Chronic tissue ischemia due to defective vascular perfusion is a hallmark feature of peripheral artery disease for which minimal therapeutic options exist. We have reported that sodium nitrite therapy exerts cytoprotective effects against acute ischemia/reperfusion injury in both heart and liver, consistent with the model of bioactive NO formation from nitrite during ischemic stress. Here, we test the hypothesis that chronic sodium nitrite therapy can selectively augment angiogenic activity and tissue perfusion in the murine hind-limb ischemia model. Various therapeutic doses (8.25-3,300 g/kg) of sodium nitrite or PBS were administered. Sodium nitrite significantly restored ischemic hind-limb blood flow in a timedependent manner, with low-dose sodium nitrite being most effective. Nitrite therapy significantly increased ischemic limb vascular density and stimulated endothelial cell proliferation. Remarkably, the effects of sodium nitrite therapy were evident within 3 days of the ischemic insult demonstrating the potency and efficacy of chronic sodium nitrite therapy. Sodium nitrite therapy also increased ischemic tissue nitrite and NO metabolites compared to nonischemic limbs. Use of the NO scavenger carboxy PTIO completely abolished sodium nitrite-dependent ischemic tissue blood flow and angiogenic activity consistent with nitrite reduction to NO being the proangiogenic mechanism. These data demonstrate that chronic sodium nitrite therapy is a recently discovered therapeutic treatment for peripheral artery disease and critical limb ischemia.wound healing ͉ endothelial cell ͉ nitric oxide ͉ peripheral artery disease ͉ tissue perfusion

Introductory ParagraphThe coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this O 2 sensing/NO bioactivity linkage postulates an essential role for the conserved hemoglobin β93Cys residue and, specifically, for S-nitrosation of β93Cys to form S-nitrosohemoglobin (SNO-Hb) 1 . The SNO-Hb hypothesis, which conceptually linked hemoglobin and NO biology, has been debated intensely in recent years 2,3 . This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe novel mouse models that express exclusively either human wild type hemoglobin or human hemoglobin in which the β93cys residue is replaced with alanine to assess the role of SNO-Hb in red cell mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red cell S-nitrosothiol levels but shifted S-nitrosothiol distribution to lower MWt species, consistent with the loss of SNO-Hb. Loss of β93cys resulted in no deficits in systemic nor pulmonary hemodynamics under basal conditions and, importantly, did not affect isolated red cell dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO-bioactivity in vivo. *Co corresponding Authors: Rakesh P Patel, PhD, Department of Pathology, University of Alabama at Birmingham, 901 19 th street south, BMR 2, room 302, Birmingham, AL 35294, E mail: E-mail: rakeshp@uab.edu. Tim M Townes, PhD, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Bldg, room 502, 720 20 th street south, Birmingham, AL 35294, E mail: E-mail: ttownes@uab.edu. # these authors contributed equally to this work Author Contributions TSI, CWS, LCW, XT, DAV and KMP were responsible for performing experiments. TSI, CWS, DAV, RPP and TMT were responsible for planning all experiments, analyzing data and writing manuscript. MBR contributed to mass spectrometry assays, LS was responsible for exercise related studies, CGK and BGB for capillary density measurements, NP and JW contributed to blood pressure measurements and NA for assessment of pulmonary hemodynamics. JR did the ES cell injections to generate the chimeras. In addition to hemoglobin oxygen affinity, blood flow is a key component of the processes that match oxygen delivery to demand. Increased blood flow in response to hypoxia is a critical physiological response which does not correlate with dissolved oxygen tensions but does correlate with hemoglobin oxygen fractional saturation 4 . These observations have led to the concept that the red blood cell (RBC) itself is a regulator of flow and to the general paradigm that RBC/hemoglobin deoxygenation is coupled to the stimulation of vasodilation 1,5,6 . Three mechanisms for this coupling have been proposed (...

Dipyridamole augments nitrite/NO production, leading to enhanced arteriogenesis activity and blood perfusion in ischaemic limbs. Together, these data suggest that dipyridamole can augment ischaemic vessel function and restore blood flow, which may be beneficial in peripheral artery disease.

Dipyridamole anti-platelet therapy has previously been suggested to ameliorate chronic tissue ischemia in healthy animals. However, it is not known if dipyridamole therapy represents a viable approach to alleviating chronic peripheral tissue ischemia associated with type 2 diabetes. Here we examine the hypothesis that dipyridamole treatment restores reperfusion of chronic hind limb ischemia in the murine B6.BKS-Leprdb/db diabetic model. Dipyridamole therapy quickly rectified ischemic hind limb blood flow to near pre-ligation levels within three days after starting therapy. Restoration of ischemic tissue blood flow was associated with increased vascular density and endothelial cell proliferation observed only in ischemic limbs. Dipyridamole significantly increased total nitric oxide metabolite levels (NOx) in tissue that were not associated with changes in eNOS expression or phosphorylation. Interestingly, dipyridamole therapy significantly decreased ischemic tissue superoxide and protein carbonyl levels identifying a dominant antioxidant mechanistic response. Dipyridamole therapy also moderately reduced diabetic hyperglycemia and attenuated development of dyslipidemia over time. Together, these data reveal that dipyridamole therapy is an effective modality for the treatment of chronic tissue ischemia during diabetes and highlights the importance of dipyridamole antioxidant activity in restoring tissue NO bioavailability during diabetes.