The effects of periodontal therapy on vascular endothelial function: A pilot trial

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1 The effects of periodontal therapy on vascular endothelial function: A pilot trial John R. Elter, DMD, PhD, a Alan L. Hinderliter, MD, b Steven Offenbacher, DDS, PhD, c James D. Beck, PhD, d Melissa Caughey, BS, b Nadine Brodala, DDS, MS, c and Phoebus N. Madianos, BDS, PhD e Research Triangle Park and Chapel Hill, NC; Athens, Greece Background Chronic periodontal infection is associated with an increased risk of coronary heart disease. Although the mechanism responsible for the relationship between periodontal disease and cardiovascular events is not fully understood, it is hypothesized that the chronic inflammatory burden of periodontal disease may lead to impaired functioning of the vascular endothelium. Methods Twenty-two otherwise healthy adults with moderate to severe periodontitis who underwent complete mouth disinfection were evaluated to determine if periodontal therapy would result in improved endothelial function and a decrease in serum inflammatory markers. Subjects had measurements of periodontal disease severity, flow-mediated (endotheliumdependent), and nitroglycerin-mediated (endothelium-independent) dilation of the brachial artery, serum C-reactive protein (CRP) and interleukin 6 (IL-6), and serum total and high-density lipoprotein cholesterol levels on 2 baseline visits separated by 1 month and, again, 1 month after treatment. Results There were no significant changes in clinical periodontal measures, flow-mediated dilation, nitroglycerinmediated dilation, CRP, IL-6, total cholesterol, or high-density lipoprotein cholesterol between the repeated baseline measurements. Periodontal treatment, however, resulted in significant improvements in periodontal pocketing, flow-mediated dilation, and serum IL-6, as well as a trend toward reduction in serum CRP; there were no significant changes in nitroglycerinmediated dilation or in cholesterol levels. Conclusions These results represent proof of concept that improvement in endothelial function, as measured by flow-mediated dilation of the brachial artery, may be possible through near-elimination of chronic oral infection and suggest that the conduct of a larger controlled trial is justified. (Am Heart J 2006;151:47.e1-47.e6.) Periodontitis is a chronic infection by predominantly Gram-negative bacteria that affects the supporting structures of the teeth. 1 Case control and cross-sectional studies, 2-4 as well as longitudinal studies, 5-21 suggest that periodontal disease may be a novel, modifiable risk factor for coronary heart disease. Although these studies evaluated different populations and used different measures of periodontal and cardiovascular disease, the results are remarkably consistent, and the longitudinal From the a SciMetrika, LLC, Research Triangle Park, NC, Departments of b Medicine, c Periodontology and d Dental Ecology, University of North Carolina at Chapel Hill, Chapel Hill, NC, and e School of Dentistry, University of Athens, Athens, Greece. This work was supported by grants 5P60DE and RR00046 from the National Institutes of Health. Submitted May 31, 2005; accepted October 3, Reprint requests: Alan L. Hinderliter, MD, Division of Cardiology, University of North Carolina at Chapel Hill, 130 Mason Farm Road, Suite #4128, CB #7075, Bioinformatics Building, Chapel Hill, NC hinderli@med.unc.edu /$ - see front matter n 2005, Mosby, Inc. All rights reserved. doi: /j.ahj studies demonstrate that periodontal disease precedes coronary events. 6,12,14-19,22 It has been hypothesized that the chronic inflammatory burden of periodontal disease and the resultant production of circulating cytokines may contribute to the development of atherosclerosis and cardiovascular disease. 5 In addition, periodontal pathogens may disseminate systemically through the blood stream and infect atherosclerotic plaques, causing inflammation and plaque instability. 23 Traditional coronary risk factors promote the development of atherosclerosis, in part through their effects on the vascular endothelium. 24 Similarly, the circulating cytokines and oral pathogens prevalent in patients with periodontitis may activate endothelial cells, increasing the expression of adhesion molecules and suppressing the production of nitric oxide. The health of the vascular endothelium can be assessed noninvasively by measuring flow-mediated dilation of the brachial artery. 25,26 Typically, endothelial dysfunction is characterized by a blunted vascular response to the increased shear stress induced by reactive hyperemia but normal arterial dilation with administration of nitroglycerin, an exogenous nitric oxide donor. 25

