Oral Diseases (2015) 21, 263–269 doi:10.1111/odi.12261 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd All rights reserved www.wiley.com
ORIGINAL ARTICLE
Vascular endothelial cadherin and vascular endothelial growth factor in periodontitis and smoking EE Sakallio glu1, U Sakallio glu1, M L€ utfio glu1, F Pamuk2, A Kantarci3 1
Department of Periodontology, Dental Faculty, Ondokuz Mayıs University, Samsun; 2Department of Periodontology, Dental Faculty, _ Aydın University, Istanbul, Turkey; 3Department of Periodontology, The Forsyth Institute, Cambridge, MA, USA
OBJECTIVE: This study investigated the vascularization in periodontal disease process via revealing: (i) vascular endothelial cadherin (VE-cadherin) and vascular endothelial growth factor (VEGF) productions in periodontitis and (ii) the impact of smoking on this phenomenon. MATERIALS AND METHODS: Fifteen smokers and 15 non-smokers with/without periodontitis were allocated by split-mouth randomization regarding their smoking and periodontal statuses. The teeth with periodontitis in smokers (group 1), without periodontitis in smokers (group 2), with periodontitis in non-smokers (group 3), and without periodontitis in non-smokers (group 4) constituted the study groups. Gingival crevicular fluid (GCF) levels of VE-cadherin and VEGF were determined by ELISA to evaluate their profiles in the groups. RESULTS: There were increased VE-cadherin levels in groups 1 and 3 compared with groups 2 and 4 (P < 0.05). Group 2 demonstrated higher VE-cadherin level than group 4 (P < 0.05). Increased VEGF was noted in groups 1 and 3 compared with groups 2 and 4 (P < 0.05) with similar levels between groups 1 and 3 and groups 2 and 4 (P > 0.05). There were no correlations between the VEcadherin and VEGF levels in all groups (P > 0.05). CONCLUSION: The results suggest that VE-cadherin and VEGF may increase in periodontitis, and smoking may uniquely cause VE-cadherin production in GCF. Oral Diseases (2015) 21, 263–269 Keywords: VE-cadherin; VEGF; smoking; periodontitis; gingival crevicular fluid
Introduction Structural and functional alterations are commonly occur in the blood vessels of periodontium due to inflammatory processes (Hock and Nuki, 1978; Sakallıo glu et al, 2006). Correspondence: Assoc. Prof. Elif Eser Sakallıoglu, Department of Periodontology, Faculty of Dentistry, Ondokuz Mayıs University, 55139 Kurupelit, Samsun, Turkey. Tel: +90362 3121919 3780, Fax: +90362 4576032, E-mail: [email protected] Received 21 October 2013; accepted 8 May 2014
New vessel formation and vascular events are critical for the pathogenic mechanisms as well as for the resolution of inflammation and regulation of healing. One of the fundamental roles of endothelial cells is to regulate the transport of fluid, solutes, macromolecules, and immune cells from vessels to the surrounding tissues by specialized transcellular systems of transport vesicles and by coordinated stabilization/destabilization of cell-to-cell contacts (Dejana et al, 2008; Harris and Nelson, 2010). Cell-to-cell contacts are provided by several specialized adhesion complexes, that is, tight junctions and adherens junctions (Villasante et al, 2008). Endothelial cells express several cell-specific cadherin proteins. Vascular endothelial (VE)-cadherin is critical for the core of adherens junctions (Villasante et al, 2008; Harris and Nelson, 2010; Pannekoek et al, 2011) playing a crucial role in the regulation of endothelial cell permeability, transendothelial leukocyte migration, and assembly of new vessels (Villasante et al, 2008; Schulte et al, 2011; Yakovlev et al, 2011). Specific features of VE-cadherin may be essential for the initiation, progression, and maintenance of the inflammatory process, and thus, may be an important marker for the pathobiology of inflammatory conditions including periodontal diseases (Villasante et al, 2008; Schulte et al, 2011; Yakovlev et al, 2011) through angiogenesis, which is critical for the homeostatic balance. A major regulator of the new vessel formation and biology is the vascular endothelial growth factor (VEGF). VEGF induces endothelial cell proliferation, secretion of proteolytic enzymes, chemotaxis, and migration. VEGF is critical for inflammation regulating vasoactive and angiogenic cytokines, and thus implicated in the pathogenesis of inflammatory diseases including periodontal disease (Johnson et al, 1999; Sakallıoglu et al, 2007; Pradeep et al, 2011). The interaction between the VEGF and the VE-cadherin involves reciprocity where VEGF is involved in the disruption of adherens junctions by inducing the dissociation of VE-cadherin/catenin complexes in the endothelium (Esser et al, 1998). This process may cause endothelial dysfunction and increased vascular permeability (Harhaj et al, 2006; Barbieri and Weksler, 2007; Barbieri et al, 2008; Dejana et al, 2008) both of which are critical during the pathological changes in the
