Advertisement

A polymorphism at IGF1 locus is associated with carotid intima media thickness and endothelium-dependent vasodilatation

      Highlights

      • The rs35767 polymorphism near IGF1 is associated with plasma IGF-1 levels.
      • GG genotype carriers have higher values of carotid intima media thickness.
      • GG genotype carriers have impaired endothelium-dependent vasodilatation.
      • IGF-1 may plays a role in the pathogenesis of atherosclerosis.

      Abstract

      Objective

      Whether IGF-1 has a protective or a detrimental role in vascular homeostasis remains unsettled. There is evidence that the C/T polymorphism rs35767 near the promoter region of the IGF1 gene located in chromosome 12 is associated with plasma IGF-1 levels. We investigated the effects of this polymorphism on circulating IGF-1 levels, carotid intima media thickness (cIMT) and endothelial-dependent vasodilation.

      Methods

      Two samples of adult nondiabetic Whites were studied. Sample 1 comprised 1124 individuals in whom cIMT was measured by ultrasonography. Sample 2 included 162 drug-naïve hypertensive individuals in whom endothelium-dependent and endothelium-independent vasodilation were assessed by intra-arterial infusion of acetylcholine (ACh), and sodium nitroprusside (SNP), respectively. IGF-1 was determined by chemiluminescent immunoassay. rs35767 polymorphism was screened using a TaqMan allelic discrimination assay.

      Results

      In sample 1, IGF-1 levels were higher in subjects carrying the T allele compared with CC carriers (178 ± 78 vs. 166 ± 60 ng/mL, respectively; P = 0.007 adjusted for age, gender, and BMI). cIMT was lower in subjects carrying the T allele compared with CC carriers (0.71 ± 0.20 vs. 0.76 ± 0.22 mm, respectively; P < 0.0001 adjusted for age, gender, and BMI). In sample 2, maximally ACh-stimulated forearm blood flow was higher in subjects carrying the T allele compared with CC carriers (343 ± 191 vs. 281 ± 125%, respectively; P = 0.02 adjusted for age, gender, and BMI).

      Conclusion

      Subjects carrying the T allele exhibited significantly higher levels of circulating IGF-1, lower values of cIMT, and higher endothelium-dependent vasodilatation compared with CC carriers. These findings support the idea that IGF-1 plays a role in the pathogenesis of atherosclerosis.

      Keywords

      Abbreviations:

