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. 2009 Mar 23;158(6):1588–1596. doi: 10.1111/j.1476-5381.2009.00417.x

Irbesartan-mediated reduction of renal and cardiac damage in insulin resistant JCR : LA-cp rats

JC Russell 1, SE Kelly 1, DF Vine 1, SD Proctor 1
PMCID: PMC2795225  PMID: 19814728

Abstract

Background and purpose:

Angiotensin II receptor antagonists (ARBs), originally developed for antihypertensive properties, have pleiotropic effects including direct vascular actions. We tested the hypothesis that the ARB irbesartan would be effective against micro- and macrovascular complications of the prediabetic metabolic syndrome using the obese, insulin-resistant JCR : LA-cp rat that exhibits micro- and macrovascular disease with ischaemic myocardial lesions and renal disease.

Experimental approach:

Obese male rats were treated with irbesartan (30 mg·kg−1·day−1, incorporated into chow) from 12 to 25 weeks of age.

Key results:

Irbesartan treatment caused no change in food intake or body weight. Fasting glycaemic control of the JCR : LA-cp rats was marginally improved, at the expense of increased plasma insulin levels (∼50%). Fasting plasma triglycerides were marginally reduced (∼25%), while cholesterol concentrations were unchanged. Elevated concentrations of adiponectin, monocyte chemotactic protein-1 and plasminogen activator inhibitor-1 were reduced along with severity of glomerular sclerosis. Macrovascular dysfunction (aortic hypercontractile response to noradrenergic stimulus and reduced endothelium-dependent relaxation) was improved and frequency of ischaemic myocardial lesions reduced (62%).

Conclusions and implications:

Irbesartan reduces markers of inflammation and prothombotic status, improves macrovascular function and reduces glomerular sclerosis and myocardial lesions in a model of the metabolic syndrome. Unlike pharmaceutical agents targeted on metabolic dysfunction, irbesartan reduced end-stage disease without major reduction of plasma lipids or insulin. The protective effects appear to be secondary to unknown intracellular mechanisms, probably involving signal transduction pathways. Understanding these would offer novel pharmaceutical approaches to protection against cardiovascular disease.

Keywords: metabolic syndrome, vascular function, glomerular sclerosis, PAI-1, MCP-1, adiponectin

Introduction

The metabolic syndrome characterized by abdominal obesity, insulin resistance and consequent hyperinsulinaemia and dyslipidaemia has become recognized as a major public health problem in prosperous societies worldwide (Després et al., 1996; Yusuf et al., 2001). Prediabetic insulin resistance and associated metabolic abnormalities have driven a developing epidemic of type 2 diabetes, atherosclerosis and cardiovascular disease (Butler et al., 2008). There is growing evidence that hyperinsulinaemia is a major determinant of vasculopathy, atherosclerosis and ischaemic cardiovascular disease associated with the metabolic syndrome (Després et al., 1996; O'Brien and Russell, 1997). There is also a major contribution from apolipoprotein B containing lipid particles (Proctor et al., 2004). In addition to the macrovascular disease, evident in coronary, cerebral and other large arteries, the metabolic syndrome is associated with microvascular complications that underlie a major portion of glomerular sclerosis and renal failure (Lorenzo et al., 2008). The fundamental role of the metabolic syndrome in cardiovascular disease and mortality has led to a focus on therapeutic intervention to reduce obesity, related insulin resistance and hyperinsulinaemia. Changes in diet, food intake and physical activity have proven relatively ineffective in the human population and may well be confounded by environmental, behavioural and genetic factors (Hegele et al., 2003). Thus, there have been significant efforts to develop effective pharmaceutical treatments, largely based on the use of animal models that mimic the metabolic and pathophysiological aspects of the metabolic syndrome (Proctor and Russell, 2006).

