INFECTION AND IMMUNITY, Nov. 1999, p. 5792–5798 Vol. 67, No.

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0019-9567/99/$04.00⫹0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Invasion of Human Coronary Artery Cells by Periodontal Pathogens

BRIAN R. DORN,1 WILLIAM A. DUNN, JR.,2 AND ANN PROGULSKE-FOX1*
Department of Oral Biology and Periodontal Disease Research Center, College of Dentistry,1 and Department of
Anatomy and Cell Biology, College of Medicine,2 University of Florida, Gainesville, Florida 32610
Received 26 May 1999/Returned for modification 1 July 1999/Accepted 30 July 1999

There is an emerging paradigm shift from coronary heart disease having a purely hereditary and nutritional
causation to possibly having an infectious etiology. Recent epidemiological studies have shown a correlation
between periodontal disease and coronary heart disease. However, to date, there is minimal information as to
the possible disease mechanisms of this association. It is our hypothesis that invasion of the coronary artery
cells by oral bacteria may start and/or exacerbate the inflammatory response in atherosclerosis. Since a few
periodontal pathogens have been reported to invade oral epithelial tissues, we tested the ability of three
putative periodontal pathogens—Eikenella corrodens, Porphyromonas gingivalis, and Prevotella intermedia—to
invade human coronary artery endothelial cells and coronary artery smooth muscle cells. In this study we
demonstrate by an antibiotic protection assay and electron microscopy that specific species and strains invade
coronary artery cells at a significant level. Actin polymerization and eukaryotic protein synthesis in metabol-
ically active cells were required since the corresponding inhibitors nearly abrogated invasion. Many intracel-
lular P. gingivalis organisms were seen to be present in multimembranous vacuoles resembling autophago-
somes by morphological analysis. This is the first report of oral microorganisms invading human primary cell
cultures of the vasculature.

Cardiovascular disease (CVD) is the leading cause of death
in the Western world. Although classical risk factors (i.e.,
smoking, obesity, and high blood pressure, etc.) can be indi-
cations of most coronary deaths, they cannot account for all
CVD-associated deaths. For example, approximately 25% of
coronary deaths in males and 15% in females occur in persons
in the lowest two quintiles of the multivariate Framingham
Heart Study risk scores (30). This has led many to speculate
that CVD may have an infectious etiology (5, 18, 47).
Periodontal disease is an inflammatory condition caused by
a chronic bacterial infection with specific gram-negative organ-
isms. Recent epidemiological data strongly suggests that peri-
odontitis is an important risk factor for coronary heart disease
(CHD). In 1989 Mattila et al. reported an association between
dental health and acute myocardial infarction in that they
found worse dental health in patients with acute myocardial
infarction than in a control population (32). In a separate
study, DeStefano et al. monitored subjects for 13 to 16 years
after a baseline dental examination (11). Of the 9,760 subjects,
patients with periodontitis were found to have a 25% increased
risk of CHD compared to patients with minimal or no peri-
odontal disease. Men under 50 with periodontitis or no teeth
were 70% more likely to develop CHD than men with no
periodontal disease. More recently, Beck et al. evaluated peri-
odontal disease and its variables as a risk factor for CHD and
stroke (1). Those authors found that for every 20% increase in
mean bone loss (the most accurate measure of periodontitis),
the incidence of total CHD increased 40%. When age and
other ascribed risk factors were adjusted, patients with more
than 40% bone loss were 2.7 times more likely to have fatal
CHD. The biological basis for this association has not yet been
elucidated. However, a possible route to the circulatory system
for periodontal bacteria exists, since studies have shown a
transient bacteremia resulting from chewing food, flossing, and
toothbrushing in persons with periodontitis (3, 41, 42).
* Corresponding author. Mailing address: Department of Oral Bi-
ology, University of Florida, Gainesville, FL 32608. Phone: (352) 846-
0770. Fax: (352) 392-2361. E-mail: apfox@dental.ufl.edu.
Poryphyromonas gingivalis is strongly implicated as an etio-
logic agent of adult periodontitis, and Prevotella intermedia is
also frequently cultured from sites of periodontitis (44, 45).
Previous studies have established that these organisms are
capable of invading oral epithelial tissue in vitro (13, 14, 29,
40). A recent study established that P. gingivalis was also able
to invade fetal bovine heart endothelial, bovine aortic endo-
thelial, and human umbilical vein endothelial cells (10). Addi-
tionally, preliminary studies have begun to find periodontal
pathogens, including P. gingivalis and P. intermedia, within ath-
eromatous tissues (24). Eikenella corrodens is also a putative
periodontal pathogen and has been shown to be an etiologic
agent of infective endocarditis (2, 16).
