Atherosclerosis Is Attenuated by Limiting Superoxide Generation in Both Macrophages and Vessel Wall Cells
Objective— We previously showed that NAD(P)H oxidase deficiency significantly reduces atherosclerosis in apoE−/− mice. The present study was designed to determine the relative contribution of monocyte/macrophage versus vascular wall cell NAD(P)H oxidase to atherogenesis in this model.
Methods and Results— Cell-specific NAD(P)H oxidase inhibition was achieved via allogenic, sex-mismatched bone marrow transplantation. Aortic atherosclerosis and superoxide production in apoE−/− mice (Control) with functional NAD(P)H oxidase in both monocytes/macrophages and vascular wall cells was compared with that in apoE−/− mice with nonfunctional monocyte/macrophage NAD(P)H oxidase (BMO) or nonfunctional vessel wall NAD(P)H oxidase (VWO). A significant decrease in superoxide production and atherosclerotic lesions was observed in BMO and VWO mice compared with control mice. Interestingly, BMO mice had significantly lower plasma oxidized LDL levels compared with control and VWO mice, whereas aortic sections of VWO mice showed decreased expression of cellular adhesion molecules compared with control and BMO mice. NAD(P)H oxidase deficiency also attenuated neointimal hyperplasia and mitogenic protein activation in apoE−/− mice after arterial injury.
Conclusions— We conclude that (1) both monocyte/macrophages and vessel wall cells play critical roles in atherogenesis; (2) decrease in atherosclerosis results from attenuated superoxide generation in monocyte/macrophages or vessel wall cells; and (3) superoxide generation may impact atherosclerosis, in part, by activating smooth muscle cell mitogenic signaling pathways.
Reactive oxygen species (ROS) derived from NAD(P)H oxidase have been strongly associated with experimental hypertension,1 cardiac hypertrophy,2 thrombosis,3 restenosis, and atherosclerosis.4 In humans, higher expression of NAD(P)H oxidase subunit proteins is associated with increased superoxide (O2·−) production and severity of atherosclerosis.5 NAD(P)H oxidase is also an important source of increased O2·− production in human diabetes mellitus, a risk factor for atherosclerosis.6 All vascular wall cells—endothelial cells, smooth muscle cells (SMCs), and fibroblasts—as well as monocytes/macrophages contain NAD(P)H oxidases which are activated under pathophysiological conditions. The resultant ROS induce redox-sensitive signaling pathways that contribute to atherogenesis.7
The phagocytic NAD(P)H oxidase contains the membrane-bound subunits gp91phox (Nox2) and p22phox, the catalytic site of the oxidase and the cytosolic components p47phox, p67phox, and G-protein Rac1 or Rac2.8 Vascular NAD(P)H oxidases are similar in structure to phagocytic NAD(P)H oxidase, but have a distinct molecular composition. Endothelial cells and adventitial fibroblasts possess all the components of the phagocytic oxidase, whereas SMCs predominantly express homologues of gp91phox, Nox1 and Nox4. Mouse SMCs also express a p67phox homologue, Noxa1.9 The activation of vascular NAD(P)H oxidases is constitutive as well as inducible in a manner similar to the neutrophil enzyme by the translocation of the cytosolic components.10 Superoxide production by vascular oxidases is much less than that in macrophages.7
Increased ROS production from both monocytes/macrophages and in vascular wall cells has been implicated in atherosclerosis. Macrophage infiltration and ROS production were markedly increased in atherosclerotic plaques of patients with unstable angina pectoris whereas plaques from patients with stable angina had fewer macrophages and lower ROS production.11 However, ROS production in unstable plaques was derived from macrophages as well as SMCs. Similarly, the severity of atherosclerotic lesions correlated well with the activation of NAD(P)H oxidases in both macrophages and SMCs of human coronary arteries.5 In contrast, little or no macrophage infiltration was observed in experimental restenosis12 and atherosclerosis,13 which implies that ROS generation was predominantly from vascular wall cells in these models.10
We have previously shown that NAD(P)H oxidase-deficient apoE−/− mice (apoE−/−/p47phox−/− mice) had significantly less atherosclerosis compared with apoE−/− mice.4,14 Further, aortic O2·− levels were lower in p47phox−/− mice compared with wild-type mice.14,15 In addition, aortic SMCs from p47phox−/− mice were found to have a decreased proliferative response to growth factors compared with wild-type mice SMCs.
