The ROS-COX-2 axis is involved in vascular dysfunction in hypertension

S Martínez, MS Avendaño, AB García-Redondo, Y Álvarez, A Aguado, V Pérez-Grirón, MJ Alonso, AM Briones, M Salaices. Universidad Autonoma de Madrid, Pharmacology 28029, Spain

Aim: This study evaluates whether there is a relationship between the contribution of reactive oxygen species (ROS) and cyclooxygenase (COX)-2-derived products on vascular responses in conductance and resistance arteries from hypertensive animals. Method: Mesenteric resistance arteries and/or aorta from C57BL6 mice and spontaneously hypertensive (SHR) and normotensive Wistar Kyoto WKY rats were used. Mice were divided into five groups: 1) Control; 2) infused with Angiotensin II (1.44 mg/Kg/day, 2 weeks, subcutaneously by osmotic minipumps); 3) infused with Angiotensin II and the NAD(P)H inhibitor apocynin (1.5 mmol/L in the drinking water); 4) infused with Angiotensin II and the mitochondria-targeted antioxidant mito-TEMPO (0.7 mg/Kg/day i.p.); 5) infused with Angiotensin II and the COX-2 inhibitor celecoxib (25 mg/Kg/day i.p.). All treatments started 24 h before Angiotensin II-infusion. SHR rats were divided into three groups: 1) control, 2) treated with the superoxide dismutase mimetic tempol (1 mmol/L, in the drinking water, 18 days) and 3) treated with celecoxib (3 weeks). Vascular function was studied by wire myography, COX-2 or NAD(P)H Oxidase subunits (NOX) gene or protein expression by RT-PCR or western blot respectively, ROS production was determined with the oxidative fluorescent dye dihydroethidium (DHE), NAD(P)H Oxidase activity was analyzed by enhanced lucigenin chemiluminiscence and blood pressure was measured by tail-cuff pletysmography. All data are expressed as mean values±SEM. Results were analyzed by using unpaired Student’s t-test or one-way or two-way ANOVA followed by Bonferroni’s post hoc test. A p<0.05 was considered significant Results: Angiotensin II increased systolic blood pressure (Control: 98.6±1.3; Angiotensin II: 154±3.5 mm Hg, P<0.0001 vs Contro,l n=7) and the vasoconstrictor response to phenylephrine and reduced acetylcholine induced relaxation. These alterations were prevented by apocynin, mito-TEMPO and celecoxib treatments (Angiotensin II+apocynin: 129.2±4.3; Angiotensin II+mito-TEMPO: 119.7±3.3; Angiotensin II+Celecoxib: 129.9±3.19 mm Hg; P<0.001 vs Angiotensin II, n= 7 for each group). In SHR, tempol treatment did not modify blood pressure; however, celecoxib treatment partially decreased blood pressure. In Angiotensin II-infused mice, mito-TEMPO and/or apocynin treatments prevented the increase in vascular COX-2 expression (Control: 100±4; Angiotensin II: 172.45±20.8, P<0.005 vs Control; Angiotensin II+Apocynin: 85±10; Angiotensin II+mito-TEMPO: 81.4±21% P<0.001 vs Angiotensin, II n=5-7). These treatments also normalized the increased participation of COX-derived contractile prostanoids and the role of TP receptors in phenylephrine responses induced by Angiotensin II. In SHR, tempol reduced the altered vascular production of prostanoids and its participation on vascular responses; however tempol did not affect COX-2 expression. In the Angiotensin II infused mice and in SHR, celecoxib treatment normalized the increased ROS production, NOX-1 expression, NAD(P)H Oxidase activity (Control: 100±42; Angiotensin II: 720±273.7, P<0.05 vs Control; Angiotensin II+Celecoxib: 50.5±19.6%, P<0.05 vs Angiotensin II n=7 for each group) and the participation of ROS in vascular responses. In the Angiotensin II infused mice, apocynin, mito-TEMPO and celecoxib improved the dimished nitric oxide (NO) production (Control: 100±17; Angiotensin II: 39.6±6.9, P<0.001 vs Control; Angiotensin II+Apocynin: 109.3±23.6; Angiotensin II+mito-TEMPO: 120.2±32; Angiotensin II+Celecoxib: 135.4±41.6 P<0.05 vs Angiotensin II n=5-7) and the modulation by NO of phenylephrine responses. Conclussion: This study provides mechanistic evidence of a circuitous relationship between COX-2 and ROS products in hypertension. The excess of ROS from NAD(P)H Oxidase and/or mitochondria and the increased vascular COX-2/TP receptor axis act in concert to decrease NO bioavailability, induce vascular dysfunction and hypertension.

Supported by MICIIN (SAF-2009-07201), INSCIII (Red RECAVA, RD06/0014/0011) and SEF-Almirall.