      SNP (single nucleotide polymorphism), cIMT (carotid intima media thickness), EDV (endothelial-dependent vasodilation), FBF (forearm blood flow)
      Numerous experimental and clinical studies suggest that insulin-like growth factor-1 (IGF-1) system may be implicated in cardiovascular disease [
      • Copeland K.C.
      • Nair K.S.
      Recombinant human insulin-like growth factor-I increases forearm blood flow.
      ,
      • Fryburg D.A.
      NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF-I possible role of nitric oxide in muscle protein synthesis.
      ,
      • Chisalita S.I.
      • Arnqvist H.J.
      Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells.
      ,
      • Haylor J.
      • Singh I.
      • el Nahas A.M.
      Nitric oxide synthesis inhibitor prevents vasodilation by insulin-like growth factor I.
      ,
      • Walsh F.
      • Barazi M.
      • Pete G.
      • Muniyappa R.
      • Dunbar J.C.
      • Sowers J.R.
      Insulin-like growth factor-I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide.
      ,
      • Tivesten Å.
      • Bollanom E.
      • Andersson I.
      • et al.
      Liver-derived insulin-like growth factor-1 is involved in the regulation of blood pressure in mice.
      ]. Infusion of IGF-1 into human brachial arteries increases blood flow through a nitric oxide (NO)-dependent mechanism [
      • Copeland K.C.
      • Nair K.S.
      Recombinant human insulin-like growth factor-I increases forearm blood flow.
      ,
      • Fryburg D.A.
      NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF-I possible role of nitric oxide in muscle protein synthesis.
      ], and, in human endothelial cells, IGF-1, interacting with its receptor, stimulates nitric oxide (NO) production [
      • Chisalita S.I.
      • Arnqvist H.J.
      Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells.
      ]. Inhibitors of NO biosynthesis prevented IGF-1-induced vasodilatation in rats [
      • Haylor J.
      • Singh I.
      • el Nahas A.M.
      Nitric oxide synthesis inhibitor prevents vasodilation by insulin-like growth factor I.
      ,
      • Walsh F.
      • Barazi M.
      • Pete G.
      • Muniyappa R.
      • Dunbar J.C.
      • Sowers J.R.
      Insulin-like growth factor-I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide.
      ], and liver-specific IGF-1 knockout mice exhibited impaired acetylcholine-induced vasorelaxation of resistance vessels [
      • Tivesten Å.
      • Bollanom E.
      • Andersson I.
      • et al.
      Liver-derived insulin-like growth factor-1 is involved in the regulation of blood pressure in mice.
      ]. Lower plasma IGF-1 concentrations have been associated with many cardiovascular risk factors, including abnormal glucose homeostasis/diabetes [
      • Sandhu M.S.
      • Heald A.H.
      • Gibson J.M.
      • Cruickshank J.K.
      • Dunger D.B.
      • Wareham N.J.
      Circulating concentrations of insulin-like growth factor-I and development of glucose intolerance: a prospective observational study.
      ,
      • Perticone F.
      • Sciacqua A.
      • Tassone E.J.
      • et al.
      One-hour post-load plasma glucose and IGF-1 in hypertensive patients.
      ] insulin resistance [
      • Succurro E.
      • Andreozzi F.
      • Marini M.A.
      • et al.
      Low plasma insulin-like growth factor-1 levels are associated with reduced insulin sensitivity and increased insulin secretion in nondiabetic subjects.
      ], lower HDL cholesterol [
      • Succurro E.
      • Arturi F.
      • Grembiale A.
      • et al.
      Positive association between plasma insulin-like growth factor-1 and HDL cholesterol levels in adult nondiabetic subjects.
      ], metabolic syndrome [
      • Sesti G.
      • Sciacqua A.
      • Cardellini M.
      • et al.
      Plasma concentration of insulin-like growth factor-I is independently associated with insulin sensitivity in subjects with different degree of glucose tolerance.
      ], metabolically abnormal obesity [
      • Marini M.A.
      • Succurro E.
      • Frontoni S.
      • et al.
      Metabolically healthy but obese women have an intermediate cardiovascular risk profile between healthy non-obese women and obese insulin resistant women.
      ], endothelial dysfunction [
      • Perticone F.
      • Sciacqua A.
      • Perticone M.
      • et al.
      Low plasma insulin-like growth factor-1 levels are associated with impaired endothelium-dependent vasodilatation in a cohort of untreated hypertensive Caucasian subjects.
      ], left ventricular hypertrophy [
      • Sesti G.
      • Sciacqua A.
      • Scozzafava A.
      • et al.
      Effects of growth hormone and insulin-like growth factor-1 on cardiac hypertrophy of hypertensive patients.
      ], inflammatory factors [
      • Succurro E.
      • Andreozzi F.
      • Sciaqua A.
      • Hribal M.L.
      • Perticone F.
      • Sesti G.
      Reciprocal association of plasma insulin-like growth factor-1 and interleukin-6 levels with cardio-metabolic risk factors in nondiabetic subjects.
      ], and nonalcoholic fatty liver disease [
      • Arturi F.
      • Succurro E.
      • Procopio C.
      • et al.
      Nonalcoholic fatty liver disease is associated with low circulating levels of insulin-like growth factor-I.
      ,
      • Hribal M.L.
      • Procopio T.
      • Petta S.
      • et al.
      Insulin-like growth factor-1, inflammatory proteins, and fibrosis in subjects with nonalcoholic fatty liver disease.
      ], all of which are potential contributors to the increased risk of cardiovascular disease associated with low plasma IGF-1 levels. Despite these and several other observations suggesting that low circulating IGF-1 levels within the normal range have a role in the development of cardiovascular diseases, prior studies examining the clinical outcomes of lower plasma IGF-1 concentrations have yielded mixed results. Cross-sectional studies using subclinical measures of atherosclerosis have reported an inverse relationship between circulating IGF-1 levels and carotid intima media thickness (cIMT) in healthy subjects [
      • Colao A.
      • Spiezia S.
      • Di Somma C.
      • et al.
      Circulating insulin-like growth factor-I levels are correlated with the atherosclerotic profile in healthy subjects independently of age.
      ] or atherosclerotic plaques in elderly subjects [
      • Janssen J.A.
      • Stolk R.P.
      • Pols H.A.
      • Grobbee D.E.
      • Lamberts S.W.
      Serum total IGF-I, free IGF-I, and IGFB-1 levels in an elderly population: relation to cardiovascular risk factors and disease.
      ], and low circulating IGF-1 concentrations have been associated with angiographically documented coronary artery disease [
      • Spallarossa P.
      • Brunelli C.
      • Minuto F.
      • et al.
      Insulin-like growth factor-I and angiographically documented coronary artery disease.
      ]. Moreover, in individuals with acute myocardial infarction, serum IGF-1 levels on admission to hospital were reduced compared with matched controls, and were associated with subsequent adverse events including death, recurrent ischemia, revascularization and sustained ventricular tachycardia [
      • Conti E.
      • Andreotti F.
      • Sciahbasi A.
      • et al.
      Markedly reduced insulin-like growth factor-I in the acute phase of myocardial infarction.
      ]. Several prospective studies have suggested that low circulating levels of IGF-1 are associated with increased risk of ischemic heart disease [
      • Juul A.
      • Scheike T.
      • Davidsen M.
      • Gyllenborg
      • Jørgensen T.
      Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: a population-based case-control study.
      ], nonfatal myocardial infarction [
      • Kaplan R.C.
      • McGinn A.P.
      • Pollak M.N.
      • et al.
      Association of total insulin-like growth factor-I, insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-3 levels with incident coronary events and ischemic stroke.
      ], ischemic heart disease mortality [
      • Laughlin G.A.
      • Barrett-Connor E.
      • Criqui M.H.
      • Kritz-Silverstein D.
      The prospective association of serum insulin-like growth factor I (IGF-I) and IGF-binding protein-1 levels with all cause and cardiovascular disease mortality in older adults: the Rancho Bernardo Study.
      ], and cardiovascular mortality [
      • Svensson J.
      • Carlzon D.
      • Petzold M.
      • et al.
      Both low and high serum IGF-I levels associate with cancer mortality in older men.
      ].
      In contrast with these findings, other studies have reported that high circulating IGF-1 concentrations are independently associated with angiographically assessed coronary heart disease [
      • Fischer F.
      • Schulte H.
      • Mohan S.
      • et al.
      Associations of insulin-like growth factors, insulin-like growth factor binding proteins and acid-labile subunit with coronary heart disease.
      ], common carotid artery IMT [
      • Kawachi S.
      • Takeda N.
      • Sasaki A.
      • et al.
      Circulating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 are associated with early carotid atherosclerosis.
      ], and coronary artery disease progression in young male survivors of myocardial infarction [
      • Ruotolo G.
      • Båvenholm P.
      • Brismar K.
      • et al.
      Serum insulin-like growth factor-I level is independently associated with coronary artery disease progression in young male survivors of myocardial infarction: beneficial effects of bezafibrate treatment.
      ]. In addition, in a cross-sectional study carried out in 6773 primary care patients, circulating IGF-1 concentrations were reported to be associated with increased risk for coronary artery disease [
      • Schneider H.J.
      • Klotsche J.
      • Saller B.
      • et al.
      Associations of age-dependent IGF-I SDS with cardiovascular diseases and risk conditions: cross-sectional study in 6773 primary care patients.
      ]. Thus, whether IGF-1 has a protective or a detrimental role in vascular homeostasis remains a subject of debate.
      In an attempt to clarify this issue, we took advantage of the opportunity to study the association of an IGF-1-raising polymorphism (rs35767) near the promoter region of the IGF1 gene located in human chromosome 12 with early signs of atherosclerosis, and with endothelial-dependent vasodilation. Previous studies have repeatedly reported that carriers of the T allele of polymorphism rs35767 have increased levels of circulating IGF-1 as compared with CC carriers [
      • Canzian F.
      • McKay J.D.
      • Cleveland R.J.
      • et al.
      Polymorphisms of genes coding for insulin-like growth factor 1 and its major binding proteins, circulating levels of IGF-1 and IGFBP-3 and breast cancer risk: results from the EPIC study.
      ,
      • Palles C.
      • Johnson N.
      • Coupland B.
      • et al.
      Identification of genetic variants that influence circulating IGF1 levels: a targeted search strategy.
      ,
      • Patel A.V.
      • Cheng I.
      • Canzian F.
      • et al.
      IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3).
      ,
      • Ollberding N.J.
      • Cheng I.
      • Wilkens L.R.
      • et al.
      Genetic variants, prediagnostic circulating levels of insulin-like growth factors, insulin, and glucose and the risk of colorectal cancer: the Multiethnic Cohort study.
      ]. In the present study, we investigated the effects of the rs35767 polymorphism on circulating IGF-1 levels, carotid artery IMT, and endothelial-dependent vasodilation in two cohorts of nondiabetic White Europeans.

