Linköping University Postprint
The periodontal pathogen Porphyromonas
gingivalis cleaves apoB-100 and increases the
expression of apoM in LDL in whole blood
leading to cell proliferation
Torbjörn Bengtsson, Helen Karlsson, Patrik Gunnarsson, Caroline Skoglund, Charlotte Elison, Per Leanderson and Mats Lindahl
N.B.: When citing this work, cite the original article.
The definitive version is available at www.blackwell-synergy.com:
Torbjörn Bengtsson, Helen Karlsson, Patrik Gunnarsson, Caroline Skoglund, Charlotte Elison, Per Leanderson and Mats Lindahl, The periodontal pathogen Porphyromonas gingivalis cleaves apoB-100 and increases the expression of apoM in LDL in whole blood leading to cell proliferation, 2008, Journal of Internal Medicine, (263), 5, 558-571.
http://dx.doi.org/10.1111/j.1365-2796.2007.01917.x.
Copyright: Blackwell Publishing www.blackwell-synergy.com
Postprint available free at:
Submitted to Journal of Internal Medicine 2007-01-17, revised 2007-05-16, revised 2007-09-25
The periodontal pathogen Porphyromonas gingivalis cleaves
apoB-100 and increases the expression of apoM in LDL in whole blood
leading to cell proliferation
Torbjörn Bengtsson1*, Helen Karlsson2, Patrik Gunnarsson1, Caroline Skoglund1, Charlotte Elison2, Per Leanderson2, Mats Lindahl2
Cardiovascular Inflammation Research Centre
1
Division of Pharmacology, Department of Medicine and Health Sciences
2
Division of Occupational and Environmental Medicine, Department Clinical and Experimental Medicine
Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden
*Corresponding author: Torbjörn Bengtsson
Division of Pharmacology, Department of Medicine and Care
Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden Fax: +46 13 149106, Phone: +46 13 222058
E-mail address: torbe@imv.liu.se
Abstract
Objective: Several studies support an association between periodontal disease and atherosclerosis with a crucial role for the pathogen Porphyromonas gingivalis. This study aims to investigate the proteolytic and oxidative activity of P. gingivalis on LDL in a whole blood system by using a proteomic approach and analyze the effects of P. gingivalis-modifed LDL on cell proliferation. Methods: The cellular effects of P. gingivalis in human whole blood were assessed using lumi-aggregometry analyzing reactive oxygen species (ROS) production and aggregation. Blood was incubated for 30 min with P. gingivalis, whereafter LDL was isolated and a proteomic approach was applied to examine protein expression. LDL-oxidation was determined by analyzing the formation of protein carbonyls. The effects of P. gingivalis-modifed LDL on fibroblast proliferation were studied using the MTS-assay.
Results: Incubation of whole blood with P. gingivalis caused an extensive aggregation and ROS-production, indicating platelet and leukocyte activation. LDL prepared from the bacteria-exposed blood showed an increased protein oxidation, elevated levels of apoM and formation of two apoB-100 N-terminal fragments. P. gingivalis-modified LDL markedly increased the growth of fibroblasts. Inhibition of gingipain R suppressed the modification of LDL by P. gingivalis.
Conclusions: The ability of P. gingivalis to change the protein expression and the proliferative capacity of LDL may represent a crucial event in periodontitis-associated atherosclerosis.
1.
Introduction
Periodontitis has been associated with an elevated risk in the development of cardiovascular disease [1-3]. Porphyromonas gingivalis is a Gram-negative anaerobic rod found in periodontal pockets and considered to be the key etiologic agent of adult periodontal disease [4]. As a consequence of injury of periodontal tissue, P. gingivalis has a chance to disseminate into the bloodstream and cause transient bacteremias. Several studies have localized the bacteria in the blood stream and in atherosclerotic plaques thus suggesting a role for P. gingivalis in atherogenesis [5, 6]. However, it is uncertain whether a P. gingivalis infection is a triggering factor of atherosclerosis or a secondary complication of a smouldering inflammatory process already present in atherosclerotic plaques. P. gingivalis expresses a broad range of virulence factors, including cysteine proteases (gingipains), lipopolysaccharides (LPS) and fimbriae [7]. Among these factors, gingipains (gingipain R and gingipain K) are of special importance due to their ability to destroy tissues and disturb host defense mechanisms. The destruction of periodontal tissue and possibly also the development of the atherosclerotic plaque is probably due to host-derived factors generated during the interaction between P. gingivalis and inflammatory cells. An increasing amount of evidence suggests that reactive oxygen species (ROS) and proteolytic enzymes (bacteria- and host-derived) are major contributors to the chronic inflammatory reactions of periodontitis and atherosclerosis [8,9].
