RCAN1 MODULATES COX-2 EXPRESSION IN THE VASCULAR WALL

AB Garcia-Redondo1, V Esteban2, AM Briones1, N Mendez-Barbero2, L Garcia-Redondo1, JM Redondo2, M Salaices1. 1Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPAZ)., 1Dpt. Farmacología. Facultad de Medicina. 28029, Spain, 2Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029, Spain

Introduction: Cyclooxygenase (COX-2) expression is regulated by several transcription factors such as NFAT, CREB and NFƘB. NFAT regulates gene expression in response to the increase in the intracellular free calcium levels [Ca2+]i. The increase in [Ca2+]i stimulates calcineurin, which dephosphorylates NFAT and induces its translocation to the nucleus. Rcan is an endogenous modulator of calcineurin activity and its expression is positively regulated by NFAT. On the other hand, Rcan can also modulate NFƘB activation by stabilizing its inhibitory protein IκBα. Importantly, NFƘB is involved in the expression of inflammatory genes. COX-2 derived prostanoids and Rcan1 are involved in several pathological processes, including tumor growth and angiogenesis, sepsis, cardiac hypertrophy, synaptic plasticity and aortic aneurism formation and restenosis.

Aim: To analyse the possible role of Rcan1 on vascular COX-2 expression and in vascular function.

Methods Vascular smooth muscle cells (VSMC) and aortic segments from Rcan1 Knockout (Rcan1-/-) and wild type (Rcan+/+) mice were used. Aortic contraction was analysed by wire myography. COX-2 and TXA2 synthase (TXAS) expression was analysed by RT-PCR and/or western blot. TXB2 levels were analysed by enzymoimmunoassay. Differences were evaluated using one or two-way ANOVA and Bonferroni’s post-hoc test (experiments with three or more groups). Statistical significance was assigned at P < 0.05.

Results: COX-2 mRNA levels and protein expression was greater in aortic VSMCs obtained from Rcan1-/- than from Rcan1+/+ mice (COX-2 mRNA; Rcan1+/+ : 1; Rcan1-/- : 11.68). Additionally, adenoviral-mediated re-expression of Rcan1.4 in Rcan1-/- VSMCs decreased COX-2 expression. These results suggest that Rcan1 negatively modulates COX-2 expression in the vascular wall. Vasoconstrictor responses induced by phenylephrine (1 nM-30 μM) were greater in aorta from Rcan1-/- compared to Rcan1+/+ mice (Emax: Rcan1+/+ : 49.14±1.8, n=16; Rcan1-/- 95.42±5.44% n=20; p<0.0001). The inhibitors of COX-2 and TXAS, etoricoxib (10 μM) and furegrelate (100 μM), respectively, reduced phenylephrine contraction only in aorta from Rcan1-/- mice (Emax: Rcan1+/+ Control: 49.14±1.8 n=16; Etoricoxib; 52.28±3.05 n=8; Furegrelate: 61.9±4.87 n=7; Rcan1-/- control: 87.25±4.3 n=13; Etoricoxib: 60.06±4.9 n=13; Furegrelate: 46.73±5.71% n=5, P<0.001). TXB2 levels but not TXAS protein expression was greater in aorta from Rcan1-/- than Rcan1+/+ mice (TXB2 levels: Rcan1+/+ : 27.63 ± 6.62 n=4; Rcan1-/- : 102.12 ± 16.6 pg/mg n=5; p<0.0001). These results suggest that COX-2-derived TXA2 participates in the increased vascular contraction to phenylephrine observed in Rcan1-/- mice. The calcineurin inhibitor, cyclosporine A (200 ng/ml), and the NFƘB inhibitor, parthenolide (1 μM) inhibited phenylephrine contraction only in aorta from Rcan1-/- mice (Emax: Rcan1+/+ Control: 49.14±1.8 n=16; CsA: 60.47±3.41 n=10; Parthenolide: 36.86±4.17 n=4; Rcan1-/- Control: 95.42±5.44 n=20; CsA: 45.5±8.57 n=5; Parthenolide: 52.30±16 n=5). Moreover, both cyclosporin A and pathenolide abolished the inhibitory effect of etoricoxib on phenylephrine-induced contraction observed in aorta from Rcan1-/- mice (Emax: Rcan1+/+ CsA+ Etoricoxib: 56.97±3.07 n=7;Parthenolide + Etoricoxib: 40.3±3.1 n=4; Rcan1-/- CsA+ Etoricoxib: 44.71 ± 11.7 n=5; Parthenolide + Etoricoxib: 63.10 ± 12.4 n=5)

Conclusions: Rcan1 negatively modulates vascular COX-2 expression and the participation of COX-2-derived products in vascular function probably through the Calcineurin/NFAT and NFƘB pathways.

Supported by ISCIII (Red RECAVA, RD06/0014/0011 and RD06/0014/0005), MICIN (SAF- 2009-07201)