ANALYSIS OF PERIODONTAL DISEASE IN ASSOCIATION WITH CORONARY

ARTERY DISEASE AND INFLAMMATORY MEDIATORS IN PATIENTS

UNDERGOING CARDIAC CATHETERIZATION

A

THESIS

Presented to the Faculty of

The University of Texas Health Science Center at San Antonio

Graduate School of Biomedical Sciences

In Partial Fulfillment

Of the Requirements

For the Degree of

MASTER OF SCIENCE

Rachel A. Schallhorn, D.D.S.

San Antonio, Texas

May 2009

ANALYSIS OF PERIODONTAL DISEASE IN ASSOCIATION WITH CORONARY ARTERY DISEASE AND INFLAMMATORY MEDIATORS IN PATIENTS UNDERGOING CARDIAC CATHETERIZATION Rachel A. Schallhorn APPROVED: Supervising professor: Brian L. Mealey, D.D.S., M.S. Devang Patel, MBChB, FACC, FSCAI Thomas Oates, D.M.D., PhD David Lasho, D.D.S., M.S. Paul McLornan, B.D.S. Date APPROVED: Interim Dean, Graduate School of Biomedical Sciences: Robert Reddick, MD

ACKNOWLEDGEMENTS

I would like to thank Drs. Brian Mealey, Devang Patel, Thomas Oates, and David

Lasho for giving me the opportunity to conduct my research under their supervision.

Each member of my supervising committee extended invaluable knowledge, advice, and

guidance throughout the investigatory process. I would like to extend special thanks to

Dr. Bysani Chandrasekar for time and effort spent both in the research laboratory and at

research metings. Without Dr. Chandrasekar’s tireless effort, my investigation would not

have been possible. I would also like to acknowledge the cardiology fellows and staff at

the University Hospital Cardiac Catheterization clinic who helped make the investigation

possible. Finally, I would like to thank my husband, Andrew, for his unfaltering support.

iii

ANALYSIS OF PERIODONTAL DISEASE IN ASSOCIATION WITH CORONARY

ARTERY DISEASE AND INFLAMMATORY MEDIATORS IN PATIENTS UNDERGOING CARDIAC CATHETERIZATION

Rachel A. Schallhorn, M.S.

The University of Texas Health Science Center at San Antonio Graduate School of Biomedical Sciences

Supervising Professor: Brian L. Mealey

Inflammation is a key component in initiation and progression of both periodontal

and cardiovascular diseases. Atherosclerosis, an inflammatory process resulting in

narrowing of the arterial lumen, can lead to development of coronary artery disease

(CAD) predisposing affected patients to acute coronary events. In addition to targeting

the classic risk factors in treatment and prevention of CAD, investigation of potential risk

iv

factors contributing to the inflammatory component of atherogenesis is at the forefront

of preventive cardiology. Interleukin-18 (IL-18) is a pro-inflammatory cytokine present

in chronically inflamed tissues, which has been positively associated with increased

inflammation and probing depths in patients with periodontitis. Additionally, IL-18 has

been associated with clinical signs of an unstable, symptomatic atherosclerotic plaque.

CXC ligand 16 (CXCL 16) is a chemokine identified in endothelial cells, smooth muscle

cells and macrophages. CXCL 16 is induced by IL-18 expression and functions as an

adhesion molecule and scavenger receptor. Investigations of the association between

periodontitis and CAD have identified both direct and indirect effects of the chronic

inflammatory lesion of periodontitis on the cardiovascular system. The primary aim of

this research study was to correlate periodontal parameters with serum levels of IL-18

and CXCL 16 to evaluate a potential indirect association between periodontitis and CAD.

The study population was a convenience sample of 51 patients presenting for

clinically indicated coronary angiography. Clinical information collected prior to

catheterization included demographics, body mass index, blood pressure, history of statin

use, smoking status, presence of cardiac risk factors such as diabetes and family history,

and serum C-reactive protein levels. CAD was classified according to presence and

extent as follows: no CAD, non-obstructive CAD (<50 % occlusion), and CAD (>50 %

occlusion) affecting 1-, 2-, 3-, or 4-vessels. Venous blood was collected at the time of

catheterization and used to determine serum levels of inflammatory mediators IL-18 and

CXCL16. Periodontal status was evaluated by a full mouth clinical periodontal exam at 6

sites per tooth. The percent of sites with probing depth (PD) ≥5 mm and ≥7 mm, clinical

attachment loss (CAL) ≥3 mm and ≥5 mm, and bleeding on probing (BOP) were

v

recorded to determine the extent and severity of periodontitis. Correlations between

periodontal parameters, levels of inflammatory mediators, and CAD status were analyzed

using the univariate analysis Spearman Rank Correlation and multiple regression.

The percent of sites with BOP exhibited a significant positive correlation with

serum levels of IL-18 in non-parametric univariate analysis (p=0.0389) indicating a

correlation between periodontal inflammation and systemic levels of IL-18. When

multiple regression analysis was performed, extent of CAL ≥3 mm (p=0.0452) and CAL

≥5 mm (p=0.0242) exhibited a significant association with IL-18. Statin use also

exhibited a significant association with IL-18 (p=0.0361). Multiple regression analysis

of CXCL16 identified a statistically significant association with CAL ≥5 mm (p=0.040)

and PD ≥7 mm (p=0.047). In addition a statistically significant association between

CXCL16 levels and smoking was also identified (p=0.0054). Multiple regression

analysis of CAD also identified a significant association with CAL ≥3 mm (p=0.0471).

Periodontal inflammatory mediators are similar to those involved in development

of the atherosclerotic lesion. Analysis of our patient population suggests a significant

association between periodontitis and serum levels of IL-18, CXCL16 and the presence

of CAD. Elevated levels of IL-18 and CXCL16 associated with the inflammatory lesion

of periodontitis may indirectly contribute to development of atherosclerosis and thus

place patients at increased risk for CAD.

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TABLE OF CONTENTS

Page

Title ……………………………………………………………………………. i

Approval ………………………………………………………………………. ii

Acknowledgements ……………………………………………………………. iii

Abstract ……………………………………………………………………….. iv

Table of Contents ………………………………………………………........... v

List of Tables ………………………………………………………………….. vi

1. INTRODUCTION AND LITERATURE REVIEW ………………….. 1

A. Periodontal Disease as an Inflammatory Disease ………… 1

B. Coronary Artery Disease as an Inflammatory Disease …….. 6

C. Periodontal Disease and Coronary Artery Disease Interrelationships ………………………………………. 8

D. Interleukin-18, CXCL16 and Inflammatory Diseases …….. 12

E. Specific Research Objectives ……………………………… 16

2. MATERIALS AND METHODS ………………………………………. 18

A. Patient Population ………………………………………… 18

B. Cardiac Catheterization and Clinical Data Collection ……… 19

C. Periodontal Examination …………………………............… 20

D. Serum Levels of Inflammatory Mediators …………............. 22

E. Data Entry and Statistical Analysis …………………............ 23

3. RESULTS ……………………………………………………………….. 26

4. DISCUSSION AND SUMMARY ………………………………............. 42

5. LITERATURE CITED ………………………………………………….. 50

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viii

LIST OF TABLES

Page

Table 1 Summary Statistics ……………………………………................. 29

Table 2 Coronary Artery Disease Status ………………………................. 30

Table 3 Periodontal Disease Parameters …………………………………. 31

Table 4 Serum Levels of Inflammatory Mediators ………………………. 35

Table 5 Non-Parametric Univariate Analysis of Serum Levels of IL-18 and Periodontal Parameters (Spearman Rank Correlation) ……… 36

Table 6 Non-Parametric Univariate Analysis of Serum Levels of CXCL16

and Periodontal Parameters (Spearman Rank Correlation) ……… 37 Table 7 Non-Parametric Univariate Analysis of CAD and Periodontal

Parameters (Spearman Rank Correlation) ……………………….. 38 Table 8 Multiple Regression Analysis of Serum IL-18 …………………... 39

Table 9 Multiple Regression Analysis of Serum CXCL16 ………………. 40

Table 10 Multiple Regression Analysis of CAD …………………………… 41

I. INTRODUCTION AND LITERATURE REVIEW

A. Periodontitis as an Inflammatory Disease

Periodontal disease is characterized as a chronic, low-grade infection involving

the supporting structures of the teeth. While bacteria are a necessary etiologic agent they

are not sufficient to establish disease. Evidence clearly demonstrates the role of

inflammation in periodontal disease initiation and progression. The host response to

bacteria within a biofilm ranges from the initial, neutrophil-dominated lesion of gingivitis

to the established, plasma cell-dominated lesion of periodontitis (Page 1976). The long-

standing lesion of periodontitis represents a chronic bacterial infection not effectively

neutralized by host immune defenses, thus perpetuating an inflammatory state. Although

active infection is generally localized to the periodontal pocket, bacteria and bacterial

products, such as lipopolysaccharide (LPS), as well as locally produced inflammatory

mediators may gain access to the systemic circulation through the ulcerated pocket

epithelium (Loos 2000, Loos 2005).

