Coupled and uncoupled NOS: separate but equal? Uncoupled NOS in endothelial cells is a critical pathway for intracellular signaling.

Endothelial dysfunction is seen early in the development of atherosclerosis, before overt vascular and structural changes. Nitric oxide (NO) is recognized as one of the major mediators of the maintenance of vascular homeostasis, and a decrease in NO bioavailability is associated with endothelial dysfunction. Endothelial NO synthase (eNOS; NOS3) catalyzes the formation of NO from L-arginine and O2 in a reaction requiring Ca –calmodulin, FAD, FMN, NADPH, and tetrahydrobiopterin (BH4). A decrease in NO bioavailability may be caused by: (1) a decrease in the expression or activity of NOS3, (2) uncoupling of NOS to produce superoxide (O2 ), or (3) degradation of NO by reacting with O2 from other enzymatic sources resulting in the formation of peroxynitrite (ONOO ). Physiologically, NOS3-derived NO inhibits leukocyte–endothelial cell adhesion, vascular smooth muscle proliferation and migration, and platelet aggregation to maintain the health of the vascular endothelium. Under a number of pathological conditions, NOS3 enzymatic activity becomes uncoupled, resulting in the production of O2 . NOS3-derived O2 has been shown to contribute to the development and progression of atherosclerosis and hypertension.1,2 In this issue of Circulation Research, Gharavi et al report that treatment of endothelial cells with oxidized phospholipids results in increased interleukin-8 (IL-8) production through the activation and uncoupling of NOS3.3 When NOS is uncoupled, electrons flowing from the reductase domain to the heme are diverted to molecular oxygen instead of to L-arginine, resulting in the formation of O2 . A number of potential mechanisms are responsible for uncoupling of NOS3, although the most consistent evidence exists for BH4 deficiency.4,5 Also, NOS3 uncoupling has been shown to occur when: (1) there is a shortage of L-arginine or Hsp90,6 (2) NOS3 is dephosphorylated on threonine residue 495,7 or (3) NOS3 is redistributed to the cytosolic fraction of the cell.8 Gharavi et al suggest that phosphorylation of NOS3 at threonine 495 is involved in the uncoupling of NOS3 in their experimental model; however, the potential contribution of additional mechanisms was not examined.3 Is NOS Involved in Atherogenesis? NO has been recognized as an antiatherogenic mediator at many points in the atherosclerotic process. Impaired aortic endothelium-dependent vasodilation caused by a dysfunctional NOS3–NO pathway is one of the early consequences associated with the major risk factors for the development of atherosclerosis such as hyperlipidemia, hypertension, diabetes, and smoking. However, in most animal models of atherosclerosis NOS3 protein expression is either unchanged or actually increased.9 This finding may be explained, in part, by evidence in the literature suggesting that NOS3 is uncoupled in atherosclerosis and hyperlipidemia to produce O2 . In contrast to NO, O2 has been demonstrated to be a proatherogenic mediator contributing to the development of atherosclerotic lesions. Recent studies in genetically engineered and knock-out (KO) mice have implicated NOS3 in the progression of atherosclerosis. NOS3-KO mice fed a high-fat diet have a reduction in atherosclerotic lesion size compared with wildtype mice.10 NOS3 also contributes to the generation of oxidized low-density lipoprotein (oxLDL) and a proatherogenic phenotype. In addition, NOS3-overexpressing apoE-KO mice have significantly larger atherosclerotic lesions compared with control apoE-KO mice.2,9 In this latter study, additional experiments demonstrated that NOS3 was uncoupled, and supplementation with BH4 reduced the lesion size. These data implicate NOS3-derived O2 in the formation of atherosclerotic lesions and support the hypothesis that BH4 depletion results in the uncoupling of NOS3. The regulation of NOS3 enzymatic activity appears to play a major role in the balance of NO and O2 in the endothelial response during atherogenesis. Gharavi et al report that the mechanism by which oxidized phospholipids stimulate the subsequent upregulation of IL-8 is through the activation, and uncoupling, of NOS3 resulting in the production of ONOO . Oxidized phospholipids are localized in blood vessels at all stages of atherosclerosis and contribute to the progression of the disease. Berliner’s group has previously shown that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) contributes to monocyte–endothelial cell adhesion and stimulates endothelial cells to synthesize chemotactic factors, including IL-8, via the activation of sterol regulatory element binding proteins (SREBPs).11,12 IL-8 has been implicated in monocyte activation and endothelial chemotaxis during angiogenesis, and IL-8 levels have been shown to be elevated in atherosclerotic lesions.13 SREBPs are transcription factors that regulate cholesterol, fatty acid, triglyceride, and phospholipid synthesis.14 Recent work by Berliner’s laboratory demonstrated that Ox-PAPC treatment of endothelial The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Vascular Biology Center, Medical College of Georgia, Augusta. Correspondence to Jennifer S. Pollock, PhD, CB 3213B, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912. E-mail [email protected] (Circ Res. 2006;98:717-719.) © 2006 American Heart Association, Inc.