Original ContributionHypoxia–reoxygenation-induced mitochondrial damage and apoptosis in human endothelial cells are inhibited by vitamin C
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.
References (82)
- et al.
Free-radical-mediated postischemic reperfusion injury in the kidney
J. Free Lett. Biol. Med.
(1986) - et al.
Rapid superoxide production by endothelial cells and their injury upon reperfusion
J. Surg. Res.
(1994) - et al.
Cells in focus: endothelial cell
Int. J. Biochem. Cell. Biol.
(2002) - et al.
Beneficial effects of alpha-lipoic acid and ascorbic acid on endothelium-dependent, nitric oxide-mediated vasodilation in diabetic patients: relation to parameters of oxidative stress
Free Radic. Biol. Med.
(2001) - et al.
Requirement for GSH in recycling of ascorbic acid in endothelial cells
Biochem. Pharmacol.
(2001) - et al.
Recycling of vitamin C from its oxidized forms by human endothelial cells
Biochim. Biophys. Acta
(2003) - et al.
Human HL-60 myeloid leukemia cells transport dehydroascorbic acid via the glucose transporters and accumulate reduced ascorbic acid
Blood
(1994) - et al.
Vitamin C contributes to inflammation via radical generating mechanisms: a cautionary note
Med. Hypotheses
(2003) - et al.
Mechanism of vitamin C inhibition of cell death induced by oxidative stress in glutathione-depleted HL-60 cells
J. Biol. Chem.
(2001) - et al.
Vitamin C prevents DNA mutation induced by oxidative stress
J. Biol. Chem.
(2002)
Ascorbic acid blunts oxidant stress due to menadione in endothelial cells
Arch. Biochem. Biophys.
Generation of oxidant stress in cultured endothelial cells by methylene blue: protective effects of glucose and ascorbic acid
Biochem. Pharmacol.
Vitamin C inhibits FAS-induced apoptosis in monocytes and U937 cells
Blood
Vitamin C inhibits hypoxia-induced damage and apoptotic signaling pathways in cardiomyocytes and ischemic hearts
Free Radic. Biol. Med.
Vitamin C recycling and function in human monocytic U-937 cells
Free Radic. Biol. Med.
Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-kappa B site and p65 homodimers
J. Biol. Chem.
Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum
Am. J. Clin. Nutr.
Determination of ascorbic acid and dehydroascorbic acid in plasma by high-performance liquid chromatography with coulometric detection—are they reliable biomarkers of oxidative stress?
Anal. Biochem.
Colony-stimulating factors signal for increased transport of vitamin C in human host defense cells
Blood
Recycling of vitamin C by a bystander effect
J. Biol. Chem.
Ischemia-reperfusion injury of retinal endothelium by cyclooxygenase- and xanthine oxidase-derived superoxide
Exp. Eye Res.
Free radical generation in human endothelial cells exposed to anoxia and reoxygenation
Transplant. Proc.
Energy metabolism of the kidney: segmental differences may determine xenobiotic actions
Toxicol. Lett.
Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia
J. Mol. Cell. Cardiol.
Inhibiting caspase-9 after injury reduces hypoxic ischemic neuronal injury in the cortex in the newborn rat
Neurosci. Lett.
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade
Cell
Mitochondrial uptake and recycling of ascorbic acid
Arch. Biochem. Biophys.
Mitochondrial recycling of ascorbic acid from dehydroascorbic acid: dependence on the electron transport chain
Arch. Biochem. Biophys.
Reactive oxygen species rapidly increase endothelial ICAM-1 ability to bind neutrophils without detectable upregulation
Blood
Reactive oxygen species and nitric oxide in myocardial ischemia and reperfusion
Z. Kardiol.
Pharmacology of the endothelium in ischemia-reperfusion and circulatory shock
Annu. Rev. Pharmacol. Toxicol.
Mechanics of endothelial cell architecture and vascular permeability
Crit. Rev. Biomed. Eng.
Neutrophil transendothelial migration and alteration in vascular permeability: focus on neutrophil-derived azurocidin
Curr. Opin. Hematol.
Modulation of endothelial hemostatic properties: an active role in the host response
Annu. Rev. Med.
The role of free radicals as mediators of endothelial cell injury in hyperhomocysteinemia
Ir. J. Med. Sci.
Pathophysiology of ischaemia-reperfusion injury
J. Pathol.
Attenuated coronary relaxation after reperfusion: effects of superoxide dismutase and TxA2 inhibitor U 63557A
Am. J. Physiol.
Adhesion of flowing monocytes to hypoxia-reoxygenation-exposed endothelial cells: role of Rac1, ROS, and VCAM-1
Am. J. Physiol. Cell. Physiol.
Time course of endothelial dysfunction and myocardial injury during myocardial ischemia and reperfusion in the cat
Circulation
Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy
Circulation
Endothelial function and oxidative stress in renovascular hypertension
N. Engl. J. Med.
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2020, Free Radical Biology and MedicineCitation Excerpt :In earlier studies, it was found that pretreatment of vitamin C significantly decreases infarct size in a primate model of focal cerebral ischemia and reperfusion [9] and is neuroprotective against global ischemia in striatum of gerbil when pretreated 1 h before occlusion [10]. A recent in vitro study indicates that vitamin C prevents hypoxia and hypoxia-reperfusion-induced damage to human endothelium [11]. In a rat model of stroke, it was noted that vitamin C reduces the adverse effects of delayed administration of tissue plasminogen activator.
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Deceased.