Flow-Induced Remodeling in Resistance Arteries From Obese Zucker Rats Is Associated With Endothelial Dysfunction
Chronic increases in blood flow increase arterial diameter and NO-dependent dilation in resistance arteries. Because endothelial dysfunction accompanies metabolic syndrome, we hypothesized that flow-mediated remodeling might be impaired in obese rat resistance arteries. Obese and lean Zucker rat mesenteric resistance arteries were exposed to chronic flow increases through arterial ligation in vivo: arteries exposed to high flow were compared with normal flow arteries. Diameter was measured in vitro in cannulated arteries using pressure arteriography. After 7 days, outward remodeling (diameter increased from 346±9 to 412±11 μm at 100 mm Hg) occurred in lean high-flow arteries. Endothelium-dependent tone was reduced in high-flow arteries from obese rats by contrast with lean animals. On the other hand, diameter enlargement occurred similarly in the 2 strains. The involvement of NO in endothelium-dependent dilation (evidenced by NO blockade) and endothelial NO synthase phosphorylation was smaller in obese than in lean rats. Superoxide anion and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunit expression (p67phox and gp91phox) increased in obese rats and were higher in high-flow than in control arteries. Acute Tempol (a catalase mimetic), catalase plus superoxide dismutase, and l-arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat normal and high-flow arteries to the level found in lean control arteries. Thus, flow-induced remodeling in obese resistance arteries was associated with a reduced endothelium-mediated dilation because of a decreased NO bioavailability and an excessive superoxide production. This dysfunction might have negative consequences in ischemic diseases in patients with obesity or metabolic syndrome.
- resistance arteries
- shear stress
- reactive oxygen species
- metabolic syndrome
The metabolic syndrome is a common health problem. Its incidence and prevalence are increasing in parallel with the enhanced prevalence and incidence of obesity and type 2 diabetes.1,2 This syndrome is not only a metabolic disorder; it is also associated with endothelial dysfunction3 and vascular remodeling leading to a reduction in arterial diameter.4 Patients with metabolic syndrome demonstrate an increasing risk of overall mortality, as well as cardiovascular morbidity and mortality.5
Basically, an increase in blood pressure or a decrease in blood flow induces a transient adjustment in vessel diameter mediated by changes in myogenic tone and by the release of vasoactive substances. On the other hand, increasing blood flow induces a vasodilation mediated by endothelium-derived NO. A long-term exposition to altered mechanical forces induces vascular remodeling to restore tensile and shear stresses.6,7 Long-term increase in shear stress enhances NO production by endothelial cells, and we have shown previously that NO has a key role in vascular remodeling of large8 and resistance arteries in response to an increased blood flow.9
Vascular disorders associated with the metabolic syndrome involve alterations in local blood flow supply.5 Restoring blood flow can be achieved using vasodilator treatments10 or exercise training, which has been shown to induce outward remodeling in resistance arteries, thus improving blood flow.11 Studies in Zucker obese rats, a model of metabolic syndrome, have demonstrated an endothelial dysfunction in several vascular beds12 associated with a reduced NO concentration13 and an increased vascular oxidative stress.12 We used this model to test the hypothesis that the adaptation, or remodeling, of resistance arteries in response to a chronic rise in blood flow would be reduced in obesity. This adaptation involves an increase in diameter and an enhanced endothelium (NO)–dependent dilation as shown in our previous studies.9,14 Thus the questions were whether resistance arteries in obese rat are able to increase their diameter and whether they are able to increase NO-dependent dilation in response to a chronic increase in blood flow. To show this, we used a model described previously in rats6,9,15 and mice.14,16 It is based on ligation of mesenteric arteries, allowing increasing blood flow in one mesenteric artery in vivo. In this model, blood flow increases by 60% in this “high-flow” (HF) artery, which is compared with control (“normal flow” [NF]) arteries located at distance.14,16
Twelve adult male obese Zucker rats and their littermate controls (lean Zuker rats), at 10 weeks old, were purchased from Charles River (L’Arbresles, France). Rats were anesthetized (sodium pentobarbital, 50 mg/kg, IP) and submitted to surgery to modify blood flow as described previously.9 Briefly, 3 consecutive first-order arteries were used. Ligatures were applied to second-order branches (Figure 1A). The artery located between the 2 ligated arteries was designed as an HF artery. Arteries located at distance of the ligated arteries were used as control (NF).
