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INVITED REVIEW

Vascular NAD(P)H oxidases: specific features, expression, and regulation

Emory University School of Medicine, Division of Cardiology, Atlanta, Georgia 30322

	ABSTRACT

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
The importance of reactive oxygen species (ROS) in vascular physiology and pathology is becoming increasingly evident. All cell types in the vascular wall produce ROS derived from superoxide-generating protein complexes similar to the leukocyte NADPH oxidase. Specific features of the vascular enzymes include constitutive and inducible activities, substrate specificity, and intracellular superoxide production. Most phagocyte enzyme subunits are found in vascular cells, including the catalytic gp91phox (aka, nox2), which was the earliest member of the newly discovered nox family. However, smooth muscle frequently expresses nox1 rather than gp91phox, and nox4 is additionally present in all cell types. In cell culture, agonists increase ROS production by activating multiple signals, including protein kinase C and Rac, and by upregulating oxidase subunits. The oxidases are also upregulated in vascular disease and are involved in the development of atherosclerosis and a significant part of angiotensin II-induced hypertension, possibly via nox1 and nox4. Likewise, enhanced vascular oxidase activity is associated with diabetes. Therefore, members of this enzyme family appear to be important in vascular biology and disease and constitute promising targets for future therapeutic interventions.

NAPDH oxidase; superoxide; blood vessels; atherosclerosis; hypertension

	THE PHAGOCYTE AND VASCULAR OXIDASES

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
The phagocyte oxidase model. Because it is closely related to the vascular enzymes, we will first briefly describe the main features of the well-characterized neutrophil NADPH oxidase, which have been reviewed previously (6, 8, 33, 41, 110, 111, 206). This multimeric enzyme is the source of ROS essential to the phagocyte bactericidal activity. Four oxidase subunits: gp91phox, p22phox, p47phox, and p67phox (phox stands for phagocyte oxidase) are required for activity, because mutations in any one of them can cause chronic granulomatous disease, an immune deficiency resulting from impaired phagocyte function. Additional components of the enzyme include p40phox and the small G proteins Rac and Rap1A. Together gp91phox and p22phox form an integral membrane complex termed cytochrome b₅₅₈, located in cytoplasmic vesicles and the plasma membrane. The catalytic subunit of this cytochrome, gp91phox, binds three prosthetic groups, one FAD and two heme molecules. Another protein complex, composed of p47phox, p67phox, and p40phox, is cytosolic and does not interact with the cytochrome in resting cells. The function of the p40phox subunit, which is not essential for oxidase activity, is controversial (8, 97, 100, 204). Two events are required for oxidase activation: exchange of GTP for GDP on the small G protein Rac and phosphorylation of the p47phox subunit by protein kinase C (PKC), which triggers a change in conformation of the cytosolic complex, possibly release of p40phox, and association with Rac. This activated cytoplasmic complex then associates with the cytochrome in the membrane to form a functional enzyme that is thought to include one copy of each phox subunit, as well as Rac and Rap1A. The reduced substrate NADPH binds to gp91phox on the cytoplasmic side of the membrane and releases two electrons, which are passed in turn to FAD, then to the first and second heme group, and finally accepted by two successive molecules of oxygen on the opposite side of the membrane to produce two molecules of superoxide radical. Because this process carries negative charges across the membrane, it is electrically balanced by conduction of protons in the same direction through a channel that is part of the oxidase or closely associated with it (42, 95, 123) (Fig. 1). Once activated, phagocytes produce large quantities of superoxide, on the order of 10 nmol·min^-1·10⁶ neutrophils^-1 during the oxidative burst (6).

View larger version (33K): [in this window] [in a new window]   Fig. 1. Proposed topology of nox proteins. Carboxy terminus of the protein is thought to form a cytoplasmic globular region harboring the FAD prosthetic group and a binding site for the NAD(P)H substrate. Amino terminus contains 6 hydrophobic regions believed to form membrane-spanning -helices. Histidine residues in helices 3 and 5 coordinate iron atoms at the center of heme molecules. Upon oxidase activation, electrons are transferred in sequence from the substrate to FAD, each of the 2 hemes, and finally to molecular oxygen on the opposite side of the membrane. Additionally, the -helices likely form a proton channel (11, 22, 95, 101, 102, 110).

Vascular oxidases. More recently it has become clear that the vascular wall also produces superoxide, mostly via enzymes similar to the neutrophil oxidase. Furthermore, it was also discovered that the catalytic subunit gp91phox is only one member of a new family of homologous proteins termed nox (for NADPH oxidase) (11, 101, 102, 186) and that most cells express multiple nox proteins (30). In EXPRESSION OF VASCULAR NAD(P)H OXIDASES IN CELLS AND TISSUES, evidence that vascular cells can express gp91phox (aka, nox2), as well as nox1, nox4, and nox5, will be presented in more detail (Fig. 2). It is thought that nox family members transfer electrons from a reduced substrate to molecular oxygen in a way similar to gp91phox. Very recent reports suggest that nox1 can interact with the phagocytic subunits p22phox (73), p47phox, and p67phox (10), as well as two novel homologues of p47phox and p67phox (10). However, it is not yet known whether these latter proteins are expressed in vascular cells. Whatever the case may be, it is worth noting that the overall oxidase activity of any cell is the sum of the activities of the multiple homologues it expresses. Therefore, the term "vascular oxidase" refers to the set of oxidases expressed in vascular cells at any given time, regardless of their molecular identities. Specific properties set these enzymes apart from the phagocyte prototype.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Dendrogram of vascular nox protein sequences. Primary sequence similarities were calculated from a multiple alignment and represented as a tree. Graph clearly defines 4 separate clusters, corresponding to the high conservation of each nox protein across species (human nox5 is the only one known so far). Also note that all nox1 and gp91phox sequences are closer to each other than to any nox4. GenBank accession numbers: nox1, human NP_008983 [GenBank] , mouse XP_142128, rat NP_446135 [GenBank] ; gp91phox, human NP_000388 [GenBank] , mouse NP_031833 [GenBank] , rat AAG31606 [GenBank] ; nox4, human NP_058627 [GenBank] , mouse NP_056575 [GenBank] , rat NP_445976 [GenBank] ; nox5, human AAG33638 [GenBank] .

	SPECIFIC FEATURES OF VASCULAR NAD(P)H OXIDASES

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
Low activity. The rate of superoxide production in vascular cells is thought to be 1–10% of that in leukocytes (67, 81, 159), as confirmed recently using highly specific electron spin resonance (ESR) methods (Table 1). To determine whether this weak activity could simply be due to a lower number of oxidase subunits per cell compared with phagocytes, mRNA and protein expression were measured. Although p22phox and p47phox mRNAs were abundant in endothelial cells, gp91phox and p67phox messages were only 1–3% as high in these cells as in leukocytes (16, 159). Similarly, expression of p22phox, p47phox, p67phox, and Rac1 proteins was significantly lower in four different endothelial cell types than in a phagocyte cell line (113). Another possible reason for the low activity of the vascular oxidase was suggested by the detection of an F416S mutation in rat endothelial cell gp91phox within the putative NADPH binding site (16). However, this substitution may be a polymorphism because it was not observed in human endothelial cDNA (58, 159) and we did not find an alternative exon in the human (GenBank NT_011657) and mouse (Celera mCG12212) genes. It is also possible that oxidase activity might be limited in vascular cells by the cytosolic concentrations of NADH and NADPH, as suggested by the increase in superoxide production observed when these substrates were added to whole cells or tissues (13, 58, 68, 69, 76, 113, 146, 148, 182, 183, 224). However, it is not clear how these molecules or reducing equivalents derived from them might cross the plasma membrane. Finally, tightly regulated signaling mechanisms of activation, detailed in EXPRESSION OF VASCULAR NAD(P)H OXIDASES IN CELLS AND TISSUES, may also be responsible for the low activity of the vascular enzyme.

