Elsevier

Biochemical Pharmacology

Volume 68, Issue 2, 15 July 2004, Pages 341-350
Biochemical Pharmacology

Effects of the selective COX-2 inhibitors celecoxib and rofecoxib on human vascular cells

https://doi.org/10.1016/j.bcp.2004.03.029Get rights and content

Abstract

Rheumatoid arthritis (RA) is associated with a reduced life expectancy considered to be partly caused by cardiovascular events. A growing concern is that accelerated atherosclerosis is driven by inflammatory mechanisms similar to those responsible for RA. Therefore, selective COX-2 inhibitors, which are widely used for the symptomatic treatment of pain and inflammation in RA, may have an impact on atherosclerotic processes. Their anti-inflammatory properties might provoke anti-atherogenic effects but on the other hand, selective inhibition of anti-thrombotic prostacyclin and COX-2 independent effects might promote the risk of increased prothrombotic activity.

In the current study, the effects of the presently marketed selective COX-2 inhibitors celecoxib and rofecoxib on vascular cells have been investigated. Celecoxib inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. At high concentrations, it induced apoptosis and the modulation of inhibitory cell cycle proteins. In contrast rofecoxib—even at high concentrations—had no effect on cell proliferation, apoptosis or cell cycle distribution indicating that celecoxib and rofecoxib do not affect the same signal transduction pathways in endothelial cells. Both drugs did not affect apoptosis induction or cell cycle proliferation in human vascular smooth muscle cells. The observed effects on endothelial cells appear to be COX-independent since both drugs selectively inhibited COX-2-activity and the applied concentrations lay beyond the IC50 for inhibition of prostacyclin production. Regarding endothelial apoptosis as a relevant event in the initiation and progression of atherosclerosis the present data put forward the hypothesis that the presently marketed COX-2 inhibitors have a different impact on atherosclerotic processes.

Introduction

Patients with rheumatoid arthritis (RA) have a reduced life expectancy compared with the general population. Cardiovascular death is considered to be the leading cause of mortality in patients with RA and seems to be responsible for about 50% of deaths in this patient group. Moreover, age-specific cardiovascular mortality in persons with RA occurs about four-fold in excess of what would be expected in persons without RA [1]. There is a growing concern and indeed evidence showing that the premature mortality due to the cardiovascular disease is caused by accelerated atherosclerosis [2], [3] promoted by inflammatory mechanisms similar to those responsible for the development of RA. Patients exhibit increased concentrations of inflammatory cytokines and cell adhesion factors as well as high concentrations of lipids and these may influence the initiation of atherosclerosis and the formation of atherosclerotic plaques [4], [5], [6].

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and inflammation in rheumatoid arthritis. The analgesic and anti-inflammatory effects and also partly the chemopreventive effects of these agents are attributable to the inhibition of cyclooxygenase (COX) enzymes, which convert arachidonic acid to prostaglandins. Two isoforms of cyclooxygenases are known, one of which, COX-1, is expressed constitutively in most tissues and is mainly responsible for the basal physiological production of prostaglandins and the other, COX-2, is rapidly induced at sites of inflammation [7]. The non-selective NSAID aspirin has been reported to inhibit vascular smooth muscle cell proliferation (a marker of atherosclerosis) by arresting the cell cycle in the G1-phase at high doses [8] and to attenuate atherosclerosis in Apo E−/− mice [9]. On the other hand, the clinical use of classical NSAIDs is often limited by side-effects such as gastrointestinal ulcers thought to result from COX-1-inhibition. Therefore, COX-2-selective inhibitors which exhibit a reduced rate of gastrointestinal side-effects have been developed and are now preferentially used for the long-term treatment of RA. Celecoxib and rofecoxib are COX-2-selective drugs approved by the FDA for the treatment of arthritis (osteoarthritis and rheumatoid arthritis for celecoxib, osteoarthritis for rofecoxib) and acute pain. Interestingly, the VIGOR [10] study suggested an increased risk of cardiovascular events [11] for rofecoxib but this finding has prompted considerable controversy. The selective inhibition of COX-2 diminishes the production of vasodilatory prostacyclin, whereas the COX-1-generated production of thromboxane remains unaffected. The resulting imbalance between pro- and anti-thrombotic factors might constitute a cardiovascular risk [2], [12]. Recent studies have shown that COX-2 is expressed by endothelial cells, smooth muscle cells and macrophages in human atherosclerotic lesions [13], [14]. It thus might be expected that COX-2-selective inhibitors would have beneficial effects on these lesions.

There is currently no evidence indicating that long-term treatment with COX-2-selective NSAIDs causes pro- or anti-atherogenic effects in RA patients. Since endothelial cell injury and turnover as well as proliferation of vascular smooth muscle cells are key factors in the pathogenesis of atherosclerosis [15], [16], in the present study, we have investigated the effects of the selective COX-2 inhibitors, celecoxib and rofecoxib, on human vascular cells. Their effects on such cells may reflect a possible role in the initiation and progression of atherosclerosis.

Section snippets

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Systems/Clonetics and cultured in endothelial growth medium (EGM) supplemented with 1% penicillin/streptomycin, 2% fetal calf serum, hydrocortisone, hEGF, hFGF, VEGF, R3-IGF-1, ascorbic acid, GA-1000 and heparin (Cell Systems/Clonetics). For cell culture experiments passages 2–4 have been used. Human vascular smooth muscle cells (hVSMC) were purchased from ATCC and cultured in Ham’s F12 medium (F12K) with the supplements

Determination of drug protein binding

A LC/MS–MS-method was used to determine celecoxib and rofecoxib protein binding in serum-containing medium. The results showed that an average of 40% of the applied celecoxib remained unbound while 60% is bound to the proteins in the medium. In contrast, rofecoxib showed only about 10% protein binding indicating that 90% of the drug remains unbound in the medium.

Effects of celecoxib and rofecoxib on cell proliferation

Sulforhodamin B proliferation experiments were performed to determine the proliferation rate of HUVEC with and without the addition of

Discussion

The effect of long-term treatment of RA patients with NSAIDs on the development and progression of atherosclerosis is not well established. Since it is becoming increasingly accepted that atherosclerosis is an inflammatory disease, there may be benefits in the administration of anti-inflammatory drugs. Several reports indicate an improvement in atherosclerotic disease after treatment of atherosclerotic mice with indomethacin, aspirin or sulindac which inhibited neointimal formation after

Acknowledgements

The authors would like to thank B.G. Woodcock for critically reading the manuscript and helpful discussion. This study was supported by a grant of the Heinrich und Fritz Riese-Stiftung of the Klinikum der Johann Wolfgang Goethe-Universität Frankfurt to Ellen Niederberger and in part by the Deutsche Forschungsgemeinschaft (DFG GR 2011/1-1).

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