Seven days after surgery, rats were anesthetized (sodium pentobarbital, 50 mg/kg, IP) for blood pressure measurement. They were then euthanized by CO2 inhalation, the gut excised, and mesenteric arteries dissected. From each rat, HF and NF arteries were isolated and divided in several segments used, respectively (from the proximal to the distal part of the artery), for diameter measurement and pharmacological, biochemical, and immunohistological analyses.
The university ethical committee approved the present protocol. The procedure followed in the care and euthanasia of the study animals was in accordance with the European Community Standards on the Care and Use of Laboratory Animals (Ministère de l’Agriculture, France, authorization No. 6422).
Pressure–Diameter Relationship and Flow-Mediated Dilation in NF and HF Arteries
A first segment of NF and HF arteries, 4-mm long and ≈300 μm ID, was cannulated at both ends and mounted in a video monitored perfusion system17 as described previously.16 Briefly, flow-mediated dilation (FMD; steps of 3 to 100 μL/min) was determined under a pressure of 75 mm Hg on preconstricted arteries (phenylephrine; 1 μmol/L). After 30 minutes, FMD was repeated in the presence of NG-nitro-l-arginine methyl ester (l-NAME; 100 μmol/L; 20 minutes). Finally, after washout, passive arterial diameter was evaluated in the absence of smooth muscle tone, that is, in the presence of EGTA (2 mmol/L) and sodium nitroprusside (SNP; 0.1 mmol/L) and in the absence of extracellular calcium.16
To determine their cross-sectional area (CSA), arteries were fixed with formaldehyde under a pressure of 75 mm Hg. CSA was determined as described previously.16
Pharmacological Profile of Isolated NF and HF Arteries
Two other arterial segments (2-mm long each) were dissected and mounted in a wire myograph (DMT).16 Cumulative concentration–response curves to phenylephrine (0.01 to 10 μmol/L) and SNP (0.001 to 10 μmol/L) were performed. In other arterial segments, 2 concentration–response curve to acetylcholine (ACh; 0.001 to 10 μmol/L) were obtained before and after incubation with one of the following agents: l-NAME (100 μmol/L), the catalase mimetic Tempol (10 μmol/L), superoxide dismutase (120 U/mL) plus catalase (80 U/mL), or tetrahydrobiopterin (10 μmol/L) plus l-arginine (100 μmol/L). SNP- and ACh-dependent dilation was performed in arteries precontracted with phenylephrine to 50% of their maximal contractile response.
Western Blot Analysis
As described previously,9 arteries were homogenized, and proteins (25 μg of total protein from each sample) were separated by SDS-PAGE using a 4% stacking gel followed by a 10% running gel. Proteins were detected with specific antibodies (Transduction Laboratories) against endothelial NO synthase (eNOS) 1:1000, phospho-eNOS 1:500, and subunits of reduced nicotinamide-adenine dinucleotide phosphate oxidase (gp91phox and p67phox) actin 1:1000 (dilutions in Tris-buffered saline Tween 20). Protein expression was visualized using the ECL-Plus chemiluminescence kit (Amersham) and normalized by actin expression.
Detection of Superoxide Anions
Another arterial segment was embedded vertically in Tissue-tek (Sakura) and frozen in isopentane. Superoxide detection was performed on transverse cross-sections 7-μm thick incubated with dihydroethydine (DHE), as described previously.18 Positive staining was visualized using confocal microscopy and QED-Image software (Solamere Technology). Image analysis was performed using Histolab (Microvision). Pixel quantification was executed as described previously.9 A positive control experiment was performed using sections of mesenteric artery treated for 2 hours with lipopolysaccharide in vitro at 37°C in a physiological salt solution. A negative control was obtained by omitting DHE or by adding Tempol to the section 15 minutes before DHE.
