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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
Correspondence: * corresponding author, stefan.hippenstiel{at}charite.de
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ABSTRACT
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Porphyromonas gingivalis dihydroceramides are found in extracts of calculus-contaminated root surfaces, diseased gingival tissue, and atherosclerotic plaques. These ceramides have been shown to promote inflammatory secretory responses in gingival fibroblasts. Little is known about their effects on the vascular system. We tested the hypothesis that P. gingivalis lipids induce apoptosis of human endothelial cells, and investigated the effects of extracted and purified P. gingivalis lipids on human umbilical vein endothelial cells. P. gingivalis phosphoglycerol dihydroceramides induced apoptosis, but not necrosis, in endothelial cells. Early apoptotic cells showed exposure of phosphatidylserine on the cell surface, followed by the cleavage of procaspases 3, 6, and 9. The release of apoptosis-inducing factor was increased, suggesting mitochondrial involvement. Different caspase inhibitors and cAMP elevation blocked DNA fragmentation. Moreover, N-acetylcysteine significantly reduced apoptosis, suggesting a role for reactive oxygen species in this process. Analysis of these data indicates that dihydroceramides may be important virulence factors of P. gingivalis.
Key Words: Porphyromonas gingivalis • ceramide • apoptosis • endothelium • caspase • periodontitis
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INTRODUCTION
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Periodontal disease is a chronic inflammatory condition of the tooth-supporting tissues. Porphyromonas gingivalis is considered to be one of the major periodontal pathogens and is found in elevated levels in the subgingival biofilm in periodontal pockets and within diseased periodontal tissues. P. gingivalis has been shown to invade endothelial cells, followed by pro-inflammatory cell activation (Walter et al., 2004). This organism produces many virulence factors, e.g., fimbriae, lipopolysaccharide, proteinases, and outer membrane proteins (Holt et al., 1999). Furthermore, P. gingivalis synthesizes free dihydroceramides and other complex lipids similar in structure to mammalian ceramides, which are known to participate in the regulation of cell differentiation, gene expression, and apoptosis (Huwiler et al., 2000). P. gingivalis complex lipids have been shown to potentiate IL-1β-mediated prostaglandin E2 secretion in gingival fibroblasts, and to alter gingival fibroblast morphology in culture (Nichols et al., 2001, 2004). They have been identified in extracts from calculus-contaminated root surfaces and diseased gingival tissue (Nichols, 1994; Nichols and Rojanasomsith, 2006), and, more significantly, in atherosclerotic plaques (Nichols et al., unpublished observations). Several epidemiological studies have suggested an association between periodontal disease and cardiovascular disease (Beck et al., 2000; Söder et al., 2005).
P. gingivalis and other periodontal pathogens were identified within atheromas by polymerase chain-reaction (Haraszthy et al., 2000), and P. gingivalis has been reported to accelerate atherosclerosis in ApoE knockout mice (Gibson et al., 2004).
Atherosclerosis is characterized inter alia by the accumulation of lipids, particularly low-density lipoprotein (LDL)-cholesterol, within the intima of the arterial wall. Also, mammalian ceramide is reportedly elevated in atherosclerotic plaques (Kinnunen and Holopainen, 2002). The development of early atherosclerotic lesions is mediated by inflammation, including the infiltration of macrophages, and, furthermore, increased apoptosis of endothelial cells is apparent in advanced lesions.
Since mammalian ceramide has been implicated as a second messenger in the development of apoptosis (Huwiler et al., 2000), and complex lipids of P. gingivalis, including dihydroceramides, have been recovered in elevated levels in carotid endarterectomy samples (Nichols et al., unpublished observations), we tested the hypothesis that extracted and purified lipids of P. gingivalis induce apoptosis of cultured human umbilical vein endothelial cells.
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MATERIALS & METHODS
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Unless specified, all chemicals used were of analytical grade and were obtained from commercial sources.