In the present study, we investigated the relative contribution of monocyte/macrophages and the vessel wall cell-derived ROS to atherosclerosis using bone marrow transplantation. Our data indicate that NAD(P)H oxidase-derived ROS generated from both monocytes/macrophages as well as vascular wall cells are important in the development of atherosclerotic lesions. Deficiency of NAD(P)H oxidase in monocytes/macrophages decreases plasma oxidized LDL levels, whereas deficiency of this enzyme in vascular wall cells attenuates the expression of cellular adhesion molecules in the aortas of apoE−/− mice. Thrombin-induced mitogenic signaling pathways are attenuated in p47phox−/− SMC compared with wild-type SMCs. Consistent with this, attenuation of neointimal hyperplasia after arterial injury and decrease in the activation of mitogenic signaling proteins in the neointima were observed in apoE−/−/p47phox−/− mice compared with wild-type. Together, these results suggest that ROS derived from monocytes/macrophages as well as vascular wall cells are involved in atherosclerosis, whereas NAD(P)H oxidase-dependent SMC mitogenic signaling pathways play an important role in restenosis.
Materials and Methods
Mice and Diet
ApoE−/−, p47phox−/−, and apoE−/−/p47phox−/− mice were obtained as described previously14 (see supplemental materials, available online at http://atvb.ahajournals.org). Bone marrow transplanted mice were maintained on rodent chow for 4 weeks and then fed a Western diet (42% fat, Harlan Teklad, TD 88137) for 12 weeks.
Bone Marrow Transplantation
Bone marrow transplantation (BMT) was performed as described in the supplemental materials.
Quantification of Atherosclerotic Lesions
Atherosclerosis in the aortas was measured by quantitation of oil red O-positive lesions (see supplemental materials).
Femoral Artery Injury and Morphometry
Transluminal femoral artery injury was performed as described4 (see supplemental materials).
Measurement of ROS Production in the Aorta
ROS levels in the aortas were measured by staining with dihydroethidium14 (see supplemental materials).
Fluorescence In Situ Hybridization
Frozen arterial sections were processed for in situ hybridization with Y chromosome paints (Applied Spectral Imaging) as described earlier.16 Images were captured with a charge-coupled device (CCD) camera.
Immunostaining of aortic and femoral artery cross sections was described online in the supplemental materials.
RNA Extraction and Real-Time RT-PCR
Detailed information regarding RNA extraction and real-time RT-PCR is available in the supplemental materials.
Analysis of Plasma Lipids
Plasma total cholesterol and triglyceride (TG) levels were measured by a quantitative enzymatic colorimetric assay (Stanbio Laboratory). Plasma Oxidized LDL levels were measured using the Oxidized LDL Competitive ELISA kit (Mercodia).
Cell Culture and Western Analysis
Isolation of aortic SMCs and Western analysis were described online in the supplemental materials.
Data are presented as mean±SEM. All data were analyzed using 1-way ANOVA followed by Newman-Keuls multiple comparison test. Differences were considered significant at P<0.05.
