© 2000 American Heart Association, Inc.
Editorials |
Cellular and Molecular Dissection of Reperfusion Injury
ROS Within and Without
From the Institute of Genetic Medicine, Departments of Pediatrics and Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Gregg L. Semenza, MD, PhD, Johns Hopkins Hospital, CMSC-1004, 600 North Wolfe St, Baltimore, MD 21287-3914. E-mail gsemenza{at}jhmi.edu
Key Words: apoptosis • ceramide • ischemia • tumor necrosis factor-
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Introduction |
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 Top Introduction References |
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Reperfusion injury in the heart can occur either as a result of transient arterial occlusion (eg, due to vasospasm or thrombus formation with spontaneous lysis) or as an iatrogenic consequence of thrombolytic or angioplastic therapy. A basic research objective is to delineate pathogenesis in order to devise effective prevention and/or treatment strategies. This, of course, applies to most biomedical research, and one general approach to this and other conditions is a reductionist model in which the responses of isolated cell types are investigated in tissue culture. This experimental approach has the advantage of distinguishing primary responses to the stimulus from those that are secondary to the responses of other cell types within the organ. Not only the target cell but also the inciting stimulus can be well defined in cell culture. Arterial occlusion leads to ischemia, which involves deprivation of energy substrates (glucose and oxygen) and accumulation of toxic metabolites (H+ and K+), as well as secondary alterations in endothelial and inflammatory cell function. In contrast, cultured cells (eg, cardiac myocytes) can be subjected to anoxia/hypoxia under highly controlled conditions. Furthermore, the expression of specific proteins can be experimentally manipulated in an attempt to establish molecular mechanisms. Two obvious limitations of tissue culture systems involve the analysis of responses that are not cell autonomous (ie, involve more than one cell type) and those that are cell-type or developmental-stage specific (eg, adult versus neonatal cardiomyocytes). Thus, the relevance of responses in tissue culture to organ function in vivo must be confirmed experimentally, eg, by the use of transgenic or knockout mouse models.
Webster et al1 have used cultured neonatal rat cardiac myocytes to explore the mechanisms of cell death triggered by hypoxia and reoxygenation. They recently reported that under their culture conditions these cells undergo apoptosis via a p53-independent pathway, in contrast to a previous study that implicated p53 in this process.2 They also used Langendorff-perfused hearts from wild-type and p53-knockout mice to provide compelling evidence that cell death induced by ischemia-reperfusion did not require p53 expression.1 To follow up this interesting but essentially negative result, the same group now reports that hypoxia-reoxygenation results in the induction of neutral sphingomyelinase activity, accumulation of ceramide, and increased c-Jun N-terminal kinase (JNK) activity.3 This cascade can be inhibited by pretreatment of cells with antioxidants, suggesting that it is triggered by reactive oxygen species (ROS) generated during reoxygenation. Ceramide is a signaling molecule, generated from sphingomyelin by the action of acidic and neutral sphingomyelinases (reviewed in Reference 44 ), that has been linked to the JNK and apoptotic pathways in a number of cell types (reviewed in Reference 55 ). However, additional studies are required to determine whether ceramide-induced JNK activation is necessary and sufficient to trigger apoptosis in cardiac myocytes. The results reported by Hernandez et al3 in this issue of Circulation Research are in agreement with a previous study linking both oxygen/glucose deprivation in cardiac myocytes and ischemia/reperfusion in the intact heart with increased ceramide production and apoptosis.6 Reoxygenation-induced ceramide production is a specific response of cardiac myocytes, as exposure of cardiac fibroblasts to ceramide induced JNK activity whereas hypoxia-reoxygenation did not.3
These results provide a basic molecular framework for understanding one
apoptotic pathway that is induced by reperfusion (Figure 1
), but multiple questions remain to be
answered by future studies: (1) Which ROS (superoxide, hydrogen
peroxide, hydroxyl radical, peroxynitrite, or others) generated by
reperfusion are responsible for the induction of neutral
sphingomyelinase activity? (2) What is the mechanism by which these ROS
are generated? Several sources of oxygen free radicals have been
identified including xanthine oxidase, the mitochondrial electron
transport chain, and NADPH oxidase. (3) What is the mechanism by which
ROS induce neutral sphingomyelinase activity? (4) What is the mechanism
by which ceramide induces JNK activity? (5) What is the mechanism by
which JNK activity triggers apoptosis in cardiac myocytes? (6)
What other apoptotic pathways are induced by
reoxygenation or other aspects of reperfusion?
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Diacylglycerol generated by phosphatidylcholine-specific phospholipase C was shown to induce ceramide production by acidic sphingomyelinase in lymphoid cells treated with tumor necrosis factor (TNF).7 The generation of diacylglycerol by phospholipase C has been implicated in mechanisms of ischemic preconditioning (reviewed in Reference 88 ), suggesting that this enzyme may be involved in reperfusion-induced ceramide production. Exposure of cardiac myocytes and other cell types to doxorubicin also induces oxygen free radical generation that leads to ceramide production, JNK activation, and apoptosis, which is mediated by acidic sphingomyelinases and can be inhibited by pretreatment with L-carnitine.9 10 Thus, elucidation of pathways of ceramide generation may lead to therapeutic strategies for preventing myocardial apoptosis in several clinical contexts.
Myocardial cell death resulting from reperfusion in vivo is not
entirely cell autonomous, as alterations in endothelial
and inflammatory cell function are also important pathogenetic factors.