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periodontal tissues. Indeed, there is evidence that a major pro-inflammatory peptide, the C-reactive protein mediates the cross-talk between VEGF and VE-cadherin (Verma et al, 2004). This interaction has been speculated to provide a therapeutic mechanism in inflammatory bowel disease and experimental colitis (Chidlow et al, 2007). While limited, these studies provide evidence that the interaction between VEGF and VE-cadherin may have a critical role in the inflammatory process due to the cross-talk between VEGF and VE-cadherin. Such a phenomenon has not been tested as a plausible mechanism for the vascularization during inflammatory periodontal diseases. A major disruption to the periodontal vasculature would be due to smoking (Bergstr€ om et al, 1988; Rezavandi et al, 2002), smoking has already been shown to be an important risk factor for periodontal diseases (Morozumi et al, 2004; Sakallıo glu et al, 2008). One consistent finding in smoking-related diseases is the existence of endothelial dysfunction leading to damage of the endothelium (Koide et al, 2005; Michaud et al, 2006; Lu et al, 2011; Schweitzer et al, 2011). Recent data have demonstrated that smoking may impair vessel health and induce vasoconstriction in gingiva (Mavropoulos et al, 2003), decrease gingival blood flow rate (Morozumi et al, 2004; Mavropoulos et al, 2007), cause higher percent of small blood vessels but similar vascular density in gingiva (Mirbod et al, 2001; S€ onmez et al, 2003), and contribute to the disruption of immune response due to repeated vasoconstrictions (Morozumi et al, 2004). The hypothesis of this study was that smoking-induced disruption of the periodontal vascularization results in changes in VEGF and VE-cadherin expression, which will explain the mechanism of endothelial damage in periodontium. Therefore, the present study aimed: (i) to evaluate GCF levels of VE-cadherin and VEGF in patients with periodontitis and (ii) to investigate the impact of smoking on VE-cadherin and VEGF production in GCF.
Materials and methods Study group characterization Participants referred to the Periodontology Department of Ondokuz Mayıs University Dental Faculty in Samsun, Turkey, were recruited in this cross-sectional study. The study protocol was confirmed by the relevant Human Ethics Committee of Ondokuz Mayıs University-Samsun, and it was carried out according to the ‘Helsinki Declaration of 1975’, as revised in 2008. The experiments were undertaken with the understanding and written consent of each participant. A study population of male smokers and nonsmokers was selected regarding the criteria: (i) to be ≥ 35 years of age and to have localized chronic periodontitis, (ii) not to have previous periodontal therapy in the last 6 months, and (iii) not to have any systemic problems and regular/current (at least 3 weeks prior to data collection) chemotherapeutic regimens. Smoking status was determined as ≥ 5 years of duration and as ≥ 15 per day of consumption in all smokers, whereas non-smoking status included individuals who never smoked before. It has already been demonstrated in many studies that the cotinine levels in serum and urine correlate with the cigarette Oral Diseases
consumption (Wewers et al, 2000; Benowitz et al, 2003; Underner et al, 2004; Johnstone et al, 2006). Therefore, we have decided not to measure the cotinine levels in patients in this study and preferred to use the extensive references. Periodontal statuses of these participants were assessed by clinical examination, and the teeth with periodontitis were matched to the contralateral teeth with no signs of inflammation and destruction. Such a split-mouth study design was carried out to perform evaluations in similar environmental and systemic conditions. Thus, four study groups were arranged according to the smoking habits of the participants and periodontal statuses of their teeth: Group 1 = the teeth with periodontitis in smokers, Group 2 = the teeth without periodontitis in smokers, Group 3 = the teeth with periodontitis in non-smokers, and Group 4 = the teeth without periodontitis in non-smokers. Fifteen smoker and 15 non-smoker participants with healthy and diseased sites were appropriate to determine the results at a = 0.05 with a 90% power. Clinical procedures Clinical records and gingival crevicular fluid (GCF) samples were collected from all participants. The clinical examination procedure to determine periodontal status included: (i) Silness-L€oe plaque index (PI) (L€oe and Silness, 1963), (ii) L€oe-Silness gingival index (GI) (Silness and L€oe, 1964), (iii) probing pocket depth (PPD), and (iv) clinical attachment level (CAL) and radiographical evaluation of alveolar bone loss (ABL). Radiographs taken by full-mouth parallel technique were evaluated for each participant. The distance between enamel-cement line and crestal bone was evaluated, and the distance > 2 mm was accepted as ABL. PI, GI, PPD, and CAL measurements were taken at six sites (mesio-, mid-, disto-buccal/labial and palatal/lingual surfaces) per tooth using a Williams’ periodontal probe. The sites demonstrating > 4 mm PPD and CAL with radiographical evidence of ABL (by parallel technique) were regarded as sites with ‘periodontitis’ and allocated for groups 1 and 3. The tooth site with the highest PPD and CAL values in groups 1 and 3 was selected. GCF was collected from this selected sites. The matching sites with < 3 mm PPD and CAL and without radiographical evidence of ABL