Inflammation in Periodontal Disease: Possible Link to Vascular Disease

Inflammation is a well-organized protective response to pathogens and consists of immune cell recruitment into areas of infection. Inflammation either clears pathogens and gets resolved leading to tissue healing or remains predominantly unresolved triggering pathological processes in organs. Periodontal disease (PD) that is initiated by specific bacteria also triggers production of inflammatory mediators. These processes lead to loss of tissue structure and function. Reactive oxygen species and oxidative stress play a role in susceptibility to periodontal pathogenic bacterial infections. Periodontal inflammation is a risk factor for systemic inflammation and eventually cardiovascular disease (CVD). This review discusses the role of inflammation in PD and its two way association with other health conditions such as diabetes and CVD. Some of the mechanisms underpinning the links between inflammation, diabetes, CVD and PD are also discussed. Finally, we review available epidemiological data and other reports to assess possible links between oral health and CVD.


INTRODUCTION
Periodontal disease (PD) is a chronic inflammatory disorder characterized by the destruction of the periodontium, or the supporting tissues of the teeth (gingival tissue, periodontal ligament, and alveolar bone). PD is highly prevalent, and approximately 50% of adults 30 years and older and 70% of adults 65 or older have a form of the disease (Eke et al., 2012). Clinically, the failure to treat PD leads to loss of teeth (Schultz-Haudt et al., 1954;Hajishengallis et al., 2011;Abusleme et al., 2013). Central to PD is dysregulation of the resolution of inflammation, resulting in characteristic chronic and progressive destruction.
Inflammation is a programmed signaling event initiated to protect organisms upon infection and/or injury. In general, infection-injury stimuli lead to release of pathogen or danger associated molecular patterns (PAMPs or DAMPs) followed by their binding to respective receptors in the host cells. Although there are multiple contributing factors to PD, one of the increasingly well-characterized triggers is the colonization of the oral cavity by pathogenic bacteria and their subsequent penetration into local epithelial lining (Darveau, 2010;Abusleme et al., 2013). This initiates an inflammatory cascade characterized by increased expression of various inflammatory mediators and adhesion molecules that collectively mobilize and recruit polymorphonuclear neutrophils (PMN), macrophages, natural killer (NK), dendritic cells (DC) etc. into the affected tissue. Under normal conditions, neutrophils and macrophages phagocytose the microbial organisms after which they undergo apoptosis at the inflamed site (Fox et al., 2010). The clearance of apoptotic cells facilitates a switch from a pro-to an anti-inflammatory macrophage phenotype (Fadok et al., 1998;Michlewska et al., 2009) and initiates the onset of the resolution of inflammation, a coordinated signaling process that restores tissue integrity and function. However, failure to switch off the inflammation cascade once the pathogenic stimulus is removed, leads chronic inflammation (i.e., an uncontrolled inflammatory response that can culminate into damage to the host tissue) and is the hallmark of several inflammatory disorder related pathologies. In PD specifically, the inflammatory response becomes chronic when pathogenic bacteria continue to propagate and cannot be controlled by the acute immune response, resulting in unresolved inflammation, destruction of local bone and soft tissue, and fibrosis (Cochrane, 2008).
Importantly, reports have consistently highlighted a role for periodontal inflammation in acceleration of various vascular pathologies and other systemic implications (Humphrey et al., 2008;Ogrendik, 2013;Ketabi et al., 2016). The destruction of local epithelium by PD pathogens can result in release of local inflammatory mediators from the periodontal pocket into the systemic circulation thus facilitating immune cell recruitment elsewhere. Also, bacteria can either indirectly (within immune cells that have ingested them) or directly circulate in the bloodstream (Lockhart et al., 2009). Therefore, under conditions where there is disposition (e.g., genetic, lifestyle) toward cardiovascular disease (CVD), the bacterial components and systemic inflammatory mediators can potentially accelerate plaque formation. To this point, PD pathogens have been detected in distant tissues and organs, particularly in the cardiovascular system (Okuda et al., 2001;Kozarov et al., 2006;Nakano et al., 2006;Pessi et al., 2013;Moreno et al., 2017). The relationship between PD and systemic diseases such as CVD has been increasingly well-characterized. Importantly, two classic meta-analyses demonstrated the correlation between PD and CVD, highlighting PD as a potential risk factor for CVD processes such as coronary artery disease (Janket et al., 2003;Khader et al., 2004). Additionally, recent evidence suggests a major role for reactive oxygen species (ROS) and proteolytic enzymes (bacteria-and host-derived) in PD and CVD such as atherosclerosis (Chistiakov et al., 2016).
CVD is an umbrella term for a number of linked pathologies, commonly defined as coronary heart disease (CHD), cerebrovascular disease, peripheral arterial disease, rheumatic and congenital heart diseases, and venous thromboembolism (Lara-Pezzi et al., 2012;Mandviwala et al., 2016). The associated risk factors include ethnicity, age, and family history of CVD, dyslipidemia, hypertension, tobacco smoke, excess body weight, physical inactivity, and diabetes mellitus. It is well established that these classic risk factors interact with cellular immune-inflammatory signaling processes to lead to endothelial dysfunction and atheromatous plaque development (Lopez-Candales et al., 2017;Lazzerini et al., 2019). Thus chronic inflammation plays a crucial role in the long-term progression of atherosclerosis. About 35-50% of the world population suffers from periodontitis as reported by World Health Organization (Petersen and Ogawa, 2012); therefore understanding any correlation or link between PD and CVD is a question that has tremendous importance given the high incidences of both diseases. This review summarizes pathophysiology of PD and examines the possibility of its link with CVD.