Functional NAD(P)H Oxidase in Bone Marrow-Derived Cells and the Vessel Wall Cells Contributes to Aortic Atherosclerosis and ROS Production
We have previously reported that reduced NAD(P)H oxidase-derived O2·− generation results in reduced atherosclerosis, using apoE−/− and apoE−/−/p47phox−/−mice.4,14 In these studies, the deficiency of NAD(P)H oxidase was neither cell- nor tissue-specific. Therefore, it was not possible to determine which among the cell types with the capacity to produce O2·− through activation of NAD(P)H oxidase were responsible for the observed decrease in atherosclerosis. To determine the relative contribution of monocyte/macrophage NAD(P)H oxidase to that of SMC and endothelial cells in atherosclerosis, we performed allogenic sex-mismatched BMT from apoE−/−/p47phox−/− mice to atherosclerosis prone apoE−/− mice and vice versa. En face oil red O staining of the aortas of apoE−/− mice (Figure 1A), which received BMT from apoE−/− mice (control) showed that 7.5% of the aortic surface area was covered with atherosclerotic lesions (Figure 1B). ApoE−/− mice that received bone marrow from apoE−/−/p47phox−/− mice, hence lacking functional NAD(P)H oxidase in bone marrow-derived cells (monocytes/macrophages, BMO), had 53% less total mean lesion area (P<0.05) than the total mean lesion area in apoE−/− mice. Similarly, apoE−/−/ p47phox−/− mice (lacking functional NAD(P)H oxidase in aortic wall cells including endothelial cells, SMCs, and fibroblasts, VWO) that received bone marrow from apoE−/− mice (functional NAD(P)H oxidase in marrow-derived monocytes/macrophages) had 66% less total mean atherosclerotic lesion area than the total mean lesion area in apoE−/− mice (P<0.01; Figure 1B). These data indicate that NAD(P)H oxidase activity in both monocytes/macrophages and aortic wall cells contributes to atherosclerotic lesion formation.
To confirm the functional role of NAD(P)H oxidase in monocytes/macrophages and vascular wall cells in atherosclerotic disease process, we measured O2·− production in situ in the control, BMO, and VWO mice aortas by incubating fresh frozen sections of aortas with DHE. Aortas from control mice had consistently increased DHE fluorescence compared with aortas from BMO and VWO mice (Figure 1C and 1D). Quantitative analysis of DHE fluorescence demonstrated a 63% decrease in O2·− production in BMO and VWO aortas compared with control aortas (P<0.05). Consistent with decreased O2·− production resulting from p47phox deficiency, no compensatory increases in the expression of other components of the NAD(P)H oxidase were observed. Specifically, the expression of the catalytic subunits of Nox1 and Nox4 isoforms and p22phox subunit of NAD(P)H oxidase were similar in BMO and VWO aortas compared with control aortas (supplemental Figure I). Together, these results indicate that absence of a functional NAD(P)H oxidase in monocytes/macrophages or vascular wall cells leads to decreased ROS production and attenuated atherosclerotic lesion formation in this murine model of atherosclerosis.
Absence of a Functional NAD(P)H Oxidase in Monocytes/Macrophages or Vascular Wall Cells Results in Decreased Macrophage Infiltration Into Atherosclerotic Lesions
To determine the effect of functional NAD(P)H oxidase on histological composition of atherosclerotic lesions, fresh frozen cross sections from the aortic arch of control, BMO, and VWO mice were immunohistochemically stained for presence of macrophages with anti-Mac-3 antibody (Figure 2A). Cross sections from control mice showed larger lesions enriched with macrophages (brown staining) and SMCs (staining not shown), whereas aortic sections from BMO and VWO mice had smaller lesions with fewer macrophages. These results indicate that a functional NAD(P)H oxidase is necessary in both the vessel wall and in the circulating cells for macrophage infiltration and SMC proliferation.
To confirm that monocytes/macrophages were derived from the donor bone marrow in the sex-mismatched allogenic transplantation used in the current study, cross sections from arterial arch in the control mice were stained with Y chromosome paint. As shown in Figure 2B, considerable number of Y chromosome-positive cells were present in macrophage enriched area of the neointima (top panel) and adventitia (bottom panel). These results indicate that absence of a functional NAD(P)H oxidase in either circulating inflammatory cells or vessel wall cells retards atherosclerotic lesion growth by curtailing the recruitment of macrophages to the endothelial surface during lesion initiation/progression. Further, these results confirm that macrophages infiltrate into the adventitia of the atherosclerotic lesions.17
NAD(P)H Oxidase in Bone Marrow-Derived Cells Plays a Critical Role in LDL Oxidation
One mechanism by which NAD(P)H oxidase promotes atherogenesis is through its effect on the composition of lipid levels.18,19 To assess whether the differences observed in the atherosclerotic burden among the 3 experimental mouse groups resulted from functionally important differences in plasma lipids, plasma cholesterol, triglyceride, and oxidized LDL (oxLDL) levels were determined in control, BMO, and VWO mice (Figure 3). Absence of NAD(P)H oxidase in either bone marrow-derived cells or vascular wall cells did not significantly affect plasma total cholesterol or triglyceride levels (Figure 3A and 3B). However, BMO mice had significantly lower (P<0.05) oxLDL levels compared with control and VWO mice (Figure 3C). These findings, together with the report that macrophage-mediated LDL oxidation is dependent on NAD(P)H oxidase,19,20 suggest that monocyte/macrophage oxidation of LDL contributes to the pathogenesis of atherosclerosis.
