Abstract 456: Rage Suppresses Angiogenesis and Blood Flow Recovery After Hind Limb Ischemia in Diabetic Mice Through Modulation of Macrophage Inflammation and Macrophage-endothelial Interactions

Peripheral vascular disease is a condition characterized by atherosclerotic narrowed arteries distal to the aorta which triggers an acute or critical limb ischemia. Development of ischemic PVD has been considered one of the principal complications of diabetes, leading to amputation of digits and limbs. Advanced glycation end products (AGE) ligands and their receptor (RAGE) have been implicated in multiple key mechanisms underlying diabetes and diabetic complications, including hypoxia and ischemia/reperfusion injury. We tested the hypothesis that vascular recovery after hind limb ischemia would be rescued by deficiency of RAGE, at least in part through modulation of macrophage dysfunction. Wild type (WT) and Ager deficient mice were rendered diabetic with streptozotocin, and subjected to unilateral hind limb ischemia. Previous results showed an increased accumulation and expression of AGEs and RAGE in ischemic muscle, especially in diabetic WT mice. Attenuated angiogenesis and impaired blood flow recovery were also observed, in parallel with reduced early inflammatory macrophage infiltration into ischemic muscle in the WT diabetic mice. We performed flow cytometry to analyze circulating monocyte subsets: pro-inflammatory Ly6G/C hi and anti-inflammatory Ly6G/C lo . Work by others reported higher levels of monocytes in diabetes in the baseline state without injury; our data indicate that the increase is mostly attributed to the pro-inflammatory Ly6G/C hi population, being significantly lower in the Ager -/- diabetic mice. After seven days of ischemia to the unilateral hind limb, lower levels of circulating pro-inflammatory Ly6G/C hi monocytes were found in both WT and Ager deficient diabetic mice. After four weeks of injury, the pro-inflammatory monocytes levels were significantly recovered to baseline levels in Ager -/- mice, whereas WT mice failed to reacquire their baseline levels. In vitro studies using murine endothelial cells and murine macrophages revealed that RAGE suppressed macrophage-endothelial cell interaction, particularly in diabetes-relevant concentrations of D-glucose. These data suggest unique ischemia-dependent mechanisms in hind limb ischemia through RAGE down-regulation of the early adaptive immune response.