The rennin-angiotensin system plays a major role in blood pressure regulation and hypertension. Angiotensin converting enzyme (ACE) inhibitors reduce production of angiotensin II (Ang II) and have proven to be very useful antihypertensive agents. They have pleiotropic effects and we have reported that both captopril and ramipril are cardioprotective in an animal model of the metabolic syndrome and cardiovascular disease (Russell et al., 1998a; 2004;). Angiotensin II receptor antagonists (ARBs) are more recently developed agents that inhibit the rennin-angiotensin pathway by antagonizing the binding of Ang II to the AT1 receptor (Alexander et al., 2008). ARBs have been viewed as useful adjuncts to the treatment of cardiovascular disease because of their antihypertensive effects. However, there are recent indications that the effects of ARBs may be broader and include direct beneficial effects on diabetic vasculopathy (Karalliedde et al., 2008; Negro, 2008).

The JCR : LA-cp rat is a unique strain that has been well established as an animal model of the metabolic syndrome, particularly in the study of the underlying mechanisms of associated cardiovascular disease (Proctor and Russell, 2006). Rats of this strain that are homozygous for the autosomal recessive cp gene (cp/cp) spontaneously develop the pathophysiological characteristics associated with the metabolic syndrome in humans (O'Brien et al., 1999; Russell et al., 1999). These include advanced intimal (atherosclerotic) lesions, myocardial ischaemic lesions and microvascular renal dysfunction (Russell and Proctor, 2008), but the animals are not hypertensive (Russell and Amy, 1986). Vascular dysfunction, at the macrovascular level, includes increased contractility and reduced endothelium-mediated vascular relaxation, and at the microvascular level is evident in albuminuria and glomerular sclerosis. Importantly, from an experimental point of view, we have shown that heterozygous (cp/+), or homozygous normal (+/+) rats are lean and metabolically normal and not distinguishable, providing a benchmark of normality.

The obese, disease-prone phenotype of the JCR : LA-cp rat is due to the cp mutation that results in a stop codon in the extracellular domain of the leptin receptor (ObR) (Wu-Peng et al., 1997), leading to absence of the ObR in membrane-bound or soluble form. Absence of the leptin feedback signal leads to significant hyperphagia, obesity and rapid development of insulin resistance between the age of 4 and 7 weeks. There is an accompanying progressive development of a very low density lipoprotein (VLDL) hypertriglyceridaemia with delayed clearance of postprandial chylomicrons (Vance and Russell, 1990; Vine et al., 2007). Adult cp/cp rats have essentially no insulin-mediated glucose uptake, but maintain euglycaemia at the expense of extreme hyperinsulinaemia, fasting and postprandial (Russell et al., 1999). High insulin levels appear, in themselves, to have pathological effects and be a major contributor to the atherosclerosis and vascular dysfunction (Richardson et al., 1998).

The hypothesis underlying this study was that irbesartan has pleitropic effects in the presence of the metabolic syndrome, beyond reduction of food intake, obesity, hyperlipidaemia or insulin resistance, and confers protection against end-stage vascular disease. Consequently, the focus of the study was on end-points of insulin/glucose metabolism and control, macrovascular function, glomerular damage and dysfunction and end-stage ischaemic lesions of the heart. The results indicate that irbesartan has beneficial effects on the vascular disease, in the absence of a significant reduction in food intake or on the principal parameters of the prediabetic metabolic syndrome.

Methods

Animals

Male JCR : LA-cp rats, cp/cp (obese) and +/? (lean; a 2:1 mix of cp/+ and +/+), were bred and maintained in our established rat colony (Russell et al., 1995). The group of lean phenotype, +/?, animals were included throughout the study to provide a benchmark of normal physiology and metabolism. Rats were housed in an isolated high-efficiency particulate air filtered caging system (Techniplast S.p.a., Buguggiate, Italy) individually and placed on a reversed light cycle 1 week prior to the start of the experimental protocol to facilitate metabolic studies during the active (dark) phase of their diurnal cycle. All food was Lab Diet 5001 (PMI Nutrition International Inc., Brentwood, MO, USA). Rats were weighed and food intake determined twice per week throughout the experimental period. All care and treatment of the rats was in accordance with the Guidelines of the Canadian Council on Animal Care and was subject to prior review and approval by the Health Sciences Animal Policy Committee of the University of Alberta.

