Links between atherosclerotic and periodontal disease

Dimitry A. Chistiakov, Alexander N. Orekhov, Yuri V. Bobryshev

PII: S0014-4800(16)00007-1
DOI: doi: 10.1016/j.yexmp.2016.01.006
Reference: YEXMP 3897

To appear in: Experimental and Molecular Pathology

Received date: 5 January 2016

Accepted date: 8 January 2016

Please cite this article as: Chistiakov, Dimitry A., Orekhov, Alexander N., Bobryshev,
Yuri V., Links between atherosclerotic and periodontal disease, Experimental and Molec-
ular Pathology (2016), doi: 10.1016/j.yexmp.2016.01.006

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 ACCEPTED MANUSCRIPT

Review Article

Links between atherosclerotic and periodontal disease

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Dimitry A. Chistiakov a, Alexander N. Orekhov b,c,d, Yuri V. Bobryshev b,e,f, *

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a
Department of Molecular Genetic Diagnostics and Cell Biology, Institute of Pediatrics, Research

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Center for Children's Health, 119991 Moscow, Russia;
b
Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences,
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Moscow 125315, Russia;
c
Institute for Atherosclerosis, Skolkovo Innovative Center, Moscow 143025, Russia;
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Department of Biophysics, Biological Faculty, Moscow State University, Moscow 119991, Russia;
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e
Faculty of Medicine, School of Medical Sciences, University of New South Wales, Kensington,
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Sydney, NSW 2052, Australia;

f
School of Medicine, University of Western Sydney, Campbelltown NSW 2560, Australia
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Running title: Dental plaque bacteria and atherosclerosis

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Correspondence to:

Dr Yuri V. Bobryshev
Faculty of Medicine
University of New South Wales
Sydney, NSW 2052
Australia
Tel/Fax: + 612 93851217
E-mail: y.bobryshev@unsw.edu.au
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ABSTRACT

Periodontal disease (PD) and cardiovascular disease (CVD) are highly prevalent in the modern
community. Both pathologies are chronic inflammatory disorders, which are influenced by multiple

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risk factors. In part, these factors such as age, smoking, and diabetes overlap between PD and CVD.
Epidemiological studies suggest that PD is strongly associated with increased CVD risk.

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Biochemical and physiological analyses involving in vitro experiments, animal models, and clinical

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studies provided evidence for the substantial impact of periodontal pathogens, their virulence
factors, and bacterial endotoxins on all general pathogenic CVD mechanisms such as endothelial

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dysfunction, systemic inflammation, oxidative stress, foam cell formation, lipid accumulation,
vascular remodeling, and atherothrombosis. Interventional studies showed moderate beneficial
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effects of PD treatment on reducing systemic inflammation and endothelial dysfunction. However,
no interventional studies were performed to assess whether periodontal therapy can primarily
prevent CVD. In summary, current data suggest for a strong contributory role of periodontal
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infection to CVD but cannot provide sufficient evidence for a role of PD as a cause for
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cardiovascular pathology.
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Keywords: Periodontitis, Atherosclerosis, Atherogenesis, Porphyromonas gingivalis, Gingipain,

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LPS, Inflammation
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1. Introduction

Teeth are surrounded by gingival tissue. Between the tooth and gingival tissue, a space
(termed gingival sulcus) can occur. The gingival sulcus is lined by sulcular epithelium that joins

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with the oral cavity epithelium at the top of the gingival margin. In the gingival sulcus, the
epithelium and crown enamel form unique surfaces colonized by oral bacteria. In this

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microenvironment, bacteria form a biofilm called dental plaque.

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Primary colonizers, which populate the gingival sulcus, involve streptococci (such as
Streptococcus oralis, S. mutans, and S. sanguis), Neisseria sp., etc. These anaerobic bacteria greatly

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decrease local availability of oxygen thereby providing an option for obligate anaerobes to populate
and grow in the gingival sulcus. In the dental plaque, primary colonizers provide substrates and
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form a background for attachment of secondary colonizers (that are predominantly anaerobic) such
as actinomycetes (Actinomyces odontolyticus and A. naeslundii), Fusobacterium nucleatum,
Veillonella sp., etc. (Rickard et al., 2003). Overall, the dental plaque microflora exceeds 500 species
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(Moore and Moore, 1994).

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In the dental plaque, bacterial composition is influenced by many factors including supply
with oxygen, pH, diet, oral hygiene, and interactions between microbes.
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Above and below the gingival margin, bacterial composition significantly differs. In healthy
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individuals, subgingival flora is mainly composed by aerobic streptococci, anaerobic actinomycetes,

and F. nucleatum (Moore and Moore, 1994). Poor oral hygiene, insufficient or lacking dental care,
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smoking, altered immunity, and other harmful factors could induce gingivitis (i.e. inflammation of
gum tissue). Subgingival plaque could play a major role in the development of gingivitis.
In gingivitis, numbers of subgingival plaque microorganisms greatly increase along with the
enrichment of the subgingival plaque microflora with gram-negative obligate anaerobes (Moore et
al., 1987) including Porphyromonas gingivalis (Socransky et al., 1991), Aggregatibacter
actinomycetemcomitans (Zambon et al., 1983), Prevotella intermedia (Yang et al., 2014),
Prevotella melaninogenica (Mandell et al., 1992), Tannerella forsythensis (Yang et al. 2004),
Fusobacterium periodontium (Kobayashi et al., 2004), and Campylobacter rectus (Gmür et al.,
2004). All these bacteria and some other microbes are pathogens involved in gingivitis and
periodontitis.

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In the subgingival plaque, bacteria form complex relations between each other and host
cells. Plaque microbes are embedded into the extracellular matrix (ECM) of the sulcular epithelium
(Gibbons, 1989). Periodontopathogens such as P. gingivalis and A. actinomycetemcomitans invade
epithelial cells via the mechanism of endocytosis (Meyer et al., 1996). In the host cells, bacteria

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amplify and spread through intercellular protrusions to other cells (Andrian et al., 2006). The
bacterial invasion is accompanied by release of proinflammatory mediators that attract

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proinflammatory immune cells to the gingival sulcus (Jotwani et al., 2001). Periodontal pathogens

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exhibit marked adaptation capacity that supports survival within the invaded cells, neutralization of
immune cells, inactivation of anti-microbial factors, and induction of host mechanisms leading to

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tissue degradation (Amano, 2007).
The deepening of the gingival sulcus caused by dental plaque growth, local inflammation,
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and bacterially mediated destruction of the gingival fiber attachment leads to the formation of the
periodontal pocket. The periodontal pocket is a preferable site for accumulation of periodontal
pathogens where they aggravate inflammation of surrounding tissues and could stimulate
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progression of gingivitis to periodontal disease (PD) (Smalley, 1994). In PD, the connective tissue
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attachment is lacking and the dental plaque expands apically along the root tissue causing
progressive loss of the alveolar bone around the tooth and finally loss of the tooth itself. In
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periodontitis, the periodontal pocket releases gingival crevicular fluid (GCF), an inflammatory
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exudate containing cell debris, bacterial degradation products, inflammatory mediators, connective
tissue fragments, enzymes, and other proteins (Subrahmanyam and Sangeetha, 2003). Inflamed
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periodontal tissue is frequently or persistently bleeding providing an option for periodontal infection
to enter the bloodstream.
Indeed, oral pathogenic bacteria and their endotoxins could disseminate into the systemic
circulation via gingival injuries and affect distant organs (Hirschfeld and Kawai, 2015). Oral
commensals may represent a serious risk for individuals with predisposive cardiac alterations (heart
valve disease, pacemaker implantation, and so on) and cause infective endocarditis (Ito, 2006). First
evidence for positive association between the poor dental health and myocardial infarction (MI)
(Mattila et al., 1989) initiated a wave of interest to study possible relations between dental
infections and cardiovascular diseases (CVD) including atherosclerosis. Despite for heterogeneity
of the studies, overall results of epidemiological studies suggest for a modest but significant
association between periodontal infections and CVD that is independent on the effects of

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confounders (Kebschull et al., 2010). Experiments in animal models and in vitro studies found
potential molecular mechanisms linking PD and atherosclerosis.

2. Epidemiological studies

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Findings from the major epidemiologic studies involving thousands of individuals were

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comprehensively discussed in recent reviews (Kebschull et al., 2010; Lockhart et al., 2012). Several

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meta-analyses were performed and a positive association between PD and cardiovascular disease
(CVD) was reported. Blaizot et al. (2009) showed that PD patients have increased risk of

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developing CVD (odds ratio (OR), 2.35 (95% confidence interval (CI): 1.87; 2.96), p < 0.0001) and
relative risk (RR), 1.34 (95% CI: 1.27; 1.42), p < 0.0001). Sfyroeras et al. (2012) found association
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between PD and ischemic stroke reporting that PD subjects have 1.47-fold and 2.63-fold adjusted
risk to develop stroke in prospective and retrospective studies respectively. In a recent meta-
analysis, Orlandi et al. (2014) observed significant association of periodontitis with increased
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carotid intima-media thickness (cIMT; a characteristic of carotid atherosclerosis) and impaired

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flow-mediated dilatation (FMD; a characteristic of endothelial dysfunction).

Smoking is a strong confounding factor that significantly influences apparent association
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between PD and CVD (Hujoel et al., 2002). Current multivariate analyses include adjustments for
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several smoking-related variables such as smoking status, a quantity of pack-years, a time after
cessation, second-hand smoking, etc. (Nasry et al., 2006; Costa et al., 2013). Indeed, correction for
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several smoking-related confounders strengthens the validity of observed association between PD

and CVD. In addition, strong association between periodontitis and CVD was repeatedly shown in
never-smokers (Okoro et al., 2005; Pussinen et al., 2007; Sim et al., 2008). These findings in turn
indicate that peridontitis represents an independent cardiocascular risk factor.
Association between PD and CVD is influenced by age, with stronger association in
younger patients aged <60-65 yrs compared with those who is older 60-65 (Demmer and
Desvarieux, 2006; Deitrich et al., 2008; Sim et al., 2008; Jimenez et al., 2009). The age-dependent
association between two diseases may not be the only a result of effect modification when the
observed relationship is caused by interaction between two or more risk factors (Ylöstalo and
Knuuttila, 2006). The independent influence of age on PD-CVD association could be supported by
data of Deitrich et al. (2008) who started the follow-up study in the 1960s, i.e. significantly earlier

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when PD-CVD association was first reported. Indeed, this finding is not a false post hoc
observation.
Recently, in a large-scale retrospective cohort study, Chou et al., (2015) reported that
patients with treated severe periodontitis aged > 60 yrs have increased risk of long-term major

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adverse cardiovascular events compared with younger subjects (<60 yrs). The association remained
after adjustment for other potential CVD-related confounders such as gender, hyperlipidemia,

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hypertension, and diabetes. This finding compromises results of earlier studies for a stronger PD-

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CVD association in younger PD individuals. However, the history of smoking was not taken into
consideration in this study (due to data unavailability in the Taiwan National Health Insurance

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Database) that may seriously alter the value of observed associations.
A significant heterogeneity in methodology of definition of periodontitis could complicate
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explanation of observed associations between PD and CVD. In most observational studies, the
status of periodontitis was accessed radiographically (loss of alveolar bone) or clinically (tooth loss,
bleeding upon probing, depth of the periodontal pocket, rate of the fiber attachment loss). Beck and
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Offenbacher (2002) showed that attachment loss, pocket depth, and bleeding upon probing are
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individually associated with levels of circulating systemic inflammatory markers such as C-reactive
protein (CRP) and soluble intercellular adhesion molecule (sICAM), i.e. biomarkers of acute phase
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inflammation and vascular stress respectively. However, among these clinical approaches, bleeding
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assessment showed the best results for association with CRP while pocket depth was superior for
association with sICAM. Further studies confirmed correlation of bleeding on probing with levels
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of systemic inflammation markers such as CRP (Bokhari et al., 2014), interleukin (IL)-18, and C-X-
C motif chemokine ligand (CXCL)-16 (Schallhorn et al., 2010) in patients with coronary artery
disease (CAD).
Indeed, since association between periodontal markers and systemic inflammation markers
(Noack et al., 2001; Pejcic et al., 2011) was shown, a possible contribution of periodontitis to
chronic inflammatory disease such as atherosclerosis may consist in PD-dependent stimulation of
systemic (i.e. vascular inflammation).
In a meta-analysis, Mustapha et al. (2007) showed that elevated systemic bacterial exposure
(mainly assessed by serological measurements of antibody responses to periodontal microbes) is
associated with increased CAD risk (OR, 1.75 (95% CI: 1.32; 2.34), p < 0.001). However, the
utility of measurements of systemic antibody titers to assess the degree of pathogen exposure is

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limited since observed correlations between serum antibody to periodontal bacteria and clinical
periodontal conditions are species-specific (Dye et al., 2009) and increased antibody titers may
reflect activation of adaptive immunity against periodontal pathogens and PD severity. When
considering systemic antibodies levels as indicator of degree of infectious exposure, it is necessary

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to take into account smoking status and tooth loss since edentulous subjects and smokers exhibit
lower antibody responses than dentate individuals and never-smokers do (Vlachojannis et al.,

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2010). To date, no experimental studies were done to evaluate carefully whether combinations of

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clinical, bacterial, and antibody-based assessments may strengthen the accuracy of exposure
definitions and prognosis of CVD-associated outcomes.

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Regarding CVD-associated outcomes, PD showed a stronger association with ischemic
stroke than with coronary heart disease (CHD) (Hujoel et al., 2000; Dietrich et al., 2008; Jimenez et
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al., 2009). These findings were based on large-scale epidemiological data such as NHANES I and
Normative Aging Study (NAS). Watt et al. (2012) reported a correlation between tooth loss and
overall CVD mortality that was mainly driven by fatal cerebrovascular events. However, the
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mechanisms explaining why periodontitis exhibits a stronger risk factor for stroke than for CAD are
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unknown.
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3. Periodontal therapy
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The main goal of intervention studies is to evaluate whether treatment of periodontitis can
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lead to improvement in CVD-related markers and systemic inflammation markers. In a case of

observing positive association, the results of intervention studies will be helpful for defining
atherogenic mechanisms that can be influenced by PD treatment and therefore increase
atheroprotection.
GCF is a source of numerous proinflammatory markers such as tumor necrosis factor
(TNF)-α, IL-1β, IL-6, and others. GCF could be taken from periodontosis-associated tooth sites and
used for proteomic/immunochemical analyses in order to monitor periodontal inflammation (Gupta,
2013). In chronic periodontitis, inflammatory messengers and inducers persistently enter the
circulatory system and contribute to elevated levels of systemic inflammatory markers such as CRP,
IL-6, neutrophil and total leukocyte counts (Loos et al., 2000; Loos, 2005; Sun et al., 2009;
Wohlfeil et al., 2012).

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Non-surgical PD treatment was shown to result in short-time elevation in levels of acute-

phase markers such as CRP and IL-6 that reflects the host response to instrumentally induced
damage and massive entrance of oral microbiota to the bloodstream (D’Aiuto et al., 2005; Tonetti et
al., 2007). In a meta-analysis, Horliana et al. (2014) found positive evidence for periodontal

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bacteremia caused by periodontal procedures in almost a half (49.4%) of treated patients.
In 6-month post-treatment follow-up studies, an overall reduction in serum levels of

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systemic inflammatory markers including IL-6, TNF-α, and CRP was observed (S’Aiuto et al.,

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2004; Tonetti et al., 2007, Paraskevas et al., 2008; Marcaccini et al., 2009). Using an evaluation
scale of 19 inflammatory markers, Behle et al. (2009) showed the heterogeneity of systemic

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inflammatory responses after periodontal treatment, with reduction (or increase) in inflammatory
markers in 25-30% patients during 1-month post-treatment follow-up. A meta-analysis performed
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by Demmer et al. (2013) showed modest reductions in systemic CRP levels in the earliest post-
treatment time point (usually 1-3 months).
In regard to CVD risk factors, Nakajima et al. (2010) observed significant reduction in
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serum CRP and IL-6 only in the subgroup of periodontally treated subjects highly susceptible to
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increased CHD risk. Similarly, Correa et al. (2010) reported decrease in systemic inflammatory
markers in type 2 diabetic patients 3 months after non-surgical periodontal therapy. In summary,
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periodontal therapy seems to have short-term beneficial effects on systemic inflammation including
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patients at CVD risk. However, it is unclear whether treatment of periodontitis has a long-term
favorable effect on systemic inflammatory markers.
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Endothelial dysfunction is caused by imbalance between vascular vasoconstrictors and

vasodilators and leads to the impairment of the endothelial function involved in the control of
vascular tone, vessel permeability, coagulation, cell adhesion, and immune function. Endothelial
dysfunction predisposes to various vascular pathologies including atherosclerosis and other CVD
(Park and Park 2015). Chronic PD contributes to endothelial dysfunction by induction of
inflammatory response, increased adhesion, reduced endothelium-dependent dilatation (EDD) and
other functional alterations of vascular endothelium (Higashi et al., 2008; Gurav et al., 2014).
Endotoxins and antigens released by periodontal bacteria play a role in pathogenic challenging
endothelial function (Bhagat et al., 1996; Maekawa et al., 2010). Stimulation of production of
endothelin-1, a potent vasoconstrictor, is one of the mechanisms by which periodontopathic bacteria
could induce EDD (Ansai et al., 2002). Periodonthal treatment alone or supplemented with

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antibiotics was shown to improve EDD (Seinost et al., 2005; Elter et al., 2006; Tonetti et al., 2007).
Overall, a recent meta-analysis showed beneficial effects of periodonthal therapy on endothelial
function in part through increasing FMD (Orlandi et al., 2014).
cIMT (a measurement of the thickness of carotid tunica media and tunica intima) is a

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surrogate marker of carotid atherosclerosis. cIMT monitoring is a valuable tool for detecting
atherosclerotic plaque and tracking regression or progression of atherogenesis. PD, especially in

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severe form, was shown to be associated with increased cIMT (Beck et al., 2001; Leivadaros et al.,

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2005; Cairo et al., 2008; Yu et al., 2014) suggesting for a predisposing role periodontitis in carotid
atherosclerosis. Tapashetti et al. (2014) showed correlation between CRP levels and cIMT in

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patients with chronic periodontitis indicating that PD-induced inflammation could contribute to
carotid atherosclerosis. In a meta-analysis, Orlandi et al. (2014) confirmed association between PD
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and increased cIMT (mean increase in c-IMT of 0.08 mm (95% CI: 0.07; 0.09)). PD therapy was
shown to significantly reduce c-IMT during 12-month post-treatment follow-up (Piconi et al., 2009;
Kapellas et al., 2014) but not pulse wave velocity (PWV) (Kapellas et al., 2014) suggesting that
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periodontal treatment could improve arterial structure but not function.