      1. Methods

      Study subjects. Two different samples of adult (≥20 years of age) nondiabetic individuals of European ancestry were studied.
      Two cohorts were enrolled: sample 1 comprised 1124 nondiabetic individuals, and sample 2 included 162 drug-naïve hypertensive individuals. cIMT was measured by ultrasonography in sample 1.
      Sample 1 comprised 1124 nondiabetic individuals consecutively recruited at the Department of Systems Medicine of the University of Rome-Tor Vergata and at the Department of Medical and Surgical Sciences of the University “Magna Graecia” of Catanzaro [
      • Scarpelli D.
      • Cardellini M.
      • Andreozzi F.
      • et al.
      Variants of the interleukin-10 promoter gene are associated with obesity and insulin resistance but not type 2 diabetes mellitus in Caucasian Italian subjects.
      ]. The inclusion criteria were: fasting plasma glucose <126 mg/dL and presence of one or more cardio-metabolic risk factors including, hypertension, dyslipidemia, and overweight/obesity. Exclusion criteria comprised: history of any malignant disease, end stage renal disease, chronic gastrointestinal diseases, chronic pancreatitis, positivity for antibodies to hepatitis C virus (HCV) or hepatitis B surface antigen (HBsAg), and use of medications able to modify glucose metabolism including corticosteroids, glucose-lowering, lipid-lowering and antihypertensive therapies. After 12-h overnight fasting, subjects underwent anthropometrical evaluation and a venous blood sample was drawn for laboratory determinations. Total serum IGF-1 concentrations were determined by chemiluminescent immunoassay (Nichols Institute Diagnostic, San Juan Capistrano, CA).
      High resolution B-mode ultrasound was used to measure IMT of the common carotid artery by using an ATL HDI 3000 ultrasound system (Advanced Technology Laboratories, Bothell, WA) equipped with a 7.5 MHz transducer, as previously described [
      • Cardellini M.
      • Marini M.A.
      • Frontoni S.
      • et al.
      Carotid artery intima-media thickness is associated with insulin-mediated glucose disposal in nondiabetic normotensive offspring of type 2 diabetic patients.
      ]. Manual measurements were conducted in plaque-free portions of the 10-mm linear segment proximal to the carotid bulb. Plaque was defined as a clearly isolated focal thickening of the intima-media layer with a thickness >1.3 mm. For each subject two measurements were performed bilaterally, and the values were averaged. Ultrasound examination was performed by a single skilled examiner who was unaware of the subjects' clinical and laboratory findings. The intra-observer variability of IMT measurements was tested in 120 randomly chosen scans; the correlations between the two readings was r = 0.95, and the absolute mean difference was 4.6 ± 2.6% A value of cIMT >0.9 mm was used as index of vascular abnormality according to the 2013 Guidelines for the management of arterial hypertension released by the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC) [
      • Mancia G.
      • De Backer G.
      • Dominiczak A.
      • et al
      2013 ESH/ESC guidelines for the management of arterial hypertension: the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC).
      ].
      In order to get further insights on the role of the rs35767 polymorphism on vascular function, an additional sample of nondiabetic individuals was analyzed. Sample 2 included 162 drug-naïve hypertensive individuals consecutively recruited at the Department of Medical and Surgical Sciences of the University “Magna Graecia” of Catanzaro as previously described [
      • Perticone F.
      • Sciacqua A.
      • Scozzafava A.
      • et al.
      Impaired endothelial function in never-treated hypertensive subjects carrying the Arg972 polymorphism in the Insulin Receptor Substrate-1 Gene.
      ]. None of the subjects had a history or clinical evidence of angina, myocardial infarction, valvular heart disease, diabetes mellitus, hypercholesterolemia, peripheral vascular disease, or coagulopathy. Forearm blood flow (FBF) measurements were performed by strain-gauge plethysmography accordingly to a previously described method [
      • Perticone F.
      • Maio R.
      • Sciacqua A.
      • et al.
      Endothelial dysfunction and CRP are risk factors for diabetes in essential hypertension.
      ,
      • Perticone F.
      • Sciacqua A.
      • Maio R.
      • et al.
      Endothelial dysfunction, ADMA and insulin resistance in essential hypertension.
      ]. FBF and blood pressure were assessed during intra-arterial infusion of saline, acetylcholine (ACh), and sodium nitroprusside (SNP). Endothelium-dependent and -independent vasodilations were measured by a dose–response curve to intra-arterial ACh (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. Each patient's FBF maximal response to ACh or SNP was considered for statistical analysis. Forearm vascular resistance (VR), expressed in arbitrary units (U), was calculated by dividing mean blood pressure at each dose point by FBF. Inter- and intra-observer variability was 3.3 and 2.7%, respectively.
      The studies were approved by Institutional Ethics Committees and written informed consent was obtained from each subject in accordance with principles of the Declaration of Helsinki.

      1.1 DNA analysis

      DNA was isolated from whole blood using commercial DNA isolation kit (Promega, Madison, WI). Screening of rs35767 polymorphism was performed using a TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA). TaqMan genotyping reaction was amplified on a GeneAmp PCR system 2700 and fluorescence was detected using an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA). Genotyping quality was tested by including 3 HapMap samples in each 96-well plate. The agreement rate with the HapMap database genotypes was >99%.

      1.2 Statistical analysis

      Due to a skewed distribution, triglycerides values were logarithmically transformed before the statistical analyses. The results for continuous variables are given as means ± SD. Categorical variables were compared by χ2-test. Differences of continuous variables between groups were tested after adjusting for confounding factors such as age, gender, adiposity, blood pressure, fasting glucose, and lipid levels by ANCOVA (general linear model). The Hardy–Weinberg equilibrium between the genotypes was evaluated by χ2 test. Genotype distributions were in Hardy–Weinberg equilibrium (P > 0.05). Power calculations were performed with Quanto version 1.2.4 (http://hydra.usc.edu/gxe; accessed 25 July 2011). The study had 83% power (for α = 0.05) to detect a 0.04 mm change in carotid IMT per allele T in sample 1 according to a dominant model. Associations between the rs35767 polymorphism and cIMT are presented as effect sizes (β and SE) per IGF-1–raising allele, estimated by multiple linear regression analysis adjusted for various confounders. A multivariable logistic regression analysis was used to determine the association between the genotypes and vascular abnormality. A P value <0.05 was considered as significant. All analyses were performed using the SPSS software program Version 16.0 for Windows.