Cell Culture
Human umbilical vein endothelial cells were isolated from human umbilical cord veins, identified, and cultured as previously described (N’Guessan et al., 2005). Only confluent monolayers in their first passage were used. The research was carried out according to the guidelines of the Declaration of Helsinki (1975), and informed consent of the participants was obtained.
Porphyromonas gingivalis Lipids
P. gingivalis ATTC strain 33277 was grown in pure culture. The lipids were extracted according to a method described by Bligh and Dyer (1959), fractionated via HPLC, and analyzed by GC/MS as described previously (Nichols et al., 2004, 2006). The total lipid extract, phosphoglycerol dihydroceramide, and phosphatidyl-ethanolamine lipid fractions were used for further experiments. The chemical structures of the phosphoglycerol dihydroceramide and phosphatidyl-ethanolamine lipids are shown in Fig. 1 . To test the biological activity of the purified lipids, we treated primary cultures of human gingival fibroblasts with bacterial lipids, and subsequently stimulated them with IL-1β. Culture supernatants were analyzed for prostaglandin secretion (for details, see Nichols et al., 2004). P. gingivalis lipids were dried under nitrogen and stored at –20°C.
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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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Cell Stimulation
The chloroform-dissolved lipids were added to the cell culture medium at 1 µg/mL for total lipid extract, and 0.1 µg/mL for the phosphoglycerol dihydroceramide and phosphatidyl-ethanolamine fractions. The lipid-supplemented media were shaken for 1 hr on a rocking platform at 37°C and 14,000 rpm.
Light- and Fluorescence-microscopy
Confluent endothelial cells grown on glass coverslips were treated with bacterial lipids for 1 hr, and cell morphology was observed by AxioCamMRc microscopy (Zeiss, Göttingen, Germany). For apoptosis analysis, cells were stimulated as indicated, washed twice, incubated for 15 min simultaneously with annexin-V-FITC and propidium iodide (PI) in binding buffer (annexin-V FITC Apoptosis detection kit; Acris GmbH, Hiddenhausen, Germany) and for 2 hrs with Anti-FITC Fluorescein/Oregon Green Alexa Fluor 488 conjugated (Invitrogen GmbH, Karlsruhe, Germany) for fluorescence stabilization, and visualized by fluorescence microscopy with an AxioCamMRc microscope (Zeiss) as described (N’Guessan et al., 2005). For quantification, annexin-V-positive-stained cells were counted in a blinded fashion.
Cell Death and Cytotoxicity
Confluent endothelial cells cultured in 96-well plates were stimulated for the indicated time periods. One hour before stimulation, the cells were pre-incubated with pan-caspase inhibitor zVAD (25 µM), caspase 6 inhibitor zVEID (0.5 µM, Calbiochem, Darmstadt, Germany), caspase 3-DEVD-CHO (10 µM), or caspase 9-inhibitor z-LEHD-FMK (7 µM, Oncogene, Glostrup, Denmark). Pre-incubation with N-acetylcysteine (Sigma-Aldrich, München, Germany) or RP-73401 (Roth, Karlsruhe, Germany) combined with forskolin (Sigma-Aldrich) was performed in the same manner.
Cytoplasmic histone-associated DNA fragments in cell lysates were measured with cell death detection ELISA, and lactate dehydrogenase (LDH) release into the supernatants of stimulated endothelial cells was determined by a Cytotoxicity Detection Kit (both Roche, Mannheim, Germany) according to the manufacturer’s instructions (N’Guessan et al., 2005).
Western Blot
For determination of procaspases or cleaved caspases, as well as the detection of apoptosis-inducing factor (AIF), endothelial cell monolayers were stimulated for 4 hrs with P. gingivalis lipids. Protein extraction and SDS-PAGE were performed as described previously (N’Guessan et al., 2005). Each lane contained 80 µg of protein. Blocked membranes were hybridized with antibodies raised against either caspase 6 (Cell Signaling, Beverly, MA, USA), procaspase 3 (Upstate Biotechnology, Lake Placid, NY, USA), procaspase 9 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or AIF (Upstate Biotechnology). In all experiments, ERK2 kinase (Santa Cruz Biotechnology) was detected simultaneously, to confirm equal protein loads. Proteins were visualized by incubation with secondary IRDye 800- or Cy5.5-labeled antibodies, with the use of an Odyssey infrared imaging system (LI-COR Inc., Lincoln, NE, USA).
