© 2001 American Heart Association, Inc.
Editorial |
NAD(P)H Oxidase: Marker of the Dedifferentiated Neointimal Smooth Muscle Cell?
From the Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Mich.
Correspondence to P.J. Pagano, PhD, Senior Staff Investigator, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202-2689. E-mail ppagano1{at}hfhs.org
In recent years, there has been intense interest in vascular NAD(P)H oxidases and their role in vascular injury and atherosclerosis. Balloon injury has been shown to cause large increases in O2– stemming from NAD(P)H oxidases,1 while other studies have shown that hypercholesterolemia and atherosclerosis can induce vascular expression of NADPH oxidase.2 3 4 This induction has been implicated in cell phenotype change and proliferation, and a similar process can be inferred to occur during vascular grafting.5 Two key studies have demonstrated that overexpression of NAD(P)H oxidase in cells leads to transformation to a proliferative phenotype,6 7 and various redox mechanisms transducing this transformation in injury have been elucidated.8 On the contrary, other reports have shown that deletion of 2 essential components of phagocyte NAD(P)H oxidase fail to inhibit lesion formation in apoE–/– mice.9 10 However, it remains to be determined whether those components and NAD(P)H oxidase do play a role under conditions in which the renin-angiotensin system is activated.11 12
In this issue of
Arteriosclerosis,
Thrombosis, and Vascular Biology, West et
al13 present exciting
findings suggesting a role for NAD(P)H oxidase–expressing cells in
intimal hyperplasia after venous bypass. The authors performed elegant
immunohistochemical analyses in an attempt to carefully
determine the source and state of differentiation of cells in the
subintimal space. They found that cells that strongly express
p22phox
(an essential NADPH oxidase component) do not express smoothelin, a
marker for highly differentiated smooth muscle
cells.14 Their intriguing
results show that whereas most cells in the neointima are
positive for 
-actin, desmin, and smoothelin (all markers for
smooth muscle cells) and yet weakly positive for
p22phox,
those that strongly express
p22phox
do not express desmin or smoothelin. These data suggest that there is a
subclass of neointimal smooth muscle cells that are
high-capacity expressors of NAD(P)H oxidase
O2– and thereby are
more proliferative in their
potential.6 15
An extension of those findings included the correlation of O2– detection with p22phox detection by comparing dihydroethidium staining with immunohistochemistry for p22phox. Areas of the subendothelium containing p22phox-expressing cells exhibited the greatest dihydroethidium staining. Although staining for both parameters is a "snapshot" in time and the coexistence of these 2 markers with a lack of smoothelin may be episodic, it nevertheless supports the contention that dedifferentiated smooth muscle cells have a greater capacity to produce O2– by NAD(P)H oxidase. This concept is consistent with these cells’ being more proliferative, because studies have shown that NAD(P)H oxidase activity causes cellular transformation to a more proliferative phenotype.6 7 Practically speaking, these data also suggest the importance of determination of smooth muscle cell differentiation in culture when assessing NAD(P)H oxidase activity.
The study also goes to significant lengths to verify NAD(P)H oxidase activity, performing measurements on intact veins and particulate fractions. The rationale for using a high concentration of lucigenin in some experiments is unjustified, based on the knowledge of lucigenin’s ability to redox-cycle.16 However, the authors do confirm their findings with a concentration of lucigenin (5 µmol/L) that has been described as being able to avert artifactual O2– production.17 Although the latter contention is still controversial, the reader should be able to come away with important new information from the disparate cofactor specificity characteristics between the 2 different lucigenin concentrations; ie, the authors show that at 250 µmol/L lucigenin, NADH-dependent activity predominates over NADPH-dependent activity (Figure 3 in West et al13 ). This may be explained by the apparent ability of NADH to facilitate lucigenin-generated O2–.18 In contrast (and, more appropriately, at 5 µmol/L lucigenin), it appears that NADPH is the preferred substrate (Figure I of West et al13 ). Recent data have shown that at lower concentrations of lucigenin and using electron spin resonance, NADPH is the preferred substrate in smooth muscle, but a substantial amount of NADH oxidase activity is still detected.19 In a homogeneous population of fibroblasts, flavoprotein oxidase–inhibiting diphenylene iodonium was capable of inhibiting NADPH- but not NADH-dependent chemiluminescence, suggesting that the oxidase in fibroblasts is NADPH-driven.20 However, the verdict is still out on vascular oxidase, because in experiments using electron spin resonance to measure O2– in endothelial cells, NADH is the preferred oxidase substrate.21 Part of the discrepancy is likely related to the presence of 1 or more oxidase isozymes, depending on the cell type and/or its proliferative state. The ambiguity of substrate specificity in populations of cultured smooth muscle cells may be indicative of the relative presence of type 1 versus type 2 smooth muscle cells.22
To those not interested in debating the enzyme kinetics of NAD(P)H oxidase, this discussion of which substrate predominates may seem superfluous. However, because of recent interesting data on the presence of at least 2 homologues of gp91phox in the vasculature7 comprising potentially 2 or more isozymes of NAD(P)H oxidase, it is important to clearly identify which isozymes exist in which cell types, including type 1 and 2 smooth muscle cells. Cofactor specificity is likely to vary, depending on which nox subunit (nox-1 or gp91phox)23 anchors the enzyme and participates in the cofactor docking site.