2 47.e2 Elter et al American Heart Journal January 2006 We hypothesized that the removal of the infectious and inflammatory burden of periodontal disease through periodontal therapy would be associated with improved endothelial function, as measured by flow-mediated dilation, and would be accompanied by a decrease in levels of the inflammatory markers C-reactive protein (CRP) and interleukin 6 (IL-6). Figure 1 Materials and methods Study subjects Volunteer subjects with untreated, severe, advanced chronic periodontitis but, otherwise, in good general health, were recruited from the community by advertisement and from the patient population of a periodontal medicine research clinic. Inclusion criteria included age N30 years (chronic periodontitis generally becomes clinically significant after 30 years of age 27 ), z1 tooth present in at least 2 quadrants, moderate to severe chronic periodontal disease (at least 4 sites with pocket depth z5 mm distributed in at least 2 quadrants and at least 2 of the 4 sites with attachment loss z3 mm), and willingness and ability to comply with the study requirements. Exclusions included pregnancy or anticipated pregnancy during the study, history of heart disease or stroke, diabetes, high blood pressure or treatment with antihypertensive medications, and any blood or bleeding disorders. The study was approved by the UNC School of Dentistry Committee for the Protection of Human Subjects and was conducted in accordance with the Helsinki Declaration of 1975 (revised in 1983). All participants provided written informed consent. Oral examination Clinical periodontal measurements were made by a single calibrated dentist examiner and included missing teeth, plaque score, and gingival index 28 on up to 32 teeth, as well as pocket depth and cementoenamel junction (CEJ) to free gingival margin on all remaining teeth. Fractional pocket depth and CEJ measurements were rounded down to the next lower whole millimeter. Attachment level was computed from pocket depth and CEJ measure on 6 sites of each tooth. Mean pocket depth and mean attachment level were calculated for each subject as the mean of all measured periodontal sites and as the extent (percentage) of sites with attachment level z3 mm and pocket depth z5 mm. Intraexaminer percent agreement and n for reliability of periodontal measures within 1 mm were N90%. Study protocol This study is a single-masked pilot clinical trial in which endothelium-dependent flow-mediated dilation of the brachial artery, endothelium-independent nitroglycerin-mediated dilation of the brachial artery, and serum markers of systemic inflammation (CRP and IL-6) were measured before and after periodontal therapy. Treatment was consistent with whole-mouth disinfection and was completed over 1 or 2 visits occurring no less than 2 weeks apart. Periodontitis was treated with scaling and root planing, periodontal flap surgery where indicated, and extraction of hopeless teeth. All periodontal treatment was performed by 1 of 2 periodontists. A timeline of study procedures is presented in Figure 1. At Baseline Evaluation 1, subjects were seen in the University of Study timeline. North Carolina General and Oral Health Center Research Clinic, where informed consent was obtained; medical and dental histories were recorded; a venous blood sample was drawn for measurement of CRP, IL-6, total cholesterol, and high-density lipoprotein (HDL) cholesterol; a complete oral examination, including intra- and interoral soft tissue screening, was performed; and a periodontal clinical examination was performed. Full-mouth radiographs were taken, and a treatment plan was devised by the treating periodontist. Within 1 week, measurements of flow-mediated and nitroglycerinmediated dilation of the brachial artery were performed in the University of North Carolina General Clinical Research Center. One month later, subjects were seen for Baseline Evaluation 2, which included a blood draw for CRP, IL-6, and cholesterol levels; a complete dental examination; and measurements of flow-mediated and nitroglycerin-mediated dilation. Subjects then received their indicated therapy over 1 or 2 visits. A posttreatment evaluation, including a blood draw, periodontal examination, and measurements of flow-mediated and nitroglycerin-mediated dilation, was conducted 1 month after the completion of therapy. Measurement of vascular reactivity Brachial artery reactivity was assessed by measuring changes in arterial diameter induced by reactive hyperemia and by nitroglycerin. Ultrasound images of the right brachial artery proximal to the antecubital fossa were acquired using a 12-MHz linear-array transducer. Baseline images were obtained after 10 minutes of supine rest. Flow-mediated (endotheliumdependent) dilation was assessed by determining the change in arterial diameter in response to reactive hyperemia. Reactive hyperemia was induced by inflating a pneumatic occlusion cuff placed around the upper arm (proximal to the segment imaged) to a suprasystolic pressure (approximately 200 mm Hg) for 5 minutes. Images of the artery were recorded for 2 minutes after cuff deflation. After 10 minutes of rest, a second baseline image was acquired. Sublingual nitroglycerin spray (0.4 mg) was then administered to determine nitroglycerinmediated (endothelium-independent) dilation, and ultrasound images were acquired for the subsequent 5 minutes. Gated end-diastolic images of the brachial artery were acquired and stored in a digital format on a personal computer. Each set of images was coded and then analyzed in random order by a blinded observer. Measurements were performed using customized software (Brachial Tools, Medical Imaging