Numerous studies evaluating local and systemic levels of inflammatory mediators

have found a positive correlation with periodontal disease. Among the studied

inflammatory mediators, those identified as having a positive association with

periodontitis include C-reactive protein (CRP), interleukin-1 (IL-1), interleukin-6 (IL-6),

interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), and prostaglandin E2 (PGE2)

among many others. Analysis of the third National Health and Nutrition Examination

Survey (NHANES III) revealed a positive correlation between extensive periodontal

disease and serum CRP levels (Slade 2000). In a cross-sectional study evaluating

1

periodontal status and subgingival periodontal pathogens, Noack and colleagues

identified significantly higher mean CRP levels in subjects with periodontal disease

versus periodontally healthy subjects (Noack 2001). Additionally, the presence of one or

more periodontal pathogens had a direct and significant correlation with mean CRP

levels. Analysis of periodontal status and CRP levels in a cohort of men enrolled in a

study of cardiovascular disease showed a significant association between advanced

periodontitis and elevated CRP levels, adjusting for cardiovascular risk factors, at two

time-points over a 20-year period (Linden 2008). An intervention trial evaluating CRP

and IL-6 in 94 patients with generalized severe periodontitis, demonstrated significant

decreases in serum levels of both inflammatory mediators over the 6 month observation

period (D’Aiuto 2004). In addition, initial assessment of the participants revealed a

significant association between more extensive periodontitis (greater than the median

number of periodontal pockets with PD ≥5 mm) and CRP levels >3 mg/L. A recent

systematic review and meta-analysis of CRP in relation to periodontitis concluded “there

is strong evidence from cross-sectional studies that plasma CRP in periodontitis is

elevated compared with controls” and “modest evidence on the effect of periodontal

therapy in lowering the levels of CRP” (Paraskevas 2008).

In addition to acute phase reactants, signaling molecules involved in orchestration

of the immune response, such as cytokines and chemokines, have also been implicated in

the pathogenesis of periodontal disease. According to the American Academy of

Periodontology Academy Report, the pathogenic mechanisms of periodontitis are not

fully understood, however inflammatory mediators consistently associated with

periodontitis include the following: IL-1 and TNFα, pro-inflammatory multifunctional

2

cytokines; IL-6, a pro-inflammatory cytokine involved in osteoclast formation and

plasma cell proliferation; IL-8, a pro-inflammatory chemokine produced by monocytes in

response to lipopolysaccharide and pro-inflammatory cytokines; and prostaglandin E2

which induces bone resorption and matrix metalloproteinase secretion (American

Academy of Periodontology 1999). A study evaluating untreated periodontitis patients

versus healthy, age-matched controls identified a positive association between

radiographic bone loss and serum levels of CRP, IL-6, and neutrophil count (Loos 2000).

In a case series of patients classified at high risk for atherosclerosis, non-surgical

periodontal therapy combined with local delivery of minocycline-HCl significantly

reduced circulating levels of both CRP and TNFα (Iwamoto 2003).

Data from studies of the local environment of a periodontal lesion parallel

findings from studies evaluating levels of inflammatory mediators in the systemic

circulation. Comparison of gingival crevicular fluid (GCF) samples collected from

healthy gingival sulci versus periodontal pockets further demonstrates the heightened

level of inflammation associated with active periodontal disease. Prostaglandin E2, a

multifunctional proinflammatory mediator, has been associated with progressive

attachment loss in both human and animal studies (Offenbacher 1993). In a study

comparing GCF IL-1β levels among patients with varying severities of periodontal

disease, increased levels of the pro-inflammatory cytokine were detected in both site-

based and patient-based analyses. Sites with increased probing depth and attachment

loss, as well as patients with severe versus mild-moderate disease, exhibited higher levels

of GCF IL-1β (Engbretson 2002). A recent study evaluated GCF levels of IL-1β, IL-11,

and IL-12 in chronic periodontitis, gingivitis, and periodontal health. Results indicated

3

chronic periodontitis patients had significantly higher levels of IL-1β and IL-12, while

IL-11 exhibited an inverse correlation with periodontal disease (Yucel 2008). An

intervention trial evaluating the effect of surgical anti-infective therapy for peri-

implantitis on local levels of TNFα identified a significant decrease in TNFα in the

crevicular fluid (Duarte 2009). Moreover decreased TNFα levels were positively and

significantly correlated with decreased probing depth and bleeding on probing.

In addition to fluid collected from the periodontal pocket, tissue and cells

collected from diseased sites exhibit similar findings. In a study of twelve patients with

periodontal disease, tissue biopsies harvested from both active and stable sites showed

elevated levels of IL-1β in sites exhibiting attachment loss over the study period

(Stashenko 1991). Local inflammatory infiltrate and mRNA expression of pro-

inflammatory cytokines has been shown to differ between diseased and healthy patients.

A research study comparing gingival biopsies between healthy patients and those with

either chronic or generalized aggressive periodontal disease, found the diseased patients

had significantly increased neutrophil infiltration and mRNA expression of IL-1β and

TNF-α (Liu 2001). In vitro studies have demonstrated cells harvested from inflamed

sites maintain their phenotypic characteristics and have increased expression of pro-

inflammatory cytokines compared with cells from non-inflammed tissue. Gingival

fibroblasts harvested from sites affected by periodontitis have been shown to produce

more IL-6 constituitively and following treatment with bacterial LPS and IL-1α (Kent

1999). Increased expression of pro-inflammatory cytokines by human gingival

fibroblasts harvested from chronic periodontitis patients has also been shown to

positively correlate with their in situ expression within the tissue from which the cells

4

were harvested. Fibroblasts from diseased tissue were shown to have higher

constituitive expression of IL-6 and IL-8 compared to those from healthy sites;

additionally, authors suggested the cells were phenotypically inclined to secrete increased

levels of the inflammatory mediators (Dongari-Bagtzoglou 1998).

Emerging evidence points to a role for more recently discovered cytokines in the

pathogenesis of periodontitis. Interleukin-17 (IL-17) is a novel, multi-functional cytokine

which is thought to be involved in both the innate and adaptive immune responses.

Available evidence promotes the role of IL-17 in autoimmunity and bone loss (Kramer

2007). Analysis of gingival biopsies from sites with minimal (0-2 mm), moderate (3-4

mm), and severe (≥5 mm) CAL revealed a significant positive correlation between CAL

and concentrations of IL-23, IL-17, IL-6, IL-1β, and TNFα (Lester 2007). In addition to

IL-17, interleukin-18 (IL-18) has been implicated in maintenance of local chronic

inflammation (Delaleu 2004). An investigation evaluating cytokine levels in human

gingival biopsies found a significant positive correlation between IL-6 and IL-18

concentrations and probing depth (Johnson 2005).

In addition to identifying an association between inflammatory mediators and

periodontal disease, a causal effect has been established in animal models. An

investigation in non-human primates, utilizing local injection of IL-1 soluble receptors

combined with experimental periodontitis, identified increased progression of

inflammation toward alveolar bone compared to non-injected control animals (Graves

1998). Conversely, inhibition of IL-1 and TNF activity utilizing soluble receptor

blockers has been shown to inhibit loss of connective tissue attachment in a similar

animal model (Delima 2001). Gene therapy has also been utilized in animal models to

5

reduce levels of inflammatory mediators and limit alveolar bone loss (Cirelli 2008).

Establishment of a cause-and-effect relationship between inflammatory cytokines and

periodontitis provides a potential target for innovative periodontal therapy.

B. Coronary Artery Disease as an Inflammatory Disease

Coronary artery disease (CAD) is a leading cause of morbidity and mortality in

the United States. A major component in development of CAD is atherosclerosis, an

inflammatory process that can result in narrowing of the affected arterial lumen

predisposing affected patients to acute coronary events. The progression of

atherosclerosis, as described by Ross (1999), consists of 4 distinct stages. Initially

endothelial dysfunction within the affected artery occurs. The result is increased

endothelial permeability to lipoproteins and other molecules in the plasma as well as up-

regulation of leukocyte and endothelial adhesion molecules. Fatty streak formation

follows endothelial dysfunction; it is characterized by accumulation of foam cells (lipid-

filled macrophages) and T lymphocytes within the vessel wall, vascular smooth muscle

cell proliferation, and platelet adherence and aggregation. Since resolution of the lesion

cannot be achieved, the inflammatory process continues ultimately forming a fibrous cap

covering the lesion. At this point the atheroma or plaque is considered an advanced,

complicated lesion. In the final stage, the fibrous cap becomes ruptured or ulcerated.

The lesion is considered an unstable fibrous plaque and the affected artery is at high risk

for thrombosis (Ross 1999). Inflammatory mediators orchestrate the complex sequence

of events leading to development of an advanced atherosclerotic lesion (Zernecke 2005).

Of the mediators involved, pro-inflammatory cytokines and chemokines play a prominent

role.

6

In addition to targeting the classic risk factors in the treatment and prevention of

cardiovascular disease (CVD), investigation of potential risk factors contributing to the

inflammatory component of atherogenesis is at the forefront of preventive medical

research. Classic risk factors include smoking, serum cholesterol and obesity, while

examples of potential risk factors include elevated levels of inflammatory markers such

as CRP and pro-inflammatory cytokines (Lowe 2001). The relationship between CRP

and cardiovascular disease has been extensively studied. A 2003 American Heart

Association and Center for Disease Control scientific statement concluded measurement

of high sensitivity-CRP, as an adjunct to assessment of “major” risk factors, may be

useful in assessing risk for adverse cardiac events (Pearson 2003). Interleukin-6 and

TNFα have also been evaluated in relationship to cardiovascular diseases, albeit to a

lesser extent than CRP. A prospective study of 14,916 apparently healthy men found

elevated levels of serum IL-6 were associated with occurrence of myocardial infarction

over the study period (Ridker 2000a). A cross-sectional study evaluating 147 patients

undergoing coronary angiography demonstrated serum levels of IL-6, fibrinogen, and

CRP positively correlated with extent of coronary stenosis (Erren 1999). In a nested

case-control study evaluating patients who previously had myocardial infarction (MI),

patients experiencing a recurrent MI within the 5 year follow-up period (cases) had

persistently elevated plasma levels of TNFα compared to controls (Ridker 2000b).

