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Porphyromonas gingivalis Dihydroceramides Induce Apoptosis in Endothelial Cells
J. Zahlten1,2, ,
B. Riep1,,
F.C. Nichols3,
C. Walter1,4,
B. Schmeck2,
J.-P. Bernimoulin1 and
S. Hippenstiel2,*
1 Institute for Periodontology and Synoptic Dentistry, Charité Centrum 3 for Dental Medicine, and
2 Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, Charité-Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany;
3 Department of Periodontology, University of Connecticut School of Dental Medicine, 263 Farmington Avenue, Farmington, CT 06030, USA; and
4 Department of Periodontology, Endodontology and Cariology, University of Basel, Hebelstrasse 3, 4056 Basel, Switzerland
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Figure 1. Chemical structure of P. gingivalis phosphoglycerol dihydroceramide and phosphatidyl-ethanolamine lipid classes. P. gingivalis lipids were extracted according to a method previously described (Bligh and Dyer, 1959), fractionated via HPLC, and analyzed by GC/MS. (A) P. gingivalis phosphoglycerol dihydroceramides contain 3-OH isobranched (iso) C17:0 fatty acid in amide linkage to saturated dihydroxy long-chain bases of either 17, 18, or 19 carbons in length, and is substituted with isoC15:0 linked to the beta hydroxyl of 3-OH iso C17:0 (Nichols et al., 2004). (B) The dominant phosphatidyl-ethanolamine lipids are substituted with isoC15:0 with or without isoC13:0 (Nichols et al., 2006).
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Figure 2. Purified complex lipids of P. gingivalis induced time- and dose-dependent DNA fragmentation in endothelial cells and early signs of apoptosis, as evidenced by increased phosphatidylserine expression. Cells were stimulated with P. gingivalis lipids (total lipid extract [TL], 1 µg/mL; phosphoglycerol dihydroceramide [PG DHC] and phosphatidyl-ethanolamine [PE] lipid fractions each 0.1 µg/mL). (A) DNA fragmentation was measured in cell lysates at 4 different time-points. The total-lipid-extract and phosphatidyl-ethanolamine led to an increase of DNA fragmentation 5 hrs post-stimulation, but only phosphoglycerol dihydroceramide induced significant apoptosis (**p < 0.001). Staurosporine (St, 1 µM) was used as a positive control. Data are expressed as means ± SD (n = 3).
(B) The supernatants were collected, and LDH release was used as an indicator of cell lysis related to necrotic cell death. A slight but insignificant increase of LDH occurred 5 hrs after stimulation with P. gingivalis lipids, as well as after staurosporine exposure of cells. The highest, but still not significant, LDH release was triggered by the total lipid extract and staurosporine, measured 10 hrs post-stimulation. The lipid-solvent chloroform (c) had no effect on either DNA fragmentation or LDH release. Data are expressed as means ± SD (n = 3).
(C) Endothelial cells were stimulated with different concentrations of phosphoglycerol dihydroceramide (PG DHC; 0.05, 0.075, and 0.1 µg/mL) for 5 hrs, and DNA fragmentation was measured in cells that had undergone lysis. Dihydroceramide led to a dose-dependent increase of apoptotic cells. A concentration of 0.075 µg/mL provoked DNA fragmentation, but a significant number of apoptotic cells was seen with a dihydroceramide dose of 0.1 µg/mL (**p < 0.001). Data are expressed as means ± SD (n = 3, with duplicate measurements).
(D) P. gingivalis-purified lipid fractions phosphoglycerol dihydroceramide and phosphatidyl-ethanolamine disturbed the endothelial cell morphology and led to increased phosphatidylserine expression on the outer cell membrane. Endothelial cells were stimulated with the total lipid extract (TL) and the different lipid fractions (phosphoglycerol dihydroceramide; phosphatidyl-ethanolamine) for 1 hr, and cell morphological changes were documented by light microscopy. Exposure of cells to dihydroceramide and phosphatidyl-ethanolamine induced loss of monolayer integrity. Dihydroceramide notably caused loss of cell adherence to the culture dish.
(E) The cells were stained simultaneously with annexin V-FITC and propidium iodide, and, for fluorescence stabilization, with Anti-FITC Fluorescein/Oregon Green Alexa Fluor 488 conjugated, and visualized by fluorescence microscopy. Incubation of cells with dihydroceramide resulted in an increased number of phosphatidylserine-positive cells, compared with the control cells treated with lipid-solvent (none; c) and with other P. gingivalis lipid preparations (total lipid; phosphatidyl-ethanolamine). Representatives of 3 independent experiments with similar results are shown.
(F) Annexin-V-stained cells were quantified by direct counting in a blinded fashion. The graph contains data from 9 counted pictures of 3 independent experiments for each group. Only phosphoglycerol dihydroceramide induced phosphatidylserine-positive cells significantly (**p < 0.001). Data are expressed as means ± SD (n = 3).
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Figure 3. Caspase activation in endothelial cells exposed to phosphoglycerol dihydroceramide lipid fraction of P. gingivalis. Endothelial cells were incubated with 0.1 µg/mL of the phosphoglycerol dihydroceramide (PG DHC) fraction for 4 hrs, and the cleavage of procaspases 3 and 9, as well as caspase 6, and the release of apoptosis-inducing factor (AIF) were detected by Western blot. Exposure of cells to dihydroceramide resulted in cleavage of procaspases 3 and 9 and an increased level of caspase 6. Moreover, AIF level was increased. The lipid-solvent chloroform had no effect (c). Simultaneous detection of ERK2 documented the equal protein load. Representative blots of 3 different experiments with similar results are shown.
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Figure 4. The DNA fragmentation induced by the phosphoglycerol dihydroceramide lipid fraction of P. gingivalis was blocked by caspase inhibitors, elevated cAMP level, or N-acetylcysteine pre-incubation. Cells were pre-incubated with different caspase inhibitors (pan-caspase inhibitor zVAD [25 µM], caspase 3 inhibitor [10 µM], caspase 6 inhibitor [0.5 µM], and caspase 9 inhibitor [7 µM]) 1 hr before stimulation with 0.1 µg/mL phosphoglycerol dihydroceramide (PG DHC) fraction. Endothelial cells were stimulated for 5 hrs, and DNA fragmentation was measured by cell death detection ELISA. The supernatants were collected, and LDH release was determined. Dihydroceramide-induced DNA fragmentation was significantly reduced (**p < 0.001) through all engaged caspase inhibitors (A). No significant LDH release was observed (B). Pre-incubation with N-acetylcysteine (NAC; 0.5 mM) and combined RP-73401/forskolin (each 0.5 µM) 1 hr before dihydroceramide exposure resulted in a significant reduction in DNA fragmentation and no significant LDH release into the supernatant (C,D) at 5 hrs. Data are expressed as means ± SD (n = 3).
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Journal of Dental Research, Vol. 86, No. 7,
635-640 (2007)
DOI: 10.1177/154405910708600710
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