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Arterial stiffness is an important indicator of mechanical properties of arteries. PWV is

related to the arterial stiffness. Increased arterial stiffness (and indeed increased PWV) is associated
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with higher atherosclerotic risk. In periodontitis patients, PWV was found to be significantly
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increased (Houcken et al., 2015). However, periodontal treatment of hypertensive PD patients did
not lead to reducing PWV 6 months after therapy (Houcken et al., 2015). In other study, Vidal et al.
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(2013) showed beneficial effects of periodontal therapy on arterial stiffness and other CVD-related
characteristics such as circulating inflammatory markers, blood pressure, and left ventricular mass
in 6-month follow-up of treated patients with refractory hypertension.
In the Periodontitis and Vascular Events (PAVE) study aimed to examine whether PD
treatment decreases CVD risk, no beneficial effects were achieved in the periodontally treated
patients with CVD history during a follow-up period of 6 to 25 months compared to those who used
community dental care, except for a trend in a less incidence of adverse events in treated subjects
(Beck et al., 2008). However, almost a half (48%) of patients randomized in the community care
group received preventive or periodontal care outside the study, a fact that could compromise the
validity of obtained results. Overall, systematic review of randomized trials performed to verify
whether periodontal therapy could prevent or manage CVD in individuals with chronic PD failed to

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reveal favorable effect of non-surgical PD treatment due to the low quality evidence (Li et al., 2014;
Henschel and Keenan 2015). In fact, the only a single randomized controlled trial (i.e. the PAVE
study) was performed to date.

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4. Periodontal pathogens in animal models of atherosclerosis

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Apolipoprotein E (ApoE)- and low density lipoprotein (LDLR)-deficient mice are

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established rodent models of human atherosclerosis. Both models were challenged with periodontal
pathogens or their endotoxins/antigens in order to study potential molecular mechanisms linking

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periodontal infection and atherosclerosis (Table 1). In overall, oral infection of atherosclerotic mice
with single or several periodontal bacteria led to advanced disease accompanied with increased
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plaque progression, enhanced macrophage infiltration, elevated levels of inflammatory markers,
lipid accumulation in the arterial wall, foam cell formation, and advances in other proatherogenic
signs (Kebschull et al., 2010).
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In mice, invasion patterns were different between periodontal pathogens. P. gingivalis DNA
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was detected in the aorta, liver, and heart (Li et al., 2002). Furthermore, in ApoE-deficient mice, P.
gingivalis was able to access brain and induce complement activation and brain inflammation while
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other periodontal pathogens (F. nucleatum, Treponema denticola, and T. forsythia) did not (Poole et
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al., 2015). P. gingivalis infection markedly promoted atherosclerosis while A.

actinomycetemcomitans had no effect on lesional size and progression but invaded liver leading to
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the stimulation of expression of proinflammatory genes, induction of hepatic macrophage

infiltration, and impaired lipid homeostasis towards the generation of proatherogenic profile
(Tuomainen et al., 2008; Hyvärinen et al., 2009). Liver plays a key role in lipid metabolism and
production of acute-phase proteins (CRP and others). Indeed, along with impairment of endothelial
function and systemic inflammation, induction of hepatic inflammation and alteration of liver-
dependent lipid metabolism may represent an additional pathway by which periodontal bacteria can
influence atherosclerosis.
Challenge with bacterial products such as BspA (virulence factor of T. forsythia), P.
gingivalis lypopolysacharide (LPS), and GroEL from F. nucleatum (a chaperonin) also promoted
atherosclerosis in mice (Gitlin and Loftin, 2009; Lee et al., 2012; Lee et al., 2014). Indeed,
endotoxins and other substances released by periodontal pathogens are involved in proatherogenic

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effects of periodontal endotoxemia (Wiesner et al., 2010). Toll-like receptors (TLRs), i.e. innate
immune receptors recognizing various pathogen-associated molecular patterns (PAMPs) play a
primary role in mediating effects of periodontal bacterial antigens and virulence factors (Bainbridge
and Darveau, 2001; Hajishengallis et al., 2002).

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Fimbrillin (FimA), a structural component of fimbriae (attachment pilli), plays a central role
in P. gingivalis colonization and virulence (Yoshimura et al., 2009). FimA-deficient P. gingivalis

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mutants were unable to up-regulate TLR2 and TLR4 and promote atherosclerosis in ApoE-deficient

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mice (Gibson et al., 2004). Both TLRs are not involved in binding bacterial fimbriae. Other pattern-
recognition receptors (PRRs) namely CD14 and CD11b/CD18 interact with fimbriae but TLR2 and

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TLR4 mediate cell activation in response to fimbriae (Hajishengallis et al., 2004). In vascular
endothelial cells (ECs), P. gingivalis FimA is involved in TLR2- and TLR4-dependent up-
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regulation of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1), vascular cell
adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin (Khlgatian et al., 2002). In monocytes,
TLR2 mediates monocyte adhesion to activated ECs in response to P. gingivalis P. gingivalis and
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transendothelial migration, a mechanism implicated in early stages of atherosclerosis (Harokopakis

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et al., 2006). Therefore, fimbrillin is crucially involved in modulating proatherogenic effects of P.

gingivalis fimbriae by induction of proinflammatory activation of ECs and recruitment of
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monocytes.
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Interestingly, P. gingivalis-mediated atherosclerosis could be prevented by prior

immunization with P. gingivalis (Miyamoto et al., 2006) or P. gingivalis 40-kDa outer membrane
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protein (OMP) (Koizumi et al., 2008). OMPs are expressed on the surface of gram-positive bacteria
and are responsible for survival of engulfed bacteria in macrophages and cell invasion.
Immunization of ApoE-deficient mice with P. gingivalis 40-kDa OMP induces humoral immune
response and production of 40-kDa OMP-specific antibodies (IgG1, IgG2b, and IgA) (Koizumi et
al., 2008) that inhibit bacterial coaggregation and biofilm formation and hence reduce P. gingivalis
pathogenicity and atherogenicity upon injection (Namikoshi et al., 2003).
Madan et al. (2007) attempted to model effects of periodontal treatment on atherosclerosis
and systemic inflammation. Since mechanical dental therapy is not acceptable in mice, the authors
used systemic administration of doxycycline (an antibiotic). In ApoE heterozygotic mice, treatment
with doxycycline had anti-atherogenic effect by reducing both P. gingivalis-mediated atherogenesis
and inflammation (Madan et al., 2007). However, these findings are not applicable in humans where

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periodontitis is mediated by dental plaque, i.e. by biofilm that greatly increases bacterial resistance
to antibiotics (Lewis, 2008). Indeed, this can explain inefficacy of systemic therapy with antibiotics
in the secondary prevention of cardiac events (Grayston et al., 2005; Stassen et al., 2008; Almalki
and Guo 2014). In mice, Madan et al. (2007) used repeated injections of P. gingivalis that did not

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lead to the formation of a stable biofilm. Hence, non-aggregated P. gingivalis bacteria are more
sensitive to an antibiotic.

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In summary, elaborating of experimental models of atherosclerosis resulted in obtaining

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useful data about pathogenic mechanisms linking periodonatal infections and atherogenesis. For
example, Arg-specific gingipain, one of two gingipain endoproteases released by P. gingivalis, was

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shown to specifically cleave apoB-100, a key protein moiety in LDL (Miyakawa et al., 2004).
Pathogen-mediated ApoB-100 degradation results in LDL aggregation, rising LDL levels,
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generation of foam cells, and enhanced lipid deposits in the arterial wall of ApoE-deficient mice
(Hashimoto et al., 2006). Protein convertase subtilisin/kexin type 9 (PCSK9) is activated in PD
patients (Miyazawa et al., 2012a). In mice, Miyazawa et al. (2012b) showed that P. gingivalis
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infection up-regulates hepatic production of PCSK9 in sterol regulatory element binding

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transcription factor 2 (Srebf2)-dependent fashion. PCSK9 binds to LDLR and enhances lysosomal
degradation of the receptor (Sasaki et al., 2014). Decrease in LDLR density on the surface of
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hepatocytes leads to elevated plasma LDL cholesterol and induces hypercholesterolemia in mice
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(Miyazawa et al., 2012b). These two examples explain how P. gingivalis invasion could contribute
to atherogenesis by altering lipid metabolism and homeostasis.
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Findings obtained in animal atherosclerotic models may have limited relevance to humans.
Investigators usually challenged hypercholesteroemic mice with repeated administration of
periodontal monoinfections that are unlikely to form stable biofilms while in humans PD is
mediated by bacterial community (i.e. dental plaque) composed of several or multiple periodontal
pathogenic species. However, using animal models of atherosclerosis remains to be very helpful for
studying pathogenic mechanisms of atherogenesis.

5. Bacteremia and endotoxemia

Bacteremia and endotoxemia are two main mechanisms by which PD pathogens may
directly influence atherogenesis and initiate pathological events linking PD and atherosclerosis.

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Bacteremia refers to the presence of bacteria in the blood while endotoxemia means the presence of
endotoxins in the circulation.
In oral cavity, enter of periodontal bacteria to the bloodstream commonly occurs after dental
treatments and may happen during chewing food (Geerts et al., 2002) and after dental hygiene

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procedures such as tooth brushing and dental flossing (Crasta et al., 2009; Zhang et al., 2013),
especially in patients with gingivitis or periodontitis. In PD, bacteremia happens due to the

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interruption of oral epithelium in the periodontal pocket and/or contacts between the subgingival

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plaque and adjacent microvessels (Nanci and Bosshardt, 2006). The extent of bacterial blood
dissemination depends on the magnitude of tissue trauma, bacterial density in the dental plaque, and

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severity of local inflammation (Wilson et al., 2007). The gingival sulcus progressing to the
periodontal pocket is the major source and portal for entry of periodontal bacteria to the circulation
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(Strom et al., 1998).
Around 300 distinct oral bacterial species were found in blood samples after single-tooth
extraction and dental brushing (Bahrani –Mouqeot et al., 2008; Lockhart et al., 2008). More
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frequent bacteria were Streptococcus viridans, A. actinomycetemcomitans, P. gingivalis,

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Micrococcus micros and species Streptococcus and Actinomyces (Horliana et al., 2014). In the
blood, oral bacteria circulate freely or within cell autophagosomes. Through the circulatory system,
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periodontal bacteria can reach distant organs. Bacteria could attach to and invade various vascular
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cells including arterial ECs and arterial smooth muscle cells (SMCs) (Dorn et al., 1999; Dorn et al.,
2001).
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In PD patients, many studies reported detecting DNA of various periodontal pathogens in

arterial atheromatous plaques removed in endarterectomy (Harazsthy et al., 2000; Kozarov et al.,
2006; Nakano et al., 2008; Gaetti-Jardim et al., 2009). In some studies, the percentage of human
lesion specimens positive for DNA of periodontal pathogens was high. For example, Harazsthy et
al. (2000) found DNA of one or more of four tested species (A. actinomycetemcomitans, P.
gingivalis, P. intermedia, and Bacteroides forsythus) in 80% (40 of 50) endarterectomy specimens.
In addition, Kozarov et al. (2005) reported obtaining viable P. gingivalis and A.
actinomycetemcomitans bacteria from atherosclerotic lesions of a PD patient.
On the other hand, many studies did not found specific DNA of any of periodontal bacteria
in atherosclerotic plaques (Cairo et al., 2004; Aimetti et al., 2007; Romano et al., 2007) while
bacterial DNA was identified in all or almost all endarterectomy specimens. In 74% of tested plaque

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samples, Nakano et al. (2006) found presence of Streptococcus mutans DNA, a caries causative
agent, while prevalence of other bacteria including periodontal pathogens was significantly less.
This finding may indicate an option for teeth decay-causing bacteria to use disrupted epithelium as
a gate for extra-oral dissemination. Due to significant inconsistency of available data, it is difficult

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to conclude whether periodontal pathogens can cause atherosclerosis in humans.
Current diagnostic approaches used for detection of bacteria in the plaque were

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comprehensively criticized due to methodological limitations (Ieven and Hoymans 2005).

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Periodontal pathogens found in the atheroma were proposed to not be directly involved in the
plaque formation. The local infection burden, which could promote CVD pathogenesis through

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inflammatory mechanisms, was suggested to serve as a more reliable cardiovascular risk marker
(Zhu et al., 2000; Georges et al., 2003).
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Endotoxins released by periodontal bacteria can induce and mediate distant inflammation
through the circulation. In severe periodontitis, serum levels of LPS due to the overgrowth of
periodontal gram-negative microorganisms are increased (Pussinen et al., 2004). In healthy patients,
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circulating LPS is mainly associated with high density lipoprotein (HDL). However, in chronic PD,
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shift of LPS toward less dense and proatherogenic lipoprotein such as LDL and very light density
lipoprotein (VLDL) occurs (Rufail et al., 2007; Kallio et al., 2008). VLDL- and LDL-associated
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LPS facilitate lipoprotein-dependent activation of macrophages and induction of expression of

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proinflammatory messengers such as IL-6, macrophage chemotactic protein-1 (MCP-1), and TNF-α
(Kallio et al., 2013). In induction of proinflammatory activation of macrophages, low levels of LPS
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were shown to act synergistically with minimally oxidized LDL (oxLDL) through derepression of
transcription factor AP-1 and activation of nuclear factor (NF)-κB that drive expression of
proinflammatory genes (Wiesner et al., 2010).
Along with the induction of the proinflammatory phenotype, LPS from periodontal microbes
promote accumulation of LDL cholesterol in macrophages and generation of foam cells through
inhibition of expression of scavenger receptor B1 (SRB1) (HDL-binding receptor) and ATP-
binding cassette transporter 1 (ABCA1) responsible for reverse cholesterol transport (Lakio et al.,
2006). Periodontitis also reduces the anti-atherogenic potential of HDL via decreasing HDL
capacity to mediate cholesterol efflux from macrophages (Pussinen et al., 2004) and altering
expression and activity of proteins involved in lipoprotein metabolism such as phospholipid transfer

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protein (PLTP), lecithin-cholesterol acyltransferase (LCAT), and cholesterol ester transfer protein
(CETP) (Pussinen et al., 2001).
Indeed, LPS from periodontal pathogens can promote atherogenesis via multiple
mechanisms that involve serum lipid redistribution towards formation of proatherogenic lipid

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profile and cooperation with LDL, VLDL, and oxLDL in induction of vascular inflammation and
lipid accumulation in macrophages.

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Bacterial products of periodontal pathogens serve as antigens that induce humoral immunity

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and production of antibodies specific to periodontal bacteria. Bacterial proteins could share
molecular homology with host proteins. This in turn may initiate cross-reactivity between pathogen-

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specific antibodies and host proteins through the mechanism of molecular mimicry.

6. Molecular mimicry
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This phenomenon results from the sequence similarities between foreign antigens and self-
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antigens that may induce cross-activation of autoreactive T- and B-lymphocytes by foreign antigens
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and induce autoimmune response (Froude et al., 1989).

Overexpression of heat shock protein (HSP)60 on the surface of vascular ECs may be
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induced by many inflammatory and non-inflammatory stimuli including exposure to LPS (Wick et
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al., 2008). Mammalian HSP60 share significant homology with bacterial chaperonin GroEL
presented in P. gingivalis and other periodontal pathogens. In PD patients, GroEL-specific humoral
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response was found to be accompanied with cross-reactivity between GroEL-specific antibodies

and endothelial HSP60 (Tabeta et al., 2000; Ford et al., 2005a) and accumulation of HSP60-reactive
T-cells in periodontal plaques (Yamazaki et al., 2002). HSP-reactive T cells were also found in
atherosclerotic lesions of PD patients (Choi et al., 2002; Ford et al., 2005b).
Interestingly, immunization of ApoE-deficient mice with two different P. gingivalis GroEL
epitopes (peptide 14 and peptide 19) led to the induction of opposite immune responses (Jeong et
al., 2015). Peptide 14 caused dendritic cell (DC)-mediated polarization of naïve T cells to
regulatory T cells (Tregs) and anti-inflammatory response while peptide 19 stimulated induction of
proinflammatory Th1 cells. In non-immunized wild-type mice, DCs activated Tregs in an antigen-
independent manner. Indeed, in ApoE-deficient mice, GroEL-specific epitopes may induce distinct
(pro- or anti-atherogenic) responses mediated by distinct DC subsets.

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Choi et al. (2011) observed positive cross-reactivity to HSP60/GroEL-specific peptide 19 in

30% of PD patients and 100% of atherosclerosis patients. Peptide 19-specific Tregs produced high
levels of anti-inflammatory IL-10 but had no expression of CD25 and forkhead box (Fox) P3, i.e.
markers specific for CD25+FoxP3+ natural Tregs and CD25+FoxP3+inducible Tregs (iTregs). These

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data argue for a role of HSP60/GroEL identity as a link between periodontitis and atherosclerosis.
Moreover, the anti-inflammatory peptide 19 has a value as a putative therapeutic tool for treatment

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of CVD and PD.