Constitutive activity and subcellular localization. As might be expected from the cytotoxicity of superoxide, the leukocyte enzyme is inactive in the absence of stimulation (6, 8). Thus, in homogenized neutrophils, it was necessary to add a cytosolic fraction with p47phox and p67phox to the membrane fraction containing the cytochrome to observe activity. In contrast, in unstimulated vascular cells, oxidase activity was observed in isolated membrane fractions (114, 145, 177, 182) and even inhibited by addition of cytosol (62, 179). This constitutive activity was explained by detection of p47phox and p67phox proteins in the membrane fraction (67, 114, 145, 175, 193), coimmunoprecipitation of the cytochrome with p47phox or p67phox (114) and p47phox with gp91phox (53), suggesting that at least part of the oxidase is preassembled in vascular cells, although translocation of cytosolic subunits to the membrane fraction was also observed upon stimulation in endothelial cells (53, 127, 136) and vascular smooth muscle cells (VSMC) (14, 151, 193, 215). Further studies of subcellular localization showed that oxidase subunits colocalized together and with cytoskeletal elements in endothelial cells (16, 53, 67, 81, 114), suggesting that the vascular oxidase is intracellular. Indeed, the gp91phox subunit from endothelial cells was detected as discrete bands on Western blots, mostly around 65 kDa, consistent with the absence of glycosylation expected for a protein located in nonplasmalemmal membranes (16). In addition, some oxidase molecules are likely located at or near the plasma membrane, as suggested by the generation of extracellular superoxide, especially in endothelial cells and adventitial fibroblasts (13, 81, 126, 127, 171, 208, 210). Taken together, these results indicate that the oxidase is present in several major pools in vascular cells. One is preassembled and likely responsible for constitutive activity, and another is activated by translocation of cytosolic subunits in a manner similar to the neutrophil enzyme. Finally, although a large part of the oxidase is intracellular, a fraction is likely present at or near the plasma membrane.

Inducible activity. Although the vascular enzyme is activated within minutes of stimulation (107, 168, 231), its activity can also be markedly upregulated in the hours after exposure to agonists. Thus the rate of superoxide production was increased two- to threefold in VSMC exposed for 4–8 h to ANG II (62, 179). This effect appears to result from increased expression of the oxidase (107). Indeed, de novo protein synthesis was required for upregulation of oxidase subunits in VSMC exposed to ANG II for 2 h (193). Upregulation by agonists of mRNA and/or protein of all major oxidase subunits, including the catalytic gp91phox, nox1, and nox4, has been observed and will be reviewed in more detail in REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE.

Intracellular superoxide production. Another specific feature of the vascular enzyme is the intracellular release of superoxide and its ROS derivatives, such as hydrogen peroxide. Intracellular superoxide formation in vascular cells was described in a number of reports using assays such as the inhibition of aconitase (14) and dihydroethidium staining (14, 40, 103, 129, 148, 181, 188). Nitroblue tetrazolium reduction may also detect both extra- and intracellular superoxide (151, 210). Intracellular hydrogen peroxide is also frequently visualized using fluorescent dyes such as dichlorofluorescein (25, 151, 193, 200, 231). It should be noted here that the proposed topology of the nox subunits implies that superoxide is not released in the cytosol but rather inside vesicles (Fig. 1), which may serve to confine this deleterious molecule. Such postulated compartments, which would be expected to also contain SOD and ROS-sensitive signaling molecules, may include the nucleus and large organelles (79, 114). Although it is possible that superoxide might be transported out of a vesicle via anion channels (72, 117), hydrogen peroxide, which can diffuse through membranes (52, 167), could easily become cytosolic or even extracellular. As noted above, superoxide is also produced outside vascular cells, presumably by plasmalemmal oxidase molecules, or from intracellular vesicles merging with the plasma membrane, most notably in the endothelium and adventitia, where it is likely to exert a paracrine signaling function (156, 208). Therefore, the formation of ROS observed both inside and outside vascular cells is consistent with the subcellular localization of the oxidase described above and the signaling function of these molecules.

Role in signaling. Another important distinctive feature of the nonphagocytic oxidases is their function in the cell. In contrast to the cytotoxic amounts of superoxide generated by phagocytes, most nonphagocytic cells produce low amounts of ROS that stimulate numerous transcription factors as well as signaling cascades via activation of kinases and inhibition of tyrosine phosphatases. The role of ROS in signaling has been reviewed extensively (47, 49, 50, 52, 63, 64, 66, 87, 99, 138, 162, 187, 190, 191, 195, 197, 217, 222).

It should be noted here that in physiological conditions, the intracellular production of ROS does not alter the redox state of cells, which have large reserves of reducing agents, notably reduced glutathione, as well as extremely effective antioxidant defense mechanisms, such as SOD, catalase, and peroxidases (52). This reducing intracellular environment actually allows agonist-induced increases in ROS to function as second messengers by limiting their effect in time and space in a manner similar to other well-known intracellular signals, such as calcium ion. Thus, because of their confinement, it is possible for ROS to promote cell proliferation (37, 65) despite the fact that transition from a differentiated to a proliferating phenotype is marked by a shift toward a more reduced overall cellular state (163). Therefore, in physiological conditions, ROS production is not accompanied by oxidative stress, but rather provides a means of finely regulating signaling in vascular cells.

The fact that all vascular cells express multiple nox homologues (see EXPRESSION OF VASCULAR NAD(P)H OXIDASES IN CELLS AND TISSUES), which are differentially regulated (see REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE, REGULATION OF VASCULAR NAD(P)H OXIDASES IN ATHEROSCLEROSIS, REGULATION OF VASCULAR NAD(P)H OXIDASES IN HYPERTENSION, and REGULATION OF VASCULAR NAD(P)H OXIDASES IN DIABETES) suggests that these oxidases serve distinct functions. Presumably this is made possible by the specific subcellular localization of each enzyme within a particular signaling domain. Indeed, in VSMC, confocal microscopy experiments suggest that nox1 is located in a punctate pattern at the cell periphery, whereas nox4 is associated with focal adhesions, which are major sites of tyrosine kinase signaling (79).

Substrate specificity. In contrast to the phagocyte oxidase, which uses NADPH exclusively as an electron donor (35), there have been many reports of possible NADH consumption by the vascular oxidase (39, 62, 131, 132), which was thus called an NAD(P)H oxidase. This observation has been criticized as an artifact due to the use of high concentrations of lucigenin in early assays of oxidase activity (113, 179, 202). However, not all assays were based on lucigenin; for example, quantification of NADH consumption (151, 232) and recent studies measuring superoxide production by ESR have also noted that some vascular oxidases can use NADH as a possible (133, 179, 182), or even preferred, substrate (177). We speculate that this apparent abnormal lack of specificity might be due to the fact that vascular cells express multiple oxidases and that nox4, unlike gp91phox and nox1, might preferentially use NADH. The following arguments support this possibility.

1. The putative NADPH binding site in human gp91phox, which comprises 50 residues in four groups (102), is more conserved in nox1 (88% identity) than in nox4 (68% identity).
   
2. In VSMC, agonists only increase an NADPH oxidase activity (179) mediated by nox1 (107).
   
3. In VSMC, serum deprivation upregulates nox4 (107) and shifts substrate preference in favor of NADH (179).
   
4. Endothelial and HEK293 cells express nox4 (30, 181) and consume NADH (173, 177).
   
5. In VSMC, nox4 expression is high (107, 180, 188, 221) and superoxide production is low, consistent with the idea that nox4 uses a low-concentration substrate [in the cytoplasm NADH 0.01 mM (161, 185) and NADPH 0.1 mM (163)].
   

However, this possibility that nox4 might specifically account for the NADH oxidase component of vascular superoxide production will have to be tested experimentally, for example by measuring the activity of purified recombinant proteins, before a definitive conclusion regarding substrate specificity can be obtained.

