Figures
Abstract
Background and Objective
Matrix metalloproteinase (MMP) -8, -9 and myeloperoxidase (MPO) are inflammatory mediators. The potential associations between MMP-8, -9, MPO and their abilities to reflect cardiovascular risk remains to be evaluated in saliva. The objective of this study was to investigate the levels and associations of salivary MMP-8, -9, MPO and tissue inhibitors of metalloproteinase (TIMP)-1 in myocardial infarction (MI) patients and controls with or without periodontitis.
Materials and Methods
200 patients with a first MI admitted to coronary care units in Sweden from May 2010 to December 2011 and 200 controls matched for age, gender, residential area and without previous MI were included. Dental examination and saliva sample collection was performed 6-10 weeks after the MI in patients and at baseline in controls. The biomarkers MMP -8, -9, MPO and TIMP-1 were analyzed by time-resolved immunofluorescence assay (IFMA), Western blot and Enzyme-Linked ImmunoSorbent Assay (ELISA).
Results
After compensation for gingivitis, gingival pockets and smoking, the mean salivary levels of MMP-8 (543 vs 440 ng/mL, p = 0.003) and MPO (1899 vs 1637 ng/mL, p = 0.02) were higher in non-MI subjects compared to MI patients. MMP-8, -9 and MPO correlated positively with clinical signs of gingival/periodontal inflammation while TIMP-1 correlated mainly negatively with these signs. The levels of latent and active forms of MMP-8 did not differ between the MI and non-MI groups. Additionally, MMP-8, MPO levels and MMP-8/TIMP-1 ratio were significantly higher in men compared to women with MI.
Conclusions
This study shows that salivary levels of the analyzed biomarkers are associated with periodontal status. However, these biomarkers could not differentiate between patients with or without a MI. These findings illustrate the importance to consider the influence of oral conditions when analyzing levels of inflammatory salivary biomarkers.
Citation: Rathnayake N, Gustafsson A, Norhammar A, Kjellström B, Klinge B, Rydén L, et al. (2015) Salivary Matrix Metalloproteinase-8 and -9 and Myeloperoxidase in Relation to Coronary Heart and Periodontal Diseases: A Subgroup Report from the PAROKRANK Study (Periodontitis and Its Relation to Coronary Artery Disease). PLoS ONE 10(7): e0126370. https://doi.org/10.1371/journal.pone.0126370
Editor: Effie C. Tsilibary, National Center for Scientific Research Demokritos, GREECE
Received: October 31, 2014; Accepted: April 1, 2015; Published: July 1, 2015
Copyright: © 2015 Rathnayake et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The study was financially supported by the Swedish National Graduate School in Odontological Science, the Swedish Dental Society, AFA Insurance, the Heart and Lung Foundation, the Stockholm County Council (ALF project), VR Research Funding and the Academy of Finland and Helsinki University Central Hospital Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have read the journal's policy and the authors of this manuscript have the following competing interests: Prof. Timo Sorsa, University of Helsinki, Finland, holds US-Patents 5736341, 5652227, 5866432 and 6143476 on/describing/addressing technology on oral fluid diagnostic MMP-8 immunoassay, and this technology has been utilized in the authors’ paper. The patent is owned by Oy Medixbiochemica Ab/Ltd, Kauniainen, Finland, and according to the contract between Prof. Timo Sorsa and Medix Biochemica Oy/Ab/Ltd Prof. Timo Sorsa has received royalties from the Medix-company. Prof. Timo Sorsa confirms that this does not alter adherence to all PLOS ONE polices on sharing data and materials.
Introduction
Periodontal disease is an inflammatory condition appearing commonly in the adult population [1]. The prevalence of periodontal diseases varies, from gingivitis (prevalence 90%) to moderate (35%) and severe (5–8%) periodontitis [2]. An association between periodontitis and cardiovascular disease (CVD) is known but the pathological mechanisms and potential links between the two diseases are not yet completely clarified [3].
