Endothelial Nitric Oxide Synthase Uncoupling and Perivascular Adipose Oxidative Stress and Inflammation Contribute to Vascular Dysfunction in a Rodent Model of Metabolic Syndrome
The metabolic syndrome represents a constellation of cardiovascular risk factors that promote the development of cardiovascular disease. Oxidative stress is a mediator of endothelial dysfunction and vascular remodeling. We investigated vascular dysfunction in the metabolic syndrome and the oxidant mechanisms involved. New Zealand obese (NZO) mice with metabolic syndrome and New Zealand black control mice were studied. NZO mice showed insulin resistance and increased visceral fat and blood pressure compared with New Zealand black mice. Mesenteric resistance arteries from NZO mice exhibited increased media:lumen ratio and media cross-sectional area, demonstrating hypertrophic vascular remodeling. Endothelium-dependent relaxation to acetylcholine, assessed by pressurized myography, was impaired in NZO mice, not affected by NG-nitro-l-arginine methyl ester, inhibitor of endothelial NO synthase, and improved by the antioxidant Tempol, suggesting reduced NO bioavailability and increased oxidative stress. Dimer:monomer ratio of endothelial NO synthase was decreased in NZO mice compared with New Zealand black mice, suggesting endothelial NO synthase uncoupling. Furthermore, vascular superoxide and peroxynitrite production was increased, as well as adhesion molecule expression. Perivascular adipose tissue of NZO mice showed increased superoxide production and NADPH oxidase activity, as well as adipocyte hypertrophy, associated with inflammatory Mac-3–positive cell infiltration. Vasoconstriction to norepinephrine decreased in the presence of perivascular adipose tissue in New Zealand black mice but was unaffected by perivascular adipose tissue in NZO mice, suggesting loss of perivascular adipose tissue anticontractile properties. Our data suggest that this rodent model of metabolic syndrome is associated with perivascular adipose inflammation and oxidative stress, hypertrophic resistance artery remodeling, and endothelial dysfunction, the latter a result of decreased NO and enhanced superoxide generated by uncoupled endothelial NO synthase.
The metabolic syndrome represents a constellation of cardiovascular risk factors of metabolic origin that promote the development of cardiovascular disease and type 2 diabetes mellitus.1 In the past few years, to identify patients with metabolic syndrome, several clinical and biochemical criteria have been established, such as visceral obesity, increased plasma glucose, atherogenic dyslipidemia, and increased blood pressure (BP).2,3 Among them, visceral obesity is widely accepted as a diagnostic prerequisite, and it is believed to be involved in the pathogenesis of the metabolic disarray itself by inducing insulin resistance.
Although the clinical association of metabolic risk factors and BP increase is well established, the underlying mechanisms are still a matter of debate. Vascular dysfunction of resistance arteries plays a key role in the development of hypertension and is mediated, at least in part, by oxidative stress.4 In the vascular system, reactive oxygen species (ROS) are produced by different cell types, such as endothelial cells, vascular smooth muscle cells, and inflammatory cells infiltrating the perivascular tissue.5,6 NADPH oxidase represents the major source of ROS in the vasculature.7 In addition, the enzyme endothelial NO synthase (eNOS), which normally is “coupled” and produces NO, under some conditions, such as in the presence of excess oxidative stress or decreased tetrahydrobiopterin, is “uncoupled” and generates superoxide (·O2−). This leads to the production of reactive nitrogen species, such as peroxynitrite, as a result of the action of ·O2− on NO. Thus, in the vascular system, oxidative stress from different sources decreases NO bioavailability, thereby promoting endothelial dysfunction, vasoconstriction, remodeling, and enhanced systemic vascular resistance, leading to BP increase.7,8
The pathogenic role of adipose tissue in BP increase is supported by clinical evidence that weight loss leads to BP reduction.9 Perivascular adipose tissue (PVAT) has been shown to have anticontractile effects via NO release and increasing hydrogen peroxide (H2O2) generation.10 Moreover, PVAT is a source of inflammatory cells that have been shown to contribute to vascular remodeling in different model of hypertension, in part through pro-oxidant mechanisms.11,12 There is increasing evidence that remodeling of adipose tissue may be associated with endoplasmic reticulum stress that leads to the unfolded protein reaction, which stimulates oxidative mechanisms and apoptosis and activates inflammatory mediators.13
The hypothesis behind this study is that metabolic syndrome induces vascular remodeling and endothelial dysfunction in resistance arteries through oxidative stress. Here we questioned whether vascular remodeling, endothelial dysfunction, oxidative stress, and inflammation occur in a rodent model of human metabolic syndrome, investigated the vascular pro-oxidant mechanisms putatively involved, specifically NADPH oxidase and eNOS uncoupling, and determined the contribution of oxidative stress and inflammation of PVAT to vascular dysfunction.