Statistical Methods
Data are shown as the mean ± SD for at least 3 independent experiments. One-way analysis of variance (ANOVA) was used for the data, and the main effects were compared by a Newman-Keuls post-test (Figs. 2C, 2F, 4). Two-way ANOVA was used for the data shown in Figs. 2A and 2B, followed by the Bonferroni post-test. P-values less than 0.05 are indicated by an asterisk (*); p values of < 0.001 are indicated by double asterisks (**). If not indicated otherwise, each test category was compared with either unstimulated control cells or ceramide vehicle-treated cells for the indicated time period.
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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 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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RESULTS
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Purified Phosphoglycerol Dihydroceramide of P. gingivalis Induced Time- and Dose-dependent Apoptosis in Endothelial Cells.
Endothelial cells were stimulated with total lipid extract, phosphoglycerol dihydroceramide, and phosphatidyl-ethanolamine lipid fractions of P. gingivalis (ATCC strain #33277). Total lipid was used at 1 µg/mL, and the phosphoglycerol dihydroceramide and phosphatidyl-ethanolamine lipid fractions were used at 0.1 µg/mL. The dihydroceramide fraction induced significant DNA fragmentation 5 hrs after lipid exposure to the endothelial cells, indicating apoptosis (Fig. 2A ), while the lipid-solvent chloroform (c) did not promote apoptosis. Lower concentrations (< 0.1 µg/mL) of dihydroceramide (Fig. 2C), as well as the total lipid extract and the phosphatidyl-ethanolamine fraction, induced apoptosis, but to a lesser, insignificant extent (Fig. 2A). The positive control staurosporine (St) strongly promoted apoptosis in endothelial cells (Hippenstiel et al., 2002; N’Guessan et al., 2005; Sheets et al., 2005). To exclude non-specific DNA fragmentation in late cell necrosis, we analyzed the supernatant of each cell culture for LDH. We noted only insignificant LDH release upon 5 hrs of stimulation with P. gingivalis lipids, as well as after exposure to staurosporine (Fig. 2B).
P. gingivalis Lipids Disturbed Endothelial Monolayer Integrity and Showed Early Signs of Apoptosis.
One hour after stimulation with P. gingivalis lipids, cell monolayers demonstrated altered morphology. The unstimulated control cells (none), the lipid-solvent chloroform-(c), and the total lipid extract-treated cells showed no morphological changes; the dihydroceramide and phosphatidyl-ethanolamine fractions altered cell morphology and resulted in partial cell detachment (Fig. 2D). At higher magnification, typical apoptotic morphological changes were observed, including cell shrinkage, formation of apoptotic bodies, and cell blebbing (data not shown). Moreover, annexin-V and propidium iodide (PI) stain displayed enhanced annexin-V binding on outward-facing phosphatidylserine, as an early sign of apoptosis in phosphoglycerol dihydroceramide- and phosphatidyl-ethanolamine-stimulated endothelial cells (fluorescence microscopy, Fig. 2E), whereas only a few propidium-iodide-positive cells, indicating necrotic cells (dihydroceramide; 0-2 PI+ cells in the range of vision, data not shown), were observed. Quantification of the phosphatidylserine-positive cells indicated a significant increase in apoptotic cells, which were treated with the dihydroceramide (Fig. 2F).
Phosphoglycerol Dihydroceramide Induced Caspase Activation and Release of AIF in the Cytoplasm.