3 American Heart Journal Volume 151, Number 1 Elter et al 47.e3 Table I. Baseline characteristics of the study sample Age (y) 42 (6) Female sex (n [%]) 12 (55) Race (n [%]) African American 6 (27) White 15 (68) Native American 1(5) Current smoker (n [%]) 5 (23) Missing teeth (n) 6.7 (5.3) Mean pocket depth (mm) 3.2 (0.7) Mean attachment level (mm) 3.2 (1.0) Extent of pocket depth N5 mm(%) 21 (13) Extent of attachment level N3 mm(%) 54 (22) Table II. Vascular reactivity at baseline and after treatment Visit FMD (%) NTG-MD (%) Baseline (4.7) 19.5 (7.7) Baseline (5.0)* 20.3 (10.6)y Pooled baseline 8.6 (4.7) 19.8 (8.6) Posttreatment 10.2 (3.9)z 21.3 (8.0) Data presented as mean (SD). FMD, Flow-mediated dilation; NTG-MD, nitroglycerin-mediated dilation. *P for difference from Baseline 1 =.197. yp for difference from Baseline 1 =.450. zp for difference from Pooled Baseline =.034. P for difference from Pooled Baseline =.365. Data presented as mean (SD) or n (%). Applications, LLC, Coralville, IA). Arterial diameter was measured from the lumen-intimal interfaces of the proximal and distal arterial walls. Data from at least 3 consecutive enddiastolic frames were averaged for each baseline measurement and from at least 3 frames at maximum dilation during reactive hyperemia and after administration of nitroglycerin. Flowmediated dilation and nitroglycerin-mediated dilation were calculated as the percent changes in diameter from baseline. Biochemical assays C-reactive protein assays were performed using the BNII Nephelometer (Dade Behring, Deerfield, IL). Interleukin 6 was assayed by high-sensitivity enzyme-linked immunofluorescent assay (R&D Systems, Minneapolis, MN). Serum total and HDL cholesterol were measured by reflectance photometry using the Cholestech LDX System (Cholestech Corp, Hayward, CA). Statistical analysis Mean flow-mediated dilation and nitroglycerin-mediated dilation measurements, serum CRP, serum IL-6, and serum cholesterol levels were compared for changes during the 1-month period from Baseline 1 to Baseline 2 (control period) and from Pooled Baseline (mean of Baseline 1 and Baseline 2) to Posttreatment using repeated measures analysis of variance. Repeated measures analysis of variance, as implemented in SAS Proc GLM (SAS Institute Inc, Cary, NC), is able to accommodate unbalanced data, where data may be missing at 1 time point. If distributions were found to violate assumptions of normality, medians were compared using repeated measures analysis of variance for ranks. Results At baseline, 28 subjects were enrolled in the trial. Six subjects withdrew from the study either immediately before or after therapy and, therefore, had no posttreatment measurements of vascular reactivity or inflammatory markers. The analyses described in this article were limited to the 22 subjects who completed the study protocol. Of those, 2 were missing serum CRP, IL-6, and cholesterol levels at Baseline 1, 1 was missing serum IL-6 at the posttreatment evaluation because of processing errors, and 2 refused administration of Table III. Inflammatory mediators at baseline and after treatment Visit Mean (SD) CRP (mg/l) Median (IQR) IL-6 (pg/ml) Mean (SD) Median (IQR) Baseline (9.5) 2.2 (3.8) 2.3 (1.8) 1.6 (2.1) Baseline (9.8) 2.3 (3.6)* 1.9 (1.7) 1.5 (0.9)y Pooled baseline 3.6 (4.5) 2.4 (3.3) 2.1 (1.3) 1.7 (1.5) Posttreatment 3.3 (5.1) 1.4 (2.8)z 1.5 (1.0) 1.1 (1.3) Data presented as mean (SD) or median (IQR). *P for difference from Baseline 1=.515. yp for difference from Baseline 1=.277. zp for difference from Pooled Baseline =.123. P for difference from Pooled Baseline =.032. nitroglycerin. The 6 subjects who left the trial did not differ significantly from the 22 who completed the study on baseline measures of flow-mediated dilation or serum inflammatory mediators; however, dropouts had worse clinical periodontal disease, compared with those who completed the pilot study (results not shown). Baseline characteristics of the 22 subjects who completed the study are summarized in Table I. Ages ranged from 31 to 55 years, with a mean of 42 years. About two thirds of the subjects were white; 55% were women, and 23% were current cigarette smokers. The participants had an average of 6.7 missing teeth, mean pocket depth of 3.2 mm, and mean attachment level of 3.2 mm. A pocket depth of z5 mm was observed in an average of 21% of sites (extent), and an attachment level of z3 mm was present in an average of 54%. During the 1-month no-treatment period between Baseline 1 and Baseline 2, there were no changes in mean percentage of sites with pocket depth z5 mm (21% [SD, 13] to 19% [SD, 11]) or in mean pocket depth (3.2 mm [SD, 0.7] to 3.1 mm [SD, 0.6]). After therapy, however, there were significant improvements in percentage of sites with pocket depth z5 mm (20% [SD, 11] to 2% [SD, 3]) and in mean pocket depth (3.2 mm [SD, 0.6] to 1.9 mm [SD, 0.5]).