      2. Results

      Clinical characteristics of sample 1 according to the SNP rs35767 near IGF1 are presented in Table 1. Because of the small number of TT individuals and the a priori hypothesis based on the dominant effect observed in previous studies [
      • Canzian F.
      • McKay J.D.
      • Cleveland R.J.
      • et al.
      Polymorphisms of genes coding for insulin-like growth factor 1 and its major binding proteins, circulating levels of IGF-1 and IGFBP-3 and breast cancer risk: results from the EPIC study.
      ,
      • Palles C.
      • Johnson N.
      • Coupland B.
      • et al.
      Identification of genetic variants that influence circulating IGF1 levels: a targeted search strategy.
      ,
      • Patel A.V.
      • Cheng I.
      • Canzian F.
      • et al.
      IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3).
      ], association between the rs35767 polymorphism and cIMT was analyzed according to both a dominant and an additive genetic model (Table 1). IGF-1 levels were significantly higher in subjects carrying the T allele as compared with carriers of the CC genotype (178 ± 78 vs. 166 ± 60 ng/mL, respectively; P = 0.007 after adjusting for age, gender, and BMI). The rs35767 polymorphism did not show any significant association with age, gender, BMI, smoking habit, waist circumference, blood pressure, fasting plasma glucose, and lipids levels (Table 1). cIMT was significantly lower in subjects carrying the T allele as compared with carriers of the CC genotype (0.71 ± 0.20 vs. 0.76 ± 0.22 mm, respectively; P < 0.0001 after adjusting for age, gender, and BMI) (Table 1). To estimate the independent contribution of the rs35767 polymorphism to cIMT, we carried out a linear regression analysis in a model which also included cardio-metabolic factors affecting vasculature including gender, age, BMI, waist circumference, smoking habit, blood pressure, fasting plasma glucose, and lipids levels. Comparison of standardized coefficients allowed the determination of the relative strength of each trait association with cIMT (listed from strongest to weakest): age (β = 0.41, P < 0.0001), BMI (β = 0.21, P < 0.0001), the rs35767 polymorphism (β = 0.11, P < 0.0001), male gender (β = 0.10, P = 0.002), smoking habit (β = 0.09, P = 0.002), waist circumference (β = 0.09, P = 0.01), and systolic blood pressure (β = 0.07, P = 0.05). These factors explained 25.6% of the variance in cIMT.
      Table 1Clinical characteristics of 1124 study subjects (sample 1) according to the SNP rs35767 near IGF1.
      VariablesCCCTTTP*P* (C/C vs. C/T + T/T)
      Male/female368/367159/17725/280.670.37
      Age (yr)46 ± 1246 ± 1246 ± 120.63§0.37§
      BMI (kg/m2)29.1 ± 6.129.2 ± 6.229.7 ± 5.80.80#0.82#
      Waist circumference (cm)97 ± 1397 ± 15101 ± 150.09#0.17#
      Current smokers (%)21.523.322.60.810.53
      SBP (mmHg)130 ± 17129 ± 18130 ± 160.910.77
      DBP (mmHg)82 ± 1181 ± 1181 ± 100.470.23
      Fasting glucose (mg/dL)92 ± 1092 ± 1093 ± 110.720.59
      Total cholesterol (mg/dL)203 ± 38200 ± 37200 ± 330.310.13
      HDL (mg/dL)51 ± 1351 ± 1450 ± 100.570.52
      Triglycerides (mg/dL)127 ± 68123 ± 66120 ± 520.68*0.43
      IGF-1 (ng/mL)166 ± 60176 ± 81186 ± 610.010.007
      Intima-media thickness (mm)0.76 ± 0.220.71 ± 0.200.69 ± 0.19<0.0001<0.0001
      Vascular abnormality (cIMT >0.9 mm), N. (%)147 (76.6%)39 (20.3%)6 (3.1%)0.002<0.0001
      Data are means ± SD. Triglycerides were log transformed for statistical analysis, but values in the table represent a back transformation to the original scale. Categorical variables were compared by χ2 test. Comparisons between the three groups were performed using a general linear model. *P values refer to results after analyses with adjustment for age, gender, and BMI. §P values refer to results after analyses with adjustment for gender. #P values refer to results after analyses with adjustment for age, and gender using a general linear model.
      197 subjects had vascular abnormality defined as a value of IMT >0.9 mm (Table 1). A lower proportion of subjects carrying the T allele exhibited vascular abnormality as compared with carriers of the CC (P < 0.0001) (Table 1). In a logistic regression model adjusted for gender, age, BMI, waist circumference, smoking habit, blood pressure, fasting plasma glucose, and lipids levels, subjects carrying the T allele had a lower risk of having vascular abnormality as compared with carriers of the CC genotype (OR 0.39, 95%CI 0.25–0.59; P < 0.0001).
      In order to get further insights on the role of the rs35767 polymorphism on vascular function, we examined the association of this SNP with endothelium-dependent and -independent vasodilation measured by strain-gauge plethysmography in an additional sample of never-treated hypertensive subjects. Clinical characteristics of sample 2 according to the SNP rs35767 near IGF1 are presented in Table 2. The rs35767 polymorphism did not show any significant association with age, gender, BMI, smoking habit, blood pressure, fasting plasma glucose, and lipids levels (Table 2). The baseline FBF and VR values did not differ between the genotypes (Table 2). Intra-arterial infusion of ACh caused a significant dose-dependent increase in FBF in the three groups of subjects; however, maximally ACh-stimulated FBF was significantly higher in subjects carrying the T allele as compared with carriers of the CC genotype (343 ± 191 vs. 281 ± 125%, respectively; P = 0.02 after adjusting for age, gender, and BMI) (Fig. 1 and Table 1). The differences in maximally ACh-stimulated FBF remained significant (P = 0.007) after adjusting for other well-known modulators of endothelial function in addition to age, gender, and BMI including smoking habit, fasting plasma glucose, total cholesterol, HDL, triglycerides, and blood pressure. Increasing doses of intra-arterial infusion of SNP induced a significant increase in FBF in the three groups of subjects, but no significant differences were observed between carriers of the CC genotype and subjects carrying the T allele (Fig. 1 and Table 1).
      Table 2Clinical characteristics of 176 study subjects (sample 2) according to the SNP rs35767 near IGF1.
      VariablesCCCTTTP*P* (C/C vs. C/T + T/T)
      Male/female77/3826/141/60.010.25
      Age (yr)50 ± 1148 ± 1153 ± 130.43§0.55§
      BMI (kg/m2)29.4 ± 5.630.1 ± 5.130.9 ± 9.80.48#0.26#
      Current smokers (%)55 (47.8)14 (35.0)2 (28.6)0.260.10
      SBP (mmHg)143 ± 18140 ± 15142 ± 150.600.32
      DBP (mmHg)89 ± 1187 ± 1083 ± 90.210.10
      Fasting glucose (mg/dL)94 ± 1092 ± 1194 ± 120.300.18
      Total cholesterol (mg/dL)208 ± 33212 ± 39199 ± 350.450.93
      HDL (mg/dL)47 ± 1351 ± 1647 ± 170.180.19
      Triglycerides (mg/dL)133 ± 65114 ± 45129 ± 370.160.11
      Baseline FBF (mL 100 mL−1 of tissue·min−1)3.1 ± 0.82.99 ± 0.753.6 ± 1.10.190.45
      Vascular resistance (U)36.1 ± 11.735.7 ± 12.528.2 ± 7.60.180.29
      FBF after ACh infusion at 7.5 μg mL−1 min−1 (% of increase over basal)67 ± 5269 ± 6291 ± 590.630.85
      FBF after ACh infusion at 15 μg mL−1 min−1 (% of increase over basal)152 ± 93162 ± 121228 ± 1140.490.42
      FBF after ACh infusion at 30 μg mL−1 min−1 (% of increase over basal)281 ± 125326 ± 186443 ± 2090.030.02
      FBF after sodium nitroprusside infusions at 0.8 μg mL−1 min−1 (% of increase over basal)78 ± 5591 ± 5587 ± 280.500.24
      FBF after sodium nitroprusside infusions at 1.6 μg mL−1 min−1 (% of increase over basal)160 ± 93175 ± 62174 ± 570.730.45
      FBF after sodium nitroprusside infusions at 3.2 μg mL−1 min−1 (% of increase over basal)298 ± 162334 ± 108334 ± 1400.610.33
      Data are means ± SD. Triglycerides were log transformed for statistical analysis, but values in the table represent a back transformation to the original scale. Categorical variables were compared by χ2 test. Comparisons between the three groups were performed using a general linear model. *P values refer to results after analyses with adjustment for age, gender, and BMI. §P values refer to results after analyses with adjustment for gender. #P values refer to results after analyses with adjustment for age, and gender using a general linear model. FBF: forearm blood flow. ACh: acetylcholine.
      Figure thumbnail gr1
      Fig. 1Responses of maximally-stimulated forearm blood flow (FBF) to intra-arterial infusions of acetylcholine and sodium nitroprusside in carriers of the CC genotype and subjects carrying the T allele.

