Intraluminal administration of matrix metalloproteinase 2 (MMP-2) increases vascular oxidative stress and the vasoconstriction induced by phenylephrine in rabbit aortic rings

A Azevedo1,3, AF Prado1,3, L Pernomian1,2, JE Tanus-Santos1, LM Bendhack2, RF Gerlach3. 1Faculty of Medicine of Ribeirão Preto, Pharmacology, Brazil, 2Faculty of Pharmaceutical Science - University of São Paulo, Physics and Chemistry, Brazil, 3Dental School of Ribeirão Preto - University of São Paulo, Morphology, Estomatology and Physiology, Brazil

Introduction: Matrix metalloproteinase 2 (MMP-2) play an important role in cardiovascular diseases. In rat mesenteric arteries, the MMP-2 may favors vasoconstriction by cleavage of peptides (Fernandez-Patron, 1999;2000). In transgenic mice (with MMP-2 overexpression) increased cardiac lipid peroxidation was observed (Zhou et al., 2007). Thus, we hypothesized that the MMP-2 could increase the vascular oxidative stress (ROS) promoting greater vasoconstriction induced by Phenylephrine (PE). Aim: the present study was to evaluate the reactive oxygen species (ROS) content and the vascular function of aortic rings after intraluminal injection of MMP-2. Methods: thoracic aorta from New Zealand rabbits (3.0 Kg, FMRP, USP Committee 120/2010) was isolated, and divided into three segments. One segment was filled (intraluminal exposure) with Krebs solution (vehicle), MMP-2 (1.2 µg/mL) or MMP-2 + doxycycline (doxy, MMP inhibitor at 100µM) at 37º C for 30 minutes. After incubation, one part of the vessels were frozen in OCT, 4 µm sections. The vascular production of ROS was assessed by fluorescence intensity (arbitrary units – U) using dihydroethidium (DHE) 1 µM, while the in situ gelatinolytic activity was measured by quantification of fluorescence intensity. For the quantification of the results, it was used the software Image J. Other part of the vessel was used for the vascular reactivity. Cumulative concentration-response curves for phenylephrine (PE) (0.1 - 10 µM/L) were performed in intact (E+) or denuded (E-) rings, in the absence or presence of L-NAME (non-selective inhibitor of nitric oxide synthase isoforms at 100 μM) added 30 min prior the E+ rings PE curve. The vascular contraction was normalized by grams of tension by dry tissue (g/g) and were analyzed the agonist maximum effect (Emax) and potency (pD2). Data represent mean ± S.E.M and the results were statistically analyzed using one-way or two-way ANOVA with Bonferroni pos-hoc (p<0,05). Results: In situ gelatinolytic activity was increased in the vessels filled with MMP-2 (19.19 + 0.93 U, n=5) compared to vehicle (10.75 + 0.45 U, n=5). Higher fluorescence intensity to DHE was observed in vessel filled with MMP-2 (20.12 + 1.59 U, n=5) compared to vehicle (11.15 + 1.32 U, n=5). In the vessels filled with doxy, decreased gelatinolytic activity was observed (11.82 + 1.31 U, n=4), as well as lower fluorescence intensity to DHE (12,58 + 2,15 U, n=3). In the vascular reactivity, E- aortic rings filled with MMP-2 increased Emax of PE (1893,28 + 122,40 g/g. n=9) versus vehicle E- (1485.73 + 76.82 g/g, n=9), but no differences were observed on Emax of E+ aortic rings (vehicle: 1416.13 + 120.87 g/g, n=7; MMP-2: 1893.28 + 122.40 g/g, n=9). The presence of L-NAME induced an increase in Emax of E+ aortic rings filled with vehicle (1817.60 + 76.37 g/g, n=7) or MMP-2 (2340.50 + 130.43 g/g, n=4). No difference of pD2 value was observed in this study. Conclusion: the addition of MMP-2 results in increased gelatinolytic activity and vascular ROS. The treatment with doxy prevents these increases, suggesting that MMP-2 may induce the formation of ROS. In E- vessels, MMP-2 increases the maximum effect of PE, suggesting a vascular smooth muscle activity of MMP-2. In the presence of L-NAME, vessels filled with MMP-2 shows a greater maximum effect of PE than vehicle vessels, suggesting more than inhibition of the vasodilator activity of the endothelium, MMP-2 may improves the vasoconstriction through activating endothelial contractile factors, such as ROS.