Experimental procedures

Rats were allowed to age within the breeding unit, without intervention, until 11 weeks of age, when they were transferred to the experimental facility and the Techniplast™ caging. After a 1-week acclimatization period, they were randomly assigned to treatment and control groups. At 24 weeks of age, a standardized meal tolerance test was performed, including collection of fasting blood. At 25 weeks of age, the rats were killed under isofluorane anaesthesia. Fed state blood was taken from the left ventricle for biochemical assays, together with the right kidney and the heart for histology, the thoracic aorta for assessment of vascular function and urine was collected from the bladder.

Meal tolerance test

The meal tolerance test was performed following a standardized protocol (Russell et al., 1999). Rats were subjected to a mock procedure 1 week previously. The rats were deprived of food for 16 h over the light (inactive) period and the test was conducted in the early part of the dark period. Conscious unrestrained rats were subjected to three blood samplings during each session. Animals were placed on a heated table to ensure vasodilatation of the tail, and 0.5 mL of blood was taken from the tip of the tail (0 min). Rats were then replaced in their cages and given a 5 g food pellet (the test meal). Timing began when 50% of the test meal had been consumed, and samples of blood were taken at 30 and 60 min for the analysis of glucose and insulin. All rats ate the full test meal within 15 min of presentation.

Drugs and chemicals

Irbesartan was provided by Sanofi-Synthelabo Recherche (Ruel-Malmaison, France). The agent was incorporated into powdered rat chow at a concentration, based on body weight and food consumption of the rats, to maintain a dose of 30 mg·kg−1·day−1, as recommended by Sanofi-Synthelabo Recherche. The chow was pelleted by extrusion through a die and air dried as previously described (Russell et al., 2000). Reagents and chemicals were obtained from Sigma Chemical (Oakville, ON, Canada).

Analytical methods

Plasma glucose was determined using a glucose oxidase assay procedure (Diagnostic Chemicals Ltd., Charlottetown, PEI, Canada) and insulin assayed by rat ELISA (Mercodia AB, Uppsala, Sweden). Colorimetric assays obtained from Wako Pure Chemicals USA, Inc. (Richmond, VA, USA) were used to determine: plasma triglyceride (L-Type TG-H), total cholesterol (Cholesterol E) and low density lipoprotein (LDL) cholesterol (L-Type LDL-C). High density lipoprotein (HDL) cholesterol was measured using direct HDL assay (Diagnostic Chemicals). Adiponectin was measured by radioimmunoassay (#MADP-60HK) by enzyme-linked immunosorbent assay using LINCOplex™ plates (Linco Research, St Charles, MO, USA). Monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) activity were measured by immunoassays (R&D Systems, Minneapolis MN, USA and Diapharma Group Inc., West Chester, OH, USA respectively). Urine albumin and creatinine measurements were performed on a Beckman Coulter LX20i analyser using immuno turbidimetric and Jaffé methods respectively.

Vascular function studies

The vascular function of aortic rings, with intact endothelium, was assessed using established methods (Russell et al., 2001). Briefly, the thoracic aorta was excised, trimmed of adhering fat and connective tissue, and cut into 3 mm-long transverse rings. Rings were mounted on stainless steel hooks under 1.5 g resting tension in 10 mL organ baths and bathed at 37°C in Krebs solution (containing in mM: NaCl 116, KCl 5.4, CaCl2 1.2, MgCl2 2, Na2PO4 1.2, glucose 10 and NaHCO3 19) and gassed with 95% O2 and 5% CO2. Tension was recorded isometrically with Grass FTO3C transducers (Grass Medical Instruments, Quincy, MA, USA) and displayed on a Digi-Med tissue force analyser (Model 210, Micro-Med, Louisville, KY, USA), linked to an IBM-compatible computer that acquired data digitally using DMSI 210/4 (Micro-Med) software.

The contractile response of endothelium-intact rings of aortae to phenylephrine (PE) was assessed through concentration–response curves for PE (1 nM to 300 mM). The basal nitric oxide (NO)-mediated relaxation of aortic rings (precontracted with PE to 80% of maximal contraction) was assessed by determining the relaxant response to different concentrations of the endothelial NO-releasing agent ACh and the nitrate donor sodium nitro prusside (SNP). Direct assessment of NO-mediated effects was also determined through addition of NG-nitro-L-arginine methyl ester (l-NAME), at 10−4 M, in order to inhibit nitric oxide synthase (NOS).