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As mentioned above, P. gingivalis produce two endopeptidases, Arg-specific and Lys-
specific gingipain. These proteases play an important role in bacterial adhesion, colonization,

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defense, and invasion through interaction with ECM proteins of host cells, degradation of ECM and
surface receptors, and proteolytic inactivation of cytokines and antibodies (O-Brien-Simpson et al.,
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2003). P. gingivalis gingipain was found to share homology with LDL modified with
malondyaldehide (MDA-LDL) and could be recognized by MDA-LDL-specific antibodies
(Turunen et al., 2012). Immunization with MDA-LDL of LDLR-deficient mice challenged with P.
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gingivalis had atheroprotective effect by reducing lipid depositions in the arterial wall and inducing
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the anti-atherogenic humoral immune response (Turunen et al., 2015). MDA-LDL and other
modified LDL are highly immunogenic, pro-inflammatory and atherogenic (Rafieian-Kopaei et al.,
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2014). Indeed, natural IgM directed against P. gingivalis infection are able to bind and neutralize
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proatherogenic MDA-LDL by recognizing molecular mimicry on bacterial epitopes and modified

lipid and protein structures.
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In PD patients, P. gingivalis infection induces production of gingipain-specific IgG

antibodies (Inagaki et al., 2003; Nguyen et al., 2004). However, it is unknown whether these
antibodies can react with MDA-LDL epitopes and thereby modulate atherogenesis in humans. Both
humans and mice contain natural IgM antibodies, a component of innate immunity, which are
constitutively expressed and bind to both microbial and altered (i.e. oxidation-specific) self-antigens
(Chou et al., 2009; Binder 2010). Oxidation-specific IgM are atheroprotective in both humans and
mice. MDA-LDL-specific IgM specifically recognizing MDA epitopes were found in sera of
healthy subjects and patients with MI and stable angina pectoris (Amir et al., 2012). It is necessary
to investigate whether these antibodies are cross-reactive with gingipain. This may provide a
molecular link between periodontal infection and human atherosclerosis.

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7. Periodontal pathogens and proatherogenic mechanisms

In the bloodstream, periodontal bacteria may interact with various vascular and circulating
immune and non-immune cells exerting different effects on each cell population (Fig. 1). Most of

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data related to the interactions between periodontal pathogens and host cells were obtained from in
vitro experiments in which cultured cells were exposed to bacteria and their products. However,

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many important findings were done in animal models and affected patients. P. gingivalis was used

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as a model organism of periodontal infection.

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7.1. Endothelial dysfunction

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In bacteremia, periodontal pathogens interact with vascular ECs in order to escape from fast
clearance by immune cells. One of the evading ways is the invasion of ECs by pathogenic bacteria
(Progulske-Fox et al., 1999). Endothelial invasion by P. gingivalis is critically mediated by
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hemagglutinin A (HagA) (Bélanger et al., 2012), hemagglutinin B (HagB) (Song et al., 2005), and
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fimbriae (FimA). Compared to fimbriae-negative bacteria, fimbriae-positive strains of P. gingivalis

exhibit profound invasive, adhesion and proatherogenic properties. FimA induces expression of
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various adhesion molecules on the surface of vascular ECs and production of inflammatory
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messengers including IL-6, MCP-1, IL-8, and cyclooxygenase-2 (COX-2) (Nassar et al., 2002;
Chou et al., 2005; Takanashi et al., 2006; Ho et al., 2009). FimA also stimulates expression of
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TLR4 that in turn sensitizes ECs to P. gingivalis LPS (Yumoto et al., 2005). Indeed, LPS and FimA
could cooperate in proinflammatory activation of arterial endothelium by P. gingivalis.
Periodontal bacteria induce proinflammatory activation of ECs with different capacity. For
example, P. gingivalis is a robust inducer of endothelial MCP-1 production while T. forsythia
modestly activates MCP-1 expression and T. denticola has no visible effects (Niu and Kolattukudy
2009). In invaded cells, P. gingivalis and A. actinomycetemcomitans influence distinctive pathways.
A. actinomycetemcomitans-associated network is related to EC function whereas P. gingivalis
mainly affects MAPK-dependent pathways (Lv et al., 2015). Compared with P. gingivalis, A.
actinomycetemcomitans (and its leukotoxin) up-regulate expression of a less number of adhesion
molecules (i.e. ICAM-1, VCAM-1, and P-selectin) and do not activate NF-κB in ECs (Assinger et

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al., 2011; Dietmann et al., 2013). Indeed, each periodontal bacterium utilizes unique mechanisms of
host-pathogen interactions.
Up-regulation of endothelial expression of adhesion and chemoattractant molecules by
periodontal bacteria stimulates attachment of monocytes and other leukocytes on the surface of ECs

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(Hajishengallis et al., 2006; Roth et al., 2007b; Hashizume et al., 2011). Recruitment of monocytes
on the endothelial surface is the important event in early atherosclerosis that precedes their

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transendothelial migration to the arterial intima and differentiation to macrophages. P. gingivalis

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LPS was shown to mediate adhesion of monocytes via TLR2-dependent mechanism (Erridge et al.,
2007; Nakamura et al., 2008). In ECs, TLR2 activation leads to the adhesion and transmigration of

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monocytes associated with stimulation of Rac1 signaling and up-regulation of adhesion molecules
ICAM-1, CD11b, and CD18 (Harokopakis et al., 2006).
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P. gingivalis is able to increase vascular permeability through gingipain-dependent
mechanism. Gingipains cleave platelet endothelial cell adhesion molecule 1 (PECAM-1 or CD31)
that leads to the progressive gap formation between adjacent ECs (Yun et al., 2005). In ECs, P.
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gingivalis-derived LPS enhances endothelial permeability through NF-κB-dependent up-regulation

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of IL-8, a key inducer of vascular permeability (Kim et al., 2013). LPS and gingipains are released
from outer membrane vesicles (OMVs).
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P. gingivalis constitutively shed OMVs. OMV production and invasion is regulated by

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fimbriae (Mantri et al., 2015). OMVs is a major virulence P. gingivalis factor (Bartruff et al., 2005).
OMVs could be internalized by recipient cells with 2-3-fold higher efficiency compared with
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bacterial cells. After 1h of exposure, 70-90% of cultured HUVECs had vesicles while 20-50% of
the host cells had internalized P. gingivalis cells (Ho et al., 2015).
Gingipains released from OMVs facilitate endothelial P. gingivalis attachment (Nassar et
al., 2002) and sensitize ECs to P. gingivalis LPS and to the pathogen itself by activation of release
of Weibel-Palade bodies (a store of vasoactive substances) (Inomata et al., 2007). Protease-
activated receptors (PARs) mediate cellular effects of gingipains. Through PAR activation,
gingipains cooperate in induction of IL-8 and MPC-1 production by ECs (Nassar et al., 2002).
OMVs were shown to inhibit endothelial nitric oxide (NO) synthase (eNOS), a key producer of NO
that plays a central role in the regulation of a proper endothelial function (Jia et al., 2015). P.
gingivalis-derived OMVs display anti-angiogenic and inhibitory effects on ECs via suppressing
their growth and proliferation and inducing apoptosis (Bartruff et al., 2005). Apoptotic properties of

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OMVs are mediated by gingipains that cleave adhesion molecules on the endothelial surface and
induce EC detachment (Baba et al., 2002; Sheets et al., 2005; Sheets et al., 2005; Roth et al.,
2007a). These findings suggest that P. gingivalis OMVs is the best offensive weapon supporting
pathogen survival, invasion, and P. gingivalis-induced endothelial dysfunction (Ho et al., 2015).

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EC apoptosis is a hallmark of atherosclerosis-related endothelial dysfunction. Endothelial
apoptosis persists during the atherosclerosis progression leading to EC detachment and denudation

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of the arterial wall. Endothelial dysfunction is the earliest vascular manifestation of atherosclerosis

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associated with other traditional CVD risk factors (Widlansky et al., 2003). In patients without
clinically manifected atherosclerosis, systemic endothelial dysfunction is associated with increased

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risk of adverse cardiac events (Heitzer et al., 2005; de Berrazueta et al., 2010).
At present, endothelial dysfunction is assessed with help of the digital pulse amplitude
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tonometry and ultrasonic evaluation of the brachial artery after flow-mediated vasodilation (FMD)
or nitroglycerin administration (Hamburg and Benjamin 2009; Onkelinx et al., 2012). In patients
with advanced PD, FMD was significantly reduced compared to the controls (Amar et al., 2003;
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Seinost et al., 2005). Mercanoglu et al. (2004) observed significant decrease in both endothelium-
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dependent dilatation (EDD) and endothelium-independent dilatation (EID) in response to

nitroglycerin. Higashi et al. (2008) reported decreased forearm blood flow responses to
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acetylcholine in hypertensive PD patients compared with hypertensive subjects without

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periodontitis. No significant differences were observed in EID in response to nitroprusside. These

studies showed the presence of endothelial dysfunction in PD patients. After periodontal therapy,
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significant improvement in endothelial finction was observed (Mercanoglu et al., 2004; Higashi et
al., 2008).

7.2. Vascular remodeling

Vascular remodeling is an adaptive response of arteries to an increase in atherosclerotic

plaque size in order to maintain constant flow (Korshunov et al., 2007). In atherosclerosis-
associated remodeling, the arterial wall is a subject of significant structural and morphological
changes. Arterial SMCs play a key role in arterial wall remodeling. They undergo hyperplasia and
switch a ‘contractile’ phenotype to ‘synthetic’ along with acquiring ability to proliferate, grow, and
migrate. Migration and proliferation of SMCs cause the neointimal formation and intimal

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thickening (Chistiakov et al., 2015). In the neointima, migrated SMCs usually produce ECM to
form a fibrous cap that covers the necrotic plaque core (Lacolley et al., 2012).
The involvement of periodontal pathogens such as P. gingivalis in the neointimal
hyperplasia was shown in mice and humans. In a murine model of aortic hyperplasia induced by EC

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injury, intravenous injection of P. gingivalis led to dramatic increase in intimal hyperplasia
associated with overexpression of S100 Ca2+-binding protein A9 (S100A9) and embryonic isoform

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of myosin heavy chain (SMemb), a marker of SMC proliferation (Hokamura et al., 2010). Inaba et

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al. (2009) showed activation of S100A9 expression and phenotypic changes in human aortic SMCs
exposed to supernatant of plasma incubated with P. gingivalis. This indicates that soluble factors

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released by bacteria can induce SMC hyperplasia. Similarly, up-regulation of S100A9 and SMemb
was observed in aortic aneurisms of PD patients (Hokamura et al., 2010; Nakano et al., 2011)
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suggesting on the involvement of P. gingivalis in intimal hyperplasia.
In vitro experiments involving cultured arterial SMCs showed the ability of P. gingivalis
invade, survive, and transmit between the cells (Dorn et al., 1999; Dorn et al., 2002; Li et al., 2008).
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In infected cells, bacteria could alter expression of multiple genes. In human aortic SMCs invaded
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with P. gingivalis, Znang et al. (2013) observed changes in expression of a total of 982 genes in part
regulated by transforming growth factor (TGF)-β and Notch pathways, i.e. signaling mechanisms
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involved in cell proliferation, growth, and hypertrophy. Gingipains released by P. gingivalis were
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shown to stimulate expression of angiopoietin-2 in aortic SMCs and inhibit production of

angiopoietin-1 (Zhang et al., 2015). Angiopoietin-2 is expressed in sites of tissue remodeling. This
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factor regulates angiogenesis through inhibiting antigiopoietin-1 and its receptor Tie-2 and
promoting migration of SMCs (Felcht et al., 2012). The ability of P. gingivalis to modulate balance
between these two angiogenic factors may be important in the context of intraplaque
neovascularization during pathological vascular wall remodeling.
Isolated rat coronary arteries exposed to LPS from P. gingivalis exhibited greater
contraction in response to endothelin-1 (Ghorbani et al., 2010). Bacterial LPS was shown to up-
regulate expression of the endothelin B receptor in SMCs that in turn sensitizes arteries to
endothelin-1-mediated vasoconstriction. In summary, P. gingivalis display opposite effects on
vascular cells. While inhibiting function, proliferation, and migration and inducing apoptosis of
ECs, this pathogen and its products support proliferation, growth, and motility of vascular SMCs,

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the evidence about a role of P. gingivalis in promoting atherogenic vascular remodeling (Hokamura
and Umemura 2010).

7.3. Foam cell formation

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Transendothelial migration of monocytes leads to the accumulation of macrophages in the

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arterial intima. In the intima, macrophages ingest lipids and transform to foam cells due to inability

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to efficiently utilize and pass away engulfed lipids. Foam cells are the main contributors of
lipid/necrotic core formation due to the death and degradation within the plaque (Hilgendorf et al.,

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2015). Periodontal pathogens such as P. gingivalis and their products (LPS, etc.) positively
contribute to all stages of monocyte fate in atherosclerosis supporting monocyte recruitment on the
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endothelial surface, intimal infiltration, differentiation to proinflammatory macrophages, and
transformation to foam cells (Roth et al., 2007b, Giacona et al., 2004; Uehara et al., 2008; Pollreisz
et al., 2010). P. gingivalis bacteria were shown to invade and survive in monocytes and
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macrophages, a phenomenon that could contribute to the transmission of the bacteria from the site
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of periodontal infection to other inflamed sites (Jayaprakash et al., 2014). Except for P. gingivalis,
the most studied periodontal pathogen, other periodontal bacteria such as T. forsythia (Lee et al.,
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2014) and A. actinomycetemcomitans (Lakio et al., 2006) were able to induce generation of foam
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cells.
Normally, macrophages could efficiently control serum lipoprotein and cholesterol
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homeostasis by well-regulated taking up and releasing lipids to the bloodstream (Graham 2015).
Periodontal pathogens and their endotoxins can disturb lipid metabolism in macrophages. In
patients with chronic PD, P. gingivalis LPS is mainly associated with proatherogenic LDL (Kallio
et al., 2008). LPS-LDL assemblies efficiently stimulate LDL-dependent proinflammatory activation
(Kallio et al., 2013) and increase uptake of LDL cholesterol by macrophages (Qi et al., 2003;
Miyakawa et al., 2004). LPS down-regulate expression of liver X receptors (LXR), which are the
key regulators of lipid catabolism in macrophages, along with suppressing ABCA1 and SRB1
(Lakio et al., 2006). In macrophages, P. gingivalis-derived LPS up-regulate CD36, a scavenger
receptor for LDL and oxLDL (Li et al., 2013; Brown et al., 2015). As a consequence, LDL
cholesterol and oxLDL accumulate in macrophages since both lipid catabolism and efflux are
suppressed by LPS.

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P. gingivalis and its vesicles not only stimulate LDL binding to macrophages but also
induce macrophages to modify native LDL that in turn increases LDL atherogenicity (Bolton et al.,
1994; Niu et al., 2000). LDL from blood exposed to P. gingivalis exhibited increased protein
oxidation and generation of two apoB-100 N-terminal fragments (likely due to the proteolitic

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activity of gingipains) (Bengtsson et al., 2008). Treatement of oxLDL-stimulated macrophages and
foam cells with P. gingivalis-derived LPS led to the stimulation of NF-κB pathway and up-

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regulation of proinflammatory cytokines IL-1β and IL-12 (Lei et al., 2011). Similarly, Groeneweg

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et al. (2006) reported that prior exposure to oxLDL enhanced proinflammatory effects of LPS on
macrophages by activating production of TNF-α, IL-6, and IL-12 whereas expression of anti-

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inflammatory IL-10 was decreased. Therefore, P. gingivalis LPS and oxLDL may act
synergistically in induction/stimulation of proinflammatory phenotype in macrophages and foam
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cells.

7.4. Oxidative stress

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Oxidative stress occurs due to the excessive production of reactive oxygen species (ROS)
that cannot be fully scavenged and detoxified by antioxidant defenses. Oxidative stress leads to
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cytotoxicity. Production of peroxides and free radicals causes damage (frequently irreversible) of
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biomolecules such as proteins, nucleic acids, and lipids and inactivation of enzymes. Some ROS act
as cellular messengers in redox signaling. Indeed, oxidative stress could impair cell signaling (Siti
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et al., 2015, Perotta and Aquila 2015). Oxidative stress plays an important role in atherogenesis,
particularly by enhancing the oxidative modification of LDL (Peluso et al., 2012). ROS-mediated
formation of oxLDL is a prerequisite for cholesterol uptake by macrophages and the generation of
foam cells (Itabe 2009).
Periodontal pathogens could contribute to atherosclerosis-related oxidative stress through
promoting oxLDL formation (Kurita-Ochiai and Yamamoto 2014). In ApoEshl mice, infection with
A. actinomycetemcomitans led to elevated oxidative stress associated with lipid peroxidation,
increase in levels of oxidative stress markers (4-hydroxynoneal and 8-oxo-2′-deoxyguanosine), and
activation of phospholipase A2, myeloperoxidase and NADPH oxidase in the aorta (Jia et al.,
2013). P. gingivalis was shown to stimulate LDL oxidation in ApoE-deficient mice (Hashimoto et
al., 2006; Bengtsson et al., 2006).

