DIFFERENT ACTIVITY OF CARVEDILOL, NITROGLYCERIN AND DOBUTAMINE AS MODULATORS OF ANGIOGENESIS

D Vicente1, G Fornaciari1, D Pascual1, MC Montesinos1,2, P D\'Ocon1. 1University of Valencia, Departmento de Farmacologia, 46100, Spain, 2University of Valencia, Centre de Reconeixement Molecular i Desenvolupament Tecnològic,46100, Spain

Aims: Research studies have identified agents which modulate the new blood vessel formation, such as NO, VEGF, FGF, and other growth factors (1), but none or little information has been provided about the role that the therapeutic agents traditionally employed in the pharmacotherapy of the ischemic myocardium play in the angiogenic process. Therefore, the aim of the present work was to determine the angiogenic activity of three drugs extensively used in patients with ischemic heart failure: nitroglycerin, carvedilol and dobutamine, as well as the involvement of NO on this activity. Methods: Aortas from 16 week old Wistar rats were dissected free of periaortic fibro-adipose tissue and sectioned transversally into 1mm rings which were seeded on 50 µl of polymerized Matrigel® in a 96 well plate, with fetal calf serum supplemented endothelial cell growth medium MV2, and incubated at 37°C and 5% CO2 for 7 days. In some experiments the medium was also enriched with hEGF, hbFGF, VEGF, R3IGF-1, ascorbic acid and hydrocortisone. Some samples were incubated with vehicle (H2O or DMSO) or nitroglycerin (0.01-1-10 µM), carvedilol (1 µM), dobutamine (10 µM), phenylephrine (1µM), DETA-NO (1-10-100 µM) and L-NAME (100 µM), renewing the medium, as well as the corresponding stimuli, the day after starting the experiment, and every two days. Image acquisition of tube formation was achieved daily with an inverted microscope (objective 2.5x). Growth kinetics was determined by measuring the sprouts length from the outer surface of the ring in the x and y axis. The statistical analysis included the two way Anova method, followed by a Student’s t-test for unpaired samples . Results: Angiogenic growth was significantly inhibited (P<0.01) by dobutamine (10 µM) (β1-adrenoceptor agonist) but was not modified by phenilephrine (1 µM) (α1-adrenoceptor agonist) or carvedilol (1 µM) (α1- and β-adrenoceptor antagonist). However, the length of the new angiogenic vessels was significantly increased by incubation with phenylephrine + Carvedilol (P<0,05). Therapeutical doses of nitroglycerine (0.01 µM) significantly inhibited the angiogenic growth (P<0.01) and the same effect was observed in rings incubated with the NO-donor DETA-NO (100 µM) (P<0.05). By the contrary, incubation with the NOS inhibitor L-NAME (100 µM) significantly increased the growth of the new vessels (P<0.05). Conclusions: Angiogenesis was negatively modulated by nitroglycerin, dobutamine and endogenous or exogenous NO, while carvedilol facilitated the formation of new vessels when α1 adrenoceptors were activated. The peculiar behavior of carvedilol could be related to its “biased agonist” activity on an alternate pathway mediated by β-arrestin-2 and ERK1/2 (2,3) which could contribute to its therapeutic usefulness in the long-term treatment of heart failure.

(1) Aplin AC, et al. Methods in Enzymology, 443: 119-136 (2008)

(2) Rajagopal S, et al. Nat Rev Drug Discovery, 9: 373-386 (2010)

(3) Tzingounis AV, et al. PNAS, 107: 21028-21033 (2010)

Study supported by a grant from Universitat de València (2011-0739). Diana Vicente received a fellowship from the Spanish Ministry of Education and Science