Matrix metalloproteinases (MMP) are calcium- dependent zinc containing endopeptidases that play an important role in normal physiological processes such as tissue development and remodeling as well as in pathological processes [4]. Twenty-three genetically distinct MMPs have been identified in humans. They have an anti-inflammatory (defensive) as well as a pathogenic (tissue destructive) role and are involved in the pathogenesis of large number of different diseases and conditions [5]. MMPs are produced in latent, non-active pro-forms and activated extra- or intracellularly depending upon the structure of the MMP molecule [6]. The main inhibitors of MMPs are tissue inhibitors of metalloproteinases (TIMPs) that restrict extracellular matrix component breakdown [7].
MMP-8, also known as collagenase-2 or neutrophil collagenase, is related to inflammatory conditions. It is expressed mainly by neutrophils [8] but also by endothelial and smooth muscle cells and macrophages in atherosclerotic lesions [9, 10]. Apoptosis of endothelial cells and release of MMP-8 promote the conversion of stable lesions to unstable lesions and lead to plaque rupture [11] and elevated MMP-8 levels have been found in rupture prone and vulnerable plaques [10]. Furthermore, salivary MMP-8 levels are associated with progressive loss of attachment in periodontitis [8, 12]. MMP-9 (gelatinase B) is increased in stimulated whole saliva from inflamed periodontal sites [13, 14]. Increased levels of MMP-9 in serum have also been shown to be a marker of cardiovascular disease [15].
Myeloperoxidase (MPO) is strongly associated with on-going inflammation [16] but has widely been used as a marker of both acute and chronic inflammatory conditions. Its main role is to generate hypochlorous acid to kill bacteria. The oxidative function of MPO also activates latent forms of proMMP-8 and -9 and inactivates TIMPs [17, 18]. MPO is associated with CVD and elevated salivary MPO levels have been demonstrated in periodontal disease [19–22].
The objective of the current study was to investigate salivary MMP-8, -9, TIMP-1 and MPO levels in relation to MI and periodontal disease.
Materials and Methods
Ethics Statement
The case-control PAROKRANK study (Periodontal disease and the relation to myocardial infarction), including the study protocols for participant recruitment, and informed consent for participants, were approved by the regional ethical review board (Dnr. 2008/152-31/2) Karolinska Institutet, Stockholm, Sweden. All participants gave their written informed consent.
Study population
Two-hundred patients, < 75 years old and admitted to a coronary care unit with a first MI were enrolled. Enrollment for this sub-study occurred between May 2010 and December 2011 at eleven participating hospitals in Sweden. The MI was diagnosed according to international definitions [23, 24]. In addition, 200 control subjects with no history of MI and matched for age, gender and postal code were recruited from the national population registry. Study participants were excluded if they had undergone cardiac valvular surgery or had language barriers preventing them to complete study procedures.
Study protocol
Study participants attended the local cardiology and dental departments for clinical and laboratory measurements, questionnaire evaluation and for extensive periodontal examination including salivary collection. Patients were examined 6–10 weeks after the MI and the matched controls in close proximity thereafter.
Dental examination and evaluation.
All participants fasted one hour prior to the dental examination. Saliva and bacterial samples were collected first and the subsequent dental examination comprised number of present teeth, soft tissue pathologies, dental caries and periodontal status, including probing pocket depth (PPD) at four sites per tooth, bleeding on probing (BOP) and present or absent plaque score. Mobility and furcation involvement were also recorded.
Collection and preparation of salivary samples.
Sample collection: Stimulated saliva samples were obtained by chewing paraffin wax up to 10 minutes. The produced saliva was collected into a graded test-tube. The saliva collection continued until 2 mL of saliva was obtained or until 10 minutes had passed. The collected amount was determined, excluding the foam. Collected samples were immediately frozen at -20°C or lower until processing. Each vial was thawed and centrifuged at 500g for 5 minutes, at 5°C. The supernatants were aliquoted into 1.5 mL Eppendorf tubes (Eppendorf, Hauppauge, NY, USA), and stored at -80°C. Each saliva aliquot was used twice for the determination of selected biomarkers.