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Indeed, PD patients display advanced lipid oxidation in the gingival crevicular fluid, blood, and
saliva (Akalin et al., 2007; Wei et al., 2010), and peroxidation level correlates with the severity of
periodontitis (Bastos et al., 2012).
Induction of the respiratory (or oxidative) burst could also contribute to oxidative stress. The

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respiratory burst is a host defense mechanism that typically occurs in immune cells such as
neutrophils, monocytes, and macrophages (called ‘professional phagocytes’) and refers to the fast

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ROS release upon contact with invaded microorganisms. In the respiratory burst, NADPH oxidases

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(NOXs) and myeloperoxidase are the major contributors to ROS production. NOXs are abundantly
present in phagocytic immune cells such as neutrophils, monocytes, and macrophages. These

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enzymes are needed to produce the oxidative burst in order to kill phagocytized microbes (Cross
and Segal 2004). MA
In PD, bacteremia induces the respiratory burst in phagocytes that is a normal reaction to
microbial invasion. Upon contact with neutrophils, P. gingivalis bacteria were shown to induce IL-8
and ROS production by neutrophils (Jayaprakash et al., 2015). Indeed, pathogen-induced
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respiratory burst may increase neutrophil-mediated oxidative damage of the surrounding tissue.
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Ramírez et al. (2014) observed increased serum levels of myeloperoxidase in subjects with
untreated chronic periodontitis suggesting for neutrophil activation. In GCF of PD patients taken
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from periodontitis sites, significantly higher levels of myeloperoxidase, lactoferrin, and glutathione
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peroxidase were detected compared with non-diseased sites indicating the local redox imbalance
and presence of oxidative stress (Wei et al., 2004). Rangé et al. (2014) reported potential
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involvement of periodontal bacteria especially T. forsythia in stimulation of neutrophils associated

with activation of myeloperoxidase and formation of neutrophil extracellular traps (NETs) within
hemorrhagic atherosclerotic carotid lesions. Interestingly, Arg-specific gingipain of P. gingivalis
was shown to increase ingestion of neutrophils by macrophages via cleavage of the antiphagocytic
signal (CD31) thereby protecting bacteria from cytotoxic effects of neutrophils (Guzik et al., 2007).
Non-phagocytic NOXs are expressed at low levels in vascular cells such as ECs and
vascular SMCs and produce low levels of ROS that are involved in intracellular signaling (Cave et
al., 2005). However, in metabolic disturbances (hyperlipidemia, hyperglycemia) or upon
stimulation by mitogenic factors and/or TGF, non-phagocytic NOXs become activated and
substantially enhance ROS generation (Li and Shah 2003).

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As mentioned above, periodontal pathogens are able to induce proinflammatory activation of ECs.
Inflammatory activation of ECs may lead to increase in NOX activity (Kuramitsu et al., 2001).
Antioxidant treatment of ECs exposed to P. gingivalis and inhibition of NOX resulted in significant
reduction of endothelial MCP-1 production (Choi et al., 2015) thereby limiting monocyte

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transmigration to the arterial intima.
Generally, periodontal pathogens are anaerobic microorganisms. However, in the presence

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of hemin or within the dental plaque, peridontal anaerobes are able to tolerate increased levels of

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oxygen (Diaz et al., 2002; Diaz and Rogers 2004). Hemin and iron are essential for P. gingivalis
viability. Iron is required for P. gingivalis growth. Iron could be acquiesced from transferrin with

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help of gingipains that degrade this protein (Brochu et al., 2001). Gingipain-mediated degradation
of ferritin is accompanied with generation of toxic hydroxyl radicals (Goulet et al., 2004).
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Hemin is needed for synthesis of the cytochrome b subunit of P. gingivalis fumarate
reductase, a key generator of metabolic energy for this pathogen. Due to periodontal bleeding, red
blood cells can extravasate to the GCF where they release hemoglobin. P. gingivalis could extract
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hemin from hemoglobin through gingipain-mediated mechanisms (Lewis 2010). Gingipains have
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hemagglutinin domains capable to capture hemin from hemoglobin in cooperation with surface
hemagglutinins (De Carlo et al., 1999). Hemin is then stored on the surface of P. gingivalis cells in
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the form of μ-oxo-heme protecting bacteria against peroxide stress and phagocyte-induced oxidative
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burst (Smalley et al., 1998). In fact, periodontal pathogens such as P. gingivalis could colonize
hemorrhagic areas of unstable lesions adjacent to the necrotic core. These regions are hypoxic and
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enriched with deposits of iron and hemin essential for P. gingivalis life (Chiu 1999). Findings
recently obtained by Rangé et al. (2014) suggest for a potential contribution of periodontal bacteria
and their endotoxins to intraplaque hemorrhages and plaque destabilization.

7.5. Plaque rupture

Plaque progression could lead to plaque stabilization or destabilization. Stable plaque is

typically characterized by a relatively small atheroma size, thick collagen-rich fibrous cap, which is
frequently mineralized, and low infiltration rate by inflammatory cells. Vulnerable plaque has a
large lipid pool, thin and poorly collagenated erosive fibrous cap (that could be disrupted),

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increased plaque inflammation, increased intralesional neovascularization, intraplaque

hemorrhages, and frequent thrombus formation (Finn et al., 2010).
Degradation of fibrous cap plays a key role in plaque rupture followed by intraluminal
exposure of the prothrombotic necrotic core and thrombogenesis (Virmani et al., 2006). Cap

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degradation is directed by matrix metalloproteinases (MMPs) that are produced by inflammatory
cells (mainly by macrophages) resided in or in the vicinity to the cap. P. gingivalis itself is able to

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degrade collagenous ECM in a gingipain-dependent manner, a common event that occurs during

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destruction of the periodontal connective tissue in PD (Baba et al., 2001; Al-Shibani and Windsor
2008). Periodontlal bacteria such as P. gingivalis, P. intermedia, and A. actinomycetemcomitans

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were shown to up-regulate production and matrix-degrading activity of various MMPs in different
cell types including ECs, monocytes, macrophages, and fibroblasts (Ding et al., 1995; Zhou and
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Windsor 2006; Madan et al., 2007; Tuomainen et al., 2008; Guan et al., 2008; Guan et al., 2009;
Zhou et al., 2012). LPS released by P. gingivalis was shown to act as a potent inducer of MMPs in
macrophages and fibroblasts (Kuo et al., 2012; Santos et al., 2013)
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Sato et al. (2009) found that exposure of fibroblasts to ultrasound-generated P. gingivalis bacterial
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extracts led to the down-regulation of expression of tissue inhibitor of metalloproteinases (TIMP)-1

and TIMP-2, i.e. natural suppressors of MMP activity. Proapoptotic and prooxidant activity of
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periodontal bacteria and their products against ECs, fibroblasts, and other cells also contributes to
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lesion instability.
In a murine model of MI-induced cardiac injury, DeLeon-Pennell et al. (2013) showed that
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LPS released by P. gingivalis increases cardiac inflammation through overexpression of MMP-9 in

macrophages involved in leftventricular post-MI tissue remodeling. Activation of MMP-9 causes
increased migration of monocytes to the site of injury where they differentiate to propinflammatory
M1 macrophages. Cardiac remodeling performed by macrophages may lead to leftventricular wall
thinning and rupture and increase risk of heart dysfunction and sudden death (DeLeon-Pennell et
al., 2014). MMP-9 controls leftventricular remodeling after MI through recruitment of monocytes
and regulating local inflammatory response by cleavage of excessive cytokines and chemokines
(DeLeon-Pennell et al., 2015). MMP-9 overproduction could be neutralized by overexpression of
TIMP-1 but P. gingivalis LPS inhibit TIMP-1 production (Muthukuru and Cutler, 2015). Indeed,
cardiac overproduction of MMP-9 impairs post-MI myocardial remodeling and may have

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deleterious effects on heart function. This explains a strong association between PD and increased
post-MI mortality.

7.6. Atherothrombosis

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Thrombosis is a normal physiological process in response to vascular injury required for

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preventing blood loss and maintaining vascular integrity. In atherosclerosis, thrombogenic

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mechanism plays a pathophysiological role. Atherothrombosis is induced in diseased arterial
regions especially in disrupted plaques. Large thrombi greatly decrease blood flow causing tissue

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hypoxia due to oxygen deprivation. Thromboembolic events are caused by freely circulating
thrombi and may lead to anoxia (i.e. complete prevention of oxygen supply) and infarction (i.e.
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tissue death) (Furie and Furie, 2008).
Normal arterial endothelium exhibits anti-coagulant and anti-adhesion properties.
Periodontal pathogens such as P. gingivalis could induce procoagulant properties in ECs through
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gingapain-dependent proteolitic degradation of endothelial thrombomodulin (Inomata et al., 2009).

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Thrombomodulin (also known as CD141) resides on the plasma membrane of ECs and converts
thrombin from a procoagulant enzyme to an anti-coagulant enzyme. Due to the lack of
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thrombomodulin activity, thrombin can bind to plateles and induce their aggregation and release of
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serotonin (Esmon et al., 1983). Dental plaque inhabitans such as P. gingivalis and S. sanguis
express on the surface the platelet aggregation-associated protein (PAAP) that stimulate platelet
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aggregation (Herzberg and Weyer 1998).

In ApoE-deficient mice, heat shock protein GroEL from F. nucleatum was shown to exhibit
procoagulant properties by inducing tissue factor (TF) and inhibiting a TF pathway inhibitor (TFPI)
activity (Lee et al., 2012). Similarly, Lalla et al. (2003) showed prothrombotic effects of P.
gingivalis infection able to stimulate aortic TF expression at early stages of atherogenesis. P.
gingivalis can also induce prothrombotic responces in human aortic SMCs by suppression of TF
pathway inhibitor (TFPI) (Roth et al., 2006; Roth et al., 2009). The procoagulant effects of P.
gingivalis on arterial SMCs may therefore play a role in the induction of atherothrombosis in
denuded areas of atherosclerotic vessels.
Thus, periodontal pathogens can contribute to the atherothrombosis. Emekli-Alturfan et al.
(2011) reported elevated plasma TF levels in PD patients with CAD compared to PD subjects

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without CAD and an inverse correlation between the salivary TF activity and the Community
Periodontal Index Treatment Needs (CPITN) scores. Indeed, this may suggest for a potential utility
of salivary TF as a marker of periodontitis and CAD. However, this finding should be
independently reevaluated in a larger group of patients because Emekli-Alturfan et al. (2011)

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measured TF activity only in 26 subjects affected with CAD + PD and in 26 cases of periodontitis.

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8. Conclusion

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Overall, epidemiological, interventional and functional studies show a significant

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association between peridontitis and CVD. Peridontal bacteria and their products could be involved
in all stages of atherogenesis. Furthermore, in vitro, animal, and clinical studies showed that
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periodontal pathogens positively influence all major proatherogenic mechanisms and hence
periodontal infection could play a contributory role in atherosclerotic pathogenesis. Observational
studies indicate that periodontal infection could increase CVD risk independently of other
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confounding factors. However, these observations cannot provide strong evidence that dental
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plaque bacteria may serve as a causal agent for CVD.

A recent consensus report of the Joint European Federation of Periodontology/American
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Academy of Periodontology (EFP/AAP) Workshop on Periodontitis and Systemic Diseases

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summarized epidemiological and interventional data on association between PD and atherosclerotic

CVD (Tonetti et al., 2013). In the report, strong and consistent evidence of longitudinal studies for
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the independent role of periodontitis as a risk CVD factor was highlighted. Moderate evidence in
favor of plausible effects of periodontal therapy on the improvement of clinical and surrogate
markers of endothelial dysfunction and decrease in systemic inflammation was stated (Tonetti et al.,
2013). However, no beneficial effect of the periodontal treatment on the lipid profile was observed.
To date, limited evidence on improving procoagulant status, blood pressure, and subclinical
atherosclerosis after PD treatment was provided (Tonetti et al., 2013).
No periodontal interventional studies on primary CVD prevention were conducted. A single
interventional trial on secondary CVD prevention was done but had serious limitations to make
reasonable conclusions. Indeed, well-constructed interventional studies involving large numbers of
subjects and long-term follow-up should be performed to evaluate the contribution of PD therapy on
prevention of well-defined clinical CVD outcomes.

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Conflict of interest disclosure

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The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.

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Acknowledgements

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This work was supported by the Russian Science Foundation (grant #14-15-00112), Russian
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Federation.
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Legend to Figure

Fig. 1. Proatherogenic and proinflammatory effects of periodontal pathogens and their products on
host vascular and blood-borne cells. Periodontitis progression leads to the destruction of

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surrounding gingival tissues and microvessels that opens a portal for dissiminating periodontal
bacteria (bacteremia) and their endotoxins (endotoxemia) in the circulation and option to influence

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atherogenesis.

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References

Aimetti M, Romano F, Nessi F. Microbiologic analysis of periodontal pockets and carotid

atheromatous plaques in advanced chronic periodontitis patients. J Periodontol.

PT
2007;78(9):1718-23.
Akalin FA, Baltacioğlu E, Alver A, Karabulut E. Lipid peroxidation levels and total oxidant status

RI
in serum, saliva andgingival crevicular fluid in patients with chronic periodontitis. J Clin

SC
Periodontol. 2007;34(7):558-65.
Almalki ZS, Guo JJ. Cardiovascular events and safety outcomes associated with azithromycin

NU
therapy: a meta-analysis of randomized controlled trials. Am Health Drug Benefits.
2014;7(6):318-28. MA
Al-Shibani N, Windsor LJ. Effects of Porphyromonas gingivalis on human gingival fibroblasts
from healthy and inflamed tissues. J Periodontal Res. 2008;43(4):465-70.
Amano A. Disruption of epithelial barrier and impairment of cellular function by Porphyromonas
D

gingivalis. Front Biosci. 2007;12:3965-74.

TE

Amar S, Gokce N, Morgan S, Loukideli M, Van Dyke TE, Vita JA. Periodontal disease is
associated with brachial artery endothelial dysfunction and systemic inflammation.
P

Arterioscler Thromb Vasc Biol. 2003;23(7):1245-9.

CE

Andrian E, Grenier D, Rouabhia M. Porphyromonas gingivalis-epithelial cell interactions in

periodontitis. J Dent Res. 2006;85(5):392-403.
AC

Ansai T, Yamamoto E, Awano S, Yu W, Turner AJ, Takehara T. Effects of periodontopathic

bacteria on the expression of endothelin-1 in gingival epithelial cells in adult periodontitis.
Clin Sci (Lond). 2002;103 Suppl 48:327S-331S.
Assinger A, Buchberger E, Laky M, Esfandeyari A, Brostjan C, Volf I. Role of MCP-1 in
cardiovascular disease: molecular mechanisms and clinical implications. Thromb Res.
2011;127(1):e20-6.
Baba A, Abe N, Kadowaki T, Nakanishi H, Ohishi M, Asao T, Yamamoto K. Arg-gingipain is
responsible for the degradation of cell adhesion molecules of human gingival fibroblasts and
their death induced by Porphyromonas gingivalis. Biol Chem. 2001;382(5):817-24.

30
 ACCEPTED MANUSCRIPT

Baba A, Kadowaki T, Asao T, Yamamoto K. Roles for Arg- and Lys-gingipains in the disruption of
cytokine responses and loss of viability of human endothelial cells by Porphyromonas
gingivalis infection. Biol Chem. 2002;383(7-8):1223-30.
Bahrani-Mougeot FK, Paster BJ, Coleman S, Ashar J, Barbuto S, Lockhart PB. Diverse and novel

PT
oral bacterial species in blood following dental procedures. J Clin Microbiol.
2008;46(6):2129-32.

RI
Bainbridge BW, Darveau RP. Porphyromonas gingivalis lipopolysaccharide: an unusual pattern

SC
recognition receptor ligand for the innate host defense system. Acta Odontol Scand.
2001;59(3):131-8.

NU
Bartruff JB, Yukna RA, Layman DL. Outer membrane vesicles from Porphyromonas gingivalis
affect the growth and function of cultured human gingival fibroblasts and umbilical vein
MA
endothelial cells. J Periodontol. 2005;76(6):972-9.
Bastos AS, Graves DT, Loureiro AP, Rossa Júnior C, Abdalla DS, Faulin Tdo E, Olsen Câmara N,
Andriankaja OM, Orrico SR. Lipid peroxidation is associated with the severity of periodontal
D

disease and local inflammatory markers in patients with type 2 diabetes. J Clin Endocrinol
TE

Metab. 2012;97(8):E1353-62.
Beck JD, Couper DJ, Falkner KL, Graham SP, Grossi SG, Gunsolley JC, Madden T, Maupome G,
P

Offenbacher S, Stewart DD, Trevisan M, Van Dyke TE, Genco RJ. The Periodontitis and
CE

Vascular Events (PAVE) pilot study: adverse events. J Periodontol. 2008;79(1):90-6.

Beck JD, Elter JR, Heiss G, Couper D, Mauriello SM, Offenbacher S. Relationship of periodontal
AC

disease to carotid artery intima-media wall thickness: the atherosclerosis risk in communities
(ARIC) study. Arterioscler Thromb Vasc Biol. 2001;21(11):1816-22.
Beck JD, Offenbacher S. Relationships among clinical measures of periodontal disease and their
associations with systemic markers. Ann Periodontol. 2002;7(1):79-89.
Behle JH, Sedaghatfar MH, Demmer RT, Wolf DL, Celenti R, Kebschull M, Belusko PB, Herrera-
Abreu M, Lalla E, Papapanou PN. Heterogeneity of systemic inflammatory responses to
periodontal therapy. J Clin Periodontol. 2009;36(4):287-94.
Bélanger M, Kozarov E, Song H, Whitlock J, Progulske-Fox A. Both the unique and repeat regions
of the Porphyromonas gingivalis hemagglutin A are involved in adhesion and invasion of host
cells. Anaerobe. 2012;18(1):128-34.

31
 ACCEPTED MANUSCRIPT

Bengtsson T, Karlsson H, Gunnarsson P, Skoglund C, Elison C, Leanderson P, Lindahl M. The

periodontal pathogen Porphyromonas gingivalis cleaves apoB-100 and increases the
expression of apoM in LDL in whole blood leading to cell proliferation. J Intern Med.
2008;263(5):558-71.

PT
Bhagat K, Moss R, Collier J, Vallance P. Endothelial "stunning" following a brief exposure to
endotoxin: a mechanism to link infection and infarction? Cardiovasc Res. 1996;32(5):822-9.

