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Type 2 diabetes severely impairs structural and functional adaptation of rat resistance arteries to chronic changes in blood flow

Eric J. Belin de Chantemèle , Emilie Vessières , Anne-Laure Guihot , Bertrand Toutain , Maud Maquignau , Laurent Loufrani , Daniel Henrion
DOI: http://dx.doi.org/10.1093/cvr/cvn334 788-796 First published online: 2 December 2008

Abstract

Aims Endothelial dysfunction in resistance arteries (RAs) leads to end-organ damage in type 2 diabetes. Remodelling of RAs in response to chronic increases in blood flow depends on the integrity of the endothelium. Since type 2 diabetes impairs endothelial sensitivity to flow and increases oxidative stress, we hypothesized that flow-induced remodelling in RAs would be impaired in diabetes. Thus, we studied the structural and functional adaptation of RAs from Zucker diabetic fatty (ZDF) and lean Zucker (LZ) rats to chronic changes in flow.

Methods and results Mesenteric RAs were alternatively ligated so that one artery was submitted to high flow (HF) and compared with normal-flow (NF) arteries located at distance. After 3 weeks, arteries were studied in vitro (n = 10 rats per group). Arterial diameter (468 vs. 394 ± 8 µm) and endothelial (acetylcholine)-dependent dilation (91 ± 8 vs. 75 ± 6% dilation) were higher in HF than in NF arteries in LZ rats. In ZDF rats, diameter (396 ± 9 vs. 440 ± 17 µm) and acetylcholine-mediated dilation (42 ± 8 vs. 75 ± 7%) were lower in HF than in NF arteries. Nevertheless, endothelial NO synthase and NADP(H) oxidase subunits (gp91, p67) expression level and superoxide production (dihydroethidium staining) were higher in HF than in NF arteries in both strains, suggesting an efficient flow-sensing process in ZDF rats. In ZDF rats, basal oxidative stress was higher compared with LZ rats: dihydroethidium staining was higher in NF and HF arteries from ZDF rats, and acetylcholine-mediated dilation was improved by an acute antioxidant (tempol) in NF and HF arteries from ZDF rats. Thus, superoxide overproduction in ZDF rats impaired NO-dependent dilation and HF remodelling. Indeed, a chronic treatment with tempol increased HF artery diameter and endothelium-dependent dilation in ZDF rats.

Conclusion In type 2 diabetic rats, a chronic increase in blood flow failed to induce outward remodelling and to improve endothelium-dependent dilation, mainly because of superoxide overproduction.

KEYWORDS
  • Blood flow
  • Shear stress
  • Resistance arteries
  • Type 2 diabetes
  • Zucker diabetic fatty rats
  • Endothelium
  • Nitric oxide
  • Reactive oxygen species

1. Introduction

Type 2 diabetes is the most frequently encountered metabolic disorder, currently affecting 5–10% of the population.1 Associated with obesity, type 2 diabetes is characterized by an insulin resistance inducing several metabolic changes, including hyperinsulinaemia, hyperglycaemia, dyslipidaemia, and hypertension, all leading to an increased risk of cardiovascular events.2 The morbidity and mortality associated with type 2 diabetes are essentially related to the vascular lesions that develop over time in this condition.3 The microcirculation is mainly involved, and as a consequence, vital organs are damaged. Although the consequences of type 2 diabetes on large elastic arteries4,5 have been extensively studied, less is known about its effects on the microcirculation. In addition, no study has yet investigated the effects of type 2 diabetes on the ability of resistance arteries to adapt their structure and function in response to a chronic rise in blood flow.

