Cardiovascular Research Advance Access published August 5, 2010

The TRIB3 R84 Variant is Associated with Increased Carotid Intima Media Thickness in vivo and with Enhanced MAPK Signalling in Human Endothelial Cells Gloria Formoso, Pamela Di Tomo, Francesco Andreozzi, Elena Succurro, Sara Di Silvestre, Sabrina Prudente, Francesco Perticone, Vincenzo Trischitta, Giorgio Sesti, Assunta Pandolfi, and Agostino Consoli

Department of Medicine and Aging Sciences University "G. d'Annunzio", Aging Research Center, Ce.S.I., "G. d'Annunzio" University Foundation, Chieti-Pescara, Italy (GF, AC)

Department of Biomedical Sciences, University "G. d'Annunzio", Aging Research Center, Ce.S.I., "G. d'Annunzio" University Foundation, Chieti-Pescara, Italy (PDT, SDS, AP)

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Catanzaro, Italy (FA,ES,FP,GS)

IRCCS Casa Sollievo della Sofferenza-Mendel Institute, Rome, Italy (SP, VT)

Research Unit of Diabetes and Endocrine Disease, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy (VT)

Address correspondence to: Prof. Agostino Consoli, MD, Dept. of Medicine and Aging Sciences Edificio CeSi, room 271, University of Chieti, via dei Vestini, 1, 66100 CHIETI (Italy) Tel +39 085 4219938 or +39 349 3600419, Fax +39 0871 541425, e.mail [email protected]

Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2010. For permissions please email: [email protected]

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Department of Experimental and Clinical Medicine, University Magna Græcia of Catanzaro,

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ABSTRACT AIM. TRIB3, a mammalian tribbles homolog, affects insulin signaling and action by inhibiting Akt phosphorylation. A TRIB3 Q84R gain-of-function polymorphism has been associated with insulin resistance both in vitro and in vivo and with several atherosclerotic phenotypes, including increased carotid Intima-Media Thickness (IMT). We wanted to replicate this latter association and, if so, to get deeper insights about the molecular mechanisms underlying the role of the TRIB3 Q84R polymorphism on atherosclerosis. METHODS AND RESULTS. In 430 Caucasians of European ancestry carotid IMT was increased in QR (n=116) and RR (n=15) as compared to QQ (n=299) subjects (P= 0.009), thus replicating similar data recently obtained among Asians. In human umbilical vein endothelial cells (HUVECs) naturally carrying the QQ genotype, 24-hour insulin stimulation, increased monocyte

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(ICAM-1) expression and MAP-kinase kinase (MEK)-Mitogen-activated Protein Kinase (MAPK) activation. Conversely, QR- and RR-HUVECs had increased un-stimulated monocyte adhesion, VCAM-1 and ICAM-1 expression and MEK-MAPK activation which did not increase further upon insulin stimulation. In addition, QQ-, QR- and RR-HUVECs showed similar basal Akt phosphorylation and NOS activity which, however, were significantly increased by insulin only in QQ cells. CONCLUSIONS. TRIB3 R84 variant is associated with increased carotid IMT also in Caucasians, thus replicating previous data obtained in Asians. In addition, in HUVECs this variant is associated with unbalanced insulin signalling. This abnormality may favour vasoreactivity, intima-media thickening and plaque formation and may, therefore, underlie the deleterious role exerted by the variant on the susceptibility to atherosclerosis.

Keywords: insulin signalling, genetic, cardiovascular diseases, endothelium, atherosclerosis.

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adhesion, Vascular Cell Adhesion Molecule-1 (VCAM-1) and Intercellular Adhesion Molecules-1

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INTRODUCTION Endothelial insulin resistance is thought to contribute to endothelial dysfunction and atherosclerosis but the underlying mechanisms are not yet fully elucidated 1. Several insulin resistance-related conditions are characterized by a specific impairment of phosphatydilinositol 3-kinase (PI3K)/Akt-dependent signalling pathways, whereas alternative insulin-signalling pathways, such as ras/mitogen activated protein kinase (MAPK)-dependent pathways, are largely unaffected 2. It is conceivable that compensatory hyper-insulinemia secondary to a selective impairment of PI3K/Akt signalling leads, to enhanced activation of MAPK-dependent pathway, tipping the balance of insulin’s vascular effects so to favour abnormal vasoreactivity (hypertension) and vascular growth (hypertrophy and plaque formation)3. Furthermore, enhanced signalling along MAPK-dependent pathways might increase endothelial leukocytes adhesion