Evidence clearly demonstrates systemic inflammation is elevated in patients with

coronary artery disease. The degree and extent to which specific inflammatory mediators

influence the disease process is not, however, clearly understood. Deciphering the

7

complex chain of events leading to atherosclerosis and identifying potential therapeutic

targets remains the focus of ongoing research.

C. Periodontal Disease and Cardiovascular Disease Interrelationships

While an association between periodontitis and coronary artery disease has been

established, the mechanism of this association has not been fully elucidated.

Investigations have focused on both the direct and indirect effects of chronic

inflammation in the periodontium on the cardiovascular system. The direct systemic

effects of periodontitis involve local elements of the periodontal pocket that have gained

access to the systemic circulation. Bacteremia and endotoxemia of oral origin can result

from various stimuli in the oral cavity and have been positively correlated with

periodontitis. A study evaluating systemic levels of endotoxin following mastication,

found a significantly increased level and prevalence of endotoxemia in patients with

severe periodontitis compared to healthy controls (Geerts 2002). An investigation of

periodontal probing identified oral bacteria in the peripheral venous blood in 40% of

periodontitis patients versus 10% of gingivitis patients (Daly 2001). Bacteremia was

significantly associated with bleeding on probing and probing depth. A mechanism by

which bacteria may affect the vasculature is through systemic release of endotoxin.

Available evidence in cardiovascular literature also implicates endotoxin in promoting

chronic inflammation and predisposing patients to atherosclerosis. Endotoxin is thought

to contribute to atherosclerosis through induction of proinflammatory cytokines,

chemokines, acute phase reactants, and adhesion molecules (Stoll 2004). Results from a

large-scale prospective study evaluating changes in carotid atherosclerosis over a 5-year

period demonstrated 40% of new atherosclerotic lesions were attributable to chronic

8

systemic infection (Kiechl 2001). Another notable observation was that the risk of

atherosclerosis tended to be higher in patients with chronic infection and a prominent

inflammatory response; authors implicate either highly virulent pathogens or an

atherogenic host response.

In addition to transiently entering the vascular system, bacterial DNA has been

isolated from atheroma specimens; however, there is conflicting evidence as to their

origin. In an analysis of 50 carotid atheroma specimens, 44% were found to contain 16S

rDNA from periodontal pathogens (Haraszthy 2000). A more recent study analyzed

subgingival plaque samples and atheroma specimens in 33 patients undergoing carotid

endarterectomy. While bacterial DNA was isolated in 31 out of 33 atheroma specimens,

no DNA from periodontal pathogens was found (Aimetti 2007). A similar investigation

identified the same periodontal pathogens in subgingival and coronary artery

atherosclerotic plaque samples in 10 out of 20 patients. Furthermore patients with

bacteria in their atherosclerotic lesions had a significantly higher mean bleeding score

(Zaremba 2007). Results from the aforementioned studies confirm that bacteria, bacterial

products, and inflammatory mediators involved in the pathogenesis of periodontitis do

not remain at the site of infection. While the effect these organisms and mediators have

upon other systems in the body is not known, it does represent a potential link between

oral and systemic disease.

The indirect association between periodontitis and systemic disease may be

mediated through upregulation of inflammatory mediators in the systemic circulation due

to localized infection. The phenomenon of increased systemic inflammation resulting

9

from a local infection is well supported in periodontal literature, as cited in section A of

the literature review.

In order to explore the systemic effect of reducing or eliminating infection and

inflammation in the oral cavity, it is necessary to examine results from intervention trials.

Published data from interventional trials demonstrates a positive systemic effect can be

achieved with periodontal treatment. Full mouth extraction, performed in adults

diagnosed with severe periodontitis and a terminal dentition, has been shown to

precipitate significant decreases in systemic levels of CRP, plasminogen activator

inhibitor, and white blood cell counts (Taylor 2006). A pilot intervention trial evaluated

the effects of traditional versus intensive initial periodontal therapy in a group of 40,

otherwise healthy, severe chronic periodontitis patients (D’Aiuto 2006). The intensively

treated group received local antimicrobial therapy in addition to scaling and root planing.

Intensive initial therapy resulted in significant decreases in IL-6, CRP, total cholesterol,

and systolic blood pressure.

A recent focus of intervention trials has been the association between periodontal

disease and endothelial dysfunction. Endothelial dysfunction is a component of coronary

artery disease and is characterized by a decreased vascular response to increased shear

stress on the vascular wall in combination with normal dilation in response to sublingual

nitroglycerine. An investigation of endothelial function in 30 severe periodontitis

patients and 31 periodontally healthy control patients identified significantly lower

baseline flow-mediated vasodilation in patients with periodontitis (Seinost 2005). Non-

surgical periodontal therapy resulted in a significant improvement in endothelial function

and a concomitant decrease in CRP levels. The results of standard versus intensive

10

periodontal therapy on endothelial dysfunction were analyzed in a randomized

controlled clinical trial (Tonetti 2007). One-hundred and twenty patients with

generalized severe periodontitis were assigned to receive either supragingival scaling

(control) or scaling and root planing with local minocycline delivery and extraction of

hopeless teeth (intensive). Results indicated scaling and root planing increased levels of

CRP and IL-6 as well as endothelial activation markers E-selectin and vonWillebrand

factor. In addition, reduced flow-mediated dilation indicating endothelial dysfunction was

seen within the first 24 hours after scaling and root planning. Re-evaluation at 2 and 6

months, however, showed flow-mediated dilation was significantly higher in the test

group, suggesting a long-term benefit from intensive periodontal therapy. The above

cited intervention trials demonstrate a direct relationship between periodontal

inflammation and endothelial dysfunction, thus lending strength to the association

between periodontal and coronary artery diseases.

Review of the evidence clearly demonstrates inflammation is a key component in

initiation and progression of both periodontal and cardiovascular disease. The pathologic

process in both cases results from tipping the delicate balance of inflammation toward a

destructive rather than protective pathway. Although much has been discovered through

clinical and laboratory investigations, the pathogenesis and interrelationship of

periodontal disease and atherosclerosis are not fully understood. Evaluating periodontal

and cardiovascular diseases is difficult due to their chronic and multifactorial nature.

Contributing factors involved in the pathogenesis of both diseases, such as smoking and

diabetes, also obscure the relationship. A review of the available evidence reveals

significant heterogeneity among research studies both in study design and definitions of

11

disease (Beck 2005); hence a strong relationship between periodontal and cardiovascular

disease cannot be readily established.

Despite the relative lack of prospective longitudinal studies and intervention

trials, available evidence supports a moderate association between periodontal disease

and atherosclerosis-induced diseases such as CAD, stroke, and peripheral vascular

disease (Scannapieco 2003). An evaluation of clinical periodontal parameters and

radiographic alveolar bone loss (ABL) in 110 patients diagnosed with coronary heart

disease versus 140 healthy controls identified a statistically significant association

between ABL >4 mm and coronary heart disease, with an odds ratio of 6.6 (Geismar

2006). Another case-control study evaluating periodontal status in 90 males, 45 with and

45 without CAD, found significantly deeper mean probing depths (PD) and greater mean

clinical attachment loss in patients with angiographically confirmed CAD

(Nonnenmacher 2007). While the majority of research studies evaluating this

relationship are cross-sectional in nature and thus cannot establish causality, they do

contribute to our current body of knowledge and provide a basis for future research.

D. Interleukin-18, CXCL16 and inflammatory diseases

Advancements in technology have led to an exponential increase in our

knowledge of the immune system and pathogenesis of disease. These innovations have

led to the discovery of previously unidentified inflammatory mediators whose roles in

inflammatory processes are currently being investigated. Among these more recently

identified inflammatory mediators are those of primary interest in this investigation:

interleukin-18 (IL-18) and CXCL16.

12

Interleukin-18 (IL-18) is a pro-inflammatory cytokine and is a member of the

interleukin-1 superfamily. It is expressed in sites of chronic inflammation. IL-18 has

been shown to induce IFNγ, both alone and synergistically with IL-12, hence promoting

the Th1 immune response (Johnson 2005). In order for inflammation to resolve, a shift

from a Th1 dominated response to a Th2 response must generally occur. Researchers

have hypothesized a sustained Th1 response leads to development of chronic

inflammation (Gemmell 2004). Upregulation of IL-18 has been identified in association

with several chronic diseases including inflammatory bowel disease (Sanches-Munoz

2008), Sjogren’s syndrome (Manoussakis 2007), systemic lupus erythematosus,

rheumatoid arthritis (Mosaad 2003), and atherosclerosis.

Immunohistochemistry of human atheroma tissue identified IL-18 and its

functional receptor localized within lesional macrophages; neither IL-18 nor its receptor

were found in non-diseased human arterial tissue (Gerdes 2002). When human

macrophages and smooth muscle cells were treated with recombinant human IL-18, they

induced IFNγ expression. Taken together, these observations suggest IL-18 is involved in

development of atherosclerotic lesions. While IL-18 is known to be involved in the early

stages of inflammation, it also appears to modulate the cytokine cascade at later points

(Gracie 2002). Increased levels of IL-18 are present during acute coronary syndromes.

