Exacerbation of endothelial dysfunction during the progression of diabetes: role of oxidative stress

Abstract

To test the deterioration of endothelial function during the progression of diabetes, shear stress-induced dilation (SSID; 10, 20, and 40 dyn/cm(2)) was determined in isolated mesenteric arteries (80-120 μm in diameter) of 6-wk (6W), 3-mo (3M), and 9-mo (9M)-old male db/db mice and their wild-type (WT) controls. Nitric oxide (NO)-mediated SSID was comparable in 6W WT and db/db mice, but the dilation was significantly reduced in 3M db/db mice and declined further in 9M db/db mice. Vascular superoxide production was progressively increased in 3M and 9M db/db mice, associated with an increased expression of NADPH oxidase. Inhibition of NADPH oxidase significantly improved NO-mediated SSID in arteries of 3M, but not in 9M, db/db mice. Although endothelial nitric oxide synthase (eNOS) expression was comparable in all groups, a progressive reduction in shear stress-induced eNOS phosphorylation existed in vessels of 3M and 9M db/db mice. Moreover, inducible NOS (iNOS) that was not detected in WT, nor in 6W and 3M db/db mice, was expressed in vessels of 9M db/db mice. A significantly increased expression of nitrotyrosine in total protein and immunoprecipitated eNOS was also found in vessels of 9M db/db mice. Thus, impaired NO bioavailability plays an essential role in the endothelial dysfunction of diabetic mice, which becomes aggravated when endothelial nitrosative stress is further activated via perhaps, an additional iNOS-mediated pathway during the progression of diabetes.

Fig. 1.

Shear stress-induced dilation in mesenteric arteries of 6-wk, 3-mo, and 9-mo-old db/db and wild-type (WT) mice in control conditions and after inhibition of nitric oxide (NO) synthesis with nitro-l-arginine methyl ester (l-NAME) (n = 12 for each group). *Significant difference between the curves of control and l-NAME, P < 0.05. #Significant difference between the curves of WT and db/db, P < 0.05.

Fig. 2.

NaNO₂ (NO donor)-induced dilation in mesenteric arteries of 6-wk, 3-mo, and 9-mo-old db/db and WT mice (n = 12 for each group).

Fig. 3.

Protein expression of Nox-1, Nox-2, and β-actin (A and B; n = 3 blots) and superoxide production in the control and in the presence of apocynin, VAS2870, and l-NAME (C; n = 6–9 for each group) in mesenteric arteries of 3- and 9-mo-old WT and db/db mice. D: effects of VAS2870 (VAS) on ANG II-induced superoxide production in mesenteric arteries of 3-mo-old db/db mice (n = 4). *Significant difference between groups, P < 0.05.

Fig. 4.

Shear stress-induced dilation in mesenteric arteries of 3- and 9-mo-old db/db and WT mice in control and after inhibition of NADPH oxidase with VAS2870 (n = 8 for each group). In the group of 3-mo-old db/db mice, additional l-NAME was administered in the presence of VAS2870. *Significant difference from control, P < 0.05.

Fig. 5.

Western blots for mesenteric arteries of 6-wk, 3-mo-, and 9-mo-old db/db and WT mice. A: expressions of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and β-actin. B and C: shear stress (20 dyn/cm², 10 min)-induced eNOS phosphorylation (peNOS). D: immunoprecipitation (IP) of eNOS and immunoblotting (IB) with anti-nitrotyrosine and eNOS antibodies. E: anti-nitrotyrosine in total proteins of mesenteric arteries is shown with 3 or 4 blots for each group. The darkness of protein bands in E reflects the level of nitrotyrosine formation. Actin was used for loading controls. *Significant difference between groups, P < 0.05.