The primary function of the microcirculation is to optimize nutrient and oxygen supply within tissues in response to the metabolic demand. For this purpose, resistance arteries are able to adapt their diameter in response to mechanical stimuli such as pressure and flow (shear stress). Mechanisms involved are, respectively, myogenic tone in response to pressure and flow-mediated dilation (FMD).6 Furthermore, resistance arteries are able to adapt to chronic increases in blood flow, leading to diameter enlargement (outward remodelling) and higher endothelium (NO)-dependent relaxation.710 This remodelling is involved in response to an increase in the metabolic demand of different tissues, in either pathological conditions such as diabetes or in growth, following exercise training or during pregnancy. The production of NO by the endothelium and the activation of matrix metalloproteinase (MMP) are required for flow-mediated remodelling, as previously shown in small resistance9 and large elastic arteries.11 In conditions involving a reduced endothelial ability to produce vasodilator agents, such as ageing, increasing chronically local blood flow has been shown to improve endothelium (NO)-dependent dilation. This was associated with reduced reactive oxygen species (ROS) production and improved endothelial nitric oxide synthase (eNOS) expression level and function.12

Type 2 diabetes is associated with an increased ROS production13 that might impair the ability of resistance arteries to adapt their structure and function in response to chronic increases in blood flow because of a decreased NO bioavailability.14 Nevertheless, as ROS, besides NO, are also required for flow-mediated remodelling,15 the effect of ROS overproduction on remodelling cannot be deduced from previous studies. Indeed, our previous study performed in obese Zucker rats has shown that flow-induced remodelling (diameter enlargement) occurred despite a large overweight and a slight hypertension and hyperglycaemia. We are thus hypothesizing that endothelial alteration and oxidative stress might compromise resistance arteries remodelling in type 2 diabetic rats.

To verify this hypothesis, we used a model of ligature of the mesenteric bed1618 allowing the comparison of resistance arteries chronically submitted to high or normal blood flow levels, in the same physiological conditions. We used Zucker diabetic fatty (ZDF) rats treated or not with the antioxidant tempol, hypothesizing that coupling the chronic rise in blood flow to a reduction in ROS level will improve the ability of resistance artery to respond to chronic changes in blood flow.

2. Methods

2.1 Animals

Twenty adult male ZDF and 20 lean Zucker (LZ) rats, 12–14 weeks old (Charles River), were anesthetized (isoflurane, 2.5%) and submitted to surgery in order to modify blood flow as described previously.9,17,19 Briefly, three consecutive first-order arteries were used. Ligatures (7-0 silk surgical thread) were applied to second-order branches of the first and third arteries (Figure 1A). The artery located between two ligated arteries was designed as high-flow (HF) artery. Other arteries located at distance were used as control (normal flow, NF) arteries. Half of the rats were simultaneously treated with 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl (tempol, 10 mg/kg per day).

Figure 1

Drawing representing the model used to increase blood flow chronically. (A) Second-order mesenteric arteries were ligated in order to increase flow in the middle artery (HF, high flow). Arteries located at distance are used as control or ‘normal flow’ (NF) arteries. The rationale of the study is present below. The production of reactive oxygen species (ROS) due to diabetes might affect the increase in diameter and the improvement of endothelium-dependent dilation induced by the chronic rise in blood flow. Changes in diameter (B) and media cross-sectional area (C) in responses to stepwise increases in pressure in high-flow and normal-flow mesenteric resistance arteries isolated from control (LZ, lean Zucker) and diabetic rats (ZDF, Zucker diabetic fatty). Mean ± SEM is presented (n = 10 per group). *P < 0.01, ZDF vs. LZ. P < 0.05, NF-LZ vs. NF-ZDF for a pressure of 75 mmHg.

Before sacrifice, glycaemia was quantified on a sample of arterial blood with a glucometer.10

Twenty-one days after surgery, animals were anesthetized (isoflurane, 2.5%). The right femoral artery was catheterized for blood pressure measurement.20 Animals were then sacrificed by CO2 inhalation, the gut excised, and mesenteric arteries gently dissected and divided in several segments used as described in what follows.

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) and with the Principles of Laboratory Animal Care (NIH publication no. 85–23, revised 1985; http://grants1.nih.gov/grants/olaw/references/phspol.htm).