RI
Binder CJ. Natural IgM antibodies against oxidation-specific epitopes. J Clin Immunol. 2010;30

SC
Suppl 1:S56-60.
Blaizot A, Vergnes JN, Nuwwareh S, Amar J, Sixou M. Periodontal diseases and cardiovascular

NU
events: meta-analysis of observational studies. Int Dent J. 2009;59(4):197-209.
Bokhari SA, Khan AA, Butt AK, Hanif M, Izhar M, Tatakis DN, Ashfaq M. Periodontitis in
MA
coronary heart disease patients: strong association between bleeding on probing and systemic
biomarkers. J Clin Periodontol. 2014;41(11):1048-54.
Bolton EJ, Jessup W, Stanley KK, Dean RT. Enhanced LDL oxidation by murine macrophage
D

foam cells and their failure to secrete nitric oxide. Atherosclerosis. 1994;106(2):213-23.
TE

Brochu V, Grenier D, Nakayama K, Mayrand D. Acquisition of iron from human transferrin by

Porphyromonas gingivalis: a role for Arg- and Lys-gingipain activities. Oral Microbiol
P

Immunol. 2001;16(2):79-87.
CE

Brown PM, Kennedy DJ, Morton RE, Febbraio M. CD36/SR-B2-TLR2 dependent pathways
enhance Porphyromonas gingivalis mediated atherosclerosis in the Ldlr KO mouse model.
AC

PLoS One. 2015;10(5):e0125126.

Cairo F, Castellani S, Gori AM, Nieri M, Baldelli G, Abbate R, Pini-Prato GP. Severe periodontitis
in young adults is associated with sub-clinical atherosclerosis. J Clin Periodontol.
2008;35(6):465-72.
Cairo F, Gaeta C, Dorigo W, Oggioni MR, Pratesi C, Pini Prato GP, Pozzi G. Periodontal pathogens
in atheromatous plaques. A controlled clinical and laboratory trial. J Periodontal Res.
2004;39(6):442-6.
Cave A, Grieve D, Johar S, Zhang M, Shah AM. NADPH oxidase-derived reactive oxygen species
in cardiac pathophysiology. Philos Trans R Soc Lond B Biol Sci. 2005;360(1464):2327-34.
Chistiakov DA, Orekhov AN, Bobryshev YV. Vascular smooth muscle cell in atherosclerosis. Acta
Physiol (Oxf). 2015;214(1):33-50.

32
 ACCEPTED MANUSCRIPT

Chiu B. Multiple infections in carotid atherosclerotic plaques. Am Heart J. 1999;138(5 Pt 2):S534-

6.
Choi JI, Chung SW, Kang HS, Rhim BY, Kim SJ, Kim SJ. Establishment of Porphyromonas
gingivalis heat-shock-protein-specific T-cell lines from atherosclerosis patients. J Dent Res.

PT
2002;81(5):344-8.
Chou MY, Fogelstrand L, Hartvigsen K, Hansen LF, Woelkers D, Shaw PX, Choi J, Perkmann T,

RI
Bäckhed F, Miller YI, Hörkkö S, Corr M, Witztum JL, Binder CJ. Oxidation-specific epitopes

SC
are dominant targets of innate natural antibodies in mice and humans. J Clin Invest.
2009;119(5):1335-49.

NU
Chou SH, Tung YC, Lin YS, Wu LS, Lin CP, Liou EJ, Chang CJ, Kung S, Chu PH. Major adverse
cardiovascular events in treated periodontitis: A population-based follow-up study from
MA
Taiwan. PLoS One. 2015;10(6):e0130807.
Chou HH, Yumoto H, Davey M, Takahashi Y, Miyamoto T, Gibson FC 3rd, Genco CA.
Porphyromonas gingivalis fimbria-dependent activation of inflammatory genes in human
D

aortic endothelial cells. Infect Immun. 2005;73(9):5367-78.

TE

Correa FO, Gonçalves D, Figueredo CM, Bastos AS, Gustafsson A, Orrico SR. Effect of
periodontal treatment on metabolic control, systemic inflammation and cytokines in patients
P

with type 2 diabetes. J Clin Periodontol. 2010;37(1):53-8.

CE

Costa FO, Cota LO, Lages EJ, Cyrino RM, Oliveira AM, Oliveira PA, Cortelli JR. Associations of
duration of smoking cessation and cumulative smoking exposure with periodontitis. J Oral
AC

Sci. 2013;55(3):245-53.
Crasta K, Daly CG, Mitchell D, Curtis B, Stewart D, Heitz-Mayfield LJ. Bacteraemia due to dental
flossing. J Clin Periodontol. 2009;36(4):323-32.
Cross AR, Segal AW. The NADPH oxidase of professional phagocytes--prototype of the NOX
electron transport chain systems. Biochim Biophys Acta. 2004;1657(1):1-22.
D'Aiuto F, Nibali L, Parkar M, Suvan J, Tonetti MS. Short-term effects of intensive periodontal
therapy on serum inflammatory markers and cholesterol. J Dent Res. 2005;84(3):269-73.
D'Aiuto F, Parkar M, Andreou G, Suvan J, Brett PM, Ready D, Tonetti MS. Periodontitis and
systemic inflammation: control of the local infection is associated with a reduction in serum
inflammatory markers. J Dent Res. 2004;83(2):156-60.

33
 ACCEPTED MANUSCRIPT

de Berrazueta JR, Guerra-Ruiz A, García-Unzueta MT, Toca GM, Laso RS, de Adana MS, Martín
MA, Cobo M, Llorca J. Endothelial dysfunction, measured by reactive hyperaemia using
strain-gauge plethysmography, is an independent predictor of adverse outcome in heart
failure. Eur J Heart Fail. 2010;12(5):477-83.

PT
DeCarlo AA, Paramaesvaran M, Yun PL, Collyer C, Hunter N. Porphyrin-mediated binding to
hemoglobin by the HA2 domain of cysteine proteinases (gingipains) and hemagglutinins from

RI
the periodontal pathogen Porphyromonas gingivalis. J Bacteriol. 1999;181(12):3784-91.

SC
Deleon-Pennell KY, Altara R, Yabluchanskiy A, Modesti A, Lindsey ML. The circular relationship
between matrix metalloproteinase-9 and inflammation following myocardial infarction.

NU
IUBMB Life. 2015;67(8):611-8.
Deleon-Pennell KY, de Castro Brás LE, Lindsey ML. Circulating Porphyromonas gingivalis
MA
lipopolysaccharide resets cardiac homeostasis in mice through a matrix metalloproteinase-9-
dependent mechanism. Physiol Rep. 2013;1(5):e00079.
DeLeon-Pennell KY, de Castro Brás LE, Iyer RP, Bratton DR, Jin YF, Ripplinger CM, Lindsey
D

ML. P. gingivalis lipopolysaccharide intensifies inflammation post-myocardial infarction

TE

through matrix metalloproteinase-9. J Mol Cell Cardiol. 2014;76:218-26.

Demmer RT, Desvarieux M. Periodontal infections and cardiovascular disease: the heart of the
P

matter. J Am Dent Assoc. 2006;137 Suppl:14S-20S.

CE

Demmer RT, Trinquart L, Zuk A, Fu BC, Blomkvist J, Michalowicz BS, Ravaud P, Desvarieux M.
The influence of anti-infective periodontal treatment on C-reactive protein: asystematic
AC

review and meta-analysis of randomized controlled trials. PLoS One. 2013;8(10):e77441.

Diaz PI, Rogers AH. The effect of oxygen on the growth and physiology of Porphyromonas
gingivalis. Oral Microbiol Immunol. 2004;19(2):88-94.
Diaz PI, Zilm PS, Rogers AH. Fusobacterium nucleatum supports the growth of Porphyromonas
gingivalis in oxygenated and carbon-dioxide-depleted environments. Microbiology.
2002;148(Pt 2):467-72.
Dietmann A, Millonig A, Combes V, Couraud PO, Kachlany SC, Grau GE. Effects of
Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells. Microb Pathog.
2013;61-62:43-50.

34
 ACCEPTED MANUSCRIPT

Dietrich T, Jimenez M, Krall Kaye EA, Vokonas PS, Garcia RI. Age-dependent associations
between chronic periodontitis/edentulism and risk of coronary heart disease. Circulation.
2008;117(13):1668-74.
Ding Y, Uitto VJ, Firth J, Salo T, Haapasalo M, Konttinen YT, Sorsa T. Modulation of host matrix

PT
metalloproteinases by bacterial virulence factors relevant in human periodontal diseases. Oral
Dis. 1995;1(4):279-86.

RI
Dorn BR, Dunn WA Jr, Progulske-Fox A. Invasion of human coronary artery cells by periodontal

SC
pathogens. Infect Immun. 1999;67(11):5792-8.
Dorn BR, Dunn WA Jr, Progulske-Fox A. Porphyromonas gingivalis traffics to autophagosomes in

NU
human coronary artery endothelial cells. Infect Immun. 2001;69(9):5698-708.
Dorn BR, Harris LJ, Wujick CT, Vertucci FJ, Progulske-Fox A. Invasion of vascular cells in vitro
MA
by Porphyromonas endodontalis. Int Endod J. 2002;35(4):366-71.
Dye BA, Herrera-Abreu M, Lerche-Sehm J, Vlachojannis C, Pikdoken L, Pretzl B, Schwartz A,
Papapanou PN. Serum antibodies to periodontal bacteria as diagnostic markers of
D

periodontitis. J Periodontol. 2009;80(4):634-47.

TE

Elter JR, Hinderliter AL, Offenbacher S, Beck JD, Caughey M, Brodala N, Madianos PN. The
effects of periodontal therapy on vascular endothelial function: a pilot trial. Am Heart J.
P

2006;151(1):47.
CE

Emekli-Alturfan E, Basar I, Malali E, Elemek E, Oktay S, Ayan F, Emekli N, Noyan U. Plasma

tissue factor levels and salivary tissue factor activities ofperiodontitis patients with and
AC

without cardiovascular disease. Pathophysiol Haemost Thromb. 2010;37(2-4):77-81.

Erridge C, Spickett CM, Webb DJ. Non-enterobacterial endotoxins stimulate human coronary artery
but not venous endothelial cell activation via Toll-like receptor 2. Cardiovasc Res.
2007;73(1):181-9.
Esmon NL, Carroll RC, Esmon CT. Thrombomodulin blocks the ability of thrombin to activate
platelets. J Biol Chem. 1983;258(20):12238-42.
Felcht M, Luck R, Schering A, Seidel P, Srivastava K, Hu J, Bartol A, Kienast Y, Vettel C, Loos
EK, Kutschera S, Bartels S, Appak S, Besemfelder E, Terhardt D, Chavakis E, Wieland T,
Klein C, Thomas M, Uemura A, Goerdt S, Augustin HG. Angiopoietin-2 differentially
regulates angiogenesis through TIE2 and integrin signaling. J Clin Invest. 2012;122(6):1991-
2005.

35
 ACCEPTED MANUSCRIPT

Finn AV, Nakano M, Narula J, Kolodgie FD, Virmani R. Concept of vulnerable/unstable plaque.
Arterioscler Thromb Vasc Biol. 2010;30(7):1282-92.
Ford PJ, Gemmell E, Hamlet SM, Hasan A, Walker PJ, West MJ, Cullinan MP, Seymour GJ.
Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of

PT
pathogens in atherosclerosis. Oral Microbiol Immunol. 2005a;20(5):296-302.
Ford P, Gemmell E, Walker P, West M, Cullinan M, Seymour G. Characterization of heat shock

RI
protein-specific T cells in atherosclerosis. Clin Diagn Lab Immunol. 2005;12(2):259-67.

SC
Froude J, Gibofsky A, Buskirk DR, Khanna A, Zabriskie JB. Cross-reactivity between
streptococcus and human tissue: a model of molecular mimicry and autoimmunity. Curr Top

NU
Microbiol Immunol. 1989;145:5-26.
Furie B, Furie BC. Mechanisms of thrombus formation. N Engl J Med. 2008;359(9):938-49.
MA
Gaetti-Jardim E Jr, Marcelino SL, Feitosa AC, Romito GA, Avila-Campos MJ. Quantitative
detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries. J
Med Microbiol. 2009;58(Pt 12):1568-75.
D

Geerts SO, Nys M, De MP, Charpentier J, Albert A, Legrand V, Rompen EH. Systemic release of
TE

endotoxins induced by gentle mastication: association with periodontitis severity. J

Periodontol. 2002;73(1):73-8.
P

Georges JL, Rupprecht HJ, Blankenberg S, Poirier O, Bickel C, Hafner G, Nicaud V, Meyer J,
CE

Cambien F, Tiret L; AtheroGene Group. Impact of pathogen burden in patients with coronary
artery disease in relation to systemic inflammation and variation in genes encoding cytokines.
AC

Am J Cardiol. 2003;92(5):515-21.
Ghorbani B, Holmstrup P, Edvinsson L, Kristiansen KA, Sheykhzade M. LPS from Porphyromonas
gingivalis increases the sensitivity of contractile response mediated by endothelin-B (ET(B))
receptors in cultured endothelium-intact rat coronary arteries. Vascul Pharmacol. 2010;53(5-
6):250-7.
Giacona MB, Papapanou PN, Lamster IB, Rong LL, D'Agati VD, Schmidt AM, Lalla E.
Porphyromonas gingivalis induces its uptake by human macrophages and promotes foam cell
formation in vitro. FEMS Microbiol Lett. 2004;241(1):95-101.
Gibbons RJ. Bacterial adhesion to oral tissues: a model for infectious diseases. J Dent Res.
1989;68(5):750-60.

36
 ACCEPTED MANUSCRIPT

Gibson FC 3rd, Hong C, Chou HH, Yumoto H, Chen J, Lien E, Wong J, Genco CA. Innate immune
recognition of invasive bacteria accelerates atherosclerosis in apolipoprotein E-deficient mice.
Circulation. 2004;109(22):2801-6.
Gmür R, Wyss C, Xue Y, Thurnheer T, Guggenheim B. Gingival crevice microbiota from Chinese

PT
patients with gingivitis or necrotizing ulcerative gingivitis. Eur J Oral Sci. 2004;112(1):33-41.
Goulet V, Britigan B, Nakayama K, Grenier D. Cleavage of human transferrin by Porphyromonas

RI
gingivalis gingipains promotes growth and formation of hydroxyl radicals. Infect Immun.

SC
2004;72(8):4351-6.
Graham A. Mitochondrial regulation of macrophage cholesterol homeostasis. Free Radic Biol Med.

NU
2015 Sep 28. pii: S0891-5849(15)00527-4.
Grayston JT, Kronmal RA, Jackson LA, Parisi AF, Muhlestein JB, Cohen JD, Rogers WJ, Crouse
MA
JR, Borrowdale SL, Schron E, Knirsch C; ACES Investigators. Azithromycin for the
secondary prevention of coronary events. N Engl J Med. 2005;352(16):1637-45.
Groeneweg M, Kanters E, Vergouwe MN, Duerink H, Kraal G, Hofker MH, de Winther MP.
D

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior

TE

exposure to oxLDL. J Lipid Res. 2006;47(10):2259-67.

Guan SM, Shu L, Fu SM, Liu B, Xu XL, Wu JZ. Prevotella intermedia induces matrix
P

metalloproteinase-9 expression in human periodontal ligament cells. FEMS Microbiol Lett.

CE

2008;283(1):47-53.
Guan SM, Shu L, Fu SM, Liu B, Xu XL, Wu JZ. Prevotella intermedia upregulates MMP-1 and
AC

MMP-8 expression in human periodontal ligament cells. FEMS Microbiol Lett.

2009;299(2):214-22.
Gupta G. Gingival crevicular fluid as a periodontal diagnostic indicator-II: Inflammatory mediators,
host-response modifiers and chair side diagnostic aids. J Med Life. 2013;6(1):7-13.
Gurav AN. Endothelial dysfunction: clinical implications in cardiovascular disease and Therapeutic
Approaches. Eur J Clin Invest. 2014;44(10):1000-9. doi: 10.1111/eci.12322.
Hajishengallis G, Sharma A, Russell MW, Genco RJ. Interactions of oral pathogens with toll-like
receptors: possible role in atherosclerosis. Ann Periodontol. 2002;7(1):72-8.
Guzik K, Bzowska M, Smagur J, Krupa O, Sieprawska M, Travis J, Potempa J. A new insight into
phagocytosis of apoptotic cells: proteolytic enzymes divert the recognition and clearance of
polymorphonuclear leukocytes bymacrophages. Cell Death Differ. 2007;14(1):171-82.

37
 ACCEPTED MANUSCRIPT

Hajishengallis G, Sojar H, Genco RJ, DeNardin E. Intracellular signaling and cytokine induction
upon interactions of Porphyromonas gingivalis fimbriae with pattern-recognition receptors.
Immunol Invest. 2004;33(2):157-72.
Hajishengallis G, Wang M, Harokopakis E, Triantafilou M, Triantafilou K. Porphyromonas

PT
gingivalis fimbriae proactively modulate beta2 integrin adhesive activity and promote binding
to and internalization by macrophages. Infect Immun. 2006;74(10):5658-66.