High plasma levels of low-density lipoprotein (LDL) are a major risk factor for coronary artery disease [10]. A variety of cell types involved in the atherosclerotic process, including leukocytes, platelets, smooth muscle cells, and endothelial cells, can modify LDL and transform the lipoprotein to a highly atherogenic form [11,12]. Modified LDL exerts several effects supporting atherogenesis including increased synthesis and secretion of adhesion molecules, leukocyte
adhesion and chemotaxis, injury to endothelial cells, enhanced foam cell formation, increased smooth muscle cell proliferation and release of inflammatory mediators [13,14]
.
Although several studies support a role for modified LDL in atherogenesis, the pathogenic stimuli that induce LDL modification and the mechanisms by which this occur in vivo are unknown. It is possible that infectious agents and their interaction with inflammatory cells modify LDL and render it atherogenic, and thereby contribute to lesion development. In support, animal models have demonstrated that the host response to infection and inflammation induces LDL oxidationin vivo [15]. We have recently shown that platelet activation by the respiratory pathogen Chlamydia pneumoniae is associated with an extensive oxidation of LDL [16].
Although prevalent modification of LDL takes place in the arterial intima, it is possible that circulating LDL is already mildly modified and expresses atherogenic structures. Indeed, there is evidence for the presence of mildly oxidized LDL in the blood stream [17,18]. It has been suggested that circulating modified LDL can damage endothelial cells and cause endothelial dysfunction and that plasma-modified LDL particles accumulate in the arterial intima in the preference to native LDL [19]. P. gingivalis penetrates the periodontal tissue and vascular endothelium and consequently encounters the cells and plasma components of the circulating blood. Indeed, P. gingivalis has in
vitro been found to trigger platelet and neutrophil activation including aggregation, secretion and
ROS production [20-22]. Consequently, interaction between periodontal bacteria, platelets and neutrophils in the vascular compartment may release reactive radicals and proteolytic enzymes transforming circulating native LDL to a modified and atherogenic form.
The mechanisms by which LDL particles are modified are far from completely understood. Degradation of apoB-100 through the action of proteases released from inflammatory and/or intimal cells may lead to instability of LDL particles and to their fusion [23]. Indeed, the apoB-100 of LDL
isolated from atherosclerotic intima was found to be proteolytically fragmented [23,24]. Fragmentation of apoB-100 can also be caused by oxidative modification of LDL [25]. Consequently, a detailed analysis of the protein and lipid expression and the type of oxidative damage of LDL particles is crucial in order to identify and clarify the role of different cell-derived proteases and oxidative agents in the modification of LDL to an atherogenic form.
The aim of this study was to investigate whether the inflammatory response to P. gingivalis in an ex
vivo human blood model transforms LDL to an atherogenic form by analyzing the protein
2.
Methods
2.1 Chemicals and buffers
The chemicals and their sources were as follows: C-reactive protein (CRP), formyl-methionyl-leucyl-phenylalanine (fMLP), L-αLysophosphatidylcholine (LPC) (Sigma Chemical Co. St Louis, MO, USA); collagen (Chrononlog Corp., Havertown, PA, USA); Rhodamine phalloidin, rabbit anti-DNPH (Molecular Probes, Eugene, OR, USA); paraformaldehyde (PFA) (Labkemi, Stockholm, Sweden); CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega
Corp., Madison, WI, USA); FITC-conjugated rabbit goat IgG (Calbiochem, UK); anti-Apolipoprotein B-48/100 IgG (Nordic Biosite AB, Täby, Sweden); goat anti-rabbit IgG peroxidase conjugate (DakoCytomation, Washington DC, PA, USA); α-cyano-4-hydroxy-cinnamic acid, 2,5 –dihydroxybenzoic acid, Bio-Rad protein assay nr: 500-0006 (Bio-Rad, Hercules, CA, USA); IPG buffer pH 3-10 NL, IPG:s 3-10 NL, dry strip cover fluid, gelbond PAG film, Pharmalyte 3-10, PD 10 columns (SephadexTM G-25 M) (Amersham Biosciences, Uppsala, Sweden); human skin fibroblasts (AG0518C; Coriell Institute for Medical Research, Camden, NJ, USA); porcine trypsin (Promega, Madison, WI, USA); calibration mixture for peptide mass fingerprinting; des-Arg-bradykinin, angiotensin I, Glu 1-fibrinopeptide B, adrenocorticotropic hormone (ACTH) clip 1-17 and clip 18-39 with masses: 904.468, 1296.6853, 1570.6774, 2093.0867 and 2465.1989, respectively (Applied Biosystems, Foster City, CA, USA).
Dulbeccos Modified Eagle Medium (supplemented with 1 mM sodium pyruvate, 1 % non-essential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin) (DMEM), Fetal Bovine Serum (FBS), Trypsin-EDTA (Gibco Invitrogen Corp. Canada); KBrsolution (density 1.100 g/mL; 0.133 g/mL KBr, 1 mg/mL EDTA); Phosphate-Buffered Saline (PBS) (pH 7.3; density 1.006 g/mL; 137 mM NaCl, 2.7 mM KCl, 8.45 mM Na2HPO4, 1,47 mM KH2PO4); TBS (500
mmol/L NaCl, 20 mmol/L Tris pH 7.4); Antibody diluent (0.5 % nonfat dry milk and 1 % Tween-20 in TBS); Washing buffer (0.5 % nonfat dry milk and 0.05 % Tween-Tween-20 in TBS); DNPH
solution (1 mM dinitrophenylhydrazine in 6 M guanidine hydrochloride and 0.5 M potassium phosphate buffer, pH 2.5).
