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Impaired Endothelial Function in Never-Treated Hypertensive Subjects Carrying the Arg⁹⁷² Polymorphism in the Insulin Receptor Substrate-1 Gene

Department of Experimental and Clinical Medicine (F.P., A.Sci., A.Sco., G.V., E.L., A.P., G.S.), "G. Salvatore" University Magna Græcia of Catanzaro, 88100 Catanzaro, Italy; and Department of Internal Medicine (M.F., R.L.), University of Rome-Tor Vergata, 00173 Rome, Italy

Address all correspondence and requests for reprints to: Francesco Perticone, M.D., Department of Medicina Sperimentale e Clinica, Policlinico Mater Domini-Via Tommaso Campanella, 88100 Catanzaro, Italy. E-mail: perticone{at}unicz.it.

	Abstract

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Some cardiovascular risk factors, such as hypertension and insulin resistance, are associated with endothelial dysfunction. Insulin regulates both in vitro and in vivo expression of endothelial nitric oxide synthase (eNOS) via a pathway involving insulin receptor substrate-1 (IRS-1) and phosphatidylinositol-3 kinase. Recently, we found that human endothelial cells obtained from carriers of the Arg⁹⁷² IRS-1 polymorphism exhibited reduced eNOS expression in response to chronic exposure to insulin. A reduction in eNOS expression would be expected to be associated with impaired endothelium-dependent vasodilation. To investigate a possible relationship between Arg⁹⁷² IRS-1 polymorphism and endothelial dysfunction in vivo, we enrolled a cohort of 100 never-treated hypertensive subjects. Endothelium-dependent and endothelium-independent vasodilation were assessed by increasing doses of acetylcholine and sodium nitroprusside. IRS-1 polymorphism was detected by PCR. The allelic frequency of the Arg⁹⁷² IRS-1 variant was 8.0%. Stratifying subjects according to IRS-1 genotype, we observed that acetylcholine-stimulated forearm blood flow was significantly (P < 0.0001) lower in Gly/Arg heterozygous carriers than in Gly/Gly carriers (11.3 ± 4.4 vs. 14.7 ± 5.9 ml/100 ml^–1 of tissue per min^–1). Sodium nitroprusside caused comparable increments in forearm blood flow in both groups (12.9 ± 2.4 vs. 13.3 ± 3.5 ml/100 ml^–1 of tissue per min^–1). Our data strongly suggest that, by inducing endothelial dysfunction, the Arg⁹⁷² IRS-1 polymorphism may contribute to the genetic predisposition to develop cardiovascular disease.

	Introduction

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ENDOTHELIAL DYSFUNCTION IS an early event in atherogenesis that precedes the thickening of the intima and the formation of atherosclerotic plaques (1, 2). It has been reported that forearm endothelial dysfunction predicts cerebro-cardiovascular events in individuals at risk for atherosclerosis such as hypertensive patients (3). Insulin has multiple physiological effects on the vascular tissues including regulation of the expression of endothelial nitric oxide synthase (eNOS) (4, 5). These effects appear to be mediated via the activation of the signaling pathway involving the insulin receptor/insulin receptor substrate-1 (IRS-1)/phosphatidylinositol-3 (PI-3) kinase (5). Accordingly, mice with a specific vascular endothelial cell insulin receptor knockout exhibit reduced eNOS mRNA levels in endothelial cells (6). IRS-1 knockout mice showed impaired endothelium-dependent vasodilatation of the aorta but a normal endothelium-independent vasodilatation (7). Molecular scanning of the IRS-1 gene in normal individuals and patients with type 2 diabetes from different ethnic groups has revealed several polymorphisms resulting in amino acid substitutions (8). Of these, the GlyArg change at codon 972 (Arg⁹⁷² IRS-1) is the most common and has been studied most extensively (9, 10, 11, 12, 13, 14, 15). Primary cells from subjects carrying this polymorphism and cell line transfected with the Arg⁹⁷² IRS-1 variant exhibited defects in binding of the p85 subunit of PI-3 kinase to IRS-1 and IRS-1-associated PI-3 kinase activity (16, 17, 18). We have recently found that human umbilical vein endothelial cells obtained from carriers of the Arg⁹⁷² IRS-1 polymorphism exhibited reduced eNOS expression in response to chronic exposure to insulin (19). An altered regulation in insulin-dependent eNOS expression in carriers of the Arg⁹⁷² IRS-1 polymorphism would be expected to be associated with in vivo endothelial dysfunction. To address this issue, we studied the distribution of Arg⁹⁷² variant in a population of never-treated hypertensive subjects in whom vascular function vasodilation was measured by strain-gauge plethysmography.