The specific features of the nonphagocytic oxidases presented above are adapted to maintaining the cellular homeostasis of ROS and allowing their signaling function. It is now clear that vascular enzymes constitutively produce low amounts of superoxide intracellularly and are also activated and upregulated by agonists. In upcoming sections, we will review the regulation of vascular oxidases in various pathophysiological conditions.

	ACTIVATION PATHWAYS OF VASCULAR NAD(P)H OXIDASES

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
The ROS originating from the vascular oxidase have been recognized as important second messengers in cellular signaling. This function of ROS is possible because the oxidase is activated by agonist stimulation in addition to being constitutive. The coupling mechanisms, which enable the vascular oxidase to respond to agonists, have long been poorly understood. However, a clearer picture has recently begun to emerge that shows similarities with the activation mechanisms of the phagocytic enzyme.

PKC. Because phosphorylation of the p47phox subunit by PKC is required for activation of the neutrophil oxidase, several studies have investigated the role of this kinase in activation of the vascular oxidase. Thus treatment of VSMC with the PKC inhibitors Ro31–8220, chelerythrine, calphostin C, or GF109203X reduced ROS production after stimulation by platelet-derived growth factor (PDGF) or ANG II (121, 168, 196, 228). Similarly, GF109203X decreased ROS-dependent phosphorylation of raf-1 and ERK1/2 by lysophosphatidylcholine (LPC) in VSMC (227). Furthermore, tumor necrosis factor- (TNF-)-induced translocation of p47phox in endothelial cells was inhibited by calphostin C and chelerythrine, as well as by three specific inhibitors of PKC- (53). One of these latter inhibitors also abolished TNF--induced phosphorylation of p47phox, association with gp91phox, and intracellular ROS production. Conversely, a constitutively active PKC- increased p47phox translocation in the absence of agonist stimulation (53). Although other PKC isoforms may be involved in other cell types, taken together these results show that PKC is an important activator of the vascular NAD(P)H oxidase. It should be noted that the effect of PKC inhibition was usually partial, especially after exposure to the agonist for longer periods (168), suggesting that the oxidase is activated by redundant pathways. However, PKC activation may have long-lasting effects on ROS production because it was also reported to upregulate oxidase subunits (107).

Phospholipase D. The possibility that lipid metabolites involved in signaling might activate the oxidase was investigated in vascular cells. Exogenous phosphatidic acid (PA) significantly increased oxidase activity in intact VSMC and homogenates (62, 194), suggesting that phospholipase D (PLD), which is activated rapidly after agonist stimulation of vascular cells and generates endogenous PA (105, 106, 194, 196), could stimulate the oxidase. Indeed, incubation of VSMC with sphinganine or suramin, which are nonspecific PLD inhibitors, reduced ANG II-induced ROS formation and signaling (194, 196, 228). In the presence of inhibitors, ROS production was restored by addition of PA. Furthermore, signals induced by exogenous PA were blocked by the flavoprotein inhibitor diphenylene iodonium (DPI), suggesting that PLD-induced signaling is dependent on the NAD(P)H oxidase (194, 196). Therefore, PLD may be one mechanism of oxidase activation, either via PA directly (125) or following conversion to diacylglycerol. Both lipid metabolites may also activate the oxidase via PKC stimulation. Finally, diacylglycerol and its metabolite monoacylglycerol can be hydrolyzed by specific lipases (169) to generate arachidonic acid (AA), another oxidase activator.

Phospholipase A₂. In vitro characterization of the phagocyte oxidase had demonstrated that it could be activated by the addition of fatty acids. Similarly, in VSMC homogenates, exogenous AA and linoleic acid specifically increased NAD(P)H oxidase activity (62, 232). Free fatty acids, together with LPC, are produced in cells after agonist activation of phospholipase A₂ (PLA₂), suggesting that this enzyme might activate the oxidase. Indeed, an inhibitor of PLA₂ reduced thrombin-induced ROS production in endothelial cells (82). Furthermore, inhibitors of AA metabolism via lipoxygenase and cytochrome P-450 monooxygenase significantly blunted ANG II- and thrombin-induced ROS production in VSMC and endothelial cells (82, 232). Exogenous LPC also stimulated NAD(P)H oxidase-mediated ROS production and signaling in VSMC and endothelial cells (77, 189, 227). These results suggest that lipid metabolites produced by PLA₂ contribute to oxidase activation by agonists in vascular cells.

Rac. Another important activator of the phagocyte enzyme is the small G protein Rac. Experiments were designed to investigate its possible effect on the vascular oxidase. Agents that stimulate oxidase activity in VSMC and endothelial cells, such as thrombin, ANG II, or depolarization, increased Rac expression, its GTPase activity, and translocation from the cytoplasmic to the membrane fraction (151, 168, 176, 214, 215). In addition, studies on the mechanism of action of statins revealed that besides their cholesterol-lowering effect, part of their antioxidant effect can be ascribed to inhibition of Rac acylation, thus depriving it of a membrane anchor. Statins inhibited ANG II- and epidermal growth factor (EGF)-induced ROS production in VSMC, as well as Rac activity and translocation to the membrane fraction. These effects were reversed specifically by the addition of mevalonate (214, 215), confirming that statins acted via inhibition of the biosynthesis of this lipid precursor. In vivo administration of statins also decreased vascular ROS production and Rac1 translocation to the membrane (214, 215). Similarly, in vivo administration of raloxifene, an estrogen receptor modulator, decreased aortic expression and activity of Rac1, as well as oxidase activity (216). To further characterize the effect of Rac on the oxidase, cells were exposed to clostridium toxins, inhibitors of small G proteins of the Rho family, such as Rac. This treatment decreased ROS production after depolarization of endothelial cells (176), as well as ANG II- and PDGF-induced ROS formation and signaling in VSMC (168, 214). More specifically, expression of dominant-negative Rac in transgenic mice or in transfected cells inhibited pressure-, PDGF-, and ANG II-induced ROS production in VSMC (98, 141, 168). Conversely, overexpression of constitutively active Rac increased basal and ANG II-induced ROS production (168). Together, these results demonstrate that Rac is an important activator of the vascular NAD(P)H oxidase of VSMC and endothelial cells.

Upstream activators of Rac. Additional experiments were undertaken to further characterize the signaling pathways that may lead to activation of Rac. Incubation of VSMC with inhibitors of phosphatidylinositol-3 kinase (PI3K), such as wortmannin and LY-294002, inhibited PDGF- and ANG II-induced ROS formation (121, 168) and ANG II-induced Rac activation (168). ANG II-induced ROS production and Rac activation were similarly inhibited by the EGF receptor blocker AG-1478 (168), suggesting that this receptor is upstream of PI3K, a notion consistent with previous studies on the PDGF receptor (9). Finally, ANG II-induced ROS formation and Rac activation were also abrogated by the Src kinase inhibitor PP1. This observation is consistent with previous reports of tyrosine kinase-dependent activation of the oxidase (176, 189) and ROS-dependent transactivation of the EGF receptor by ANG II via Src (198). These results suggest the existence of a positive feedback loop whereby ANG II increases ROS formation, which in turn activates EGF receptors and sustains oxidase activity (Fig. 3). A feed-forward mechanism, allowing oxidants to specifically activate the NAD(P)H oxidase, was also observed in VSMC and fibroblasts exposed to exogenous hydrogen peroxide (115).