      3. Discussion

      There is some evidence from cell biology and clinical research suggesting that IGF-1 may have beneficial actions on the vasculature [
      • Copeland K.C.
      • Nair K.S.
      Recombinant human insulin-like growth factor-I increases forearm blood flow.
      ,
      • Fryburg D.A.
      NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF-I possible role of nitric oxide in muscle protein synthesis.
      ,
      • Chisalita S.I.
      • Arnqvist H.J.
      Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells.
      ,
      • Haylor J.
      • Singh I.
      • el Nahas A.M.
      Nitric oxide synthesis inhibitor prevents vasodilation by insulin-like growth factor I.
      ,
      • Walsh F.
      • Barazi M.
      • Pete G.
      • Muniyappa R.
      • Dunbar J.C.
      • Sowers J.R.
      Insulin-like growth factor-I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide.
      ,
      • Tivesten Å.
      • Bollanom E.
      • Andersson I.
      • et al.
      Liver-derived insulin-like growth factor-1 is involved in the regulation of blood pressure in mice.
      ], but prior studies investigating major outcomes of low plasma IGF-1 concentrations have yielded conflicting results [
      • Colao A.
      • Spiezia S.
      • Di Somma C.
      • et al.
      Circulating insulin-like growth factor-I levels are correlated with the atherosclerotic profile in healthy subjects independently of age.
      ,
      • Janssen J.A.
      • Stolk R.P.
      • Pols H.A.
      • Grobbee D.E.
      • Lamberts S.W.
      Serum total IGF-I, free IGF-I, and IGFB-1 levels in an elderly population: relation to cardiovascular risk factors and disease.
      ,
      • Spallarossa P.
      • Brunelli C.
      • Minuto F.
      • et al.
      Insulin-like growth factor-I and angiographically documented coronary artery disease.
      ,
      • Conti E.
      • Andreotti F.
      • Sciahbasi A.
      • et al.
      Markedly reduced insulin-like growth factor-I in the acute phase of myocardial infarction.
      ,
      • Juul A.
      • Scheike T.
      • Davidsen M.
      • Gyllenborg
      • Jørgensen T.
      Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: a population-based case-control study.
      ,
      • Kaplan R.C.
      • McGinn A.P.
      • Pollak M.N.
      • et al.
      Association of total insulin-like growth factor-I, insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-3 levels with incident coronary events and ischemic stroke.
      ,
      • Laughlin G.A.
      • Barrett-Connor E.
      • Criqui M.H.
      • Kritz-Silverstein D.
      The prospective association of serum insulin-like growth factor I (IGF-I) and IGF-binding protein-1 levels with all cause and cardiovascular disease mortality in older adults: the Rancho Bernardo Study.
      ,
      • Johnsen S.P.
      • Hundborg H.H.
      • Sorensen H.T.
      • et al.
      Insulin-like growth factor (IGF) I, -II, and IGF binding protein-3 and risk of ischemic stroke.
      ,
      • Svensson J.
      • Carlzon D.
      • Petzold M.
      • et al.
      Both low and high serum IGF-I levels associate with cancer mortality in older men.
      ,
      • Fischer F.
      • Schulte H.
      • Mohan S.
      • et al.
      Associations of insulin-like growth factors, insulin-like growth factor binding proteins and acid-labile subunit with coronary heart disease.
      ,
      • Kawachi S.
      • Takeda N.
      • Sasaki A.
      • et al.
      Circulating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 are associated with early carotid atherosclerosis.
      ,
      • Ruotolo G.
      • Båvenholm P.
      • Brismar K.
      • et al.
      Serum insulin-like growth factor-I level is independently associated with coronary artery disease progression in young male survivors of myocardial infarction: beneficial effects of bezafibrate treatment.
      ,
      • Schneider H.J.
      • Klotsche J.
      • Saller B.
      • et al.
      Associations of age-dependent IGF-I SDS with cardiovascular diseases and risk conditions: cross-sectional study in 6773 primary care patients.
      ]. Previous studies have shown that the rs35767 polymorphism near the IGF1 gene is associated with serum IGF-1 levels [
      • Canzian F.
      • McKay J.D.
      • Cleveland R.J.
      • et al.
      Polymorphisms of genes coding for insulin-like growth factor 1 and its major binding proteins, circulating levels of IGF-1 and IGFBP-3 and breast cancer risk: results from the EPIC study.
      ,
      • Palles C.
      • Johnson N.
      • Coupland B.
      • et al.
      Identification of genetic variants that influence circulating IGF1 levels: a targeted search strategy.
      ,
      • Patel A.V.
      • Cheng I.
      • Canzian F.
      • et al.
      IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3).
      ,
      • Ollberding N.J.
      • Cheng I.
      • Wilkens L.R.
      • et al.
      Genetic variants, prediagnostic circulating levels of insulin-like growth factors, insulin, and glucose and the risk of colorectal cancer: the Multiethnic Cohort study.
      ]. These observations coupled with the accessibility of carefully characterized cohorts of nondiabetic individuals have provided the rationale for addressing the question of whether the rs35767 polymorphism could be associated with cIMT, a reliable indicator of early atherosclerosis [
      • O’Leary D.H.
      • Polak J.F.
      • Kronmal R.A.
      • et al.
      Carotid artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults.
      ], and endothelial dysfunction, an early event in the developmental process leading to atherosclerosis. We found that, in a cohort of subjects of European ancestry, subjects carrying the T allele exhibited significantly higher levels of circulating IGF-1, lower values of cIMT, and lower risk of having vascular abnormality (defined as a value of IMT >0.9 mm) as compared with carriers of the CC genotype. The association between the rs35767 polymorphism near IGF1 and cIMT was not affected by other confounding cardio-metabolic risk factors such as gender, age, adiposity, smoking habit, blood pressure, fasting plasma glucose, and lipids levels. These risk factors have also been associated with both low IGF-1 levels [
      • Sandhu M.S.
      • Heald A.H.
      • Gibson J.M.
      • Cruickshank J.K.
      • Dunger D.B.
      • Wareham N.J.
      Circulating concentrations of insulin-like growth factor-I and development of glucose intolerance: a prospective observational study.
      ,
      • Perticone F.
      • Sciacqua A.
      • Tassone E.J.
      • et al.
      One-hour post-load plasma glucose and IGF-1 in hypertensive patients.
      ,
      • Succurro E.
      • Andreozzi F.
      • Marini M.A.
      • et al.
      Low plasma insulin-like growth factor-1 levels are associated with reduced insulin sensitivity and increased insulin secretion in nondiabetic subjects.
      ,
      • Succurro E.
      • Arturi F.
      • Grembiale A.
      • et al.
      Positive association between plasma insulin-like growth factor-1 and HDL cholesterol levels in adult nondiabetic subjects.
      ,
      • Sesti G.
      • Sciacqua A.
      • Cardellini M.
      • et al.
      Plasma concentration of insulin-like growth factor-I is independently associated with insulin sensitivity in subjects with different degree of glucose tolerance.
      ,
      • Marini M.A.
      • Succurro E.
      • Frontoni S.
      • et al.
      Metabolically healthy but obese women have an intermediate cardiovascular risk profile between healthy non-obese women and obese insulin resistant women.
      ,
      • Perticone F.
      • Sciacqua A.
      • Perticone M.
      • et al.
      Low plasma insulin-like growth factor-1 levels are associated with impaired endothelium-dependent vasodilatation in a cohort of untreated hypertensive Caucasian subjects.
      ,
      • Sesti G.
      • Sciacqua A.
      • Scozzafava A.
      • et al.
      Effects of growth hormone and insulin-like growth factor-1 on cardiac hypertrophy of hypertensive patients.
      ,
      • Succurro E.
      • Andreozzi F.
      • Sciaqua A.
      • Hribal M.L.
      • Perticone F.
      • Sesti G.