	Subjects and Methods

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Study population

A total of 100 hypertensive subjects, consecutively recruited from the Department of Internal Medicine of the University Hospital of Catanzaro, participated in this study. All subjects were Caucasian and underwent physical examination and review of their medical history. All participants had never been treated with antihypertensive drugs. Causes of secondary hypertension were excluded by clinical and biochemical tests. None of the patients had a history or clinical evidence of angina, myocardial infarction, valvular heart disease, diabetes, hyperlipidemia, peripheral vascular disease, coagulopathy, or any disease predisposing them to vasculitis or Raynaud’s phenomenon. All subjects were normal glucose tolerant as assessed by the oral glucose tolerance test. Insulin sensitivity was estimated by using the previously validated homeostasis model assessment (HOMA) index (20), calculated from the fasting glucose and insulin concentrations according to the following formula: HOMA = [insulin (µU/ml) x glucose (mmol/liter)]/22.5. The study was approved by the institutional ethics committee, and informed written consent was obtained from each subject in accordance with principles of the Declaration of Helsinki.

Blood pressure (BP) measurements

Readings of clinic BP were obtained in the left arm of the supine patients, after 5 min of quiet rest, with a mercury sphygmomanometer. A minimum of three BP readings was taken on three separate occasions at least 2 wk apart. Patients with a clinic BP 140 mm Hg systolic and/or 90 mm Hg diastolic were defined as hypertensive.

Vascular function

All studies were performed at 0900 h after subjects had fasted overnight, with the subjects lying supine in a quiet, air-conditioned room (22–24 C). The protocol that has been previously described by Panza et al. (1) and subsequently used by our group (3) was used for the present study. All patients underwent measurement of forearm blood flow (FBF) and BP during intraarterial infusion of saline, acetylcholine (ACh), and sodium nitroprusside (SNP) at increasing doses. ACh (Sigma, Milan, Italy) was diluted with saline immediately before infusion. SNP (Malesci, Florence, Italy) was diluted in 5% glucose solution immediately before each infusion and protected from light with aluminum foil. All participants rested 30 min after artery cannulation to reach a stable baseline before data collection; measurements of FBF and vascular resistance (VR), expressed in units, were repeated every 5 min until stable. Endothelium-dependent and endothelium-independent vasodilation were assessed by a dose-response curve to intraarterial ACh infusions (7.5, 15, and 30 µg/ml^–1·min^–1, each for 5 min) and SNP infusions (0.8, 1.6, and 3.2 µg/ml^–1·min^–1, each for 5 min), respectively. The sequence of administration of ACh and SNP was randomized to avoid any bias related to the order of drug infusion. The drug infusion rate, adjusted for forearm volume of each subject, was 1 ml/min.

DNA analysis

Genomic DNA was isolated from peripheral blood according to standard procedures. The glycine to arginine substitution at codon 972 of the human IRS-1 sequence was detected by digestion of PCR products with restriction enzyme BstNI as previously described (21).

Statistical analysis

ANOVA was performed for clinical and biological data, and the differences between means were compared using the unpaired Student’s t test. The vasodilating responses to ACh and SNP were compared by ANOVA for repeated measurements, and when analysis was significant, the Tukey test was applied. Differences were also tested after adjusting for factors reported to impair endothelium-dependent vasodilation, such as age, gender, body mass index (BMI), lipid levels, and BP values, by analysis of covariance. Parametric data are reported as mean ± SD. Differences were considered to be significant at P < 0.05. All comparisons were performed using the statistical package SPSS 10.0 for Windows (SPSS, Inc., Chicago, IL).

	Results

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Study population

In keeping with previous results observed in subjects with features of the insulin-resistance syndrome (10), the allelic frequency of the Arg⁹⁷² IRS-1 variant in the study population was 8.0%. Clinical, biochemical, and hemodynamic characteristics of Gly/Gly and heterozygous carriers for the Arg⁹⁷² IRS-1 variant are reported in Table 1. No significant differences in age, gender, BMI, heart rate (HR), systolic BP, diastolic BP, fasting glucose levels, total cholesterol, high-density lipoprotein cholesterol, triglycerides, microalbuminuria, and fibrinogen were observed between the heterozygous and Gly/Gly carriers. By contrast, fasting insulin levels and insulin resistance, estimated as HOMA index, were significantly higher in heterozygous carriers for the Arg⁹⁷² IRS-1 variant compared with Gly/Gly carriers (P < 0.0001).

The baseline FBF and VR values did not differ between the heterozygous and Gly/Gly carriers (Table 1). Intraarterial infusion of ACh caused a significant dose-dependent increase in FBF and decrease in forearm VR in both groups of subjects. As shown in Fig. 1, carriers of the Arg⁹⁷² IRS-1 variant exhibited a significantly lower FBF compared with Gly/Gly carriers, as estimated by either two-way ANOVA (P < 0.0004) or the area under the curve (P < 0.02). The differences in ACh-stimulated FBF remained significant (P < 0.004) after adjusting for well-known modulators of endothelium function including age, gender, BMI, serum glucose, serum cholesterol, serum triglycerides, and systolic BP. VR decreased in a dose-dependent manner in the two groups, and changes in VR mirrored the changes in FBF. The VR values at the three incremental doses of ACh were 24.1 ± 7.1, 15.9 ± 5.3, and 8.9 ± 3.5 U, and 25.4 ± 7.6, 19.3 ± 6.3, and 11.5 ± 3.8 U for Gly/Gly carriers and heterozygous carriers for the Arg⁹⁷² IRS-1 variant, respectively. Changes in VR in response to ACh infusion were significantly less pronounced in carriers of the Arg⁹⁷² IRS-1 variant compared with Gly/Gly carriers (P < 0.03). Intraarterial infusion of ACh caused no change in BP or HR values in both groups of patients.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Responses of FBF to intraarterial infusions of ACh and SNP in Arg972 IRS-1 polymorphism carriers and Gly/Gly carriers.

