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INVITED REVIEW
Emory University School of Medicine, Division of Cardiology, Atlanta, Georgia 30322
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ABSTRACT |
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 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...CONCLUSIONREFERENCES |
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NAPDH oxidase; superoxide; blood vessels; atherosclerosis; hypertension
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THE PHAGOCYTE AND VASCULAR OXIDASES |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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.
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SPECIFIC FEATURES OF VASCULAR NAD(P)H OXIDASES |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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.
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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.
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.
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ACTIVATION PATHWAYS OF VASCULAR NAD(P)H OXIDASES |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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).
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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.
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EXPRESSION OF VASCULAR NAD(P)H OXIDASES IN CELLS AND TISSUE |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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.
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REGULATION OF VASCULAR NAD(P)H OXIDASES IN CELL CULTURE |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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.
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REGULATION OF VASCULAR NAD(P)H OXIDASES IN ATHEROSCLEROSIS |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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 NG-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.
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REGULATION OF VASCULAR NAD(P)H OXIDASES IN HYPERTENSION |
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TOPABSTRACTTHE 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...CONCLUSIONREFERENCES |
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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.
-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.