Azelnidipine, a new calcium channel blocker, inhibits endothelial inflammatory response by reducing intracellular levels of reactive oxygen species

https://doi.org/10.1016/j.ejphar.2006.07.030Get rights and content

Abstract

Oxidized low-density lipoprotein (ox-LDL) plays an important in the development of atherosclerosis by stimulating the production of reactive oxygen species in endothelial cells, and thereby up-regulating vascular cell adhesion molecule-1 (VCAM-1). The objectives of the present study were to determine the effects of azelnidipine, a new calcium channel blocker, on the expression of VCAM-1 induced by 7-ketocholesterol, components of ox-LDL, and tumor necrosis factor-α (TNF-α). The scavenging activities of azelnidipine against superoxide, hydroxyl, and carbon-centered radicals were determined by electron spin resonance assay. The levels of intracellular reactive oxygen species were determined fluorometrically with the use of dichlorodihydrofluorescein diacetate (H2DCF-DA). Human aortic endothelial cells and U937 were used as endothelial cells and monocytic cells, respectively. The surface expression and mRNA levels of VCAM-1 were determined by enzyme immunoassay and RT-PCR performed on endothelial cell monolayers stimulated with 7-ketocholesterol or TNF-α. The numbers of monocytic cells adhering on the stimulated endothelial cells were counted in the microscopic fields. Translocation of p65 protein to the nucleus was estimated by fluorescence microscopy. Azelnidipine, but not nifedipine, reduced the signal intensity of 1,1-diphenyl-2-picrylhydrazyl radicals. Azelnidipine scavenged hydroxyl radicals, but not superoxide radicals. Intracellular levels of reactive oxygen species and RelA (p65) nuclear translocation in stimulated endothelial cells were reduced by azelnidipine. Azelnidipine significantly inhibited the expression of protein and mRNA of VCAM-1, and prevented the U937 cell adhesion to endothelial cells treated with 7-ketocholesterol or TNF-α. These results suggest that azelnidipine works as an anti-atherogenic agent by inhibiting the reactive oxygen species-dependent expression of VCAM-1 induced by 7-ketocholesterol and TNF-α.

Introduction

The adhesive interaction between monocytes and the endothelium plays an important role in regulating the migration of monocytes to the subendothelial space. This localized accumulation of monocytes is mediated by the endothelial expression of specific adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or platelet-endothelial cell adhesion molecule-1 (PECAM-1). Several lines of evidence support the idea that VCAM-1 plays a crucial role in the development of atherosclerosis and plaque instability. For example, the expression of VCAM-1 has been consistently observed in atherosclerotic plaques (Davies et al., 1993, O'Brien et al., 1993). In apolipoprotein E-deficient mice, it has been shown that VCAM-1 appears to be localized over the surface of endothelial cells in lesion-prone sites and precedes lesion formation (Nakashima et al., 1998), and that additional knockout of the gene for VCAM-1 domain 4 results in significant reductions both monocyte adherence in the aortic root and fatty streak formation (Dansky et al., 2001). In addition, a soluble form of VCAM-1 has been detected in human serum, and its levels have been shown to increase in patients with atherosclerosis (Blann and McCollum, 1994, Peter et al., 1997). These data suggest that modulation of the VCAM-1 expression on endothelial cells might be a useful strategy for the treatment of atherosclerosis.

Although our understanding of the mechanism of VCAM-1 activation and signal transduction is still fragmentary, increased oxidative stress may be an important stimulus for the up-regulation of VCAM-1. It has been reported that ox-LDL, oxysterols, tumor necrosis factor-α (TNF-α) (Shimozawa et al., 2004), angiotensin II (Costanzo et al., 2003), shear stress (McNally et al., 2003), and hyperglycemia (Schiekofer et al., 2000) play a role in the production of intracellular reactive oxygen species, which results in enhanced expression of VCAM-1. Several years ago, we became interested in determining whether antioxidants would be able to inhibit the adhesion of monocytes to endothelial cells by inhibiting and enhancing the interaction between reactive oxygen species and integrins. In the previous studies investigating this question, we demonstrated that vitamin E, tocopherols and tocotrienols significantly inhibited the leukocyte–endothelial interaction via inhibition of the expression of adhesion molecules located on both leukocytes and endothelial cells (Yoshikawa et al., 1998, Naito et al., 2003, Naito et al., 2005).

Dihydropyridine-type calcium channel blockers are among the most commonly used drugs for the treatment of hypertension. In the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT), the calcium channel blocker amlodipine provided significant clinical benefits compared with placebo, including a marked reduction in cardiovascular morbidity and a reduction in the progression of carotid atherosclerosis (Pitt et al., 2000). However, the molecular mechanisms by which calcium channel blockers reduce or inhibit atherosclerosis are not fully understood. One of the mechanisms that have been proposed to explain the beneficial effect of calcium channel blockers on endothelial function is antioxidant activity. Azelnidipine is a newly developed long-acting calcium channel blocker with a hypotensive effect comparable to that of other common calcium channel blockers. Recently, Shinomiya et al. (2004) reported that azelnidipine (10–100 nM) more effectively inhibited the production of isoprostane from cultured human arterial endothelial cells stimulated with hydrogen peroxide compared to other calcium channel blockers (nifedipine and amlodipine). Yamagishi et al. (2004) also showed that azelnidipine inhibited interleukin-8 expression in endothelial cells through its anti-oxidative properties. However, the direct reactions between free radicals and these calcium channel blockers have not been investigated. Therefore, this study aimed to compare azelnidipine with nifedipine with respect to the following: 1) their reactivities with oxygen-derived free radicals and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical; 2) their effects on the concentrations of intracellular reactive oxygen species in human aortic endothelial cells stimulated by 7-ketocholesterol or tumor necrosis factor-α (TNF-α); and 3) their effects on the activation of transcription factor-κB (NF-κB), monocyte adherence to endothelial cells, and the expression of VCAM-1 induced by prooxidant signals.

