Elevated pressure causes endothelial dysfunction in mouse carotid arteries by increasing local angiotensin signaling

Abstract

Experiments were performed to determine whether or not acute exposure to elevated pressure would disrupt endothelium-dependent dilatation by increasing local angiotensin II (ANG II) signaling. Vasomotor responses of mouse-isolated carotid arteries were analyzed in a pressure myograph at a control transmural pressure (PTM) of 80 mmHg. Acetylcholine-induced dilatation was reduced by endothelial denudation or by inhibition of nitric oxide synthase (NG-nitro-L-arginine methyl ester, 100 μM). Transient exposure to elevated PTM (150 mmHg, 180 min) inhibited dilatation to acetylcholine but did not affect responses to the nitric oxide donor diethylamine NONOate. Elevated PTM also increased endothelial reactive oxygen species, and the pressure-induced endothelial dysfunction was prevented by the direct antioxidant and NADPH oxidase inhibitor apocynin (100 μM). The increase in endothelial reactive oxygen species in response to elevated PTM was reduced by the ANG II type 1 receptor (AT1R) antagonists losartan (3 μM) or valsartan (1 μM). Indeed, elevated PTM caused marked expression of angiotensinogen, the precursor of ANG II. Inhibition of ANG II signaling, by blocking angiotensin-converting enzyme (1 μM perindoprilat or 10 μM captopril) or blocking AT1Rs prevented the impaired response to acetylcholine in arteries exposed to 150 mmHg but did not affect dilatation to the muscarinic agonist in arteries maintained at 80 mmHg. After the inhibition of ANG II, elevated pressure no longer impaired endothelial dilatation. In arteries treated with perindoprilat to inhibit endogenous formation of the peptide, exogenous ANG II (0.3 μM, 180 min) inhibited dilatation to acetylcholine. Therefore, elevated pressure rapidly impairs endothelium-dependent dilatation by causing ANG expression and enabling ANG II-dependent activation of AT1Rs. These processes may contribute to the pathogenesis of hypertension-induced vascular dysfunction and organ injury.

Keywords: ANG II type 1 receptors; angiotensin II; endothelium; hypertension.

Fig. 1.

Effects of a transient increase in transmural pressure (P_TM) on acetylcholine-induced dilatation in mouse isolated carotid arteries. Dilatation was analyzed at a P_TM of 80 mmHg during KCl (34 mM) constriction in arteries that had been maintained at 80 mmHg (P80) or exposed to a transient increase in P_TM (150 mmHg, 180 min) (P150). Top: effects of elevated P_TM in untreated arteries. Middle and bottom: influence of the direct antioxidant and NADPH oxidase inhibitor apocynin (100 μM) in arteries exposed to 150 mmHg or maintained at 80 mmHg, respectively. Data are expressed relative to baseline diameter and presented as means ± SE (n = 6). B, baseline; C, KCl constriction. Vertical arrows indicate statistical comparisons between maximal responses, whereas horizontal arrows indicate comparisons between −log 30% dilatation of the KCl constriction (EC₃₀) values (***P < 0.001; **P < 0.01; NS, not significant).

Fig. 2.

Effects of elevated P_TM on endothelial reactive oxygen species activity and the expression of ANGogen in mouse carotid arteries. A: reactive oxygen species activity was assessed using 5-(and 6)-chloromethyl-29,79-dichlorodihydro-fluorescein diacetate (DCDHF) fluorescence in isolated arteries maintained at 80 mmHg or exposed to 150 mmHg for 15 min (P150₁₅) or 180 min (P150₁₈₀). Effects of elevated P_TM were assessed under control conditions and after ANG II type 1 receptor antagonism by losartan (3 μM). Representative images (green, DCDHF; blue, Draq5) and combined data with means ± SE (n = 3 to 4) are presented. Fluorescence is expressed as detector units. B: angiotensinogen (ANGogen) expression was assessed in arteries maintained at a P_TM of 80 mmHg or exposed to elevated P_TM (150 mmHg, 180 min). A representative immunoblot and combined density data (relative to β-actin expression) with means ± SE (n = 6) are presented. The mean at 150 mmHg was set as 100%, and the intensity of all measurements expressed relative to that value. ***P < 0.001; **P < 0.01.

Fig. 3.

Effects of inhibiting angiotensin-converting enzyme (1 μM perindoprilat or 10 μM captopril) on acetylcholine-induced dilatation in mouse isolated carotid arteries. Dilatation was analyzed at a P_TM of 80 mmHg during KCl (34 mM) constriction in arteries that had been maintained at 80 mmHg (P80) or exposed to a transient increase in P_TM (150 mmHg, 180 min) (P150). Data are expressed relative to baseline diameter and presented as means ± SE (n = 6). Vertical arrows indicate statistical comparisons between maximal responses, whereas horizontal arrows indicate comparisons between −log EC₃₀ values. **P < 0.01 significant difference between perindoprilat and control; ###P < 0.001, significant difference between captopril and control. For ease of presentation, control represents a combined control group (n = 12).

Fig. 4.

Effects of inhibiting AT₁Rs (3 μM losartan or 1 μM valsartan) on acetylcholine-induced dilatation in mouse isolated carotid arteries. Dilatation was analyzed at a P_TM of 80 mmHg during KCl (34 mM) constriction in arteries that had been maintained at 80 mmHg (P80) or exposed to a transient increase in P_TM (150 mmHg, 180 min) (P150). Data are expressed relative to baseline diameter and presented as means ± SE (n = 6). Vertical arrows indicate statistical comparisons between maximal responses, whereas horizontal arrows indicate comparisons between −log EC₃₀ values. ***P < 0.01, significant difference between losartan and control; ###P < 0.001; #P < 0.05, significant differences between captopril and control. For ease of presentation, control represents a combined control group (n = 12).