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smooth muscle cells (vSMC) migration into the intima 3. All these abnormalities are implicated in the development and progression of macro- and microvascular complications 4. Several genetic single nucleotide polymorphisms (SNPs) have been associated with 5-7

; among these is the TRIB3 Q84R SNP (rs2295490), which affects

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and in vivo 8. In line with its potential atherogenic role, the R84

endothelial insulin resistance insulin action both in vitro

variant was found to associate with several cardiovascular risk factors 8, including enhanced carotid intima-media thickness (IMT) 11. As a matter of fact, data obtained in cellular models indicate that the TRIB3 R84 variant is, indeed, a gain of function mutation which increases the ability of TRIB3 to impair insulin-mediated Akt phosphorylation and downstream insulin signalling

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. Of note,

such inhibitory effect was observed also in human umbilical vein endothelial cells (HUVECs) naturally carrying the R84 variant, which are characterized not only by impaired insulin signalling at the level of Akt phosphorylation but also, and most importantly in this specific context, by reduced insulin-induced nitric oxide (NO) production

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. Whether R84 HUVECs are also

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molecules expression, thus favouring monocytes diapedesis within the vessel, and promote vascular

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characterized by abnormal activation of MAPK-dependent pathways and whether this may play an atherogenic role has never been investigated so far. On the basis of this background, we first investigated whether the association between the TRIB3 R84 variant and carotid IMT

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could be replicated. Having succeeded in the replication

attempt, we moved on to investigate whether HUVECs carrying the R84 variant, which are characterized by impaired insulin ability to stimulate Akt-activation and NO production, show, conversely, upregulation of alternative insulin signalling pathways, such as MAPK-dependent ones, potentially leading to endothelial dysfunction and vSMC proliferation and migration and thus to

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development of intima hyperplasia 12.

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METHODS Genotyping of the Q84R polymorphism Genomic DNA was isolated from peripheral blood or from umbilical cord arteries using the Wizard Genomic DNA Purification kit (Protégé, Milan, Italy) according to manufacturer’s protocol. Genotyping was performed by RFLP method, as previously reported 8. Ultrasound measurement of IMT of the common carotid artery The study group consisted of 430 nondiabetic Caucasians of European ancestry participating to the CATAnzaro MEtabolic RIsk factors Study (CATAMERIS), an observational study carried out

at the Department of Experimental and Clinical Medicine, University Magna Græcia of

Catanzaro, and focused on cardio-metabolic risk factors

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. Subjects, aged 21-73 years, were

excluded if they had history of diabetes (fasting glucose levels >126 mg/dl), chronic gastrointestinal

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history of alcohol or drug abuse, liver or kidney failure, and treatments able to modify glucose metabolism including hypertensive drugs. After a 12-h overnight fast, subjects underwent anthropometrical evaluation and a venous blood sample was drawn for laboratory determinations. Body mass index (BMI) was calculated as body weight (kilograms) divided by the square of height (meters). Waist circumference was measured to the nearest 0.5 cm at the midpoint between the iliac crest. Three consecutive measurements of clinic blood pressure (BP) were obtained in the left arm of the supine patients, after five minutes of quiet rest, with a mercury sphygmomanometer. High resolution B-mode ultrasound was used to measure common carotid artery IMT by using an ATL HDI 3000 ultrasound system (Advanced Technology Laboratories, Bothell, WA) equipped with a 7.5 MHz transducer, as previously described 14, 15. Manual measurements were conducted in plaquefree portions of the 10-mm linear segment proximal to the carotid bulb. For each patient two measurements were performed bilaterally, and the values were averaged, which presented the mean of IMT of the common carotid arteries. The ultrasound study was performed by an experienced examiner who was unaware of the subjects’ clinical and laboratory findings.