were regarded as ‘nonperiodontitis/control’ sites and allocated for groups 2 and 4. In groups 2 and 4, GCF was collected from the nonperiodontitis/control sites. All clinical parameters were measured 1 day before GCF sampling to eliminate possible quantitative and/or qualitative alterations in GCF due to the examination methods. For GCF sampling, the teeth were isolated with cotton rolls and air-dried for 5 s. A standard paper strip (Periopaper; Ora Flow Inc., Amityville, NY, USA) was gently inserted into the gingival crevice (i.e., standard depth of 1 mm) and left there for 30 s. Volumetric analysis of the fluid collected in the strip was performed immediately by a volume quantifying device (Periotronâ 8000; Pro Flow Inc., Amityville, NY, USA), and the strips were stored at 80°C for the laboratory
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procedures. Two investigators calibrated for the measurements of the clinical examination and GCF collection. Laboratory procedures GCF levels of VE-cadherin and VEGF were determined and compared among the study groups. GCFs was first eluded from the paper strip by a protocol described elsewhere (Curtis et al, 1998), and the obtained GCF samples were utilized in the biochemical analyses of VE-cadherin and VEGF. VE-cadherin and VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available VE-cadherin (Human VE-cadherin ELISA; Bender MedSystems GmbH, Vienna, Austria) and VEGF (Human VEGF ELISA; Bender MedSystems GmbH) kits. These laboratory procedures were performed by a single investigator blinded to the study. The measured marker concentrations were corrected for GCF volume, and VE-cadherin concentrations were expressed in nanogram per milliliter (ng ml 1), whereas VEGF concentrations were expressed in picogram per milliliter (pg ml 1). The limits of detection were 0.10 ng ml 1 for VE-cadherin and 7.9 pg ml 1 for VEGF. Statistical analysis Calculation of sample size was performed regarding the difference between VEGF levels of GCF in healthy and periodontitis sites (Prapulla et al, 2007) as the study outcome, as no similar data were available for VE-cadherin. Normality of the data was analyzed by Shapiro–Wilk test and, multiple comparison of the groups was performed by Kruskal–Wallis and Friedman tests. Wilcoxon and Mann– Whitney-U tests (with Bonferroni correction) were utilized for the comparison between 2 groups. All results were expressed as median (min.-max.), and the differences were determined as significant at P = 0.05 (independent groups) and P = 0.05 (dependent groups) levels. Spearman’s correlation test was utilized to evaluate the correlation between VE-cadherin and VEGF in each study group. These calculations were performed using a statistical software package program (SPSS 12.0v; IBMCo., Chicago, IL, USA).
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Results Thirty smokers and non-smokers with an age range of 35– 58 years were allocated as the study population. Two teeth in each participant, thus 15 teeth in each group were included in the study. Data on clinical parameters and GCF volumes of the groups are presented in Table 1. Study groups of teeth with periodontal disease (groups 1 and 3) showed significantly higher levels of plaque regardless of the smoking status (Table 1). Gingival tissues around teeth with periodontal disease (groups 1 and 3) showed clinical signs of inflammation compared with healthy sites (Table 1). Smoking was not associated with PPD or CAL (Table 1). GCF volume significantly increased at sites with periodontitis (groups 1 and 3) (Table 1). VE-cadherin concentration was higher in the periodontitis sites of smokers and non-smokers (groups 1 and 3) than in the non-periodontitis sites of smokers and nonsmokers (groups 2 and 4) (P < 0.05) with similar levels between the periodontitis sites of smokers and non-smokers (groups 1 and 3) (P > 0.05) (Figure 1a). It was also found to be higher in the non-periodontitis sites of smokers than in the non-periodontitis sites of non-smokers (groups 2 and 4) (P < 0.05) (Figure 1a). Total amount of VE-cadherin was higher in the periodontitis sites of smokers and non-smokers (groups 1 and 3) than in the non-periodontitis sites of smokers and non-smokers (groups 2 and 4) (P < 0.05) (Figure 1b). However, it was found to be similar in non-periodontitis sites of smokers and non-smokers (groups 2 and 4) (P > 0.05) (Figure 1b). VEGF concentration was higher in the periodontitis sites of smokers and non-smokers (groups 1 and 3) than in the non-periodontitis sites of smokers and non-smokers (groups 2 and 4) (P < 0.05), and it was similar between the periodontitis and non-periodontitis sites of both smokers and non-smokers (groups 1 and 3; groups 2 and 4) (P > 0.05) (Figure 2a). Total VEGF amount was higher in the periodontitis sites of smokers and non-smokers (groups 2 and 4) (P < 0.05), and it was similar between the
Table 1 Distribution and comparison of the clinical examination parameters in the study groups. Groups (n = 15) Group 1 (smoker + periodontitis) median (min.–max.) PI Pa GI Pa PPD (mm) Pa PAL (mm) Pa GCF volume (µl) Pa
2 (0–2) 1 (1–2) 5 (5–7) 6 (5–9) 0.82 (0.28–1.27)
Group 2 (smoker + non-periodontitis) median (min. –max.)