View larger version (30K): [in this window] [in a new window]   Fig. 3. Activation pathways of NAD(P)H oxidase in vascular smooth muscle cells by the prototypical agonist ANG II. Initial stimulation of the AT1 receptor (AT1R) by ANG II activates phospholipase C (PLC) enzymes, in part via the -subunit of heterotrimeric G proteins (G). PLC produces inositol trisphosphate (IP3) and diacylglycerol (DG). Both IP3-mediated release of calcium from intracellular stores and DG contribute to activation of protein kinase C (PKC), which phosphorylates the p47phox (p47) subunit, thus activating the NAD(P)H oxidase (nox). Additionally, calcium ions activate phospholipase A2 (PLA2), which produces lysophosphatidylcholine (LPC) and arachidonic acid (AA). Both compounds can enhance oxidase activity. ANG II also activates the Src kinase via -subunits released from heterotrimeric G proteins. Src transactivates the EGF receptor (EGFR) and activates protein tyrosine kinases (PTK) upstream of the small G protein Rac and phospholipase D (PLD). Rac also participates in activation of nox. During the sustained phase of oxidase activation, PLD produces phosphatidic acid (PA), which is converted to DG. Together, DG and prolonged calcium influx from calcium channels enhance PKC activity. AA may also be generated from DG by lipases. In a self-sustained activation loop, reactive oxygen species (ROS) released by the oxidase activate Src, thus maintaining transactivation of EGFR, which is upstream of phosphatidylinositol-3 kinase (PI3K) and Rac. The oxidase releases superoxide (O2·-) in an intracellular compartment. Superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD). Both oxidants can induce downstream signaling.

It is apparent that multiple signaling pathways contribute to activating the vascular NAD(P)H oxidase (Fig. 3) over a broad time frame, beginning with receptor stimulation and lasting hours and possibly days. These mechanisms provide ample opportunity for a fine control of ROS production in the cell as required for important second messengers with potential deleterious effects. However, the complexity of these pathways of oxidase activation leaves room for multiple disturbances to take place, as may be the case in various pathological conditions that will be reviewed in upcoming sections.

	EXPRESSION OF VASCULAR NAD(P)H OXIDASES IN CELLS AND TISSUE

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
Early investigations of the origin of vascular ROS demonstrated that a major source of superoxide is an NAD(P)H oxidase. This enzymatic activity was found in all layers of the vessel wall, in the endothelium (131, 147), the media (132), and the adventitia (146), as well as in cultured VSMC (62). Because this enzyme shared some features with the neutrophil oxidase, researchers attempted to demonstrate the presence of phagocyte NADPH oxidase subunits in the vessel wall, particularly the major four: gp91phox, p22phox, p47phox, and p67phox, as reviewed recently (201).

Intima and adventitia. In the endothelium, expression of the mRNA for all four subunits was soon demonstrated, as well as the presence of p47phox and p67phox proteins (89). These results were confirmed by reports of the cloning of endothelial cDNAs almost identical to the phagocytic sequences (16, 17, 58, 67, 159) and demonstration of the presence of the four proteins by Western blotting (127, 136) and immunocytochemistry (81, 114). Similarly, in the adventitia, the four major phagocytic subunits were detected by immunohistochemistry (145, 171, 208, 210). Furthermore, p67phox was cloned from adventitial fibroblasts (144). Detailed information on the endothelial and adventitial oxidases can be found in recent reviews (7, 157). Evidence that endothelial p47phox and gp91phox, as well as adventitial p67phox and gp91phox subunits are functional will be presented in REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE and REGULATION OF VASCULAR NAD(P)H OXIDASES IN HYPERTENSION. Recently p22phox and gp91phox proteins were detected by immunohistochemistry in 25–30% of intimal smooth muscle cells in nondiseased areas of human aorta (90). This is one of the few observations of gp91phox expression in smooth muscle.

Media. In the media, the situation is more complicated, because all phagocytic subunits are not always detected. Only p22phox and p47phox seem to be expressed consistently. The presence of p22phox subunit in VSMC was demonstrated by molecular cloning (55) and Western blotting (16, 58, 193). Furthermore, p22phox was also found in the vascular media by in situ hybridization (54) and immunohistochemistry (5, 76, 88, 188, 220). Similarly, expression of p47phox RNA and protein was also demonstrated in VSMC (151, 164). Evidence that p22phox and p47phox are functional in VSMC will be presented in REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE. The other major phagocytic oxidase subunits p67phox and gp91phox were either very low or undetectable in aortic VSMC and media (90, 107, 141, 151, 181, 193). The absence of functional gp91phox was supported by experiments showing that basal and growth factor-induced oxidase activity and proliferation of aortic VSMC were unaffected by disruption of this gene (14, 182). Surprisingly, unlike cells from large vessels, VSMC from human resistance arteries expressed all major phagocytic NADPH oxidase subunits both as mRNA and protein (141, 193), including p67phox and gp91phox, which appear to be functional, because p67phox translocated from the cytosolic to the particulate fraction upon ANG II stimulation and gp91phox antisense oligonucleotides blocked superoxide production (193). The expression of gp91phox, rather than nox1 (see below), in microvascular smooth muscle suggests that these two homologues serve different functions, possibly due to different subcellular localizations and coupling to different effector mechanisms.

Expression of new homologues. Recent studies have begun exploring expression of the newly discovered gp91phox homologues in the vasculature. The nox4 subunit was abundantly expressed in all cell types as RNA (90, 107, 133, 181, 188, 193, 221) as well as protein in aortic lysates (221), but was most abundant in the vascular media as seen by immunohistochemistry (181, 188). In contrast, the nox1 mRNA was expressed, but notably less abundant than nox4 message, in aortic and A7r5 VSMC (58, 60, 92, 107, 181, 186, 193, 221) and only detected in whole vessels by using RT-PCR (96, 188). Surprisingly, nox1 was not expressed in VSMC from resistance arteries (193). Evidence that nox1 is functional in aortic VSMC will be presented in REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE. So far, there is only one report of nox1 RNA expression in human endothelial cells and cardiac fibroblasts (181). With the use of RT-PCR, nox5 was detected in human (12), but not rat (92), VSMC. In a recent study, duox1 was detected by RT-PCR in human aortic media and intimal lesions (90). Finally, the other gp91phox homologues, nox3 and duox2 (102), were not detected in VSMC (107) and their expression has not been reported in other vascular cells.

In summary, despite some controversy regarding the expression of a particular subunit or homologue in a cell type or tissue, especially when nonquantitative techniques are used such as immunohistochemistry are used, a consensus seems to emerge from the results obtained by different authors using a variety of approaches (Table 2). All the major subunits of the phagocytic NADPH oxidase, including gp91phox, appear to be expressed in endothelial and adventitial cells, as well as in VSMC from resistance arteries. In contrast, in the VSMC of large arteries, p67phox is low or absent and nox1 appears to substitute for gp91phox. The nox4 homologue appears to be expressed in all cell types.

	REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
Because they alter the function of cells and tissues, signal transduction pathways must be finely controlled. This is why the activity and expression of numerous proteins in signaling cascades are regulated by retroactive loops. To determine if the vascular NAD(P)H oxidase is likely involved in agonist-induced signaling, it is therefore convenient to investigate its regulation in cultured cells exposed to various stimuli.

p22phox. Many investigators have found that oxidase subunits are indeed frequently upregulated by treatments leading to increased ROS production. The p22phox mRNA was elevated in VSMC exposed to platelet-derived products, including PDGF and transforming growth factor- (TGF-) (57), or to TNF- (39, 122). Similarly, p22phox message was also upregulated in endothelial cells exposed to ANG II, phorbol myristate acetate (PMA), pulsatile strain, and shear stress (85, 122, 160, 174). Conversely, treatments that decrease oxidant production in endothelial cells, such as activators of peroxisome proliferator-activated receptor (PPAR) and statins, downregulated p22phox mRNA (85, 86). To confirm the involvement of this subunit in oxidase function and signaling, studies were carried out using antisense RNA or oligonucleotides, as well as electroporation of anti-p22phox antibodies. This inhibition of p22phox expression decreased ANG II-, TNF--, platelet-derived products-, thrombin-, and AA-induced superoxide and hydrogen peroxide production and signaling in VSMC (25, 39, 57, 200, 205, 231). Therefore, these studies showed that p22phox is a required component of the smooth muscle NAD(P)H oxidase.