Atherosclerosis develops due to the inflammatory response
to endothelial cell injury and dysfunction and is likely a chronic
process (39). It is our hypothesis that frequent bacteremias
could provide a chronic insult to the vasculature and that inva-
sion of the cells of the arterial wall by oral bacteria could con-
tribute to the injury that initiates and/or exacerbates atheroscle-
rosis. Therefore, we tested the ability of these organisms to invade
primary cultures of human coronary artery endothelial cells
(HCAEC) and coronary artery smooth muscle cells (CASMC).
MATERIALS AND METHODS
Bacterial strains and growth conditions. P. gingivalis 381 and W50 (a gift of
M. A. Curtis) were grown in brain heart infusion broth (Difco Laboratories,
Detroit, Mich.) supplemented with 0.5% yeast extract (Difco), 0.1% cysteine,
hemin (5 ␮g/ml), and menadione (5 ␮g/ml). P. intermedia 17 and 25611 (gifts of
K.-P. Leung and W. E. Nesbitt) were grown in Todd-Hewitt broth (Difco)
supplemented with 0.5% yeast extract, 0.075% cysteine, hemin (5 ␮g/ml), and
menadione (0.05 mg/ml). Both P. gingivalis and P. intermedia were grown in a
Coy anaerobic chamber with an atmosphere of 5% CO2, 10% H2, and 85% N2.
E. corrodens 23834 (obtained from the American Type Culture Collection, Ma-
nassas, Va.) was grown in BY broth in a humidified atmosphere containing 10%
CO2. Escherichia coli MC1061 (a gift of A. S. Bleiweis) was grown in Luria-
Bertani medium, consisting of Bacto Tryptone (10 g/liter; Difco), Bacto yeast
extract (5 g/liter), and NaCl (10 g/liter).
Cell culture. KB cells (derived from a human oral epidermoid carcinoma),
HCAEC, and CASMC were used in this study. The KB cells (ATCC CCL-17)
were maintained in Eagle’s minimum essential medium (Mediatech, Herndon,
Va.) supplemented with 10% fetal bovine serum (HyClone Laboratories, Inc.,
Logan, Utah), 200 mM L-glutamine (Sigma Chemical Co., St. Louis, Mo.), and
100 mg of penicillin-streptomycin (Sigma) per ml. The HCAEC (Clonetics, Inc.,

5792
VOL. 67, 1999 P. GINGIVALIS INVASION OF CORONARY ARTERY CELLS
TABLE 1. Comparison of invasion of coronary artery cells by
periodontal pathogens and a negative control
CFU/ml recovered after
antibiotic treatmenta
Strain
HCAEC CASMC
5
P. gingivalis 381 (4.6 ⫾ 0.6) ⫻ 10 (2.7 ⫾ 1.4) ⫻ 105
P. gingivalis W50 (9.5 ⫾ 0.3) ⫻ 103 (2.0 ⫾ 0.7) ⫻ 104
P. intermedia 17 (2.0 ⫾ 0.3) ⫻ 103 (3.5 ⫾ 2.1) ⫻ 103
P. intermedia 25611 0 11 ⫾ 19
E. corrodens 23834 446 ⫾ 274 320 ⫾ 99
E. coli MC1061 2.2 ⫾ 3.9 6.6 ⫾ 3.5
a
Values represent the means and standard deviations for triplicate samples of
lysates from the infection of 105 cells by 107 bacteria from at least three inde-
pendent experiments.
San Diego, Calif.) were maintained in microvascular endothelial growth medi-
um-2, consisting of endothelial cell basal medium-2 supplemented with fetal
bovine serum, hydrocortisone, human recombinant fibroblast growth factor, vas-
cular endothelial growth factor, recombinant insulin growth factor-1, ascorbic
acid, human recombinant epidermal growth factor, gentamicin, and amphoteri-
cin (Clonetics). The CASMC (Clonetics) were maintained in smooth muscle
growth medium, consisting of smooth muscle basal medium-2 supplemented with
insulin, human recombinant fibroblast growth factor, fetal bovine serum, human
recombinant epidermal growth factor, gentamicin, and amphotericin (Clonetics).
