Dipyridamole reverses peripheral ischemia and induces angiogenesis in the Db/Db diabetic mouse hind-limb model by decreasing oxidative stress

Abstract

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.

Figure 1

Dipyridamole therapy restores diabetic ischemic hind limb blood flow as measured by laser Doppler. Dipyridamole treatment (200 mg/kg) significantly increases and maintains ischemic hind limb blood flow by day 3 post-ischemia compared to vehicle control therapy. n=8 per cohort, *p<0.05.

Figure 2

Dipyridamole therapy promotes ischemic angiogenesis in the diabetic hind limb. Panels A and B illustrate CD31 (endothelial stain; red), Ki67 (proliferating cell stain; green), and DAPI (nuclear stain; blue) staining of non-ischemic and ischemic gastrocnemius tissue at day 7 from vehicle control treated mice. Panels C and D show CD31 (endothelial stain; red), Ki67 (proliferating cell stain; green), and DAPI (nuclear stain; blue) staining of non-ischemic and ischemic gastrocnemius tissue at day 7 from dipyridamole treated mice. Images are representative of 30 sections per tissue and treatment.

Figure 3

Quantitative measurement of dipyridamole mediated vascular density and cell proliferation. Panel A reports the quantitative measurement of vascular density (CD31:DAPI ratio) from images of ischemic (Isch) and non-ischemic (NI) vehicle control and dipyridamole treated diabetic mice at day 7. Panel B shows the quantitative measurement of cell proliferation (Ki67:DAPI) ratio from images of Isch and NI vehicle control and dipyridamole treated diabetic mice at day 7. n=20 per cohort, *p<0.05.

Figure 4

Dipyridamole therapy increases diabetic tissue NOx levels. Tissue NOx levels were measured from vehicle control and dipyridamole treated ischemic (Isch) and non-ischemic (NI) limbs at day 7. n=8 per cohort, *p<0.05.

Figure 5

Dipyridamole therapy does not alter diabetic total eNOS or eNOS phospho-Ser 1177 levels. Panel A illustrates a representative western blot of total eNOS between vehicle control and dipyridamole tissues compared to GAPDH. Panel B shows a representative phospho-eNOS Ser1177 western blot between vehicle control and dipyridamole treatments compared to GAPDH. Panel C reports western blot densitometry for total eNOS expression normalized to GAPDH. Panel D reports the western blot densitometry for phospho-eNOS expression normalized to GAPDH. n=6 per cohort, *p<0.05.

Figure 6

Dipyridamole therapy decreases diabetic tissue superoxide formation and protein oxidation. Panel A reports tissue superoxide production in ischemic (Isch) and non-ischemic (NI) limbs of dipyridamole and vehicle control treated diabetic mice. Panel B shows tissue protein carbonyl levels in Isch and NI limbs of dipyridamole or vehicle control treated diabetic mice. n=6 per cohort, *p<0.05.