Original articleAngiotensin II induces Fat1 expression/activation and vascular smooth muscle cell migration via Nox1-dependent reactive oxygen species generation
Introduction
Vascular remodeling is critical in the pathogenesis of clinically important cardiovascular disorders [1], [2]. Angiotensin II (Ang II) activates signaling responses via the Ang II type 1 receptor (AT1R), thereby mediating the generation of reactive oxygen species (ROS), inflammation, proliferation, migration, hypertrophy, and fibrosis, all of which are important in vascular remodeling in cardiovascular disease [3], [4], [5], [6].
Among the several sources of ROS in vascular smooth muscle cells (VSMCs), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role triggering the activation of many redox-sensitive pathways, such as mitogen-activated protein kinases (MAPKs), which are involved in proliferation events [7], [8]. The NADPH oxidase, or Nox, family comprises seven isoforms of ROS-generating enzymes, Nox1-5 and DUOX (dual oxidase) 1 and 2. These enzymes differ not only in their ability to generate specific ROS, but also in their tissue distribution, subcellular regulation, requirement for cofactors, and oxidase subunits. Of the seven family members, four have been identified as important sources of ROS in the vasculature: Nox1, Nox2, Nox4, and Nox5. Nox1 is expressed in all layers of the vascular wall, and Ang II increases both its expression and activity, leading to augmented Nox1-derived ROS generation [3], [9], [10], [11].
Cadherins are transmembrane adhesion proteins characterized as major mediators of cell homeostasis. Cadherins are involved in calcium (Ca2 +)-dependent cell–cell adhesion, intracellular junction assembly, and tissue morphogenesis during development and vascular remodeling [12], [13], [14]. Previous studies from our laboratory showed that the vascular expression of Fat1 cadherin, an atypical cadherin, is significantly increased after arterial balloon injury and stimulation with growth factors or Ang II [14], [15]. In addition, we found that Fat1 is involved in the regulation of key cellular functions for vascular remodeling, including migration and proliferation. Accordingly, the present study tested the hypothesis that Ang II leads to an increase in Fat1 protein expression and activity via redox-sensitive pathways, focusing on Nox1-derived ROS generation. We also sought to determine whether Fat1 activation plays a role in Ang II-induced redox-mediated VSMC migration.
Section snippets
Animals
Housing conditions and experimental protocols were in accordance with the Ethical Animal Committees from the University of Sao Paulo and the Albert Einstein College of Medicine of Yeshiva University. Animals were housed under standard laboratory conditions with free access to food and water. Vascular smooth muscle cells were harvested from the aortas of male Sprague–Dawley rats (200 to 250 g, Charles River).
Cell culture
Primary culture of rat aortic VSMCs was prepared as previously described [16] and
Ang II induces Fat1 and Nox1 mRNA and protein expression increase
The effect of Ang II on Fat1 expression is demonstrated in Fig. 1. Ang II increased Fat1 mRNA (Fig. 1A) and protein levels (Fig. 1B) in VSMCs in a time-dependent manner. Increased protein expression of Fat1 was inhibited by the AT1R antagonist valsartan and the antioxidant agent, apocynin (Fig. 1C). To further evaluate the contribution of ROS to Ang II induced Fat1 expression increase, VSMCs were subjected to Nox1 gene knockdown. Downregulation of Nox1 inhibited the effects of Ang II on Fat1
Discussion
Major findings of the present study demonstrate that: 1) Ang II through AT1R-dependent redox mechanisms increases the expression and activity of Fat1, 2) Nox1 and Erk1/2 are upstream signaling components involved in Ang II-induced short and long term Fat1 regulation, and 3) Fat1 expression is necessary for Ang II-mediated VSMC migration.
The cadherin superfamily of cell surface proteins includes classical, proto-, atypical, and other cadherin families that share characteristic extracellular Ca2 +
Acknowledgment
Sources of funding
This study was funded by FAPESP (Foundation for Research Support of the State of Sao Paulo 2011/01785-6; 2011/22035-5) to RT and by the National Institutes of Health Grants HL088104 and HL104518 to NS.
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