Cerebral vasospasm is the major cause of morbidity and mortality in the patients after aneurysmal subarachnoid hemorrhage (SAH). The pathogenesis of cerebral vasospasm is unclear, in spite of the intensive investigations in the past years. Even though multiple factors are proposed to be involved in cerebral vasospasm, oxyhemoglobin (OxyHb), the main components of erythrocytes, is probably responsible for the prolonged vasoconstriction and most of the cytotoxicity in vascular wall especially in endothelial cells. Auto-oxidation from OxyHb to met-hemoglobin releases free radicals that in turn may cause damage to vessel wall tissue especially to endothelial cells.
It has been reported that craters, blebs and vacuoles occurred in endothelial cell in cerebral arteries during vasospasm that may lead to detachment of endothelial cells. However, little is known about the pattern of cell death, as a final stage of cell damage, induced by OxyHb in endothelial cells. This study investigated the pattern of cell death induced by OxyHb in cultured bovine aortic endothelial cells using specific methods such as DNA laddering, transmission electron microscopy, and Western blotting of poly (ADP-ribose) polymerase cleavage.
Materials and Methods
Cell density study
Analysis of DNA-fragmentation (DNA ladder)
Transmission Electron Microscopy
DNA degradation - DNA ladder
Transmission Electron Microscopy - Apoptotic body
Discussion and Conclusion
Summary Structural changes in the arterial wall after SAH have been reported and believed to contribute to cerebral vasospasm. Detachment and vacularization of endothelial cells and necrotic changes of the medial smooth muscle cells are the most striking histological alterations. Whether these pathological changes are secondary to the prolonged contraction or the results of direct effects of subarachnoid blood clot is still debatable. There is a line of evidence showing OxyHb produces direct cytotoxicity in smooth muscle or endothelial cells. In feline cerebral arteries, OxyHb caused myonecrosis, transformation of nerve endings, invasion of myointimal cells into the tunica intima, changes in endothelial cell basement membrane, and the detachment of endothelial cells.
Other pathological changes induced by OxyHb such as craters, blebs and vacuoles in endothelial cells and vacuoles, nuclear pyknosis, and mitochondria degeneration in smooth muscle cells were documented in rat basilar artery. In addition, OxyHb produce d vaculation and detachment in cultured bovine internal carotid endothelial cells, under a similar culturing condition as reported in this study. The direct cytotoxicity of OxyHb to endothelial or smooth muscle cells may play a role in the pathogenesis of cerebral vasospasm. However, little is known about the pattern of cell death, necrosis or apoptosis, as a final stage of cell damage, in either smooth muscle or endothelial cells.
We have demonstrated in this study that OxyHb induced concentration- and time-dependent decrease in cell density (as shown by cell detachment) in cultured bovine aortic endothelial cells. This result is consistent with the reports by others using endothelial cells from bovine internal carotid artery. The pattern of cell death induced by OxyHb is clearly apoptosis in endothelial cells as shown by three selective methods to identify apoptosis. (1) DNA ladder is the hallmark of apoptotic changes. OxyHb produced concentration- and time-dependent DNA fragmentation in endothelial cells. (2) Apoptotic body is another feature of apoptosis that can be demonstrated by transmission electron microscopy. OxyHb induced condensation and margination of chromatin, nuclear fragmentation and convolution of the cellular membrane, crowded and condensed apoptotic bodies in endothelial cells. (3) Western blotting of cleavage of 116 kDa PARP to 85 kDa, an apoptosis-related fragment, is also a selective marker for apoptosis. OxyHb cl early cleaved 116 kDa PARP to 85 kDa in endothelial cells.
Two fundamentally different forms of cell death, necrosis and apoptosis, have been defined in terms of morphology and biochemistry. Necrosis is a degenerative phenomenon produced by major environmental change such as severe ischemia, extremes of temperature, and mechanical trauma. The characteristics of necrosis are swelling of cytoplasm and organelle, balloon-like degeneration and early rupture of plasma membrane. Lyzosomal enzymes released after necrosis cleaved DNA randomly into fragments that displaying a smear pattern.
Apoptosis, in contrast, is an active biological process of eliminating unwanted cells involved in the regulation of cell number under physiological and pathological condition. Apoptosis is associated with early and prominent condensation of nuclear chromatin, decreasing cell size and ultimate cleavage of DNA by a specific endonuclease into mono-, and polynucleosomal fragments. The present study shows that exposure of endothe lial cells to OxyHb results in concentration- and time-dependent decrease of cell density. Dying cells exhibit condensation of nuclei and apoptotic bodies, inter-nucleosomal DNA fragmentation, and PARP cleavage associated with apoptosis.
The mechanism of apoptosis induced by OxyHb in endothelial cells is not clear. One of the possible explanations for OxyHb-induced apoptosis is free radical generation. Normally, OxyHb undergoes a slow, but spontaneous oxidation-reduction reaction, in which the oxygen is reduced to superoxide. This reaction is the basis for the oxidation of OxyHb to met-hemoglobin and is the main source of superoxide and hydrogen peroxide. Those free radicals may initiate and propagate lipid peroxidation by the Haber-Weiss reaction and Fenton chemistry. Free radicals are important factors for the induction of apoptosis in other tissues.
Another possible mechanism for apoptosis is the elevation of intracellular Ca2+ concentration. Sustained elevation of intracellular Ca2+ stimulates an endogenous endonuclease activity and initiates apoptosis in other tissues. Erythrocyte lysate increases [Ca2+]i by release Ca2+ from internal stores and promote Ca2+ influx from voltage-independent Ca2+ channels in cerebral endothelial cells. Other components of blood clot may be involved in apoptosis. Extracellular ATP, another main components of erythrocytes, causes apoptosis in pulmonary artery endothelial cells, as assessed by morphological changes and inter-nucleosomal DNA degradation. Interestingly, both OxyHb and ATP can be released from blood clot and they act not only as vasoconstrictors but also as promoters for apoptosis. TGF-b1, which is major component of platelets, accelerates non-muscle compaction, inhibits bcl-2 expression and induces apoptosis in cultured umbilical vein endothelial cells.
Since Kerr et al proposed the term apoptosis for cell death in 1972, apoptotic changes have not been observed in endothelial cells in spastic arteries after SAH. One possibility is that, once apoptosis occurs in endothelial cells, those damaged cells may be detached from the internal elastic lamina and be washed away by blood flow. Another possibility is that simply no attention was paid to look for apoptotic changes in any layers of the spastic arteries. Vascular endothelial cells are not only a physical and metabolic barrier but play an important role in the regulation of vascular tone. Endothelial cells generate both vasoconstricting and vasodilating agents and those agents such as endothelin8 and nitric oxide have been documented to contribute to cerebral vasospasm. Destruction of endothelial cells also exposes smooth muscle cells directly to blood flow that may contain vasoconstrictors such as OxyHb.
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|Zhang, J.; Ogihara, K.; Zubkov, AY.; Bernanke, DH.; Lewis, AI.; Parent, AD.; (1998). Oxyhemoglobin Induced Apoptosis In Cultured Endothelial Cell. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/|
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