Expanded materials and methods are provided in the online Data Supplement (please see http://hyper.ahajournals.org).
New Zealand obese (NZO/HILtJ [NZO]) and New Zealand lean (New Zealand black (NZB/BINJ [NZB]) mice were obtained from Jackson Laboratory (Bar Harbor, ME). The NZO mouse has been characterized by others as a model of human metabolic syndrome14–16 (please see the online Data Supplement). NZO mice exhibit polygenic obesity associated with hyperglycemia, hyperinsulinemia, increased serum cholesterol and triglyceride levels, and elevated BP, when compared with their control, the NZB mice.
Systolic BP and heart rate were determined by the tail-cuff technique using the Visitech systems BP-2000.17
Functional Vascular Studies
Second-order branches of the mesenteric artery were dissected and studied on a pressurized myograph, as described previously.6,18 Endothelium-dependent relaxation was assessed by measuring the dilatory response to acetylcholine (10−9 to 10−4 mol/L) and bradykinin (10−10 to 10−6 mol/L) in norepinephrine precontracted vessels (5×10−5 mol/L). Endothelium-independent relaxation was assessed with sodium nitroprusside (10−8 to 10−3 mol/L) in precontracted vessels. NO availability and ROS production were evaluated by a acetylcholine concentration-response curve repeated after a 20-minute incubation with the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 10−4 mol/L) and the antioxidant and ·O2− dismutase mimetic Tempol (10−3 mol/L), respectively. Concentration-response curves to norepinephrine (10−9 to 10−4 mol/L), endothelin-1 (10−10 to 10−6 mol/L), and angiotensin II (10−9 to 10−5 mol/L) were performed in the presence or absence of PVAT to evaluate the effect of the PVAT on vascular contractility.
Vascular and Perivascular Adipose ·O2− Production and NADPH Oxidase Activity
Vascular ·O2− production was assessed on aorta and aortic and mesenteric PVAT with the ·O2−-sensitive fluorescent dye dihydroethidium (2 μmol/L). NADPH oxidase activity was assessed both in aorta and in the mesenteric artery, as well as in aortic and mesenteric PVAT, by lucigenin chemiluminescence assay, as described previously.19
Mesenteric artery extracts were prepared with sodium dodecyl sulfate sample buffer in nondenaturating conditions. Samples were loaded on 7.5% polyacrylamide gels and subjected to electrophoresis. Buffers and gels were cooled to 4°C, and the buffer tank was placed in an ice bath during electrophoresis. Endothelial eNOS monomer and dimer were detected by Western blotting analysis.
Plasma Nitrite Measurement
Plasma nitrites were evaluated with a colorimetric assay, as described previously.20
Two-way repeated-measures ANOVAs were used to evaluate differences in concentration-response curves between groups, followed by Bonferroni post hoc analysis. Unpaired t test was performed to evaluate all of the other variables. P<0.05 was considered statistically significant. Results are presented as mean±SEM.
Table 1 summarizes the metabolic and cardiovascular variables of NZO and NZB control mice. NZO mice had increased body weight, a greater amount of abdominal fat, and visceral adipocyte hypertrophy (please see Figure S1 in the online Data Supplement) compared with NZB mice. As well, subcutaneous adipose tissue was increased in NZO mice (data not shown). Tibia length was not different between groups. Plasma insulin was higher in NZO mice than in NZB mice. NZO mice showed increased heart, kidney, liver, and spleen weight. Systolic BP was slightly but significantly higher in NZO mice compared with NZB mice.
PVAT and Vascular Remodeling of Resistance and Conduit Vessels
Table 2 summarizes the vascular phenotype in NZO and NZB control mice. NZO mice exhibited increased PVAT and perivascular adipocyte hypertrophy in both aorta and mesenteric arteries compared with those of controls (Figure S1). Media cross-sectional area was increased in aorta of NZO mice compared with NZB mice. Mesenteric resistance arteries from NZO mice exhibited hypertrophic vascular remodeling, as shown by increased media thickness, media:lumen ratio (M/L ratio), and media cross-sectional area.