4 47.e4 Elter et al American Heart Journal January 2006 Table IV. Serum cholesterol levels at baseline and after treatment Visit Total cholesterol (mg/dl) HDL cholesterol (mg/dl) Baseline (37) 63 (17) Baseline (37)* 66 (20)y Pooled baseline 227 (30) 64 (17) Posttreatment 224 (46)z 65 (22) Data presented as mean (SD). *P for difference from Baseline 1 =.434. yp for difference from Baseline 1 =.529. zp for difference from Pooled Baseline =.571. P for difference from Pooled Baseline =.294. Measures of vascular reactivity at baseline and after periodontal therapy are shown in Table II. There were no significant differences in flow-mediated dilation or nitroglycerin-mediated dilation between the 2 baseline measurements. The mean absolute difference in flow-mediated dilation between baseline measurements was 2.0% (SD, 1.9), suggesting reproducibility similar to that reported by other laboratories. 29,30 After periodontal therapy, however, mean flow-mediated dilation improved from a pooled baseline value of 8.6% (SD, 4.7) to 10.2% (SD, 3.9) ( P =.034), whereas nitroglycerinmediated dilation remained unchanged. Levels of inflammatory markers at baseline and after treatment are summarized in Table III. Because serum CRP values at all visits were skewed by extreme values from 3 subjects, nonparametric analysis of variance on ranks was performed. The median serum CRP was 2.2 mg/l (interquartile range [IQR], 3.8) at Baseline 1 and 2.3 mg/l (IQR, 3.6) at Baseline 2 ( P =.515). With treatment, median serum CRP decreased from a pooled baseline value of 2.4 mg/l (IQR, 3.3) to 1.4 mg/l (IQR, 2.8) ( P =.123). Serum IL-6 values were also skewed at each visit, so medians were compared for IL-6 as well. Median serum IL-6 levels were similar on the 2 baseline examinations (1.6 pg/ml [IQR, 2.1] vs 1.5 pg/ml [IQR, 0.9] [ P =.277]). After treatment, median IL-6 decreased to 1.1 pg/ml (IQR, 1.3) ( P =.032). Table IV shows serum cholesterol levels at baseline and after periodontal therapy. Total cholesterol and HDL cholesterol levels were similar on the 2 baseline visits, and there was no significant difference between pooled baseline values and levels after treatment (for total cholesterol, 227 mg/dl [SD, 30] to 224 mg/dl [SD, 46] [ P =.571]; for HDL cholesterol, 64 mg/dl [SD, 17] to 65 mg/dl [SD, 22] [ P =.294]). No subjects changed smoking status or began vasoactive medications during the period of observation. Discussion This pilot study provides important evidence that near-complete elimination of periodontal infection by comprehensive local periodontal therapy can result in improved endothelial function and a decrease in the levels of mediators of the acute-phase inflammatory response. Comparison with previous studies Few previous studies have evaluated the relationship between periodontal disease and endothelial function. Amar et al 31 compared flow-mediated dilation and nitroglycerin-mediated dilation of the brachial artery, as well as serum levels of CRP, in 26 subjects with advanced periodontal disease and 29 controls. Subjects with periodontal disease had lower values for flow-mediated dilation (7.8% [SD, 4.6] vs 11.7% [SD, 5.3]) and higher CRP levels (2.3 [SD, 2.3] vs 1.0 [SD, 1.0]) than did controls; vascular responses to nitroglycerin were similar in the 2 groups. Merconaglu, et al 32 observed a marked increase in flow-mediated dilation, from 8.4% (SD, 4.0) to 17.7% (SD, 5.7), in 28 patients who underwent full-mouth scaling and root planing. An increase in nitroglycerin-mediated (endotheliumindependent) reactivity (from 13.3% [SD, 6.3] to 24.9% [SD, 7.3]) was also noted. More recently, Seinost et al 33 also reported improved flow-mediated dilation (from 6.1% [SD, 4.4] to 9.8% [SD, 5.7]) but no change in nitroglycerin-mediated dilation 3 months after a course of periodontal treatment which included both mechanical and systemic antibiotic therapy. Our study confirms that treatment of periodontal disease leads to increased flow-mediated dilation. It also demonstrates that improvement in endothelial function can be observed as early as 1 month after treatment and occurs in the absence of systemic antibiotic therapy, suggesting that it is the result of a localized intervention. As reported by Seinost, et al, 33 we found no change in nitroglycerin-mediated vascular reactivity, suggesting that the increased flow-mediated dilation is due to an improvement