PERIODONTAL DISEASE
The inflammation of tissues in gingivitis and periodontitis is caused by a host of bacteria (Schultz-Haudt et al., 1954). The bacterial species present in the gingival margin are Porphyromonas gingivalis, Treponema denticola, and Tannnerella forsythia, all of which are Gram negative. Also present are Gram positive bacteria like Streptococcus sanguis, Streptococcus oralis, Streptococcus mutans, Actinomyces naeslundii, and Actinomyces odontolyticus (Abusleme et al., 2013). This is followed by appearance of secondary bacteria such as Fusobacterium nucleatum (Kolenbrander et al., 1989). Aggregates of bacterial colonies form and Gram-positive and Gram-negative bacilli become embedded in the extracellular matrix (Gibbons, 1989). Indeed more than 700 bacterial species have been reported to be detected in dental plaques (Moore, 1987;Gao et al., 2018). Bacterial species normally act as symbiotic communities with the host but shifts of the oral microbiome often associated with "poor" host health can lead to dysbiosis, an imbalance that is responsible for the development of microbe-related PD (Socransky et al., 1998;Darveau, 2010).
Several of these bacteria are also present in healthy individuals; however it is their relative abundance due to with poor oral hygiene (Hoare et al., 2019), tobacco consumption etc. that drives the selection and prevalence of pathogenic bacteria in subgingival margin that lead to the onset of periodontitis (Schultz-Haudt et al., 1954;Socransky et al., 1998). Increased oxidative stress with smoking, lifestyle diseases and aging also plays a role; indeed a strong association between oxidative stress and PD has been reported (Chapple and Matthews, 2007). This occurs either due to diminution of antioxidants or an exaggerated inflammatory response post periodontal infection. This is described in the next section. Bacterial plaque formation leads to increases in PAMPs causing a rise in local inflammation, causing increased flow of gingival crevicular fluid (GCF). This in turn provides protein rich nutrients that increase the proliferation of the Gramnegative bacteria. The dental plaque biofilm of bacteria in the periodontal crevice induces clinical signs of inflammation. The progression of PD is driven primarily by the proliferation of P. gingivalis which facilitates increase in harmful microbiota. The next step is the secondary bacteria F. nucleatum's role in the subgingival biofilm as this bacterium interacts with other bacterial species found in the biofilm. F. nucleatum serves as a bridge between the early colonizers like Streptococcus sp. and the late colonizers like P. gingivalis. The innate immune response is the recruitment of PMN and the NK cells that is driven by the subgingival bacterial community present in the periodontal pocket. As the microorganisms are abundant, PMN recruitment and phagocytosis is followed by extensive PMN apoptosis or necrosis. A cytokine rich proinflammatory environment consists of tumor necrosis factor (TNF)-α, interleukins (IL)-1, IL-4, IL-10, interferon (IFN-γ) and transforming growth factor (TGF-β).
These signaling molecules stimulate the activation of enzymes and transcription factors that in turn recruit more immune cells and degrade the surrounding tissues by maintaining a continual loop of local inflammation (Cekici et al., 2014; Figure 1). This is also accompanied by an adaptive immune response as antigen uptake and processing is carried out by DC and presented to naive T cells. DCs direct CD4+ T cells to differentiate to T-cell subsets such as T helper cells types 1, 2, and 17, and regulatory T cells (Song et al., 2018). CD4+ T cells produce the bone resorption promoting cytokine, Receptor activator of nuclear factor-κB (RANK-L; Tang et al., 2007) leading to bone loss.

