Mas-Mediated Antioxidant Effects Restore the Functionality of Angiotensin Converting Enzyme 2-Angiotensin-(1–7)-MasAxis in Diabetic Rat Carotid

Abstract

We hypothesized that endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species during type I-diabetes impairs carotid ACE2-angiotensin-(1–7)-Mas axis functionality, which accounts for the impaired carotid flow in diabetic rats. We also hypothesized that angiotensin-(1–7) chronic treatment of diabetic rats restores carotid ACE2-angiotensin-(1–7)-Mas axis functionality and carotid flow. Relaxant curves for angiotensin II or angiotensin-(1–7) were obtained in carotid from streptozotocin-induced diabetic rats. Superoxide or hydrogen peroxide levels were measured by flow cytometry in carotid endothelial cells. Carotid flow was also determined. We found that endothelial AT1-activated NAD(P)H oxidase-driven generation of superoxide and hydrogen peroxide in diabetic rat carotid impairs ACE2-angiotensin-(1–7)-Mas axis functionality, which reduces carotid flow. In this mechanism, hydrogen peroxide derived from superoxide dismutation inhibits ACE2 activity in generating angiotensin-(1–7) seemingly by activating I Cl,SWELL, while superoxide inhibits the nitrergic Mas-mediated vasorelaxation evoked by angiotensin-(1–7). Angiotensin-(1–7) treatment of diabetic rats restored carotid ACE2-angiotensin-(1–7)-Mas axis functionality by triggering a positive feedback played by endothelial Mas receptors, that blunts endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species. Mas-mediated antioxidant effects also restored diabetic rat carotid flow, pointing to the contribution of ACE2-angiotensin-(1–7)-Mas axis in maintaining carotid flow.

Vasoprotective effects of neurocompensatory response to balloon injury during diabetes involve the improvement of Mas signaling by TGFβ1 activation

“…Restenosis could persistently damage the cerebral irrigation by carotid pathways during type I-diabetes since carotid flow is drastically reduced by the enhanced local resistance resultant from diabetic endothelial and muscular dysfunction [9,11,12]. Nevertheless, our previous findings show that this circulatory complication is counterregulated by balloon neurocompensation, which prevents further damages in carotid cerebral irrigation by restoring contralateral carotid flow.…”

“…The fasting glucose levels were determined from rat tail blood samples prior to (day 0) or 48 h (day 2) after STZ injection by using a one-touch glucometer (LifeScan Inc., Milpitas, CA, USA). Rats were considered diabetic when presented a glycemia higher than 300 mg/dL (Table 1) [9,11,12]. Twenty-eight days after STZ-injection [22] balloon injury was performed in the left (ipsilateral) carotid artery of anesthetized rats [4,[7][8][9].…”

“…Isolated common carotid arteries were longitudinally sectioned and endothelial cells were mechanically isolated by gentle friction with a plastic stem in plates containing Hanks' solution (composition in mmol/L: CaCl 2 1.6; MgSO 4 1.0; NaCl 145.0; KCl 5.0; NaH 2 PO 4 0.5; dextrose 10.0; HEPES 10.0, pH 7.4). The cell suspensions were centrifuged at 1.375 ×g for 5 min [8,9,11,12,25] and the supernatant was stored at − 20°C until use. Each n comprised a pool of fifteen carotid arteries.…”

“…Likewise, to investigate if the H 2 O 2 levels were generated from the dismutation of superoxide anion (O 2 − ) (eventually generated by Nox), carotid arteries were also pretreated with tiron (O 2 − scavenger, 0.1 mmol/L) [8,9,11,12] for 30 min before sample homogenization.…”

“…Changes in the isometric tension were measured by connecting the arterial rings to an isometric force transducer (Letica Scientific Instruments, Barcelona, Spain) [7][8][9]11,12,25]. The viability of carotid rings was tested after 60 min of stabilization at a resting tension of 1 g by adding the appropriate molar concentration of phenylephrine that produces 50% of the maximum contraction response (EC 50 ) in each experimental group (phenylephrine EC 50 = 0.1 μmol/L for carotid rings from normoglycemic intact or operated rats, or phenylephrine EC 50 = 10 nmol/L for carotid rings from diabetic intact or operated rats), as previously determined by cumulative concentration-response curves for phenylephrine.…”