2.2 Pressure-diameter relationship and flow-dependent dilation in normal-flow and high-flow arteries

A first arterial segment was cannulated at both ends and mounted on a video-monitored perfusion system.21 Briefly, arterial segments were bathed in a physiological salt solution (PSS) and arterial diameter measured continuously (LSI, Burlington, VT, USA).

Arteries were then submitted to 75 mmHg of pressure and contracted with phenylephrine. Intraluminal flow was subsequently increased by step in order to induce FMD. After 30 min of recovery, FMD was repeated in the presence of N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 µmol/L, 20 min). Finally, arteries were bathed in a Ca2+-free PSS containing EGTA (2 mmol/L) and sodium nitroprusside (SNP, 10 µmol/L). Arteries were then submitted to a stepwise increase in pressure in order to determine the passive diameter, i.e. in the absence of tone. Flow-induced dilation was expressed as per cent dilation of the precontraction.22

2.3 Pharmacological profile of isolated normal-flow and high-flow arteries

Arterial segments were mounted on a wire-myograph (DMT, Aarhus, Denmark).23 Briefly, two tungsten wires (25 µm diameter) were inserted into the lumen of the arteries and fixed to a force transducer and a micrometer, respectively. Arteries were bathed in a PSS and wall tension was applied as described previously.24 Arteries viability was tested using a potassium-rich solution (KCl, 80 mmol/L). A cumulative concentration–response curve (CRC) to ACh was then performed. Thirty minutes after washout, a second CRC to ACh was repeated in the presence of L-NAME (100 µmol/L, 20 min), indomethacin (10 µmol/L), or tempol (10 µmol/L).

2.4 Histology

At the end of the functional analysis on the arteriograph, the artery was bathed in Ca2+-free PSS containing 10 µmol/L SNP. Pressure was set at 75 mmHg, and the artery was fixed in a 4% buffered formaldehyde solution as described previously.10 Sections (7 µm thick) were stained with orcein. External diameter, lumen diameter, and media thickness were determined and analysed (Histolab; Microvision, France) for cross-sectional area calculation as described previously.25

2.5 Western blot analysis

The remaining NF arteries of each mesenteric vascular bed were pooled and then homogenized. Proteins (25 µg 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, eNOS 1:1000, Cav-1 1:4000, p67 and gp91 and actin 1:1000 in T–TBS–BSA 5%). Protein expression was visualized using the ECL-Plus Chemiluminescence kit (Amersham).26

2.6 Detection of reactive oxygen species using confocal microscopy

Other NF and HF arterial segments were embedded vertically in Tissue-tek (Sakura) and frozen in isopentane. ROS detection was performed on transverse cross-sections 7 µm thick incubated with dihydroethidium (DHE) as described previously.10 Briefly, DHE, in the presence of superoxide, is oxidized to fluorescent ethidium bromide. Ethidium bromide is trapped by intercalation with DNA, and the number of fluorescent nuclei indicates the relative level of superoxide production. Positive staining was visualized using confocal microscopy and QED-image software (Solamere, Salt Lake City, UT, USA).10,25

2.7 Statistical analysis

Results are expressed as means ± SEM. Significance of the difference between arteries was determined by ANOVA (one-factor ANOVA or ANOVA for consecutive measurements, when appropriate). 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.

3. Results

3.1 Physiological parameters

In order to determine the metabolic status of the LZ and ZDF rats, we measured their body weight, glycaemia, and blood pressure. Rat body weight was significantly higher in ZDF than in LZ rats (441 ± 17 vs. 325 ± 11 g, P < 0.05) and was not significantly affected by the chronic treatment with tempol in both ZDF (428 ± 20 vs. 441 ± 17 g) and LZ rats (312 ± 14 vs. 325 ± 11 g).

Similarly, blood glucose was significantly enhanced in ZDF compared with LZ rats (302 ± 23 vs. 112 ± 13 mg/dL). Blood glucose was not modified by the chronic treatment with tempol in both LZ (117 ± 10 mg/dL) and ZDF rats (298 ± 20 mg/dL).