p67phox, p40phox, and p47phox. To ascertain the molecular composition of the vascular NAD(P)H oxidase, investigators measured the regulation of the cytosolic phox subunits. ANG II upregulated p67phox mRNA and increased oxidase activity in endothelial cells (160) and adventitial fibroblasts (144). Immunodepletion of p67phox in these latter cells blocked oxidase activity, which was rescued by addition of recombinant protein (145), indicating that p67phox is a functional part of the adventitial enzyme. In VSMC from resistance arteries, ANG II upregulated the proteins of all phagocytic oxidase subunits, including p40phox (193). This is the first suggestion that this latter subunit may be functional in vascular cells, although its role has not yet been determined. In VSMC, thrombin increased superoxide and hydrogen peroxide formation and upregulated p47phox mRNA and protein (14, 151). In endothelial cells, ANG II upregulated the p47phox message (160). The p47phox protein was also upregulated in endothelial cells by PMA and downregulated by the ROS-decreasing PPAR activators (85). These results suggest that p47phox is functional in endothelial and smooth muscle cells. To confirm its role, investigators took advantage of the p47phox knockout model. In VSMC and endothelial cells from p47phox^-/- mice, signaling and ROS production by a variety of stimuli, such as pressure, PMA, PDGF, thrombin, and ANG II, were significantly reduced (14, 24, 28, 103, 109, 112, 141). Similarly, ANG II-induced ROS production and signaling were inhibited by electroporation of anti-p47phox antibodies in VSMC (164). Conversely, overexpression of functional p47phox in VSMC or endothelial cells from p47phox^-/- mice rescued agonist-induced superoxide production (109, 112). These results show that p47phox is an essential part of the vascular NAD(P)H oxidase, in both VSMC and endothelial cells.

gp91phox. To investigate the possible signaling function of a gp91phox-based oxidase, the regulation of this subunit was measured in endothelial cells. Agents that increase endothelial cell superoxide production, such as ANG II, endothelin-1, and oxidized low-density lipoproteins (LDL), significantly upregulated gp91phox mRNA (48, 159, 160) and protein (160). Conversely, statins and estrogens reduced superoxide formation as well as gp91phox message and protein expression in endothelial cells (159, 207), and long-term treatment with an ANG II type 1 (AT₁) receptor blocker downregulated gp91phox message in human arteries (160). The gp91phox subunit appears to be functional because superoxide production was inhibited by antisense oligonucleotides in endothelial cells (199) and by gene knockout in fibroblasts (115) and aortic segments (58). All phagocyte oxidase subunits, including gp91phox, were expressed and upregulated by ANG II in VSMC from resistance arteries (193), supporting the notion that the gp91phox-based oxidase is functional when expressed in vascular cells and is responsive to agonists that promote ROS formation. However, as described in EXPRESSION OF VASCULAR NAD(P)H OXIDASES IN CELLS AND TISSUE, gp91phox is not present in VSMC and vascular media from large arteries.

nox1 and nox4. Recent studies have begun investigating the regulation of nox1 and nox4 in VSMC. Stimuli that promote the formation of superoxide in these cells, such as ANG II, PGF₂, serum, PDGF, LDL, and PMA, upregulated nox1 mRNA (92, 107, 186, 221). Conversely, treatment with atorvastatin decreased both ROS production and nox1 message expression (215). Because VSMC from large arteries express little or no gp91phox, these results suggest that nox1 is the agonist-coupled catalytic subunit of the oxidase in these cells. To confirm that nox1-based oxidase was truly functional, nox1 antisense mRNA or anti-nox1 ribozymes were expressed in VSMC. In both instances, the treatments reduced nox1 expression and inhibited agonist-induced superoxide production and redox-sensitive signaling (92, 107). Therefore, nox1 appears to be an essential part of the oxidase and a functional substitute for gp91phox in VSMC. A possible role for the nox4 subunit in VSMC signaling was also investigated in two recent studies (107, 221). However, opposite results were found, most likely due to differences in cell lines and exposure to serum before the experiment. Whereas expression was increased by ANG II and serum in one report (221), it was lowered by ANG II, serum, and PDGF in the other (107). Although this discrepancy will have to be resolved in additional experiments, recent in vivo studies tend to support the view that nox4 is downregulated during vascular growth (181, 188). However, cell proliferation was decreased by nox4 in NIH 3T3 cells (56, 173) and increased in melanoma cells (26), suggesting that its effects depend very much on the cellular context.

Overall, these results demonstrate a good correlation between ROS production in vascular cells and expression of NAD(P)H oxidase subunits, suggesting that all cells in the vascular wall express one or more functional NAD(P)H oxidase(s). Although data from the literature tend to be fragmentary, it may be worth noting that agonists appear to regulate only a subset of subunits (Table 3). For example, in VSMC from large arteries, ANG II upregulates nox1, but not p22phox (T. Fukui and K. K. Griendling, personal communication), whereas PDGF upregulates both. The reason for this difference is still unclear, because the two agonists increase oxidase activity to similar degrees (107, 179). Obviously a better understanding of oxidase subunit interaction, intracellular localization, and agonist coupling will be required to resolve such issues. Nevertheless, the fact that many agents implicated in vascular disease upregulate gp91phox and nox1 suggests that these oxidases may be associated with pathological conditions, as detailed in the following sections.

	REGULATION OF VASCULAR NAD(P)H OXIDASES IN ATHEROSCLEROSIS

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
Atherosclerosis is a vascular pathology of major clinical importance. Although its causes are believed to be multifactorial, a number of investigations have implicated ROS in its development and have been reviewed extensively (19, 34, 47, 75, 93, 108, 128, 135, 139, 149, 150, 180, 184, 229). Because ROS may potentially be generated in vascular tissue through a number of biochemical pathways, recent studies have attempted to establish the contribution of NAD(P)H oxidases in atherosclerosis by investigating the regulation of its subunits during the development of the disease.

Models. Different animal models have been used for this purpose, which fall into two main categories. The first consists of hyperlipidemic models, such as monkeys fed a high-fat diet (76), rabbits (88, 129, 212), and genetic models of mice (14, 83, 96) with or without a high-fat diet. The second category comprises nonhyperlipidemic models of vascular lesions, such as grafting of a vein segment in the carotid artery (220), nonocclusive banding of rabbit aorta (148), and balloon injury of rat aorta (151), carotid artery (188), or pig coronary artery (171). These acute injury models resemble atherosclerosis inasmuch as they induce intimal hyperplasia, but they are not associated with nearly as much inflammation as chronic human atherosclerotic lesions in which leukocytes are an important source of NADPH oxidase. To address this possible limitation, a few studies used samples of human vessels obtained during surgery or from autopsies (4, 5, 90, 115, 181).

Characterization. In these reports, lesion development was confirmed histologically by a marked thickening of the intimal layer of the vessels (4, 5, 14, 76, 88, 129, 148, 151, 181, 188, 220). This neointima resulted from the proliferation of cells that appeared closely related to smooth muscle cells, because they expressed typical VSMC markers, such as smooth muscle myosin heavy chain, smoothelin, -actin, and calponin (5, 14, 88, 181, 188, 220), suggesting that they were myocytes or myofibroblasts. Although the most advanced lesions were accompanied by notable inflammation and infiltration of macrophages (4, 5, 76, 90, 181), which are a major source of ROS, it is important to note that in some models (171, 188, 220) or during the early stages of the disease, in contrast to chronic human atherosclerosis, little or no macrophage proliferation was observed, which implies that ROS were generated by cells native to the vascular wall, presumably within the smooth muscle cells or myofibroblasts undergoing proliferation in the neointima.

