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Blockade of the apoptotic machinery by cyclosporin A redirects cell death toward necrosis in arterial endothelial cells: regulation by reactive oxygen species and cathepsin D¹

CHUM Research Centre, University of Montréal, Montréal, Québec, Canada; and
^* Guy-Bernier Research Centre, Maisonneuve-Rosemont Hospital, University of Montréal, Montréal, Québec, Canada

²Correspondence: CRCHUM, labo y-3624, 1560 Sherbrooke est, Montréal, QC, H2L 4M1, Canada. E-mail: marie-josee.hebert.chum{at}ssss.gouv.qc.ca

SPECIFIC AIMS

Blockade of the mitochondrial permeability transition pore (mPTP) by cyclosporin A (CsA) inhibits apoptosis in a wide array of cell types, including endothelial cells (EC). However, use of CsA in humans is associated with development of endothelial injury in the arterial vasculature. As mounting evidence suggests that blockade of the apoptotic machinery might reveal back-up death programs, we investigated whether inhibition of the apoptotic machinery by CsA promotes nonapoptotic forms of cell death in arterial EC.

PRINCIPAL FINDINGS

1. Morphological characteristics of cell death induced by cyclosporin A in normal and DNA damaged human umbilical artery endothelial cells (HUAEC)
The viability of HUAEC was significantly decreased after exposure to clinically relevant concentrations of CsA (1 to 100 µg/mL) for 6–24 h (Fig. 1 A). Using Hoechst 33342 (HT) and propidium iodide staining (PI), we found a significant increase in the percentage of cells bearing the morphological characteristics of necrosis in HUAEC exposed to CsA (1 and 10 µg/mL) for 24 h, without changes in the percentage of apoptotic cells (Fig. 1C, E ). Mitomycin C (MMC), a DNA damaging agent, significantly increased the percentage of apoptotic cells without increasing necrosis (Fig. 1C, E ). Concomitant exposure to MMC and CsA significantly inhibited apoptosis but significantly increased necrotic features (Fig. 1C, E ) in HUAEC. Mitochondrial dysfunction and decreased ATP/ADP ratios were found in HUAEC exposed to CsA and CsA + MMC but not in HUAEC exposed to MMC alone (Fig. 1D, E ). Representative micrographs of HUAEC exposed to two different pronecrotic positive controls (heating at 65°C and exposure to H₂O₂) show staining patterns similar to that observed in HUAEC exposed to CsA (Fig. 1E ).

View larger version (50K): [in this window] [in a new window]   Figure 1. Cyclosporin A protects against apoptosis, but enhances necrosis of HUAEC. A) Viability of HUAEC exposed to CsA (1, 10, and 100 µg/mL) for up to 24 h. *P < 0.05 vs. normal control, n 6. B) BrdU incorporation in subconfluent HUAEC exposed for 6, 16, and 24 h to CsA (1, 10, and 100 µg/mL). *P < 0.01 vs. N, n 10. C) % of apoptotic and necrotic cells in HUAEC exposed for 24 h to CsA (1 and 10 µg/mL), MMC (0.01 mg/mL), or both. *P < 0.04 vs. normal control (N), P < 0.05 vs. MMC, n 6. D) Ratio of ATP/ADP in HUAEC exposed for 0, 6, 16, and 24 h to CsA (10 µg/mL), MMC (0.01 mg/mL), or both. *P < 0.01 vs. normal control (N), n 6. E) Representative findings observed with HT/PI staining (upper panels) and DIOC6(3)/PI staining (lower panels) in adherent HUAEC exposed to normal medium (N), CsA (10 µg/mL), MMC (0.01 mg/mL), CsA+MMC, and two positive controls for necrosis: heating to 65°C for 30 min and H2O2 (20 mM) for 45 min. Necrotic cells are characterized by positive PI staining, cell swelling, absence of chromatin condensation, and loss of mitochondrial function.

Hence, these results suggest that CsA induces a necrotic response in normal arterial endothelial cells and, upon induction of DNA damage, shifts development of cell death from apoptosis to primary necrosis.

2. Classical effectors of apoptosis and caspases implicated in nonapoptotic programmed cell death are not involved in endothelial cell death induced by cyclosporin A
CsA-induced necrosis developed in the absence of p53 induction (data not shown). Preincubation with DEVD-CHO, an inhibitor of caspase-3, did not inhibit necrosis in HUAEC exposed to CsA but significantly reduced apoptosis induced by MMC (data not shown). Caspase-8 and caspase-1 were recently implicated in regulation of nonapoptotic programmed cell death, yet we found no evidence of caspase-8 or caspase-1 activation in HUAEC exposed to CsA (data not shown).

3. Evidence that oxidative stress and subsequent lysosomal damage are molecular regulators of cyclosporin-induced endothelial necrosis
We then tested the role of reactive oxygen species as potential regulators of CsA-induced necrosis. Flow cytometry analysis of HUAEC stained with dihydroethidium (DHE) showed that exposure to CsA induces a dose-dependent increase in superoxide production (Fig. 2 A). Superoxide dismutase, 1,3-dimethyl-2-thiourea (1,3 DT), a hydroxyl radical scavenger, and tiron, a scavenger of superoxide and hydroxyl radicals, significantly attenuated CsA-induced cell death (Fig. 2B ). However, uric acid, a scavenger of peroxynitrite, did not modulate CsA-induced cell death whereas inhibition of NO formation with L-NAME markedly enhanced CsA-induced cell death (Fig. 2B ).