2.2 Aggregation and ROS-production in whole blood
Heparinized human blood was obtained by venous puncture from healthy, adult volunteers from the Blood Bank at Linköping University Hospital. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2-4). The effects of P.
gingivalis on aggregation and reactive oxygen species (ROS) production in whole blood were
measured simultaneously in two samples using a lumi-aggregometer model 560 (Chrononlog Corp, Haverton, PA, USA), as previously described [26]. The blood (0.5 mL) was diluted with KRG (0.5 mL), containing luminol (100 µmol/L, final concentration) and horse radish peroxidase (4 U/mL, final concentration), in a plastic cuvette with a siliconized stirring bar. Platinium electrodes were inserted and the samples were preincubated and stirred (1000 rpm) for 5 min at 37°C. After reaching equilibrium, the instrument was calibrated and the blood samples were stimulated with P. gingivalis (5x106/mL or 5x107/mL). The extent of platelet aggregation was evaluated by measuring the maximum amplitude of the change in impedance. The system was calibrated so that a 5-ohm change in impedance determined a 7.5 mm deflection of the pen. The chemiluminescence data are based on peak values and are expressed as arbitrary units (AU).
2.3 LDL and HDL preparation from whole blood stimulated with P. gingivalis
Heparinized human blood or, in some cases, plasma was allowed to equilibrate for 10 min at 37°C and thereafter unstimulated or stimulated for 30 min under stirring (800 rpm) with different doses (5x106 – 5x107/mL) of P. gingivalis, which have been untreated or treated for 10 min with 0.1 mM leupeptin. LDL and, in some cases, HDLwere then prepared from human blood by two-step short time/fast speed ultracentrifugation procedures as previously described [27, 28]. Briefly, after centrifugation of the blood samples (1500 x g for 15 min at room temperature), the plasma was
collected and LDL (density 1.019-1.063 g/mL) was prepared by sequential density-gradient ultracentrifugation, according to the method described by Da Silva et al. [29] with slight modifications. EDTA (1 mg/mL) and sucrose (0.5 %) was added to plasma to prevent oxidation and aggregation, respectively. Plasma was adjusted to a density of 1.22 g/mL (LDL) or 1.24 g/mL (HDL) with solid KBr and PBS (1.006 g/mL) (LDL) or KBr/phosphate buffer solution (1.063 g/mL) (HDL) was added above the plasma in centrifuge tubes (Beckman Coulter; Quick-Seal Polyallomer, 16 x 76 mm, 13.5 mL) without mixing of the phases. Ultracentrifugation was performed in a Beckman XL-90 equipped with a Ti 70 rotor (fixed angle, Beckman) at 290 000 x g for 2 h at 4° C (LDL) or 4 h at 15° C. (HDL) The distinct LDL or HDL bands were extracted by penetrating the side of the tube with a syringe and transferred to new centrifuge tubes. KBr solution (1.10 g/mL for LDL or 1.24 g/mL for HDL) was added and the preparations were then centrifuged for .2 h at 4° C or 15° C respectively. LDL and HDL almost depleted of albumin and other plasma contaminants were then extracted from the top of the tube and desalted with desalting buffer (CH5NO3, 12 mM, pH 7.1) in PD 10 columns. Protein concentration of the
preparations was determined with the Bio-Rad protein assay by the method of Bradford [30]. If stored, LDL/HDL was stored dark under nitrogen gas at a maximum of three weeks after preparation. To obtain oxidized LDL (UV-LDL), native LDL was placed 10 cm from a UC tube (254 nm, TUV 15W/G15 T8, Philips) and illuminated during 15 minutes.
2.4 Analysis of carbonyls on LDL and plasma proteins
Carbonyls on LDL were analyzed with a slightly modified version of an ELISA method that earlier has been described by Buss et al [31]. Briefly, LDL samples (1 mg/ml) were diluted 10 or 2 times, respectively, in DNPH solution. After 45 minutes incubation in room temperature (RT), the derivatized samples were diluted further in carbonate buffer to a final dilution of 1:800 for plasma and 1:80 for LDL before aliquots of 100 µl were transferred in duplicate to a 96-well microplate
(Nunc MaxiSorp, no 436110). Proteins were allowed to immobilize during 1 h before the wells were washed with antibody diluent and then blocked with 0.5% nonfat dry milk in TBS (200 µl). The wells were incubated for 30 minutes at RT with 100 µl rabbit anti-DNPH antibody (diluted 1:5000), followed by 30 min incubation with 100 µl goat anti-rabbit IgG peroxidase conjugate (diluted 1:3000). After washing, 100 µl of chemiluminescence substrate (ECL+, GE Healthcare Amersham, Little Chalfont, UK) were added to each well and after 10 minutes incubation at RT, the luminescence was read with a chemiluminescence plate reader (LUMIStar, BMG Labtechnologies, Germany). UV-C exposed LDL served as positive controls and the signal from a blank without protein (PBS in DNPH solution) were subtracted from all sample readings.