Results are expressed as mean±SEM. Significance of the difference between arteries was determined by ANOVA (1-factor ANOVA or ANOVA for consecutive measurements for the comparison of concentration–response curves). Means were compared by paired t test or by the Bonferroni test for multigroup comparisons. Values of P<0.05 were considered to be significant.
Mean arterial blood pressure was significantly higher in obese (102±2 mm Hg; n=12) versus lean rats (91±2 mm Hg; n=10). Similarly, body weight was significantly higher in obese versus lean rats (391±16 versus 322±7 g; n=12 per group).
After 7 days of ligation, NF and HF artery passive diameters were determined in vitro in response to intraluminal pressure ranging from 10 to 150 mm Hg. Passive diameter was significantly higher in HF than in NF arteries in lean rats (412±11 versus 346±9 μm; n=12; for a pressure of 100 mm Hg; P<0.05). This diameter enlargement occurred similarly in obese rats (348±14 versus 294±11 μm; n=12 per group; P<0.05; pressure was 100 mm Hg; Figure 1B shows diameter measurements for pressure values ranging from 10 to 150 mm Hg). Arterial diameter was significantly smaller in obese than in lean rats for both NF and HF arteries (Figure 1B). Passive diameter in NF and HF rats was significantly lower in obese versus lean rats. The ligation also induced an increase of CSA in HF arteries, which was significant in lean rats only. CSA was significantly higher in obese HF arteries than in lean HF arteries (Figure 1C).
In both NF and HF arteries, ACh-induced dilation was significantly lower in obese versus lean rats. In obese rats, ACh-induced dilation was significantly lower in HF than in NF arteries (Figure 2A). ACh-induced dilation was similar in all of the groups after NO synthase inhibition with l-NAME (Figure 2B and 2C). The expression level of eNOS was higher in HF than in NF arteries in both lean and obese rats (Figure 2D). Nevertheless, eNOS phosphorylation was not significantly higher in HF than in NF arteries (Figure 2E). The ratio of phospho-eNOS/eNOS was significantly reduced in obese rats compared with lean rats in NF and HF arteries. In addition, it was lower in HF arteries compared with NF arteries in obese rats (Figure 2F).
Superoxide formation, evaluated by ethidium bromide–enhanced fluorescence (Figure 3A), was significantly increased in HF compared with NF arteries. Moreover, in obese rats, superoxide levels were significantly higher than in lean rats in both HF and NF arteries (Figure 3B). Positive and negative control experiments confirmed that DHE staining is efficient in sections of arterial segments (Figure S1, available at http://hyper.ahajournals.org). Expression levels of the reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits gp91phox and p67phox were higher in obese than in lean rats in both NF and HF arteries (Figure 3C and 3D). In addition, they were augmented in all of the HF arteries compared with NF arteries.
The relation between excess reactive oxygen species (ROS) production and endothelial dysfunction was confirmed using Tempol (Figure 3E) acutely or the combination of superoxide dismutase plus catalase (Figure 3F). In these conditions, ACh-induced dilation was no longer different between HF and NF arteries, as well as between obese and lean rats (compared with Figure 2A). Similarly, the combination of l-arginine plus tetrahydrobiopterin restored endothelium-dependent dilation in obese rat NF and HF arteries to the control level (lean NF arteries; Figure 3G). Tempol, in the addition of l-arginine plus tetrahydrobiopterin, had no additional effect on endothelium-dependent dilation than l-arginine plus tetrahydrobiopterin alone (Figure 3H).
Phenylephrine- and KCl-induced contraction, as well as SNP-induced dilation, was similar in obese and lean rats. Similarly, they were not significantly affected by the chronic changes in blood flow (Figure S2).
In lean rats, FMD was significantly higher in HF than in NF arteries. In obese rats, no difference in FMD was observed between the NF and HF arteries (Figure S3A). In both NF and HF arteries, FMD was lower in obese than in lean rats. l-NAME reduced FMD in both groups. In lean rats, the attenuation of FMD by l-NAME was significantly higher in HF than in NF arteries, whereas, in obese rats, FMD attenuation by l-NAME was similar in NF and HF arteries. Moreover, in HF and NF arteries, the effect of l-NAME was lower than in the corresponding arteries in lean rats (Figure S3B). Arterial diameter before dilating arteries with flow was similar in the 4 groups of arteries (n=12 per group): 158±26 μm (lean NF), 153±26 μm (lean HF), 163±21 μm (obese NF), and 161±22 μm (obese HF).