Determination of biomarker levels in saliva
The MMP-8 concentrations were determined by an immunofluorescence assay (IFMA). The monoclonal MMP-8 specific antibodies 8708 and 8706 (Medix Biochemica, Kauniainen, Finland) were used as a catching antibody and a tracer antibody, respectively. The tracer antibody was labeled using europium-chelate [25]. The assay buffer contained 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM CaCl2, 50 μM ZnCl2, 0.5% BSA, 0.05% sodium azide and 20 mg/L diethylenetriaminepentaacetic acid (DTPA). Samples were diluted in assay buffer and incubated for one hour, followed by incubation for one hour with tracer antibody. Enhancement solution was added and after 5 min fluorescence was measured using a 1234 Delfia Research Fluorometer (Wallac, Turku, Finland). The specificity of the monoclonal antibodies against MMP-8 corresponded to that of polyclonal MMP-8. The interassay coefficient of variation (CV) % was 7.3%. The detection limit for the assay is 0.08 ng/mL. Salivary MMP-9, TIMP-1 and MPO levels were determined using ELISA kits according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA; Amersham Biosciences, Buckinghamshire, UK; Immundiagnostik, Bensheim, Germany, respectlively) [26]. Furthermore, salivary samples were examined by Western blotting using polyclonal antibodies against MMP-8. The immunoblot was quantitated by densitometric computer scanning, and the data expressed as arbitrary units [27].
Statistical analysis
Statistical analyses were performed using Statistical Package for Social Sciences (SPSS) version 22 (SPSS Inc., Chicago, IL). Differences were considered significant at a probability level of p < 0.05. To compare the ratio of categorical variables Chi-squared test was used. The amounts of the analyzed biomarkers are expressed as median and interquartile range and the significance of the differences calculated with Mann-Whitney U test. However, to allow compensation for smoking, gender and periodontal pockets, when comparing biomarker levels in patients with and without MI, a General Linear Model test was performed and the values are expressed as mean ± SD. The resulting p-values were verified with a logarithmic transformation of the data. Correlations were calculated by using Spearman´s correlation tests.
Results
The results are based on examination of patients 6–10 weeks after the MI and the matched controls at baseline. The mean age was 61±8 in both groups and 84% were male. Tobacco use or incidence of hypertension and diabetes did not differ between the study groups. Only the total pathogenic periodontal pocket depth differed between the groups, median 48 mm in the MI group compared to 38 mm in the non-MI group (p < 0.01) (Table 1).
The mean salivary levels of MMP-8 and MPO were significantly higher in non-MI subjects after adjustment for smoking, BOP, PPD 4–5 mm and ≥ 6 mm (MMP-8; 543 vs 440 ng/mL, p = 0.003) and MPO; 1899 vs 1637 ng/mL, p = 0.02), (Table 2). In addition, the different enzyme forms (pro- and active) of MMP-8 obtained as densitometric units from scanning Western blot images did not differ between MI and non-MI patients. Active MMP-8 levels were higher in the non-MI group but it didn’t reach the significant level (7.12 (16.6) vs 9.12 (18.1), p = 0.06) (Table 3).
The correlations of salivary MMP-8, -9, MPO, TIMP-1 are shown in Table 4. Most of the analyzed biomarkers correlated significantly with the other biomarkers. TIMP-1 showed a negative correlation with MMP-8, -9 and MPO levels.
A weak but significant positive correlation between most of the analyzed biomarkers and the included clinical variables was seen in both study groups (Table 5). There was a negative correlation between TIMP-1 and clinical variables.
The association between biomarkers and gender showed that MMP-8, MPO and MMP-8/TIMP-1 ratio were significantly higher in men with MI. MMP-8 levels in men without MI were also significantly higher compared to women without MI. There were no differences between smoker and non-smokers (Table 6).
Discussion
We analyzed salivary levels of MMP-8 and -9, MPO, and TIMP-1 in MI and non-MI subjects with regard to their periodontal conditions. Salivary levels of MMP-8 were slightly higher in the non-MI group. This result differs to some extent, from earlier studies. Furuholm et al. [28] showed that patients referred for open heart surgery had significantly higher salivary concentrations of MMP-8 compared to matched controls after adjusting for number of teeth. No information about the reason for the open heart surgery or medical treatment was available. A more recent study showed a lower concentration of MMP-8 in saliva collected 3–4 days after the MI [27] compared to controls, which is in line with our findings. However, the same study also showed that the percentage of active MMP-8 was higher in the MI group. This differed from our study where a tendency of more active MMP-8 in the non-MI group was seen, although the same techniques and anti MMP-8 antibody were used as in Buduneli and co-authors study [27].