Type 2 diabetes significantly increased mean blood pressure (92 ± 2 mmHg in LZ vs. 111 ± 2 mmHg in ZDF, P < 0.05). However, mean blood pressure was not significantly affected by the chronic treatment with tempol, in both the LZ (94 ± 3 mmHg) and ZDF (113 ± 2 mmHg) rats.

3.2 Arterial diameter and structure

Passive arterial diameter in HF and NF arteries was determined in order to assess the arterial remodelling induced by the chronic increase in blood flow. In LZ rats, HF arteries diameter was significantly higher than in NF arteries (Figure 1B). In contrast, in ZDF rats, HF arteries diameter did not increase compared with NF arteries. In addition, it was significantly lower than in NF arteries for an intraluminal pressure of 75 mmHg (P < 0.05). Thus arterial outward remodelling did not occur in ZDF rats. The chronic rise in blood flow significantly increased the media cross-sectional area of HF arteries from the LZ rats only. Nevertheless, type 2 diabetes induced an increase in the media cross-sectional area of the NF artery. Furthermore, no difference in media cross-sectional area was observed between NF and HF arteries from ZDF rats. (Figure 1C).

3.3 Endothelium-dependent relaxation

The vasodilator function of the endothelium was assessed by measuring the response of NF and HF arteries to stepwise increases in flow and to increasing concentrations of ACh. In LZ rats, FMD (Figure 2A) and ACh-induced relaxation (Figure 2B) were significantly higher in HF than in NF arteries. In contrast, in ZDF rats, increasing blood flow chronically reduced endothelium-dependent dilation as evidenced by a decrease in FMD (Figure 2A) and ACh-induced relaxation in HF compared with NF arteries (Figure 2B). As a control, endothelium-independent dilation in response to sodium-nitroprusside was performed in the same arteries. This dilation was not affected by the chronic rise in blood flow (no difference between NF and HF arteries) or by diabetes (no significant change between LZ and ZDF rats) (Figure 2C). On the other hand, the contraction induced by phenylephrine was higher in ZDF than in LZ rats and higher in HF than in NF arteries (Figure 2D).

Figure 2

Flow (A), acetylcholine (B), and sodium nitroprusside (C)-mediated dilation obtained in high-flow (HF) and normal-flow (NF) mesenteric resistance arteries isolated from lean Zucker (LZ) and Zucker diabetic fatty (ZDF) rats. (D) Phenylephrine-induced contraction in the same arteries. (E) Level of pre-contraction obtained in arteries of the different groups before the application of flow, acetylcholine, or sodium nitroprusside. Mean ± SEM is presented (n = 10 per group). #P < 0.01, Zucker diabetic fatty vs. lean Zucker rats within normal-flow or high-flow groups. *P < 0.01, high flow vs. normal flow within each group.

The pre-contraction of NF and HF arteries before inducing dilation with flow, Ach, or SNP was not significantly different among groups (Figure 2E).

3.4 Effect of acute endothelial nitric oxide synthase and cyclooxygenase blockade on endothelium-dependent dilation

To determine the role played by eNOS and NO in endothelium-dependent dilation, CRCs to ACh were performed in the presence of the eNOS inhibitor L-NAME. In LZ rats, L-NAME significantly suppressed ACh-induced relaxation in HF arteries, whereas it reduced, but not suppressed, the ACh-mediated relaxation in NF arteries (Figure 3A). In ZDF rats, L-NAME significantly suppressed ACh-induced relaxation in NF arteries but not in HF arteries. In HF arteries, ACh-induced dilation was reduced by half for the three highest concentrations, whereas it was not significantly affected with the lowest concentrations (Figure 3B). After the addition of indomethacin, no significant dilation could be detected in response to ACh in LZ rats NF arteries, whereas the dilation was improved by indomethacin in ZDF rats NF arteries. Indomethacin had no further effect in HF arteries isolated from either LZ or ZDF rats (Figure 3A and B).