In order to evaluate the association between IL-18 and later stages of atherosclerosis, IL-

18 expression was analyzed in 40 human carotid atherosclerotic plaques and correlated to

intraplaque ulceration and clinical symptoms (Mallat 2001). Results revealed IL-18

expression was significantly higher when clinical signs of an unstable, symptomatic

plaque were evident and when plaque ulcerations were present.

13

The association between IL-18 and periodontal disease has also been

investigated, albeit to a lesser extent than in the cardiovascular literature. While

laboratory studies have linked IL-18 expression to periodontal pathogens, clinical studies

have correlated IL-18 levels with the presence and severity of periodontal disease. An in

vitro study, evaluating cytokine expression in human monocytes, demonstrated exposure

to Porphyromonas gingivalis resulted in IL-18 expression (Hamedi 2008). Clinical

investigations have evaluated IL-18 levels in periodontal tissues and GCF. Johnson and

Serio (2005) correlated levels of IL-18 in gingival biopsies to parameters of periodontal

inflammation and probing depth. Interleukin-18 was significantly higher in tissue

harvested from sites with severe disease (PD >6 mm) compared to healthy sites. Assays

of GCF in 10 periodontitis and 10 gingivitis patients demonstrated IL-18 levels were

significantly higher in patients with periodontitis (Orozco 2006). A more recently

published study reached a similar conclusion. Pooled subgingival plaque and GCF

samples from deep (PD ≥5 mm) and shallow (PD ≤3 mm) sites of 18 periodontitis

patients were analyzed; investigators found significantly higher IL-18 levels compared to

gingivitis patients (Figueredo 2008). No association was identified between IL-18 and

the presence of red complex bacteria. Available evidence clearly demonstrates an

association between periodontal disease and local expression of IL-18. The effect

periodontal inflammation has on systemic IL-18 levels, and the sequelae of the

relationship, is yet to be determined.

CXCL16 is a pro-inflammatory chemokine. It has been shown to promote cell-

cell adhesion, act as a scavenger receptor promoting foam cell formation, and induce

smooth muscle cell proliferation. CXCL16 exerts its effects through transmembrane

14

receptor CXCR6. Lipopolysaccharide has been shown to upregulate CXCR6, the

receptor for CXCL16, in human aortic smooth muscle cells resulting in delayed but

persistent increases in level of expression (Patel 2006). Results from this investigation

promote the idea that endotoxemia or chronic low levels of endotoxin may contribute to

atherogenesis. CXCL16 and CXCR6 have been found to be expressed in both human

and murine atherosclerotic lesions. An investigation evaluating the effect of IFNγ on

human monocytes found IFNγ can induce upregulation of CXCL16 which then results in

increased ox-LDL uptake by the monocytes (Wuttge 2004). Monocytic uptake of ox-

LDL results in foam cell formation, a component of early atherosclerosis.

Less evidence is available linking CXCL16 with periodontal disease. Hosokawa

and colleagues (2001) investigated CXCL16 and CXCR6 mRNA expression within

diseased and healthy periodontal tissues. Although CXCL16 mRNA was detected in

healthy periodontal tissue, it was more heavily expressed within diseased tissue. Further

analysis of the diseased tissue demonstrated CXCL16 was strongly expressed by

fibroblasts and CXCR6-positive cells were generally distributed near the sulcular

epithelium. Moreover, harvested human gingival fibroblasts were shown to significantly

upregulate CXCL16 mRNA expression when treated with pro-inflammatory cytokines

IL-1β, TNF-α, and IFN-γ (Hosokawa 2007). Considering the available evidence,

CXCL16 is likely involved in the pathogenesis of both atherosclerosis and periodontitis.

It is hypothesized that circulating levels of CXCL16 may serve as a marker to

predict future development of coronary heart disease (CHD) (Sheikine 2008).

Conflicting evidence exists, however, suggesting CXCL16 may have an atheroprotective

effect. In a laboratory study using a murine model, investigators found CXCL16-

15

deficient animals had increased inflammation and accelerated atherosclerosis. While

CXCL16 levels were found to increase in the aortic arch under pro-atherogenic

conditions, authors suggest scavenger receptor activity of CXCL16 may in fact be

atheroprotective (Aslanian 2006). In human subjects, significantly lower levels of serum

CXCL16 were identified in stable angina patients versus healthy controls; again

suggesting a relationship between atherosclerosis and lack of CXCL16 and its protective

activity (Sheikine 2006).

CXCL16 may work synergistically with IL-18 in the pathogenesis of

atherosclerosis. Results from a laboratory study evaluating the effects of IL-18 in rat

aortic smooth muscle cells, demonstrated IL-18 induces smooth muscle cell proliferation

in a CXCL16-dependent manner (Chandrasekar 2005). The aforementioned study

implicates IL-18-CXCL16 cross-talk in atherogenesis. Laboratory studies evaluating

atherogenesis in immunodeficient mice have found that the proatherogenic role of IL-18

may partially depend on IFNγ-mediated upregulation of CXCL16 (Tenger 2005). It is

evident both IL-18 and CXCL16 have a role in coronary artery disease and possibly

periodontal disease as well. The extent to which the inflammatory mediators influence

the disease process, however, is unknown.

E. Specific Research Objectives

The association between periodontal disease and cardiovascular disease has long

been recognized, however the mechanisms behind this association remain obscure.

Numerous studies have been published over the past few decades including case-control,

cross-sectional, and longitudinal observational studies. The majority of studies have

identified a low to moderate, yet significant, level of association between periodontitis

16

and cardiovascular disease. A major goal in treatment of any chronic disease is

identifying and attempting to control exposure to factors placing patients at risk for

development of the disease. Increased levels of circulating pro-inflammatory mediators

have been identified as potential risk factors for development of coronary artery disease

and associated adverse cardiac events. Further elucidation of the indirect relationship

between periodontal and cardiovascular diseases may lead to improved medical care for

at-risk patients.

The purpose of this research study was to examine the relationship between

periodontal disease and coronary artery disease in association with pro-inflammatory

mediators. Therefore, the specific aims were as follows:

1. To investigate the association between periodontal disease and serum levels of

inflammatory mediators IL-18 and CXCL16.

2. To investigate the association between periodontal disease and coronary artery

disease.

3. To investigate the association between inflammatory mediators IL-18, CXCL16, and

coronary artery disease.

The overall goal of the study was to investigate specific systemic inflammatory

consequences of periodontal disease and their potential indirect role in contributing to the

development of coronary artery disease and predisposing affected patients to adverse

cardiac events. A positive correlation between periodontal disease and the inflammatory

markers IL-18, and CXCL16 could represent a common pathogenic mechanism between

periodontal disease and coronary heart disease and might infer increased risk for

development of adverse cardiac events in patients with periodontitis.

17

II. MATERIALS AND METHODS

A. Patient Population

The study population was a convenience sample consisting of patients presenting

for clinically indicated coronary angiography at the University Hospital cardiology clinic

of the University of Texas Health Sciences Center San Antonio (UTHSCSA). An

ongoing research study was in progress at the facility with a specific aim to examine the

relationship between IL-18, CXCL 16 and CAD; the patients receiving a periodontal

examination are a subset of this larger patient population. The study protocol was

approved by the Institutional Review Board of UTHSCSA. Each patient read and signed

a detailed consent form prior to participation in the study.

Patients were recruited during their pre-catheterization appointment, which is

generally scheduled approximately one week prior to the cardiac catheterization. A

cardiology fellow performed pre-catheterization examinations and identified patients

meeting the inclusion criteria for participation in the research study. Patients consenting

to participate received a periodontal examination during their pre-catheterization

appointment. At the time of cardiac catheterization the presence, severity, and extent of

CAD was determined and recorded. A blood sample was obtained from the patient at the

time of catheterization for analysis of inflammatory mediators.

Exclusion criteria for participation in the study included the following: patients

requiring endocarditis prophylaxis, patients unable or unwilling to undergo diagnostic

angiography, patients unable or unwilling to participate in the research study, patients

with active systemic infections evident by fevers and leukocytosis, patients on antibiotics

recovering from systemic infection, immunosuppressed patients due to HIV/AIDS,

18

chemotherapy, chronic steroid use, patients with autoimmune disorders such as SLE,

rheumatoid arthritis, scleroderma, or other significant connective tissue diseases, patients

admitted for chest pain or acute coronary syndromes associated with the use of cocaine.

Patients with active infection, systemic conditions outlined above, or use of certain

medications including antibiotics or steroids were excluded due to the effect these

variables can have on systemic cytokine levels.

B. Cardiac Catheterization and Clinical Data Collection

Clinical data collection was performed at the time of the pre-catheterization

examination. Clinical data collected include patient characteristics and results of

pertinent laboratory tests and are listed as follows: age, gender, race, smoking status,

patient reported family history of cardiovascular disease, body mass index (BMI) as

calculated from height and weight, history of statin medication use, blood pressure,

triglycerides, total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein

(HDL), serum CRP, and hemoglobin A1c values for diabetic patients. Smoking status, as

determined by patient report, was classified into four categories as follows: 1= never

smoke, 2= quit more than one year prior, 3= quit four weeks to one year prior, and 4=

active smoker. Information regarding patient characteristics was recorded at the pre-

catheterization appointment. Laboratory values were collected from recent tests, which

had been recorded in the patients’ electronic medical records.