Cells were cultured in 75-cm2 flasks at 37°C in a humidified atmosphere of 5%
CO2. Both the HCAEC and CASMC were obtained cryopreserved at the third
passage and were passaged an additional two or three times before use.
Invasion assay. For the invasion assays, the bacteria were grown in broth, centri-
fuged at low speed, and resuspended in antibiotic-free medium to a concentration of
7
10 cells/ml as determined spectrophotometrically (Shimadzu UV-1201; VWR, Mar-
ietta, Ga.). Approximately 105 human cells per well in a 24-well tissue culture plate
were washed three times with phosphate-buffered saline (PBS) prior to incubation
with 1.0 ml of the bacterial suspension at 37°C aerobically for 90 min. In order
to more closely approximate in vivo conditions, the bacteria were not centrifuged
onto the cells to promote intimate contact. The medium was removed from in-
fected cells after 90 min, and the cells were washed three times with PBS. Me-
dium containing gentamicin (300 ␮g/ml) and metronidazole (200 ␮g/ml) was then
added to each well to kill any extracellular bacteria, and the plates were incubated
for 60 min aerobically at 37°C. Finally the medium was removed, and the cells were
washed three times with PBS and lysed by a 20-min incubation with 1.0 ml of
sterile distilled water at 37°C under aerobic conditions. Dilutions of the lysates of
cells infected with P. gingivalis, P. intermedia, and E. corrodens 23834 were plated
in triplicate on tryptic soy agar (Difco) plates supplemented with 5.0% sheep
blood, 0.5% yeast extract, hemin (5 ␮g/ml), and menadione (5 ␮g/ml). Plates of
P. gingivalis and P. intermedia were cultured anaerobically, and those of E.
corrodens 23834 was cultured in a humidified atmosphere containing 10% CO2.
The dilutions of the lysates of E. coli MC1061 were cultured on Luria-Bertani
plates at 37°C aerobically. CFU of invasive bacteria were then enumerated. Each
assay was performed in duplicate wells and was performed independently at least
three times. Viability of cells was examined by trypan blue exclusion. Controls for
the antibiotic were tested by adding 107 bacteria to unseeded wells.
Temperature dependence. KB cells were infected as described above, and the
bacteria were incubated concurrently at 37 and 4°C.
Treatment with cycloheximide and cytochalasin D. The effects of cyclohexi-
mide (Sigma) and cytochalasin D (Sigma) on invasion were also investigated.
Invasion assays were performed as described above with the exception of the
presence of these inhibitors. Cycloheximide (1 mg/ml in ethanol) was preincu-
bated with the human cells for 4 h prior to addition of the bacteria and was
present during the assay. Cytochalasin D (5 ␮g/ml in dimethyl sulfoxide) was
preincubated with the human cells for 0.5 h before addition of the bacteria and
was present during the assay. The inhibitors were tested at the appropriate
concentration for adverse effects on the human cells by trypan blue exclusion and
by examining the confluency of the monolayer. Cycloheximide, cytochalasin D,
dimethyl sulfoxide, and ethanol were tested for possible toxicity to the bacteria
by viable counting.
Transmission electron microscopy. Following 90 min of incubation of eukary-
otic cells with bacteria, the cells were washed two times with PBS, fixed in 2%
PBS-buffered glutaraldehyde at room temperature for 1 h, centrifuged, and
washed with PBS (pH 7.3). Three drops of 3% low-gelling agarose were then
added to the pellet and allowed to solidify at 4°C for 10 min. The agarose-
embedded pellet was then washed twice for 10 min in PBS, incubated in 1%
osmium tetroxide for 1 h, and washed three times in distilled H2O. The washed
cell pellets were dehydrated in a graded series of ethanol solutions and stained
overnight en bloc with 2% uranyl acetate. Finally, the pellets were infiltrated and
embedded in EM Bed-812 (Electron Microscopy Sciences, Ft. Washington, Pa.).
Thin sections were cut, poststained with uranyl acetate and lead citrate, and
examined in a Hitachi 7000 transmission electron microscope.