RI
Hamburg NM, Benjamin EJ. Assessment of endothelial function using digital pulse amplitude

SC
tonometry. Trends Cardiovasc Med. 2009;19(1):6-11.
Harokopakis E, Albzreh MH, Martin MH, Hajishengallis G. TLR2 transmodulates monocyte

NU
adhesion and transmigration via Rac1- and PI3K-mediated inside-out signaling in response to
Porphyromonas gingivalis fimbriae. J Immunol. 2006;176(12):7645-56.
MA
Hashimoto M, Kadowaki T, Tsukuba T, Yamamoto K. Selective proteolysis of apolipoprotein B-
100 by Arg-gingipain mediates atherosclerosis progression accelerated by bacterial exposure.
J Biochem. 2006;140(5):713-23.
D

Hashizume T, Kurita-Ochiai T, Yamamoto M. Porphyromonas gingivalis stimulates monocyte

TE

adhesion to human umbilical vein endothelial cells. FEMS Immunol Med Microbiol.
2011;62(1):57-65.
P

Hayashi J, Saito I, Ishikawa I, Miyasaka N. Effects of cytokines and periodontopathic bacteria on

CE

the leukocyte function-associated antigen 1/intercellular adhesion molecule 1 pathway in

gingival fibroblasts in adult periodontitis. Infect Immun. 1994;62(12):5205-12.
AC

Heitzer T, Baldus S, von Kodolitsch Y, Rudolph V, Meinertz T. Systemic endothelial dysfunction

as an early predictor of adverse outcome in heart failure. Arterioscler Thromb Vasc Biol.
2005;25(6):1174-9.
Henschel M, Keenan AV. Insufficient evidence of effect of periodontal treatment on prevention or
management of cardiovascular disease. Evid Based Dent. 2015;16(1):17-8.
Haraszthy VI, Zambon JJ, Trevisan M, Zeid M, Genco RJ. Identification of periodontal pathogens
in atheromatous plaques. J Periodontol. 2000;71(10):1554-60.
Herzberg MC, Weyer MW. Dental plaque, platelets, and cardiovascular diseases. Ann Periodontol.
1998;3(1):151-60.

38
 ACCEPTED MANUSCRIPT

Higashi Y, Goto C, Jitsuiki D, Umemura T, Nishioka K, Hidaka T, Takemoto H, Nakamura S, Soga

J, Chayama K, Yoshizumi M, Taguchi A. Periodontal infection is associated with endothelial
dysfunction in healthy subjects and hypertensive patients. Hypertension. 2008;51(2):446-53.
Hilgendorf I, Swirski FK, Robbins CS. Monocyte fate in atherosclerosis. Arterioscler Thromb Vasc

PT
Biol. 2015;35(2):272-9.
Hirschfeld J, Kawai T. Oral inflammation and bacteremia: implications for chronic and acute

RI
systemic diseases involving major organs. Cardiovasc Hematol Disord Drug Targets.

SC
2015;15(1):70-84.
Ho MH, Chen CH, Goodwin JS, Wang BY, Xie H. Functional advantages of Porphyromonas

NU
gingivalis vesicles. PLoS One. 2015;10(4):e0123448.
Ho YS, Lai MT, Liu SJ, Lin CT, Naruishi K, Takashiba S, Chou HH. Porphyromonas gingivalis
MA
fimbriae-dependent interleukin-6 autocrine regulation by increase of gp130 in endothelial
cells. J Periodontal Res. 2009;44(4):550-6.
Hokamura K, Inaba H, Nakano K, Nomura R, Yoshioka H, Taniguchi K, Ooshima T, Wada K,
D

Amano A, Umemura K. Molecular analysis of aortic intimal hyperplasia caused by

TE

Porphyromonas gingivalis infection in mice with endothelial damage. J Periodontal Res.

2010;45(3):337-44.
P

Hokamura K, Umemura K. Roles of oral bacteria in cardiovascular diseases--from molecular

CE

mechanisms to clinical cases: Porphyromonas gingivalis is the important role of intimal

hyperplasia in the aorta. J Pharmacol Sci. 2010;113(2):110-4.
AC

Horliana AC, Chambrone L, Foz AM, Artese HP, Rabelo Mde S, Pannuti CM, Romito GA.
Dissemination of periodontal pathogens in the bloodstream after periodontal procedures: a
systematic review. PLoS One. 2014;9(5):e98271.
Houcken W, Teeuw WJ, Bizzarro S, Alvarez Rodriguez E, Mulders TA, van den Born BJ, Loos
BG. Arterial stiffness in periodontitis patients and controls. J Hum Hypertens. 2016;30(1):24-
9.
Hujoel PP, Drangsholt M, Spiekerman C, DeRouen TA. Periodontal disease and coronary heart
disease risk. JAMA. 2000;284(11):1406-10.
Hujoel PP, Drangsholt M, Spiekerman C, DeRouen TA. Periodontitis-systemic disease associations
in the presence of smoking - causal or coincidental? Periodontol 2000. 2002;30:51-60.

39
 ACCEPTED MANUSCRIPT

Hyvärinen K, Tuomainen AM, Laitinen S, Bykov IL, Törmäkangas L, Lindros K, Käkelä R,

Alfthan G, Salminen I, Jauhiainen M, Kovanen PT, Leinonen M, Saikku P, Pussinen PJ.
Chlamydial and periodontal pathogens induce hepatic inflammation and fatty acid imbalance
in apolipoprotein E-deficient mice. Infect Immun. 2009;77(8):3442-9.

PT
Ieven MM, Hoymans VY. Involvement of Chlamydia pneumoniae in atherosclerosis: more
evidence for lack of evidence. J Clin Microbiol. 2005;43(1):19-24.

RI
Inaba H, Hokamura K, Nakano K, Nomura R, Katayama K, Nakajima A, Yoshioka H, Taniguchi K,

SC
Kamisaki Y, Ooshima T, Umemura K, Murad F, Wada K, Amano A. Upregulation of S100
calcium-binding protein A9 is required for induction of smooth muscle cell proliferation by a

NU
periodontal pathogen. FEBS Lett. 2009;583(1):128-34.
Inagaki S, Ishihara K, Yasaki Y, Yamada S, Okuda K. Antibody responses of periodontitis patients
MA
to gingipains of Porphyromonas gingivalis. J Periodontol. 2003;74(10):1432-9.
Inomata M, Into T, Ishihara Y, Nakashima M, Noguchi T, Matsushita K. Arginine-specific
gingipain A from Porphyromonas gingivalis induces Weibel-Palade body exocytosis and
D

enhanced activation of vascular endothelial cells through protease-activated receptors.

TE

Microbes Infect. 2007;9(12-13):1500-6.

Inomata M, Ishihara Y, Matsuyama T, Imamura T, Maruyama I, Noguchi T, Matsushita K.
P

Degradation of vascular endothelial thrombomodulin by arginine- and lysine-specific cysteine

CE

proteases from Porphyromonas gingivalis. J Periodontol. 2009; 80(9):1511-17.

Itabe H. Oxidative modification of LDL: its pathological role in atherosclerosis. Clin Rev Allergy
AC

Immunol. 2009;37(1):4-11.
Ito HO. Infective endocarditis and dental procedures: evidence, pathogenesis, and prevention. J
Med Invest. 2006;53(3-4):189-98.
Jayaprakash K, Demirel I, Khalaf H, Bengtsson T. The role of phagocytosis, oxidative burst and
neutrophil extracellular traps in the interaction between neutrophils and the periodontal
pathogen Porphyromonas gingivalis. Mol Oral Microbiol. 2015;30(5):361-75.
Jayaprakash K, Khalaf H, Bengtsson T. Gingipains from Porphyromonas gingivalis play a
significant role ininduction and regulation of CXCL8 in THP-1 cells. BMC Microbiol.
2014;14:193.

40
 ACCEPTED MANUSCRIPT

Jeong E, Kim K, Kim JH, Cha GS, Kim SJ, Kang HS, Choi J. Porphyromonas gingivalis HSP60
peptides have distinct roles in the development of atherosclerosis. Mol Immunol.
2015;63(2):489-96.
Jia R, Kurita-Ochiai T, Oguchi S, Yamamoto M. Periodontal pathogen accelerates lipid

PT
peroxidation and atherosclerosis. J Dent Res. 2013;92(3):247-52.
Jia Y, Guo B, Yang W, Zhao Q, Jia W, Wu Y. Rho kinase mediates Porphyromonas gingivalis

RI
outer membrane vesicle-induced suppression of endothelial nitric oxide synthase through

SC
ERK1/2 and p38 MAPK. Arch Oral Biol. 2015;60(3):488-95.
Jimenez M, Krall EA, Garcia RI, Vokonas PS, Dietrich T. Periodontitis and incidence of

NU
cerebrovascular disease in men. Ann Neurol. 2009;66(4):505-12.
Jotwani R, Palucka AK, Al-Quotub M, Nouri-Shirazi M, Kim J, Bell D, Banchereau J, Cutler CW.
MA
Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis:
in situ, in vivo, and in vitro studies. J Immunol. 2001;167(8):4693-700.
Kapellas K, Maple-Brown LJ, Jamieson LM, Do LG, O'Dea K, Brown A, Cai TY, Anstey NM,
D

Sullivan DR, Wang H, Celermajer DS, Slade GD, Skilton MR. Effect of periodontal therapy
TE

on arterial structure and function among aboriginal australians: a randomized, controlled trial.
Hypertension. 2014;64(4):702-8.
P

Kallio KA, Buhlin K, Jauhiainen M, Keva R, Tuomainen AM, Klinge B, Gustafsson A, Pussinen
CE

PJ. Lipopolysaccharide associates with pro-atherogenic lipoproteins inperiodontitis patients.

Innate Immun. 2008;14(4):247-53.
AC

Kallio KA, Hyvärinen K, Kovanen PT, Jauhiainen M, Pussinen PJ. Very low density lipoproteins
derived from periodontitis patients facilitate macrophage activation via lipopolysaccharide
function. Metabolism. 2013;62(5):661-8.
Kebschull M, Demmer RT, Papapanou PN. "Gum bug, leave my heart alone!" -epidemiologic and
mechanistic evidence linking periodontal infections and atherosclerosis. J Dent Res.
2010;89(9):879-902.
Khlgatian M, Nassar H, Chou HH, Gibson FC 3rd, Genco CA. Fimbria-dependent activation of cell
adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells. Infect
Immun. 2002;70(1):257-67.

41
 ACCEPTED MANUSCRIPT

Kim SR, Jeon HJ, Park HJ, Kim MK, Choi WS, Jang HO, Bae SK, Jeong CH, Bae MK.
Glycyrrhetinic acid inhibits Porphyromonas gingivalis lipopolysaccharide-induced vascular
permeability via the suppression of interleukin-8. Inflamm Res. 2013;62(2):145-54.
Kobayashi N, Ishihara K, Sugihara N, Kusumoto M, Yakushiji M, Okuda K. Colonization pattern

PT
of periodontal bacteria in Japanese children and their mothers. J Periodontal Res.
2008;43(2):156-61.

RI
Koizumi Y, Kurita-Ochiai T, Oguchi S, Yamamoto M. Nasal immunization with Porphyromonas

SC
gingivalis outer membrane protein decreases P. gingivalis-induced atherosclerosis and
inflammation in spontaneously hyperlipidemic mice. Infect Immun. 2008;76(7):2958-65.

NU
Korshunov VA, Schwartz SM, Berk BC. Vascular remodeling: hemodynamic and biochemical
mechanismsunderlying Glagov's phenomenon. Arterioscler Thromb Vasc Biol.
MA
2007;27(8):1722-8.
Kozarov EV, Dorn BR, Shelburne CE, Dunn WA Jr, Progulske-Fox A. Human atherosclerotic
plaque contains viable invasive Actinobacillus actinomycetemcomitans and Porphyromonas
D

gingivalis. Arterioscler Thromb Vasc Biol. 2005;25(3):e17-8.

TE

Kozarov E, Sweier D, Shelburne C, Progulske-Fox A, Lopatin D. Detection of bacterial DNA in

atheromatous plaques by quantitative PCR. Microbes Infect. 2006;8(3):687-93.
P

Kuo PJ, Tu HP, Chin YT, Lu SH, Chiang CY, Chen RY, Fu E. Cyclosporine-A inhibits MMP-2
CE

and -9 activities in the presence of Porphyromonas gingivalis lipopolysaccharide: an

experiment in human gingival fibroblast and U937 macrophage co-culture. J Periodontal Res.
AC

2012;47(4):431-8.
Kuramitsu HK, Qi M, Kang IC, Chen W. Role for periodontal bacteria in cardiovascular diseases.
Ann Periodontol. 2001;6(1):41-7.
Kurita-Ochiai T, Yamamoto M. Periodontal pathogens and atherosclerosis: implications of
inflammation and oxidative modification of LDL. Biomed Res Int. 2014;2014:595981.
Lacolley P, Regnault V, Nicoletti A, Li Z, Michel JB. The vascular smooth muscle cell in arterial
pathology: a cell that can take on multiple roles. Cardiovasc Res. 2012;95(2):194-204.
Lakio L, Lehto M, Tuomainen AM, Jauhiainen M, Malle E, Asikainen S, Pussinen PJ. Pro-
atherogenic properties of lipopolysaccharide from the periodontal pathogen Actinobacillus
actinomycetemcomitans. J Endotoxin Res. 2006;12(1):57-64.

42
 ACCEPTED MANUSCRIPT

Lalla E, Lamster IB, Hofmann MA, Bucciarelli L, Jerud AP, Tucker S, Lu Y, Papapanou PN,
Schmidt AM. Oral infection with a periodontal pathogen accelerates early atherosclerosis in
apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol. 2003;23(8):1405-11.
Lee HR, Jun HK, Choi BK. Tannerella forsythia BspA increases the risk factors for atherosclerosis

PT
in ApoE(-/-) mice. Oral Dis. 2014;20(8):803-8.
Lei L, Li H, Yan F, Li Y, Xiao Y. Porphyromonas gingivalis lipopolysaccharide alters

RI
atherosclerotic-related gene expression in oxidized low-density-lipoprotein-induced

SC
macrophages and foam cells. J Periodontal Res. 2011;46(4):427-37.
Leivadaros E, van der Velden U, Bizzarro S, ten Heggeler JM, Gerdes VE, Hoek FJ, Nagy TO,

NU
Scholma J, Bakker SJ, Gans RO, ten Cate H, Loos BG. A pilot study into measurements of
markers of atherosclerosis inperiodontitis. J Periodontol. 2005;76(1):121-8.
MA
Lewis JP. Metal uptake in host-pathogen interactions: role of iron in Porphyromonas gingivalis
interactions with host organisms. Periodontol 2000. 2010;52(1):94-116.
Lewis K. Multidrug tolerance of biofilms and persister cells. Curr Top Microbiol Immunol.
D

2008;322:107-31.
TE

Li C, Lv Z, Shi Z, Zhu Y, Wu Y, Li L, Iheozor-Ejiofor Z. Periodontal therapy for the management

of cardiovascular disease in patients with chronic periodontitis. Cochrane Database Syst Rev.
P

2014;8:CD009197.
CE

Li L, Messas E, Batista EL Jr, Levine RA, Amar S. Porphyromonas gingivalis infection accelerates
the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model.
AC

Circulation. 2002;105(7):861-7.
Li L, Michel R, Cohen J, Decarlo A, Kozarov E. Intracellular survival and vascular cell-to-cell
transmission of Porphyromonas gingivalis. BMC Microbiol. 2008;8:26.
Li JM, Shah AM. ROS generation by nonphagocytic NADPH oxidase: potential relevance
indiabetic nephropathy. J Am Soc Nephrol. 2003;14(8 Suppl 3):S221-6.
Li XY, Wang C, Xiang XR, Chen FC, Yang CM, Wu J. Porphyromonas gingivalis
lipopolysaccharide increases lipid accumulation by affecting CD36 and ATP-binding cassette
transporter A1 in macrophages. Oncol Rep. 2013;30(3):1329-36.
Lockhart PB, Bolger AF, Papapanou PN, Osinbowale O, Trevisan M, Levison ME, Taubert KA,
Newburger JW, Gornik HL, Gewitz MH, Wilson WR, Smith SC Jr, Baddour LM; American
Heart Association Rheumatic Fever, Endocarditis, and Kawasaki Disease Committee of the

43
 ACCEPTED MANUSCRIPT

Council on Cardiovascular Disease in the Young, Council on Epidemiology and Prevention,

Council on Peripheral Vascular Disease, and Council on Clinical Cardiology. Circulation.
2012;125(20):2520-44.
Lee HR, Jun HK, Choi BK. Tannerella forsythia BspA increases the risk factors for atherosclerosis

PT
in ApoE(-/-) mice. Oral Dis. 2014;20(8):803-8.
Lee HR, Jun HK, Kim HD, Lee SH, Choi BK. Fusobacterium nucleatum GroEL induces risk

RI
factors of atherosclerosis in human microvascular endothelial cells and ApoE(-/-) mice. Mol

SC
Oral Microbiol. 2012;27(2):109-23.
Lockhart PB, Brennan MT, Sasser HC, Fox PC, Paster BJ, Bahrani-Mougeot FK. Bacteremia

NU
associated with toothbrushing and dental extraction. Circulation. 2008;117(24):3118-25.
Loos BG. Systemic markers of inflammation in periodontitis. J Periodontol. 2005;76(11
MA
Suppl):2106-15.
Loos BG, Craandijk J, Hoek FJ, Wertheim-van Dillen PM, van der Velden U. Elevation of systemic
markers related to cardiovascular diseases in the peripheral blood of periodontitis patients. J
D

Periodontol. 2000;71(10):1528-34.
TE

Lv J, Zhu YX, Liu YQ, Xue X. Distinctive pathways characterize A. actinomycetemcomitans and P.
gingivalis. Mol Biol Rep. 2015;42(2):441-9.
P

Madan M, Bishayi B, Hoge M, Messas E, Amar S. Doxycycline affects diet- and bacteria-
CE

associated atherosclerosis in an ApoE heterozygote murine model: cytokine profiling

implications. Atherosclerosis. 2007;190(1):62-72.
AC

Maekawa T, Takahashi N, Honda T, Yonezawa D, Miyashita H, Okui T, Tabeta K, Yamazaki K.