OXIDATIVE STRESS IN PD and CVD
Reactive oxygen species and the resultant oxidative stress plays an important role in onset and progression of PD (Chapple and Matthews, 2007). This occurs via multiple mechanisms. First, is the ROS production that occurs with periodontal infection, as inflammatory cells are recruited to the infection site with chronic or aggressive periodontitis. Numerous reports have shown that PMN in the population diagnosed with PD generate significantly more ROS (upon stimulation) as compared to PMN of healthy controls (Aboodi et al., 2011;White et al., 2014;Ling et al., 2016). While this is indicative of a hyperreactive phenotype of neutrophils in the PD affected, it also suggests that high oxidative stress arising from the excessive ROS could increase local gingival oxidative stress which in turn would drive more inflammation. Alteration of the gingival crevicular environment increase susceptibility to periodontal pathogenic bacteria. Second, is the antioxidant status of PD affected individuals; several studies have shown that the low levels of anti-oxidants (that may be associated with high levels of ROS) in the GCF activated the local periodontal inflammation and caused oxidative injury and destruction of the tissue (Tsai et al., 2005;Chapple and Matthews, 2007;Konopka et al., 2007). Figure 2 shows the feed forward loop of infection induced ROS and oxidative stress that in turn drives more inflammation and changes the local gingival tissue environment making it more susceptible to infection. Lifestyle diseases also play a part in this ROS oxidative stress inflammation cascade.
As is well established, inflammation and oxidative stress are pivotal events that lead to CVD (Mandviwala et al., 2016;Cervantes Gracia et al., 2017) and seem to be the common link between the onset of tissue destruction in periodontitis and systemic inflammation (Wang et al., 2017). Indeed several lifestyle and age related conditions associated with CVD (such as diabetes, hypertension etc.) that lead to high oxidative stress (as assessed by markers of ROS and lipid peroxidation) can also increase susceptibility to PD (Dhadse et al., 2010). When periodontitis susceptible patients are exposed to the bacterial antigen, their main two immune responses in the form of neutrophil recruitment and proteolytic enzymes production further release ROS at the gingival site, thus perpetuating oxidative stress and tissue damage (Scott and Krauss, 2012;Cortes-Vieyra et al., 2016). As periodontitis progresses, periodontal inflammation produces ROS that diffuses into the blood stream (Sobaniec and Sobaniec-Lotowska, 2000;Tomofuji et al., 2007). As a result, various moieties in the blood get oxidized and induce an oxidative stress on other organs via the circulating blood causing circulating oxidative stress (Yagi, 1987;Tomofuji et al., 2007; Figure 3). Thus, it can be inferred that the bacteria present in the periodontal pocket suppress detoxification of ROS by consuming the antioxidants present in the pockets  of the oral cavity (Ekuni et al., 2009). The consequence of the lowered levels of antioxidants enables ROS to enter into systemic circulation from the periodontal tissues (Wang et al., 2017).
Regular smoking, diabetes mellitus, insufficient and appropriate nutrition, and aging have all been mentioned as risk factors for both PD and CVD (Yanbaeva et al., 2007;Graves and Kayal, 2008;Dhadse et al., 2010). All the above lifestyle diseases have been known to increase the circulating oxidative stress; indeed increase in levels of malondialdehyde (MDA) and 4-hydroxynonenal (HNE) locally and systemically with PD and with CVD have been observed, thus suggesting an association between local and systemic oxidative stress diseases (Celec et al., 2005;Guentsch et al., 2008;Hendek et al., 2015). Similarly, levels of antioxidants such as SOD and glutathione decrease in the (GCF) and saliva due to smoking in both, healthy individuals and patients with periodontitis (Guentsch et al., 2008;Agnihotri et al., 2009).

