Abstract-Ischemia is a stimulus for production of angiogenic cytokines that activate local vascular cells and mobilize angiogenic cells to the circulation. These responses are impaired in elderly patients with peripheral arterial disease. Hypoxia-inducible factor (HIF)-1 mediates adaptive responses to ischemia, including production of angiogenic cytokines. In this study, we demonstrate that aging and HIF-1 loss-of-function impair the expression of multiple angiogenic cytokines, mobilization of angiogenic cells, maintenance of tissue viability, and recovery of limb perfusion following femoral artery ligation. We show that HIF-1 directly activates transcription of the gene encoding stem cell factor and that mice lacking the cognate receptor C-KIT have impaired recovery from ischemia. Administration of AdCA5, an adenovirus encoding a constitutively active form of HIF-1␣, improved the recovery of perfusion in older mice to levels similar to those in young mice. Injection of AdCA5 into nonischemic limb was sufficient to increase the number of circulating angiogenic cells. These results indicate that HIF-1 activity is necessary and sufficient for the mobilization of angiogenic cells and that HIF-1␣ gene therapy can counteract the pathological effects of aging in a mouse model of limb ischemia.

SummaryWe present here the analysis of fluid-phase endocytosis (FPE) in human blood monocytes and monocyte-derived dendritic cells (MDDC) facilitated by our serendipitous identification of rottlerin as an efficient inhibitor of dendritic cell FPE (IC 50 of 0Á4 lM). Rottlerin was found to be an excellent tool for FPE analysis: rapid-acting, irreversible and selective for FPE (as opposed to receptor-mediated endocytosis) at concentrations of 3 lM and below. The inhibitory effect was not due to toxicity or visible change in membrane ruffles, but affects on cytoskeletal reorganization were evident in MDDC treated with relevant rottlerin concentrations during adhesion. A marked increase in FPE was observed in 1 hr interleukin (IL)-4 and granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated monocytes. Moreover, rottlerin inhibited the augmented FPE of 1-day cytokine treated monocytes and their augmented ability to induce T cell proliferative responses to tetanus toxoid. We conclude that rottlerin is a useful tool for investigating FPE and its functional importance.

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-1α, 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.