Model Of Critical Limb Ischemia Research Articles

Adenoviral transfer of HIF-1α enhances vascular responses to critical limb ischemia in diabetic mice

Diabetes is a major risk factor for ischemic disease. Treatment options for diabetic patients with peripheral arterial disease when revascularization is not possible are limited, resulting in a high incidence of limb amputation. We evaluated the therapeutic potential of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, in a diabetic model of critical limb ischemia. Diabetic db/db and nondiabetic db/+ mice were subjected to unilateral femoral artery ligation. Limb perfusion, tissue viability, and motor function were more severely impaired in db/db mice. Intramuscular injection of AdCA5 into the ischemic limb of db/db mice increased the recovery of limb perfusion and function, reduced tissue necrosis, rescued the diabetes-associated impairment of circulating angiogenic cells, enhanced endothelial nitric oxide synthase activation, and increased vessel density and luminal area in the ischemic limb.

A018 Modulation of macrophage activation state protects tissue from necrosis during critical limb ischemia in thrombospondin-1-deficient mice

Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. Using a genetic model of tsp-1-/- mice subjected to femoral artery excision, we report that tsp-1-/- mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1-/- and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1-/- mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1-/- mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1-/- mice, thereby demonstrating that macrophages mediated tissue protection in these mice. This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

Fibroblast growth factor-2 gene transfer can stimulate hepatocyte growth factor expression irrespective of hypoxia-mediated downregulation in ischemic limbs.

Hepatocyte growth factor (HGF) is a potent angiogenic polypeptide that stimulates angiogenesis. Transcriptional regulation of HGF, however, has not been fully defined, with the exception of the hypoxia-mediated downregulation in cultured cells. In the present study, we report that angiogenic growth factors, including HGF, were upregulated in a murine model of critical limb ischemia in vivo, a finding that was in conflict with previous in vitro data. Mice deficient in basic fibroblast growth factor-2 (FGF-2) showed reduced induction of HGF protein in ischemic muscles, and overexpression of FGF-2 via gene transfer stimulated endogenous HGF, irrespective of the presence of ischemia. In culture, FGF-2 rapidly stimulated HGF mRNA, and a sustained expression was evident in the time course in vascular smooth muscle cells and fibroblasts. FGF-2-mediated induction of HGF was fully dependent on the mitogen-activated protein kinase pathway yet was not affected by either hypoxia or a protein kinase A inhibitor. In the early expression, FGF-2 directly stimulated HGF mRNA without the requirement of new protein synthesis, whereas sustained induction of HGF in the later phase was partly mediated by platelet-derived growth factor-AA. Furthermore, in vivo overexpression of FGF-2 significantly improved the blood perfusion, and the effect was abolished by systemic blockade of HGF in ischemic limbs. This is the first demonstration of a regulational mechanism of HGF expression via FGF-2 that was independent of the presence of hypoxia. The harmonized therapeutic effects of FGF-2, accompanied with the activity of endogenous HGF, may provide a beneficial effect for the treatment of limb ischemia.