5793
SEM. For scanning electron microscopy (SEM) analysis, HCAEC were incu-
bated without bacteria and with P. gingivalis 381 for 15, 30, and 45 min. Following
the infection, the cells were washed two times with PBS and fixed in 2% PBS-
buffered glutaraldehyde for 30 min. After fixation, the cells were dipped in PBS,
washed for 5 min in fresh PBS, and then incubated in 4% osmium tetroxide for
5 min. Following postfixation, the cells were dipped in distilled water, washed for
5 min in fresh distilled water, and then dehydrated in a graded series of ethanol
solutions (50, 70, 95, 100, and 100%) for 5 min each. After dehydration, the cells
were incubated twice for 5 min in hexamethyldisilizane (Sigma) and air dried for
30 min before being mounted and sputter coated with gold. The samples were
then viewed with a Hitachi S-4000 field emission scanning electron microscope.
Digital prints were produced with a SemAges digital imaging acquisition system
(Advanced Database Systems, Boulder, Colo.).
The effect of cycloheximide preincubation was also analyzed by SEM. HCAEC
were preincubated with 1 mg of cycloheximide per ml for 4 h. Following the
preincubation, the HCAEC were infected for 15 min with P. gingivalis 381, after
which they were washed two times with PBS and fixed in 2% PBS-buffered
glutaraldehyde. These cells were then processed for SEM in the same manner as
for the aforementioned samples.
RESULTS
In vitro invasion assay. For bacterial invasion studies, pri-
mary cultures of HCAEC and CASMC were infected with
the aforementioned periodontal pathogens, and invasion was
quantitated by the standard antibiotic protection assay as mod-
ified for these organisms (28). The results show that certain
strains of these periodontal pathogens do invade both HCAEC
and CASMC (Table 1). Interestingly, the bacteria varied in
their ability to invade, even among different strains of the same
species. For example, P. gingivalis 381 was able to invade by
1 to 2 orders of magnitude more than P. gingivalis W50. P. inter-
media 17 was found to be invasive, whereas P. intermedia 25611
was not able to invade under the same conditions. Thus, under
the conditions used here, certain strains of P. gingivalis and
P. intermedia were able to invade coronary artery cells.
E. corrodens 23834 showed a minimal ability to invade, since
the number of CFU recovered per milliliter of lysate was great-
er than that for the negative bacterial control, E. coli MC1061,
and the antibiotic control (data not shown), but the number of
CFU was not of the same magnitude as that of the other spe-
cies. Even increasing the number of E. corrodens 23834 organ-
isms by 10-fold resulted in only 3.2 times greater CFU. There-
fore, it is possible that certain cells phagocytosed the E. corrodens,
as opposed to an active invasion by this bacterial species.
Effects of inhibitors. The effects of temperature and meta-
bolic inhibitors on bacterial invasion were investigated to elu-
cidate possible mechanisms of invasion (Tables 2 and 3). Per-
forming the incubation portion of the invasion assay at 4°C
instead of 37°C produced a large reduction in the number of
CFU in all cases except for E. corrodens 23834. This is further
evidence that E. corrodens 23834 did not actively invade the
TABLE 2. Effect of temperature, cycloheximide, and cytochalasin D
treatment on bacterial invasion of HCAEC
% Inhibition of invasion of HCAEC a:
Strainb
At 4°C With cycloheximidec With cytochalasin Dd
P. gingivalis 381 97.3 99.9 99.6
P. gingivalis W50 97.1 99.8 99.9
P. intermedia 17 93.6 99.3 99.8
E. corrodens 23834 60.0 98.5 97.0
a
Values represent the percentage of invasion that diminished in the presence
of inhibitors compared to invasion without inhibitors present.
b
P. intermedia 25611 and E. coli MC1061 were not included, since there was
no invasion to inhibit.
c
Cycloheximide (1 mg/ml) was preincubated with HCAEC for 4 h and was
present during the assay.
d
Cytochalasin D (5 ␮g/ml) was preincubated with HCAEC for 0.5 h and was
present during the assay.
5794 DORN ET AL.
TABLE 3. Effect of temperature, cycloheximide, and cytochalasin D
treatment on bacterial invasion of CASMC
% Inhibition of invasiona:
Strainb
At 4°C With cycloheximidec With cytochalasin Dd
P. gingivalis 381 99.92 99.99 97.5
P. gingivalis W50 97.6 99.7 99.8
P. intermedia 17 77.8 99.7 99.7
E. corrodens 23834 2.0 97.3 96.0
a
Values represent the percentage of invasion that diminished in the presence
of inhibitors compared to invasion without inhibitors present.
b
P. intermedia 25611 was not included, since there was no invasion to inhibit.
c
Cycloheximide (1 mg/ml) was preincubated with CASMC for 4 h and was
present during the assay.
d
Cytochalasin D (5 ␮g/ml) was preincubated with CASMC for 0.5 h and was
present during the assay.
cell lines, since metabolism nearly stops at 4°C. Additionally,
the cell membranes lose their fluidity at 4°C, making invasion
nearly impossible.
