Published online before print April 19, 2004, doi: 10.1161/01.HYP.0000127811.48554.12
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(Hypertension. 2004;43:1168.)
© 2004 American Heart Association, Inc.
Editorial Commentaries |
Death Receptor Induced Apoptosis
A New Mechanism of Homocysteine-Mediated Endothelial Cell Cytotoxicity
From the Molecular Cardiology/Whitaker Cardiovascular Research Institute, Boston University School of Medicine, 715 Albany Street, Boston, Mass.
Correspondence to Kenneth Walsh, PhD, Molecular Cardiology/Whitaker Cardiovascular Institute Boston University School of Medicine 715 Albany Street, W611 Boston, MA 02118.E-mail kxwalsh{at}bu.edu
The hypothesis that homocysteine (Hcy) is atherogenic was proposed more than 30 years ago.1 People with inherited disorders of Hcy and methionine metabolism due to rare mutations such as in the cystathionine ß-synthase gene present with a severe form of hyperhomocysteinemia that is characterized by premature atherosclerosis and thrombotic disease. A common variant in the metylenetetrahydrofolate reductase gene, 677C to T, is associated with decreased enzyme activity leading to mild hyperhomocysteinemia in humans.2 This mutation is present in the homozygous state in 10% to 15% of North American and European populations.2 Epidemiological studies in the general population have indicated a positive correlation between an elevation of total plasma Hcy and stroke, myocardial infarction, peripheral vascular disease, and venous thromboembolism.3 Whereas a significant association between Hcy levels and clinical cardiovascular events has not been observed in all prospective studies, hyperhomocysteinemia is now considered by many to be an independent risk factor for atherosclerotic vascular disease. Furthermore, the belief that hyperhomocysteinemia may be not only a marker but also a cause for atherosclerosis and thrombosis has intensified research in this area.
Hcy is a thiol amino acid, but only a small fraction (<2%) circulates in the thiol form. The reminder is a mixture of disulfide derivatives, including protein-bound disulfides. Hyperhomocysteinemia is usually defined as an elevation of plasma Hcy above 15 µmol/L. This elevation may be caused by genetic defects, renal insufficiency, certain drugs, or nutritional insufficiencies of cofactors required for Hcy metabolism, including folate, vitamin B2 (riboflavin), vitamin B6 (pyridoxal phosphate), or vitamin B12 (methylcobalamin). Because plasma Hcy can be lowered by oral supplementation of folic acid or B vitamins, the treatment of hyperhomocysteinemia as a strategy to prevent cardiovascular disease and its complications is currently being tested in several large randomized prospective clinical trials.3
Hcy is cytotoxic to vascular endothelial cells and impairs normal cellular function. One of the hallmarks of endothelial cell damage by Hcy is the impairment of vasorelaxation in both human patients and animal models.4 What are the potential mechanisms by which Hcy promotes endothelial cell injury? Hcy is reported to impair the response to oxidative stress in endothelial cells.5,6 It has been suggested that autophosphorylation of Hcy’s thiol group generates reactive oxygen species (ROS) that induce endothelial dysfunction. However, cysteine, a related amino acid that is present at much higher concentrations and is more readily auto-oxidized, fails to induce endothelial cell injury and is not a cardiovascular risk factor. These considerations indicate that the toxicity of Hcy is caused by other mechanisms. A recently proposed mechanism of Hcy-induced vascular injury involves endoplasmic reticulum (ER) stress and activation of the unfolded protein response caused by incorrect protein folding.7 Hcy is reported to cause ER stress by disrupting disulfide bond formation and causing misfolding of proteins traversing the ER. Hcy induces the expression of GADD153, a basic region leucine zipper transcription factor involved in ER stress-induced cell death; the ER chaperones GRP78 and GRP94; and Herp, a protein involved in the degradation of misfolded ER proteins.8 Other potential mechanisms involve Hcy-induced increases in the procoagulant activity of endothelial cells by enhancing tissue factor expression, increasing Factor V and XII activity, and inhibition of protein C activity.4 It has also been demonstrated that Hcy alters mitochondrial function, promoting ROS stress.9
In this issue of Hypertension, Suhara et al present evidence suggesting a link between Hcy toxicity and Fas-mediated apoptosis. Their findings demonstrate the importance of FLICE inhibitory protein (FLIP), an endogenous caspase-8 inhibitor in the death receptor pathway, in Hcy-induced apoptotic cell death (Figure). Their data also show that Hcy upregulates the receptor Fas on the endothelial cell surface through a NF-