View larger version (36K): [in this window] [in a new window]   Figure 2. Cyclosporin A-induced superoxide formation and lysosomal damage in HUAEC. A) FACS analysis of HUAEC stained with DHE showing a dose-dependent increase in generation of superoxide after exposure to CsA (1 and 10 µg/mL) for 3 and 6 h and at 6 h with MMC (0.01 mg/mL) (*P<0.01 vs. normal (N), n 6) 2,3-Dimethoxy-1-naphthoquinone (DMNQ) is a positive control for induction or reactive oxygen radicals. Representative FACS overlays for data obtained at 6 h are shown. B) % inhibition of CsA-induced cell death (CsA 10 µg/mL for 24 h) in the presence of Tiron (0,1 µM), Superoxide dismutase (SOD) (500 U/mL), 1,3-dimethyl-2-thiourea (1,3 DT) (0,1 mM), and uric acid (1 mM).(*P<0.01, n16 per conditions). C) Lysosomal dysfunction evaluated with acridine orange staining in HUAEC exposed for 16 h to normal medium (N), CsA 10 (µg/mL), CsA + Tiron (0,1 µM), or MMC (0.01 mg/mL) (*P=0.0001 vs. N, n5). Lower panel: representative findings in HUAEC treated for 16 h and stained with acridine orange. Lysosomal dysfunction (showing green fluorescence instead of red) is found in HUAEC exposed to CsA but not in HUAEC exposed to CsA + Tiron or MMC (*P=0.0001 vs. N, n5). D) % inhibition of CsA-induced cell death (CsA 10 µg/mL for 24 h) in presence of pepstatin A (100 µM), ZFA-FMK (100 µM), and ALLN (100 µM), *P < 0.0001, n 16.

We also evaluated the importance of lysosomal damage in CsA-induced necrosis using acridine orange staining. Lysosomal dysfunction peaked after 16 h of exposure to CsA and coincubation with tiron significantly attenuated development of CsA-induced lysosomal damage (Fig. 2C ). Exposure to a proapoptotic stimulus (MMC) did not induce lysosomal dysfunction (Fig. 2C ). Pepstatin A, an inhibitor of cathepsin D activity, significantly decreased CsA-induced endothelial cell death whereas inhibition of cathepsin B and L did not (Fig. 2D ).

CONCLUSIONS AND SIGNIFICANCE

CsA binds cyclophilin D, a mitochondrial matrix protein associated with the adenine nucleotide translocator, and thus prevents mitochondrial membrane permeabilization and apoptosis in a wide variety of cell types including endothelial cells. In the present paper we provide evidence that inhibition of the apoptotic machinery by CsA activates nonapoptotic forms of cell death in EC. We quantified concomitantly the various types of cell death (apoptosis and necrosis) developing upon exposure to CsA using adherent endothelial cells to avoid trypsinization, which may alter cell membrane integrity. We used arterial endothelial cells to reproduce the characteristic arterial cytotoxicity associated with use of CsA in humans. We tested the effect of CsA on endothelial cell death using clinically relevant concentrations. Area under the curve (AUC) CsA concentrations of 1 to 5 µg/mL are recommended in the immediate post-transplant period. Hence we studied CsA concentrations ranging from 1 to 10 µg/mL.

We report that CsA, while inhibiting apoptosis, enhances a form of cell death that bears the morphological and biochemical features of primary necrosis i.e., cell and nuclear swelling, absence of chromatin condensation, loss of cell membrane integrity, loss of mitochondrial function, and decreased ATP/ADP ratio. Concomitant exposure of HUAEC to CsA and a proapoptotic stimulus inhibited development of apoptosis but redirected cell death toward necrosis. We found that classical regulators of apoptosis such as p53 and caspase-3 are not involved in the pathways that govern cyclosporin-induced necrosis.

Reactive oxygen radicals and lysosomal proteases have been implicated in regulation of apoptotic and nonapoptotic forms of cell death. We show that CsA increased generation of superoxide and led to delayed lysosomal damage. Scavengers of superoxide and/or hydroxyl radicals prevented development of lysosomal damage and significantly decreased CsA-induced EC death. These results show that necrosis induced by CsA retains the character of a program in the sense that selective biochemical inhibitors can block CsA-induced cell death without interfering with the initial triggering event. We propose that oxidative stress is a central regulatory event inducing lysosomal damage and activation of cathepsin D, which in turn amplifies development of endothelial cell death (Fig. 3 ).

View larger version (20K): [in this window] [in a new window]   Figure 3. Mechanisms of CsA-induced endothelial necrosis. CsA increases generation of reactive oxygen species, which in turn induce lysosomal damage and necrosis. CyD: cyclophilin D, mitochondrial permeability transition pore: mPTP.

Our results provide novel insights into the molecular regulation of cell death that develops in presence of inhibitors of mitochondrial membrane permeabilization. Our results help resolve an ongoing debate on the role of CsA on endothelial apoptosis, as contradictory results have been reported. Using a method that clearly discriminates the various stages of apoptosis from necrosis, we confirmed the anti-apoptotic activity of CsA and provide conclusive evidence that CsA-induced cell death is a nonapoptotic form of programmed cell death bearing the morphological characteristics of necrosis yet under molecular regulation by oxidative stress and the lysosomal protease cathepsin D. Finally, our results suggest novel pathways that could be inhibited for prevention of CsA-induced endothelial damage.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0500fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0500fje

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