Group 3 (non-smoker + periodontitis) median (min. –max.)
1 (0–2) 2 (2–3) 0.001 for groups 1–2, 1–4, 2–3 and 3–4 1 (0–1) 2 (2–3) 0.001 for groups 1–2, 1–4, 2–3, and 3–4 2 (1–3) 5 (5–8) 0.001 for groups 1–2, 1–4, 2–3 and 3–4 2 (1–3) 6 (5–9) 0.001 for groups 1–2, 1–4, 2–3 and 3–4 0.08 (0.01–0.25) 1.12 (0.55–1.60) 0.001 for groups 1–4 and 2–3; 0.002 for groups 1–2 and 3–4
Group 4 (non-smoker + non-periodontitis) median (min. –max.) 1 (0–2) 1 (0–2) 2 (1–3) 2 (1–3) 0.09 (0.03–0.28)
PI, plaque index; GI, gingival index; PPD, probing pocket depth; GCF, Gingival crevicular fluid. a Statistically significant values. Oral Diseases
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0.35 0.3
(a) 30 0.29 (0.20-0.57)
0.28 (0.20-0.51) 0.2 (0.19-0.31)
0.25 0.2 0.15 0.1
0.19 (0.01-0.21)
0.05 0
VEGF CONCENTRATION (pg ml–1))
(a) VE-CADHERIN CONCENTRATION (pg ml–1)
266
22.66 (2.63-43.91)
27.58 (14.-81.54)
25 20 15
0.55 (0.03-2.32)
9.23
(0.01-15.38)
10 5 0
0.35 0.3 0.25
(b) 30
0.29 (0.13-0.66) 0.23 (0.10-0.44)
0.2 0.15 0.1 0.05
0.02 (0.001-0.05)
0.02 (0.01-0.06)
0
TOTAL VEGF AMOUNT (pg)
TOTAL VE-CADHERIN AMOUNT (pg)
(b)
25
periodontitis and non-periodontitis sites of both smokers and non-smokers (groups 1 and 3; groups 2 and 4) (P > 0.05) (Figure 2b). Spearman’s correlation test demonstrated no correlations between the VE-cadherin and VEGF levels of periOral Diseases
22.66 (2.63-43.91)
20 15 10 5 0
Figure 1 (a) Distribution and comparison of VE-cadherin concentrations [median (min.-max.)] in the study groups. P = 0.004 for periodontitis sites and non-periodontitis sites of smokers (groups 1–2). P = 0.001 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of non-smokers (groups 2–3), non-periodontitis sites of smokers and nonsmokers (groups 2–4), and periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.851 for periodontitis sites of smokers and non-smokers (groups 1–3). (b) Distribution and comparison of VEcadherin total amounts [median (min.-max.)] in the study groups. P = 0.002 for periodontitis sites and non-periodontitis sites of smokers (groups 1–2). P = 0.000 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of non-smokers (groups 2–3). P = 0.003 for periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.110 for periodontitis sites of smokers and non-smokers (groups 1– 3). P = 0.340 for non-periodontitis sites of smokers and non-smokers (groups 2–4).
27.58 (14.05-59.98)
0.55 (0.03-2.32)
0.66 (0.001-3.21)
Figure 2 (a) Distribution and comparison of VEGF concentrations [median (min.-max.)] in the study groups. P = 0.001 for periodontitis and non-periodontitis sites of smokers (groups 1–2). P = 0.000 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of nonsmokers (groups 2–3). P = 0.001 for periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.663 for periodontitis sites of smokers and non-smokers (group 1–3), and non-periodontitis sites of smokers and non-smokers (groups 2–4). (b) Distribution and comparison of VEGF total amounts [median (min.-max.)] in the study groups. P = 0.002 for periodontitis and non-periodontitis sites of smokers (groups 1–2). P = 0.000 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of non-smokers (groups 2–3). P = 0.003 for periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.176 for periodontitis sites of smokers and non-smokers (group 1–3). P = 0.853 for non-periodontitis sites of smokers and non-smokers (groups 2–4).
odontitis sites in smokers (r = 0.265) and non-smokers (r = 0.338) as well as in the non-periodontitis sites of smokers (r = 0.006) and non-smokers (r = 0.114).