Cardiac catheterization was performed at University Hospital in the cardiology

unit based upon clinical indications. According to the American College of Cardiology

and American Heart Association Practice Guidelines, clinical indications for coronary

angiography include, but are not limited to, the following: non-invasive test results

19

indicate high risk for adverse outcomes, severely symptomatic angina patients who are

receiving medical treatment, patients who have been resuscitated from sudden cardiac

death, and patients who have sustained monomorphic ventricular tachycardia or

nonsustained polymorphic ventricular tachycardia (Scanlon 1999). The majority of

patients enrolled in the present study were referred for coronary angiography based upon

results from a recent, non-invasive stress test. A minority of the patients enrolled in the

study were referred for coronary angiography as a preoperative evaluation prior to

noncardiac surgery.

Results from the coronary angiography were obtained from the patients’

electronic health records as it was not feasible for the investigator to be present during

each of the catheterization procedures. The coronary angiography procedure evaluated

the presence and extent of coronary artery stenosis, left ventricular end-diastolic pressure,

left ventricular ejection fraction, and the treatment decision based upon catheterization

results. If occlusion of a vessel was identified, it was recorded by the percent of stenosis.

Stenosis less than 50% was diagnosed as non-obstructive CAD and ≥50% stenosis was

classified as CAD. When CAD was present, the number of vessels affected were

recorded, which represented extent of the disease.

C. Periodontal Examination

Periodontal examination was performed during the pre-catheterization

appointment prior to cardiac catheterization. Patients were assigned an identification

number at the time of periodontal examination; the same number was used on clinical

data collection forms and for labeling blood samples. All examinations were performed

by Dr. Rachel Schallhorn utilizing a portable headlamp to aid in visualization. Full

20

mouth periodontal examinations were performed evaluating all teeth present, excluding

third molars. A University of North Carolina (UNC) probe was utilized to evaluate six

sites per tooth for probing pocket depth (PD), recession, and bleeding on probing (BOP).

Bleeding on probing was recorded using a dichotomous index (present or absent).

Clinical attachment level (CAL) was calculated by adding recession and PD. A

dichotomous, visual plaque index was recorded at four sites per tooth.

Information collected from the periodontal exam was recorded on a periodontal

charting form and later entered into an electronic spreadsheet. When evaluating PD, two

thresholds were set representing moderate disease with PD ≥5 mm and severe disease

with PD ≥7 mm. Probing depth is a standard measure of periodontal disease. Deep PD is

associated with increased numbers of periodontal pathogens within the subgingival

plaque (Socransky 1998) and progressive loss of attachment (Claffey 1990). The

inflammation of the supporting periodontal tissues generally leads to loss of connective

tissue attachment and alveolar bone. A standard measure for attachment loss and alveolar

bone destruction is clinical attachment level (CAL). Loss of clinical attachment reflects a

history of destructive periodontitis and does not necessarily indicate active inflammatory

disease. In order to measure active inflammation, BOP was evaluated. Bleeding on

probing has consistently been associated with active inflammation and disease

progression, both clinically (Schaetzle 2003) and histologically (Greenstein 1984). For

each patient, periodontal disease parameters were analyzed as the percent of sites

involved. Analyzing patients by frequency distributions, rather than mean values, takes

into account the site specific nature of periodontal disease. Epidemiologic studies have

shown severe periodontitis more frequently affects posterior teeth (Albandar 1999).

21

Calculating mean values can negate localized deep PD if shallow PD are also present,

and a patient with severe disease could be potentially misrepresented.

D. Serum Levels of Inflammatory Mediators

Serum levels of inflammatory mediators were determined using blood samples

obtained from patients during the cardiac catheterization. The catheterization procedure

necessitates placement of a sheath into the femoral artery; after successful completion of

the procedure, 10 mililiters of blood were collected from the sheath access. Once blood

samples were collected they were stored at -80°C in the research laboratory of Dr. Bysani

Chandrasekar at UTHSCSA. Serum was obtained by centrifugation of whole blood and

analyzed in two separate batches. Sera from the first 15 patients were assayed after one

year of patient recruitment; samples from the remaining participants were assayed after

patient recruitment was concluded (after approximately 2 years of patient recruitement).

All sample analysis was performed by Dr. Bysani Chandrasekar.

Quantification of serum levels of IL-18 and CXCL 16 was performed utilizing

enzyme linked immunosorbent assay (ELISA). Serum levels of IL-18 were analyzed

using a quantitative test kit for human IL-18 manufactured by MBI International

Corporation. Serum levels of CXCL16 were analyzed using a quantitative test kit for

human CXCL16 known as Quantikine® manufactured by R&D Systems, Incorporated.

Both immunoassays employ a sandwich technique utilizing two monoclonal antibodies,

which bind to different epitopes on the inflammatory mediator being evaluated. The

procedures for quantification of IL-18 and CXCL16 are nearly identical with the

exception of small variations in incubations times (30-60 minutes).

22

The basic steps for the immunoassays utilized in the research study are described

in the following text. Serum samples are first diluted; appropriate dilution factors are

determined by the investigator. Samples and standards are placed into wells coated with

anti-human IL-18 antibody and incubated at room temperature (20-25°C) for 60 minutes.

Well contents are aspirated and the wells are washed four times with a buffer solution.

Then, a peroxidase-conjugated anti-human IL-18 antibody is placed into the wells and

incubated at room temperature for 60 minutes. Well contents are aspirated and the wells

are again washed. A peroxidase substrate is then placed into the wells and incubated at

room temperature for 30 minutes. Following incubation, an acid solution is added to

each well in order to stop the enzyme reaction and stabilize the developed color. The

optical density of each well is measured using a wavelength of 450 nm on a microplate

reader. Mean absorbance values of each standard are calculated and used to create a

standard curve. The standard curve is utilized to determine the IL-18 concentrations of

the samples; each sample must be multiplied by the dilution factor to obtain the true

concentration.

E. Data Entry and Statistical Analysis

All data collected was initially recorded on clinical forms and later entered into

electronic spreadsheets. A spreadsheet was created specifically for entering the data from

each periodontal exam. The periodontal spreadsheet was set up as an electronic

periodontal chart into which the values for PD, CAL, BOP, and plaque were entered.

Equations were set up within the spreadsheet in order to calculate frequency distributions

for the five parameters utilized to characterize periodontal disease status. A master

spreadsheet was created which includes patient demographics and clinical information,

23

periodontal disease parameters, CAD, and serum levels of inflammatory mediators.

Data was arranged within the master spreadsheet for input and analysis using a statistical

software package.

Statistical analysis was performed using GraphPad InStat analytical software with

a primary aim to evaluate the interrelationships between the inflammatory mediators IL-

18 and CXCL 16 and periodontal disease. Secondary aims were to evaluate the

relationship between the aforementioned inflammatory mediators and CAD, in addition

to the direct relationship between periodontal disease and CAD. Five periodontal

parameters were analyzed as frequency distributions including the following: percent of

sites with PD ≥ 5 mm and ≥ 7 mm, percent of sites with CAL ≥ 3 mm and ≥ 5 mm, and

percent of sites with BOP. The two thresholds set for PD (≥ 5 mm, ≥ 7 mm) and CAL (≥

3 mm, ≥ 5 mm) represent moderate and severe disease. Frequency distribution represents

extent of disease. Utilizing frequency distributions provided a set of continuous variables

for each periodontal parameter within the study population. Serum levels of

inflammatory mediators were recorded as raw values, although log values were also

calculated and used in statistical analysis. Coronary artery disease status was classified

according to the presence and percent of vessel occlusion; patients with vessels having

less than 50% occlusion were categorized as non-obstructive CAD while those having

vessels with 50% or greater occlusion were categorized as CAD. Patients with CAD

were further categorized according to the number of vessels affected. For statistical

calculations, CAD status was ranked as follows: 0= no CAD, 1= non-obstructive CAD,

2= 1-vessel obstructive CAD, 3= 2-vessel obstructive CAD, 4= 3-vessel obstructive

CAD.

24

Spearman rank correlation, a non-parametric univariate analysis, was performed

to analyze the association between levels of inflammatory mediators and individual

periodontal disease parameters. The analysis was also perfomed to analyze the

association between periodontal disease and CAD. A non-parametric statistical analysis

was chosen due to the relatively small sample size and because the data characterizing

periodontal disease and CAD did not exhibit a normal distribution. The univariate

analyses of the periodontal disease parameters and serum levels of inflammatory

mediators, however, do not take into account the effect of other parameters known to

influence inflammation and the disease processes including BMI, smoking, diabetes, and

statin use.

In order to evaluate the effect of periodontal disease on inflammation and CAD

while simultaneously accounting for other factors known to contribute to the disease

process, multiple regression analyses were performed. The variables analyzed as

outcomes include serum levels of IL-18 and CXCL16 as well as CAD status. Variables

included in the regression equation include the following: age, male gender, BMI, race,

smoking, statin use, each of the five outlined periodontal disease parameters, CAD (when

analyzing IL-18 and CXCL16), and serum levels of IL-18 and CXCL16 (when analyzing

CAD).