      Reciprocal association of plasma insulin-like growth factor-1 and interleukin-6 levels with cardio-metabolic risk factors in nondiabetic subjects.
      ,
      • Arturi F.
      • Succurro E.
      • Procopio C.
      • et al.
      Nonalcoholic fatty liver disease is associated with low circulating levels of insulin-like growth factor-I.
      ,
      • Hribal M.L.
      • Procopio T.
      • Petta S.
      • et al.
      Insulin-like growth factor-1, inflammatory proteins, and fibrosis in subjects with nonalcoholic fatty liver disease.
      ,
      • Colao A.
      • Spiezia S.
      • Di Somma C.
      • et al.
      Circulating insulin-like growth factor-I levels are correlated with the atherosclerotic profile in healthy subjects independently of age.
      ], and cardiovascular outcomes. The present data are in agreement with those of a prior study showing that a cytosine-adenine repeat polymorphism in the promoter region of the IGFI gene was associated with low circulating levels of IGF-1 and increased cIMT in hypertensive subjects [
      • Schult A.F.
      • Janssen J.A.
      • Deinum J.
      • et al.
      Polymorphism in the promoter region of the insulin-like growth factor 1 gene is related to carotid intima-media thickness and aortic pulse wave velocity in subjects with hypertension.
      ], confirming the potential role of lower IGF-1 levels in the pathogenesis of vascular abnormalities, in particular in subjects with hypertension.
      In human endothelial cells, IGF-1 stimulates NO production, contributing to the regulation of vascular function [
      • Chisalita S.I.
      • Arnqvist H.J.
      Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells.
      ], and infusion of IGF-1 in humans increases FBF through a NO-dependent mechanism [
      • Copeland K.C.
      • Nair K.S.
      Recombinant human insulin-like growth factor-I increases forearm blood flow.
      ,
      • Fryburg D.A.
      NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF-I possible role of nitric oxide in muscle protein synthesis.
      ]. The effects of IGF-1 on NO production are also mediated via the regulation of endothelial NO synthase expression [
      • Wickman A.
      • Jonsdottir I.H.
      • Bergstrom G.
      • Hedin L.
      GH and IGF-1 regulate the expression of endothelial nitric oxide synthase (eNOS) in cardiovascular tissues of hypophysectomized female rats.
      ]. Thus, a potential mechanism whereby low circulating IGF-1 levels associated with the rs35767 polymorphism could contribute to vascular damage is by impairing endothelium-dependent vasodilatation. We found that, in a cohort of never-treated hypertensive subjects, subjects carrying the T allele showed a higher endothelium-dependent vasodilatation as compared with carriers of the CC genotype. Importantly, the association remains significant after adjustment for the confounding effects of well-known modulators of endothelium function including age, gender, adiposity, smoking, fasting plasma glucose, lipids levels, and blood pressure. By contrast, no significant differences in endothelium-independent vasodilatation were observed between CC genotype carriers and subjects carrying the T allele.
      To the best of our knowledge, this is the first evidence demonstrating an association between a functional polymorphism at IGF1 gene and impaired vasodilator response to intra-arterial infusion of ACh. Our results are consistent with those observed in liver-specific IGF-1 knockout mice, which showed impaired endothelium-dependent vascular response to ACh [
      • Tivesten Å.
      • Bollanom E.
      • Andersson I.
      • et al.
      Liver-derived insulin-like growth factor-1 is involved in the regulation of blood pressure in mice.
      ]. However, we cannot exclude that the association between the rs35767 polymorphism and impaired vasodilator response to intra-arterial infusion of ACh is an accidental result due to the high number of comparisons.
      The strengths of the present study include the homogeneous population, the use of a gold standard technique for assessment of endothelial-dependent vasodilation [
      • Perticone F.
      • Sciacqua A.
      • Scozzafava A.
      • et al.
      Impaired endothelial function in never-treated hypertensive subjects carrying the Arg972 polymorphism in the Insulin Receptor Substrate-1 Gene.
      ,
      • Perticone F.
      • Maio R.
      • Sciacqua A.
      • et al.
      Endothelial dysfunction and CRP are risk factors for diabetes in essential hypertension.
      ,
      • Perticone F.
      • Sciacqua A.
      • Maio R.
      • et al.
      Endothelial dysfunction, ADMA and insulin resistance in essential hypertension.
      ], the exclusion of confounding conditions such as previous cardiovascular disease, history of malignant disease, and end stage renal disease, the inclusion of subjects without the confounding effects of medical therapy, known to modulate endothelial function such as aspirin, statins, beta-blockers, angiotensin II receptor blockers, and angiotensin converting enzyme inhibitors, the strict quality control of data collection by a trained staff following a standardized protocol, and the centralized assays of biochemical variables.
      Nevertheless, some limitations should be acknowledged in the interpretation of our results. Although cIMT is a simple and non-invasive measure of structural changes in the arterial wall and has been widely used as a surrogate marker of atherosclerotic disease [
      • O’Leary D.H.
      • Polak J.F.
      • Kronmal R.A.
      • et al.
      Carotid artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults.
      ], increasing evidence suggests that maximum IMT of internal carotid artery, and, particularly, carotid plaque had a higher diagnostic accuracy for the prediction of future coronary artery disease events [
      • Polak J.F.
      • Pencina M.J.
      • Pencina K.M.
      • O'Donnell C.J.
      • Wolf P.A.
      • Sr D'Agostino RB.
      Carotid-wall intima-media thickness and cardiovascular events.
      ,
      • Inaba Y.
      • Chen J.A.
      • Bergmann S.R.
      Carotid plaque, compared with carotid intima-media thickness, more accurately predicts coronary artery disease events: a meta-analysis.
      ]. A second limitation is that metabolic and cardiovascular risk factors, including circulating IGF-1 concentration, were measured once. Although such an approach is common in clinical research, intra-individual variation in cardio-metabolic variables cannot be taken into account. Furthermore, although the present findings are clinically and biologically plausible, causality cannot be inferred due to the cross-sectional nature of the study, which precludes us from drawing conclusions about the causal relationships between the rs35767 polymorphism and major cardiovascular outcomes. A further limitation of this study is represented by the robustness of our P-values which do not reach a genome-wide level of significance and are, therefore, still compatible with a false-positive result. Moreover, this is an observational study based on outpatients recruited at a referral university hospital, representing subjects at risk for cardio-metabolic disease, and, therefore, may not be extendible to the general population. Additionally, the study included only non-diabetic subjects, thus excluding from the analysis individuals at very high risk of cardiovascular disease such as patients with type 2 diabetes. Finally, our findings may apply only to white Europeans, and should not be extended to other ethnic groups. Indeed, differences exist in cardio-metabolic risk among different ethnic groups with Mexican Americans and African Americans having a less favorable cardio-metabolic risk profile than Whites, likely due to socio-demographic, lifestyle, anthropometric, and genetic characteristics. Therefore, the present findings should be considered hypothesis generating and requiring confirmation by further prospective studies including individuals of other ethnic groups. Nevertheless, we consider our findings important in attempting to understand the pathophysiological interaction between the rs35767 polymorphism near IGF1 and cardiovascular disease.

