Migracion Celular En Respuesta Al Óxido Nítrico

Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration
´ ´ ´ ´ Esther Lopez-Rivera*†, Tania R. Lizarbe*†, Monica Martınez-Moreno‡, Jose Miguel Lopez-Novoa§, ´ ´ ´ ´ Alicia Rodrıguez-Barbero§, Jose Rodrigo¶, Ana Patricia Fernandez¶, Alberto Alvarez-Barrientos*, Santiago Lamas*† , ´ and Carlos Zaragoza*†
*Fundacion Centro Nacional de InvestigacionesCardiovasculares, Ronda de Poniente 5 Tres Cantos, 28760 Madrid, Spain; §Departamento de Fisiologia y ´ Farmacologia, Instituto ‘‘Reina Sofia’’ de Investigacion Nefrologica, Universidad de Salamanca, Avenida Campo Charro, 37007 Salamanca, Spain; ¶Instituto ´ ´ ´ Ramon y Cajal, Consejo Superior de Investigaciones Cientıficas, Avenida Del Dr. Arce 37, 28002 Madrid, Spain; †Centro de Investigaciones Biologicas, ´´ Instituto ‘‘Reina Sofıa’’ de Investigaciones Nefrologicas, Consejo Superior de Investigaciones Cientıficas, Ramiro de Maeztu 9, 28040 Madrid, Spain; and ´ ´ ´ ‡Departamento de Bioquimica y Biologia Molecular, Facultad de Quimica, Universidad Complutense de Madrid, 28040 Madrid, Spain Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved January 7,2005 (received for review November 4, 2004)

angiogenesis

endothelium

vasular remodeling

ound healing is an orchestrated cascade of enzymatic activities that converge toward damage repair. Wound healing involves inflammation and angiogenesis and is tightly regulated by cytokines (1). Vascular endothelial growth factor (VEGF) is a critical cytokine involved in angiogenesis, and nitricoxide (NO) is a downstream effector (2, 3). VEGF increases NO levels in endothelial cells (ECs) by activating endothelial NO synthase (eNOS NOS3) (4–7). Recently, the role of eNOS in EC migration has been demonstrated in vivo and in vitro (8, 9) but the precise mechanism by which NO regulates migration is unknown. ECs migrate as a result of an injury and during angiogenesis from preexistingvessels. The process is tightly regulated by matrix turnover, in which matrix metalloproteinases (MMPs) play a pivotal role (10–12). We have previously reported that NO induces MMP-13 expression and activity in bovine aortic ECs (BAECs) (13, 14). MMPs are extracellular matrix-degrading endopeptidases. MMP expression and activity can be found in physiological and pathological situations, such as tissuedevelopment, atherosclerosis, ovarian function, arthritis, osteoarthritis, cancer, angiogenesis, and wound healing (15). MMP-13 was initially discovered in mammalian cell carcinomas and is also expressed by several cell types, including endothelium (13, 16–18). Here, we present evidence that MMP-13 is a downstream effector of NO-activated EC movement. We have developed a wound-healing model in BAECsand aortic cells from eNOS WT and eNOS-deficient mice. We show that aortic ECs lacking MMP-13 experience delayed migration, and aortic ECs from eNOS null mice present delayed cell migration and a significant decrease in MMP-13 expression. In addition, we present data showing that MMP-13 exists in association with caveolin-1 in resting cells, and that this complex is disrupted in the presencewww.pnas.org cgi doi 10.1073 pnas.0408217102

W

Animals. WT C57BL 6 mice and (C57BL 6,129) eNOS null mice were purchased from The Jackson Laboratory and housed in our own animal facilities in isolated rooms. Cells. BAECs were incubated in gelatin or collagen type I as described (14). Murine aortic ECs (MAECs) were cultured from aortas extracted from anesthetized animals after death. The aortaswere sectioned into 2-mm pieces, deposited in Matrigel solution, and fed with fresh growth medium for 7 days [DMEM HAM’s medium (19), 20% FBS, 0.05 mg ml penicillin streptomycin, and 2.5 g ml amphotericin]. The tissue was removed, and 500 l of BD Cell recovery solution was added to each culture. The Matrigel layer was removed and poured on ice for 1 h. The solution was centrifuged at 4°C,...