Figure 3

Effects of N(omega)-nitro-l-arginine methyl ester (L-NAME) (100 µmol/L, LN) and indomethacin (10 µmol/L, indo) on acetylcholine-induced dilation in high-flow (HF) and normal-flow (NF) mesenteric resistance arteries isolated from lean Zucker (LZ) (A) and Zucker diabetic fatty (ZDF) rats (B). (C) Endothelial nitric oxide synthase (eNOS) and caveolin-1 (CAV-1) expression levels measured in high-flow (HF) and normal-flow arteries from lean Zucker and Zucker diabetic fatty rats. A representative blot obtained with arteries isolated from a Zucker diabetic fatty and a lean Zucker rat is represented on the right side of the bar graph. Mean ± SEM is presented (n = 10 per group). #P < 0.01, Zucker diabetic fatty vs. lean Zucker rats within high-flow and normal-flow groups. *P < 0.01, high flow vs. normal flow in each group.

3.5 Caveolin-1 and endothelial nitric oxide synthase protein expression levels

As shown previously by our group,9,10 eNOS and caveolin-1 expression levels increased in HF arteries compared with NF vessels (Figure 3C). There was no significant difference in expression levels between LZ and ZDF rats, suggesting that a change in eNOS or caveolin-1 expression level cannot explain the changes in NO-dependent dilation.

3.6 Effect of acute reactive oxygen species reduction on endothelium-dependent dilation

In order to test the effect of ROS on NO-dependent dilation, we measured the acute effect of the antioxidant tempol on ACh-induced relaxation (Figure 4A and B). In LZ rats, the treatment with tempol did not affect ACh-induced dilation in NF or HF arteries (Figure 4A). On the other hand, in ZDF rats, tempol increased significantly ACh-induced relaxation in both NF and HF arteries (Figure 4B).

Figure 4

Effect of acute reactive oxygen species (ROS) reduction (acute tempol infusion) on acetylcholine-induced dilation in high-flow (HF) and normal-flow (NF) mesenteric resistance arteries isolated from lean Zucker (LZ) (A) and Zucker diabetic fatty (ZDF) rats (B). The expression level of the NADP(H)-oxidase subunit gp91 and p67 was measured by western blot (C). A representative blot obtained with arteries isolated from a Zucker diabetic fatty and a lean Zucker rat is represented on the right side of the bar graph. Mean ± SEM is presented (n = 10 per group). &P < 0.01, effect of tempol on acetylcholine-induced dilation. *P < 0.01, high flow vs. normal flow in each group (A and B). #P < 0.01, Zucker diabetic fatty vs. lean Zucker rats within normal-flow or high-flow group (C).

3.7 NADP(H) oxidase (gp91 and p67) protein expression levels

We have shown previously that the NADP(H)-oxidase subunits gp91 and p67 expression levels increase in HF arteries.10 The expression levels of these two proteins increased significantly in HF arteries compared with NF vessels in both LZ and ZDF rats (Figure 4C). In addition, there was a significant increase in gp91 and p67 expression levels in ZDF rats compared with LZ rats in both NF and HF arteries (Figure 4C).

3.8 Effect of a chronic treatment with tempol on flow-induced remodelling

In order to test the effect of oxidative stress on the vascular response to a chronic increase in flow, ZDF rats were chronically treated with tempol. In tempol-treated rats, HF arteries diameter was significantly higher than in non-treated rats (Figure 5A). No effect of tempol was observed on the diameter of NF arteries in ZDF rats (Figure 5A). Cross-sectional area was not significantly affected by tempol (Supplementary material online, Panel A).

Figure 5

Effect of a chronic treatment of Zucker diabetic fatty (ZDF) rats with tempol on passive arterial diameter (A) and acetylcholine-induced dilation (B) in high-flow (HF) and normal-flow (NF) mesenteric resistance arteries. In situ detection of superoxide (ROS) using dihydroethidium (DHE) staining (C) was performed in arteries and quantified (bar graph on the right). In a negative control, dihydroethidium was omitted and a positive control was obtained from a rat treated with lipopolysaccharide. &P < 0.01, effect of chronic tempol. *P < 0.01, high flow vs. normal flow in each group. #P < 0.01, Zucker diabetic fatty vs. lean Zucker (LZ) rats within normal-flow or high-flow group.

