RESUMEN
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-Lepr(db/db) diabetic model. Dipyridamole therapy quickly rectified ischemic hind-limb blood flow to near preligation levels within 3 days of the start of 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 in tissue, which were not associated with changes in endothelial NO synthase 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.
Asunto(s)
Diabetes Mellitus Experimental/prevención & control , Dipiridamol/farmacología , Modelos Animales de Enfermedad , Miembro Posterior/irrigación sanguínea , Isquemia/prevención & control , Neovascularización Fisiológica , Estrés Oxidativo , Animales , Miembro Posterior/efectos de los fármacos , Miembro Posterior/metabolismo , Hipercolesterolemia/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación , Receptores de Leptina , Superóxidos , Factor A de Crecimiento Endotelial Vascular , Vasodilatadores/farmacologíaRESUMEN
AIMS: Anti-platelet agents, such as dipyridamole, have several clinical benefits for peripheral artery disease with the speculation of angiogenic potential that could preserve ischaemic tissue viability, yet the effect of dipyridamole on ischaemic arteriogenesis or angiogenesis is unknown. Here we test the hypothesis that dipyridamole therapy augments arteriolar vessel development and function during chronic ischaemia. METHODS AND RESULTS: Mice were treated with 200 mg/kg dipyridamole twice daily to achieve therapeutic plasma levels (0.8-1.2 microg/mL). Chronic hindlimb ischaemia was induced by permanent femoral artery ligation followed by measurement of tissue perfusion using laser Doppler blood flow along with quantification of vascular density, cell proliferation, and activation of nitric oxide (NO) metabolism. Dipyridamole treatment quickly restored ischaemic hindlimb blood flow, increased vascular density and cell proliferation, and enhanced collateral artery perfusion compared with control treatments. The beneficial effects of dipyridamole on blood flow and vascular density were dependent on NO production as dipyridamole did not augment ischaemic tissue reperfusion, vascular density, or endothelial cell proliferation in endothelial NO synthase (eNOS)-deficient mice. Blood and tissue nitrite levels were significantly higher in dipyridamole-treated mice compared with controls and eNOS(-/-) mice, verifying increased NO production that was regulated in a PKA-dependent manner. CONCLUSION: 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.
Asunto(s)
Dipiridamol/farmacología , Isquemia/tratamiento farmacológico , Neovascularización Fisiológica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Enfermedad Crónica , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dipiridamol/sangre , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Isquemia/metabolismo , Isquemia/fisiopatología , Flujometría por Láser-Doppler , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neovascularización Fisiológica/fisiología , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/genética , Nitritos/metabolismo , Enfermedades Vasculares Periféricas/tratamiento farmacológico , Enfermedades Vasculares Periféricas/metabolismo , Enfermedades Vasculares Periféricas/fisiopatología , Inhibidores de Agregación Plaquetaria/sangreRESUMEN
The 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 oxygen-sensing-NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin beta-chain (betaCys93) and, specifically, for S-nitrosation of betaCys93 to form S-nitrosohemoglobin (SNO-Hb). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years. This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the betaCys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell-mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of betaCys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood 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.
Asunto(s)
Eritrocitos/fisiología , Hemoglobinas/fisiología , Hipoxia/fisiopatología , Vasodilatación/fisiología , Animales , Eritrocitos/patología , Hemodinámica , Hemoglobinas/genética , Hemoglobinas/metabolismo , Humanos , Hipoxia/patología , Ratones , Nitratos/análisis , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitritos/análisis , Nitritos/metabolismo , Nitrosación , Oxígeno/metabolismo , S-Nitrosotioles/análisis , S-Nitrosotioles/metabolismoRESUMEN
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 mug/kg) of sodium nitrite or PBS were administered. Sodium nitrite significantly restored ischemic hind-limb blood flow in a time-dependent 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.