2.5 Two-dimensional gel electrophoresis
Two-dimensional gel electrophoresis (2-DE) was performed using IPGphor and Multiphor (Pharmacia Biotech) as described in detail previously [27,32], essentially by the method of Görg
et.al [33]. In the first dimension, samples containing 300µg protein each were applied by in-gel
rehydration for 12 h using low voltage (30 V) in pH 3-10 NL IPGs. The proteins were then focused at 53000 Vh at maximum voltage of 8000 V. The second dimension (SDS-PAGE) was performed by transferring the proteins to a homogenous (T=14%, C=1.5%) home cast gel on gel bond running at 40-800 V, 10ºC, 20-40 mA over night. Separated proteins were then fixed and visualized by silver staining [34].
2.6 Tryptic digestion, mass spectrometry and database search
Peptides obtained after tryptic digestion were mixed 1:1 with matrix, in this case α-cyano 4-hydroxycinnamic acid (0.01 mg/ml) or dihydroxybenzoic acid (0.02 mg/ml) in 70% acetonitrile /0.3% TFA and then spotted on a stainless steel target plate. Analyses of peptide masses were performed using MALDI-TOF MS (Voyager DE PRO, Applied Biosystems, Foster City, CA, USA) equipped with 337 nm N2 laser operated in reflector mode with delayed extraction. Peptide
masses (plus one H+) corresponding to the 40 most intense peaks in the spectra were submitted to database searches. NCBI and SWISS-PROT were used with PeptIdent or MS-Fit as search engines. Restrictions were human species, mass tolerance < 50 ppm, maximum one missed cleavage by trypsin, methionine oxidation (MSO) and cysteine modification by carbamidomethylation.
The visualized protein spots were quantified as optical density per total protein intensity on the 2-DE gel [31] and the differences in protein expression were analyzed using PDQuest 2-2-DE gel analysis software, version 7.1 (Bio-Rad Laboratories,U.S.).
2.7 Cell culture and proliferation assay
Human skin fibroblasts were maintained at 37°C in 5 % CO2 and humidified atmosphere and
grown in DMEM. In order to investigate the proliferation responses, fibroblasts (passage 15-19) were seeded in 96-well plates at a density of 2000 cells/well in medium with 10 % FBS for 24 h. To make the cells quiescent they were maintained in medium without FBS for 24 h and the medium was thereafter replaced with 0.1 % medium and LDL was added at a final concentration of 50 μg/mL for another 24 h. The medium was then replaced with new 0.1 % medium and proliferation responses were monitored using CellTiter 96® AQueous One Solution Cell
Proliferation Assay, followed by measurements of absorbance at 490 nm in a 96-well plate reader (SpectraMax 340, Microplate Reader, Molecular Devices Corp., Sunnyvale, California).
2.8 The uptake of LDL in fibroblasts
To study the uptake of LDL, cellular morphology and the actin cytoskeleton, fibroblasts were cultured on glass coverslips in 24-multiwell plates as described above and then exposed to native or P. gingivalis–modified LDL. After 3 hours, the cells were washed in PBS (pH 7.3) and fixed for 30 minutes in ice-cold 4% paraformaldehyde. F-actin was stained by incubation in a mixture of
rhodamine phalloidin (0.6 µg/mL) and lysophosphatidylcholine (100 µg/mL) for 30 minutes in the dark [35]. LDL was stained by incubation with 5 µg/mL of goat anti-apolipoproteinB-48/100 antibodies and 10 µg/mL of FITC-conjugated rabbit anti-goat antibodies. The cells were mounted upside-down and analysed in a Zeiss Axioscope (Carl Zeiss, Oberkochen, Germany). Digital images were generated with a Carl Zeiss ZVS-47E camera and Easy Image Measurement 2000 software (ver. 2.3, Bergström Instruments AB, Solna, Sweden).
2.9 Statistical analyses
Values are expressed as mean plus standard error of the mean (SEM). Unpaired, two-tailed, Students t-test or Wilcoxon paired rank test was used to asses differences between means and p<0.05 was considered as statistically significant. Significance is denoted * (p < 0.05), ** (p < 0.01) and *** (p < 0.001).
3.