Porphyromonas gingivalis antigens and interleukin-6 stimulate the production of monocyte
chemoattractant protein-1 via the upregulation of early growth response-1 transcription in
human coronary artery endothelial cells. J Vasc Res. 2010;47(4):346-54.
Mandell RL, Dirienzo J, Kent R, Joshipura K, Haber J. Microbiology of healthy and diseased
periodontal sites in poorly controlled insulin dependent diabetics. J Periodontol.
1992;63(4):274-9.
Mantri CK, Chen CH, Dong X, Goodwin JS, Pratap S, Paromov V, Xie H. Fimbriae-mediated outer
membrane vesicle production and invasion of Porphyromonas gingivalis. Microbiologyopen.
2015;4(1):53-65.

44
 ACCEPTED MANUSCRIPT

Marcaccini AM, Meschiari CA, Sorgi CA, Saraiva MC, de Souza AM, Faccioli LH, Tanus-Santos
JE, Novaes AB, Gerlach RF. Circulating interleukin-6 and high-sensitivity C-reactive protein
decrease after periodontal therapy in otherwise healthy subjects. J Periodontol.
2009;80(4):594-602.

PT
Mattila KJ, Nieminen MS, Valtonen VV, Rasi VP, Kesäniemi YA, Syrjälä SL, Jungell PS,
Isoluoma M, Hietaniemi K, Jokinen MJ. Association between dental health and acute

RI
myocardial infarction. BMJ. 1989;298(6676):779-81.

SC
Mercanoglu F, Oflaz H, Oz O, Gökbuget AY, Genchellac H, Sezer M, Nişanci Y, Umman S.
Endothelial dysfunction in patients with chronic periodontitis and its improvement after initial

NU
periodontal therapy. J Periodontol. 2004;75(12):1694-700.
Meyer DH, Lippmann JE, Fives-Taylor PM. Invasion of epithelial cells by Actinobacillus
MA
actinomycetemcomitans: a dynamic, multistep process. Infect Immun. 1996;64(8):2988-97.
Muthukuru M, Cutler CW. Resistance of MMP9 and TIMP1 to endotoxin tolerance. Pathog Dis.
2015 Jul;73(5). pii: ftu003.
D

Miyamoto T, Yumoto H, Takahashi Y, Davey M, Gibson FC 3rd, Genco CA. Pathogen-accelerated

TE

atherosclerosis occurs early after exposure and can be prevented via immunization. Infect
Immun. 2006;74(2):1376-80.
P

Miyakawa H, Honma K, Qi M, Kuramitsu HK. Interaction of Porphyromonas gingivalis with low-

CE

density lipoproteins: implications for a role for periodontitis in atherosclerosis.

J Periodontal Res. 2004;39(1):1-9.
AC

Miyazawa H, Honda T, Miyauchi S, Domon H, Okui T, Nakajima T, Tabeta K, Yamazaki K.

Increased serum PCSK9 concentrations are associated with periodontal infection but do not
correlate with LDL cholesterol concentration. Clin Chim Acta. 2012a;413(1-2):154-9.
Miyazawa H, Tabeta K, Miyauchi S, Aoki-Nonaka Y, Domon H, Honda T, Nakajima T, Yamazaki
K. Effect of Porphyromonas gingivalis infection on post-transcriptional regulation of the low-
density lipoprotein receptor in mice. Lipids Health Dis. 2012;11:121.
Moore WE, Moore LV. The bacteria of periodontal diseases. Periodontol 2000. 1994;5:66-77.
Moore LV, Moore WE, Cato EP, Smibert RM, Burmeister JA, Best AM, Ranney RR. Bacteriology
of human gingivitis. J Dent Res. 1987;66(5):989-95.

45
 ACCEPTED MANUSCRIPT

Mustapha IZ, Debrey S, Oladubu M, Ugarte R. Markers of systemic bacterial exposure in

periodontal disease and cardiovascular disease risk: a systematic review and meta-analysis. J
Periodontol. 2007;78(12):2289-302.
Nahid MA, Rivera M, Lucas A, Chan EK, Kesavalu L. Polymicrobial infection with periodontal

PT
pathogens specifically enhances microRNA miR-146a in ApoE-/-mice during experimental
periodontal disease. Infect Immun. 2011;79(4):1597-605.

RI
Nakajima T, Honda T, Domon H, Okui T, Kajita K, Ito H, Takahashi N, Maekawa T, Tabeta K,

SC
Yamazaki K. Periodontitis-associated up-regulation of systemic inflammatory mediator level
may increase the risk of coronary heart disease. J Periodontal Res. 2010;45(1):116-22.

NU
Nakamura N, Yoshida M, Umeda M, Huang Y, Kitajima S, Inoue Y, Ishikawa I, Iwai T. Extended
exposure of lipopolysaccharide fraction from Porphyromonas gingivalis facilitates
MA
mononuclear cell adhesion to vascular endothelium via Toll-like receptor-2 dependent
mechanism. Atherosclerosis. 2008;196(1):59-67.
Nakano K, Inaba H, Nomura R, Nemoto H, Takeda M, Yoshioka H, Matsue H, Takahashi T,
D

Taniguchi K, Amano A, Ooshima T. Detection of cariogenic Streptococcus mutans in

TE

extirpated heart valve and atheromatous plaque specimens.J Clin Microbiol. 2006;44(9):3313-
7.
P

Nakano K, Inaba H, Nomura R, Nemoto H, Takeuchi H, Yoshioka H, Toda K, Taniguchi K, Amano

CE

A, Ooshima T. Distribution of Porphyromonas gingivalis fimA genotypes in cardiovascular

specimens from Japanese patients. Oral Microbiol Immunol. 2008;23(2):170-2.
AC

Nakano K, Wada K, Nomura R, Nemoto H, Inaba H, Kojima A, Naka S, Hokamura K, Mukai T,

Nakajima A, Umemura K, Kamisaki Y, Yoshioka H, Taniguchi K, Amano A, Ooshima T.
Characterization of aortic aneurysms in cardiovascular disease patients harboring
Porphyromonas gingivalis. Oral Dis. 2011;17(4):370-8.
Namikoshi J, Otake S, Maeba S, Hayakawa M, Abiko Y, Yamamoto M. Specific antibodies
induced by nasally administered 40-kDa outermembrane protein of Porphyromonas gingivalis
inhibits coaggregation activity of P. gingivalis. Vaccine. 2003;22(2):250-6.
Nassar H, Chou HH, Khlgatian M, Gibson FC 3rd, Van Dyke TE, Genco CA. Role for fimbriae and
lysine-specific cysteine proteinase gingipain K in expression of interleukin-8 and monocyte
chemoattractant protein in Porphyromonas gingivalis-infected endothelial cells. Infect
Immun. 2002;70(1):268-76.

46
 ACCEPTED MANUSCRIPT

Nasry HA, Preshaw PM, Stacey F, Heasman L, Swan M, Heasman PA. Smoking cessation advice
for patients with chronic periodontitis. Br Dent J. 2006;200(5):272-5.
Nguyen KA, DeCarlo AA, Paramaesvaran M, Collyer CA, Langley DB, Hunter N. Humoral
responses to Porphyromonas gingivalis gingipain adhesin domains in subjects with chronic

PT
periodontitis. Infect Immun. 2004;72(3):1374-82.
Niu J, Kolattukudy PE. Role of MCP-1 in cardiovascular disease: molecular mechanisms and

RI
clinical implications. Clin Sci (Lond) 2009;117(3):95-109.

SC
Niu XL, Xia Y, Hoshiai K, Tanaka K, Sawamura S, Nakazawa H. nducible nitric oxide synthase
knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density

NU
lipoprotein oxidation. Nitric Oxide. 2000;4(4):363-71.
Noack B, Genco RJ, Trevisan M, Grossi S, Zambon JJ, De Nardin E. Periodontal infections
MA
contribute to elevated systemic C-reactive protein level. J Periodontol. 2001;72(9):1221-7.
O-Brien-Simpson NM, Veith PD, Dashper SG, Reynolds EC. Porphyromonas gingivalis
gingipains: the molecular teeth of a microbial vampire. Curr Protein Pept Sci. 2003;4(6):409-
D

26.
TE

Okoro CA, Balluz LS, Eke PI, Ajani UA, Strine TW, Town M, Mensah GA, Mokdad AH. Tooth
loss and heart disease: findings from the Behavioral Risk Factor Surveillance System. Am J
P

Prev Med. 2005;29(5 Suppl 1):50-6.

CE

Onkelinx S, Cornelissen V, Goetschalckx K, Thomaes T, Verhamme P, Vanhees L. Reproducibility

of different methods to measure the endothelial function. Vasc Med. 2012;17(2):79-84.
AC

Orlandi M, Suvan J, Petrie A, Donos N, Masi S, Hingorani A, Deanfield J, D'Aiuto F. Association

between periodontal disease and its treatment, flow-mediated dilatation and carotid intima-
media thickness: a systematic review and meta-analysis. Atherosclerosis. 2014;236(1):39-46.
Paraskevas S, Huizinga JD, Loos BG. A systematic review and meta-analyses on C-reactive protein
in relation toperiodontitis. J Clin Periodontol. 2008;35(4):277-90.
Park KH, Park WJ. Endothelial dysfunction: clinical implications in cardiovascular disease and
therapeutic approaches. J Korean Med Sci. 2015;30(9):1213-25.
Pejcic A, Kesic LJ, Milasin J. C-reactive protein as a systemic marker of inflammation in
periodontitis. Eur J Clin Microbiol Infect Dis. 2011;30(3):407-14.

47
 ACCEPTED MANUSCRIPT

Peluso I, Morabito G, Urban L, Ioannone F, Serafini M. Oxidative stress in atherosclerosis

development: the central role of LDL and oxidative burst. Endocr Metab Immune Disord
Drug Targets. 2012;12(4):351-60.
Pereira RB, Vasquez EC, Stefanon I, Meyrelles SS. Oral P. gingivalis infection alters the vascular

PT
reactivity in healthy and spontaneously atherosclerotic mice. Lipids Health Dis. 2011;10:80.
Perrotta I, Aquila S. The role of oxidative stress and autophagy in atherosclerosis. Oxid Med Cell

RI
Longev. 2015;2015:130315.

SC
Piconi S, Trabattoni D, Luraghi C, Perilli E, Borelli M, Pacei M, Rizzardini G, Lattuada A, Bray
DH, Catalano M, Sparaco A, Clerici M. Treatment of periodontal disease results in

NU
improvements in endothelial dysfunction and reduction of the carotid intima-media thickness.
FASEB J. 2009;23(4):1196-204. MA
Pollreisz A, Huang Y, Roth GA, Cheng B, Kebschull M, Papapanou PN, Schmidt AM, Lalla E.
Enhanced monocyte migration and pro-inflammatory cytokine production by Porphyromonas
gingivalis infection. J Periodontal Res. 2010;45(2):239-45.
D

Poole S, Singhrao SK, Chukkapalli S, Rivera M, Velsko I, Kesavalu L, Crean S. Active invasion of
TE

Porphyromonas gingivalis and infection-induced complement activation in ApoE-/- mice

brains. J Alzheimers Dis. 2015;43(1):67-80.
P

Progulske-Fox A, Kozarov E, Dorn B, Dunn W Jr, Burks J, Wu Y. Porphyromonas gingivalis

CE

virulence factors and invasion of cells of the cardiovascular system. J Periodontal Res.
1999;34(7):393-9.
AC

Pussinen PJ, Alfthan G, Jousilahti P, Paju S, Tuomilehto J. Systemic exposure to Porphyromonas

gingivalis predicts incident stroke. Atherosclerosis. 2007;193(1):222-8.
Pussinen PJ, Jauhiainen M, Vilkuna-Rautiainen T, Sundvall J, Vesanen M, Mattila K, Palosuo T,
Alfthan G, Asikainen S. Periodontitis decreases the antiatherogenic potency of high density
lipoprotein. J Lipid Res. 2004;45(1):139-47.
Pussinen PJ, Metso J, Malle E, Barlage S, Palosuo T, Sattler W, Schmitz G, Jauhiainen M. The role
of plasma phospholipid transfer protein (PLTP) in HDL remodeling in acute-phase patients.
Biochim Biophys Acta. 2001;1533(2):153-63.
Pussinen PJ, Vilkuna-Rautiainen T, Alfthan G, Palosuo T, Jauhiainen M, Sundvall J, Vesanen M,
Mattila K, Asikainen S. Severe periodontitis enhances macrophage activation via increased
serum lipopolysaccharide. Arterioscler Thromb Vasc Biol. 2004;24(11):2174-80.

48
 ACCEPTED MANUSCRIPT

Qi M, Miyakawa H, Kuramitsu HK. Porphyromonas gingivalis induces murine macrophage foam

cell formation. Microb Pathog. 2003;35(6):259-67.
Rafieian-Kopaei M, Setorki M, Doudi M, Baradaran A, Nasri H. Atherosclerosis: process,
indicators, risk factors and new hopes. Int J Prev Med. 2014;5(8):927-46.

PT
Ramírez JH, Parra B, Gutierrez S, Arce RM, Jaramillo A, Ariza Y, Contreras A. Biomarkers of
cardiovascular disease are increased in untreated chronic periodontitis: a case control study.

RI
Aust Dent J. 2014;59(1):29-36.

SC
Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS. Bacterial coaggregation: an
integral process in the development of multi-species biofilms. Trends Microbiol.

NU
2003;11(2):94-100.
Rangé H, Labreuche J, Louedec L, Rondeau P, Planesse C, Sebbag U, Bourdon E, Michel JB,
MA
Bouchard P, Meilhac O. Periodontal bacteria in human carotid atherothrombosis as a potential
trigger for neutrophil activation. Atherosclerosis. 2014;236(2):448-55.
Romano F, Barbui A, Aimetti M. Periodontal pathogens in periodontal pockets and in carotid
D

atheromatous plaques. Minerva Stomatol. 2007;56(4):169-79.

TE

Roth GA, Aumayr K, Giacona MB, Papapanou PN, Schmidt AM, Lalla E. Porphyromonas
gingivalis infection and prothrombotic effects in human aortic smooth muscle cells. Thromb
P

Res. 2009;123(5):780-4.
CE

Roth GA, Moser B, Huang SJ, Brandt JS, Huang Y, Papapanou PN, Schmidt AM, Lalla E.
Infection with a periodontal pathogen induces procoagulant effects in human aortic
AC

endothelial cells. J Thromb Haemost. 2006;4(10):2256-61.

Roth GA, Ankersmit HJ, Brown VB, Papapanou PN, Schmidt AM, Lalla E. Porphyromonas
gingivalis infection and cell death in human aortic endothelial cells. FEMS Microbiol Lett.
2007a;272(1):106-13.
Roth GA, Moser B, Roth-Walter F, Giacona MB, Harja E, Papapanou PN, Schmidt AM, Lalla E.
Infection with a periodontal pathogen increases mononuclear cell adhesion to human aortic
endothelial cells. Atherosclerosis. 2007b;190(2):271-81.
Rufail ML, Schenkein HA, Koertge TE, Best AM, Barbour SE, Tew JG, van Antwerpen R.
Atherogenic lipoprotein parameters in patients with aggressive periodontitis. J Periodontal
Res. 2007;42(6):495-502.

49
 ACCEPTED MANUSCRIPT

Santos J, Marquis A, Epifano F, Genovese S, Curini M, Grenier D. Collinin reduces

Porphyromonas gingivalis growth and collagenase activity and inhibits the
lipopolysaccharide-induced macrophage inflammatory response and osteoclast differentiation
and function. J Periodontol. 2013;84(5):704-11.

PT
Sasaki M, Terao Y, Ayaori M, Uto-Kondo H, Iizuka M, Yogo M, Hagisawa K, Takiguchi S,
Yakushiji E, Nakaya K, Ogura M, Komatsu T, Ikewaki K. Hepatic overexpression of idol

RI
increases circulating protein convertase subtilisin/kexin type 9 in mice and hamsters via dual

SC
mechanisms: sterol regulatory element-binding protein 2 and low-density lipoprotein
receptor-dependent pathways. Arterioscler Thromb Vasc Biol. 2014;34(6):1171-8.

NU
Sato Y, Kishi J, Suzuki K, Nakamura H, Hayakawa T. Sonic extracts from a bacterium related to
periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases
MA
TIMP-1 and TIMP-2. Int Endod J. 2009;42(12):1104-11.
Seinost G, Wimmer G, Skerget M, Thaller E, Brodmann M, Gasser R, Bratschko RO, Pilger E.
Periodontal treatment improves endothelial dysfunction in patients with severe periodontitis.
D

Am Heart J. 2005;149(6):1050-4.
TE

Schallhorn RA, Patel DN, Chandrasekar B, Mealey BL. Periodontal disease in association with
systemic levels of interleukin-18 and CXC ligand 16 in patients undergoing cardiac
P

catheterization. J Periodontol. 2010;81(8):1180-6.

CE

Sfyroeras GS, Roussas N, Saleptsis VG, Argyriou C, Giannoukas AD. Association between
periodontal disease and stroke. J Vasc Surg. 2012;55(4):1178-84.
AC

Sheets SM, Potempa J, Travis J, Fletcher HM, Casiano CA.Gingipains from Porphyromonas
gingivalis W83 synergistically disrupt endothelial cell adhesion and can induce caspase-
independent apoptosis. Infect Immun. 2006;74(10):5667-78.
Sheets SM, Potempa J, Travis J, Casiano CA, Fletcher HM. Gingipains from Porphyromonas
gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells.
Infect Immun. 2005;73(3):1543-52.
Sim SJ, Kim HD, Moon JY, Zavras AI, Zdanowicz J, Jang SJ, Jin BH, Bae KH, Paik DI, Douglass
CW. Periodontitis and the risk for non-fatal stroke in Korean adults. J Periodontol.
2008;79(9):1652-8.
Siti HN, Kamisah Y, Kamsiah J. The role of oxidative stress, antioxidants and vascular
inflammation in cardiovascular disease (a review). Vascul Pharmacol. 2015;71:40-56.