We found in obese Zucker rats that a chronic increase in blood flow in resistance arteries induced a diameter enlargement associated with a reduced vasorelaxing capacity of the endothelium by contrast with lean rats in which remodeling was associated with an increased vasorelaxing capacity of the endothelium. The reduced NO-dependent dilation found in obese rats was most likely the consequence of an excessive ROS production.
The obese Zucker rat is a model of metabolic syndrome.19 Arterial diameter was smaller in obese than in lean rats (Figure 1). This is in agreement with previous studies performed in obese rat skeletal muscle arteries.20,21 In addition, we found that endothelium-dependent dilation was lower in obese rats compared with lean animals (Figure 2). Indeed, both ACh and FMD were decreased in obese versus lean rats (Figures 2 and S3). This might reflect a reduced involvement of NO, because l-NAME was less efficient in obese than in lean rats (Figure S3B), though this issue remains to be confirmed, because no direct measurement of NO production was performed. Nevertheless, we found that eNOS expression and phosphorylation were reduced in obese compared with lean rats. Furthermore, the ratio of phospho-eNOS/eNOS was reduced in obese rats (Figure 2). This ratio is an index of eNOS activity, especially when eNOS expression level is different among groups.22 Our observations are in agreement with previous studies demonstrating an endothelial dysfunction in obese Zucker rat, especially in the NO pathway.20,23 An excessive ROS production might also cause this low NO bioavailability. This was evidenced by a rise in DHE staining and NADPH oxidase (gp91 and p67) expression in mesenteric arteries, as well as by the restoration of ACh-induced dilation by antioxidant treatments, as shown in Figure 3. This is in agreement with previous studies performed in gracilis muscle arteries isolated from high- glucose–fed rats18 and in mice with type 2 diabetes (db/db mice)24 in which NO-dependent dilation is reduced because of an excessive ROS production. Interestingly, we found that tetrahydrobiopterin added to the bath of obese NF and HF arteries also restored ACh-induced dilation to the control level. Thus, the endothelial disorder found in obese rats was not only because of an excessive production of ROS, but these superoxide anions might, at least in part, provide from eNOS uncoupling as described in diabetic mice resistance arteries.18 As described previously in a review article,25 in hypercholesterolemia and diabetes, eNOS itself can produce ROS when its cofactor tetrahydrobiopterin availability is reduced.
The endothelium dysfunction found in obese rats selectively affected the endothelium, because we found that SNP, a direct NO donor, induced similar relaxation in obese and in lean rats (Figure S2), indicating that the responsiveness of smooth muscle cells to NO was not affected. This is in agreement with a previous study performed in large arteries.26 Similarly, phenylephrine-induced contraction was similar in obese and lean rats, suggesting that smooth muscle cell contractility was also preserved.