Results
3.1 P. gingivalis stimulates aggregation and ROS-production in human whole blood
The capacity of Porphyromonas gingivalis to activate cells in whole blood was investigated by analyzing aggregation and ROS-production with lumiaggregometry. Human whole heparinized blood was incubated with P. gingivalis (5x107 CFU/mL) at 37°C under stirring for 30-60 min. P.
gingivalis caused an extensive aggregation and ROS production as measured by changes in
impedance and luminol-dependent chemiluminescence (CL), respectively, with a maximal effect at 5x107 bacteria/mL (Figure 1). The aggregatory response was irreversible, started after 3-5 min and reached maximum amplitude after approximately 30 min (Figure 1A). The P. gingivalis–induced aggregation was comparable with the response triggered by the strong platelet activator collagen (2 µg/mL), but significantly higher than the aggregation induced by the bacterial peptide fMLP (1 µmol/L) (Figure 1B). Furthermore, P. gingivalis stimulated an extensive and long-lasting production, which correlated kinetically with the aggregatory response (Figure 1A). The ROS-production was approximately 7 and 4 times higher than the ROS-production induced by collagen and fMLP, respectively (Figure 1B). No aggregation or CL was observed in stirred, unstimulated blood.
P. gingivalis has been shown to trigger platelet aggregation and neutrophil ROS production in pure
cell suspensions [20-22]. Consequently, the P. gingivalis–stimulated aggregation and ROS-production in whole blood probably reflect activation of platelets and leukocytes, respectively.
3.2 The effects of P. gingivalis on the protein expression of LDL
The protein expression of LDL, isolated from untreated or P. gingivalis-treated human whole blood, was studied by using a proteomic approach, as previously described [27]. Separation of proteins in native LDL, i.e. isolated from untreated blood, with 2-DE revealed about 20 proteins. By using trypsin digestion, MALDI-TOF-MS and peptide mass fingerprinting, the major part of these protein spots were identified as apolipoproteins, including apoA-I, apoA-IV, apoE and apoJ. In some LDL
A
0 10 20 30 40 50 0 25 50 75 Aggregation ROS-productionP. gingivalis
Time (min)B
Figure 1: Porphyromonas gingivalis-induced aggregation and reactive oxygen species
(ROS)-production in whole blood. Heparinized human whole blood diluted 1:1 in buffer, was
preincubated at 37°C for 5 min at stirring conditions, and then monitored for impedance and luminol-amplified chemiluminescence triggerd by P. gingivalis (5x107 CFU/mL), collagen (2 µg/mL) or fMLP (1 µM). The results are presented as representative recordings (A) and the mean±SEM of five separate experiments based on the maximal amplitude of change in impedance and chemiluminescence, respectively (B). The P. gingivalis-induced aggregation was significantly higher (p<0.001) than the response triggered by fMLP, and the P. gingivalis-induced
ROS-production was significantly higher (p<0.001) than the response triggered by collagen and fMLP, respectively. Student t-test was used for statistical analysis. AU=arbitrary units.
preparations low expression of apoM was detected. There were few protein spots over 100 kDa and due to its large size (500 kDa) and hydrophobic characteristics, the most abundant lipoprotein of LDL, apoB-100, was not detected in the 2-DE pattern. Protein mapping of LDL isolated from P.
gingivalis-stimulated blood revealed an extensive expression of apoM (Figure 2B). Quantification
of the apoM spots on the 2-DE gel as optical density per total protein intensity is shown in Figure 2C. Two of three subjects demonstrated a marked increase in apoM expression, whereas apoM in the third subject was elevated both in native and bacteria-modified LDL. Furthermore, P. gingivalis promoted degradation of apoB-100 into two distinct fragments that were separated by 2-DE (Figure 3 A, B). The molecular masses and pI of the two N-terminal fragments were 20 kDa and 7.2, and 15 kDa and 7.9, respectively. The identity of two apoB-fragments was determined with peptide mass fingerprinting (Table 1). Quantification of the apoB spots on the 2-DE gel as optical density per total protein intensity is demonstrated in Figure 3E. In the presence of more P. gingivalis (i.e. >5x107/ml), further degradation of apo B into smaller fragments was observed.
In order to clarify whether the modification of LDL in whole blood was due to proteases from P.
gingivalis or from activated leukocytes or platelets, the effects of P. gingivalis on LDL were
analyzed in human plasma. We found that increased apoB-fragmentation also occurred in plasma (Figure 3C, D, F) and that this effect was antagonized by the gingipain R-inhibitor leupeptin. On the contrary, transfer of apo M from HDL to LDL was not found in plasma treated with P. gingivalis. Some P. gingivalis proteins have been reported to bind to LDL particles [36], however, we were not able to detect any components of P. gingivalis among the proteins in the LDL 2-DE pattern.
3.3 P. gingivalis mediates the formation of carbonyls in LDL
Protein carbonyl formation is a good biomarker for oxidation of proteins, including lipoproteins [37,38]. To investigate whether the P. gingivalis-induced ROS-production in whole blood caused an oxidation of LDL protein moieties, determination of protein carbonyls were performed. When
C
Figure 2: Increased expression of apoM in LDL from human whole blood treated with
Porphyromonas gingivalis (5x107
CFU/mL). Proteins in LDL from untreated whole blood (A; native) and from whole blood incubated with P. gingivalis (B) were separated with 2-DE and silver stained. Proteins indicated (arrows) were identified as apoM by mass spectrometry
analysis. The apoM spots on the 2-DE gels of LDL preparations from three different subjects were quantified as optical density per total protein intensity (OD/TPI) (C).