50
 ACCEPTED MANUSCRIPT

Smalley JW. Pathogenic mechanisms in periodontal disease. Adv Dent Res. 1994;8(2):320-8.
Smalley JW, Silver J, Marsh PJ, Birss AJ. The periodontopathogen Porphyromonas gingivalis
binds iron protoporphyrin IX in the mu-oxo dimeric form: an oxidative buffer and possible
pathogenic mechanism. Biochem J. 1998;331 ( Pt 3):681-5.

PT
Socransky SS, Haffajee AD, Smith C, Dibart S. Relation of counts of microbial species to clinical
status at the sampled site. J Clin Periodontol. 1991;18(10):766-75.

RI
Song H, Bélanger M, Whitlock J, Kozarov E, Progulske-Fox A. Hemagglutinin B is involved in the

SC
adherence of Porphyromonas gingivalis to human coronary artery endothelial cells. Infect
Immun. 2005;73(11):7267-73.

NU
Stassen FR, Vainas T, Bruggeman CA. Infection and atherosclerosis. An alternative view on an
outdated hypothesis. Pharmacol Rep. 2008;60(1):85-92.
MA
Strom BL, Abrutyn E, Berlin JA, Kinman JL, Feldman RS, Stolley PD, Levison ME, Korzeniowski
OM, Kaye D. Dental and cardiac risk factors for infective endocarditis. A population-based,
case-control study. Ann Intern Med. 1998;129(10):761-9
D

Subrahmanyam MV, Sangeetha M. Gingival crevicular fluid a marker of the periodontal disease
TE

activity. Indian J Clin Biochem. 2003;18(1):5-7.

Sun XJ, Meng HX, Shi D, Xu L, Zhang L, Chen ZB, Feng XH, Lu RF, Ren XY. Elevation of C-
P

reactive protein and interleukin-6 in plasma of patients with aggressive periodontitis. J

CE

Periodontal Res. 2009;44(3):311-6.

Tabeta K, Yamazaki K, Hotokezaka H, Yoshie H, Hara K. Elevated humoral immune response to
AC

heat shock protein 60 (hsp60) family in periodontitis patients. Clin Exp Immunol.
2000;120(2):285-93.
Tapashetti RP, Guvva S, Patil SR, Sharma S, Pushpalatha HM. C-reactive protein as predict of
increased carotid intima media thickness in patients with chronic periodontitis. J Int Oral
Health. 2014;6(4):47-52.
Takahashi Y, Davey M, Yumoto H, Gibson FC 3rd, Genco CA. Fimbria-dependent activation of
pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial
cells. Cell Microbiol. 2006;8(5):738-57.
Tonetti MS, D'Aiuto F, Nibali L, Donald A, Storry C, Parkar M, Suvan J, Hingorani AD, Vallance
P, Deanfield J. Treatment of periodontitis and endothelial function. N Engl J Med.
2007;356(9):911-20.

51
 ACCEPTED MANUSCRIPT

Tonetti MS, Van Dyke TE; working group 1 of the joint EFP/AAP workshop. Periodontitis and
atherosclerotic cardiovascular disease: consensus report of the Joint EFP/AAP Workshop on
Periodontitis and Systemic Diseases. J Periodontol. 2013;84(4 Suppl):S24-9.
Tuomainen AM, Jauhiainen M, Kovanen PT, Metso J, Paju S, Pussinen PJ. Aggregatibacter

PT
actinomycetemcomitans induces MMP-9 expression and proatherogenic lipoprotein profile in
apoE-deficient mice. Microb Pathog. 2008;44(2):111-7.

RI
Turunen SP, Kummu O, Harila K, Veneskoski M, Soliymani R, Baumann M, Pussinen PJ, Hörkkö

SC
S. Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to
malondialdehyde modified low-density lipoprotein. PLoS One. 2012;7(4):e34910.

NU
Turunen SP, Kummu O, Wang C, Harila K, Mattila R, Sahlman M, Pussinen PJ, Hörkkö S.
Immunization with malondialdehyde-modified low-density lipoprotein (LDL) reduces
MA
atherosclerosis in LDL receptor-deficient mice challenged with Porphyromonas gingivalis.
Innate Immun. 2015;21(4):370-85.
Uehara A, Imamura T, Potempa J, Travis J, Takada H. Gingipains from Porphyromonas gingivalis
D

synergistically induce the production of proinflammatory cytokines through protease-

TE

activated receptors with Toll-like receptor and NOD1/2 ligands in human monocytic cells.
Cell Microbiol. 2008;10(5):1181-9.
P

Vidal F, Cordovil I, Figueredo CM, Fischer RG. Non-surgical periodontal treatment reduces
CE

cardiovascular risk in refractory hypertensive patients: a pilot study. J Clin Periodontol.

2013;40(7):681-7.
AC

Virmani R, Burke AP, Farb A, Kolodgie FD. Pathology of the vulnerable plaque. J Am Coll
Cardiol. 2006;47(8 Suppl):C13-8.
Vlachojannis C, Dye BA, Herrera-Abreu M, Pikdöken L, Lerche-Sehm J, Pretzl B, Celenti R,
Papapanou PN. Determinants of serum IgG responses to periodontal bacteria in a nationally
representative sample of US adults. J Clin Periodontol. 2010;37(8):685-96.
Watt RG, Tsakos G, de Oliveira C, Hamer M. Tooth loss and cardiovascular disease mortality risk -
results from the Scottish Health Survey. PLoS One. 2012;7(2):e30797.
Wei PF, Ho KY, Ho YP, Wu YM, Yang YH, Tsai CC. The investigation of glutathione peroxidase,
lactoferrin, myeloperoxidase and interleukin-1beta in gingival crevicular fluid: implications
for oxidative stress in human periodontal diseases. J Periodontal Res. 2004;39(5):287-93.

52
 ACCEPTED MANUSCRIPT

Wei D, Zhang XL, Wang YZ, Yang CX, Chen G. Lipid peroxidation levels, total oxidant status
and superoxide dismutase inserum, saliva and gingival crevicular fluid in chronic
periodontitis patients before and after periodontal therapy. Aust Dent J. 2010;55(1):70-8.
Wick MC, Mayerl C, Backovic A, van der Zee R, Jaschke W, Dietrich H, Wick G. In vivo imaging

PT
of the effect of LPS on arterial endothelial cells: molecular imaging of heat shock protein 60
expression. Cell Stress Chaperones. 2008;13(3):275-85.

RI
Widlansky ME, Gokce N, Keaney JF Jr, Vita JA. The clinical implications of endothelial

SC
dysfunction. J Am Coll Cardiol. 2003;42(7):1149-60.
Wiesner P, Choi SH, Almazan F, Benner C, Huang W, Diehl CJ, Gonen A, Butler S, Witztum JL,

NU
Glass CK, Miller YI. Low doses of lipopolysaccharide and minimally oxidized low-density
lipoprotein cooperatively activate macrophages via nuclear factor kappa B and activator
MA
protein-1: possible mechanism for acceleration of atherosclerosis by subclinical endotoxemia.
Circ Res. 2010;107(1):56-65.
Wilson W, Taubert KA, Gewitz M, Lockhart PB, Baddour LM, Levison M, Bolger A, Cabell CH,
D

Takahashi M, Baltimore RS, Newburger JW, Strom BL, Tani LY, Gerber M, Bonow RO,
TE

Pallasch T, Shulman ST, Rowley AH, Burns JC, Ferrieri P, Gardner T, Goff D, Durack DT.
Circulation. 2007;116(15):1736-54.
P

Wohlfeil M, Scharf S, Siegelin Y, Schacher B, Oremek GM, Sauer-Eppel H, Schubert R, Eickholz

CE

P. Increased systemic elastase and C-reactive protein in aggressive periodontitis (CLOI-D-

00160R2). Clin Oral Investig. 2012;16(4):1199-207.
AC

Wu T, Trevisan M, Genco RJ, Dorn JP, Falkner KL, Sempos CT. Periodontal disease and risk of
cerebrovascular disease: the first national health and nutrition examination survey and its
follow-up study. Arch Intern Med. 2000;160(18):2749-55.
Yamazaki K, Ohsawa Y, Tabeta K, Ito H, Ueki K, Oda T, Yoshie H, Seymour GJ. Accumulation of
human heat shock protein 60-reactive T cells in the gingival tissues of periodontitis patients.
Infect Immun. 2002;70(5):2492-501.
Yang HW, Huang YF, Chou MY. Occurrence of Porphyromonas gingivalis and Tannerella
forsythensis in periodontally diseased and healthy subjects. J Periodontol. 2004;75(8):1077-
83.
Yang NY, Zhang Q, Li JL, Yang SH, Shi Q. Progression of periodontal inflammation in adolescents
is associated with increased number of Porphyromonas gingivalis, Prevotella intermedia,

53
 ACCEPTED MANUSCRIPT

Tannerella forsythensis, and Fusobacterium nucleatum. Int J Paediatr Dent. 2014;24(3):226-

33.
Ylöstalo PV, Knuuttila ML. Confounding and effect modification: possible explanation for
variation in the results on the association between oral and systemic diseases.

PT
J Clin Periodontol. 2006;33(2):104-8.
Yoshimura F, Murakami Y, Nishikawa K, Hasegawa Y, Kawaminami S. Surface components of

RI
Porphyromonas gingivalis. J Periodontal Res. 2009;44(1):1-12.

SC
Yu H, Qi LT, Liu LS, Wang XY, Zhang Y, Huo Y, Luan QX. Association of carotid intima-media
thickness and atherosclerotic plaque with periodontal status. J Dent Res. 2014;93(8):744-51.

NU
Yumoto H, Chou HH, Takahashi Y, Davey M, Gibson FC 3rd, Genco CA. Sensitization of human
aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial
MA
fimbria-dependent invasion. Infect Immun. 2005;73(12):8050-9.
Yun PL, Decarlo AA, Chapple CC, Hunter N. Functional implication of the hydrolysis of platelet
endothelial cell adhesion molecule 1 (CD31) by gingipains of Porphyromonas gingivalis for
D

the pathology of periodontal disease. Infect Immun. 2005;73(3):1386-98.

TE

Zambon JJ, Christersson LA, Slots J. Actinobacillus actinomycetemcomitans in human periodontal

disease. Prevalence in patient groups and distribution of biotypes and serotypes within
P

families. J Periodontol. 1983;54(12):707-11.

CE

Zhang W, Daly CG, Mitchell D, Curtis B. Incidence and magnitude of bacteraemia caused by
flossing and by scalingand root planing. J Clin Periodontol. 2013;40(1):41-52.
AC

Zhang B, Elmabsout AA, Khalaf H, Basic VT, Jayaprakash K, Kruse R, Bengtsson T, Sirsjö A. The
periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular
smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell
proliferation. BMC Genomics. 2013;14:770.
Zhang B, Khalaf H, Sirsjö A, Bengtsson T. Gingipains from the periodontal pathogen
Porphyromonas gingivalis play a significant role in regulation of angiopoietin 1 and
angiopoietin 2 in human aortic smooth muscle cells. Infect Immun. 2015;83(11):4256-65.
Zhou J, Windsor LJ. Porphyromonas gingivalis affects host collagen degradation by affecting
expression, activation, and inhibition of matrix metalloproteinases. J Periodontal Res.
2006;41(1):47-54.

54
 ACCEPTED MANUSCRIPT

Zhou J, Zhang J, Chao J. Porphyromonas gingivalis promotes monocyte migration by activating

MMP-9. J Periodontal Res. 2012;47(2):236-42.
Zhu J, Quyyumi AA, Norman JE, Csako G, Waclawiw MA, Shearer GM, Epstein SE. Effects of
total pathogen burden on coronary artery disease risk and C-reactive protein levels. Am J

PT
Cardiol. 2000;85(2):140-6.

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Figure 1
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Table 1. Effects of challenge of murine atherosclerosis models with periodontal pathogens and
their products

Recipient Pathogen Findings Reference

mice
LDLR- P. gingivalis Natural anti-MDA-LDL IgM recognizes Turunen et al.,

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deficient epitopes of P. gingivalis gingipain. (2012)
Immunization with P. gingivalis induced IgM,
response to MDA-LDL, and decreased aortic

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lipid accumulation
LDLR- P. gingivalis Immunization of P. gingivalis-challenged Turunen et al.,
deficient mice with MDA-LDL induced humoral (2015)

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immune response, IgM and IgG production,
and led to diminished atherosclerosis
C57BL/6 P. gingivalis P. gingivalis infection stimulates PCSK9 Miyazawa et al.,
expression through activation of Srebf2 (2012b)

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associated with degradation of LDLR and
hypercholesterolemia
ApoE- Immunization with Peptide 14 plays proatherogenic role (through
MA Jeong et al.,
deficient peptides from P. induction of Th1 cells) while peptide 19 plays (2015)
gingivalis GroEL anti-atherogenic role (through induction of
Tregs)
ApoE- P. gingivalis P. gingivalis could access murine brain and Poole et. al.
deficient F. nucleatum induce complement activation and brain (2015)
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Treponema denticola inflammation

Tannerella forsythia
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(monoinfection and
polyinfection)
ApoE- T. forsythia or BspA T. forsythia or BspA causes accelerated Lee et al.,(2014)
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deficient (from T. forsythia) atherosclerosis, foam cell formation, down-

regulation of LXRα, LXRβ, and ABCA1,
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increase in serum CRP and LDL, decrease in

serum HDL
ApoE- GroEL from F. GroEL causes accelerated atherosclerosis, Lee et al.,
deficient nucleatum decrease in serum HDL, increase in serum (2012)
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CRP, IL-6, and LDL, foam cell formation,

induction of proinflammatory (MCP-1, IL-8)
and adhesion (ICAM-1, VCAM-1) in
endothelial cells
ApoE- P. gingivalis P. gingivalis infection impairs vascular Pereira et al.,
deficient function through enhanced vasoconstriction (2011)
and altered α-adrenoceptor-mediated vascular
response
ApoE- P. gingivalis Periodontal infection increased miR-146 Nahid et al.,
deficient T. denticola associated with down-regulation of targets (2011)
T. forsythia IRAK1 and TRAF6
(monoinfection and
polyinfection)
ApoE- LPS from P. gingivalis LPS increases atherosclerosis and stimulates Gitlin and
deficient TNF-α production by macrophages in Loftin, (2009)
COX2/PGE2-dependent manner
ApoE- A. A. actinomycetemcomitans infection causes Hyvärinen et
deficient actinomycetemcomitans hepatic infiltration by inflammatory cells, up- al., (2009)
regulation of expression of proinflammatory
genes, and increase in serum amyloid A

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ApoE- P. gingivalis P. gingivalis stimulates plaque development Koizumi et al.,

deficient 40-kDa OMP from P. associated with elevated levels of (2008)
gingivalis inflammatory cytokines and induction of
OMP-specific antibodies. Nasal immunization
with 40-kDa OMP reduces plaque progression
and levels of inflammatory markers

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ApoE- A. Infection does not influences plaque size and Tuomainen et
deficient actinomycetemcomitans progression but induces elevated CRP levels, al., (2008)
atherogenic lipid profile (increased VLDL and
LDL and decreased LDL), and increase in

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aortic MMP-9 expression
ApoE +/- P. gingivalis Treatment of P. gingivalis-induced Madan et al.,

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atherosclerosis with doxycycline results in (2007)
reduction of plaque area, percentage of
aromathous lesions, levels of proinflammatory
cytokines, and MMP-9

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ApoE- P. gingivalis Arg-gingipain from P. gingivalis specifically Hashimoto et
deficient cleaves ApoB-100 inducing formation of al., (2006)
foam cells, lipid accumulation, increase in
LDL cholesterol, decrease in HDL
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cholesterol, and atherosclerosis progression
ApoE- P. gingivalis P. gingivalis accelerates early atherosclerosis Miyamoto et al.,
deficient accompanied with increased macrophage (2006)
infiltration, atheroma development, and
stimulation of innate immunity but without
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up-regulation of systemic inflammation. This

local proatherogenic inflammation could be
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prevented by P. gingivalis immunization

ApoE- P. gingivalis P. gingivalis fimbriae-deficient mutant Gibson et al.,
deficient (FimA-) cannot induce up-regulation of TLR2 (2004)
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and TLR4 and promote atherosclerosis

ApoE- P. gingivalis P. gingivalis infection promotes early Lalla et al.,
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deficient atherosclerosis through endothelial activation (2003)

(increased expression of VCAM-1 and TF)
and increased serum IL-6
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ApoE- P. gingivalis P. gingivalis increases lesion size, plaque Li et al., (2002)

deficient macrophage accumulation; ribosomal DNA of
the pathogen was found in the aorta, liver, and
heart

Abbreviations: ABCA1, ATP-binding cassette transporter 1; ApoE, apolipoprotein E; BspA, T.

forsythia virulence factor; CRP, C-reactive protein; FimA, fimbrillin, bacterial attachment pillus;
GroEL, chaperonin; HDL, high density lipoprotein; HSP60, heat shock protein 60; ICAM-1,
intercellular adhesion molecule-1; IgG, immunoglobulin G; IL, interleukin; IRAK1, interleukin-1
receptor-associated kinase 1; LDL, low density lipoprotein; LDLR, LDL receptor; LXRα, liver X
receptor α; OMP, outer membrane protein; MCP-1, monocyte chemotactic protein-1; MDA-LDL,
malondialdehyde-LDL; miR-146, microRNA-146; MMP-9, matrix metalloproteinase-9; PCSK9,
proprotein convertase subtilisin/kexin type 9; Srebf2, sterol regulatory element-binding
transcription factor 2; TF, tissue factor; TLR, Toll-like receptor; TRAF6, TNF receptor-
associated factor 6; Tregs, regulatory T cells; VCAM-1, vascular cell adhesion molecule-1;
VLDL, very light density protein.

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