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Discussion The study results suggest that higher GCF levels of VEcadherin and VEGF are associated with periodontal disease. Smoking further increases VE-cadherin expression in the GCF. On the other hand, data also suggest that increased VE-cadherin levels were found in the periodontitis sites compared with the non-periodontitis sites, independent of the smoking status. This finding is within lines of the previous reports demonstrating that the diseased and/or inflammatory conditions that increase the vascular permeability may affect the structure and organization of vascular junctions (Corada et al, 1999). One possible explanation for such an increase would be due to a physical phenomenon through which the permeability of the vessel wall increases cytokines such as histamine, TNF-a, platelet-activating factor. During this process, VEGF may induce the tyrosine phosphorylation of VE-cadherin and its binding partner, b-catenin (Dejana et al, 2008). Consequently, an endothelial cell retraction may occur, therefore reducing the endothelial junctional strength and causing an extraordinary opening in the intercellular gaps (Esser et al, 1998; Andriopoulou et al, 1999). Dramatic increase in permeability and vascular fragility via administration of anti-VE-cadherin in mouse model was observed (Corada et al, 1999). The relation between VE-cadherin and vascular permeability in inflamed endothelial cells in cell cultures (Sheets et al, 2005), and a loss of cell adhesion (Takeichi et al, 2008), the barrier breakdown and increased paracellular permeability (Shen et al, 2011) as a result of altered expression of VE-cadherin were also reported in different tissues. Therefore, alterations in VE-cadherin expression (as VEGF expression) may be an important stage in the initiation and progression of inflammation. Our results demonstrated higher VEGF levels in the periodontitis sites of both smokers and non-smokers compared with the matching non-periodontitis sites. VEGF may regulate the periodontal inflammation by (i) promoting a vascular network, (ii) increasing the extent of inflammatory process, and (iii) inducing angiogenesis into the spaces created by the destruction of periodontal tissues based on the previous reports (Johnson et al, 1999; Sakallıo glu et al, 2007; Pradeep et al, 2011) and the findings from the current study. VEGF may also be effective on the disruption of adherens junctions, as it induces the dissociation of VE-cadherin/catenin complexes in endothelium and causes destabilization in the adherens junction complexes to modify vascular permeability (Harhaj et al, 2006; Barbieri and Weksler, 2007; Barbieri et al, 2008; Dejana et al, 2008; Pannekoek et al, 2011). Indeed, VEGF has been shown to produce significant changes in the VE-cadherin concentration of endothelial cell cultures as well as alterations in actin fibers resulting in increased capillary hyperpermeability (Villasante et al, 2008). VE-cadherin was found to be increased in the non-periodontitis sites of smokers compared with the non-periodontitis sites of non-smokers with similar levels in the periodontitis sites of both smokers and non-smokers. Deterioration of VE-cadherin may be evaluated as a sign of endothelial degeneration (Michaud et al, 2006; Barbieri
and Weksler, 2007; Barbieri et al, 2008), as it is known as the main component of adherens junctions in endothelial cells. This has been demonstrated as a barrier dysfunction with the disruption of VE-cadherin in mouse cardiac endothelium due to tobacco smoke (Barbieri et al, 2008). An in vitro study of endothelial progenitor cells obtained from the peripheral blood of smokers further reported significant downregulation of VE-cadherin expression (Michaud et al, 2006). It has also been revealed that vascular damage caused by the disassembly of VE-cadherin complex induced endothelial dysfunction (Harris and Nelson, 2010; Schulte et al, 2011; Yakovlev et al, 2011). Because VE-cadherin is an essential protein in the maintenance of intact vasculature by providing the integrity of endothelial lining cells (Harris and Nelson, 2010), it may only be detected in serum or body fluids whenever a destructive process leads to its release. The VE-cadherin increase in GCF may accordingly be a consequence of the adherens junction degradation on endothelium secondary to smoking. However, the similarity between the periodontitis sites suggests that this phenomenon may be masked within an established inflammatory process, and thus, an additive effect of smoking on the VE-cadherin increase may not be noted in the chronic inflammatory diseases or lesions such as periodontitis. This assumption, however, requires further testing in larger clinical and in vivo experimental models. Similar VEGF concentration in the periodontitis and non-periodontitis sites of both smokers and non-smokers was an another interesting finding in this study. Although insufficient data are available on the effect of smoking on the VEGF profile of healthy periodontium, increased (Kuwano et al, 1993; Hiroshima et al, 2002; Hashimoto et al, 2005) and/or decreased (Blomberg et al, 1997; Bocchino et al, 1997; Liu et al, 2003) VEGF levels have been detected on the pulmonary tissues and secretions of smokers and, tobacco-related