Reperfusion results in the production of chemotactic
cytokines by vascular endothelial cells that
result in the binding of polymorphonuclear leukocytes (PMNs) (via
cell surface adhesion molecules) and their subsequent migration through
the vessel into surrounding tissue. To investigate these processes,
Kokura et al,11 in a study also reported in this issue of
Circulation Research, cocultured human umbilical vein
endothelial cells (HUVECs) with PMNs and T lymphocytes
isolated from peripheral blood. Their studies suggest that
a complex interplay between these three cell types occurs after
anoxia-reoxygenation in which hydrogen peroxide
produced by HUVECs stimulates T cells to secrete TNF-, which induces
the expression by HUVECs of the cell surface molecule E-selectin, a
mediator of PMN adhesion (Figure 2).
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As in the study by Hernandez et al,3 the production of ROS after reoxygenation appears to trigger the cascade described by Kokura et al,11 but in the latter system the effects of ROS are not limited to the cell type within which they are produced. In both cases, however, many of the details regarding the mechanisms and direct effects of ROS production are yet to be delineated. Transgenic mice that overexpress copper-zinc superoxide dismutase manifest dramatically reduced myocardial reperfusion injury and reperfusion-induced superoxide generation (as demonstrated by electron paramagnetic resonance spin trapping) in the hearts of nontransgenic mice is not observed in the hearts of transgenic mice.12 These results are consistent with the hypothesis that superoxide production triggers the pathological processes described in these two reports. Finally, it is not clear how ischemic preconditioning blocks the pathophysiological processes described in these two reports.3 11 Recent experiments have demonstrated that delayed (late phase or second window) preconditioning does not occur in Nos2 knockout mice, which lack expression of inducible nitric oxide synthase.13 It will be of great interest to determine whether nitric oxide directly or indirectly affects either oxygen radical generation or a subsequent step in the pathways that are described in these two reports.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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1. Webster KA, Discher DJ, Kaiser S, Hernandez O, Sato B, Bishopric NH. Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53. J Clin Invest. 1999;104:239–252.[Medline] [Order article via Infotrieve]
2. Long X, Boluyt MO, Hipolito ML, Lundberg MS, Zheng JS, O’Neill L, Cirielli C, Lakatta EG, Crow MT. p53 and the hypoxia-induced apoptosis of cultured neonatal rat cardiac myocytes. J Clin Invest. 1997;99:2635–2643.[Medline] [Order article via Infotrieve]
3.
Hernandez OM, Discher DJ, Bishopric NH, Webster KA.
Rapid activation of neutral sphingomyelinase by
hypoxia-reoxygenation of cardiac myocytes.
Circ Res. 2000;86:198–204.
4. Mathias S, Pena LA, Kolesnick R. Signal transduction of stress via ceramide. Biochem J. 1998;335:465–480.
5. Basu S, Kolesnick R. Stress signals for apoptosis: ceramide and c-Jun kinase. Oncogene. 1998;17:3277–3285.[Medline] [Order article via Infotrieve]
6. Bielawska AE, Shapiro JP, Jiang L, Melkonyan HS, Piot C, Wolfe CL, Tomei LD, Hannun YA, Umansky SR. Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion. Am J Pathol. 1997;151:1257–1263.[Abstract]
7.
Schutze S, Potthoff K, Machleidt T, Berkovic D,
Wiegmann K, Kronke M. TNF activates NF-
B by
phosphatidylcholine-specific phospholipase C-induced "acidic"
sphingomyelin breakdown. Cell. 1992;71:765–776.[Medline]
[Order article via Infotrieve]
8. Das DK, Engelman RM, Maulik N. Oxygen free radical signaling in ischemic preconditioning. Ann N Y Acad Sci. 1999;874:49–65.[Medline] [Order article via Infotrieve]
9.
Andrieu-Abadie N, Jaffrezou JP, Hatem S, Laurent G,
Levade T, Mercaider JJ. L-Carnitine prevents
doxorubicin-induced apoptosis of cardiac myocytes: role of
inhibition of ceramide generation. FASEB J. 1999;13:1501–1510.
10.
Mas VM, Bezombes C, Quillet-Mary A, Bettaieb A,
D’Orgeix AD, Laurent G, Jaffrezou JP. Implication of radical oxygen
species in ceramide generation, c-Jun N-terminal kinase activation and
apoptosis induced by daunorubicin. Mol Pharmacol. 1999;56:867–874.
11.
Kokura S, Wolf RE, Yoshikawa T, Granger DN, Aw TY.
T-lymphocyte–derived tumor necrosis factor exacerbates
anoxia-reoxygenation–induced
neutrophil-endothelial cell adhesion. Circ
Res. 2000;86:205–213.
12.
Wang P, Chen H, Qin H, Sankarapandi S, Becher MW, Wong
PC, Zweier JL. Overexpression of human copper, zinc-superoxide
dismutase (SOD1) prevents postischemic injury. Proc
Natl Acad Sci U S A. 1998;95:4556–4560.
13.
Guo Y, Jones WK, Xuan Y-T, Tang X-L, Bao W, Wu W-J, Han
H, Laubach VE, Ping P, Yang Z, Qiu Y, Bolli R. The late phase of
ischemic preconditioning is abrogated by targeted disruption of
the inducible NO synthase gene. Proc Natl Acad Sci U S A. 1999;96:11507–11512.
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