Endothelial Function of Resistance Arteries
Mesenteric arteries from NZO mice showed significantly impaired relaxation responses to acetylcholine and bradykinin compared with NZB mice (Figure 1A and 1B, respectively), indicating endothelial dysfunction. Endothelium-independent relaxation responses to sodium nitroprusside were similar in both groups, indicating integrity of the vascular smooth muscle cell layer (Figure 1C). In mesenteric arteries from NZB mice, relaxation to acetylcholine was significantly reduced by eNOS inhibition with l-NAME but unaffected by the antioxidant and ·O2− dismutase mimetic Tempol (Figure 2A and 2C, respectively). On the contrary, in mesenteric arteries from NZO mice, relaxation to acetylcholine was unaffected by l-NAME but was restored by Tempol (Figure 2B and 2D, respectively).
Mechanisms of Vascular Oxidative Stress and Inflammation in NZO and NZB Control Mice
In mesenteric arteries of NZO mice, eNOS phosphorylation and expression were increased, indicating enhanced eNOS activity in NZO mice compared with NZB mice (Figure 3A). However, the eNOS dimer:monomer ratio was decreased in NZO mice compared with NZB mice, which indicates the existence of eNOS uncoupling (Figure 3B). As a result of increased reaction between ·O2− and NO, vascular nitrotyrosine, a marker of peroxynitrite production, was increased in mesenteric arteries of NZO mice. Plasma nitrite levels were also greater in NZO mice (Figure 3C and 3D, respectively). Activity of vascular NADPH oxidase was similar in NZO and NZB mice (please see Figure S2). Increased inflammation was present in mesenteric arteries in NZO mice, as shown by increased expression of VCAM-1 (vascular adhesion molecule-1), PECAM-1 (platelet endothelial cell adhesion molecule-1), and ICAM-1 (intercellular adhesion molecule-1), compared with NZB mice (Figure 4A and 4B).
Impaired Perivascular Adipose Anticontractile Properties in NZO Mice
Vascular contractility to norepinephrine, endothelin-1, and angiotensin II was assessed in mesenteric resistance arteries of NZB and NZO mice, with and without PVAT. Vasoconstriction to all 3 of the vasoactive agents decreased in mesenteric arteries of NZB mice in the presence of PVAT compared with arteries without PVAT (Figure 5A, 5C, and 5E). On the contrary, vasoconstriction to norepinephrine, endothelin-1, and angiotensin II in mesenteric arteries from NZO mice was not affected by PVAT, indicating loss of the physiological anticontractile properties of perivascular fat (Figure 5B, 5D, and 5F). The presence of PVAT did not significantly affect the relaxation response to acetylcholine or bradykinin, in either NZB or NZO mice, indicating a lack of an acute effect of PVAT on endothelium-dependent vasodilation (please see Figure S3).
Perivascular Adipose Oxidative Stress and Inflammation
Perivascular adipose ·O2− production was increased in NZO mice compared with NZB mice, as well as in the vasculature (Figure 6A, left). NZO mice showed enhanced Mac-3 staining in aortic and mesenteric PVAT, suggesting macrophage infiltration (Figure 6A, right). Perivascular adipose mRNA levels of TNF-α (tumor necrosis factor-α) were increased in NZO mice, suggesting a proinflammatory phenotype of macrophages. As well, mRNA levels of MCP-1 (monocyte chemotactic peptide-1) were increased and adiponectin was decreased in NZO mice, indicating proinflammatory mechanisms in the PVAT (Figure 6B). NADPH oxidase activity was increased in aortic and mesenteric PVAT of NZO mice (Figure 6C). Expression of GTPase Rac1, cofactor of NADPH oxidase, was significantly greater in mesenteric PVAT of NZO mice compared with NZB mice (Figure 6D). The expression of the antioxidant ·O2− dismutase (-1, -2, and -3), which catalyzes the transformation of ·O2− in H2O2, was lower in mesenteric PVAT of NZO mice compared with NZB mice (Figure 6D).