      Competing interest

      All authors declare that they have no conflict of interest.

      Authors' contribution

      G.S. designed the study, acquired, analyzed, and interpreted the data, and wrote the manuscript G.C.M. analyzed and interpreted the data, and reviewed the manuscript, M.P. and A.G., analyzed and interpreted the data, A.S., M.A.M. F.A. and F.P. acquired, and interpreted the data.

      Disclosure statement

      The authors have nothing to disclose.

      References

        • Copeland K.C.
        • Nair K.S.
        Recombinant human insulin-like growth factor-I increases forearm blood flow.
        J Clin Endocrinol Metab. 1994; 79: 230-232
        • Fryburg D.A.
        NG-monomethyl-L-arginine inhibits the blood flow but not the insulin-like response of forearm muscle to IGF-I possible role of nitric oxide in muscle protein synthesis.
        J Clin Invest. 1996; 7: 1319-1328
        • Chisalita S.I.
        • Arnqvist H.J.
        Insulin-like growth factor I receptors are more abundant than insulin receptors in human micro- and macrovascular endothelial cells.
        Am J Physiol Endocrinol Metab. 2004; 286: E896-E901
        • Haylor J.
        • Singh I.
        • el Nahas A.M.
        Nitric oxide synthesis inhibitor prevents vasodilation by insulin-like growth factor I.
        Kidney Int. 1991; 39: 333-335
        • Walsh F.
        • Barazi M.
        • Pete G.
        • Muniyappa R.
        • Dunbar J.C.
        • Sowers J.R.
        Insulin-like growth factor-I diminishes in vivo and in vitro vascular contractility: role of vascular nitric oxide.
        Endocrinology. 1996; 137: 1798-1803
        • Tivesten Å.
        • Bollanom E.
        • Andersson I.
        • et al.
        Liver-derived insulin-like growth factor-1 is involved in the regulation of blood pressure in mice.
        Endocrinology. 2002; 143: 4235-4242
        • Sandhu M.S.
        • Heald A.H.
        • Gibson J.M.
        • Cruickshank J.K.
        • Dunger D.B.
        • Wareham N.J.
        Circulating concentrations of insulin-like growth factor-I and development of glucose intolerance: a prospective observational study.
        Lancet. 2002; 359: 1740-1745
        • Perticone F.
        • Sciacqua A.
        • Tassone E.J.
        • et al.
        One-hour post-load plasma glucose and IGF-1 in hypertensive patients.
        Eur J Clin Invest. 2012; 42: 1325-1331
        • Succurro E.
        • Andreozzi F.
        • Marini M.A.
        • et al.
        Low plasma insulin-like growth factor-1 levels are associated with reduced insulin sensitivity and increased insulin secretion in nondiabetic subjects.
        Nutr Metab Cardiovasc Dis. 2009; 19: 713-719
        • Succurro E.
        • Arturi F.
        • Grembiale A.
        • et al.
        Positive association between plasma insulin-like growth factor-1 and HDL cholesterol levels in adult nondiabetic subjects.
        Eur J Endocrinol. 2010; 163: 75-80
        • Sesti G.
        • Sciacqua A.
        • Cardellini M.
        • et al.
        Plasma concentration of insulin-like growth factor-I is independently associated with insulin sensitivity in subjects with different degree of glucose tolerance.
        Diabetes Care. 2005; 28: 132-137
        • Marini M.A.
        • Succurro E.
        • Frontoni S.
        • et al.
        Metabolically healthy but obese women have an intermediate cardiovascular risk profile between healthy non-obese women and obese insulin resistant women.
        Diabetes Care. 2007; 30: 2145-2147
        • Perticone F.
        • Sciacqua A.
        • Perticone M.
        • et al.
        Low plasma insulin-like growth factor-1 levels are associated with impaired endothelium-dependent vasodilatation in a cohort of untreated hypertensive Caucasian subjects.
        J Clin Endocrinol Metab. 2008; 93: 2806-2810
        • Sesti G.
        • Sciacqua A.
        • Scozzafava A.
        • et al.
        Effects of growth hormone and insulin-like growth factor-1 on cardiac hypertrophy of hypertensive patients.
        J Hypertens. 2007; 25: 471-477
        • Succurro E.
        • Andreozzi F.
        • Sciaqua A.
        • Hribal M.L.
        • Perticone F.
        • Sesti G.
        Reciprocal association of plasma insulin-like growth factor-1 and interleukin-6 levels with cardio-metabolic risk factors in nondiabetic subjects.
        Diabetes Care. 2008; 31: 1886-1888
        • Arturi F.
        • Succurro E.
        • Procopio C.
        • et al.
        Nonalcoholic fatty liver disease is associated with low circulating levels of insulin-like growth factor-I.
        J Clin Endocrinol Metab. 2011; 96: E1640-E1644
        • Hribal M.L.
        • Procopio T.
        • Petta S.
        • et al.
        Insulin-like growth factor-1, inflammatory proteins, and fibrosis in subjects with nonalcoholic fatty liver disease.
        J Clin Endocrinol Metab. 2013; 98: E304-E308
        • Colao A.
        • Spiezia S.
        • Di Somma C.
        • et al.
        Circulating insulin-like growth factor-I levels are correlated with the atherosclerotic profile in healthy subjects independently of age.
        J Endocrinol Invest. 2005; 28: 440-448
        • Janssen J.A.
        • Stolk R.P.
        • Pols H.A.
        • Grobbee D.E.
        • Lamberts S.W.
        Serum total IGF-I, free IGF-I, and IGFB-1 levels in an elderly population: relation to cardiovascular risk factors and disease.
        Arterioscler Thromb Vasc Biol. 1998; 18: 277-282
        • Spallarossa P.
        • Brunelli C.
        • Minuto F.
        • et al.
        Insulin-like growth factor-I and angiographically documented coronary artery disease.
        Am J Cardiol. 1996; 77: 200-202
        • Conti E.
        • Andreotti F.
        • Sciahbasi A.
        • et al.
        Markedly reduced insulin-like growth factor-I in the acute phase of myocardial infarction.
        J Am Coll Cardiol. 2001; 38: 26-32
        • Juul A.
        • Scheike T.
        • Davidsen M.
        • Gyllenborg
        • Jørgensen T.
        Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: a population-based case-control study.
        Circulation. 2002; 106: 939-944
        • Kaplan R.C.
        • McGinn A.P.
        • Pollak M.N.
        • et al.