Histology

Kidneys were cut through the hilum on the long axis, fixed in formalin, subjected to conventional processing and sectioning, followed by haematoxylin and eosin (H&E) staining. The extent of glomerular sclerosis was determined using a similar process to that of Schäfer et al. (2004) and guided by the interpretation of Ferrario and Rastaldi (2006). Four fields of view of the right kidney of each rat were recorded at ×2 magnification on a digital camera system. Histological images were visualized using Photoshop (V7.0, Adobe Systems Inc., San Jose, CA, USA), examined blind, and all glomeruli in each field (minimum of 40 per kidney) rated as normal or sclerotic (Russell and Proctor, 2007). Results are expressed as % of glomeruli that exhibited sclerosis.

Hearts were cut transversely into four blocks, fixed in formalin, subjected to conventional processing, embedded in a single paraffin block and sectioned, followed by H&E staining. The heart sections were examined blind by an experienced observer and the number of ischaemic lesions identified in each of the sections summed for each heart. The lesions were categorized by four stages as previously described (Russell et al., 1995).

Statistical analysis

Results are expressed as mean ± SEM and were analysed using SigmaStat (Jandel Scientific, San Rafael, CA, USA) and plotted using SigmaPlot (Systat Software, Inc. San Jose, CA, USA) and Prism (Graphpad, San Diego, CA, USA). Results were compared using one-way analysis of variance (anova) followed by multiple comparison tests. Concentration–response curves were analysed using the program ALLFIT (DeLean et al., 1978), which fits the complete data set to the logistic equation and permits independent testing of differences between individual parameters. A value of P < 0.05 was taken as being statistically significant.

Results

Food intake and body weight

Figure 1 shows food intake and body weights of cp/cp rats, control and irbesartan-treated, over the period from 12 to 24 weeks of age with data from +/? control rats shown for reference. Irbesartan did not alter food intake or body weight of cp/cp rats.

Figure 1.

Figure 1

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Food intake and body weight of JCR : LA-cp rats during treatment from 12 to 24 weeks of age. Values are mean ± SEM, 10 rats per group. There were no significant differences between control and irbesartan-treated cp/cp rats (P > 0.05).

Insulin and glucose metabolism

Fasting (0 min) and postprandial (30 and 60 min during the meal tolerance test) plasma insulin and glucose concentrations are shown in Figure 2. The +/? control rats had modestly lower fasting glucose concentrations than the cp/cp controls in the presence of markedly lower plasma insulin levels. Irbesartan-treated cp/cp rats had significantly higher fasting insulin levels than the cp/cp controls (P < 0.01), accompanied by lower glucose concentrations (P < 0.05 vs. cp/cp control). At the 30- and 60-min postprandial time points, +/? rats continued to have very low insulin levels and significantly lower glucose concentrations (P < 0.05 and P < 0.001). The apparently lower glucose concentrations and higher insulin levels of the irbesartan-treated cp/cp rats through the 30 and 60 min time points were not significant compared with cp/cp controls (P > 0.05).

Figure 2.

Figure 2

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Plasma insulin (upper panel) and glucose (lower panel) concentrations in the meal tolerance test. Values are mean ± SEM, 10 rats per group. *P < 0.05; **P < 0.01; ***P < 0.001 versus cp/cp control.

Plasma lipids

Fasting plasma lipid concentrations of the rats are shown in Figure 3. As previously reported, HDL and total cholesterol levels were significantly lower in the +/? control rats than in cp/cp controls (Vance and Russell, 1990). Irbesartan treatment had no effect on cholesterol lipoprotein fractions of cp/cp rats. However, plasma TG levels of irbesartan-treated rats were modestly decreased compared with cp/cp controls (P < 0.05).

Figure 3.

Figure 3

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Fasting plasma lipid concentrations in irbesartan-treated JCR : LA-cp rats. Values are mean ± SEM, 10 rats per group. *P < 0.05; **P < 0.0001 versus cp/cp control. HDL, high density lipoprotein; LDL, low density lipoprotein.