	Discussion

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There is evidence from cell biology and experimental research suggesting that insulin regulates expression of eNOS via a PI-3 kinase-dependent pathway (4, 5, 6, 7, 19). These observations coupled with the accessibility of a carefully characterized cohort of never-treated hypertensive subjects have provided the rationale for addressing the question of whether the Arg⁹⁷² polymorphism of IRS-1 could be associated with endothelial dysfunction, an early event in the developmental process leading to atherosclerosis (3). We found that, in a population of never-treated hypertensive subjects, carriers of the Arg⁹⁷² polymorphism showed an impaired endothelium-dependent vasodilatation compared with Gly/Gly carriers. By contrast, no significant differences in endothelium-independent vasodilatation were observed between Arg⁹⁷² polymorphism carriers and Gly/Gly carriers. This is the first evidence demonstrating an association between Arg⁹⁷² polymorphism and impaired vasodilator response to intraarterial infusion of ACh in human essential hypertension. Our results are consistent with those observed in IRS-1-deficient mice, which showed an impaired endothelium-dependent vasodilatation of the aorta but a normal endothelium-independent vasodilatation (7). The functional effect of the Arg⁹⁷² IRS-1 polymorphism on insulin signaling has been extensively characterized by a series of in vitro studies (16, 17, 18, 19). Thus, it has been demonstrated that the presence of the GlyArg change at codon 972 of IRS-1 caused a specific defect in binding of the p85 regulatory subunit of PI-3 kinase to the IRS-1 variant, resulting in a decrease in IRS-1-associated PI-3 kinase activity. A potential mechanism whereby the Arg⁹⁷² polymorphism could contribute to impair endothelium-dependent vasodilatation is by affecting eNOS expression via a defective PI-3 kinase activation. Accordingly, we have recently observed that human umbilical vein endothelial cells obtained from carriers of the Arg⁹⁷² polymorphism showed a reduced eNOS expression in response to chronic exposure to insulin (19). However, because insulin resistance, estimated as HOMA index, was significantly higher in hypertensive carriers for the Arg⁹⁷² IRS-1 polymorphism, we cannot exclude the possibility that endothelial dysfunction observed in carriers of the Arg⁹⁷² variant was secondary to metabolic abnormalities induced by whole-body insulin resistance rather than a direct effect of the IRS-1 variant on endothelial cells.

Previous studies have shown that the frequency of the Arg⁹⁷² IRS-1 polymorphism was significantly higher in patients with angiographic evidence of coronary artery disease compared with control individuals (22). When adjusted for other risk factors, the relative risk of coronary artery disease associated with the Arg⁹⁷² IRS-1 polymorphism was 2.93-fold higher than in Gly/Gly carriers, and it increased to 6.97-fold in obese subjects and to 27.3-fold in subjects with clinical features of insulin-resistance syndrome (22). Interestingly, obese carriers of the Arg⁹⁷² IRS-1 polymorphism are characterized by decreased insulin sensitivity associated with a clustering of metabolic cardiovascular risk factors, including elevated fasting levels of plasma glucose, serum triglyceride, and plasma tissue plasminogen activator and its inhibitor, thus suggesting that the polymorphism potentiates obesity-linked insulin resistance (23). Finally, a recent genome-wide linkage analysis of the acute coronary syndrome has mapped a susceptibility locus on chromosome 2q33-q37.3, which harbors the gene encoding IRS-1 (24). Taken together, these observations strongly suggest that, by inducing endothelial dysfunction, the Arg⁹⁷² IRS-1 polymorphism may contribute to the genetic predisposition to develop cardiovascular disease.

	Footnotes

 
This work was supported in part by grants from European Community "EuroDiabetesGene" Grants QLG1-CT-1999-00674 (to G.S.) and PRIN-COFIN 2000 MM06A92341-002 (to F.P.).

Abbreviations: ACh, Acetylcholine; BMI, body mass index; BP, blood pressure; eNOS, endothelial nitric oxide synthase; FBF, forearm blood flow; HOMA, homeostasis model assessment; HR, heart rate; IRS-1, insulin receptor substrate 1; PI-3, phosphatidylinositol-3; SNP, sodium nitroprusside; VR, vascular resistance.

Received December 16, 2003.

Accepted March 29, 2004.

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