Evidence of superoxide production. Diseased vessels were also characterized by impaired endothelium-dependent relaxation, suggesting that nitric oxide might be inactivated by elevated superoxide (76, 129, 148, 212). To test this possibility, the production of superoxide in the vessel wall was visualized with dyes, and specificity was verified by inhibition of the signal with DPI, the superoxide scavenger tiron, or cell-permeable SOD (115, 129, 148, 181). In control vessels, nitroblue tetrazolium reduction revealed that superoxide was generated mostly in the adventitia (14, 145, 171, 210). However, a significant part of this adventitial superoxide appears to be extracellular, because it was reduced by unmodified exogenous SOD (171, 208, 210). When vessels were stained with dihydroethidium, which detects intracellular superoxide, cells in all layers of control or minimally diseased vessels appeared to generate ROS constitutively (14, 76, 88, 148, 181, 188). In diseased arteries, superoxide production was markedly increased throughout the vessel wall and, notably, in the neointima (76, 88, 129, 148, 188).

Source of superoxide. To establish the identity of the enzyme(s) responsible for superoxide production in control and diseased vessels, assays were carried out using various substrates and inhibitors. Thus ROS production was markedly reduced by DPI (14, 115, 148, 212, 220), but not by inhibitors of other candidate enzymes, such as the xanthine oxidase inhibitor oxypurinol, the mitochondrial inhibitor rotenone, or the NOS inhibitor N^G-monomethyl-L-arginine (220) [except in hyperlipidemic rabbits in which oxypurinol partially inhibited aortic vascular superoxide production (212)]. Conversely, ROS production was increased by addition of the substrates NADH or NADPH (76, 148, 171, 220), but not by succinate, arachidonate, or xanthine, which are precursors of other possible ROS-producing pathways. These results suggest that the enzyme(s) responsible for the major part of ROS production are NAD(P)H oxidase(s). Interestingly, this activity was increased in diseased vessels (76, 129, 148, 212, 220). The oxidase(s) responsible for ROS production were then further characterized by measuring their expression. As described in EXPRESSION OF VASCU-LAR NAD(P)H OXIDASES IN CELLS AND TISSUES, evidence suggests that, before the development of disease, ROS are constitutively generated in the adventitia and endothelium by a gp91phox-based enzyme that produces superoxide both intra- and extracellularly. ROS appear to be also generated in the VSMC of the media, mostly intracellularly, presumably by nox4, the most highly expressed subunit in this location.

p22phox. During the early proliferative stage of the lesion, p22phox mRNA was sharply upregulated (188). The p22phox protein, which was barely detectable in controls (90, 188), became much easier to detect in all layers of diseased vessels, including the media and neointima (4, 5, 76, 88, 90, 181, 188, 220). This increase in p22phox expression in the vessels was not due to an infiltration of macrophages (188, 220). However, as expected, in advanced lesions the recruitment of inflammatory phagocytes increased p22phox expression even more (4, 5, 76, 90, 181). These results suggest that p22phox, in association with a catalytic subunit, may be involved in vascular lesion formation. This conclusion is supported by the observation that decreased expression of p22phox either by probucol or by termination of a high-fat diet was accompanied by a decrease in superoxide production (76, 88).

p67phox and p47phox. The p67phox subunit was upregulated in arteries after the development of intimal hyperplasia, even in the absence of macrophage infiltration (171, 220). Surprisingly, p67phox was not detected in VSMC (151) and so far has almost exclusively been localized in the adventitia of atherosclerotic vessels (171) and in some intimal VSMC of aortic lesions (90), where this subunit is thought to be an essential part of a gp91phox-based oxidase by analogy to the leukocyte enzyme. Although p47phox was quite low in the media of controls, it was also upregulated in diseased vessels (14, 76, 151, 171). The p47phox protein was detected by immunohistochemistry in the adventitia, the media, and the neointima, as well as in areas of macrophage infiltration (14, 76, 151, 171). Conversely, p47phox expression was reduced on termination of a high-fat diet (76). Furthermore, crossing p47phox^-/- with hyperlipidemic ApoE^-/- mice resulted in a spectacular reduction in aortic lesion formation in the descending aorta, regardless of diet and without altering serum lipid content (14). This improvement was not observed in the ascending aorta, where advanced lesions appear very early, suggesting that additional mechanisms besides ROS intervene in this extreme case (14, 83). Disruption of the p47phox gene also resulted in decreased superoxide production in vessels (83) and inhibition of proliferation in VSMC (14). These experiments clearly establish the important role of p47phox and the NAD(P)H oxidase in the development of vascular lesions similar to atherosclerosis.

gp91phox. The relationship between vascular disease and the catalytic subunits of the oxidase was also studied. Thus gp91phox, which is mostly expressed in the adventitia of control arteries, was upregulated in established lesions with developed neointima (148, 188) even in the absence of macrophages (188). Furthermore, gp91phox was recently detected in intimal VSMC of human aortas, and the proportion of these cells that expressed gp91phox increased from 28 to 68% according to the severity of the lesions (90). However, gp91phox was not upregulated during the early stage of the disease (188) as might be expected, since the early development of vascular lesions is characterized by proliferation of cells related to VSMC, which do not express gp91phox in either culture or arterial media (32, 58, 81, 90, 107, 181, 188, 193, 210). However, gp91phox may have a role in lesion development, because ROS formation in the endothelium has been implicated in expression of chemotactic and adhesion molecules, leading to recruitment of monocytes from the circulation (64, 128, 135, 150, 180), and extracellular release of ROS by the adventitia may enhance smooth muscle proliferation via a paracrine effect (157, 208) and inactivate NO. Surprisingly, vascular lesions were not affected by deletion of the gp91phox gene in one study (96), but this result may not be definitive, because observations were limited to the ascending aorta, which is not a sensitive model of early disease (14). In summary, although the involvement of this subunit in lesion development needs to be further characterized, it is certainly important later, when advanced lesions are infiltrated by macrophages, which destabilize the plaque, especially at the shoulder region (5, 76, 181).

nox1. Expression of the nox1 subunit was also investigated in vascular lesions. Because nox1 is typically much less abundant than other NAD(P)H oxidase subunits such as nox4 (Table 2), it was not detected in some studies by RT-PCR (148) or by in situ hybridization (188) and has not yet been observed by immunohistochemistry. However, nox1 mRNA was markedly upregulated at the early stage of restenosis after balloon injury (188). Furthermore, nox1 message was increased in VSMC by proatherosclerotic LDL and ANG II (107, 221) and upregulated in both minimally and terminally diseased human coronary arteries (D. Sorescu and K. K. Griendling, personal communication), although it is not clear why it was not also increased in arteries with intermediary lesions. Nevertheless, upregulation of nox1 in early vascular lesions is consistent with its proliferative role observed in vitro (186) and suggests that it may be part of the mechanism of lesion formation. Incidentally, it should be noted that the concomitant upregulations of nox1 mRNA in injured vessels, as well as p22phox and p47phox proteins observed in media and neointima, suggest that nox1 may associate with these subunits to form a functional enzyme in smooth muscle, which could be responsible for the proliferation of the neointima. However, further studies using transgenic and knockout mice will be required to fully characterize the role of nox1 in atherosclerosis.

nox4. Finally, the nox4 subunit was abundant in all vascular cells, as determined by quantitative RT-PCR (181). In the vessel wall, nox4 protein was readily detected in the media by immunohistochemistry (181, 188). The expression of nox4 was not altered during the early stage of restenosis (188) or after aortic banding (148). However, it was upregulated in the neointima after proliferation ended, during the redifferentiation phase (188), and in moderately advanced human lesions (181). These observations support the notion that nox4 may be responsible for the low constitutive oxidase activity measured in nonproliferative cells and are consistent with in vitro studies (56, 107, 173).

In summary, these studies confirm the involvement of ROS in atherosclerosis models and establish the important contribution of the NAD(P)H oxidase(s), and most notably the p47phox subunit, in such pathological processes. In contrast to nox4, which may not be involved at the proliferative stage, p22phox, nox1, and possibly gp91phox may also prove to be essential for lesion development. This latter subunit, which is expressed in intimal VSMC and is part of the leukocyte oxidase, is involved in chronic human lesion formation and plaque destabilization to a greater degree than in acute animal models of vascular injury (61, 90, 116, 158). Additional studies will be required to demonstrate the causal role of these last three subunits in this important vascular disease.