3.9 Chronic treatment with tempol restored ACh-induced dilation

In tempol-treated ZDF rats, ACh-induced dilation was similar in HF and NF arteries. In addition, L-NAME totally suppressed ACh-induced dilation in both HF and NF arteries (Figure 5B). In LZ rats treated with tempol, ACh-induced dilation was higher in HF than in NF arteries and totally suppressed by L-NAME (Supplementary material online, Panel E). FMD in tempol-treated rats followed the same pattern (Supplementary material online, Panel F), and eNOS expression level was not significantly affected by the treatment (Supplementary material online, Panel B).

3.10 Effect of a chronic treatment with tempol on reactive oxygen species production

ROS detection using DHE-staining showed a positive ROS level in the NF and HF arteries of the ZDF rats. This staining was significantly higher in HF than in NF arteries. In tempol-treated ZDF rats, no positive nucleus could be detected, similar to negative-control arteries (DHE omitted). On the other hand, in a positive-control artery (isolated from an LPS-treated rat), the majority of the nuclei were fluorescent (Figure 5C). Chronic tempol increased gp91 and p67 expression levels (Supplementary material online, Panels C and D).

4. Discussion

In the present study, we found that type 2 diabetes impaired the ability of mesenteric arteries to remodel and to improve NO-dependent dilation in response to a chronic increase in blood flow. Indeed, ROS production, high in ZDF rats, was further increased in HF arteries, thus inducing an additional reduction of the endothelium-dependent relaxation instead of enhancing the endothelial function as observed in LZ rats. Nevertheless, a chronic antioxidant treatment restored the ability of mesenteric arteries from ZDF rats to increase their diameter and endothelium-dependent dilation in response to a chronic rise in blood flow.

In physiological conditions, a chronic rise in blood flow, in resistance arteries, enhances vascular diameter and improves endothelium-dependent dilation.10,17,22 This remodelling is essential to adjust organ perfusion during physiological processes such as development,27 pregnancy,28 or exercise training,29 as well as during pathological processes, mainly ischaemic diseases. A similar remodelling also occurs in response to vasodilator treatments.30,31 Indeed, this remodelling is also called arteriogenesis32 and the model used in the present study has the advantage to involve resistance arteries and to allow the study of the effects of blood flow, on the arterial wall, independent of pressure or metabolic changes and without ischaemia.

In type 2 diabetes, the endothelium is less capable of inducing vasodilatation, especially in resistance arteries, which control end-organs blood flow.33 In addition, the outward hypertrophic remodelling observed in arteries from type 2 diabetic animals3437 might cause the increased contractility of the smooth muscle and the higher myogenic response observed in patients suffering type 2 diabetes.34,38 Our findings are in agreement with these previous observations. In ZDF rats mesenteric arteries (NF and HF arteries), we found that hypertrophy (high cross-sectional area) was associated with a higher phenylephrine-mediated constriction.

In order to improve endothelium-dependent dilation, and consequently local blood flow, vasodilator treatments, therapies improving insulin sensitivity, or exercise is commonly used. These treatments are associated with a higher eNOS expression, which is, at least in part, the consequence of a chronic rise in blood flow.9,10,22 The latter has also been shown to increase eNOS expression level and NO-dependent dilation in ageing, a situation associated with reduced endothelium responsiveness.12 This latter study has reported that a chronic rise in flow, using the model described in the present study, improves endothelium (NO)-dependent dilation in 8-month-old rats.12 Nevertheless, no study has yet investigated the effect of a chronic rise in blood flow in resistance arteries in type 2 diabetes. It is reasonable to speculate that this should also increase arterial diameter and/or endothelium-dependent tone as reported in healthy young animals39 and in old rats.12