Cytokines and thrombosis

As shown in Figure 4, MCP-1 levels were significantly elevated in the cp/cp rats compared with +/? rats (59%, P < 0.01) and were significantly reduced by irbesartan treatment (30%, P < 0.05). The markedly elevated plasma levels of PAI-1 (fourfold, P < 0.01) were also reduced by irbesartan (46%, P < 0.01). In addition, elevated plasma levels of the adipose tissue-related cytokine adiponectin (2.8-fold, P < 0.001) were lowered significantly (30%, P < 0.01) by irbesartan.

Figure 4.

Figure 4

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Concentrations of adiponectin, MCP-1 and PAI-1 in plasma of irbesartan-treated cp/cp rats. Values are mean ± SEM, 7–10 animals per group for adiponectin and PAI-1 and 4–6 animals for MCP-1. *P < 0.05; **P < 0.01; ***P < 0.001 versus cp/cp control. MCP-1, monocyte chemoattractant protein-1; PAI-1, plasminogen activator inhibitor-1.

Vascular function

The contractile response of aortic rings to the adrenoceptor agonist PE and the relaxant response to ACh are shown in Figure 5 and Table 1. Aortae from cp/cp rats have enhanced PE-mediated contractility compared with +/? rats. Hypercontractility was reduced in aortae from irbesartan-treated rats, in both the absence and presence of the inhibitor of NO synthesis, l-NAME (P < 0.001 and P < 0.05 respectively). Irbesartan had no effect on the EC50 for PE (Table 1). ACh-mediated relaxation of PE precontracted aortic rings of cp/cp rats is significantly impaired, with reduced maximal relaxation compared with +/? control rats. Aortic rings from irbesartan-treated rats had increased maximal ACh-mediated relaxation, and increased sensitivity to ACh, indicated by a 65% decrease in EC50 (P < 0.001). In addition, the EC50 for SNP-mediated relaxation was significantly reduced in aortae from irbesartan-treated rats.

Figure 5.

Figure 5

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Effect of irbesartan treatment on aortic vascular function in cp/cp rats. (A) PE-mediated contractile response in the absence of l-NAME. (B) PE-mediated contractile response in the presence of l-NAME, 10−4 M. (C) ACh-mediated concentration–relaxant response of PE pre-contracted aortic rings. (D) SNP-mediated relaxant response of PE pre-contracted aortic rings. Values are mean ± SEM, 10 rats per group. The concentration–response curves were calculated using the logistic equation and the ALLFIT program, with values of the parameters shown in Table 1, together with statistical significance of differences. l-NAME, NG-nitro-L-arginine methyl ester; PE, phenylephrine; SNP, sodium nitro prusside.

Table 1.

Vascular function of JCR : LA-cp rats: effects of irbesartan

+/? rats
 cp/cp rats
Control Control Irbesartan
PE contractility
Maximum (g) 1.82 ± 0.07*** 2.77 ± 0.10 2.18 ± 0.09***
EC50 (M X10−8) 2.62 ± 0.57** 6.02 ± 1.35 5.46 ± 1.35
PE contractility – L-NAME
Maximum (g) 2.40 ± 0.08 2.85 ± 0.12 2.31 ± 0.15*
EC50 (M X10−8) 2.71 ± 0.06 3.70 ± 1.02 2.16 ± 0.10
ACh-relaxation
Maximum (%) 79.4 ± 2.6*** 62.9 ± 1.7 71.8 ± 1.4***
EC50 (M X10−8) 2.61 ± 0.52 3.58 ± 0.62 1.26 ± 0.15***
SNP-relaxation
Maximum (%) 130 ± 18 115 ± 5 109 ± 4.2
EC50 (M X10−7) 4.6 ± 3.7 4.26 ± 0.97 2.05 ± 0.42*
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Data are expressed as mean ± SEM; 10 rats per group; each rat 24 weeks of age.

*

P < 0.05;

**

P < 0.01;

***

P < 0.001 versus values for cp/cp control rats.

l-NAME, NG-nitro-L-arginine methyl ester; PE, phenylephrine; SNP, sodium nitro prusside.