Interestingly, the 2-fold greater chemiluminescence between vein grafts and surgically manipulated jugular veins (Figure 1 of West et al13 ) is normalized by incubation with exogenous superoxide dismutase. This result suggests that, contrary to what has been reported for smooth muscle cells,24 O2– production in the injured state is primarily to the extracellular space. This finding alone may suggest a phenotypic difference of the prevailing cell type in the neointima. The study also shows a large increase in p67phox in vein graft versus jugular vein homogenates, which correlates with O2– production. Although it will be necessary to knock out or immunodeplete p67phox to conclusively implicate its role in higher NAD(P)H oxidase activity, the data might suggest that smooth muscle–like cells in the neointima have a gp91phox-based NAD(P)H oxidase that has been shown to require p67phox.20 25 26
Role of a Dedifferentiated Smooth Muscle Cell in Neointima Formation
Early studies provided evidence of the varied origins of medial cells in different regions of the vascular tree.27 This varied embryological origin may explain the differential ability of some medial smooth muscle cells to proliferate and migrate, while others are more quiescent and participate in contraction.28 It appears that this functional discrepancy is related to the maturity of a smooth muscle cell, which can be determined by the progressive expression of differentiation markers.28 Numerous studies over the past 3 decades have established the concept of the migration of cells from the vascular media and proliferation in the neointima in vascular injury and atherogenesis.29 30 31 Recent characterizations of smooth muscle differentiation states using a variety of markers should provide greater insight into medial composition as well as phenotypic modulation of smooth muscle cells during this process.28 32 33
Smooth Muscle or Smooth Muscle–Like?
In the current study, deeper layers of
neointimal cells expressed smoothelin and some desmin,
indicating that they were smooth muscle cells. Expression of -actin,
coupled with a lack of smoothelin and desmin expression in
subendothelial cells, suggests that they have been
phenotypically modulated and are smooth muscle–like. However, how
should "smooth muscle–like" be defined in this scenario? Are these
subendothelial cells less differentiated smooth muscle
cells, or are they some other type of cell? One such cell that is often
confused with smooth muscle cells during injury and in cell culture is
the myofibroblast, which normally expresses -actin (albeit less
strongly) but not desmin and has the ability to
contract.34 These cells,
which are derived from fibroblasts during injury, could be candidate
cells and seem to contribute in part to the neointimal
population. In fact, Holifield et
al22 described 2 discrete
populations of medial cells (types 1 and 2). Type 2 cells appear
indistinguishable from adventitial fibroblasts and
neointimal cells.
Recently, the adventitia has been shown to play a role in the vascular response to injury. In the porcine model of coronary artery balloon injury used by Shi et al,35 a dissection that was produced by severe balloon injury led to migration of intima-bound myofibroblasts along medial fissures. However, dissection of the media is not necessary, because direct adventitial injury can cause neointimal lesions even in the absence of deendothelialization.36 37 38 39 Furthermore, Shi et al recently showed that in carotid artery/vein grafts, neointimal proliferation is preceded by activation/proliferation of adventitial fibroblasts, modulation to myofibroblasts, and migration to the neointima.5 Finally, Li et al40 elegantly showed that in response to moderate luminal injury, transfected carotid adventitial fibroblasts expressing ß-galactosidase migrate to the neointima. Although these studies do not clarify the relative contribution of medial versus adventitial cells, they do support the concept that adventitial fibroblasts contribute to neointima formation.
In summary, the current data support the presence of a
subclass of proliferative neointimal cells expressing high
amounts of NAD(P)H oxidase in vascular grafts. These cells are likely
to be derived from at least 2 loci, the vascular media and adventitia.
The relative contribution of each vascular segment and cell type is
difficult to assess at this time because of the need for more careful
characterization of the smooth muscle–like cells in the intima
and their origin. Future scrutiny of the presence of various markers of
smooth muscle differentiation, such as smooth muscle myosin heavy chain
or
SM22,41 42
will likely allow better discrimination of this
etiology.
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