A
B
40 kDa
a
b
c
d
25
e
3
pI
6
3
pI
6
Table 1: Identification of apo B-100 fragments in LDL after incubation of
human whole blood with Porphyromonas gingivalis (5x107 CFU/mL)
Protein pI a) Mass
(Da) a) Masses matched b) Amino acid position
Apo B-100 (fragment) Apo B-100 (fragment) 7.2 7.9 20000 15000 700.3958 837.4060 1139.5101 1212.5629 1228.5127 Met-ox 1666.8895 1674.9266 2153.9827 2210.1458 2282.1325 1139.5069 1228.5003 Met-ox 1674.8951 2153.9606 2210.1629 2282.0994 46-50 94-100 101-110 118-128 118-128 159-174 80-93 52-71 140-157 51-71 101-110 118-128 80-93 52-71 140-157 51-71
a) Apparent masses and pIs of the fragments determined after 2-DE (cf. figure 1) b) Peptide mass values [MH] + (m/z) matched to apo B-100 with a mass accuracy
< 50 ppm using MALDI-TOF MS.
whole blood was incubated with 5x107 P. gingivalis/mL, the amount of carbonyls in LDL increased with 239%, compared to the carbonyl content in native LDL (Figure 4). Inhibition of gingipain R with leupeptin (0.1 mM) markedly counteracted the bacteria-induced carbonyl formation.
3.4 The effects of P. gingivalis-modified LDL on cell proliferation
The proliferative effects of LDL on fibroblasts were examined by using the MTS-assay. We found that incubation with 50 μg/mL of P. gingivalis-modified LDL for 24h significantly (p<0.001) increased the proliferation of fibroblasts, whereas native LDL was ineffective (Figure 5). The proliferative effects were markedly reduced when LDL was prepared from blood that has been
Figure 3: Apo B-100 fragmentation in LDL from human whole blood or plasma treated with
Porphyromonas gingivalis (5x107
CFU/mL). Proteins in LDL from untreated whole blood (A; native), whole blood (B) incubated with P. gingivalis, untreated plasma (C) or plasma incubated with P. gingivalis (D) were separated with 2-DE and silver stained. The two proteins indicated (a and b) were identified as fragments of apo B-100 by mass spectrometry analysis (Table 1); a) apoB-100 fragment: pI 7.2, Mr 20000 b) apoB-100 fragment: pI 7.9, Mr 15000. The apoB
fragments on the 2-DE gels of LDL preparations from whole blood from three different subjects (E) and from plasma from two different subjects (F) were quantified as optical density per total protein intensity (OD/TPI) and are presented as the mean±SEM.
Figure 4: Porphyromonas gingivalis induced carbonyl formation. Human whole blood was
untreated (control) or treated with P. gingivalis (5x107 CFU/mL) for 30 min at 37°C at stirring conditions, whereafter the plasma was collected and the LDL was prepared. In some experiments,
P. gingivalis was pretreated with leupeptin (0.1 mM) for 10 min at 37°C. The effects of P. gingivalis on carbonyls on LDL were analyzed and are expressed as percentage of the carbonyl
content of native plasma and LDL (control). The results are presented as the mean±SEM of four separate experiments. Student t-test was used for statistical analysis.
Figure 5: Porphyromonas gingivalis–modified LDL increases cell proliferation. The MTS assay
was used to analyze changes in fibroblast growth after incubating the cells for 24 hours in the absence (control) or presence of 50 µg/mL of native LDL (N-LDL) or P. gingivalis-modified LDL (P.g-LDL). In some experiments, P. gingivalis was pretreated with leupeptin (0.1 mM) for 10 min at 37°C. The results are presented as the mean±SEM of five separate experiments. Student t-test was used for statistical analysis.
incubated with P. gingivalis treated with leupeptin (0.1 mM). Isolated native LDL modified by UV caused a significant (p<0.05) proliferation comparable with that obtained by P. gingivalis-modified LDL.
The cellular uptake of LDL was visualised by fluorescence microscopy. As shown in figure 6, LDL was taken up efficiently by fibroblasts and was localized in intracellular vesicles. We found that P. gingivalis-modified LDL was internalized in large vesicles, which were clearly different from the smaller vesicles formed during uptake of native LDL.
B
A
Figure 6: The uptake of native and Porphyromonas gingivalis–modified LDL in fibroblasts.
Fibroblasts were incubated on glass coverslips in multiwell plates in the presence of native (A) or P. gingivalis-modiifed LDL (B) for three hours and then stained for LDL, with primary anti-Apolipoprotein B-48/100 antibodies and secondary FITC-conjugated antibodies, and for F-actin with rhodamine phalloidin. The cells were mounted upside-down and analysed in a Zeiss
Axioscope (Carl Zeiss, Oberkochen, Germany). Digital images were generated with a Carl Zeiss ZVS-47E camera and Easy Image Measurement 2000 software (ver. 2.3, Bergström Instruments AB, Solna, Sweden). Bar 10 µm.