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diseases associated with malabsorption, chronic pancreatitis, history of any malignant disease,

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The in vivo protocol was approved by the ethical committee of University Magna Græcia of Catanzaro and informed written consent was obtained from all participants. All investigations were performed in accordance with the principles of the Declaration of Helsinki. Materials M199 endothelial growth medium, Fetal Calf Serum (FCS), glutamine, phosphate buffered saline (PBS) and 0.05% trypsin/0.02% EDTA were purchased from Mascia Brunelli (Milan, Italy) and tissue-culture disposables from Hiwaki Glass (Tokio, Japan). Anti-MEK, anti-ERK1/2, antiAkt, anti-Ser473-Akt antibodies were from Cell Signalling Technology (Beverly, MA, USA).AntiVCAM-1 and Anti-ICAM-1 were from Santa Cruz. Anti-Beta Actin Mouse Monoclonal antibody was from Sigma, (Saint Louis, USA). L-(3H)-arginine was purchased from PerkinElmer Italia S.p.a. (Milan, Italy). N-nitro l-arginine methyl ester (L-NAME), Tris/HCl, KF, EDTA, Dowex AGWX8-

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MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK), was from Sigma-Aldrich Logistic (Germany). Cell cultures The protocol for umbilical cords harvesting was approved by the ethical committee of Pescara Town Hospital, in accordance with the principles of the Declaration of Helsinki. Umbilical cords were obtained from randomly selected healthy mothers delivering at the Pescara Town Hospital who had signed a written consent form. Umbilical cord arteries were screened for the polymorphism of interest as described below. Primary HUVECs were obtained and cultured as previously described

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. In all experiments cells were used between the 3rd and 5th passage. All

experiments were repeated at least 3 times. Each time, different cell lines from different donors were used for each genotype group (Q84Q, Q84R and R84R). Preparation of U937 cells and adhesion assays For adhesion assays, HUVECs were grown to confluence in six-well tissue culture plates. HUVECs were then cultured for 16 hours in serum-deprived medium and incubated for 24 hours

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200, TNF-α were from Sigma Chemicals (St. Louis, MO, USA). PD98059, a specific inhibitor of

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with or without insulin (100 nmol/L) or TNFα (1 ng/ml). In selected experiments, PD98059 (25 µmol/L) was added 1 hour before the adhesion assay. Adhesion assay was performed as previously described 17 Insulin signalling studies and adhesion molecules protein level. HUVECs were cultured for 16 hours in serum-deprived medium and incubated for 24 hours with or without insulin (100 nmol/L). ). In selected experiments, PD98059 (25 µmol/L) was added 1 hour before insulin stimulation. Thereafter, cells were lysed and total cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with specific primary antibodies followed by incubation with peroxidase-conjugated secondary antibodies. Proteins were detected by using enhanced chemiluminescence, and band densities were quantified by densitometry. To normalize for protein levels, the blots were stripped and reprobed with primary

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and ICAM-1 protein level were divided by those of β-actin content and the ratio indicated as arbitrary units. Cytofluorimetric analysis HUVECs were cultured for 16 hours in serum-deprived medium. The subconfluent HUVEC monolayers were then cultured for 24 hours with or without insulin (100 nmol/L). After the indicated incubation time, cells were harvested and incubated at RT for 30 min with fluorescent VCAM-1 and ICAM-1 antibodies (FITClabeled anti-VCAM-1, FITC-labeled anti-ICAM-1, BioLegend (San Diego, CA, USA). Cells were then washed and fixed with PBS containing 2% paraformaldehyde at RT for 15 min. VCAM-1 and ICAM-1 surface expression was measured by fluorescence activated cell sorting (FACS) analyzer equipped with a single 488-nm argon laser (FACSCalibur, BD Bioscences Franklin Lakes, NJ, USA). A minimum of 5,000 cells per sample was analyzed. Data were analyzed using CELLQuest 3.2.1.fl software (BD Biosciences) and expressed as percentage of positive cells for VCAM-1 and/or ICAM-1.

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antibodies against the total unphosphorylated form of the appropriate protein. Densities of VCAM-1

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Nitric Oxide Synthase activity Endothelial Nitric Oxide Synthase (eNOS) activity was determined by measuring the conversion of L-(3H)-arginine into L-(3H)-citrulline as described by Pandolfi et al.16. In selected experiments, L-NAME (1 mmol/L) was added 40 minutes before L-(3H)-arginine. Statistical analysis (in vitro experiments) Results are expressed as mean±SD of at least 3 different experiments. Differences were assessed by Student t test and by 2-way ANOVA test. Significance was defined as P