Renal function and glomerular sclerosis

Urinary albumin excretion was markedly elevated in the cp/cp rats at 24 weeks of age (P < 0.001), as shown in Figure 6, and was accompanied by increased severity of glomerular sclerosis (P < 0.05). Treatment of cp/cp rats with irbesartan did not significantly reduce the albumin/creatinine ratio. However, the fraction of glomeruli that were sclerotic was significantly reduced in the irbesartan-treated rats compared with controls (P < 0.05).

Figure 6.

Figure 6

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Urinary albumin/creatinine ratio, as an index of renal vascular function (A), and fractional glomerular sclerosis (B) of control and irbesartan-treated rats. Values are mean ± SEM, 10 rats per group. *P < 0.05; ***P < 0.001 versus cp/cp control.

Ischaemic myocardial lesions

As shown in Figure 7, there were relatively few Stage 1 lesions (areas of early ischaemia or necrosis) or Stage 3 lesions (foci of chronic inflammatory cells without cell dropout) in the hearts of any of the groups of rats. There were no significant differences between the groups. Stage 2 lesions (areas with strong inflammatory cell infiltration and cell lysis) were infrequent in the hearts of +/? control rats and frequent in the hearts of cp/cp control rats (P < 0.001). Irbesartan treatment of cp/cp rats reduced the frequency of Stage 2 lesions to the range seen in the hearts of +/? rats (P < 0.01). Stage 4 lesions were relatively infrequent in these 24-week-old hearts reflecting the cumulative nature of these mature scarred lesions and the relatively young age of the rats. No Stage 4 lesions were seen in irbesartan-treated hearts, but the low frequency of these lesions in these younger rats makes this observation non-significant (P > 0.05).

Figure 7.

Figure 7

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Frequency of ischaemic myocardial lesions in irbesartan-treated and control rats. Lesion stages as described in the Methods. Values are mean ± SEM, 10 rats in each group. **P < 0.01; ***P < 0.005 versus cp/cp control. No Stage 4 lesions were detected in the hearts of irbesartan-treated cp/cp rats (†), but the difference was not statistically significant (P > 0.05).

Discussion

The absence of any effect of irbesartan on food intake or body weight is consistent with it being a specific antagonist of a peptide hormone receptor (i.e. an ARB). The reduction in fasting glucose concentrations may be interpreted as reflecting an improvement in glucose control. This is consistent with the physiological adaptation of the untreated cp/cp rat that maintains euglycaemia in the presence of the developing extreme insulin resistance through a compensating hyperinsulinaemia (Russell et al., 1999). However, the present results do not indicate a major increase in insulin sensitivity, which has been seen in this model with other agents (Russell et al., 2000). Given that hyperinsulinaemia plays a major role in the development of atherosclerosis and myocardial damage in the cp/cp rat, the substantial increases in plasma insulin seen in the irbesartan-treated rat cannot be viewed as beneficial.

The modest 20% reduction in fasting plasma TG concentrations caused by irbesartan treatment of cp/cp rats must reflect a reduction in VLDL, but the mechanism while clearly a pleiotropic effect is not clear. Consistent with the primary cholesterol bearing lipoprotein fraction in the rat being HDL, there was no effect on cholesterol levels. TG-rich particles and remnants play an important role in atherogenesis (Proctor et al., 2004) and the reduction in TG may play a role in vascular protection seen with irbesartan treatment of the cp/cp rat, but we feel this is too small to account for all of the beneficial effects seen.

Adiponectin (also known as Acrp30 or GBP28) is a cytokine, exclusively secreted by adipocytes, and there is an unresolved debate over the role of adiponectin and whether it is involved in cardiovascular disease (Empana, 2008). However, there is growing evidence that insulin resistance and hyperinsulinaemia stimulate adiponectin secretion (Fernández-Veledo et al., 2008) and our data show markedly elevated plasma adiponectin levels in the cp/cp rat. Plasma concentrations in the cp/cp rat are approximately sevenfold higher than those reported for fa/fa Zucker diabetic fatty rats, which are type 2 diabetic, as opposed to being insulin-resistant, and have essentially normal insulin levels (Szöcs et al., 2008). While the mechanistic relationships remain obscure, it is clear that adiponectin, at least in the cp/cp rat, is implicated in the metabolic status and susceptibility to vascular disease, possibly acting as an inflammatory cytokine. The reduction of adiponectin levels observed in irbesartan treated cp/cp rats is, thus, consistent with the associated reduction in micro- and macrovascular disease. At some level, it appears that irbesartan reduces the pro-inflammatory state of the cp/cp rat, through a pathway not directly related to obesity and insulin metabolism. It remains unclear whether the role of adiponectin is causal or merely as a marker.

