Atherosclerosis 198 (2008) 320–331 Metalloproteinase inhibition ameliorates hypertension and prevents vascular dysfunction and remodeling in renovascular hypertensive rats Michele M. Castro a , Elen Rizzi a , L´ ıvia Figueiredo-Lopes a , Karla Fernandes a , Lusiane M. Bendhack b , Dimitrius Leonardo Pitol c , Raquel F. Gerlach c , Jose E. Tanus-Santos a,∗ a Department of Pharmacology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirao Preto, SP, Brazil b Laboratory of Pharmacology, Faculty of Pharmaceutical Sciences of Ribeirao Preto, Brazil c Department of Morphology, Estomatology and Physiology, Dental School of Ribeirao Preto, University of Sao Paulo, Av. Bandeirantes 3900, 14049-900 Ribeirao Preto, SP, Brazil Received 27 March 2007; received in revised form 8 October 2007; accepted 16 October 2007 Available online 3 December 2007 Abstract Altered activity of matrix metalloproteinases (MMPs) is implicated in the vascular remodeling of hypertension. We examined whether increased MMP-2 expression/activity plays a role in the vascular remodeling and dysfunction found in the two-kidney, one-clip (2K-1C) hypertension. Sham operated or 2K-1C hypertension rats were treated with doxycycline 30 mg/(kg day) (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and independent relaxations. Quantitative morphometry of structural changes, collagen, and elastin contents in the aortic wall were studied in hematoxylin/eosin, Sirius Red, and Orceine stained aortic sections, respectively. Aortic MMP-2 levels were determined by gelatin zymography and aortic MMP-2 proteolytic activity was measured using DQ gelatin as the substrate after MMP-2 was captured by a specific antibody and immobilized on a microplate. Aortic MMP-2/tissue inhibitor of metalloproteinases (TIMP)-2 mRNA levels were determined by real time RT-PCR. Doxycycline attenuated 2K-1C hypertension (215 ± 8 mmHg versus 167 ± 13 mmHg in 2K-1C rats and 2K-1C + doxy rats, respectively; P < 0.01) and prevented the 35% reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Doxycycline prevented the increases in media thickness, and was associated with lower media/lumen and cross-sectional areas (all P < 0.01). Doxycycline also prevented excessive collagen and elastin deposition in the vascular wall. Increased MMP-2 and Pro-MMP-2 levels and MMP-2 activity were found in the aortas of 2K-1C rats (all P < 0.05). A 21-fold increase (P < 0.001) in the ratio of MMP-2/TIMP-2 mRNA expression was found in the 2K-1C group, whereas this ratio remained unaltered in 2K-1C + doxy rats. Our results suggest that MMP-2 plays a role in 2K-1C hypertension and its structural and functional vascular changes, which were attenuated by doxycycline. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: 2K-1C hypertension; Doxycycline; Metalloproteinases; Renovascular hypertension; Vascular dysfunction 1. Introduction Hypertension is a major health problem associated with structural and functional modifications of the vasculature. Recent studies have suggested that altered expression and ∗ Corresponding author. Tel.: +55 16 3602 3163; fax: +55 16 3633 2301. E-mail address: tanus@fmrp.usp.br (J.E. Tanus-Santos). activity of key members of a group of zinc-dependent endopeptidases called matrix metalloproteinases (MMPs) may be implicated in the vascular remodeling of many car- diovascular conditions including hypertension [1]. Indeed, MMPs play an important role in monocyte invasion and in vascular smooth muscle cell migration [2], thereby contribut- ing to the progress of many commonly found vascular lesions [1,3], especially those involving increased degradation 0021-9150/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2007.10.011