Rac, PAK, and eNOS ACTion.

Rac1 is a member of the Rho family of GTPases, which includes Rho, Rac, and cdc42 subfamilies.1,2 On activation by guanine nucleotide exchange factors, the Rho family GTPases exchange a GDP molecule for a GTP and act as molecular switches to transduce signals in response to various extracellular stimuli.2 Apart from their characteristic role in the regulation of cytoskeletal rearrangement and cell adhesion, the Rho family proteins have been found to regulate polarization, endocytosis, intracellular trafficking, cell cycle progression, differentiation, and gene transcription.1 Rho family GTPases play a role in endothelial permeability, polarization, leukocyte adhesion, and production of reactive oxygen species, including the superoxide anion (O 2). More specifically, activation of endothelial cells with growth factors such as vascular endothelial growth factor or by fluid shear stress has been shown to activate Rac1, which, in turn, promotes cytoskeletal rearrangement and changes in cell motility.6,7 Endothelial activation by growth factors or shear stress also results in the activation of NADPH oxidases (NOXs) and endothelial NO synthase (eNOS), which are responsible for the production of O 2 and NO, respectively.8–10 Because the modulation of endothelial NOXs by Rac1 has been well demonstrated,11–13 it is believed that Rac1 influences endothelial function, as measured by NO bioavailability, by enhancing the production of O 2 and not by direct modulation of eNOS. This assumes mutually exclusive mechanisms for the production of O 2 and NO and that a decreased bioavailability of NO on Rac1 activation is a result of O 2-dependent peroxynitrite production.8,14 Any association with or direct link between Rac1 activation and regulation of eNOS, therefore, has remained unexplored despite the observation that Rac1 and eNOS are both required for endothelial motility, proliferation, survival, and angiogenesis.6,7 The normally functioning endothelium maintains vascular homeostasis. It is intrinsically antithrombotic, antiinflammatory, and responsive to vasodilators. The dysfunctional endothelium loses these protective properties and, as such, negatively participates in vascular pathology. Endothelial dysfunction has been demonstrated in the metabolic syndrome, dyslipidemic states, types 1 and 2 diabetes mellitus, and hypertension and is associated with cardiovascular events, most notably of a coronary nature.15 It is thought to be the earliest stage in vascular pathological development. A critical and master regulator that maintains the protective features of normal endothelial function is NO.8 Thus, endothelial dysfunction is commonly considered a defect in NO production and/or bioavailability. This has led to extensive work, performed in many laboratories, dissecting the complex mechanisms that control eNOS expression and activity. Apart from the coenzymes, cosubstrates and cofactors required for normal eNOS function, many other eNOSinteracting proteins have been shown to modulate eNOS function under various conditions.16 These include several G protein–coupled receptors, porin, dynamin-2, caveolin-1, NOS-interacting protein, and heat shock protein-90.14 Phosphorylation on several critical serine/threonine residues (with defined upstream kinases), myristoylation, and intracellular translocation have also been described to alter eNOS function.14,16 However, a functional relationship between Rho family GTPases and eNOS has only begun to emerge. With the discovery that statins, hydroxymethylglutaryl-coenzyme A reductase inhibitors, can increase NO production by effects on RhoA, and can reduce NOX-dependent O 2 production via Rac1,17,18 a potential link has been proposed between Rho family GTPases and eNOS function. A recent report by Selvakumar et al demonstrating that Rac1 both interacts with eNOS and regulates eNOS activity has further corroborated the hypothesis that Rac1 may directly modulate eNOS function.13 In an article published in this issue of Circulation Research, Sawada et al provide significant new insights on the functional link between activated Rac1 and the modulation of eNOS function in the endothelium.19 Using an endothelialspecific Rac1 haploinsufficient (EC-Rac1 / ) mouse and Rac-1–modulated cell lines, the authors demonstrate that Rac1 is required for the maintenance of eNOS mRNA expression and stabilization, eNOS enzymatic activity, and NO production in the endothelium (see Figure). The attenuation of Rac1 expression resulted in impaired endotheliumdependent vasodilation, mild hypertension, and retarded angiogenesis, all consistent with an NO-deficient state. Aortic rings from EC-Rac1 / mice demonstrated an elevated phenylephrine-induced contraction and attenuated acetylcholineinduced relaxation. Decreased blood flow recovery to hindlimb ischemia was seen in the EC-Rac1 / mice. Moreover, aortic explants from EC-Rac1 / mice implanted into Matrigel also showed impaired capillary sprouting, rescued either by exogenous addition of the eNOS substrate L-arginine or by the NO donor S-nitrosoglutathione. The use of a Rac1 pharmacological inhibitor or dominant negative Rac1 expression in endothelial cell lines displayed that active Rac1 regulates eNOS promoter activity but not the phosphorylation of either eNOS or its upstream kinase, Akt. Dominant negative p21-regulated kinase (PAK) expression blocked The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Sections of Cardiovascular Medicine and Immunobiology, Raymond and Beverly Sackler Foundation, and Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, Conn. Correspondence to Jeffrey R. Bender, MD, The Anlyan Center S469, 300 Cedar St, New Haven, CT 06510. E-mail [email protected] (Circ Res. 2008;103:328-330.) © 2008 American Heart Association, Inc.