In many cases, programmed cell death is induced by the activation of caspase cascades, resulting in caspase activation. Therefore, we analyzed whether the P. gingivalis lipids could induce caspase cleavage, i.e., caspase activation. Endothelial cells were incubated with 1 µg/mL total lipid and 0.1 µg/mL of the phosphoglycerol dihydroceramide and phosphatidyl-ethanolamine fractions. Caspase-cleavage was detected by Western blot, with specific antibodies for procaspases 3 and 9, as well as a caspase 6 antibody. Four hours post-incubation with phosphoglycerol dihydroceramide, procaspases 3 and 9 were cleaved, and the caspase 6 level was markedly increased (Fig. 3). Western blot analysis did not show involvement of caspases 3, 6, and 9 after incubation with total lipid and the phosphatidyl-ethanolamine fraction (data not shown). The cleavage of caspases 9 observed with dihydroceramide-exposed endothelial cells suggests involvement of mitochondria-dependent factors in P. gingivalis dihydroceramide-induced apoptosis (Susin et al., 1999). Phosphoglycerol dihydroceramide treatment of endothelial cells induced significant cytoplasmic AIF release (Fig. 3). The lipid-solvent chloroform (c) showed neither induced caspase cleavage nor increased cytoplasmic AIF.
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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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Apoptosis Induced by Phosphoglycerol Dihydroceramide was Blocked by Caspase Inhibition and cAMP Elevation.
Next, we evaluated phosphoglycerol dihydroceramide lipid effects on caspase-dependent DNA fragmentation. For this, we pre-incubated endothelial cells for 1 hr with zVAD, a pan-caspase inhibitor, and specific inhibitors specifically blocking caspase 3, 6, and 9 activity, followed by treatment with dihydroceramide. After 5 hrs, we measured DNA fragmentation using cell death detection ELISA. The pan-caspase inhibitor zVAD, as well as the specific inhibition of caspases 3, 6, and 9, significantly reduced phosphoglycerol dihydroceramide-induced DNA fragmentation (Fig. 4A). To exclude DNA fragmentation triggered by toxic effects of the inserted inhibitors, we measured LDH levels in the culture supernatants. No significant LDH release was found (Fig. 4B). Furthermore, we investigated the potential beneficial effect of cAMP augmentation on phosphoglycerol dihydroceramide-induced DNA fragmentation. Endothelial cells degrade cAMP by phosphodiesterase-isoenzymes 3 and 4 (Suttorp et al., 1993). A pre-incubation for 1 hr with 0.5 µM of the adenylyl-cyclase activator forskolin and 0.5 µM of the phosphodiesterase 4 inhibitor RP-73401 significantly decreased the dihydroceramide-induced DNA fragmentation after 5 hrs (Fig. 4C). A comparable decrease in DNA fragmentation 5 hrs post-ceramide exposure occurred after pre-incubation of the endothelial cells with the reactive oxygen species (ROS) scavenger N-acetylcysteine (0.5 mM) (Fig. 4C). LDH release was not elevated in the culture supernatants (Fig. 4D).
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DISCUSSION
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P. gingivalis dihydroceramides are considered to be virulence factors at periodontally diseased sites (Nichols and Rojanasomsith, 2006). Several lines of evidence have indicated a link between periodontal and cardiovascular disease (Mattila et al., 1993; Beck et al., 2000; Desvarieux et al., 2003; Söder et al., 2005). In particular, observations in humans (Haraszthy et al., 2000) and animal models (Gibson et al., 2004; Brodala et al., 2005) have suggested that infections with the periodontal pathogen P. gingivalis may contribute to the development of atherosclerosis.
P. gingivalis dihydroceramides can be detected in carotid atherosclerotic lesions (Nichols et al., unpublished observations); therefore, we tested the hypothesis that P. gingivalis ceramides induce endothelial cell death, thereby potentially contributing to the development of vascular lesions. Phosphoglycerol dihydroceramides, but not the phosphatidyl-ethanolamine lipid fraction or the total lipid extract of P. gingivalis, induced endothelial cell apoptosis in vitro. Interestingly, Sphingobacterium phosphoceramides, which show structural similarities to P. gingivalis dihydroceramides, are also capable of inducing apoptosis, as was shown in human myeloid leukemia cells (Minamino et al., 2003). Apoptosis was also stimulated by synthetic sphingosine, as was endogenous ceramide formation in endothelial and other cells (Obeid et al., 1993; Hisano et al., 1999). However, the mechanisms on how ceramide induces apoptosis have not been established, although activation of caspase 3 was demonstrated in several studies (Huwiler et al., 2000; Minamino et al., 2003; Shikata et al., 2003).