The first main result of the present study is that endothelium-dependent dilation was not improved but further reduced after submitting ZDF rats arteries to a chronic rise in blood flow. Indeed, in contrast with lean rats, HF arteries from ZDF rats exhibited a reduced endothelium-dependent dilation compared with NF arteries, showing that a further endothelial dysfunction occurred. The defect observed in ZDF rats was not due to a change in smooth muscle response to NO, as the relaxation induced by the NO-donor SNP was not affected. Furthermore, eNOS expression was higher in HF than in NF arteries in both ZDF and LZ rats. Nevertheless, despite an increased eNOS expression in HF arteries in ZDF rats, the involvement of NO in the dilation was severely reduced as evidenced by the absence of the effect of L-NAME on acetylcholine-induced dilation. On the other hand, in LZ rats, L-NAME strongly reduced acetylcholine-induced dilation and this effect was higher in HF than in NF arteries, in agreement with previous studies.10,12 The expression level of eNOS and caveolin-1, modulated by HF as shown previously,9 was increased in HF arteries from ZDF rats. This suggests that the initial response of the arteries to the rise in flow or shear stress was maintained. The defect inducing a reduced dilation is thus located downstream eNOS. The cycloxygenase inhibitor indomethacin suppressed the remaining dilation after L-NAME in LZ rats NF arteries, in agreement with previous observations in the same vessel.40 In ZDF rats NF arteries, vasoconstrictor prostanoids are most probably produced in response to ACh, in agreement with previous studies showing that vasoconstrictor cycloxygenase derivatives reduce endothelium-mediated dilation in diabetes.41 In HF arteries, isolated from both LZ and ZDF rats, indomethacin had no effect, as L-NAME suppressed the dilation totally. This might be related to the increased eNOS expression found in HF arteries, which may act on COX expression and/or activity although this issue remains controversial42 and requires further investigation.

In order to find the cause of the paradoxically low endothelium (NO)-dependent dilation found in ZDF rats HF arteries, we investigated the effect of reducing ROS level, on the dilation, using tempol, which catalyses the transformation of ROS into H2O2.26 Tempol, acutely applied to isolated arteries, increased ACh-induced dilation in NF and HF arteries of ZDF rats. Tempol did not affected ACh-dependent relaxation in LZ rats. Nevertheless, a chronic treatment with tempol improved both ACh- and FMD in HF arteries. Thus, the reduction in NO-dependent dilation observed in ZDF rats arteries was the consequence of an excessive ROS production counteracting NO bioavailability. In the HF artery, the reduction in NO-dependent dilation was the result of the initial scavenging of NO by ROS, as seen in NF arteries, plus the additional increase in ROS level due to the chronic rise in blood flow.

The cause of this excessive ROS production may be multiple. In type 2 diabetes, ROS production is abnormally high and interacts with NO-dependent dilation in resistance arteries.43 In HF artery, a further increase in ROS level was observed. We found that NADP(H)-oxidase subunits expression (gp91 and p67, Figure 4) and DHE staining (Figure 5) were higher in HF than in NF in ZDF rats. This difference between NF and HF vessels is in agreement with our previous studies analysing NF and HF arteries isolated from other rat strains.10,26 Our data are also in agreement with a study analysing carotid arteries submitted to a rise in blood flow through arterio-venous fistulae.15 Thus, in ZDF rats, high basal ROS level was associated with high NADP(H) oxidase expression level, which was further increased by the chronic rise in flow. As a consequence, the rise in eNOS expression and the associated higher NO production in HF arteries were counteracted by this excessively high ROS level.

The hyperphagia and overweight, which characterize ZDF rats, in addition to diabetes, can also induce endothelium dysfunction. In a previous study, we have shown that rats with a similar overweight but without major hyperglycaemia (obese Zucker rats) present an impairment of their endothelial function, similar to that observed in ZDF rats.10 Nevertheless, the effects of the chronic increase in blood on the endothelium-dependent dilation were lower on the arteries from obese Zucker rats than on the HF artery of the ZDF rats. Thus, obesity per se might explain part of the dysfunction found in HF arteries in ZDF rats, whereas diabetes is certainly the cause of the most important loss of dilation.