The reason for the discrepancy between the findings in our study and previous studies is not clear but possible explanations could be the time between the MI and saliva sampling, and the medication taken by the MI patients. The patients in this investigation were taking a number different medications, such as statins, angiotensin-converting enzyme inhibitors (ACEI), beta blockers, anti-inflammatory drugs and thrombocyte inhibitory agents (ASA) that could exert an effect on salivary levels of MMP-8 and -9, MPO and TIMP-1. According to previous studies ACEI and statins could potentially interfere with the expression of MPO, MMP-8 and -9. It has been described that ACE inhibitors can decrease MMP-9 levels in both acute and chronic phases of systemic conditions while mechanisms of statin use include inhibition of MMP-9 levels [29–31].
The present study did not only correlate salivary biomarkers to MI but also to the periodontal status. The clinical signs of periodontal inflammation, i.e. gingival inflammation (BOP) and PPD were more or less the same between the study groups. Three of the measured biomarkers, MMP-8, MMP-9 and MPO correlated with BOP, PPD and clinical periodontal diagnosis. TIMP-1 generally showed a negative correlation with the clinical signs of oral inflammation. These results are in agreement with resent studies [32, 33].
Yamamoto et al. showed that MMP-9 plays an important role in the onset and prognosis of MI [34]. In another study several inflammatory mediators and the relation to severity of CVD were studied in serum collected from patients with aortic sclerosis. Elevated general MMP expression was detected and MMP-9 and TIMP-1 had a positive correlation with each other [35]. In our current study MMP-9 correlated positively with MMP-8 and MPO but had a negative correlation with TIMP-1 levels. In previous animal studies, TIMP-1 was shown to inhibit both the activity of MMP-9 during infarct healing and in the protection against infarct plaque rupture after MI [36].
MPO in plasma is associated with an increased risk of CVD [37]. To our knowledge there are no studies of MPO in saliva in relation to MI but one cross-sectional study relating salivary MPO to ischemic stroke has been presented [33]. According to previous reports, MPO exerts distinct protective and surrogate roles during inflammation, such as inactivation of the pathogenic microbes and could oxidatively activate latent proMMP-8 and -9 and inactivate TIMPs [22, 38]. In fact non-proteolytic oxidative activation of proMMP-8 could be directly induced by MPO-derived hypochlorous acid, which is likely to represent the most direct mechanism for triggered neutrophils to endogenously activate MMP-8 [38]. Our results showed that MPO strongly correlated with MMP-8.
MMP-8 is currently regarded among the key biomarkers of inflammation [8, 39]. According to previous studies, salivary MMP-8 levels are higher in coronary artery disease patients and periodontitis patients [39, 40]. Furuholm and co-authors suggested that increased salivary levels of MMP-8 could reflect periodontal disease activity in patients with coronary artery disease compared to systemically healthy controls [28]. It has also been suggested that it may reflect cardiovascular disease, such as MI [28, 41].
It has been demonstrated that GCF levels of MPO and MMP-8 and associations between them were related to development and treatment responses in patients with chronic periodontitis. This indicates an interaction between the MPO oxidative pathway and MMP-8 activation and this cascade might be useful as a potential biomarker for assessing treatment outcomes [38].
The strong correlation between the measured salivary biomarkers and clinical conditions in the mouth illustrates the importance of considering oral inflammation when analyzing salivary biomarkers for systemic diseases. However, in the present study, MMP-8 and MPO were higher in the non-MI group also after compensation for periodontal inflammation and smoking, which might be explained, as previously discussed, by the extensive use of medication in the MI group at time of sample collection, 6–10 weeks after the MI.
In the current investigation, the analyzed biomarkers showed no relation with smoking. Smoking has been reported to increase as well as decrease levels of inflammatory mediators in oral fluids [16, 42]. The impact of smoking on salivary biomarkers of acute inflammation may be explained by a direct effect on inflammatory cells, both on their presence and activity and also the GCF volume per se. Smoking affect the expression and degranulation of MMPs and TIMP-1 [42, 43].