Metabolic syndrome, with a prevalence of 40% in the adult population, is a critical medical issue in developed countries and requires the development of new therapies targeting more than one cardiovascular risk factor at a time.3 The availability of animal models allows for investigation of the pathophysiological mechanisms leading to metabolic syndrome. Here we demonstrated that the NZO mouse, a reliable model of human metabolic syndrome, presents vascular dysfunction caused by reduced NO bioavailability because of uncoupling of eNOS and increased ROS and reactive nitrogen species production. Moreover, we showed that perivascular fat contributes to vascular dysfunction, through reduced anticontractile effects, and we suggest inflammation and oxidative stress as possible mechanisms. Our findings extend recent data in the human metabolic syndrome,21 providing new pathophysiological mechanisms and a rodent model for future study.
In agreement with previous data,14–16 we showed that the NZO mouse exhibited visceral obesity, hyperinsulinemia, and BP increase, fulfilling the most commonly used criteria to define the metabolic syndrome in patients. Moreover, NZO mice presented increased heart, liver, and spleen weight, suggesting cardiac hypertrophy, steatohepatitis, and systemic inflammation, as described in humans.
Previous data have shown vascular dysfunction in conduit and small arteries in patients with metabolic syndrome.21,22 Similarly, we demonstrated vascular remodeling and endothelial dysfunction in NZO mice. Increased ROS production is involved in the mechanisms leading to vascular disease, reducing NO bioavailability and, therefore, promoting endothelial dysfunction, vasoconstriction, and increased vascular resistance. Indeed, vascular ·O2− production was increased in NZO mice compared with controls, and endothelium-dependent relaxation was impaired and was unaffected by inhibition of eNOS, indicating reduced bioavailability of NO. Moreover, endothelial dysfunction was improved by pretreatment with an antioxidant. This suggests a prominent role for ROS in the decreased NO bioavailability. In the vascular system of different models of hypertension and diabetes mellitus, ROS have been shown to be produced by different enzymatic sources, NADPH oxidase and uncoupled eNOS being the most important.23,24 Vascular NADPH did not contribute to increased ROS formation in NZO mice, suggesting different mechanisms of oxidative stress in metabolic syndrome compared with hypertension and diabetes mellitus. The dimer:monomer ratio is an indirect marker of eNOS uncoupling, because the normal function of eNOS requires its dimerization.25 We demonstrated eNOS uncoupling in our model of metabolic syndrome, suggesting its role as a source of vascular oxidative stress, in particular, ·O2− and peroxynitrite, which result from the reaction between NO and ·O2−. Recently, oral supplementation with tetrahydrobiopterin, an essential cofactor of eNOS, has been shown to lead to improvement of endothelial dysfunction in fructose-fed rats, a model of metabolic syndrome.26 However, an inverse relationship was shown recently between plasma and vascular biopterins in patients with coronary artery disease, raising concerns as to their role as biomarkers.27 Thus, it will be of interest in the future to address the role of tetrahydrobiopterin in this model and to investigate its potential as a therapeutic and diagnostic tool. Taken together, these findings suggest that uncoupling of eNOS is a major mechanism of oxidative stress in the metabolic syndrome, although we cannot exclude the contribution of other vascular sources of oxidative stress, such as xanthine oxidase, cyclooxygenase, and mitochondria, which deserve future study.
NZO mice exhibited hypertrophic vascular remodeling of both aorta and mesenteric resistance arteries. Eutrophic remodeling results from persistent vasoconstriction and extracellular matrix deposition, in the absence of growth response, whereas hypertrophic remodeling implies vascular smooth muscle cell growth and proliferation.28 In hypertension, reduction of oxidative stress has been shown to prevent hypertrophic remodeling,6 which suggests a key role for oxidative stress in vascular growth. Therefore, hypertrophic vascular remodeling in NZO mice is possibly because of both increased vasoconstriction and vascular smooth muscle cell proliferation and growth. Vascular oxidant mechanisms are likely to have contributed to remodeling of resistance arteries in NZO mice beyond the hemodynamic effects of BP increase.
