Original Contribution
Hypoxia–reoxygenation-induced mitochondrial damage and apoptosis in human endothelial cells are inhibited by vitamin C

https://doi.org/10.1016/j.freeradbiomed.2005.01.017Get rights and content

Abstract

Hypoxia and hypoxia–reperfusion (H-R) play important roles in human pathophysiology because they occur in clinical conditions such as circulatory shock, myocardial ischemia, stroke, and organ transplantation. Reintroduction of oxygen to hypoxic cells during reperfusion causes an increase in generation of reactive oxygen species (ROS), which can alter cell signaling, and cause damage to lipids, proteins, and DNA leading to ischemia–reperfusion injury. Since vitamin C is a potent antioxidant and quenches ROS, we investigated the role of intracellular ascorbic acid (iAA) in endothelial cells undergoing hypoxia–reperfusion. Intracellular AA protected human endothelial cells from H-R-induced apoptosis. Intracellular AA also prevents loss of mitochondrial membrane potential and the release of cytochrome C and activation of caspase-9 and caspase-3 during H-R. Additionally, inhibition of caspase-9 activation prevented H-R-induced apoptosis, suggesting a mitochondrial site of initiation of apoptosis. We found that H-R induced an increase in ROS in endothelial cells that was abrogated in the presence of iAA. Our results indicate that vitamin C prevents hypoxia and H-R-induced damage to human endothelium.

Introduction

Hypoxia and reoxygenation injury are common causes of mortality due to myocardial ischemia, circulatory shock, stroke, and transplantation of organs [1], [2], [3]. It has become increasingly evident that ROS play a significant role in reoxygenation injury. During hypoxia and reperfusion (H-R) vascular endothelium is a primary site of ROS generation and target of injury [4]. Endothelial cells function as a permeability barrier, regulating leukocyte migration and inhibiting thrombosis [5], [6], [7], [8]. These cells are damaged by proinflammatory cytokines, bacterial endotoxins, and atherogenic factors such as homocysteine and oxidized lipoproteins [9]. Cellular models of H-R have provided useful tools for the study of ROS-mediated mechanisms of cellular dysfunction. The molecular mechanisms of reperfusion injury on vascular endothelium are not well understood [10].

Since ROS play an important role in H-R, antioxidants have been used to ameliorate consequent cellular injury [11], [12], [13]. Vitamin C, a strong antioxidant that quenches ROS, has also been used to reduce endothelial dysfunction in conditions such as diabetes, hyperhomocysteinemia, coronary artery disease, hypercholesterolemia, and renovascular hypertension [14], [15], [16], [17], [18]. However, experimental studies have shown that high physiological concentrations of vitamin C are required to prevent ROS-mediated vascular dysfunction [19].

The role of vitamin C in endothelial cells undergoing H-R has not been analyzed [20], [21]. Certain specialized cells transport vitamin C directly as AA via sodium-dependent cotransporters localized on the cell membrane [22]. However, all cells can transport the oxidized form of vitamin C, dehydroascorbic acid (DHA), via facilititative glucose transporters (GLUTs) [23]. It has been shown that loading cells with vitamin C by treatment with DHA circumvents the prooxidant effects of AA in cell culture [24], [25]. Loading cells with vitamin C by treatment with DHA protects against FAS-mediated apoptosis in monocytes, menadione-induced oxidant stress in endothelial cells, and hydrogen peroxide-induced cell death in HL-60 leukemic cells and protects against oxidant-induced DNA mutations [26], [27], [28], [29], [30], [31]. In vivo, DHA administration has resulted in amelioration of ischemia-induced infarct size in experimental stroke models [32], [17].

We have examined the role of vitamin C in reducing hypoxia and H-R-induced injury in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC). Our results indicate that increased intracellular concentrations of vitamin C in endothelial cells substantially reduced hypoxia and H-R-induced apoptosis.

Section snippets

Cell culture

HUVEC and HCAEC were obtained from Clonetics Corporation (Cambrex Inc., East Rutherford, NJ). HUVEC were grown in endothelial basal medium supplemented with 2% fetal calf serum (FCS), human epidermal growth factor, insulin growth factor-1, fibroblast growth factor, vascular growth factor, hydrocortisone (1.0 μg/ml), gentamicin (50 μg/ml), and heparin. HCAEC were maintained in EBM2-MV medium with 5% FCS and the growth factor supplements. Cells were used at passages 5 or less.

DHA uptake in HUVEC and HCAEC

Cells were grown in

Endothelial cells transport vitamin C as DHA via facilitative glucose transporters

To load cells with vitamin C we used the oxidized form of vitamin C, which is taken up by endothelial cells to a greater extent than ascorbate. The accumulation of vitamin C in HUVEC was dependent on the concentration of DHA (Fig. 1A). Cells incubated with 500 μM DHA for 30 min accumulated 23 mM intracellular AA. To establish that DHA uptake in these cells was via the facilitative glucose transporters, competition studies were performed using D-DOG and the nontransportable stereoisomer L-DOG.

Discussion

Endothelial cells undergo apoptosis in response to a variety of pathophysiological conditions including hypoxia, proinflammatory cytokines, bacterial endotoxins, and atherogenic risk factors such as homocysteine and lipoproteins. All these cellular stresses have the generation of oxidative damage in common. Understanding the role of ROS in cellular processes paves the way for pharmacological intervention using antioxidants such as vitamin C. Organs used for transplantation also experience long

Acknowledgments

Dr. David W. Golde, our mentor who was the motivating inspiration behind this work, died on August 9, 2004. We appreciate the technical assistance of Alicia Pedraza, Oriana Borquez-Ojeda, and Jeffrey R. Gardner. This work was supported by Grants from the NIH (CA30388), New York State Department of Health #M030407), and the Lebensfeld Foundation.

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