AGEs formation blocker, 50 mg/kg). After 8 weeks of treatment, hindlimb ischemia was induced by right femoral artery ligature. Plasma AGE levels were strongly elevated in diabetic mice when compared with control mice (579 +/- 21 versus 47 +/- 4 pmol/ml, respectively; P < 0.01). Treatment with aminoguanidine reduced AGE plasma levels when compared with untreated diabetic mice (P < 0.001). After 28 days of ischemia, ischemic/nonischemic leg angiographic score, capillary density, and laser Doppler skin-perfusion ratios were 1.4-, 1.5-, and 1.4-fold decreased in diabetic mice in reference to controls (P < 0.01). Treatment with aminoguanidine completely normalized ischemia-induced angiogenesis in diabetic mice. We next analyzed the role of proteolysis in AGE formation-induced hampered neovascularization process. After 3 days of ischemia, MMP-2 activity and MMP-3 and MMP-13 protein levels were increased in untreated and aminoguanidine-treated diabetic mice when compared with controls (P < 0.05). Despite this activation of the MMP pathway, collagenolysis was decreased in untreated diabetic mice. Conversely, treatment of diabetic mice with aminoguanidine restored collagenolysis toward levels found in control mice. In conclusion, blockade of AGE formation by aminoguanidine normalizes impaired ischemia-induced angiogenesis in diabetic mice. This effect is probably mediated by restoration of matrix degradation processes that are disturbed as a result of AGE accumulation.

Fig. 1.

(A) Ischemic/nonischemic angiographic score ratio. (B) Ischemic/nonischemic capillary density ratio. (C) Ischemic/nonischemic foot blood flow expressed as a ratio of blood flow in ischemic limb to that in nonischemic limb. Values are mean ± SEM; n = 7 per group. *, P < 0.05 versus control (C) group at day 28; †, P < 0.05 versus STZ-treated animals (SPTZ) at day 28; ≠, P < 0.05 versus STZ and aminoguanidine-treated mice (SPTZ+AG) at day 28. SPTZ+AG+MMPi, doxycycline (nonselective MMP inhibitor) and aminoguanidine-treated diabetic mice.

Fig. 2.

(A Left) Representative gelatin zymographic analysis of protein extract from untreated animals and diabetic mice treated with or without aminoguanidine (AG) at day 3 after ischemia. (Right) Representative reverse-zymography analysis of protein extract from untreated animals and diabetic mice treated with or without aminoguanidine at day 3 after ischemia. (B) Densitometric analysis of zymographic (Left) and reverse-zymographic (Right) gels in ischemic and nonischemic leg at day 3 after femoral artery occlusion in mice. (C) Densitometric analysis of zymographic (Left) and reverse-zymographic (Right) gels in ischemic and nonischemic leg at day 28 after femoral artery occlusion in mice. Values are mean ± SEM; n = 7 per group. *, P < 0.05 versus ischemic control (C) mice. SPTZ, STZ-treated animals; SPTZ + AG, STZ and aminoguanidine-treated mice.

Fig. 3.

(A) Representative Western blot of MMP-3 and actin protein content in ischemic and nonischemic leg at day 3 after femoral artery occlusion. Quantitative evaluation of MMP-3 expressed as a percentage of nonischemic control (C) at days 3(B) and 28 (C) after ischemia is shown. Values are mean ± SEM; n = 7 per group. *, P < 0.05 versus ischemic control mice. SPTZ, STZ-treated animals; SPTZ+AG, STZ and aminoguanidine-treated mice.

Fig. 4.

(A) Representative Western blot of MMP-13, three-fourths cleaved collagen, and actin protein content in ischemic and nonischemic leg at day 3 after femoral artery occlusion. (B) Quantitative evaluation of MMP-13 (Left) and three-fourths cleaved collagen (Right) protein levels expressed as a percentage of nonischemic control (C) at day 3 after ischemia. (C) Quantitative evaluation of MMP-13 protein content expressed as a percentage of nonischemic control at day 28 after ischemia. Values are mean ± SEM; n = 7 per group. *, P < 0.05 versus ischemic control mice; †, P < 0.05 versus ischemic diabetic mice. nd, not detected; SPTZ, STZ-treated animals; SPTZ+AG, STZ and aminoguanidine-treated mice.

Fig. 5.

Representative photomicrographs of ischemic muscle sections from control (Cont) and treated mice stained with antibody directed against AGEs (A, AGEs appear in green), with Sirius red (B, collagen fibers appear in red), with antibody against AGEs and collagen [C, AGEs appear in green, collagen appears in red, and colocalization (Merge) is revealed by yellow staining; ischemic muscle section of diabetic mice], and with antibody directed against cleaved collagen (D, positive staining appears in brown). (Magnification, ×20.) SPTZ, STZ-treated animals; SPTZ+AG, STZ and aminoguanidine-treated mice.