	REGULATION OF VASCULAR NAD(P)H OXIDASES IN HYPERTENSION

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
The concept that superoxide might be implicated in hypertension emerged from observations that vascular cells in culture produce ROS, and from the suggestion that superoxide could impair endothelium-dependent relaxation by inactivation of the potent endothelial vasodilator nitric oxide. The pathophysiology of ROS in hypertension has been reviewed in detail (74, 104, 124, 154, 165, 166, 192, 235).

Models. The relationship between superoxide and hypertension was studied in animal models in which blood pressure is elevated by diverse mechanisms, including 1) long-term infusion of subpressor doses of vasoconstrictor agonists, such as ANG II (18, 32, 54, 103, 133, 140, 143, 153, 155, 209, 211) or norepinephrine (NE) (18, 153); 2) overexpression of human renin in transgenic animals (43, 221); 3) administration of the mineralocorticoid deoxycorticosterone associated with a high-salt diet (DOCA salt) (20, 21, 178, 224), a model of low plasma renin and ANG II; 4) one- or two-kidney, one-clip rats (46, 78); and 5) spontaneously hypertensive rats (SHR) (29, 134, 170, 213, 215, 224, 233, 234).

Superoxide production. Production of superoxide was then measured in hypertensive models. Although rats infused with ANG II or NE for 5 days had identical increases in blood pressure, only ANG II increased vascular superoxide production, suggesting that ROS may be implicated in blood pressure regulation specifically by this agonist (18, 153). Elevation in vascular superoxide was not only confirmed after ANG II infusion (32, 103, 133, 140, 155, 211), but also observed in other models of hypertension, such as the two-kidney, one-clip (78); the one-kidney, one-clip (46); the DOCA salt (20, 21, 178, 224); the renin transgenic (43); and the SHR (134, 137, 213, 215, 224, 233). Furthermore, the time courses of increase in blood pressure and aortic superoxide production with age were similar in the SHR model (224). Hypertension was accompanied by impaired endothelium-dependent and -independent relaxations (43, 78, 133, 134, 153, 178, 213, 233, 234), suggesting that blood pressure might be elevated at least in part through inactivation of endothelial nitric oxide, an effect that would be expected from superoxide.

Role of superoxide. To investigate the possibility that superoxide itself might be causing the elevation in blood pressure and impaired vascular relaxation, it was important to determine if hypertension could be reduced by antioxidants. Thus in vivo administration of membrane-permeant SOD markedly reduced the elevation in blood pressure in SHR (137) and after ANG II infusion (18, 54) and had no effect on the response to NE, which is not accompanied by superoxide production (18). These results suggest that a significant portion of hypertension in SHR and ANG II infusion is mediated by superoxide. Similarly, ANG II-induced vascular ROS production and hypertension were also inhibited by administration of the SOD mimetic tempol or the antioxidant vitamin E (140, 143) and by overexpression of the human SOD gene in mice (209). In vivo antioxidants successfully reduced vascular superoxide production and blood pressure in other models of hypertension as well, such as tempol in the one-kidney, one-clip (46) and the SHR models (170), tempol or pyrrolidinedithiocarbamate in the DOCA-salt rats (21), the superoxide scavenger tiron in the renin transgenic (43), and the inhibitor of NADPH oxidase assembly apocynin in the DOCA-salt rat (20). Endothelium-dependent and -independent relaxations were also improved in vitro by incubation of vessels from DOCA-salt rats with antioxidants such as SOD (178) and worsened by incubation of vessels from renin transgenic mice with the SOD inhibitor diethyldithiocarbamate (43). In addition, treatments that decrease vascular superoxide production, such as tempol and AT₁ receptor antagonists, also inhibited medial thickening and improved vasodilatory responses (46, 233, 234), indicating that ROS may be involved in vascular remodeling associated with the disease. These observations are consistent with in vitro studies showing that activation of the oxidase by various agonists can induce VSMC hypertrophy and proliferation (64) and also suggest that superoxide is an important component both in the development and in the maintenance of chronic hypertension.

Source of superoxide. Additional experiments, similar to those described above in the section on atherosclerosis, using inhibitors and substrates, were performed to characterize the enzymatic source of elevated ROS in hypertension. Free radical formation in vessel homogenates was blocked by SOD (54, 234), confirming that it was derived from superoxide and by DPI, but not by inhibitors of xanthine oxidase, NOS, arachidonate metabolism, and the mitochondrial electron chain (20, 32, 78, 153). Apocynin also decreased superoxide production in isolated vessels (20). Conversely, superoxide production was increased by NADH or NADPH, but not by xanthine, arachidonate, or succinate (54, 78, 133, 153, 224, 233, 234). Vascular oxidase activity was minimally affected by removal of the endothelial layer (20, 234), as confirmed in later studies showing that superoxide is generated throughout the vessel wall (103, 133, 155). However, the endothelium may significantly amplify superoxide production in hypertension due to the uncoupling of endothelial NOS by oxidants, which makes this enzyme produce superoxide rather than nitric oxide, as observed in the SHR and DOCA-salt models (104). Taken together, these results indicate that vascular NAD(P)H oxidase(s) are the primary superoxide source in vessels from hypertensive animals.

p22phox. To further assess the role of superoxide in hypertension, it was necessary to characterize the molecular composition of the enzyme(s) responsible for its generation. When it became clear that p22phox was part of the vascular enzyme (55, 200), the regulation of this subunit was investigated in hypertension. The p22phox mRNA was upregulated in aorta in the chronic ANG II infusion (54, 133), DOCA-salt (20), and SHR models (70, 213, 215, 234). Elevation in p22phox mRNA was observed in all layers of the aorta (54) without phagocyte infiltration in the vessel wall (20, 54, 234). Upregulation of p22phox message was also reported in lymphoblasts from hypertensive patients (152). Conversely, a concomitant decrease in blood pressure, vascular oxidase activity, and expression of p22phox mRNA was observed by treatment of ANG II-infused rats with the PKC inhibitor chelerythrine (133) or with an AT₁ receptor antagonist (54, 234). Similar effects were obtained by treatment of SHR with atorvastatin (213, 215). These results demonstrate an association between p22phox upregulation and hypertension, but do not indicate whether one event is causing the other. However, it is unlikely that elevated blood pressure caused the upregulation of p22phox, because NE infusion had no effect on superoxide production (18, 153). Furthermore, increased ANG II could explain p22phox upregulation in some, but not all, cases. Thus AT₁ receptor antagonists inhibited p22phox increase (234) and superoxide production (213, 234) in the SHR, but had no effect on blood pressure in the low plasma ANG II DOCA-salt model (178). Taken together, these results suggest that a role for p22phox in some forms of hypertension is likely, but additional evidence will be required, for example using transgenic and knockout animals, before a definitive conclusion can be obtained.

gp91phox. Regulation of the essential phagocytic subunit gp91phox was also investigated in experimental hypertension. Its mRNA was markedly upregulated in the aorta of stroke-prone SHR (134) and after ANG II infusion in the rat (133). In addition, both gp91phox message and protein were increased in the aortic adventitia after ANG II infusion in mice (32, 211). Conversely, in vivo administration of chelerythrine, which decreased ANG II-induced hypertension, also inhibited gp91phox upregulation (133). To establish more firmly the role of this subunit in blood pressure regulation, gp91phox^-/- mice were infused with ANG II for 6 days. Although basal blood pressure was lowered, the absence of gp91phox did not reduce the hypertensive effect of the agonist (211). However, disruption of the gp91phox gene did inhibit ANG II-induced adventitial nitrotyrosine accumulation and increased aortic media thickness, supporting a role of the adventitial oxidase in nitric oxide inactivation and vascular remodeling via a paracrine effect on the underlying smooth muscle (156, 208). Because gp91phox is normally expressed in vascular smooth muscle of resistance arteries (193), in addition to the adventitia and endothelium, disruption of its gene would be expected to have a more significant impact on blood pressure regulation. Perhaps a longer experiment is required before its effect on vascular remodeling becomes functionally evident.