RISK FACTORS (PD AND VASCULAR INFLAMMATION)
Vascular inflammation involves the onset of a signaling cascade that is triggered by endothelial signaling which leads to increase in cellular adhesions molecules, cytokines and chemokines. This leads to recruitment and adherence of immune cells. The atherogenic process starts with endothelial dysfunction and the accumulation of several plasma low density lipoproteins (LDL) in the subendothelial space. The accumulation of LDL correlates with classical risk factors, such as smoking, hypertension, and metabolic dysregulation in obesity and diabetes mellitus (Gimbrone et al., 2000). As these risk factors are largely associated with PD too, it is reasonable to conclude that common biochemical signaling pathways play a role in vulnerability to both CVD and PD. All the major risk factors associated with PD either activate pathogen initiated inflammation signals (bacteria like P. gingivalis and A. actinomycetemcomitans B. forsythus, P. intermedia, P. micros, and F. nucleatum (Lovegrove, 2004;Saito et al., 2008) or a life style (diabetes mellitus, obesity, aging, smoking, vascular disease) driven inflammatory cascade (Jensen et al., 1991;Grossi and Genco, 1998;Cohen, 2000;Merchant et al., 2003;Humphrey et al., 2008;Hujoel, 2009;Kumar et al., 2011;Nakamura et al., 2011;Ozcaka et al., 2011). Nutrition and oral health are closely linked. This is because oxidative stress and antioxidant balance which drives ROS induced inflammation signals, can be regulated by diets rich in antioxidants (Muniz et al., 2015). Diets that lead to obesity such as high carbohydrates and sugars have been implicated in dental caries and PD as these drive plaque formation and accelerate inflammation thus causing dental tissue oxidative damage and decay (Hujoel, 2009). Changes in dietary intake influence the extent of PD. As seen in the dietary intake of adolescents between 11 and 18 years old, the decrease in consumption of raw fruits and non-potato vegetables with concomitant increase in uptake of soft drinks, led to increased PD (Chaffee and Weston, 2010).

PD AND CVD: IS THERE A LINK?
It is not clear if there is a direct and common thread between PD and CVD; however the fact that people with PD have a two or three times higher risk of a cardiovascular event (stroke, heart attack etc.) seems to point to a cluster of shared risk factors between the two (Sanz et al., 2020). The "inflammation" link seems to be a key contributor to both.
For instance, when infected with P. gingivalis, the host innate immune system responds by activating inflammation consisting of the NLRP3 inflammasome (pro-inflammatory IL-1β, IL-18) (Lamkanfi and Dixit, 2009;Xue et al., 2015). Patients with chronic PD and aggressive PD expressed significantly higher levels of NLRP3 in gingiva (Xue et al., 2015;Ran et al., 2017). In wild type mice with P. gingivalis infection, the increase in expression of NLRP3 inflammation cascade in the gingival tissue was matched with a concomitant increase in caspase-1 activity in the macrophages found in peritoneum; this was not observed in NLRP3 deficient mice (Yamaguchi et al., 2017). This suggests that the NLRP3 inflammasome activated in periodontitis has effects on the systemic organs. NLRP3 inflammasome has also been shown to be highly expressed and activated with systemic vascular disease (Satoh et al., 2014) although it is not clear whether NLRP3 from P. gingivalis (Yamaguchi et al., 2017) is directly involved. However, excess ROS, glucose, ATP, ceramides, sphingosine, cholesterol crystals, uric acid and oxidized LDL (all of which are associated with CVD) have been known to activate NLRP3 inflammasome (Duewell et al., 2010;Jiang et al., 2012;Luheshi et al., 2012).
Epidemiologic data till date that suggest an association between PD and CVD (Humphrey et al., 2008;Blaizot et al., 2009;Tonetti, 2009)  Periodontal treatment resulted in decreased serum CRP and IL-6 at 24-week follow-up Periodontal treatment resulted in reduced acetylcholine-stimulated vasodilation Delivery of a nitric oxide synthase inhibitor before and after PD treatment resulted in similar acetylcholine-stimulated vasodilation, suggesting role of nitric oxide bioavailability in mechanism of endothelial dysfunction in patients with PD Higashi et al., 2009 To evaluate endothelial function and the effects of periodontal treatment on patients with CAD and PD 101 patients with CAD (48 with PD and 53 without PD) -Experimental group: subject to periodontal treatment -Control group: subject to community-based periodontal care -Periodontal treatment resulted in decreased serum CRP and IL-6 at follow-up -Periodontal treatment resulted in reduced acetylcholine-stimulated vasodilation -Delivery of a nitric oxide synthase inhibitor before and after PD treatment resulted in similar acetylcholine-stimulated vasodilation, suggesting role of nitric oxide bioavailability in mechanism of endothelial dysfunction in patients with PD Offenbacher et al., 2009 Impact of periodontal treatment on serum CRP levels, clinical PD parameters, and cardiovascular endpoints in patients with PD Three hundred three patients with PD and previous history of CVD -Experimental group: subject to periodontal treatment (scaling and root planning -Control group: not subject to periodontal treatment

