Fig. 1. Effect of streptozotocin treatment on the vasorelaxant response of phenylephrine-constricted aortic rings to (A) the endothelium-dependent vasodilator acetylcholine (ACh, endothelium-intact) and (B) the endothelium-independent vasodilator nitroglycerin (NTG, endothelium-denuded). Data are expressed as mean ± SE of 16–28 experiments. (A) *P < 0.001 vs. EC₅₀ of control. (B) §P < 0.05 vs. similar concentration of NTG in control rings.

Fig. 2. Fluorescent photomicrographs of aortic sections of control and diabetic animals incubated with the superoxide-sensitive dye dihydroethidum (red staining). The autofluorescence of the elastic laminae is green. E, endothelium; M, medium; and A, adventitia. Data are representative of similar results obtained from 3 to 4 animals.

Fig. 3. Effect of the streptozotocin treatment (“Diabetes”) on the NADPH oxidase activity in aorta and heart of rats. Membrane fractions of (A) heart and (B) aorta from control and diabetic animals were stimulated with 200 μM NADPH, heart membranes also in the presence or absence of 10 μM DPI, and the lucigenin (5 μM)-derived chemiluminescence (CL) (counts/min/mg) was detected (“basal”: CL in the absence of NADPH). Data are expressed as mean ± SE of 4–7 animals. *P < 0.001 vs. group without DPI, †P < 0.05 vs. control.

Fig. 4. Effect of streptozotocin treatment (“Diabetes”) on the expression of nox1 and nox4 in rat aorta. (A) Original Western blots representing 4 control and 4 diabetic animals. Equal protein loading was verified by α-actinin immunostaining. (B) Densitometric quantification of blots (n = 6–12 animals).*P < 0.001 vs. control.

Fig. 5. Effect of streptozotocin treatment (“Diabetes”) on NOSIII expression. (A) Immunofluorescent staining of NOSIII (red) in aortic cryosections of control and diabetic animals. The autofluorescence of the elastic laminae is green. E, endothelium; M, media; and A, adventitia. Data are representative of similar results obtained from 3 to 4 animals. (B) Densitometric quantification of Western blots using a NOSIII antibody. Data are expressed as mean ± SE of 12 animals. *P < 0.05 vs. control.

Fig. 6. Expression of Ser₂₃₉-P-VASP in aortic tissue of control and of diabetic rats. (A) Original blot of each of two representative control and diabetic animals in the presence or absence of 1 μM ACh. (B) Densitometric quantification of Western blots. Data are expressed as mean ± SE of 5 animals. *P < 0.05 vs. control.

Fig. 7. Effect of streptozotocin treatment on ACh-stimulated vascular NO production, assessed by EPR spin trapping using colloid Fe(DETC)₂. (A) Original EPR spectra obtained from ACh (1 μM, 1 h)-stimulated aortas of a representative control and a diabetic animal. “g 2.035” indicates the position of the g value characteristic for the NOFe(DETC)₂ complex. The horizontal arrow indicates the direction of the increase in magnetic field strength; its length represents an increase in 2.5 mT. (B) NO formation as calculated from the EPR signal amplitude expressed as picomoles NO per square centimeter endothelial surface per minute. Data are expressed as the mean ± SE of 3–4 animals. *P < 0.05 vs. control.

Animal body weights and blood glucose levels at the time of sacrifice

Data are expressed as the mean ± SE.

Detection of ROS in isolated rat heart mitochondria, of XO activity in cytosolic fraction and of XO activity in plasma by chemiluminescence and the cytochrome c assay