Monocyte chemoattractant protein-1 is the principal chemotactic factor for the migration of monocytes/macrophages and mediation of chronic inflammation (Dragomir and Simionescu, 2006). Our data show higher MCP-1 levels in the cp/cp rat in comparison with +/? rats, consistent with the effect of hyperinsulinaemia on MCP-1 secretion (Fernández-Veledo et al., 2008). Greater expression of MCP-1 in the cp/cp rat is also consistent with our earlier observations of widespread activation and endothelial adherence of macrophages in atherosclerosis-prone adult cp/cp rats (Russell et al., 1995; 1998a; Schepers et al., 2006). In addition, Takebayashi et al. (2006) have shown a potentially important association between MCP-1 and urinary albumin excretion in non-obese type 2 diabetic patients, analogous to our observations in the cp/cp rat. Our results show a reduced incidence of glomerular sclerosis with irbesartan treatment, without any change in urinary albumin loss. This is an apparently contradictatory finding as glomerular sclerosis is viewed as the end-stage of microvascular damage, first evident in capillary dysfunction. The reduction in glomerular damage in the irbesartan-treated rats may, thus, be mediated through effects on a process separate from that leading to glomerular capillary permeability and albuminuria.

Humans exhibiting the metabolic syndrome have approximately twofold higher plasma PAI-1 levels (Bahia et al., 2006), even in the absence of evidence of type 2 diabetes. The cp/cp rat has also been shown to have elevated circulating levels of PAI-1 (Schneider et al., 1998), consistent with the finding of both macro- and micro-thrombi in the arterial system (Russell et al., 1998b). The reduction in PAI-1 levels in irbesartan-treated cp/cp rats indicates a reduction in the prothrombotic status and should be beneficial in terms of reduced obstructive vascular disease. Reduced incidence of glomerular sclerosis and reduced frequency of Stage 2 lesions in the heart is consistent with this concept.

Irbesartan treatment had significant effects on the macrovascular dysfunction of the cp/cp rats. The reduction in the PE-stimulated contractile response was evident in the absence of the NOS inhibitor l-NAME, and present, although less marked in the presence of l-NAME. Together with the marked changes in the relaxant response to ACh on irbesartan treatment, and the smaller but still significant changes in SNP-stimulated relaxation, these results suggest irbesartan improves the status of the endothelium and, to a lesser degree, the vascular smooth muscle cells (VSMCs). The reduction in the relaxant EC50 for SNP, a direct NO donor, further indicates a change in the VSMC. In addition, the increased relaxation response to ACh and reduced EC50 indicate an improvement in endothelial cell function and NO release. Paradoxically, these changes occurred in the presence of an increase in hyperinsulinaemia, which is a critical mediator of vascular dysfunction and cardiovascular disease in prediabetic and type 2 diabetic states (Russell et al., 1995; O'Brien and Russell, 1997). The improvement in vascular function, together with reduced PAI-1 levels, may be expected to lead to a reduction in vasospastic and thrombotic events and ischaemic damage to organs, such as the heart.