The endothelial dysfunction observed in obese rats led us to investigate the capacity of resistance arteries to adapt in response to a chronic increase in blood flow. This adaptation or remodeling involves a rise in diameter associated with an increased endothelium (NO)-dependent dilation.9,16
First, flow-induced outward remodeling occurred in the same proportion in obese and lean rats (Figure 1B). The diameter enlargement, which we found in both groups, was equivalent to that found in Wistar rat mesenteric arteries.6,7,9,27 However, the increase in media CSA accompanying the diameter enlargement in lean rats was not found in obese animals (Figure 1C). This might be the consequence of the pre-existing arterial wall hypertrophy observed in obese rat mesenteric arteries. A similar arterial wall hypertrophy has been described in a previous study performed in skeletal muscle arteries.21 The diameter enlargement induced by the chronic rise in blood flow depends on the production of NO9 and ROS.28 In agreement with these studies, we observed an increase in eNOS expression and NO efficacy (Figure 2), as well as a rise in ROS production (Figure 3) in HF arteries in lean rats. In obese rats, the rise in ROS production was larger and the NO bioavailability was lower than in lean rats. Nevertheless, the diameter enlargement was equivalent in obese and lean rats. Although the rise in ROS and the decrease in NO bioavailability are probably linked,18 the rise in diameter induced by flow may occur in conditions where the production of NO is reduced. In a previous study,29 a partial NO blockade with l-NAME failed to prevent flow-induced remodeling in rats, whereas we have shown that a higher dose of l-NAME, or the absence of eNOS, prevented the diameter enlargement.9 On the other hand, that the excessive ROS production found in obese rats did not oppose the remodeling is in agreement with a previous study showing that ROS play a key role in flow-mediated remodeling (diameter enlargement) in the carotid artery.28 This latter study has shown that NO and ROS associate to form peroxynitrites, which then activate matrix metalloproteinases and arterial remodeling. Nevertheless, in resistance arteries, the involvement of ROS and peroxynitrites has not yet been shown directly and, thus, remains to be demonstrated.
Second, we found that the chronic increase in blood flow induced a larger endothelium dysfunction in obese rat HF arteries than in obese NF arteries. By contrast, in lean rats, endothelium-dependent dilation to ACh and flow was higher in HF than in NF arteries. Thus, as compared with lean arteries, obese HF arteries presented a strong endothelial dysfunction. Because smooth muscle cells were intact in obese rats (no change in response to phenylephrine or SNP; Figure S2), this reduced NO-dependent dilation was probably because of an increased inactivation of NO. It is most likely that this is the consequence of the excessive ROS production found in these arteries (Figure 3). We found that the increase in superoxide generation and the expression level of gp91 and p67 was higher in obese HF arteries than in lean HF arteries. This excessive superoxide production might explain the altered NO-dependent dilation. This was confirmed by the restoration of ACh-induced dilation in obese HF arteries by Tempol and by catalase plus superoxide dismutase (Figure 3). Such an antioxidant treatment totally suppressed the differences between lean and obese rats and between HF and NF arteries (Figure 3). In addition, we found that tetrahydrobiopterin added to the bath of obese HF arteries restored dilation to the level found in lean arteries. Tempol added to the bath in addition to tetrahydrobiopterin had no additional effect, suggesting that a large part of the endothelial dysfunction was because of eNOS uncoupling as described previously in diabetes.18 Thus, the rise in ROS production accompanying flow-induced remodeling (diameter enlargement plus increased endothelium-dependent dilation) had a negative impact on NO-mediated dilation in obese rats.
The current study predicts impaired microvascular regulation and structural abnormality during metabolic syndrome. Although a “structural” remodeling occurred after increasing chronically blood flow, the arteries presented a reduced diameter and an exaggerated alteration in endothelium-dependent dilation. This could support elevated vascular resistance, moderate hypertension, or limited oxygenation of host organs. In addition, the endothelial alteration after chronic rise in blood flow might have negative consequences in the long term when patients suffering from metabolic syndrome are recommended to practice exercise or are given vasodilator treatments to improve blood flow.10,11
In conclusion, we identified an endothelial dysfunction occurring after high blood flow–induced remodeling in addition to the pre-existing endothelial dysfunction characterizing the obese. This finding might be important to consider in the treatment of obese patients suffering vascular disorders. Treatments improving endothelium-dependent dilation, such as angiotensin I–converting enzyme inhibitors,30 might be preferred for the treatment of cardiovascular diseases, such as hypertension, in obese subjects.
We thank the local animal care unit of the University of Angers and Jérôme Roux, Pierre Legras, and Dominique Gilbert for their kind help in treating the rats.
Sources of Funding
C.B. received a studentship from the Research and Development/Canadian Institute of Health and Research. P.M. is a scholar from the Fonds de Recherche en Santé du Québec. Centre National de la Recherche Scientifique, INSERM, University of Angers, supported this work.
C.B. and E.B.d.C. contributed equally to this article.
- Received February 9, 2007.
- Revision received February 27, 2007.
- Accepted April 26, 2007.
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