        Association of total insulin-like growth factor-I, insulin-like growth factor binding protein-1 (IGFBP-1), and IGFBP-3 levels with incident coronary events and ischemic stroke.
        J Clin Endocrinol Metab. 2007; 92: 1319-1325
        • Laughlin G.A.
        • Barrett-Connor E.
        • Criqui M.H.
        • Kritz-Silverstein D.
        The prospective association of serum insulin-like growth factor I (IGF-I) and IGF-binding protein-1 levels with all cause and cardiovascular disease mortality in older adults: the Rancho Bernardo Study.
        J Clin Endocrinol Metab. 2004; 89: 114-120
        • Johnsen S.P.
        • Hundborg H.H.
        • Sorensen H.T.
        • et al.
        Insulin-like growth factor (IGF) I, -II, and IGF binding protein-3 and risk of ischemic stroke.
        J Clin Endocrinol Metab. 2005; 90: 5937-5941
        • Svensson J.
        • Carlzon D.
        • Petzold M.
        • et al.
        Both low and high serum IGF-I levels associate with cancer mortality in older men.
        J Clin Endocrinol Metab. 2012; 97: 4623-4630
        • Fischer F.
        • Schulte H.
        • Mohan S.
        • et al.
        Associations of insulin-like growth factors, insulin-like growth factor binding proteins and acid-labile subunit with coronary heart disease.
        Clin Endocrinol (Oxf). 2004; 61: 595-602
        • Kawachi S.
        • Takeda N.
        • Sasaki A.
        • et al.
        Circulating insulin-like growth factor-1 and insulin-like growth factor binding protein-3 are associated with early carotid atherosclerosis.
        Arterioscler Thromb Vasc Biol. 2005; 25: 617-621
        • Ruotolo G.
        • Båvenholm P.
        • Brismar K.
        • et al.
        Serum insulin-like growth factor-I level is independently associated with coronary artery disease progression in young male survivors of myocardial infarction: beneficial effects of bezafibrate treatment.
        J Am Coll Cardiol. 2000; 35: 647-654
        • Schneider H.J.
        • Klotsche J.
        • Saller B.
        • et al.
        Associations of age-dependent IGF-I SDS with cardiovascular diseases and risk conditions: cross-sectional study in 6773 primary care patients.
        Eur J Endocrinol. 2008; 158: 153-161
        • Canzian F.
        • McKay J.D.
        • Cleveland R.J.
        • et al.
        Polymorphisms of genes coding for insulin-like growth factor 1 and its major binding proteins, circulating levels of IGF-1 and IGFBP-3 and breast cancer risk: results from the EPIC study.
        Br J Cancer. 2006; 94: 299-307
        • Palles C.
        • Johnson N.
        • Coupland B.
        • et al.
        Identification of genetic variants that influence circulating IGF1 levels: a targeted search strategy.
        Hum Mol Genet. 2008; 17: 1457-1464
        • Patel A.V.
        • Cheng I.
        • Canzian F.
        • et al.
        IGF-1, IGFBP-1, and IGFBP-3 polymorphisms predict circulating IGF levels but not breast cancer risk: findings from the Breast and Prostate Cancer Cohort Consortium (BPC3).
        PLoS One. 2008; 3: e2578
        • Ollberding N.J.
        • Cheng I.
        • Wilkens L.R.
        • et al.
        Genetic variants, prediagnostic circulating levels of insulin-like growth factors, insulin, and glucose and the risk of colorectal cancer: the Multiethnic Cohort study.
        Cancer Epidemiol Biomarkers Prev. 2012; 21: 810-820
        • Scarpelli D.
        • Cardellini M.
        • Andreozzi F.
        • et al.
        Variants of the interleukin-10 promoter gene are associated with obesity and insulin resistance but not type 2 diabetes mellitus in Caucasian Italian subjects.
        Diabetes. 2006; 55: 1529-1533
        • Cardellini M.
        • Marini M.A.
        • Frontoni S.
        • et al.
        Carotid artery intima-media thickness is associated with insulin-mediated glucose disposal in nondiabetic normotensive offspring of type 2 diabetic patients.
        Am J Physiol Endocrinol Metab. 2007; 292: E347-E352
        • Mancia G.
        • De Backer G.
        • Dominiczak A.
        • et al
        2013 ESH/ESC guidelines for the management of arterial hypertension: the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC).
        Eur Heart J. 2013; 34: 2159-2219
        • Perticone F.
        • Sciacqua A.
        • Scozzafava A.
        • et al.
        Impaired endothelial function in never-treated hypertensive subjects carrying the Arg972 polymorphism in the Insulin Receptor Substrate-1 Gene.
        J Clin Endocrinol Metab. 2004; 89: 3606-3609
        • Perticone F.
        • Maio R.
        • Sciacqua A.
        • et al.
        Endothelial dysfunction and CRP are risk factors for diabetes in essential hypertension.
        Diabetes. 2008; 57: 167-171
        • Perticone F.
        • Sciacqua A.
        • Maio R.
        • et al.
        Endothelial dysfunction, ADMA and insulin resistance in essential hypertension.
        Int J Cardiol. 2010; 142: 236-241
        • O’Leary D.H.
        • Polak J.F.
        • Kronmal R.A.
        • et al.
        Carotid artery intima and media thickness as a risk factor for myocardial infarction and stroke in older adults.
        N Engl J Med. 1999; 340: 14-22
        • Schult A.F.
        • Janssen J.A.
        • Deinum J.
        • et al.
        Polymorphism in the promoter region of the insulin-like growth factor 1 gene is related to carotid intima-media thickness and aortic pulse wave velocity in subjects with hypertension.
        Stroke. 2003; 34: 1623-1627
        • Wickman A.
        • Jonsdottir I.H.
        • Bergstrom G.
        • Hedin L.
        GH and IGF-1 regulate the expression of endothelial nitric oxide synthase (eNOS) in cardiovascular tissues of hypophysectomized female rats.
        Eur J Endocrinol. 2002; 147: 523-533
        • Polak J.F.
        • Pencina M.J.
        • Pencina K.M.
        • O'Donnell C.J.
        • Wolf P.A.
        • Sr D'Agostino RB.
        Carotid-wall intima-media thickness and cardiovascular events.
        N Engl J Med. 2011; 365: 213-221
        • Inaba Y.
        • Chen J.A.
        • Bergmann S.R.
        Carotid plaque, compared with carotid intima-media thickness, more accurately predicts coronary artery disease events: a meta-analysis.
        Atherosclerosis. 2012; 220: 128-133