4.
Discussion
Porphyromonas gingivalis has been recognized as a key pathogen in periodontal disease and as a
risk factor for atherosclerosis [4-6]. During translocation from periodontal tissues to the coronary artery wall, P. gingivalis encounters various types of blood cells and plasma constituents. In order to elucidate the effects of inflammatory and thrombotic responses to P. gingivalis on LDL, human whole blood was exposed to the bacterium followed by analysis of protein expression, oxidation and mitogenic properties of the isolated LDL.
We found that P. gingivalis caused an extensive aggregation and ROS-production in whole blood, presumably reflecting both platelet and leukocyte activation. In support, P. gingivalis has been shown to stimulate platelet aggregation and neutrophil ROS production, respectively, in pure cell suspensions [20-22]. These effects are attributed bacterium-derived gingipain R activating protease-activated receptors (PARs) [39,40]. Furthermore, P. gingivalis triggers the release of cytokines and prostaglandins from circulating leukocytes [41]. Consequently, the spreading of P. gingivalis infection into the circulating blood may cause inflammatory and aggregatory reactions involving production and release of inflammatory mediators, which may modify and transform LDL to an atherogenic form.
Substantial evidence suggests that oxidative damage to vascular cells and oxidation of LDL contribute to atherosclerosis [42]. Oxididative modifications of phosphatidylcholines in LDL have been found to be a marker of subclinical atherosclerosis [43] and formation of nitrated or chlorinated amino acids or fragmentation of LDL are examples of other oxidative modifications. Another type of oxidative modifications is the formation of carbonyls, a process that could result in degradation, protein inactivation and cellular dysfunction [44]. In this study, we found that the P.
gingivalis-induced ROS production in whole blood was associated with increased amounts of
carbonyls on LDL. This was attributed protease activity of gingipain R since leupeptin inhibited ROS-production and protein carbonyl formation in LDL triggered by P. gingivalis. Protein carbonylation could be generated via the formation of reactive aldehyde products such as malondialdehyde and 4-hydroxynonenal that are capable to attach covalently to amino groups of lysine residues in apolipoprotein B [45]. The fact that LDL-carbonylation could play an important role in atherosclerosis is further supported by the fact that hydrazine compounds, that bind carbonyls, can inhibit the glycation of LDL and prevent the formation of “foam cells” in vitro. This implicates that carbonyl-scavenging compounds might inhibit LDL glycation and intracellular accumulation of cholesterol ester and “foam cell” formation also in vivo [46].
P. gingivalis was shown to promote degradation apoB-100 into two distinct N-terminal fragments.
Apo B fragmentation can be caused by both proteolytic activity and oxidation on LDL [23, 47], and both mechanisms may be operating in our experimental model. Taking into account the data from the MS analyses, molecular masses and isoelectric points of the fragments, 15k/7.9 and 20k/7.2, our results are in agreement with protease activity on apoB-100 by gingipain R at position Arg158 and Arg207, respectively, but not with the proteolytic activity of gingipain K. Furthermore, specific inhibition of gingipain R with leupeptin reduced the P. gingivalis-induced degradation of apoB. A specific role for bacterial proteases was also supported by the finding that P. gingivalis also produced apoB fragments in human plasma, devoid of inflammatory cells as sources of proteases and oxygen radicals. These results correlate with a recent report demonstrating apoB-100-fragmentation by western blotting when exposing isolated LDL to P. gingivalis [36] and Hashimoto et al [48] have recently found in an animal model that degradation of apoB-100 by gingipain R plays a crucial role in the development of atherosclerosis by P. gingivalis infection. The degradation of apoB-100 likely alters the surface charge of the LDL-particles and thereby increases their
aggregation. The aggregated form of LDL is considered to be internalized by macrophages in the arterial wall to form foam cells [23]. In correlation, Miyakawa et al [36] show that LDL-aggregates, formed by the proteolytic activity of P. gingivalis, stimulate foam cell formation. Interestingly, apoB-degraded LDL, e.g. formed by the proteolytic activity of P. gingivalis, is not recognized by the LDL (apoB/apoE) receptor in the liver and is thereby accumulated in the circulation [48].