In order to confirm that excessive ROS production in ZDF rats prevented endothelium-dependent dilation to be improved despite high eNOS expression level, ZDF rats were chronically treated with tempol. After a chronic tempol infusion, acetylcholine-mediated dilation was increased to control level (NF arteries in LZ rats) and completely blocked by L-NAME, showing that NO-induced dilation was fully restored (Figure 5B). Thus, the combination of a chronic rise in blood flow, increasing eNOS expression, with an antioxidant treatment allowed improving endothelium-dependent dilation in ZDF rats.

Another key finding of the present study is that the chronic rise in blood flow in ZDF rats did not induce the expected increase in arterial diameter. A possible explanation is that type 2 diabetes has already induced an outward hypertrophic remodelling3437 and that a further rise in diameter is not possible. Nevertheless, after a chronic treatment with tempol, a further increase in diameter was observed, although the rise was not equivalent to that found in LZ rats (present study) or in other rats strains.9,10 The flow-sensing process was probably not affected in ZDF rats, as the response to the chronic rise in flow was ‘normal’ with a rise in eNOS and caveolin-1 expression equivalent to that observed in LZ rats. This observation rules out, at least in part, a possible reduction in flow sensing by advanced glycation end-products (AGEs), which have been shown to reduce the activity of several processes in type 2 diabetes. AGEs have been reported to alter the matrix proteins collagen, vitronectin, and laminin, through AGE–AGE intermolecular covalent bonds, or cross-linking.37,44 Furthermore AGE cross-linking on type I collagen and elastin causes an increase in the area of extracellular matrix, resulting in increased stiffness of the vasculature.44 Finally, the absence of an increase in diameter in HF arteries cannot be the consequence of the overweight observed in ZDF rats, as in a previous study performed in obese but not diabetic Zucker rats we have shown that outward remodelling occurred normally.10

In the present study, ROS reduction restored in part flow-induced outward remodelling. Indeed, the excessive ROS production found in HF arteries may affect the remodelling process per se. Indeed, if the association of NO plus ROS is important for the remodelling, an excessive ROS level might reduce NO availability to a level low enough to prevent ONOO production and MMP activation, both essential for flow-induced remodelling.9,15,26 This is supported by our observation showing that L-NAME was totally unable to block acetylcholine-dependent dilation in ZDF rats HF arteries (Figure 3B).

The consequences of the present study are multiple. Vasodilator treatments in type 2 diabetes not only have to reduce hypertension but they are also expected to improve local blood flow supply in order to prevent end-organ damage. This latter effect might be more efficient with treatments possessing antioxidant properties or if they are associated with antioxidant. Similarly, exercise is recommended to patients suffering type 2 diabetes,45 based on the observation that exercise improves local blood flow as shown previously in gracilis muscle resistance arteries.46 Our finding suggests that this recommendation would benefit an association with a reduced oxidative stress. Indeed, our finding provides a rationale for the epidemiological observations showing that associating exercise with healthy diet has a better chance to improve NO availability and reduces oxidative stress.47 We have shown previously that a reasonable quantity of vegetal polyphenols with antioxidant properties had a beneficial effect of post-ischaemic revascularization of the rat hindlimb after femoral ligation. This revascularization involves arteriogenesis,48 which is equivalent to HF-remodelling.

In summary, increased ROS production induced by type 2 diabetes and by the rise in shear stress seriously altered the ability of resistance arteries to adapt their structure and function in response to a chronic increase in blood flow. This impairment was reversed by an antioxidant treatment suggesting that a vasodilator treatment should have antioxidant properties in order to be fully efficient in diabetic patients.

Funding

This work was supported in part by the Foundation for Medical Research (FRM: Fondation pour la Recherche Médicale), Paris, France.

Acknowledgements

E.J.B. was a post-doctoral fellow of the Centre National d’Etudes Spatiales (CNES), France.

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. The authors gratefully acknowledge the assistance of Mr James Mintz in the correction of the English of this manuscript.

Conflict of interest: none declared.

References

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