have monitored PD via indices of clinical
Periodontal treatment resulted in a significant improvement in PD status at 6 months as assessed by reduction of probing depth, but no difference in attachment levels, bleeding upon probing, or extent of subgingival calculus Periodontal treatment resulted in significant decrease of the odds of being in the high-risk (>3 mg/L) CRP group at 6 months, with obesity nullifying such effect

(Continued)
Frontiers in Physiology | www.frontiersin.org attachment level, pocket depth, bleeding on probing and decayed-missing-filled teeth and CVD by degree and number of obstructed coronary arteries, observed an association between PD and CVDs (Ketabi et al., 2016). Table 1 shows the clinical trials which investigated the relationship between the two diseases. While risk factors as discussed in the earlier section play a crucial role in the onset of CVD, increasing number of CVD patients do not harbor the classical risk factors. Low grade infection such as in periodontal infection could be a potential cause for CVD in these cases; indeed several studies show that PD as a risk factor for CVD and, in particular, atherosclerosis (Bartova et al., 2014;Toregeani et al., 2016). Potential links between PD and CVD could be via two mechanisms (Figure 3).

Systemic Inflammation: Systemic inflammatory markers such
as C-reactive protein (CRP), IL-6 etc. have shown direct correlation with specific indices of CVD such as carotidintima media thickness, or MI (myocardial infarct size) (Ali et al., 2006). Chronic periodontal infection is characterized by elevation of CRP and inflammatory cytokines in the systemic circulation (Loos et al., 2000), so it is possible that systemic inflammation in patients with PD can potentially accelerate endothelial dysfunction, plaque buildup and CHD events. 2. Vascular Infection: There have been reports that identify bacterial species in blood after dental procedures suggesting gingiva as a portal via which oral bacterial pathogens can enter the systemic circulation (Bahrani-Mougeot et al., 2008;Lockhart et al., 2009). As a result bacteremia of dental origin seems to play a role in the appearance of bacterial endocarditis (Mang-de la Rosa et al., 2014) and periodontal bacterial components colonize human atheromatous plaques (Haraszthy et al., 2000;Fiehn et al., 2005).

CONCLUSION
While both PD and CVD have manifestations of classic inflammation, a causative link between them has not been established. However, oxidative stress arising from lifestyle diseases play a crucial role in progression of both PD and CVD, indicating that host influence in terms of an imbalance between ROS production and endogenous antioxidant levels can increase susceptibility in individuals. Understanding how oxidative stress and inflammation overlap in PD and CVD for high risk and older populations is of great public health importance because of the high prevalence of PD. Although both these pathologies arise from the same risk factors and show a similar systemic inflammation profile, it is not clear how these diseases intersect. Therefore it cannot be concluded that therapeutic periodontal interventions can prevent heart disease or stroke. Nevertheless, controlling the overall inflammation status by implementing a good periodontal maintenance program could presumably control the progression of CVD in periodontitis patients.

AUTHOR CONTRIBUTIONS
OP drafted the article and prepared the figures. PA drafted the article. MM drafted the article and prepared the table. SC developed the concept and outline, drafted the article, and formalized the final version. All authors approved the submission.