A common strategy among invasive bacteria is to trigger the
host cell to undergo cytoskeletal rearrangements mediated by
actin polymerization (19). Cytochalasin D, an actin polymer-
ization inhibitor, also significantly reduced invasion in all cases.
These data indicate that actin polymerization of the cytoskel-
eton in a metabolically active cell is needed for invasion by
these bacteria. Cytochalasin D has been shown to inhibit a
majority of invasive bacteria (4, 20, 21, 28, 35).
Cycloheximide, a eukaryotic protein synthesis inhibitor, also
reduced the CFU recovered at 2.5 h postinfection, presumably
because it prevents coronary artery cell synthesis of the pro-
teins required during attachment and/or invasion. This data
differs from that of a previous study, which reported that using
10-fold less cycloheximide than that used here did not inhibit
invasion of gingival epithelial cells by P. gingivalis 33277 (28).
Transmission electron microscopy. Electron microscopy of
infected cells also showed evidence of invasion (Fig. 1 and 2).
As demonstrated in Fig. 1A to C, numerous P. gingivalis and
P. intermedia organisms were evident intracellularly. Most ex-
tracellular bacteria, especially in the case of P. gingivalis, ap-
pear to be concentrated at certain spots along the cellular
membrane, while the rest of the membrane remained relatively
free of bacteria (Fig. 1A). This could be due to bacterial ag-
gregation and subsequent attachment and/or invasion by the
aggregate as a whole. It could also indicate the presence of
cellular receptors that are expressed only at certain areas on
the surfaces of the cellular membrane. In several of the micro-
graphs, bacteria can be seen dividing within the coronary artery
cell (Fig. 1C and E). This indicates that the bacteria are met-
abolically active during invasion and may be able to persist in
the coronary artery cells for at least short periods of time.
The transmission electron micrographs also showed several
interesting cellular features. Many of the micrographs showed
a concentration of rough endoplasmic reticulum around inter-
nalized P. gingivalis (Fig. 2A). The internalized P. gingivalis or-
ganisms appear to be in thin membranous vacuoles, which also
appeared to include cytoplasmic ground substance, surrounded
by ribosomes (an association with the rough endoplasmic re-
ticulum), indicating residence within autophagosomes (15).
These bacteria thus might exploit the cell’s autophagic mech-
anism to establish a favorable intracellular niche, similar to
Legionella pneumophila in macrophages and Brucella abortus in
nonphagocytic cells (36, 46).
The transmission electron microscopy study of infection of
approximately 105 HCAEC by 1010 E. corrodens 23834 cells
demonstrated that the majority of HCAEC did not contain any
INFECT. IMMUN.
internalized bacteria. This would support the hypothesis that
E. corrodens 23834 did not invade either cell line or was able to
invade only a small subset of the cell population under these
conditions. The HCAEC that did internalize E. corrodens
23834 contained multiple bacterial cells within the cytoplasm
which appeared to be in large vacuoles (Fig. 1D), which is not
the case with P. gingivalis and P. intermedia invasion (Fig. 1A to
C and 2A and B).
SEM. The SEMs revealed that the HCAEC monolayer was
strikingly uniform, such that it was difficult to differentiate the
borders between cells. The HCAEC surface surrounding the
aggregate in Fig. 3B was representative of the monolayer at 15
min of infection and the monolayer with no bacteria added
(Fig. 3A).
The SEMs also demonstrated an interaction between aggre-
gates of P. gingivalis 381 and the HCAEC monolayer. After 15
min of infection, aggregates of P. gingivalis 381 could be seen
attached to the HCAEC monolayer (Fig. 3B). This attachment
appeared to be mediated by an endothelial cell-derived struc-
ture. Therefore, cycloheximide-treated HCAEC were also an-
alyzed by SEM. Structures as seen in Fig. 3B were not visible
with any P. gingivalis 381 organisms associated with cyclohex-
imide-treated cells. When P. gingivalis 381 could be seen at-
tached to cycloheximide-treated HCAEC, it appeared that the
attachment structure was either absent or greatly reduced in
size compared with untreated HCAEC (Fig. 3C). Additionally,
the protein heads on the surface of the HCAEC (Fig. 3A and
B) were not nearly as abundant on the cycloheximide-treated
cells. This data suggests that cycloheximide inhibits the specific
attachment by P. gingivalis 381 to the monolayer by preventing
the synthesis of a host-derived structure, thereby hindering the
initial step in invasion.