According to our results, phosphoglycerol dihydroceramide induced activation of caspases 3, 6, and 9, and inhibition of these caspases significantly reduced phosphoglycerol dihydroceramide-mediated apoptosis in endothelial cells.
In addition to caspase 3, caspases 6 and 9 are involved in ceramide-dependent cell death induced by phosphoglycerol dihydroceramide lipids of P. gingivalis. The prominent role of caspase 9 in P. gingivalis ceramide-induced endothelial apoptosis suggests the involvement of mitochondrial proteins. Increased levels of cytosolic AIF in phosphoglycerol dihydroceramide-exposed cells support this hypothesis as well.
The production of reactive oxygen species (ROS) is suggested to contribute to ceramide-mediated perturbation of endothelial cell behavior (Li et al., 2002). P. gingivalis dihydroceramide-induced apoptosis was reduced after the pre-incubation of endothelial cells with the ROS scavenger N-acetylcysteine. In addition, cAMP elevation greatly reduced P. gingivalis ceramide-related apoptosis. Since both measures also reduced the endothelial apoptosis induced by other bacteria [e.g., pneumococci (N’Guessan et al., 2005)], the elucidation of the contributing pathways (i.e., ROS signaling, cAMP action in infected cells) could pave the way to new anti-apoptotic strategies in endothelial cells.
A previous report demonstrated that P. gingivalis phosphorylated dihydroceramides promote pro-inflammatory reactions (Nichols et al., 2004), and this process, together with the induction of endothelial cell apoptosis, has been proposed
to contribute to the development of atherosclerosis. Altered endothelial cell function and apoptosis have been proposed to contribute to inflammatory vessel diseases, including atherosclerosis (Stefanec, 2000; Hippenstiel and Suttorp, 2003).
In summary, phosphoglycerol dihydroceramides of P. gingivalis induced apoptosis in cultured human endothelial cells. Dihydroceramide-related apoptosis is associated with caspase 3, 6, and 9 activation. Furthermore, P. gingivalis dihydroceramides induced AIF release, and the elevation of intracellular cAMP inhibited endothelial cell apoptosis, suggesting mitochondrial involvement. Moreover, reducing oxidative stress in endothelial cells by pre-incubation with ROS scavenger N-acetylcysteine reduced phosphoglycerol dihydroceramide-induced apoptosis. Therefore, P. gingivalis phosphoglycerol dihydroceramides may be important virulence factors in the development of periodontal as well as cardiovascular diseases.
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ACKNOWLEDGMENTS
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This investigation was supported by University research funding of the Charité and by the Deutsche Forschungsgemeinschaft to J.Z., B.R., C.W., and J.-P.B. (GRK 325), and to S.H. and B.S. by the BMBF competence network CAPNETZ-C15.
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FOOTNOTES
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 authors contributing equally to this study 
Received for publication December 2, 2005.
Revision received December 14, 2006.
Accepted for publication February 12, 2007.
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Journal of Dental Research, Vol. 86, No. 7,
635-640 (2007)
DOI: 10.1177/154405910708600710
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N Pischon, E Rohner, A Hocke, P N'Guessan, H C Muller, G Matziolis, V Kanitz, P Purucker, B-M Kleber, J-P Bernimoulin, et al.
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[Abstract]
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S. Amar, S.-c. Wu, and M. Madan
Is Porphyromonas gingivalis Cell Invasion Required for Atherogenesis? Pharmacotherapeutic Implications
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TOP
ABSTRACT
INTRODUCTION