H2O2 Mediates VEGF- and Flow-Induced Dilations of Coronary Arterioles in Early Type 1 Diabetes: Role of Vascular Arginase and PI3K-Linked eNOS Uncoupling

Figures

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  Figure 1. The role of NO in mediating flow- and VEGF-induced dilations was investigated in coronary arterioles isolated from control and diabetic pigs. (A): The elevated luminal flow, driven by the longitudinal pressure gradient (Δ pressure) across the vessel, caused vasodilation in normal pigs (n = 9). This vasodilation was compromised in the vessels isolated from diabetic pigs (n = 6). Administration of NOS inhibitor L-NAME (10 µmol/L) abolished the flow-induced vasodilation in both the control (n = 9) and diabetic pigs (n = 6). (B): Coronary arterioles from the control pigs (n = 7) dilated to VEGF in a concentration-dependent manner. VEGF-induced vasodilation was compromised by diabetes (n = 5). Administration of L-NAME abolished the vasodilation in both the control (n = 7) and diabetic (n = 5) vessels. The resting vascular tones were not altered before and after L-NAME in either control (63 ± 1% vs. 62 ± 1%) or diabetic (55 ± 3% vs. 58 ± 4%) vessels. * p < 0.05 vs. control, # p < 0.05 vs. diabetes.

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  Figure 2. The role of H2O2 in mediating flow- and VEGF-induced dilations was investigated in coronary arterioles isolated from control and diabetic pigs. (A): The vasodilation induced by a stepwise increase in pressure gradient (Δ pressure), i.e., flow, in control pigs (n = 6) was not affected by H2O2 scavenger catalase (1000 units/mL; n = 6). The blunted flow-induced dilation in diabetic vessels (n = 8) was further inhibited by catalase (n = 8). (B): The vasodilation induced by VEGF was not affected by catalase in the control pigs (n = 5). In diabetic pigs (n = 6), the vasodilation to VEGF was blunted and the remaining dilation was abolished by catalase. The resting vascular tones were not altered before and after catalase in either the control (61 ± 2% vs. 62 ± 2%) or diabetic (60 ± 2% vs. 60 ± 2%) vessels. * p < 0.05 vs. control, # p < 0.05 vs. diabetes.

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  Figure 3. Responses of coronary arterioles to NO donor sodium nitroprusside and H2O2. (A) Both the control (n = 5) and diabetic (n = 5) vessels dilated to sodium nitroprusside in a concentration-dependent manner without differences. (B) H2O2 produced similar dilations in the control (n = 5) and diabetic (n = 6) vessels and catalase (1000 units/mL) abolished the dilations in the control (n = 5) and diabetic (n = 6) vessels in the same manner. * p < 0.05, control/diabetes vs. catalase treatment.

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  Figure 4. The impacts of L-arginine availability and arginase activity on flow-induced dilation were evaluated in coronary arterioles from pigs with and without diabetes. (A) Administration of L-arginine (3 mmol/L; n = 6) did not alter flow-induced dilation in the control vessels (n = 6) but the impaired vasodilation by diabetes (n = 5) was restored by L-arginine (n = 5). (B) The blunted flow-induced dilation in diabetic vessels (n = 6) was reversed by arginase inhibitor nor-NOHA (0.1 mmol/L; n = 6). Co-incubation with nor-NOHA and L-NAME abolished the flow-induced dilation of coronary arterioles in diabetic pigs (n = 4). The resting vascular tones were not altered before and after L-arginine in either control (59 ± 4% vs. 58 ± 3%) or diabetic (62 ± 3% vs. 63 ± 4%) vessels. nor-NOHA also did not alter the resting tone in diabetic vessels (62 ± 3% vs. 62 ± 4%). * p < 0.05 vs. diabetes.

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  Figure 5. Effect of PI3K inhibition on flow- and VEGF-induced coronary arteriolar dilations. (A) The dilation of coronary arterioles to increased luminal flow (Control; n = 4) was attenuated by diabetes (n = 4) and administration of PI3K inhibitor wortmannin (0.1 µmol/L) abolished dilation of the control (n = 5) and diabetic (n = 5) vessels. (B) The VEGF-induced vasodilation (Control; n = 5) was abolished by wortmannin (n = 5) and the dilation of the diabetic vessels (n = 5) was also abolished by wortmannin (n = 5). The resting vascular tones were not altered before and after wortmannin in either control (64 ± 2% vs. 64 ± 2%) or diabetic (62 ± 3% vs. 65 ± 2%) vessels. * p < 0.05 vs. diabetes.

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  Figure 6. Effect of early diabetes on the mRNA expressions of SOD1, catalase, and GPX in isolated coronary arterioles. (A) The expressions of antioxidant genes (SOD1, catalase, and GPX) were determined by RT-PCR and the expressions were normalized to the 18S transcripts. (B) The quantitative analysis showed no difference in the antioxidant gene expressions between the control (n = 3) and diabetic (n = 3) coronary arterioles.

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  Figure 7. The diagram summarizes the pathways involved in the compensatory generation of H2O2 for NO deficiency in coronary arterioles due to arginase activation in the early stages of type 1 diabetes. L-Arginine is used by eNOS and arginase for the synthesis of NO and L-ornithine, respectively. In healthy coronary arterioles, shear stress and VEGF elicit endothelium-dependent NO-mediated vasodilation via activation of VEGR2/PI3K signaling. In the early stages of diabetes, arginase is activated and competes with eNOS for L-arginine. The reduction in L-arginine availability compromises the production of NO but causes PI3K-linked eNOS uncoupling and consequently promotes superoxide production and H2O2 formation via superoxide dismutase. The compromised NO-mediated vasodilation in early diabetes is compensated by H2O2. The inhibitors used for probing the signaling pathway are indicated.