Section snippets

Reagents

A monoclonal antibody for VCAM-1 was purchased from Becton Dickson (San Jose, CA). Azelnidipine was a gift from Sankyo Co., Ltd. (Tokyo, Japan). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was purchased from LABOTEK (Tokyo, Japan). 1,1′-Diphenyl-2-picrylhydrazyl (DPPH), N,N-dimethylformamide (DMF), xanthine oxidase, ferrous sulfate (FeSO4), RPMI 1640 medium, 7-ketocholesterol, and nifedipine were obtained from Sigma Chemical (St. Louis, MO). Diethylenetriaminepentaacetic acid (DETAPAC),

Azelnidipine but not nifedipine reduced DPPH radical

Fig. 1 shows a typical ESR spectrum for DPPH. In the presence of azelnidipine in ethanol/PBS solution, the signal intensities of DPPH decreased as shown in Fig. 1A. The scavenging effect of azelnidipine was expressed as the IC50, which is the concentration required to reduce by 50% the signal intensity of DPPH. The IC50 value of azelnidipine was 0.79 mM, as calculated using the data in Fig. 1B. Nifedipine at a concentration of 5 mM did not affect the signal intensity of DPPH radical in

Discussion

In the present study, we demonstrated for the first time that azelnidipine inhibited 7-ketocholesterol- or TNF-α-induced NF-κB activation and VCAM-1 expression in human aortic endothelial cells by reducing the intracellular reactive oxygen species levels on the basis of the following evidence. (1) 7-Ketocholesterol or TNF-α significantly enhanced intracellular reactive oxygen species generation in endothelial cells, and these enhancements were significantly inhibited by azelnidipine. (2)

Acknowledgment

This work was supported by a Grant-in-Aid for Scientific Research (15390178 TY) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a grant from the Bio-oriented Technology Research Advancement Institution, and by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan.

References (32)

  • A.J. Brown et al.

    Oxysterols and atherosclerosis

    Atherosclerosis

    (1999)
  • Y. Naito et al.

    Tocotrienols reduce 25-hydroxycholesterol-induced monocyte–endothelial cell interaction by inhibiting the surface expression of adhesion molecules

    Atherosclerosis

    (2005)
  • S.K. Peng et al.

    Cholesterol oxidation derivatives and arterial endothelial damage

    Atherosclerosis

    (1985)
  • M. Arita et al.

    A new Ca-antagonist, azelnidipine, reduced blood pressure during exercise without augmentation of sympathetic nervous system in essential hypertension: a randomized, double-blind, placebo-controlled trial

    J. Cardiovasc. Pharmacol.

    (1999)
  • A.D. Blann et al.

    Circulating endothelial cell/leukocyte adhesion molecules in atherosclerosis

    Thromb. Haemost.

    (1994)
  • G.A. Boissonneault et al.

    Oxysterols, cholesterol biosynthesis, and vascular endothelial cell monolayer barrier function

    Proc. Soc. Exp. Biol. Med.

    (1991)
  • J.W. Chen et al.

    Ginkgo biloba extract inhibits tumor necrosis factor-{alpha}-induced reactive oxygen species generation, transcription factor activation, and cell adhesion molecule expression in human aortic endothelial cells

    Arterioscler. Thromb. Vasc. Biol.

    (2003)
  • A. Costanzo et al.

    Endothelial activation by angiotensin II through NFkappaB and p38 pathways: involvement of NFkappaB-inducible kinase (NIK), free oxygen radicals, and selective inhibition by aspirin

    J. Cell. Physiol.

    (2003)
  • H.M. Dansky et al.

    Adhesion of monocytes to arterial endothelium and initiation of atherosclerosis are critically dependent on vascular cell adhesion molecule-1 gene dosage

    Arterioscler. Thromb. Vasc. Biol.

    (2001)
  • M.J. Davies et al.

    The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis

    J. Pathol.

    (1993)
  • K.K. Griendling et al.

    NAD(P)H oxidase: role in cardiovascular biology and disease

    Circ. Res.

    (2000)
  • M. Ishiyama et al.

    A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet

    Biol. Pharm. Bull.

    (1996)
  • T. Jinno et al.

    Calcium channel blocker azelnidipine enhances vascular protective effects of AT1 receptor blocker olmesartan

    Hypertension

    (2004)
  • P. Lesnik et al.

    Impact of a combination of a calcium antagonist and a beta-blocker on cell- and copper-mediated oxidation of LDL and on the accumulation and efflux of cholesterol in human macrophages and murine J774 cells

    Arterioscler. Thromb. Vasc. Biol.

    (1997)
  • N. Marui et al.

    Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells

    J. Clin. Invest.

    (1993)
  • J.S. McNally et al.

    Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress

    Am. J. Physiol., Heart Circ. Physiol.

    (2003)
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