Expression of Cellular Adhesion Molecules at Luminal Surface of the Aorta Is Regulated by NAD(P)H Oxidase in the Vascular Wall Cells
Adherence of circulating blood monocytes to the vessel wall is one of the earliest events in atherogenesis, and an NAD(P)H oxidase inhibitor decreased atherosclerosis by attenuating cytokine-induced expression of cellular adhesion molecules (CAMs) and the adherence of monocytes to the endothelium.21 To determine whether the decrease in atherosclerosis observed in BMO and VWO mice resulted from inhibition of CAM expression, mRNA levels of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin, and P-selectin from aortas were analyzed by real-time RT-PCR (supplemental Figure II). VWO mice had significantly decreased expression of VCAM-1, ICAM-1 and P-selectin in their aortas compared with control and BMO mice. E-selectin expression levels were not significantly different among the 3 groups, though lower values were observed in the aortas of VWO mice.
We then performed immunohistochemical analysis of aortic cross sections to confirm the changes observed in mRNA expression of the CAMs (Figure 4). Immunoreactive VCAM-1 expression was abundant in the endothelium and medial SMCs of control and BMO mouse aortas, whereas it was sparse and confined to endothelium in the aortas of VWO mice. Similarly, ICAM-1 expression was low and restricted to endothelium in VWO mice compared with that in control and BMO mice wherein it was detected in both the endothelium and adventitia. Immunoreactive E-selectin was highly expressed in the endothelium and neointima of control and BMO mice, whereas it was absent in the aortic cross sections of VWO mice. P-selectin was expressed in the endothelium of control and BMO mouse arteries and absent in the aorta of VWO mice. Together, these data indicate that NAD(P)H oxidases in monocytes/macrophages and vascular wall cells affect atherogenesis by at least 2 distinct mechanisms: (1) monocyte/macrophages induce LDL oxidation in a NAD(P)H oxidase-dependent manner; and (2) vascular wall cells express cell adhesion molecules in a NAD(P)H oxidase-dependent manner.
NAD(P)H Oxidase Deficiency Attenuates Neointimal Hyperplasia and Mitogenic Protein Activation in ApoE-−/− Mice After Arterial Injury
NAD(P)H oxidase is necessary for neointimal hyperplasia after arterial injury. We previously demonstrated a significant decrease in neointimal lesions in p47phox−/− mice compared with wild-type mice in response to arterial injury.4 Here we investigated whether deficiency of apoE overrides the protection against neointimal hyperplasia afforded by a nonfunctional NAD(P)H oxidase. As expected, a statistically significant increase in neointimal hyperplasia (P<0.05) was observed in the femoral arteries of apoE−/− mice compared with that seen in sham operated mice, 4 weeks after injury (supplemental Figure III). Neointimal hyperplasia also increased in the femoral arteries of apoE−/−/p47phox−/− mice in response to injury, but the increase was less than that in apoE−/− mice and not statistically significant from that seen in sham operated apoE−/−/p47phox−/− mice.
Immunohistochemical staining of femoral artery cross sections for smooth muscle α-actin demonstrated that neointimal lesions are predominantly made up of SMCs4. To determine the molecular basis of SMC proliferation, we compared the activation of redox-sensitive mitogenic protein expression22,23—janus kinase 2 (JAK2), extracellular signal regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK), and signal transducer and activator of transcription 3 (STAT3)—in wire-injured femoral arteries of apoE−/− and apoE−/−/p47phox−/− mice (Figure 5). JAK2 and ERK1/2 phosphorylation increased substantially in the neointima of femoral arteries of apoE−/− mice than that in apoE−/−/p47phox−/− mice 4 weeks after injury. Similarly, significant increase in the expression of phosphorylated p38 MAPK and STAT3 was observed in the medial and neointimal SMCs of apoE−/− mice than that seen in apoE−/−/p47phox−/− mice. Together, these results indicate that NAD(P)H oxidase-stimulated activation of mitogenic signaling pathways is necessary for SMC proliferation after endothelial denudation injury in mice, even on a hypercholesterolemic background. Thus, SMC proliferation is likely an additional contributor to atherogenesis in apoE−/− mice impacted by NAD(P)H oxidase activity, together with LDL oxidation and adhesion molecule expression.