Strengths and limitations of this study
Our finding regarding measured salivary enzymes (MMP-8, -9 and MPO), inhibitor (TIMP-1) and their molar ratios shows statistically significant associations between each other and to MI, as well as correlation to periodontal status. Salivary samples from patients were collected 6–10 weeks following the MI and the acute inflammatory response evoked by the MI had receded at this time. In addition, most patients were treated with CVD drugs at this time and this will likely have affected the analyzed biomarkers compared to if analysis had been performed at time of the MI.
Conclusion
This study shows that salivary levels of the analyzed biomarkers are associated with periodontal status. However, these biomarkers could not differentiate between patients with or without a MI. These findings illustrate the importance to consider the influence of oral conditions when analyzing levels of inflammatory salivary biomarkers.
Acknowledgments
We gratefully acknowledge the PAROKRANK Steering Group: Professor Ulf de Faire: Departments of Medicine and Cardiovascular Epidemiology, Karolinska Institutet, Professor Bertil Lindahl: Department Medical Sciences, Uppsala University, Professor Åke Nygren: Clinical Sciences, Karolinska Institutet, Dr. Per Näsman: Center for Safety Research, KTH Royal Institute of Technology and Dr. Elisabet Svenungsson: Department of Medicine, Karolinska Institutet. We thank all participants, the operators who performed the clinical examinations of the study population, Mrs Hedi Husu for excellent technical assistance and Dr. Per-Erik Isberg, Department of Statistics, Lund University, Sweden for advice regarding the statistical analyses.
Author Contributions
Conceived and designed the experiments: NR AG AN LR B. Klinge TS B. Kjellström. Performed the experiments: TS TT. Analyzed the data: NR AG TS. Contributed reagents/materials/analysis tools: NR AG B. Klinge LR AN B. Kjellström TS. Wrote the paper: NR TS AG AN B. Kjellström TT B. Klinge.
References
- 1. Richards D (2014) Review finds that severe periodontitis affects 11% of the world population. Evid Based Dent 15:70–71. pmid:25343387
- 2. Hugoson A, Norderyd O (2008) Has the prevalence of periodontitis changed during the last 30 years? J Clin Periodontol 35:338–345. pmid:18724861
- 3. Bahekar AA, Singh S, Saha S, Molnar J, Arora R (2007) The prevalence and incidence of coronary heart disease is significantly increased in periodontitis: a meta-analysis. Am Heart J 154:830–837. pmid:17967586
- 4. Uitto VJ, Overvall Cm, McCulloch C (2003) Proteolytic host enzymes in gingival crevicular fluid. Periodontol 2000 31:77–104. pmid:12656997
- 5. Amalinei C, Caruntu ID, Ginsca SE, Balan RA (2010) Matrix metalloproteinase involvement in pathological condition. Rom J Morphol Embryol 52:215–228.
- 6. Nagase H (1997) Activation mechanisms of matrix metalloproteinases. Biol Chem 378:151–160. pmid:9165065
- 7. Verstappen J, Von den Hoff JW (2006) Tissue inhibitors of metalloproteinases (TIMPs): their biological functions and involvement in oral disease. J Dent Res 85:1074–1084. pmid:17122157
- 8. Sorsa T, Tjäderhane L, Konttinen YT, Lauhio A, Salo T, Lee HM, et al. (2006) Matrix metalloproteinases: Contribution to pathogenesis, diagnosis and treatment of periodontal inflammation. Ann Med 38:306–321. pmid:16938801
- 9. Lenglet S, Mach F, Montecucco F (2013) Role of matrix metalloproteinase-8 in atherosclerosis. Mediators Inflamm 659282. pmid:23365489