diseases causing pathological angiogenesis such as neoplasies and angiogenic squamose dysplasia have been described before in pulmonary tissues (Keith et al, 2000; Heeschen et al, 2001). In the present study, smoking was not found to be directly associated with GCF levels of VEGF, and the results suggest that VEGF is likely to increase only in inflammatory conditions. Increased VEGF levels in both GCF and periodontal tissues were showed at the periodontal disease (Pradeep et al, 2011; Bletsa et al, 2012; Tian et al, 2013) and diabetes (Guneri et al, 2004; Sakallıoglu et al, 2007; Aspriello et al, 2009; Keles et al, 2010). In this respect, further larger scale studies are clearly required to investigate the role of VEGF in pathogenesis of periodontal disease at especially smoking status because of its regulator role in vascular integrity, dysfunction, and hyperpermeability. Contrary to our hypothesis, we could not identify any correlation between VEGF and VE-cadherin both in the periodontitis and non-periodontitis sites of smokers and non-smokers. This finding does not preclude the fact that there may be a mechanistic regulation due to the limitations of the current study. Even if the power analysis was performed prior to the study and intended patient number was recruited, the variation between the groups still suggests that a larger study needs to be designed and
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conducted. On the other hand, statistical analyses demonstrate significant differences, which suggest that the results are reliable as a basis for future studies. One limitation of the current study is obviously the tissue level expression of the studied markers and their correlations to the GCF levels. This shortcoming requires an immunohistochemical analysis and testing of additional markers to elaborate the mechanistic link between the analytes studied. Therefore, we have refrained from making any mechanistic suggestions and present the data as associative findings. Future studies are needed to identify a possible link at various stages of the inflammatory process, because inflammatory markers may be found altered at smoking status than those in non-smoking status. This has been becoming an important area of research in periodontology and many milieu such as saliva, GCF, and serum have been tested for potential samples for diagnostic and prognostic markers of disease. On the other hand, this field is at its infancy especially due to the need for indepth understanding of high-throughput technologies. Another limitation to this end is the difficulty in designing longitudinal studies that will demonstrate the progression of gingivitis to periodontitis in humans. Due to obvious ethical concerns, only reversible gingivitis can be induced experimentally in humans while non-reversible periodontitis can only be tested in animals in vivo. Therefore, studies on human periodontitis are associative in nature, and biomarkers of inflammation can only be inferred upon based on the cross-sectional study designs and/or longitudinal treatment protocols. Our study is a cross-sectional evaluation of the existing periodontitis in humans and cannot address if and how the analytes studied are critical for the progression of disease. Therefore, longitudinal testing of these molecules (and others) in response to periodontal therapy is required for validation of their ‘biomarker’ status. Within these limitations, it can be concluded that (i) VE-cadherin and VEGF may increase in GCF due to the periodontal inflammatory process and (ii) VE-cadherin and VEGF may be utilized to confirm periodontal inflammation or active process. Acknowledgement The authors declare that they have no conflict of interest and that there is no source of funding for this research.
Author contributions Dr. E.E. Sakallioglu: Creation of the study hypothesis and coordination of the experimental design. Dr. M. Lutfioglu: Patient selection and statistical analyses. Dr. U. Sakallıoglu: Coordination of the study phases and manuscript editing. Dr. F. Pamuk: Laboratory procedures and ELISA. Dr. A. Kantarcı: Control of the experimental procedures and manuscript editing.
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cells within the bronchial epithelium of smokers. Cancer 95: 1539–1545. Hock JM, Nuki K (1978). A vital microscopy study of morphology of normal and inflamed gingiva. J Periodontol 49: 343– 350. Johnson RB, Serio FG, Dai X (1999). Vascular endothelial growth factors and progression of periodontal diseases. J Periodontol 70: 848–852. Johnstone E, Berowitz N, Cargill A et al (2006). Determinants of the rate of nicotine metabolism and effects on smoking behavior. Clin Pharmacol Ther 80: 319–330. Keith RL, Miller YE, Gemmill RM et al (2000). Angiogenic squamous dysplasia in bronchi of individuals at high risk for lung cancer. Clin Cancer Res 6: 1616–1625. Keles GC, Cetinkaya BO, Eroglu C, Simsek SB, Kahraman H (2010). Vascular endothelial growth factor expression levels of gingiva in gingivitis and periodontitis patients with/without diabetes mellitus. Inflamm Res 59: 543–549. Koide M, Nishizawa S, Yamamoto S, Yamaguchi M, Namba H, Terakawa S (2005). Nicotine exposure, mimicked smoking, directly and indirectly enhanced protein kinase C activity in isolated canine basilar