Furthermore, we found that P. gingivalis-modified LDL had acquired increased amounts of apoM. The clinical significance of this is, however, uncertain. ApoM is a protein mainly associated with high-density lipoprotein (HDL), but also present in LDL and triglyceride-rich lipoproteins [49,50]. The protein is structurally related to the lipocalin protein super family [51] and is expressed in liver hepatocytes and kidney tubular cells [52]. Some reports suggest that apoM is involved in host defense responses [53,54]. Other results indicate that apoM plays a part in lipid or lipoprotein metabolism. Thus, leptin, a regulator of lipoprotein metabolism, inhibits apoM expression in the human hepatic cell line, HepG2 [55]. Moreover, Wolfrum et al. [56] have shown that over expression of apoM is coupled with reduced atherosclerosis in mice and suggested that apoM is required for pre-β-HDL formation and cholesterol efflux. In line with this, purified apoM containing HDL particles have been shown to be more effective in reversed cholesterol transport and have more anti-oxidative effects than HDL without apoM [57]. Interestingly, results also indicate that apoM may be transferred between lipoprotein particles. Thus, in normal mice, like in humans, apoM is mainly found in HDL, while in LDL-receptor deficient and apoE deficient mice apoM is mainly located in LDL and VLDL [58]. However, in experiments with P. gingivalis in plasma we were not able to demonstrate a translocation of apo M from HDL to LDL. Consequently, this suggests that apoM may be produced and delivered by blood cells or that the cells are important for the transfer to occur.
The P. gingivalis–modified LDL induced proliferation of human fibroblasts, approximately 150 % of control and significantly higher compared to the effects of native LDL. This mitogenic effect of LDL is a consequence of the protease activity of P. gingivalis, since inhibition of gingipain R antagonized the increased cell proliferation. A higher proliferative effect of modified LDL has previously been reported [13]. Zettler et al. [59] demonstrated an increased proliferation of both fibroblasts and SMC in response to oxidized LDL, whereas native LDL only had minor effects. Klouche et al. [60] obtained similar results on SMC growth when using enzymatically modified, but non-oxidized, LDL. Consequently, these findings show that both oxidized and enzymatically modified LDL induce cell proliferation. However, there are also studies reporting anti-proliferative effects of oxidized LDL [61]. It is evident that the way by which LDL is modified has implications for its cellular effects.
Our finding that P. gingivalis-modified LDL was localized in large vesicles in fibroblasts, compared to the much smaller vesicles formed during uptake of native LDL, indicates different pathways for the internalization and processing of native and modified LDL. Indeed, it has been demonstrated that aggregated LDL forms larger vesicles, than native LDL, in vascular smooth muscle cells [62]. Furthermore, apoB-modified LDL has previously been shown to accumulate in secondary lysosomes rather than in endosomes [63]. This suggests that LDL modified by P.
gingivalis-induced proteolysis of apoB reaches in an aggregated form the secondary lysosomes, but
is inefficiently degraded, leading to intracellular accumulation within this compartment.
P. gingivalis could potentially induce LDL modification in whole blood through two different
mechanisms. Firstly, direct proteolytic action of gingipains and other released proteases and lipases on LDL. Our results indicate that the two fragments of apoB-100 found are formed by P. gingivalis infection through C-terminal cleavage of arginines, thus suggesting a direct role of of gingipain R.
Further studies using purfied gingipains to investigate this mechanism are warranted. However, already in this study this interpretation is supported by the inhibitory effects of leupeptin on P.
gingivalis-induced effects and that P. gingivalis stimulates increased formation of apoB fragments
in plasma, i.e. in the absence of blood cells. In correlation, Hashimoto et al [48] have recently found in an animal model that degradation of apoB-100 by gingipain R plays a crucial role in the development of atherosclerosis by P. gingivalis infection. Secondly, activation of neutrophils and platelets lead to release of ROS and proteases. P. gingivalis has the ability to induce platelet aggregation and secretion, which is mainly attributed to gingipains and their activation of protease-activated receptors, PAR-1 and PAR-4, expressed on platelets [39]. In similar, P. gingivalis stimulates ROS-production in neutrophils, which appear to be dependent on both a gingipain-mediated activation of PAR-2 and a LPS-gingipain-mediated activation of Toll-like receptor 1 and 2 [40, unpublished observations]. In this study, we found that leupeptin inhibited P. gingivalis-induced ROS-production and protein oxidation of LDL, thus suggesting a role for gingipain-induced activation of PARs in the modification of LDL.
In summary, we show that the periodontal pathogen P. gingivalis modifies LDL in an ex vivo human blood model by degrading apoB-100 and increasing the expression of apoM and that this modified LDL increases the proliferation of fibroblasts. The physiological environment in a whole blood system considerates bacterial interactions with different immune cells and plasma constituents and reduces confounding factors associated with isolation procedures. In conclusion, this study suggests that P. gingivalis during translocation in circulating blood modifies vascular LDL to an atherogenic form and supports a role of periodontal disease in the development of atherosclerosis.
Acknowledgements
This study was supported by the Swedish Research Council (grants 71X-12668), the King Gustav V 80-year Foundation, Swedish Fund for Research without Animal Experiments, Trygg-Hansa Research Foundation, the strategic reasearch areas ”Cardiovascular Inflammation Research Centre” and ”Materials in Medicine” at Linköping University and the Östergötland county council.
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.
Correspondence address Torbjörn Bengtsson
Division of Pharmacology, Department of Medicine and Health Sciences
Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden Fax: +46 13 149106, Phone: +46 13 222058, E-mail address: torbe@imv.liu.se
