There are conserved structural properties of NOX enzymes that are common to all family members. Starting from the COOH terminus, these conserved structural features include 1) an NADPH-binding site at the very COOH terminus, 2) a FAD-binding region in proximity of the most COOH-terminal transmembrane domain, 3) six conserved transmembrane domains, and 4) four highly conserved heme-binding histidines, two in the third and two in the fifth transmembrane domain (Fig. 1). Given the additional NH2-terminal transmembrane domain, the histidines are in the fourth and sixth transmembrane domains in DUOX proteins. Additional features, such as EF hands, an additional NH2-terminal transmembrane domain, and/or a peroxidase homology domain, are limited to some of the family members and are discussed in the respective sections.
.FIG1 Proposed structure of the core region of NADPH oxidase (NOX) enzymes. No crystal structure data of NOX enzymes are presently available. A consensus regarding basic features of the core region of NOX enzymes has emerged based on indirect data. NOX enzymes are thought to be single electron transporters, passing electrons from NADPH to FAD, to the first heme, to the second heme, and finally to oxygen. Enlarged circles represent amino acids that are conserved through human NOX1, NOX2, NOX3, and NOX4. Most data suggest that the new NOX family members are also selective for NADPH over NADH. However, some researchers feel that the issue is not entirely resolved and therefore apply the term NADH oxidase when referring to NOX family members.
VASCULAR NADPH OXIDASE
The first Nox enzyme to be cloned and characterised was termed gp91phox (or Nox2), because it is the catalytic unit of the phagocytic respiratory burst oxidase, which is important for non-specific host defence against invading microbes. Nox1, the first of what is now a family of gp91phox homologues, was discovered and shown to play a role in vascular smooth muscle cell (VSMC) growth.2 Since then, the Nox family has expanded to seven members: Nox1-5 and two longer homologues, Duox1 and Duox2, which have additional peroxidase domains. It is now clear that cardiovascular tissues express multiple Nox homologues (table 1), and that the complement of Nox enzymes varies not only among cell types, but also according to location in the vascular tree.Direct measurements of NADPH/NADH oxidase activity in blood vessels or in blood vessel-derived cells were performed in the mid 1990s, concluding that such enzymes are a major source of ROS in the vascular system. These measurements also quickly established that there were clear differences between the properties of the phagocyte NADPH oxidase and the enzymatic activity observed in vascular preparations, which led to the widespread use of the term vascular NADPH oxidase. However, there is in fact no vascular specific NOX isoform, but rather a complex expression of different NOX isoforms in different cells and regions of the vascular system.
tabella1
Table 1
Expression of Nox mRNAs in the cardiovasculature
Nox1 Nox2 Nox3 Nox4 Nox5 Duox
VSMCs from conduit vessels + − − +++ +* +
VSMCs from resistance vessels − + ND ND ND ND
EC + + ND ++++ +* ND
Cardiac myocytes ND ++ ND + ND ND
Cardiac fibroblasts + + ND +++ ND ND
. NOX2, then called gp91phox is the catalytic subunit of the phagocyte NADPH oxidase; gp91phox/Nox2 forms a heterodimer with p22phox to constitute flavocytochrome b558. . NOX1 and NOX2 genes appear to be the result of a relatively recent gene duplication, as the number and the length of the exons is virtually identical between the two genes .Similarly, at the protein level, there is a high degree of sequence identity (∼60%) between NOX1 and NOX2 . The human and mouse NOX1 gene is located on the X chromosome. In studies using a cell-free system, NOX1 is selective for NADPH over NADH as a substrate. After the initial discovery of NOX1, it was not immediately obvious whether NOX1 was indeed a superoxide generating enzyme. While one group reported a very low level of superoxide generation in NOX1-transfected cells without the need of a stimulus, other groups did not observe such ROS generation by NOX1 alone .The discovery of colon homologs of the cytosolic subunits of the phagocyte NADPH oxidase resolved the issue: superoxide generation by NOX1 depends on cytosolic subunits. NOX1 has the same functional mechanism of phagocyte NOX ; the catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac).
Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases 2003
The novel cytosolic subunits were named NOXO1 (NOX organizer 1 = p47phox homolog) and NOXA1 (NOX activator 1 = p67phox homolog). While expression systems using the mouse proteins suggest a constitutive activity of the NOX1/NOXO1/NOXA1 system, studies using human proteins show only a weak constitutive activity, and full activation depends on activation through the PKC activator phorbol 12-myristate 13-acetate (PMA). In addition to its dependence on cytosolic subunits, NOX1 requires the membrane subunit p22phox. There is now also ample evidence for an involvement of the small GTPase Rac in the regulation of NOX1 activity . Rac binds to the TPR domain of the activator subunit NOXA1, but in analogy with NOX2, Rac activation of NOX1 might be a two-step process that also includes a direct binding to NOX1. The GTP binding protein Rac is also recruited to the membrane upon cell stimulation and is required for activation of the complex . Finally, the most recently discovered subunit p40phox appears to be modulatory, rather than obligatory.