artery, resulting in enhancement of arterial contraction. J Cereb Blood Flow Metab 25: 292–301. Kuwano K, Bosken CH, Pare PD, Bai TR, Wggs BR, Hogg JC (1993). Small airways dimensions in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 148: 1220– 1225. Liu ZJ, Snyder R, Soma A et al (2003). VEGF-A and avb3 integrin synergistically rescue angiogenesis via N-Ras and P13K signaling in human microvascular endothelial cells. FASEB J 17: 1931–1933. L€ oe H, Silness J (1963). Periodontal disease in pregnancy. I. Prevalence and Severity. Acta Odontol Scand 21: 533–551. Lu Q, Sakhatsky P, Grinnell K et al (2011). Cigarette smoke causes lung vascular barrier dysfunction via oxidative stressmediated inhibition of RhoA and focal adhesion kinase. Am J Physiol Lung Cell Mol Physiol 301: L847–L857. Mavropoulos A, Aars H, Brodin P (2003). Hyperaemic response to cigarette smoking in healthy gingiva. J Clin Periodontol 30: 214–221. Mavropoulos A, Brodin P, R€osing CK, Aass AM, Aars H (2007). Gingival blood flow in periodontitis patients before and after periodontal surgery assessed in smokers and nonsmokers. J Periodontol 78: 1774–1782. Michaud SE, Dussault S, Haddad P, Groleau J, Rivard A (2006). Circulating endothelial progenitor cells from healthy smokers exhibit impaired functional activities. Atherosclerosis 187: 423–432. Mirbod SM, Ahing SI, Pruthi VK (2001). Immunohistochemical study of vestibular gingival blood vessel density and internal circumference in smokers and non-smokers. J Periodontol 72: 1318–1323. Morozumi T, Kubota T, Sato T, Okuda K, Yoshie H (2004). Smoking cessation increases gingival blood flow and gingival crevicular fluid. J Clin Periodontol 31: 267–272. Pannekoek WJ, van Dijk JJ, Chan OY et al (2011). Epac1 and PDZ-GEF cooperate in Rap1 mediated endothelial junction control. Cell Signal 23: 2056–2064. Pradeep AR, Prapulla DV, Sharma A, Sujatha PB (2011). Gingival crevicular fluid and serum vascular endothelial growth factor: their relationship in periodontal health, disease and after treatment. Cytokine 54: 200–204. Prapulla DV, Sujatha PB, Pradeep AR (2007). Gingival crevicular VEGF levels in periodontal health and disease. J Periodontol 78: 783–1787.
Rezavandi K, Palmer RM, Odell EM, Scott DA, Wilson RF (2002). Expression ICAM-1 and E-selectin in gingival tissues of smokers and non-smokers with periodontitis. J Pathol Med 31: 69–64. Sakallıo glu EE, Ayas B, Sakallıo glu U, Acßıkg€ oz G, C glayan F ß a (2006). Osmotic pressure and vasculature of gingiva in periodontal disease. Arch Oral Biol 51: 505–511. Sakallıo glu EE, Aliyev E, L€ utfio glu M, Yavuz U, Acßıkg€ oz G (2007). Vascular endothelial growth factor (VEGF) levels of gingiva and gingival crevicular fluid in diabetic and systemically healthy periodontitis patients. Clin Oral Investig 11: 115–120. Sakallıo glu EE, L€ utfio glu M, Sakallıo glu U, Dıraman E, Pamuk F, Odyakmaz S (2008). Local peptidergic innervation of gingiva in smoking and non-smoking periodontitis patients. J Periodontol 79: 1451–1456. Schulte D, K€ uppers V, Dartsch N et al (2011). Stabilizing the VE-cadherin-catenin complex blocks leukocyte extravasation and vascular permeability. EMBO J 30: 4157–4170. Schweitzer KS, Hatoum H, Brown MB et al (2011). Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides. Am J Physiol Lung Cell Mol Physiol 301: L836–L846. Sheets SM, Potempa J, Travis J, Casiano CA, Fletcher HM (2005). Gingipains from Porphyromonas gingivalis W83 induce cell adhesion molecule cleavage and apoptosis in endothelial cells. Infect Immun 73: 1543–1552. Shen W, Li S, Chung SH et al (2011). Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-b1induced permeability of centrally derived vascular endothelium. Eur J Cell Biol 90: 323–332. Silness J, L€ oe H (1964). Periodontal disease in pregnancy. Iı. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 22: 121–135. € S€ onmez S, Canda T, Ozkara E, Ak D (2003). Quantitative evaluation of the vasculature and fibronectin localization in gingival connective tissue of smokers and non-smokers. J Periodontol 74: 822–830. Takeichi O, Hama S, Iwata K, Ito K (2008). Confocal immunolocalization of VE-cadherin- and CXC chemokine-expressing endothelial cells in periapical granulomas. Int Endod J 41: 401–407. Tian Y, Li JL, Hao L et al (2013). Association of cytokines, high sensitive C-reactive protein, VEGF and beta-defensin-1 gene polymorphisms and their protein expressions with chronic periodontitis in the Chinese population. Int J Biol Markers 28: 100–107. Underner M, Ingrand P, Favreau M, Mura P, Meurice JC (2004). Usefulness of biological markers in the evaluation of smoking at the first visit of a smoking cessation program. Rev Mal Respir 21: 705–710. Verma S, Kuliszewski MA, Li SH et al (2004). C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 109: 2058–2067. Villasante A, Pacheco A, Pau E, Ruiz A, Pellicer A, Garcia-Velasco JA (2008). Soluble vascular endothelial-cadherin levels correlate with clinical and biological aspects of severe ovarian hyperstimulation syndrome. Hum Reprod 23: 662–667. Wewers ME, Ahijevych KL, Dhatt RK et al (2000). Cotinine levels in Southeast Asian smokers. Nicotine Tob Res 2: 85–91. Yakovlev S, Gao Y, Cao C et al (2011). Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrinderived fragments. J Thromb Haemost 9: 1847–1855.
