Role of calcium channels in oxyhemoglobin-induced apoptosis in endothelial cells

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Object. Oxyhemoglobin (OxyHb) released from hemolysed erythrocytes has been considered to be responsible for cerebral vasospasm after subarachnoid hemorrhage. The authors previously reported that OxyHb produced apoptosis in cultured vascular endothelial cells. The change in intracellular Ca++ homeostasis was expected to be one of the possible mechanisms of the cytotoxic effects of OxyHb. This study was undertaken to investigate the protective effects of Ca++-channel blockers on OxyHb-induced apoptosis.

Methods. Cultured bovine coronary artery and brain microvascular endothelial cells (passages 5–9) were used. A cell density study, immunohistochemical staining, and DNA fragmentation analysis were performed to confirm apoptosis. Various concentrations (1–50 µM) of OxyHb were used for 24- to 72-hour incubations with and without Ca++-channel blockers.

Oxyhemoglobin produced cytotoxicity leading to cell detachment from the culture dish in time- and concentration-dependent manners. The highest dose (50 µM) of OxyHb produced cell detachment after a 24-hour incubation, and the lower doses (1–10 µM) produced cell detachment after 48 to 72 hours. Immunohistochemical analysis showed that apoptosis occurred in cells that were still attached to the side of the culture dish after 48 to 72 hours of OxyHb treatment (5 µM). The OxyHb (10 µM) produced DNA ladders at 48 to 72 hours. Three Ca++-channel blockers were used to prevent the toxic effect of OxyHb. The voltage-dependent Ca++-channel blocker nicardipine (1 µM), the voltage-independent Ca++-channel blocker econazole (10 µM), and the inorganic Ca++-channel blocker lanthanum (100 µM) all failed to prevent cell detachment or DNA ladders produced by OxyHb. These results were similar in both cell lines.

Conclusions. Oxyhemoglobin produced apoptotic changes in cultured vascular endothelial cells, and Ca++-channel blockers did not prevent OxyHb-induced apoptosis.

Article Information

Address reprint requests to: John H. Zhang, M.D., Ph.D., Department of Neurosurgery, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. email: jzhang@neurosurgery.umsmed.edu.

© AANS, except where prohibited by US copyright law.

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Figures

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    Bar graphs demonstrating the effects of OxyHb on endothelial cell density in BCAECs (A) and BBMvECs (B). Endothelial cells were incubated in culture medium with OxyHb (1, 5, 10, or 50 µM) for 24, 48, or 72 hours. After incubation, the numbers of adherent cells were calculated by using a hemocytometer and expressed as a percentage of the mean number of control cells. * p < 0.05 and ** p < 0.01 compared with control cells (which themselves would be 100%).

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    Photomicrographs demonstrating representative immunohistochemical staining of anti-bax (A and B), anti-bcl-2 (C and D), and anti-fas (E and F) antibodies in BCAECs treated with 5 µM OxyHb (A, C, and E) or saline control (B, D, and F) for 72 hours. The OxyHb (5 µM) increased bax (A) and fas (E) expression and decreased bcl-2 expression (C) in BCAECs (arrows). Nuclei of typical apoptotic cells are condensed and cell membranes are shrunk (arrowheads). Original magnifications × 200.

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    Bar graphs showing the effects of Ca++-channel blockers on endothelial cell density of OxyHb-induced cytotoxicity. Endothelial cells were preincubated with or without each Ca++-channel blocker (nicardipine, 1 µM; econazole, 10 µM; or lanthanum, 100 µM) for 30 minutes at 37°C before final addition of OxyHb (1, 5, 10, or 50 µM). Cells with Ca++-channel blockers and OxyHb were incubated for 24, 48, or 72 hours. A: The BCAECs. B: The BBMvECs. * p < 0.05 and ** p < 0.01 compared with cells treated only with OxyHb.

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    Results of DNA electrophoresis showing the effects of Ca++-channel blockers on OxyHb-induced DNA ladders. Endothelial cells (BCAECs [A] and BBMvECs [B]) were incubated with OxyHb (10 µM) and Ca++-channel blockers for 72 hours. The DNA markers are shown in Lane 1. Lane 2 is a saline-treated control group. Lane 3 is an OxyHb-treated group. Lanes 4 through 6 are groups treated with OxyHb and a Ca++-channel blocker (nicardipine, econazole, or lanthanum, respectively).

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