Microvascular vasodilator properties of the angiotensin II type 2 receptor in a mouse model of type 1 diabetes

Figure 1. Metabolic profile of the animals. Glucose tolerance (A), insulin tolerance (B), pancreas insulin content (C) and diabetes induction shown as percentage of mice responding to streptozotocin injection (D) were determined in wild-type (WT) and AT2R−/y (KO) mice. Glycaemia (E), weight loss (F) and mortality (G) were determined 5, 20 and 45 days after injection of streptozotocin on day zero (0) in WT and KO mice. Mean ± SEM is presented (n = 8 mice per group). *p < 0.05 KO versus WT.

Figure 2. COX and oxidative stress pathways. The mRNA expression level of COX-2 (A), COX-1 (B), p67phox (C), p22phox (D), p67phox (E) and MnSOD (F) was determined in the aorta isolated from wild-type (WT) and AT2R−/y (KO) mice using Q-RT-PCR. Mice were either treated with streptozotocin (STZ, black bars) or not (control, grey bars). Mean ± SEM is presented (n = 8 mice per group). COX-2 expression level (G) was determined using immunohistochemistry and reactive oxygen levels (ROS, H) using dihydroethidium staining in kidneys isolated from WT and AT2R−/y mice. *p < 0.05, STZ versus control; #p < 0.05, KO versus corresponding WT.

Figure 3. Angiotensin II receptors and eNOS expression levels. AT2R (A), AT1aR (B), AT1bR (C), NOS3 (D) and NOS2 (E) mRNA expression levels were determined in the aorta isolated from wild-type (WT) and AT2R−/y (KO) mice using Q-RT-PCR. Mice were treated with streptozotocin (STZ, black bars) or were not treated (control, grey bars). Mean ± SEM is presented (n = 8 mice per group). Data is expressed relative to the housekeeping gene (GAPDH, control). *p < 0.05, STZ versus control.

Figure 4. Blood 8-isoprostane and creatinine in mice. 8-isoprostane (A) and creatinine (B) blood levels were determined in wild-type (WT) and AT2R−/y (KO) mice whether treated or not with streptozotocin (STZ): Mean ± SEM is presented (n = 8 mice per group). *p < 0.05: effect of STZ; #p < 0.05: WT versus KO.

Figure 5. AT2R-dependent dilatation in mesenteric resistance arteries. Vasodilatation was induced by the AT2R agonist CGP42112 (0.1 μmol/L, A,C) and by angiotensin II (10 nmol/L) in the presence of the AT1R blocker candesartan (100 nmol/L, B,D) in mesenteric arteries isolated in wild-type (WT) and AT2R−/y (KO) mice whether treated or not (CONT) with streptozotocin (STZ). Vasodilatation was repeated after incubation of the arteries with L-NAME (0.1 mmol/L, LN). Endothelium-dependent (acetylcholine)-mediated dilatation (E) and KCl (80 mmol/L)-mediated contraction (F) were measured in arteries of WT and KO mice. Mean ± SEM is presented (n = 6 mice per group). *p < 0.05: effect of STZ. Typical recordings show vasodilatation induced by angiotensin II in the presence of candesartan. After stabilizing the response, acetylcholine (ACh) was added to the bath in order to measure maximum vasodilatation. Recordings were obtained from a control WT mouse (left recording) and in diabetic WT mice (right recording).

Figure 6. AT2R-dependent dilatation in isolated and perfused mouse kidneys. Vasodilatation induced by the AT2R agonist CGP42112 (0.1 μmol/L, A) and by angiotensin II (10 nmol/L) in the presence of the AT1R blocker candesartan (100 nmol/L, B) was determined in the perfused kidneys isolated from wild-type (WT) and AT2R−/y mice whether treated or not (CONT) with streptozotocin (STZ). AT2R-mediated vasodilatation was repeated after incubation of the arteries with the AT2R blocker PD123319 (1 μmol/L, C). Kidney perfusion pressure (D), phenylephrine (10 μmol/L)-mediated contraction (E) and endothelium-dependent (acetylcholine)-mediated dilatation (F) were measured in WT and KO mice. Mean ± SEM is presented (n = 6 mice per group). *p < 0.05: effect of STZ. Typical recordings show the vasodilatation induced by angiotensin II in the presence of candesartan. Recordings were obtained in a control (non-diabetic) WT mouse (left recording) and in a diabetic WT mouse (right recording).