- 10. Herman MP, Sukhova GK, Libby P, Gerdes N, Tang N, Horton DB, et al. (2001) Expression of neutrophil collagenese (matrix metalloproteinase-8) in human artheroma: a novel collagenolytic pathway suggested by transcriptional profiling. Circulation 104:1899–1904. pmid:11602491
- 11. Naruko T, Ueda M, Haze K, van der Wal AC, van der Loos CM, Itoh A, et al. (2002) Neutrophil infiltration of culpit lesions in acute coronary syndromes. Circulation 106:2894–2900. pmid:12460868
- 12. Tschesche H, Haerian A, Kinane DF, Konttinen YT, Sorsa T (1996) Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients. J Clin Periodontol 23:1127–1132. pmid:8997658
- 13. Meschiari CA, Marcaccini AM, Santos Moura BC, Zuardi LR, Tanus-Santos JE, Gerlach RF (2013) Salivary MMPs, TIMPs, and MPO levels in periodontal disease patients and controls. Clin Chim Acta 421:140–146. pmid:23501330
- 14. Gursoy UK, Könönen E, Huumonen S, Tervahartiala T, Pussinen PJ, Suominen AL, et al. (2013) Salivary type I collagen degradation end-products and related matrix metalloproteinases in periodontitis. J Clin Periodontol 40:18–25. pmid:23078613
- 15. Yabluchanskiy A, Ma Y, Iyer RP, Hall ME, Lindsey ML (2013) Matrix metalloproteinase-9: Many shades of function in cardiovascular disease. Physiology (Bethesda) 28:391–403. pmid:24186934
- 16. Leppilahti JM, Hernández-Ríos PA, Gamonal JA, Tervahartiala T, Brignardello-Petersen R, Mantyla P, et al. (2014) Matrix metalloproteinases and myeloperoxidase in gingival crevicular fluid provide site-specific diagnostic value for chronic periodontitis. J Clin Periodontol 41:348–56. pmid:24382144
- 17. Saari H, Suomalainen K, Lindy O, Konttinen YT, Sorsa T (1990) Activation of latent human neutrophil collagenase by reactive oxygen species and serine proteases. Biochem Biophys Res Commun 171:979–987. pmid:2171513
- 18. Spallarossa P, Garibaldi S, Barisione C, Ghigliotti G, Altieri P, Tracchi I, et al. (2008) Postprandial serum induces apoptosis in endothelial cells: Role of polymorphonuclear-derived myeloperoxidase and metalloproteinase-9 activity. Atherosclerosis 198:458–467. pmid:18177875
- 19. Ikitimur B, Karadag B (2010) Role of myeloperoxidase in cardiology. Future Cardiol 6:693–702. pmid:20932115
- 20. Loria V, Dato I, Graziani F, Biasucci LM (2008) Myeloperoxidase: a new biomarker of inflammation in ischemic heart disease and acute coronary syndromes. Mediators Inflamm 135625. pmid:18382609
- 21. Smith QT, Hinrichs JE, Melnyk RS (1986) Gingival crevicular fluid myeloperoxidase at periodontitis sites. J. Periodontal Research 21:45–55. pmid:3007721
- 22. Sakamoto W, Fujii Y, Kanehira T, Asano K, Izumi H (2008) A novel assay system for myeloperoxidase activity in whole saliva. Clin Biochem 41:584–590. pmid:18237554
- 23. Thygesen K, Alpert JS, White HD: on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Redefinition of Myocardial Infarction (2007) Universal definition of myocardial infarction. Circulation 116:2634–2653. pmid:17951284
- 24. Thygesen K, Alpert JS, Jaffe AS, Simoons ML, Chaitman BR, White HD: the Writing Group on behalf of the Joint ESC/ACCF/AHA/WHF Task Force for the Universal Definition of Myocardial Infarction (2012) Third Universal Definition of Myocardial Infarction. Circulation 126:2020–2035. pmid:22923432
- 25. Hemmilä I, Dakubu S, Mukkala VM, Siitari H, Lovgren T (1984) Europium as a label in time-resolved immunofluorometric assays. Anal Biochem 137: 335–343. pmid:6375455
- 26. Rautelin HI, Oksanen AM, Veijola LI, Sipponen PI, Tervahartiala TI, Sorsa TA, et al. (2009) Enhanced systemic matrix metalloproteinase response in Helicobacter pylori gastritis. Ann Med 41:208–215. pmid:18979291