Macrovascular disease in the cp/cp rat leads to atherosclerosis (Proctor and Russell, 2006), thrombotic disease and ischaemic lesions (Russell et al., 2005). The Stage 1 lesions, seen in hearts of cp/cp rats, are areas of necrosis and early ischaemic damage. They have a short lifespan and progress to Stage 2 or 3 lesions with infiltration of chronic inflammatory cells. The number of Stage 1 lesions present, at any point in time, is small and differences between groups difficult to demonstrate. Stage 2 lesions represent the longer active stage of resolution of lesions of significant size, and while infrequent in +/? rats, are frequent in the cp/cp rats. Irbresartan treatment reduced the frequency of these early end-stage lesions in cp/cp rats to the range seen in the lean normal +/? rats, indicating an effective reduction in the development of end-stage ischaemic lesions. Stage 3 lesions, as small foci of chronic inflammatory cell activity, are short lived, relatively infrequent and nonspecific. Stage 4 lesions reflect the persistent, or remnant scarred lesions that were of a sufficient size to be visible after resolution and contraction of the collagen structure, accumulating with aging of the rat. The small number of Stage 4 lesions present in the relatively young cp/cp rats in this study precluded statistical analysis.

A variety of pharmaceutical interventions have been studied in the cp/cp rat. ACE inhibitors, endopeptidase inhibitors, insulin sensitizers and ethanol have all been shown to reduce hyperinsulinaemia, hypertriglyceridaemia, vascular dysfunction and myocardial lesions (Russell and Proctor, 2008). Recently, we have shown that these substances also reduce renal microvascular dysfunction and damage (Russell et al., 2004; Proctor et al., 2007). A common element to the beneficial effects of interventions that are protective against vascular disease has been a significant reduction of insulin resistance and hyperinsulinaemia. In contrast to other agents, irbesartan induced only a modest, and probably biologically unimportant, reduction in TG levels and actually increased insulin levels. This suggests that irbesartan is protective against cardiovascular disease through previously unrecognized pleiotrophic effects. The reduction in markers of inflammation and thrombotic susceptibility, improved macrovascular function and reduced renal and myocardial end-stage disease are consistent with irbesartan having a direct effect on the VSMC, possibly through intracellular signalling pathways. Recently, it has been demonstrated that inhibition of the rennin-angiotensin system combined with calcium channel antagonists reduces plasma levels of inflammatory markers, including MCP-1, and increases adiponectin levels (Ogawa et al., 2008). Similarly, Vieitez et al. (2008) have shown that irbesartan lowers the expression of cytokines related to inflammation in the kidney of the cytotoxic streptozotocin model of type 1 diabetes. These findings are consistent with our earlier finding that calcium channel antagonists have antiatherogenic and cardioprotective activity in the cp/cp rat (Russell et al., 1997). The available evidence suggests not only that MCP-1 and adiponectin mediate effects through AMPK (Ogawa et al., 2008; Sharma et al., 2008), but also that AMPK metabolism is abnormal in the cp/cp rat (Atkinson et al., 2003) and that the underlying mechanisms are complex. Negro (2008) has extensively reviewed the role of the renin-angiotensisn system and endothelium in vascular dysfunction and their relationship with many of the factors identified as being involved in the vascular, myocardial and renal disease in the cp/cp rat (Russell and Proctor, 2006; 2007;). He particularly emphasized the crucial role of Ang II in the endothelial dysfunction and NO deficiency seen in the cp/cp rat. Further, he suggested that irbesartan-mediated ‘beneficial effects beyond blood pressure control’ follow from ‘blockade of the renin-angiotensin system’. These mechanisms potentially offer novel pharmaceutical approaches to protection against cardiovascular disease associated with the prediabetic metabolic syndrome.

Acknowledgments

This work was supported financially by Sanofi-Aventis, Rueil-Malmaison, France and the Heart and Stroke Foundation of Alberta and the Northwest Territories. The expert technical assistance of Mss Kristina MacNaughton and Sharon Sokolik is gratefully acknowledged.

Glossary

Abbreviations:

Ang II

angiotensin II

ARB

angiotensin II receptor antagonist

H&E

haematoxylin and eosin

HDL

high density lipoprotein

l-NAME

NG-nitro-L-arginine methyl ester

LDL

low density lipoprotein

MCP-1

monocyte chemoattractant protein-1

NO

nitric oxide

ObR

leptin receptor

PAI-1

plasminogen activator inhibitor-1

PE

phenylephrine

TG

triglyceride

VLDL

very low density lipoprotein

VSMC

vascular smooth muscle cell

Conflict of interest

The authors state no conflict of interest.

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