Elsevier

Atherosclerosis

Volume 198, Issue 1, May 2008, Pages 22-28
Atherosclerosis

Anti-inflammatory and anti-oxidant properties of telmisartan in cultured human umbilical vein endothelial cells

https://doi.org/10.1016/j.atherosclerosis.2007.09.013Get rights and content

Abstract

Purpose

To study whether telmisartan, an angiotensin II (AII) receptor blocker (ARB), modulates endothelial inflammation and oxidative cell damage induced by AII-independent stimuli in cultured human umbilical vein endothelial cell (HUVEC)s.

Methods

Endothelial inflammation, as reflected by increased VCAM-1 and ICAM-1 expression (ELISA), was induced by TNF-α, an inflammatory cytokine, and cell damage (COMET and MTT assay) by hydrogen peroxide, a reactive oxygen species. Losartan, another ARB, its active metabolites (EXP-3174, EXP-3179), dexamethasone, a synthetic steroid, and pyrrolidine dithiocarbamate (PDTC), an anti-oxidant, were the controls. The contribution of PPAR-γ agonism was assessed through synthetic PPAR-γ agonists and antagonists and the antagonism for AII-type 1 receptor-mediated stimuli by evaluating the interference against cell death induced by AII (MTT assay), a pro-apoptotic peptide that induces oxidative stress. The in vitro scavenging properties for oxyradicals were quantified by the TOSC assay.

Results

Telmisartan and PDTC reduced TNF-α-stimulated VCAM-1 in a concentration-dependent manner while losartan, EXP-3174, EXP-3179 and dexamethasone were ineffective. All compounds did not modify ICAM-1 expression. PPAR-γ agonists or antagonists did not interfere with the effect of telmisartan. Both ARBs antagonized AII-induced cell death but only telmisartan reduced hydrogen peroxide-induced cell damage. Telmisartan scavenged selectively hydroxyl radicals without affecting peroxyl radicals and peroxynitrite.

Conclusions

Telmisartan modulates pleiotropically TNF-α induced VCAM-1 expression and oxidative damage in vascular endothelium, possibly by acting as a hydroxyl radical scavenger. Those anti-inflammatory and antioxidant properties may contribute to the therapeutic effect, although the applicability of these data to the clinical situations has to be verified.

Introduction

Increased expression of vascular cell adhesion molecule (CAM)s [1] and increased reactive oxygen species (ROS) generation by activated cell membrane-associated NAD(P)H oxidase and other pro-oxidant enzymes [2], are important contributing factors to the atherosclerotic process. The renin–angiotensin system through angiotensin (A) II, its main effector, promotes both processes at the endothelial level in synergy with other pro-atherogenic biological systems [3]. Therefore, vascular endothelium represents an ideal target for anti-atherosclerotic actions of AII receptor-blocker (ARB)s [4], possibly through mechanisms unrelated to AII type 1 receptor (AT1R) blockade [5]. In this context, telmisartan (TEL), a lipophilic, highly selective ARB [6], is receiving attention because of an array of pharmacological properties unrelated to AT1R antagonism per se including partial agonism for proliferator-activated receptor (PPAR)-γ nuclear receptor system and other pleiotropic actions including modulation of oxidative stress and inhibition of pro-inflammatory stimuli [7].

This study intended to analyze in greater detail the pleiotropic effects of TEL with particular reference to its AII-independent anti-inflammatory and anti-oxidant potential. For this reason, we evaluated the effect of the drug on vascular (VCAM-1) and intercellular (ICAM-1) cell adhesion molecules expressed in response to tumour necrosis factor (TNF)-α [8], an AII-unrelated pro-inflammatory cytokine pathophysiologically involved in the development of human cardiovascular disease [9]. Losartan (LOS), a hydrophilic ARB, its active metabolites EXP-3174, EXP-3179 [10], dexamethasone (DEX), a synthetic anti-inflammatory steroid [11], were the controls for TEL. As an additional comparator, we used pyrrolidine dithiocarbamate (PDTC), an antioxidant with inhibiting properties for VCAM-1 expression [12]. We also tested the role of partial PPAR-γ agonism and AT1-R antagonism by TEL in our experimental system, and tested its effect against hydrogen peroxide, a ROS sensitive to TNF-α stimulation and an important signalling molecule in cardiovascular cells [13]. Finally, we quantified the in vitro scavenging capacity for oxyradicals of the drug [14]. The data were generated in cultured human umbilical vein endothelial cells (HUVEC)s.

Section snippets

Endothelial cell culture

HUVECs were isolated by digestion with 0.1% collagenase (specific activity: 316 U/ml, Gibco, Invitrogen) [15] and grown to confluence at 37° in 5% CO2 humidified incubator, on 25-cm2 tissue culture flasks previously coated with 1% gelatine in supplemented culture medium (M199 with 10% heat inactivated foetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM l-glutamine, 10 mM HEPES pH 7.4, heparin 12 U.I./ml, 1% retinal derived growth factor, Sigma). Following trypsin treatment, the cells

TNF-α induced CAM expression

TNF-α raised VCAM-1 expression from a baseline of 0.07 ± 0.03 to 0.63 ± 0.11 AU (p < 0.001, n = 5). TEL decreased TNF-α-stimulated VCAM-1 expression concentration-dependently by 68% at its peak, a pattern similar to that elicited by PDTC, an antioxidant compound; LOS was ineffective (Fig. 1, top panel). EXP-3174, EXP-3179 behaved as LOS; DEX reduced VCAM-1 expression by 14% (p < 0.05) as a maximum (data not shown).

TNF-α raised ICAM-1 from 0.31 ± 0.08 to 0.84 ± 0.07 (p < 0.001, n = 6) and TEL and LOS (10−4 M for

Discussion

To our knowledge, this study is the first to produce evidence for AII-independent anti-inflammatory and anti-oxidant properties of TEL in HUVECs. A first original result was the inhibition of the VCAM-1 expressed in response to TNF-α, a cytokine targeting ECs and a potent stimulus for the surface expression of adhesion molecules [8], a fundamental component of acute inflammatory processes and a close associate of human atherosclerosis [1], [22]. That anti-inflammatory action of TEL in HUVECs

Acknowledgements

The work was supported by funds from Università di Pisa (Fondi di Ateneo) and an unrestricted grant from Boheringer Ingelheim (Italy) that had no role in study design, data collection, data analysis, data interpretation, or writing of the report. We are grateful to Dr. Schupp, Institut fur Pharmakologie und Toxikologie, Universitatsmedizin Berlin, Germany for the kind gift of EXP-3174, EXP-3179. Boheringer Ingelheim, GlaxoSmithKline, Merck,Sharp&Dohme provided freely Telmisartan, Rosiglitazone

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    These authors contributed equally to the work.

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