25

III. RESULTS

A. Patient Recruitment and Demographics

Complete data sets were obtained and analyzed for 51 patients, although 55

patients received a periodontal exam. Four out of the 55 patients who received a

periodontal exam did not have a blood sample available for cytokine analysis. Two

patients experienced complications during the cardiac catheterization and a blood sample

could not be collected. The other two patients’ blood samples were collected, but were

not delivered to the research laboratory for storage and cytokine analysis. Of the 51

patients 24 were Caucasian, non-Hispanic (47%), 22 were Hispanic (43%), and 5 were

African-American (10%) (Table 1). The patient age range was 30 to 78 years with an

average age of 55.43 years.

B. Clinical Characteristics

In addition to patient demographics, multiple clinical cardiovascular parameters

were evaluated prior to cardiac catheterization. Evaluation of smoking status revealed

that approximately half (49%) of the patients had never smoked, while 13.7% had quit

more than one year prior and 11.8% had quit one month to one year prior to cardiac

catheterization. Approximately one quarter (25.5%) of the patient population were active

smokers at the time of cardiac catheterization. More than half of the patients (56.8%)

were on statin therapy. The average body mass index was 34.66 kg/m2 with a range of

19.15 to 56.09 kg/m2. A large percentage of the patient population had type 2 diabetes,

60.8%. The average hemoglobin A1c value was 7.11% indicating a fair level of control,

26

although the range was 4.90% to 14.70% demonstrating a great variability of glycemic

control within the population.

C. Coronary Artery Disease Status

Patients were categorized according to their coronary artery disease (CAD) status

into five groups (Table 2). The majority of the patients (66.7%) exhibited some degree of

CAD, either obstructive or non-obstructive. Non-obstructive CAD was defined as less

than 50% occlusion of any number of coronary arteries. Coronary artery disease was

considered present if at least 50% occlusion of a vessel was evident at cardiac

catheterization and was categorized according to the number of vessels affected. Results

of cardiac catheterization are as follows: 33.3% had no CAD, 13.7% had non-obstructive

CAD, 9.8% had 1-vessel obstructive CAD, 15.7% had 2-vessel obstructive CAD, and

27.5% had 3-vessel obstructive CAD.

D. Periodontal Disease Status

Periodontal disease was categorized by both extent and severity (Table 3).

Overall the prevalence of severe disease was relatively low in the patient population.

Probing depth was analyzed as the percent of sites with values ≥ 5 mm and ≥ 7 mm. The

average percentage of sites with PD ≥ 5 mm was 12.7% with a range of 0-76.2% and the

average percentage of sites with PD ≥ 7 mm was 2.5% with a range of 0-42.9%. Clinical

attachment loss was analyzed as the percent of sites affected; thresholds used included

CAL ≥ 3 mm and ≥ 5 mm. The average percentage of sites with CAL ≥ 3 mm was

53.5% with a range of 3.7-100% and the average percentage of sites with CAL ≥ 5 mm

was 19.7% with a range of 0-92.1%.

27

According to the American Academy of Periodontology (AAP), periodontal

disease is classified according to severity of clinical attachment loss. Moderate disease is

defined as 3-4 mm of attachment loss, while severe disease is defined as ≥5 mm of

attachment loss. In following the definition of disease endorsed by the AAP, generalized

moderate disease was relatively prevalent in the study population. Alternatively,

epidemiologic research focusing on the systemic effect of periodontal disease has often

utilized extent of disease as a primary parameter. The rationale is that the systemic effect

of periodontal disease depends upon the level of exposure; thus more extensive disease

will have increased systemic involvement, while minimally extensive disease will have

little to no systemic effect. Beck and colleagues diagnosed periodontitis by presense of

CAL ≥3 mm and utilized the extent of disease to define severity as follows: <10% none

or mild, 10% to <30% moderate, and ≥ 30% severe (Beck 2001). According to the

aforementioned definition of disease, the current study population overall was affected

with severe disease as evidenced by an average extent of CAL ≥3 mm of 53.5%.

Bleeding on probing was dichotomized according to presence or absence and

analyzed according to the percentage of sites affected. The average percent of sites with

BOP was 23.4% with a range of 1.2-82.7%. Bleeding on probing was utilized as an

indicator of active inflammation within the periodontal tissues. The large range

demonstrates some patients had a minimal extent, while others had more generalized

inflammation. Overall the inflammatory status of the patient population, evidenced by

the extent of bleeding on probing, was moderate.

28

TABLE 1

SUMMARY STATISTICS

Total patients 51 Average age (years) 55.43 (SD=9.617, range 30-78) Race

Caucasian, non-Hispanic 47% (n=24) Hispanic 43% (n=22) African-American 10% (n=5)

Average body mass index (kg/m2) 34.66 (SD=8.457, SEM=1.184, range=19.15-56.09)

Smoking status Never-smoked 49% (n=25) Quit >1 year prior 13.7% (n=7) Quit 4 weeks-1 year prior 11.8% (n=6) Active smoker 25.5% (n=13)

Statin use 56.8% (n=29) Diabetes 60.8% (n=31)

Average HbA1c 7.11% (SD=2.073, SEM=0.3723, range=4.90-14.70)

29

TABLE 2

CORONARY ARTERY DISEASE STATUS

Coronary artery disease (CAD) categories

% patient population

No CAD 33.3% (n=17) Non-obstuctive CAD (<50% occlusion, any # vessels) 13.7% (n=7) 1-vessel CAD (>50% occlusion) 9.8% (n=5) 2-vessel CAD (>50% occlusion) 15.7% (n=8) 3-vessel CAD (>50% occlusion) 27.5% (n=14)

30

TABLE 3

PERIODONTAL DISEASE PARAMETERS

Periodontal parameter Average % per patient

Standard deviation

Standard error of mean

Range

% sites PD ≥ 5 mm 12.7% 17.44 2.44 0 - 76.2% % sites PD ≥ 7 mm 2.5% 6.70 0.94 0 - 42.9% % sites CAL ≥ 3 mm 53.5% 30.82 4.32 3.7 – 100% % sites CAL ≥ 5 mm 19.7% 23.71 3.32 0 - 92.1% % sites with BOP 23.4% 18.68 2.62 1.2 - 82.7%

31

E. Serum Levels of Inflammatory Mediators

Serum levels of inflammatory mediators exhibited a large range of values among

the patient population (Table 4). Due to the large distribution, log values for the

inflammatory mediators were calculated and analyzed in addition to the raw values. The

average serum level of IL-18 was 335.92 (pg/mL) with a range of 50-1138. The average

serum level of CXCL 16 was 857.22 (ng/mL) with a range of 368-2248.

F. Periodontitis and Inflammatory Mediators

Univariate Analyses

Nonparametric univariate analyses were performed using Spearman Rank

Correlation to evaluate the relationships between each periodontal disease parameter and

the inflammatory mediators and CAD. Analyzing serum levels of IL-18 in relationship to

periodontal parameters revealed a statistically significant correlation with the percent of

sites with BOP (p=0.039) (Table 5). None of the other periodontal parameters were

significantly related to IL-18. Log transformation of IL-18 values did not change the

level of significance for any of the calculations. Analyzing serum levels of CXCL 16 in

relation to individual periodontal parameters did not reveal any statistically significant

relationships (Table 6). Log transformation of CXCL 16 values did not change the level

of significance for any of the calculations. Analysis of CAD status in relation to

periodontal parameters did not reveal any statistically significant correlations (Table 7).

Multivariate Analyses

Multiple regression analyses were performed to evaluate the serum levels of

inflammatory mediators and CAD in relation to multiple variables in order to account for

confounding factors that may have affected the measured outcomes. Results of analysis

32

of serum IL-18 levels revealed a significant association with statin use (p=0.036), CAL

≥3 mm (p=0.045), and CAL ≥5 mm (p=0.024) (Table 8). Results indicate statin use and

periodontal attachment loss are both associated with serum IL-18 levels, however the

positive or negative nature of the associations between the variables and outcomes could

not be reliably determined due to multicollinearity and resultant wide confidence

intervals within the statistical model. The R2 for the multiple regression analysis was

42.45% indicating that 42.45% of the variability among the data is explained by the

multiple regression equation. It is important to note there is 57.55% residual variability

not explained by the statistical model.

Analysis of CXCL16 as the outcome of the multiple regression analysis identified

a different relationship with the studied variables (Table 9). CXCL16 levels were

significantly related to smoking, PD ≥7 mm, and CAL ≥5 mm. Smoking demonstrated a

statistically significant association with CXCL16 levels (p=0.0054). The measures of

severe periodontal disease, PD ≥7 mm and CAL ≥5 mm, demonstrated a statistically

significant association with CXCL16 (p=0.047 and p=0.040 respectively). Overall,

results indicate both smoking and severe periodontal disease were associated with levels

of CXCL16. Similarly to multiple regression analysis of IL-18, multicollinearity was

also present within this statistical model preventing confident determination of the

positive or negative nature of the associations. The R2 for the multiple regression

analysis was 65.98% indicating the percentage of the variability among the data

explained by the multiple regression equation. Residual variability of 34.02% was not

explained by the statistical model.

33

G. Periodontitis and Coronary Artery Disease

Univariate Analyses

Analysis of individual periodontal disease parameters in relation to CAD status

did not identify any statistically significant correlations (Table 7).

Multivariate Analyses

Multiple regression analysis of CAD revealed a statistically significant

association with CAL ≥3 mm (p=0.047) (Table 10). No other statistically significant

associations were found between CAD and evaluated parameters. Multicollinearity was

again present within the statistical model. Results of the analysis indicate periodontal

attachment loss was related to CAD. The R2 for the multiple regression analysis was

54.03% indicating amount of variability among the data explained by the multiple

regression equation. Residual variability unexplained by the statistical model was

45.97%.

