COX-2 derived prostanoids, reactive oxygen species and GRK2 underlie the functional, structural and mechanical vascular alterations in hypertension

Resumen

The Renin-Angiotesin-Aldosterone System (RAAS) is responsible of the functional and structural alterations of the vasculature associated to hypertension. RAAS activation induces vascular inflammation characterized by marked upregulation of enzymes such as cyclooxygenase 2 (COX-2) and increased reactive oxygen species (ROS) production that might modulate nitric oxide (NO) bioavailability. In addition, G protein-coupled receptor kinases 2 (GRK2) are involved in the desensitization of G protein coupled receptors (GPCR) such as Angiotensin II (Ang II) type 1 receptors (AT1) and in the regulation of NO production. The main aim of this Thesis was to investigate the role of oxidative stress from NADPH Oxidase and mitochondria, COX-2 derived prostanoids and GRK2 in the vascular functional and structural abnormalities observed in hypertension. In line with this general aim, the Thesis was divided into four specific aims: 1. Knowing the pivotal role of aldosterone in the regulation of vascular tone and in hypertension development, in the first article we analyzed the effect of three weeks aldosterone treatment on noradrenaline-induced vasoconstriction in mesenteric resistance vessels from normotensive (WKY) and spontaneously hypertensive (SHR) rats. We also studied the role of endothelium-derived mediators, such as COX-2-derived prostanoids, NO and ROS in the aldosterone effect. In vessels with endothelium, the contractile responses to noradrenaline were similar in both strains irrespective of the presence or the absence of aldosterone. However, aldosterone treatment diminished the protective effect of the endothelium on noradrenaline contraction. This was probably due to decreased NO availability produced by increased ROS production. In addition, aldosterone increased the participation of COX-2-derived products and TP receptor in the noradrenaline responses. Importantly, the effect of aldosterone on NO, ROS and prostanonoids was greater in SHR compared to WKY. In conclusion, long-term treatment of rats with aldosterone despite of not affecting noradrenaline contraction, alters the role of endothelium on these responses being this effect more pronounced in SHR than in WKY. Aldosterone treatment alters the release of COX-2-derived prostanoids, NO and ROS that might participate on the above mentioned endothelial modulation. 2. Previous articles of our group and others demonstrated that ROS and COX-2-derived products are involved in vascular responses in hypertension. We next questioned whether a reciprocal relationship between ROS producing enzymes (NADPH Oxidase and mitochondria) and COX-2 might modulate vascular functional responses of conductance and resistance arteries in hypertension. For this purpose, we used two different models of hypertension, Ang II infused mice and SHR rats. Animals were treated with the antioxidant and NADPH Oxidase inhibitor apocynin, the mitochondrial-targeted superoxide dismutase (SOD) 2 mimetic mito-TEMPO, the SOD analogue tempol, or the COX-2 inhibitor celecoxib. Antioxidant treatments diminished the increased vascular COX-2 expression, prostanoids production and the participation of COX-derived contractile prostanoids and TP receptor in phenylephrine responses observed in arteries from both hypertensive models. On the other hand, celecoxib treatment normalized the increased ROS production, NADPH Oxidase expression and activity, SOD2 expression and the participation of ROS in vascular responses in both hypertensive models. Importantly, apocynin, mito-TEMPO, and celecoxib treatments reduced high blood pressure and increased the diminished NO production and the modulation of phenylephrine responses by NO in the Ang II model. Our results demonstrate that the excess of ROS from NADPH Oxidase and/or mitochondria and the increased vascular COX-2/TP receptor axis act in concert to induce vascular dysfunction and hypertension. 3. Alterations in the structure and mechanical properties of the arteries are important contributors to high blood pressure. Prostanoids from COX-2, microsomal prostaglandin E synthase (mPGES)-1 and prostaglandin E2 receptors (EP) contribute to vascular remodeling in different cardiovascular pathologies but its role in hypertension is not known. We explored the contribution of the COX-2/mPGES-1/EP1 receptor axis in vascular remodeling and function in conductance and resistance arteries from Ang II infused mice and SHR. Both hypertensive models were treated with celecoxib and the Ang II infused mice were also treated with the EP1 receptor antagonist SC19220. In addition, we used COX-2 deficient (COX-2-/-) mice infused or not with Ang II. Pharmacological blockade of COX-2 did not improve the altered structural parameters (lumen diameter and wall:lumen ratio) observed in arteries from hypertensive animals. However, celecoxib treatment and COX-2 deficiency normalized the increased vascular stiffness and the diminished wall distensibility observed in mesenteric resistance arteries from both hypertension models. These effects were probably due to decreased fibrosis since diminished collagen deposition, improved elastin structure and decreased conective tissue growth factor, tenascin-C and plasminogen activator inhibitor-1 gene expression were observed after COX-2 inhibition. In hypertensive animals vascular mPGES-1 gene was increased which was normalized by celecoxib treatment. The inhibition of EP1 receptor did not modify the structural parameters. Nevertheless, it normalized the alterations in vascular stiffness, wall distensibility, collagen levels and altered elastin structure observed in Ang II infused mice. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses to phenylphrine and the endothelial dysfunction induced by Ang II. These results suggest that the COX-2/mPGES-1/EP1 axis modulates extracellular matrix deposition, mechanical properties and vascular function in hypertension. 4. GRK2 is the most abundant GRK in vessels and it plays a determinant role in the control of systemic vascular responses by regulating not only GPCR, such as AT1, but also the AKt-eNOS pathway. Levels or activity of GRK2 are increased in different tissues from animal models of hypertension or in patients and correlate with high blood pressure in humans. In the last article we investigated if GRK2 is involved in the functional, structural and mechanical vascular alterations associated to hypertension. Given the lack of selective inhibitors for GRK2, we investigated the effects elicited by GRK2 inhibition in vascular responses using global adult hemizygous GRK2 mice (GRK2+/-). Partial GRK2 deficiency increased the vasodilator responses to acetylcholine and isoproterenol in mesenteric resistance arteries and aorta although no differences in vascular structure and mechanics or in resting blood pressure were observed when compared to wild type (WT) mice. After Ang II infusion, GRK2+/- mice were partially protected against hypertension and showed improved vascular structure (wall:lumen ratio) and mechanical properties (vascular stiffness) when compared to WT littermates. In addition, Ang II infusion increased GRK2 levels and vasoconstrictor responses to phenylephrine in WT but not in GRK2+/- vessels and decreased vasodilator responses to acetylcholine and vascular pAkt and eNOS levels more in WT than in GRK2+/-. Vascular NO production and the modulation of vasoconstrictor responses by endothelial-derived NO were greater in GRK2+/- mice infused with Ang II. Our results describe an important role for GRK2 in systemic hypertension and further establish that an inhibition of GRK2 could be a beneficial treatment for this condition. In conclusion, the results shown in this Thesis demonstrate that COX-2-derived prostanoids, ROS and GRK2 acting alone or in reciprocal relationship, are key components involved in the functional, structural and mechanical alterations observed hypertension both in conductance and resistance arteries. Whether pharmacological blockade of these targets might have beneficial effects in hypertension associated vascular damage in humans warrants future investigations.