By 30 min of infection of untreated HCAEC, gross distor-
tions in the monolayer could be observed in conjunction with
aggregates of P. gingivalis 381 (Fig. 3D and E), whereas indi-
vidual bacteria observed along the monolayer did not produce
any visible effects on the HCAEC (data not shown). The dis-
tortions of the cell monolayer included extensions of the cells
stretching to engulf the invading bacteria (Fig. 3D). In addi-
tion, many individual cells could be seen tearing away, thus
disrupting the HCAEC monolayer (Fig. 3D and E). We con-
sidered the possibility that the distortions of the monolayer
were artifacts produced by the fixation process. However, the
distortions were not seen at 15 min of infection or in unin-
fected cells. Additionally, the distortions occurred only in re-
gions where aggregates of P. gingivalis 381 were present.
After 45 min of infection, the HCAEC monolayer had se-
vere tears all along the monolayer with and without the pres-
ence of P. gingivalis 381 (Fig. 3F). There was a significant
difference in the integrity of the monolayer in HCAEC in-
fected for 45 and 15 min.
DISCUSSION
Invasion of the endothelial and smooth muscle cells of the
arterial wall by bacterial pathogens could initiate and/or exac-
erbate the inflammatory response of atherosclerosis. A strong
association between CHD and Chlamydia pneumoniae, a gram-
negative respiratory pathogen, has also been reported (6, 27).
At the molecular level, C. pneumoniae has been shown to
infect and to replicate in endothelial cells, smooth muscle cells,
and macrophages in vitro (23, 26). Whereas C. pneumoniae
needs to be transported from the lung to the arteries via mac-
rophages, oral organisms are introduced into the bloodstream
multiple times daily in individuals with periodontitis via chew-
ing and toothbrushing. Therefore, the oral cavity represents a
VOL. 67, 1999 P. GINGIVALIS INVASION OF CORONARY ARTERY CELLS 5795

FIG. 1. Transmission electron micrographs of internalized bacteria in HCAEC. (A) P. gingivalis 381 (arrows). (B) P. intermedia 17 (arrows). (C) P. intermedia 17
dividing (arrows). (D) Internalized E. corrodens 23834 after infection with 1010 organisms. (E) E. corrodens 23834 dividing within HCAEC (magnification, ⫻25,000).

potentially large reservoir of gram-negative pathogenic organ-
isms that could interact with cardiovascular tissues.
Heart disease is the most common systemic pathology in
patients with periodontal disease (34). Atherosclerosis, like
periodontal disease, is mediated by the inflammatory process.
Severe inflammation of the coronary arteries could even lead
to acute myocardial infarction (MI). Bacteria may have a role
in this process by chronically stimulating cytokines such as
interleukin-1, tumor necrosis factor alpha, and/or C-reactive
protein (CRP), an acute-phase reactant (47). Curiously, signif-
icantly elevated levels of CRP have been found in patients
suffering from MI and periodontal disease, and these elevated
5796 DORN ET AL. INFECT. IMMUN.

CRP levels may be predictive of the first MI (8, 17, 37). Early
in atherogenesis of the response-to-injury hypothesis of ath-
erosclerosis, there is increased adherence of leukocytes (38).
The T cells within atherosclerotic lesions are activated T cells,
which suggests specific antigenic stimulation (31). If our hy-
pothesis is true, this adherence of activated leukocytes may be
the cell-mediated response to an intracellular pathogen.
The differences in the abilities of species and strains to
invade indicate that invasion depends on specific bacterium-
cell interactions and does not occur with all oral bacteria.
Certain strains of bacteria likely possess adhesins and/or other
characteristics that promote internalization by the coronary
artery cells. The data presented here indicate that P. gingivalis
381 has one or more characteristics that would differentiate it
from P. gingivalis W50 and P. intermedia 17, since it invades
HCAEC and CASMC more readily. P. gingivalis W50 has a
lower ability to adhere to oral epithelial cells (14). This de-
crease in invasion of HCAEC may thus be due to a lower
percentage of strain W50 binding to the cell surface. The
ability of P. gingivalis W50 to invade HCAEC and CASMC as
reported here is greater than its ability to invade KB cells and
an immortalized human umbilical vein endothelial cell line
(12). These strains can also be differentiated from P. intermedia
25611 in that strain 25611 was not able to invade at any level.