Confirmation of Redox-Sensitive Activation of Mitogenic Proteins in SMCs In Vitro
We next examined whether the NAD(P)H oxidase-dependent activation of SMC mitogenic proteins observed in vivo occurs in arterial SMCs in vitro. We treated SMCs isolated from wild-type and p47phox−/− mice with thrombin to investigate redox-sensitive activation of mitogenic proteins. The choice of thrombin for in vitro studies is based on the observation that continued and marked increase in thrombin concentration occurs after arterial injury,24 and thrombin regulates the expression/activation of proteins involved in SMC migration and proliferation in NAD(P)H oxidase-dependent manner.4,22
Western blot analysis of thrombin-stimulated SMC lysates with a phospho-specific JAK2 antibody showed a significant increase in JAK2 tyrosine phosphorylation at 1 minute (2.38±0.28 fold increase, P<0.01) followed by a gradual decline (Figure 6A). In contrast, thrombin had no significant effect on JAK2 phosphorylation in p47phox−/− SMCs. Similarly, ERK1/2 phosphorylation increased significantly in thrombin-treated wild-type SMCs, reaching a maximum stimulation at 5 minutes (17.0±1.33 fold increase, P<0.001). There was no change in ERK1/2 phosphorylation in p47phox−/− SMCs treated with thrombin (Figure 6B). Consistent with the report of Brandes et al,15 thrombin induced a significant increase in p38 MAPK activation (3.0±1.2 fold increase, P<0.05) at 10 minutes after treatment in wild-type SMCs whereas no change in the phosphorylation of this protein was observed in p47phox−/− cells treated with thrombin (Figure 6C). Together, these results indicate NAD(P)H oxidase-derived ROS production modulates the activation of proteins involved in SMC proliferation and, in this manner, likely contributes to neointimal hyperplasia under pathophysiological conditions.
Increased O2·− production plays an important role in the initiation and progression of atherosclerosis. Investigations in cultured cells, animal models, and human studies have led to the identification of NAD(P)H oxidases as a major source of O2·−.4,5,7,11,25 Increased O2·− generation promotes atherosclerosis by several potential mechanisms. Superoxide anion promotes atherogenesis through the formation of oxidized lipids, particularly oxLDL. Monocytes/macrophages, the major inflammatory cell component of atherosclerotic lesions, induce LDL oxidation,26 a process previously shown to be dependent on NAD(P)H oxidase-derived O2·−.18
In addition to LDL oxidation, NAD(P)H oxidase-derived O2·− has been implicated in endothelial dysfunction,27 increased expression of CAMs,28 and enhanced proliferation and migration of SMCs4,22—factors that promote atherosclerosis. Superoxide also reacts with nitric oxide to produce peroxynitrite, a highly oxidizing atherogenic molecule which activates matrix metalloproteinases and facilitates vascular remodeling.29 Decreased peroxynitrite production and delayed vascular remodeling in response to increased shear stress has been observed in p47phox−/− mice compared with the wild-type mice.
We report here that NAD(P)H oxidase activity in monocytes/macrophages and vascular wall cells contributes equally to atherosclerotic lesion formation. Absence of functional NAD(P)H oxidase in bone marrow-derived monocytes/macrophages or vascular wall cells significantly decreased total aortic atherosclerotic lesion area in apoE−/− mice fed a Western diet. The protective effect against atherosclerosis in these 2 groups of mice correlated with decreased aortic O2·− production. These results extend our previous studies4,14 indicating that NAD(P)H oxidase plays an important role in atherogenesis. However, it is important to note that neither aortic atherosclerosis or O2·− production were abrogated, either in the present investigation or in our previous studies,4,14 by the deficiency of p47phox in apoE−/− mice. The remnant ROS production in these mice might have come from other sources including Nox4-NAD(P)H oxidase, xanthine oxidase, cytochrome p450, and mitochondria. It is plausible that the remnant ROS production in the background of hypercholesterolemia could have resulted in the residual atherosclerosis observed in the apoE−/−/p47phox−/− mice.
