Elsevier

Atherosclerosis

Volume 183, Issue 2, December 2005, Pages 259-267
Atherosclerosis

Intermittent high glucose enhances ICAM-1, VCAM-1 and E-selectin expression in human umbilical vein endothelial cells in culture: The distinct role of protein kinase C and mitochondrial superoxide production

https://doi.org/10.1016/j.atherosclerosis.2005.03.015Get rights and content

Abstract

In this study the effects of stable and intermittent high glucose concentrations on ICAM-1, VCAM-1 and E-selectin production, PKC activity and PKCβI, βII and δ isoforms expression in cultured HUVEC have been examined. In stable high glucose ICAM-1, VCAM-1 and E-selectin concentration and mRNA expression increased, and this effect was even more evident in intermittent high glucose. PKC activity increased in fluctuating glucose compared to stable high glucose, due to an over-expression of βI, βII and δ isoforms. ICAM-1, VCAM-1 and E-selectin, after the adding of total PKC inhibitor bisindolylmaleimide-I (BIMI-I) and LY379196, a specific inhibitor of PKCβ, were equally reduced. 8-Hydroxydeoxyguanosine (8-OHdG), a sensitive indicator of oxidative damage to DNA, increased in stable and even more in intermittent high glucose and was reduced by both BIMI-I and LY379196. However, when thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial complex II and the SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) were added, all adhesion molecules, any PKC isoforms expression and 8-hydroxydeoxyguanosine were normalized in both constant and oscillating glucose.

In conclusion intermittent high glucose induces a greater expression of the adhesion molecules than stable high glucose; this effect seems to be related to an activation of PKCβ, but completely dependent from mitochondrial free radicals over-production.

Introduction

Diabetes is associated with an increased risk of atherosclerosis. Vascular disorders, due to progression of atherosclerosis, are major causes of morbidity and mortality in diabetic patients [1]. Several studies tried to explain the precise mechanism regulating the initiation and the progression of the vascular lesions in diabetic patients, but it remains still unclear.

Adhesion molecules are thought to participate in the pathogenesis of atherosclerosis [2]. They are proteins which regulate the interaction between endothelium and leukocytes, and an increase in their expression on the endothelial surface causes increased adhesion of leukocytes and particularly monocytes, which are well known as one of the first steps in the process leading to atheroma [2]. In particular, endothelial cells in human atherosclerotic lesions have been shown to over-express intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin [3].

The effects of high glucose concentrations on the expression of adhesion molecules in endothelial cells have been widely investigated. An increase of ICAM-1 has been showed in human aortic endothelial cells cultured in high glucose [4], and these data are consistent with finding showing that high glucose is a potent promoter of leukocyte adhesion to endothelial cells under flow conditions, depending on up-regulation of E-selectin, ICAM-1 and VCAM-1 [5]. In another study, a significant increase in PBMCs adherence to endothelial cells exposed to high glucose for 8 weeks was revealed [6]. These cells exhibited an increased expression of VCAM-1, and the adhesion of leukocytes was completely abrogated by anti-VCAM-1 antibodies and by staurosporine, an inhibitor of PKC activity, suggesting that the up-regulation of VCAM-1 via a PKC pathway was essential for the adherence of leukocytes to endothelial cells monolayer in high glucose [6].

Previous studies demonstrated that increased glucose concentration increases PKC activity and expression in vivo and in vitro [7]. The activation of PKC regulates various vascular functions by modulating enzymatic activities such as cytosolic phospholipase A2 and Na+ K+ ATPase, and gene expression including extracellular matrix components and contractile proteins [7]. In a previous study, we demonstrated that intermittent high glucose is more dangerous than constant high glucose medium for human umbilical vein endothelial cells (HUVECs) in culture, because in the former condition there was a marked increase in cellular apoptosis [8]. Recently, we also reported that PKC activation is involved in such phenomenon [9].

Nishikawa et al. have recently showed that the causal link between elevated glucose and hyperglycemic damage is the increased production of superoxide (O2) by the mitochondrial electron transport chain [10]. This early and key event activates all the proposed pathways involved in the pathogenesis of diabetic complications, particularly the ones related to PKC [10]. In our recent report, we showed that PKC activation, during the exposure of endothelial cells to both constant and intermittent glucose, was related to free radicals production [9]. Two antioxidants, SOD and SOD mimetic MnTBAP were able to preserve PKC from activation in both conditions of high glucose [9]. However, the possibility in this case that the source of free radicals was an over generation by mitochondria was not tested.

In the present study, we examined the effects of intermittent and stable high glucose concentrations on the expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin. In order to verify a possible involvement of the PKC, we measured PKC activity and the expression of PKC βI, βII and δ, the isoforms which appear to be preferentially activated in high glucose conditions [11]. Moreover, we also evaluated the possible involvement of PKC activation in the adhesion molecules expression during high glucose, measuring the expression of ICAM-1, VCAM-1 and E-selectin after the adding of the PKC inhibitor bisindolylmaleimide-I [9] and LY379196 [9], a specific inhibitor of PKCβ isoforms. To test the possible involvement of free radicals, and particularly the hypothesis that free radicals are generated at the mitochondrial level, experiments have also been performed adding thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial complex II [10] and the SOD mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) [9], [10]. As an index of oxidative damage 8-hydroxydeoxyguanosine (8-OHdG) has been measured. The oxidized nucleoside 8-hydroxydeoxyguanosine (8-OHdG) is known as a sensitive indicator of oxidative damage to DNA [12], [13].

Section snippets

Materials and methods

Unless otherwise specified reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA).

Results

The results of six different experiments for each experimental condition were analysed through a 14-day period.

Discussion

Several lines of evidence suggest that endothelial dysfunction and damage are early steps in the pathophysiology of vascular complications in diabetes mellitus [15]. Hyperglycemia is the central initiating factor for all types of diabetic microvascular disease [16], [17], and it may also be involved in the pathogenesis of macrovascular complications [18].

Although in normal subjects serum glucose concentration is strictly controlled within a narrow range, in diabetic patients the serum glucose

Acknowledgment

This work was supported by an unrestricted grant from Novartis Pharma, Basel, Switzerland.

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