OBJECTIVE Pulmonary Hypertension (PH) is a progressive disease arising from remodeling and narrowing of pulmonary arteries (PA) resulting in high pulmonary blood pressure and ultimately right ventricular failure. Elevated production of reactive oxygen species (ROS) by NADPH oxidase 4 (Nox4) is associated with increased pressure in PH. However, the cellular location of Nox4 and its contribution to aberrant vascular remodeling in PH remains poorly understood. Therefore, we sought to identify the vascular cells expressing Nox4 in PA and determine the functional relevance of Nox4 in PH. APPROACH AND RESULTS Elevated expression of Nox4 was detected in hypertensive PA from 3 rat PH models and human PH using qRT-PCR, Western blot, and immunofluorescence. In the vascular wall, Nox4 was detected in both endothelium and adventitia and perivascular staining was prominently increased in hypertensive lung sections, colocalizing with cells expressing fibroblast and monocyte markers and matching the adventitial location of ROS production. Small molecule inhibitors of Nox4 reduced adventitial ROS generation and vascular remodeling as well as ameliorating right ventricular hypertrophy and non-invasive indices of PA stiffness in monocrotaline (MCT)-treated rats as determined by morphometric analysis and high resolution digital ultrasound. Nox4 inhibitors improved PH in both prevention and reversal protocols and reduced the expression of fibroblast markers in isolated PA. In fibroblasts, Nox4 over-expression stimulated migration and proliferation and was necessary for matrix gene expression. CONCLUSIONS These findings indicate that Nox4 is prominently expressed in the adventitia and contributes to altered fibroblast behavior, hypertensive vascular remodeling and the development of PH.

Nitric oxide (NO ⅐ ) is a short-lived physiological messenger. Its various biological activities can be preserved in a more stable form of S-nitrosothiols (RS-NO). Here we demonstrate that at physiological NO ⅐ concentrations, plasma albumin becomes saturated with NO ⅐ and accelerates formation of low-molecular-weight (LMW) RS-NO in vitro and in vivo. The mechanism involves micellar catalysis of NO ⅐ oxidation in the albumin hydrophobic core and specific transfer of NO ؉ to LMW thiols. Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating LMW thiols. Results suggest that the hydrophobic phase formed by albumin serves as a major reservoir of NO ⅐ and its reactive oxides and controls the dynamics of NO ⅐ -dependant processes in the vasculature. NO ⅐ is synthesized by various types of cells and involved in numerous biological functions, including vasodilation, platelet aggregation, neurotransmission, and inflammation (1, 2). Heme proteins such as guanylyl cyclase and free radical species-e.g., ( ⅐ O 2 Ϫ )-serve as NO ⅐ primary targets (1-3). Another physiologically significant component of NO ⅐ biochemistry involves the formation of thionitrite esters with cysteine (Cys) or Cys residues (Snitrosothiols; RS-NO). Low-molecular-weight (LMW) RS-NO (e.g., S-nitrosoglutathione) and nitroso-derivatives of proteins such as albumin and hemoglobin exert NO-like activity in vivo. They cause arterial and venous smooth muscle relaxation, inhibit platelet aggregation, and activate guanylyl cyclase (4-8).Vasoactive S-nitrosothiols are known to be generated in vivo (6-10), although the actual amount of these species in circulation is debated (refs. 11 and 12, and references therein). Because RS-NO are relatively stable and can release NO ⅐ when required, via reactions with transition metal ions or other reducing agents (13-15), they are envisioned as a buffering system that controls intra-and extracellular activities of NO ⅐ and magnify the range of its action. Once formed, circulating RS-NO can deliver NO into cytosol via specific mechanisms (16) or directly transfer the nitrosyl cation (NO ϩ ) to another thiol via the so-called transnitrosation reaction that ensures the dynamic state of RS-NO in vivo (7,17). S-nitrosylation of protein free Cys residues modulates activities of various regulatory factors and enzymes and represents a widespread signaling mechanism (refs. 18 and 19, and references therein).Despite the growing interest in the role of nitrosothiols in biological systems, there is still uncertainty about how they form in vivo. In the absence of an electron acceptor, NO ⅐ is unable to react with nucleophiles under oxygen free conditions, implying that metabolites of NO ⅐ oxidation, such as N 2 O 3 , are actual nitrosating agents (20-23). However, considering the low concentration and short life span of NO ⅐ in vivo, the third-order reaction of NO ⅐ with O 2 (Eq. 1) (k Ϸ 4 ϫ 10 6 M Ϫ2 ⅐sec Ϫ1 ) (20, 21) seems to be too slow to account for any detectable amount o...