After bacterial invasion, the induction of autophagy by the
host cell increases the pool of free amino acids. The autophagy
exhibited by the HCAEC and CASMC could be a mechanism
that is induced and exploited by the bacteria. The bacteria may
induce autophagy to use the amino acids for their own metab-
olism or to inhibit host protein synthesis in order to increase
survivability (43). A pool of free amino acids could be espe-
cially beneficial for P. gingivalis, which requires short peptides
for carbon and energy (48). Further studies have been initiated
to conclusively determine if P. gingivalis is localized within
autophagosomes and, if so, to investigate the role of autophagy
in the P. gingivalis intracellular parasitism of HCAEC. Prelim-
inary studies indicate that P. gingivalis trafficks to autophago-
somes following invasion in HCAEC (unpublished data).
The SEMs demonstrate that P. gingivalis first attaches via a
host cell-derived structure. Further time points of the SEMs
demonstrate a severe disruption of the endothelial monolayer
after infection with P. gingivalis 381. A disruption of the endo-
thelial layer such as that observed in the SEMs could constitute
the insult to start the atherosclerotic process. Interestingly, the
disruptions were observed to occur only in association with ag-
gregates of P. gingivalis 381 and not in association with a single
bacterium. The SEMs of the engulfment of the aggregates ap-
pear to be similar to those of invasomes of Bartonella henslae
(9). However, the transmission electron micrographs do not
show the same host cell morphology during engulfment of P.
gingivalis 381 as seen with the engulfment of aggregates of B.
henslae. Invasion of HCAEC by aggregates of P. gingivalis may
be clinically relevant, since dental plaque is a host-associated
microbial biofilm (7). Most likely, aggregates of P. gingivalis in
addition to other periodontal pathogens, including P. intermedia,
would be introduced to the bloodstream after mild traumas.
In summary, this study demonstrates that certain pathogenic
periodontal bacterial strains invade human coronary artery cells
in vitro. Bacterial invasion could constitute a chronic insult to the
arterial wall. These findings raise the possibility that a chronic in

FIG. 2. Transmission electron micrographs of bacteria internalized by

CASMC. (A) P. gingivalis 381 surrounded by a large amount of rough endoplas-
mic reticulum. (B) P. intermedia 17. (C) No internalized bacteria after infection
with E. coli MC1061.
 FIG. 3. SEMs of a time course infection of HCAEC by P. gingivalis 381. (A) Uninfected HCAEC monolayer. (B) After 15 min of incubation, an aggregate of
P. gingivalis 381 can be seen attached to the HCAEC by a host-associated pseudopod (arrow). (C) Attachment of P. gingivalis 381, after 15 min of incubation, to a
monolayer of HCAEC preincubated with 1 mg of cycloheximide per ml for 4 h. (D) HCAEC breaking away from the monolayer and engulfing an aggregate of
P. gingivalis 381 after 30 min of incubation. (E) HCAEC tearing away from a neighboring cell after a 30-min incubation. (F) An aggregate of P. gingivalis 381 straddling
a tear between neighboring HCAEC after 45 min of incubation.

5797
5798 DORN ET AL.
vivo infection of the coronary artery wall by pathogens such
as P. gingivalis and P. intermedia could be a factor in CHD.
Many studies have thus far shown an association between peri-
odontal disease and CHD (1, 11, 22, 32, 33), whereas only one
study has shown no relationship (25). Whether this association
is a causal relationship or a “bystander effect” has yet to be
determined. This is the first of a series of studies to determine
whether a molecular link between the two diseases is possible.
ACKNOWLEDGMENTS
This study was supported by a University of Florida Periodontal
Disease Research Center grant and National Institute of Dental and
Craniofacial Research grant DE 07496.
We thank W. E. Nesbitt, K.-P. Leung, and J. E. Beem for providing
P. intermedia strains and advice; E. V. Kozarov, W. A. McArthur, and
S. Sugrue for useful discussion; J. Burks and J. Whitlock for their
assistance; A. Shawley for help and advice with cell culture; and R.
Davis, S. Whittaker, and the University of Florida Electron Micros-
copy Core Laboratory of the Interdisciplinary Center for Biotechnol-
ogy Research for the electron micrographs.
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