Perivascular fat has been shown to regulate vascular tone through release of vasoactive mediators.10 Impairment of this ability may have detrimental effects on the vascular system by increasing vascular tone in resistance arteries leading to enhanced peripheral resistance and, consequently, BP elevation. We demonstrated that, in NZO mice, the physiological anticontractile properties of PVAT were abolished. Two mechanisms are known to mediate the vasoactive effect of PVAT: an endothelium-dependent pathway involving increase of NO release and an endothelial-independent pathway involving release of H2O2.29 In NZO mice, we suggest the presence of an impaired regulation of H2O2 production in PVAT, as a consequence of increased ·O2− production and decreased expression of ·O2− dismutase, which catalyzes the dismutation of ·O2− in H2O2. In vascular disease, ·O2− has been widely studied for its ability to directly attenuate the biological activity of NO. However, the short half-life and radius of diffusion of ·O2− drastically limit the role of this ROS as an important paracrine hormone in vascular biology. However, its metabolite, H2O2, is able to exert a paracrine vasoactive effect in the vasculature. Vascular H2O2 has been shown to elicit vasoconstriction in hypertension and cardiovascular disease, whereas PVAT-derived H2O2 seems to have vasorelaxing properties.7,29,30 Other than this controversy probably related to the different models and vascular beds studied, we found blunted ·O2− dismutase expression in PVAT of NZO mice, which could be an expression of impaired regulation of H2O2 production that then modulates vascular tone of resistance arteries in this model of metabolic syndrome.
Inflammation has been shown recently to play a central role in the vasoactive properties of the perivascular fat.21 We showed the presence of hypertrophy of adipose tissue in the visceral, subcutaneous, and perivascular compartments, and PVAT in NZO mice was characterized by enhanced homing of macrophages and increased oxidative stress. Recent findings have shown that PVAT and adventitia are critical sources of vascular inflammatory cells, which, through NADPH oxidase–dependent ·O2− production, play a role in the pathogenesis of hypertension and vascular remodeling.11,12 However, the pathophysiological mechanism by which PVAT and adventitia may mediate vascular dysfunction has not been clarified. The decreased release of adiponectin by adipocytes, induced in inflammatory pathophysiological conditions, such as metabolic syndrome, has been suggested as a potential mediator.21,31 Indeed, adiponectin was decreased in PVAT in the present model of metabolic syndrome. Another possible mechanism is that PVAT may represent a reservoir of inflammatory cells, which may migrate into the vascular wall and decrease NO bioavailability through paracrine release of ROS. Indeed, in NZO mice, we found increased vascular expression of adhesion molecules and MCP-1 (monocyte chemotactic peptide-1), which may exert a chemotactic effect, thereby inducing inflammatory cell infiltration into perivascular tissue.32 As well, inflammatory cells in the PVAT may represent a source of inducible NO synthase, which could contribute to some of the vascular pathophysiological mechanisms. Thus, it is possible to imagine that in this model of PVAT may play a role in the pathogenesis of BP increase, although the mechanisms by which perivascular adipose inflammation and oxidative stress may affect endothelial function and NO bioavailability need further investigation. In vivo approaches are required to achieve an effective manipulation of the PVAT in pathophysiological conditions, which would add insights on the mechanistic tie-in among perivascular adipose inflammation, oxidative stress, and vascular pathophysiology.
The present study characterizes vascular dysfunction in a rodent model of the human metabolic syndrome. The availability of animal models that present the complexity of human disease and the association of different cardiovascular risk factors, as in the metabolic syndrome, allow the study of some of the different pathophysiological mechanisms involved. The NZO mouse exhibited endothelial dysfunction attributed to eNOS uncoupling and increased ROS production. PVAT promoted vascular dysfunction, through decreased anticontractile effects, which may contribute to increase vascular tone in resistance arteries and BP values. However, the pathogenic role of adipose tissue needs to be further investigated to clearly establish the contribution of perivascular adipose proinflammatory and pro-oxidant mechanisms in vascular pathophysiology. The availability of a reliable animal model, such as the NZO mouse, may allow for future investigation and development of new therapies for subjects presenting the metabolic syndrome.
Sources of Funding
The work of the authors was supported by a medical school grant from Merck-Frosst Canada, by the Canadian Institutes of Health Research grant 82790, a Canada Research Chair on Hypertension and Vascular Research from the Canada Research Chair Program of the Government of Canada, and the Canada Fund for Innovation (all to E.L.S.). T.E. received a fellowship from the Heart and Stroke Foundation of Canada.
C.M. and T.E. contributed equally to this article.
- Received June 24, 2009.
- Revision received July 15, 2009.
- Accepted September 14, 2009.
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