While both NOX1 and NOX2 have also been described in vascular smooth muscle, there appears to be a distinct anatomical distribution: the NOX1 message is mainly expressed in large conduit vessels, while NOX2 mRNA is more strongly expressed in resistance vessels. Little is known about ROS generation and NOX expression in veins, but one study suggests a predominant role of NOX2 in venous ROS generation .The function of NOX-derived ROS in the vascular system is complex and depends not only on the NOX isoform but also on the cell type. 2004. Novel NADH oxidases in the cardiovascular system
All three layers of the vascular wall [intima (i.e., endothelial cells), media (i.e., smooth muscle cells), and adventitia (i.e., fibroblasts and macrophages)] express NOX family members. NOX4 is the predominant isoform in endothelial cells, NOX1 and NOX4 in smooth muscle cells, NOX4 in fibroblasts. NOX-derived superoxide (O2–) avidly reacts with nitric oxide (NO). The effects of NOX in the vascular system might therefore be at least in part due to depletion of the vasorelaxant NO. NOX-derived ROS also affect the extracellular matrix (ECM), influence gene expression, and might be involved in cell proliferation and differentiation
The message for NOX1 is most highly expressed in vascular smooth muscle cells endothelial cells (colon epithelium uterus, placenta, prostate, osteoclasts, as well as in several cell lines, such as the colon tumor cell lines.In addition to its constitutive expression in a variety of tissues), the NOX1 message is induced under many circumstances. In vascular smooth muscle, platelet-derived growth factor (PDGF) and prostaglandin F2α can induce NOX1; taken together, these results suggest that hypertrophy of vascular smooth muscle cells caused by PGF is mediated by NOX1 induction and the resultant overproduction of O(2)(-) by NADPH oxidase. Infact, Superoxide and hydrogen peroxide produced by this enzyme regulate expression of various proinflammatory and proproliferative genes, including PAI-1, MCP-1, ICAM-1, and VEGF,6 and mediate fibroblast proliferation and connective tissue deposition. NADH oxidase activity in vascular smooth muscle has most often been linked to proliferation and migration. PDGF stimulated proliferation is mediated by a Nox1-containing oxidase, while PDGF induced migration is dependent upon an as yet unidentified Nox homologue.
NADPH oxidase is involved in prostaglandin F2alpha-induced hypertrophy of vascular smooth muscle cells: induction of NOX1 by PGF2alpha2002
transactivation of the EGF receptor and PI3k pathway is involved in NOX1 upregulation2005
NOX UPREGULATION UPON ANGIOTENSIN II AND ALDOSTERONE STIMOLATION
Several studies suggest that NOX1 is upregulated at the mRNA and protein level upon angiotensin II stimulation , while the data on NOX4 expression and angiotensin II stimulation are less conclusive . ROS-dependent increase in blood pressure is thought to be a key function of NOX in the vasculature. Angiotensin II induces NOX1 expression . Many studies provide evidence that the vascular Nox isoforms Nox1 and Nox4 appear to be involved in vascular oxidative stress in response to risk factors like angiotensin II (Ang II) in vitro as well as in vivo. Nox mRNA and protein levels were quantified by real-time RT-PCR and Western blotting, respectively. Nox1 and Nox4 were expressed in the vascular smooth muscle cell (VSMC) aortas and kidneys of rats. Upon exposure of cells to Ang II (1 muM, 4 h), Nox1 and Nox4 mRNA levels were increased 6-fold and 4-fold, respectively. Neither the vasoconstrictor endothelin 1 (up to 500 nM, 1-24 h) nor Lipopolysaccharide (up to 100 ng/ml, 1-24 h) had any effect on Nox1 and Nox4 expression in these cells. Consistent with these observations made in vitro, aortas and kidneys of transgenic hypertensive rats overexpressing the Ren2 gene [TGR27] had significantly higher amounts of Nox1 and Nox4 mRNA and of Nox4 protein compared to tissues from normotensive wild-type animals. In conclusion, Nox4 and Nox1 are upregulated by the renin-angiotensin system. Increased superoxide production by upregulated vascular Nox isoforms may diminish the effectiveness of NO and thus contribute to the development of vascular diseases. Nox1 and Nox4 could be targeted therapeutically to reduce vascular reactive oxygen species production and thereby increase the bioavailability of NO.
Source: Upregulation of the vascular NADPH-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo. 2001
Aldosterone can induce the expression of Nox1, which is upregulated by the activation of the Src and activating transcription factor 1 (ATF1), but can not be suppressed by the inhibitors of the epidermal growth factor receptor (EGFR) or Matrix Metalloproteinase (MMP). Aldosterone triggers ATF1 phosphorylation in dose dependent fashion
Activation of Src-ATF1 pathway is involved in upregulation of Nox1, a catalytic subunit of NADPH oxidase, by aldosterone 2011,Endocrine journal
Strong data in favor of a role of NOX-derived ROS as a hypertensive signaling element come from studies demonstrating a decreased systolic blood pressure response to angiotensin II in p47phox-deficient mice. . A decrease in basal blood pressure and blood pressure response to angiotensin II is also found in NOX1-deficient mice
Department of Rehabilitation and Geriatrics, Geneva Medical Faculty, 1211 Geneva 4, Switzerland Decreased blood pressure in NOX1-deficient mice 2006
Studies on blood pressure in NOX2-deficient mice are less conclusive: angiotensin II-induced hypertension is moderately decreased or not changed .Thus, taken together, these studies suggest a predominant role for NOX1, possibly functioning together with p47phox as an organizer subunit, in ROS-dependent blood pressure elevations. In the vasculature, NOX-derived ROS are thought to be involved in nitric oxide inactivation. Serum NOvalues were negatively correlated with systolic blood pressure ≥160 mm Hg in men (r=-0.523, P=0.002).
Association between serum nitric oxide metabolites and hypertension in a general population.2011
CONCLUSION
Despite the potential importance of the Nox enzymes in cardiovascular physiology and pathophysiology, we are only in the early stages of understanding their myriad functions and mechanisms of regulation. Tremendous progress has been made in the few years since the discovery of the first gp91phox homologue, but a clearer picture of the function of the Nox enzymes awaits the development of homologue specific transgenic and knockout animals.