B dependent mechanism. Furthermore, Hcy leads to the inactivation of the serine-threonine kinase PKB/Akt and downregulates FLIP expression, thereby sensitizing endothelial cells to death via the extrinsic apoptotic pathway. This study provides a framework to explain the cytotoxic effect of Hcy on cell viability and function because Akt signaling is central to both processes.10
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Fas ligand (FasL) is a type II membrane protein that induces apoptotic cell death in cells that bear the Fas receptor. The Fas receptor is a member of the tumor necrosis factor receptor family that is ubiquitously expressed by most cell types. In contrast, FasL is much more tissue-restricted in its expression and is primarily found on the cell surface of inflammatory and vascular cells. Binding of the trimeric FasL induces Fas receptor clustering and leads to the oligomerization of caspase-8. Caspase-8 oligomerization promotes proteolytic self-activation and triggers the proteolytic activation cascade of additional caspase family members, leading to apoptosis. The susceptibility of vascular smooth muscle cells (VSMCs) to Fas-mediated cell death in vitro and in vivo has been extensively documented.11 In contrast, vascular endothelial cells are normally resistant to Fas-mediated apoptosis and remain resistant even when Fas expression is upregulated by exposure to interferon.11,12 Endothelial cells naturally express low levels of endogenous FasL on their cell surface and can be genetically modified to express high levels of Fas ligand both in vitro and in vivo, with no detrimental effect on viability or vascular function.11,12 The marked differences in the sensitivity of vascular cells to Fas-induced apoptosis is mediated, at least in part, by the expression of cellular FLIP that functions as a dominant-negative inhibitor of caspase-8 function. FLIP isoforms are abundantly expressed in endothelial cells, but their downregulation can lead to Fas-mediated cell suicide (or fratricide).
The phosphatidylinositol (PI) 3-kinase/Akt signaling axis functions downstream of growth factor and matrix attachment signals to promote endothelial cell viability and other proangiogenic cellular responses.10 It has been shown previously that PI 3-kinase/Akt signaling is an important determinant of endothelial sensitivity to Fas-mediated death signals through its ability to modulate the expression of FLIP.13 FLIP is downregulated under conditions that lead to diminished PI 3-kinase/Akt signaling whereas activation of PI 3-kinase/Akt signaling promotes FLIP expression in endothelial cells. It has also been shown that Akt signaling promotes FLIP expression in tumor cells. The regulation of FLIP by Akt signaling has recently been shown to be mediated by the forkhead transcription factor FOXO3a in endothelial cells.14
Endothelial cells become susceptible to Fas-mediated cell death under other conditions that lead to Akt inactivation.13 For example, oxidized lipids inactivate Akt, promote FLIP downregulation, and induce Fas-mediated apoptosis in endothelial cells.15 Furthermore, anoikis leads to Akt downregulation, caspase-8 cleavage, and FLIP downregulation.14,16 Thus, the Akt-forkhead-FLIP regulatory axis may be an important regulator of endothelial cell viability in response to multiple environmental stimuli. Collectively, these findings suggest that the status of FLIP expression within endothelial cells may contribute to the generation of vascular lesions and control the balance between blood vessel growth and regression.
Finally, several recent studies have demonstrated a connection between NF-B activation and hyperhomocysteinemia. In this regard it has been shown that Hcy activates the NF-B–mediated inflammatory response, including the expression of adhesion molecules, monocyte chemoattractant protein 1, and interleukin-8, resulting in monocyte and T cell recruitment to the plaque. Because the number of inflammatory cells bearing surface FasL is more abundant in unstable lesions,17 it is tempting to speculate that the Hcy-mediated increase in endothelial cell sensitivity to Fas-induced apoptosis may contribute to endothelial cytotoxicity and promote lesion progression.
The study of Suhara et al in this issue of Hypertension defines the Akt-signaling-death receptor regulatory pathway of apoptosis as a novel target for Hcy-induced endothelial cytotoxicity in vitro. The study also provides a mechanistic link between Hcy-associated NF-B activation and the apoptosis of endothelial cells. Cell culture experiments in this study were carried out with Hcy concentrations ranging from 0.2 to 2.0 mmol/L. Because Hcy levels in serum are typically much lower, further work is required to apply these findings to the clinical situation. However, these new findings can be tested with in vivo models to assess the clinical importance of this mechanism.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
References
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