in endothelial function rather than a nonspecific enhancement of the vascular smooth muscle response. Our observation that serum inflammatory markers are decreased by periodontal therapy is consistent with the findings of D Aiuto, et al, 34 who measured serum inflammatory markers in 94 subjects before and after nonsurgical therapy for severe generalized periodontitis. Compared with baseline values, median IL-6 was decreased at 2 and 6 months after therapy, and a significant decrease in CRP was evident only at 6 months. Significance of the findings Endothelial injury with resulting dysfunction is an early event in experimental studies of atherogenesis. Traditional markers of cardiovascular risk, such as age, hyperlipidemia, hypertension, diabetes, sedentary lifestyle, and tobacco use, as well as novel risk factors

5 American Heart Journal Volume 151, Number 1 Elter et al 47.e5 such as inflammation and hyperhomocysteinemia, are associated with impaired flow-mediated dilation of the brachial artery. 35 Modification of these risk factors, in turn, induces improvement in endothelial function. Endothelial dysfunction may be viewed as the common pathway between clinical risk factors and the development of atherosclerosis. Recent research has also demonstrated that impaired brachial artery flow-mediated dilation is a predictor of adverse cardiovascular events. 36,39,40 These data support the utility of flow-mediated dilation as a surrogate marker for atherosclerosis, and as a promising index for evaluating novel strategies of risk reduction. Although observational studies suggest an association between periodontitis and cardiovascular disease, the explanation for this relationship is not fully understood. Periodontal disease and atherosclerosis share important risk factors, such as tobacco use, diabetes, and obesity, and are therefore likely to coexist in many patients. Alternatively, individuals who exhibit a pronounced inflammatory response to environmental influences may be predisposed to the development of both periodontal disease and atherosclerotic vascular lesions. Our finding that periodontal therapy improves endothelial function, however, is consistent with a possible causative role for periodontal disease in the pathogenesis of atherosclerosis, perhaps mediated through systemic inflammation and endothelial dysfunction. Limitations of the study Our study has several important limitations. As a pilot study, power to detect differences was not an important consideration, and the inability to demonstrate a statistically significant change in CRP levels may have been due to the small sample size. The treatment used to achieve complete disinfection of the mouth, which included periodontal surgery and extraction of hopeless of teeth, was very extensive. Although this aggressive therapeutic strategy was feasible for a small pilot study to establish proof of principle, it is not often used in the community and would be difficult to incorporate into a larger, more definitive trial. In addition, the follow-up period of 1 month was short. C-reactive protein values may not show improvements after elimination of infection for up to 1 year, and the time course of improvement in endothelial function is not known. Finally, our observational pilot study did not include a control group of untreated patients. Although there were no apparent beneficial effects of study participation on endothelial function or inflammatory markers during the 1-month no-treatment period and there were no changes in lipid levels, smoking status, or medication use over the period of observation, changes in other unrecognized factors may have contributed to the observed improvements in flow-mediated dilation. Conclusions Our pilot trial suggests that treatment of periodontal infection may lead to a decrease in inflammatory markers and improved function of the vascular endothelium. Additional intervention studies to clarify the role of periodontal disease in the pathogenesis of atherosclerosis and to examine the systemic benefits of periodontal therapy are warranted. References 1. Offenbacher S. Periodontal diseases: pathogenesis. Ann Periodontol 1996;1: Mattila K, Nieminen M, Valtonen V, et al. Association between dental health and acute myocardial infarction. BMJ 1989;298: Mattila K, Valle M, Niemenin M, et al. 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