34

TABLE 4

SERUM LEVELS OF INFLAMMATORY MEDIATORS

Inflammatory mediator Average

per subject

Standard deviation

Standard error

of mean

Range

Raw serum levels IL-18 (pg/mL) 335.92 223.85 31.35 50 - 1138 Log values IL-18 (pg/mL) 5.59 0.72 0.10 3.91 - 7.04 Raw serum levels CXCL16 (ng/mL) 857.22 407.08 57.00 368 - 2248 Log values CXCL16 (ng/mL) 6.65 0.45 0.064 5.91 - 7.72

35

TABLE 5

NON-PARAMETRIC UNIVARIATE ANALYSIS OF SERUM LEVELS OF IL-18 AND PERIODONTAL PARAMETERS

(Spearman Rank Correlation)

Periodontal parameter r value 95% confidence interval Two-tailed P value

% sites with PD ≥5 mm 0.14 -0.15 – 0.40 0.34 % sites with PD ≥7 mm 0.073 -0.21 – 0.35 0.61 % sites with CAL ≥3 mm -0.0015 -0.28 – 0.28 0.99 % sites with CAL ≥5 mm -0.015 -0.30 – 0.27 0.92 % sites with BOP 0.29 0.0074 – 0.53 0.039*

*statistically significant

36

TABLE 6

NON-PARAMETRIC UNIVARIATE ANALYSIS OF SERUM LEVELS OF CXCL 16 AND PERIODONTAL PARAMETERS

(Spearman Rank Correlation)

Periodontal parameter r value 95% confidence interval Two-tailed P value

% sites with PD ≥5 mm 0.010 -0.27 – 0.29 0.94 % sites with PD ≥7 mm 0.099 -0.19 – 0.37 0.49 % sites with CAL ≥3 mm 0.069 -0.22 – 0.35 0.63 % sites with CAL ≥5 mm 0.098 -0.19 – 0.37 0.49 % sites with BOP 0.099 -0.19 – 0.37 0.49

37

TABLE 7

NON-PARAMETRIC UNIVARIATE ANALYSIS OF CORONARY ARTERY DISEASE AND PERIODONTAL PARAMETERS

(Spearman Rank Correlation)

Periodontal parameter r value 95% confidence interval Two-tailed P value

% sites with PD ≥5 mm -0.020 -0.30 – 0.27 0.89 % sites with PD ≥7 mm -0.088 -0.36 – 0.20 0.54 % sites with CAL ≥3 mm 0.19 -0.10 – 0.45 0.19 % sites with CAL ≥5 mm 0.11 -0.19 – 0.38 0.47 % sites with BOP -0.22 -0.47 – 0.075 0.13

38

TABLE 8

MULTIPLE REGRESSION ANALYSIS OF SERUM LEVELS OF IL-18

R2 = 42.45%

Variable Coefficient Standard error

95% confidence interval

t ratio P value

Constant 1001.50 684.17 -442.11 – 2445.10 1.46 0.16 Age 2.48 6.21 -10.63 – 15.59 0.40 0.69 Male 168.12 123.32 -92.08 – 428.32 1.36 0.19 BMI -12.50 9.52 -32.58 – 7.58 1.31 0.21 Race -12.85 74.72 -170.51 – 144.81 0.17 0.87 CRF-smoking 55.75 59.00 -68.75 – 180.24 0.95 0.36 CAD 49.63 39.66 -34.06 – 133.31 1.25 0.23 Statin use -338.50 148.71 -652.29 – 24.72 2.28 0.036* HbA1c -52.96 36.63 -130.24 – -24.33 1.45 0.17 % sites with PD ≥5 mm

3.48 5.94 -9.05 – 16.01 0.59 0.57

% sites with PD ≥7 mm

-10.91 9.67 -31.32 – 9.49 1.13 0.27

% sites with CAL ≥3 mm

-7.47 3.45 -14.76 – -0.18 2.16 0.045*

% sites with CAL ≥5 mm

10.57 4.27 1.56 – 19.58 2.48 0.024*

% sites with BOP

4.32 5.16 -6.58 – 15.21 0.84 0.41

*statistically significant

39

TABLE 9

MULTIPLE REGRESSION ANALYSIS OF SERUM LEVELS OF CXCL 16

R2 = 65.98%

Variable Coefficient Standard error

95% confidence interval

t ratio P value

Constant 944.06 882.36 -917.72 – 2805.80 1.07 0.30 Age -10.43 8.01 -27.33 – 6.48 1.30 0.21 Male -42.19 159.04 -377.77 – 293.39 0.27 0.79 BMI 25.43 12.28 -0.47 – 51.33 2.07 0.054 Race 24.26 96.37 -179.08 – 227.59 0.25 0.80 CRF-smoking

-242.39 76.09 -402.95 - -81.83 3.19 0.0054*

CAD -26.19 51.15 -134.11 – 81.74 0.51 0.62 Statin use 58.28 191.79 -346.41 – 462.96 0.30 0.76 HbA1c 10.51 47.24 -89.17 – 110.18 0.22 0.83 % sites with PD ≥5 mm

-12.54 7.66 -28.70 – 3.62 1.64 0.12

% sites with PD ≥7 mm

-26.66 12.47 -52.97 - -0.34 2.14 0.047*

% sites with CAL ≥3 mm

2.36 4.46 -7.04 – 11.75 0.53 0.60

% sites with CAL ≥5 mm

12.25 5.51 0.63 – 23.87 2.22 0.040*

% sites with BOP

-1.94 6.66 -15.99 – 12.10 0.29 0.77

*statistically significant

40

TABLE 10

MULTIPLE REGRESSION ANALYSIS OF CORONARY ARTERY DISEASE

R2 = 54.03%

Variable Coefficient Standard error

95% confidence interval

t ratio P value

Constant 0.23 4.41 -9.11 – 9.58 0.053 0.96 Age -0.025 0.039 -0.11 – 0.057 0.64 0.53 Male -0.62 0.76 -2.23 – 1.00 0.81 0.43 BMI 0.018 0.067 -0.12 – 0.16 0.27 0.79 Race -0.038 0.45 -0.98 – 0.91 0.085 0.93 CRF-smoking -0.56 0.44 -1.49 – 0.37 1.28 0.22 Statin use 1.42 0.96 -0.61 – 3.45 1.49 0.16 HbA1c 0.42 0.21 -0.024 – 0.86 2.01 0.062 % sites with PD ≥5 mm

0.021 0.038 -0.060 – 0.10 0.55 0.59

% sites with PD ≥7 mm

-0.040 0.065 -0.18 – 0.098 0.61 0.55

% sites with CAL ≥3 mm

0.045 0.021 0.00064 – 0.089 2.15 0.047*

% sites with CAL ≥5 mm

-0.044 0.030 -0.11 – 0.019 1.48 0.16

% sites with BOP

-0.053 0.029 -0.11 – 0.0079 1.85 0.084

Serum IL-18 0.0018 0.0014 -0.0012 – 0.0047 1.29 0.22 Serum CXCL 16

-0.00072 0.0011 -0.0031 – 0.0016 0.64 0.53

* statistically significant

41

IV. DISCUSSION AND SUMMARY

Periodontal disease, like coronary artery disease, is a result of the chronic

inflammatory process. Results of this investigation demonstrate an association between

clinical parameters of periodontitis and the specific inflammatory mediators IL-18 and

CXCL16. Interleukin-18 and CXCL16 are associated with CAD as well; however, their

specific roles in the disease process are not yet fully understood. IL-18 is a pro-

inflammatory cytokine involved in chronic inflammation. It has been identified within

human atherosclerotic lesions and is also present in diseased periodontal tissue. Much

less is known about the chemokine CXCL16. It is expressed in human atherosclerotic

lesions, although emerging evidence suggests it may have atheroprotective functions.

Research has also implicated CXCL16 in the pathogenesis of periodontal disease.

CXCL16 has been identified within diseased periodontal tissues and is expressed by

human gingival fibroblasts.

The relationships between parameters of periodontal disease and the measured

outcomes, serum levels of IL-18 and CXCL16 and CAD, were evaluated using both

univariate and multivariate analyses. Univariate analyses identified a significant

correlation between serum IL-18 and the extent of BOP; no other statistically significant

correlations were found among the evaluated variables and outcomes. The correlation

between IL-18 and the extent of BOP supports the hypothesis that periodontal

inflammation is associated with increased systemic inflammation. The relationship

between IL-18 and BOP was no longer evident, however, when multiple regression

analysis was performed. The dissolution of the relationship can be explained by the

effect of confounding factors. Confounding factors, including smoking and diabetes,

42

were prevalent among our patient population. In contrast, multiple regression analysis

identified a statistically significant association between periodontal disease and both

inflammatory mediators as well as CAD.

Three multiple regression analyses were performed analyzing IL-18, CXCL16,

and CAD as outcomes. When evaluating an outcome known to be affected by multiple

variables, a multiple regression analysis provides better statistical strength as it analyzes

the effect of multiple variables in relation to each other. When evaluating serum IL-18

levels, a statistically significant association was found between IL-18 and both threshold

measures of CAL. Analysis of serum CXCL16 also identified a statistically significant

association with periodontal disease; however, the relationship was slightly different.