Resting monocytes circulating in blood respond to an activating stimulus such as oxLDL, changes in vascular flow or infection, and adhere to activated endothelial cells in the vessel wall. The activated monocytes then typically extravasate into the subendothelial spaces where they mature into macrophages and contribute to lesion development by oxidizing LDL. Monocyte recruitment is critical for atherosclerosis as lesion development is significantly decreased when monocytes are prevented from entering the vessel wall.30,31
The presence of fewer macrophages in atherosclerotic lesions in BMO and VWO mice is either a reflection of the smaller lesion size or a result of disruption of NAD(P)H oxidase activity in monocytes/macrophages or vascular wall cells, respectively. Absence of a functional NAD(P)H oxidase in the endothelial cells and SMCs in VWO mice may have contributed to decreased lesion size by attenuating expression of CAMs and the subsequent monocyte/macrophage infiltration.17 Nonfunctional NAD(P)H oxidase in bone marrow cells may have reduced the recruitment of monocytes into atherosclerotic lesions by significantly decreasing oxLDL levels.32 It is also possible that absence of functional NAD(P)H oxidase decreases monocyte adhesive mechanisms at the endothelial interface. In this context, it is worth noting that an inhibitor of NAD(P)H oxidase attenuated the adhesion of leukocytes on recombinant VCAM and P-selectin and reduced atherosclerotic lesion size in apoE−/− mice.21
Our current data on the presence of fewer macrophages in atherosclerotic lesions of BMO and VWO mice compared with that in control mice is seemingly contradictory to our previous observation that there was no difference in the homing of wild-type and p47phox-deficient macrophages to atherosclerotic lesions in apoE−/− mice.14 However, these results may have been influenced by the 2 different methods used in these studies. For example, alterations in the expression of CAMs that can occur during monocyte/macrophage isolation, labeling, and injection in homing studies may modify the interaction between circulating monocytes and the vascular wall.33
Neointimal hyperplasia in response to arterial injury was significantly less in apoE−/− mice that lack p47phox than in apoE−/− mice and was characterized by decreased levels of activated SMC mitogenic proteins, JAK2, ERK1/2, p38 MAPK, and STAT3 in neointimal SMCs (Figure 5).22,23 In vitro analysis confirmed the redox-sensitive regulation of these proteins in SMC (Figure 6). These data provide a molecular explanation for the observed decrease in proliferation of p47phox−/− SMCs compared with wild-type SMCs in response to agonist treatment.14 Our current results are in consonance with our previous findings that absence of p47phox protects against injury-induced arterial neointimal hyperplasia.4 In addition, these results indicate that absence of functional NAD(P)H oxidase protects against restenosis even on a hyperocholesterolemic background.
In summary, the studies reported here confirm that NAD(P)H oxidase modulates the atherogenic phenotype in apoE−/− mice, and importantly, provide evidence for equal contribution of monocyte/macrophage and vessel wall NAD(P)H oxidase to aortic ROS production and atheroscleorsis in apoE−/− mice. Furthermore, these data indicate that monocyte/macrophage NAD(P)H oxidase significantly affects LDL oxidation in vivo. Inhibition of NAD(P)H oxidase attenuates restenosis in response to arterial injury in apoE−/− mice, and the reduction in neointimal hyperplasia is possibly attained by the downregulation of SMC intracellular mitogenic signaling pathways activated by growth factors and cytokines.
We are grateful to Marcy Kingsbury, The Scripps Research Institute, for help with fluorescence in situ hybridization.
Sources of Funding
This study was supported by NIH grant HL57352.
A.E.V., Z.S.H., and N.R.M. contributed equally to this work.
Original received March 20, 2007; final version accepted August 24, 2007.
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