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ORIGINAL ARTICLE
Vascular endothelial cadherin and vascular endothelial growth factor in periodontitis and smoking EE Sakallio glu1, U Sakallio glu1, M L€ utfio glu1, F Pamuk2, A Kantarci3 1
Department of Periodontology, Dental Faculty, Ondokuz Mayıs University, Samsun; 2Department of Periodontology, Dental Faculty, _ Aydın University, Istanbul, Turkey; 3Department of Periodontology, The Forsyth Institute, Cambridge, MA, USA
OBJECTIVE: This study investigated the vascularization in periodontal disease process via revealing: (i) vascular endothelial cadherin (VE-cadherin) and vascular endothelial growth factor (VEGF) productions in periodontitis and (ii) the impact of smoking on this phenomenon. MATERIALS AND METHODS: Fifteen smokers and 15 non-smokers with/without periodontitis were allocated by split-mouth randomization regarding their smoking and periodontal statuses. The teeth with periodontitis in smokers (group 1), without periodontitis in smokers (group 2), with periodontitis in non-smokers (group 3), and without periodontitis in non-smokers (group 4) constituted the study groups. Gingival crevicular fluid (GCF) levels of VE-cadherin and VEGF were determined by ELISA to evaluate their profiles in the groups. RESULTS: There were increased VE-cadherin levels in groups 1 and 3 compared with groups 2 and 4 (P < 0.05). Group 2 demonstrated higher VE-cadherin level than group 4 (P < 0.05). Increased VEGF was noted in groups 1 and 3 compared with groups 2 and 4 (P < 0.05) with similar levels between groups 1 and 3 and groups 2 and 4 (P > 0.05). There were no correlations between the VEcadherin and VEGF levels in all groups (P > 0.05). CONCLUSION: The results suggest that VE-cadherin and VEGF may increase in periodontitis, and smoking may uniquely cause VE-cadherin production in GCF. Oral Diseases (2015) 21, 263–269 Keywords: VE-cadherin; VEGF; smoking; periodontitis; gingival crevicular fluid
Introduction Structural and functional alterations are commonly occur in the blood vessels of periodontium due to inflammatory processes (Hock and Nuki, 1978; Sakallıo glu et al, 2006). Correspondence: Assoc. Prof. Elif Eser Sakallıoglu, Department of Periodontology, Faculty of Dentistry, Ondokuz Mayıs University, 55139 Kurupelit, Samsun, Turkey. Tel: +90362 3121919 3780, Fax: +90362 4576032, E-mail: [email protected] Received 21 October 2013; accepted 8 May 2014
New vessel formation and vascular events are critical for the pathogenic mechanisms as well as for the resolution of inflammation and regulation of healing. One of the fundamental roles of endothelial cells is to regulate the transport of fluid, solutes, macromolecules, and immune cells from vessels to the surrounding tissues by specialized transcellular systems of transport vesicles and by coordinated stabilization/destabilization of cell-to-cell contacts (Dejana et al, 2008; Harris and Nelson, 2010). Cell-to-cell contacts are provided by several specialized adhesion complexes, that is, tight junctions and adherens junctions (Villasante et al, 2008). Endothelial cells express several cell-specific cadherin proteins. Vascular endothelial (VE)-cadherin is critical for the core of adherens junctions (Villasante et al, 2008; Harris and Nelson, 2010; Pannekoek et al, 2011) playing a crucial role in the regulation of endothelial cell permeability, transendothelial leukocyte migration, and assembly of new vessels (Villasante et al, 2008; Schulte et al, 2011; Yakovlev et al, 2011). Specific features of VE-cadherin may be essential for the initiation, progression, and maintenance of the inflammatory process, and thus, may be an important marker for the pathobiology of inflammatory conditions including periodontal diseases (Villasante et al, 2008; Schulte et al, 2011; Yakovlev et al, 2011) through angiogenesis, which is critical for the homeostatic balance. A major regulator of the new vessel formation and biology is the vascular endothelial growth factor (VEGF). VEGF induces endothelial cell proliferation, secretion of proteolytic enzymes, chemotaxis, and migration. VEGF is critical for inflammation regulating vasoactive and angiogenic cytokines, and thus implicated in the pathogenesis of inflammatory diseases including periodontal disease (Johnson et al, 1999; Sakallıoglu et al, 2007; Pradeep et al, 2011). The interaction between the VEGF and the VE-cadherin involves reciprocity where VEGF is involved in the disruption of adherens junctions by inducing the dissociation of VE-cadherin/catenin complexes in the endothelium (Esser et al, 1998). This process may cause endothelial dysfunction and increased vascular permeability (Harhaj et al, 2006; Barbieri and Weksler, 2007; