Endogenous β-adrenoceptors differentially regulate contraction and angiogenesis of rat aorta via ERK1/2 and p38

M Perez-Aso1, N Flacco1, N Carpena1, MC Montesinos1,2, P D\'Ocon1, MD Ivorra1. 1University of Valencia. Pharmacy Faculty, Pharmacology, Spain, 2University of Valencia, Centre de Reconeixement Molecular i Desenvolupament Tecnològic, Spain

Introduction: Recent studies show that β-adrenoceptors (β-ARs) activate mitogen-activated protein kinase (MAPK: ERK1/2 and p38) in different cell lines (Evans et al. Br J Pharmacol, 159:1022-1038, 2010). However, little is known about the effect of β-AR in this signaling pathway and their functional role in vascular tissues. First, we sought to determine the effect of the β-AR agonist isoprenaline on ERK1/2 and p38 activation, both in rat aorta and in endothelial (EC) and aortic smooth muscle isolated cells (SMC). Secondly, we studied the role of these kinases in β-AR-mediated vasodilatation. Finally, we analyzed the role of β-ARs on angiogenesis.

Methods: Primary EC were isolated from male Wistar rat aorta by cell expansion in Matrigel®, while vascular SMC were isolated by collagenase digestion. Cells were characterized by confocal immunocytochemystry. Aortic rings or cultured isolated cells were incubated with isoprenaline (0.1-10µM) for 5 minutes in the presence or absence of the β-AR antagonist propranolol (1µM). Phosphorylation of ERK1/2 and p38 was analyzed by Western blot. Functional assays were performed by determining the relaxation-response curves to isoprenaline (1 nM to 10 µM) on phenylephrine (1 µM) precontracted aortic rings in the presence or absence of ERK1/2 or p38 inhibitors (U0126 or SB203580, respectively, 10µM). For in vitro angiogenesis assay, aortic explants were seeded on Matrigel® in fully supplemented EGM-2 media in the presence or absence of U0126, SB203580, propranolol and/or isoprenaline for 4 days, and stained with Calcein AM. Newly formed tubes were observed by fluorescent microscopy. One-way ANOVA and Student’t test was performed.

Results: Isoprenaline did not alter ERK1/2 phosphorylation in aortic rings; however it decreased ERK1/2 activation in EC (68.5±4.5% of control integrated optical density, IOD, n=4, P<0.05) while increasing it in SMC (201.1±32.4% of control IOD, n=7, P<0.05). In contrast, isoprenaline promoted p38 phosphorylation in aortic rings and in both cultured cell types. Isoprenaline effects were reversed by propranolol. Propranolol produced a slight increase on p-ERK in EC. Functional studies showed that ERK1/2 inhibition potentiated the relaxation effect of isoprenaline both in the presence (Control:pEC50=7.14±0.13, n=4; U0126:pEC50=7.61±0.09, n=4, P<0.05) and absence of endothelium (Control:pEC50=6.81±0.09, n=5; U0126:pEC50=7.43±0.05, n=4, P<0.001), whereas inhibition of p38 had no effect. In the angiogenesis assay, isoprenaline moderately decreased the new-tubule formation; propranolol not only prevented this inhibitory effect, but it stimulated angiogenesis (136.7±15.3% of control sprout area, n=4, P<0.05). The ERK1/2 inhibitor produced a decrease on new-tubule formation, while a great stimulation was observ ed in the presence of the p38 inhibitor; these results evidenced a proangiogenic role of ERK1/2 and an inhibitory angiogenic effect of p38. No additional effect of isoprenaline on angiogenic inhibition or stimulation induced by ERK1/2 or p28 inhibitors, respectively, was detected.

Conclusions: Endogenous β-ARs differentially modulate ERK1/2 and p38 activity in the smooth muscle or endothelium of aorta. β-ARs exert an antiangiogenic effect, decreasing the endothelial-driven new tubule formation, probably throughout p38 activation and/or ERK1/2 inhibition in the endothelial cells. An inhibitory effect of ERK1/2 on β-ARs mediated vasodilatation in intact and endothelium-denuded aorta has also been evidenced. Supported by SAF2007-62120 and Fondos Feder.