- 27. Buduneli E, Mäntylä P, Emingil G, Tervahartiala T, Pussinen P, Barış N, et al. (2011) Acute myocardial infarction is reflected in salivary matrix metalloproteinase-8 activation level. J Periodontol 82:716–725. pmid:21091346
- 28. Furuholm J, Sorsa T, Qvarnström M, Janket SJ, Tervahartiala T, Nuutinen P, et al. (2006) Salivary matrix metalloproteinase-8 in patients with and without coronary heart disease may indicate an increased susceptibility to periodontal disease. J Periodontal Res 41:486–489. pmid:16953826
- 29. Yokota T, Osanai T, Hanada K, Kushibiki M, Abe N, Oikawa K, et al. (2010) Effects of telmisartan on markers of ventricular remodeling in patients with acute myocardial infarction: comparison with enalapril. Heart Vessels 25:460–468. pmid:20922537
- 30. Guan W, Kozak A, Fagan SC (2011) Drug repurposing for vascular protection after acute ischemic stroke. Acta Neurochir Suppl 111:295–308. pmid:21725771
- 31. Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, et al. (2000) Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction. J Clin Invest 106:55–62. pmid:10880048
- 32. Meschiari CA, Marcaccini AM, Santos Moura BC, Zuardi LR, Tanus-Santos JE, Gerlach RF (2013) Salivary MMPs, TIMPs, and MPO levels in periodontal disease patients and controls. Clin Chim Acta 5:140–146.
- 33. Palm F, Lahdentausta L, Sorsa T, Tervahartiala T, Gokel P, Buggle F, et al. (2013) Biomarkers of periodontitis and inflammation in ischemic stroke: A case-control study. Innate Immun 17:511–518.
- 34. Yamamoto D, Takai S (2009) Pharmacological implications of MMP-9 inhibition by ACE inhibitors. Curr Med Chem 16:1349–1354. pmid:19355890
- 35. Rugina M, Caras I, Jurcut R, Jurcut C, Serbanescu F, Salageanu A, et al. (2007) Systemic inflammatory markers in patients with aortic sclerosis. Roum Arch Microbiol Immunol 66:10–16. pmid:18928057
- 36. Heymans S, Luttun A, Nuyens D, Theilmeier G, Creemers E, Moons L, et al. (1999) Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med 1135–1142. pmid:10502816
- 37. Schindhelm RK, van der Zwan LP, Teerlink T, Scheffer PG (2009) Myeloperoxidase: a useful biomarker for cardiovascular disease risk stratification? Clin Chem 55:1462–70. Epub 2009 Jun 25. pmid:19556446
- 38. Hernandez M, Gamonal J, Tervahartiala T, Mäntylä P, Rivera O, Dezerega A, et al. (2010) Associations between matrix metalloproteinase-8 and -14 and myeloperoxidase in gingival crevicular fluid from subjects with progressive chronic periodontitis: A Longitudinal Study. J. Periodontol 81:1644–1652. pmid:20653434
- 39. Sorsa T, Tervahartiala T, Leppilahti J, Hernandez M, Gamonal J, Tuomainen AM, et al. (2011) Collagenase-2 (MMP-8) as a point-of-care biomarker in periodontitis and cardiovascular diseases. Therapeutic response to non-antimicrobial properties of tetracyclines. Pharmacol Res 63:108–113. pmid:20937384
- 40. Rathnayake N, Åkerman S, Klinge B, Lundegren N, Jansson H, Tryselius Y, et al. (2013) Salivary biomarkers of oral health: a cross-sectional study. J Clin Periodontol 40:140–147. pmid:23174014
- 41. Mäntylä P, Buduneli E, Emingil G, Tervahartiala T, Pussinen PJ, Bariş N, et al. (2012) Acute myocardial infarction elevates serine protease activity in saliva of patients with periodontitis. J Periodontal Res 47:345–353. pmid:22181012
- 42. Mäntylä P, Stenman M, Kinane D, Salo T, Suomalainen K, Tikanoja S, et al. (2006) Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test. J Periodontal Res 41:503–512. pmid:17076774
- 43. Gomes SC, Piccinin FB, Oppermann RV, Susin C, Marcantonio RA (2009) The effect of smoking on gingival crevicular fluid volume during the treatment of gingivitis. Acta Odontol Latinoam 22: 201–206. pmid:20302219