The two variables reflecting severe periodontitis, PD ≥7 mm and CAL ≥5 mm, were

associated with CXCL16 levels. Analysis of CAD identified a statistically significant

association with CAL ≥3 mm. Results support the research hypothesis and thus

demonstrate and association between periodontal destruction, systemic inflammation, and

coronary artery disease. The individual relationships between each inflammatory

mediator and periodontal disease, however, are different. IL-18 was related to moderate

and severe loss of clinical attachment, while CXCL16 was only associated with measures

of severe disease.

Determination of the positive or negative nature of the associations between the

outcomes and variables included in the multiple regression analysis was an important

issue encountered during the study. Placing both CAL ≥3 mm and ≥5 mm within the

analyses resulted in counting the CAL ≥5 mm category twice, as CAL ≥3 mm includes

those measurements greater than 5 mm. A similar situation was encountered for PD

43

measurement. Additionally, PD and CAL often correlate with each other. With the

exception of psuedopocketing and recession, CAL is generally present when a deep PD

exists at a site. Essentially four measures of periodontal disease status were included in

the multiple regression analysis. Inclusion of four independent variables representing

periodontal disease resulted in multicollinearity within the statistical model. Due to

multicollinearity, the confidence intervals of periodontal disease variables were wide;

thus determination of the nature of the associations, either positive or negative, was

unreliable. Solutions to minimize the impact of multicollinearity on the results of

multiple regression analyses include removing variables that provide redundant

information or increasing sample size. While placing two measures for both PD and

CAL in the multiple regression analysis does provide some redundant information, it also

allows categorization of patients into moderate and severe disease categories. One

solution could be to combine PD and CAL in categorization of disease creating two

variables instead of four.

A significant controversy within periodontal research involves the definition of

periodontal disease. While probing depth and bleeding on probing can be considered

measures of active disease and inflammation, they do not provide much information on

past history of disease. Conversely, clinical attachment loss is a commonly utilized

measure of inflammatory periodontal disease; however, it provides no information

regarding current disease activity or inflammatory status. Ideally periodontal disease is

diagnosed by assessing the simultaneous occurrence of multiple clinical factors present

within a patient. Traditional measures of periodontal disease utilized in clinical practice

to diagnose patients include clinical attachment loss, probing depth, bleeding on probing,

44

and radiographic alveolar bone loss. When evaluating patients in an epidemiologic

research study however, examination and diagnosis of patients following traditional

methods is not generally feasible due to financial and time constraints.

Clinical attachment loss does, however, provide information regarding past

disease activity assuming the loss of attachment was a result of inflammation. While

periodontal attachment loss is generally a sequela of the local inflammatory process, it is

intuitive that minimally extensive CAL is likely not associated with generalized severe

inflammation. Evidence from epidemiologic studies has clearly shown that patients with

regular dental care and minimal inflammation can lose periodontal attachment in the form

of marginal soft tissue recession (Loe 1978). Results from our study show the most

prevalent periodontal disease parameter was CAL ≥3 mm. In an adult population with an

average age of approximately 55 years, some level of clinical attachment loss can be

expected in every individual regardless of whether they have a significant history of

inflammatory periodontal disease. Results of the multiple regression analysis reveal a

significant relationship between each of the three outcome variables and CAL. Although,

statistically, the relationship between these parameters was found to be significant, a

possibility exists that the relationship was found because CAL ≥3 mm had a high

prevalence within the study population. Conversely, analyzing CAL by its extent rather

than using patient average values strengthens the association. The relationship

demonstrated by the statistical analyses relates extent of disease, in the form of CAL, to

the outcome measures. It is less likely that a patient with a negative history of significant

inflammatory periodontal disease would have very extensive CAL.

45

It is interesting to note that complete edentulism has been significantly

associated with coronary artery disease in multiple studies. In a study of males with and

without CAD, CAD was significantly more prevalent among edentulous as compared to

dentate patients (Pussinen 2003). Additionally, in a group of 256 patients referred for

open heart surgery, 35% were edentulous as compared to 15% of age- and sex-matched

controls with no history of CAD (p <0.001) (Meurman 2003). Tooth loss is not an

uncommon sequela of the various forms of dental disease; complete edentulism may in

fact reflect a history of extensive, severe periodontal disease. Extensive CAL has also

been associated with symptomatic CAD in the literature. An investigation comparing the

periodontal status of patients with acute coronary syndrome (ACS) versus stable patients

undergoing diagnostic coronary angiography identified a positive correlation between the

extent of CAL ≥5 mm and the severity of CAD in asymptomatic patients (Gotsman

2007). Compared to patients with mild CAD, patients with severe CAD exhibited

increased periodontal destruction, in the form of more extensive clinical attachment loss.

Patients with ACS had characteristics consistent with active periodontal infection and

inflammation. Effectually the loss of clinical attachment observed as a result of

periodontitis represents an accumulation of insults upon the periodontium, just as

atherosclerosis may be considered an adverse outcome related to local vascular

environmental insults.

Suggestions for future research would include utilization of combined measures

of periodontal disease to categorize patients. Combining measures of periodontal disease

would allow for more definitive identification of patients with periodontal disease. For

example, it would be less controversial as to whether a patient is affected with

46

periodontal disease if he or she had both CAL and deep PD. Additionally, composite

measures could be developed, in a similar manner to that proposed by Lynch and

colleagues (2006), including not only PD and CAL but also BOP and alveolar bone

levels. Another suggestion for future research would include an increased sample size to

both minimize the impact of multicollinearity and allow analysis of smaller differences

within the patient population. Another method to limit the effect of multicollinearity is to

reduce the number of independent variables in the multiple regression analysis. As CAD

and periodontitis are both multifactorial diseases, removing variables from the analysis,

which are known to contribute to the etiology of both disease processes, would weaken

the results. Additionally, IL-18 and CXCL16 are novel cytokines meaning they have

been more recently discovered and information regarding their normal distribution within

a patient population is not available. Since little is known regarding distributions of IL-

18 and CXCL16, a power analysis was not performed to determine the target sample size.

However, once more is known about IL-18 and CXCL16 a power analysis would be

beneficial in designing future research studies.

The effects of chronic inflammation in the body are generally not clinically

evident until an acute exacerbation of the disease occurs. For example, myocardial

infarction is a significant acute outcome of the chronic inflammatory disease of

atherosclerosis. Current research aims to explore the association between inflammation

and CAD. Although analysis of the relationships between IL-18, CXCL16 and CAD was

not one of the outlined research objectives, they were evaluated in the multiple regression

analyses. Review of the available evidence has demonstrated a relationship between the

studied inflammatory mediators and CAD. IL-18 has been identified in human

47

atheromas and correlated with atherosclerotic plaque ulcerations as well as clinical

symptoms (Gerdes 2002, Mallat 2001). Additionally increased serum IL-18 levels have

been identified in patients with acute coronary syndrome. Although CXCL16 is known

to mediate cellular processes involved in atherosclerosis, there is a paucity of human data

available regarding the nature of its association with CAD. While CXCL16 has been

identified in human atheroma specimens, conflicting evidence suggests it may be

atheroprotective (Aslanian 2006, Sheikine 2006). Multiple regression analysis of the data

in this research study found no association between either IL-18 or CXCL16 and CAD.

It is important to note the difference between the patient populations in this

research study versus those in the aforementioned studies. Our patient population

consisted of stable patients who were not experiencing acute coronary syndrome, ergo

coronary angiography provided an analysis of the chronic effect of CAD

(atherosclerosis). Since different inflammatory mediators are involved in mediating

chronic and acute inflammation, results from studies measuring chronic versus acute

outcomes may not be comparable. In addition, when IL-18 and CXCL16 have been

correlated with CAD, the studies were evaluating local levels of the inflammatory

mediators within atherosclerotic lesions. The local and systemic inflammatory profiles

may not coincide in patients with atherosclerosis not experiencing acute symptoms.

Finally, the small sample size in this research study would be unable to identify relatively

small differences if they existed.

While the effects of chronic periodontitis may result in periodontal abscess

formation and tooth loss, the disease does not have the same associated morbidity and

mortality as CAD. It is unlikely that periodontal inflammation will directly precipitate an

48

adverse cardiovascular event such as a myocardial infarction. However, in patients who

are at risk for or are affected with coronary artery disease, minimizing exposure to factors

that may contribute to or exacerbate the ongoing disease process is important in

prevention of disease progression.

Although a certain level of skepticism is necessary when evaluating the results of

this and other cross-sectional studies which aim to establish a relationship between one

variable and a multi-factorial disease such as CAD, the statistical analyses performed in

this study demonstrate a significant relationship between measures of periodontitis and

systemic inflammation. While the results of this research study do not provide

unequivocal information regarding the complex relationship, they do contribute to the

body of knowledge and may provide a basis for future investigations.

49

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57

VITA

Rachel Amanda Schallhorn was born on October 28, 1980 to the Reverend Robert

and Frances Schallhorn in Chicago, Illinois. Following graduation from Elkhart

Memorial High School in 1998, she attended Capital University in Columbus, Ohio

where she majored in biology and minored in chemistry. In 2002 she received a Bachelor

of Arts degree graduating summa cum laude. After completing her undergraduate

education, she attended the University of Michigan School of Dentistry and received a

Doctor of Dental Surgery degree in 2006. Dr. Schallhorn entered a three-year

periodontics residency program at the University of Texas Health Science Center in San

Antonio, Texas in 2006. She was admitted into the University of Texas Graduate School

of Biomedical Sciences in San Antonio in 2007.