p67phox and p47phox. To approach the role of the vascular NAD(P)H oxidase in hypertension from a different angle, regulation of its cytosolic subunits p67phox and p47phox was also investigated. The p67phox protein was upregulated in aortic adventitia after ANG II infusion in mice for 7 days (32), consistent with the increase in gp91phox noted above. In an intriguing study, expression of p47phox protein in young normotensive SHR was upregulated in the macula densa of the kidney, suggesting that overexpression of the NAD(P)H oxidase in the juxtaglomerular apparatus might trigger the development of hypertension (29). In another interesting study, a cell-permeant decoy peptide was designed to incorporate a p47phox binding sequence from gp91phox (155). In preliminary tests using aortic rings, the peptide abolished ANG II-induced superoxide production. After intraperitoneal administration, the decoy peptide blocked vascular superoxide production and inhibited by 44% the increase in blood pressure induced by a 7-day ANG II infusion in mice (155). It should be noted that the decoy peptide might saturate the site on p47phox required for interaction with other catalytic subunits, such as nox1 or nox4. A similar result was obtained in a recent study showing that disruption of the p47phox gene in mice infused with ANG II for 7 days abolished the elevation in vascular superoxide and markedly reduced the increase in blood pressure (103). Comparison of these results to those obtained with gp91phox knockout mice described above (211) suggests that p47phox is an essential component of a vascular enzyme likely based on a new catalytic moiety, such as nox1 or nox4, and that this NAD(P)H oxidase controls part of the development of ANG II-induced hypertension.

nox1 and nox4. A few studies have begun investigating the regulation of nox1 and nox4 in hypertension. Treatment of SHR with atorvastatin, which inhibits vascular ROS formation, decreased nox1 mRNA (215). In rats made hypertensive by overexpression of the human renin gene or by infusion of ANG II for 7 days, the nox1 mRNA was markedly upregulated, whereas nox4 message and protein were also increased to a lesser degree in aorta and kidney (133, 221). After in vivo administration of chelerythrine, the increases in nox1 message and blood pressure due to ANG II infusion were markedly blunted (133). These promising observations suggest that the subunits nox1 and nox4 may be involved in blood pressure regulation. However, direct investigations with the help of specific inhibitors, such as gene knockouts, will be required to definitely ascertain their functions.

Given the evidence presented above, it is apparent that both redox-dependent and -independent pathways significantly contribute to the pathophysiology of hypertension. Although still not fully characterized, a vascular NAD(P)H oxidase comprising p47phox, and possibly p22phox, nox1, and nox4, appears to be required for the oxidative mechanisms involved in the development of the disease. Furthermore, a gp91phox-based oxidase may be implicated in the chronic aspect of hypertension, via inactivation of nitric oxide and remodeling of the vasculature.

	REGULATION OF VASCULAR NAD(P)H OXIDASES IN DIABETES

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
It is well known that diabetes is associated with vascular and tissue damage. A few studies have begun exploring the role of ROS in this process and the relationship between diabetes and the vascular NAD(P)H oxidase (47, 165). Several in vitro studies have shown that high glucose and advanced glycation end products increase ROS formation in VSMC (84), endothelial cells (218), and isolated arterial segments (31). These effects were blocked by DPI and a PKC inhibitor, suggesting that they result from activation of an NAD(P)H oxidase (84, 218). Furthermore, in isolated arteries, high glucose increased superoxide formation and p22phox mRNA, and both effects were inhibited by atorvastatin (31). In addition, advanced glycation end products also increased neutrophil oxidase activity, an effect that is likely to increase inflammation and tissue damage in diabetes (223).

Additional evidence was obtained in vivo, with experimental models of diabetes, such as genetically diabetic rats (a model of type 2 diabetes) (94), rats treated with streptozotocin to destroy pancreatic insulin-producing cells (a model of type 1 diabetes) (38, 80, 91, 142), as well as with human patients with type 2 diabetes (68). The disease was accompanied by increased vascular superoxide production, decreased tissue glutathione, impaired endothelial-dependent relaxation, and increased NAD(P)H oxidase activity as determined using inhibitors of candidate superoxide-producing enzymes (68, 80, 91, 94). Because NOS inhibitors decreased superoxide production and NOS dysfunction was corrected with tetrahydrobiopterin (68, 80), it appears that part of the increase in superoxide was due to uncoupled NOS and decreased nitric oxide production (68, 80). Superoxide increase was inhibited by the PKC inhibitor chelerythrine, consistent with the known activation mechanism of the NAD(P)H oxidases via phosphorylation of p47phox (68, 80). Finally, the mRNAs of the major phagocytic NAD(P)H oxidase subunits p22phox and gp91phox were upregulated in vessels from diabetic animals (80, 91, 94), as well as p22phox, p47phox, and p67phox proteins in vessels from diabetic patients (68). Similarly, p47phox protein was also increased in the kidney of streptozotocin-treated rats (142).

Therefore, these results indicate an association between vascular NAD(P)H oxidase activity and diabetes. Additional information regarding the identity of the subunits involved, their locations, mechanisms of activation, and regulation will be required to ascertain their causal role in this important disease.

	CONCLUSION

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 
It is now clear that the NAD(P)H oxidases are complicated enzymes in several respects. First, at least three homologous catalytic subunits, gp91phox, nox1, and nox4, may be simultaneously expressed in vascular cells. Second, interaction of catalytic and regulatory subunits, the latter of which have not yet been fully characterized in the case of nox1 and nox4, is required for activity. Third, vascular tissue is composed of endothelial cells, VSMC, and fibroblasts, each with different profiles of subunit expression that vary in different vascular beds. Fourth, expression and activity of the oxidases are regulated by agonists and increased in vascular disease. Despite this complexity, with the use of p47phox and gp91phox gene knockout models and other specific inhibitors, the studies summarized above were able to demonstrate that the NAD(P)H oxidases are essential to superoxide and ROS production in normal vascular cells and tissue, both basally and after agonist stimulation. Furthermore, it was shown that the oxidase subunits p47phox and probably also p22phox and nox1 have a causal role in the development of atherosclerotic lesions and also partly in hypertension. An association between oxidase activity and diabetes has been noted as well. Future developments in this rapidly evolving field of investigation are expected to bring a clearer picture regarding the relative importance of the oxidases and other processes involved in the development of vascular disease. However, because the phagocytic subunits have an essential role in immunity and are conserved in the vasculature, they may not be ideal candidates for therapy, at least in the early phase of vascular lesion formation in the absence of inflammation. Although much work remains to be done to fully establish the role of the new nox subunits in pathology, nox1, which is almost absent in normal vascular tissue and upregulated in disease, holds great promise as a target for the future development of specific pharmacological treatment of vascular pathology.

	ACKNOWLEDGMENTS

 
We thank Dr. S. I. Dikalov for providing most of the data in Table 1, Drs. L. L. Hilenski and K. K. Griendling for critically reading the manuscript, and Barbara Merchant for editorial assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: B. Lassègue, Emory Univ., Cardiology, 1639 Pierce Dr., WMB 319, Atlanta, GA 30322 (E-mail: medbpl{at}emory.edu).

	REFERENCES

TOP ABSTRACT THE PHAGOCYTE AND VASCULAR... SPECIFIC FEATURES OF VASCULAR... ACTIVATION PATHWAYS OF VASCULAR... EXPRESSION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... REGULATION OF VASCULAR NAD(P)H... CONCLUSION REFERENCES

 

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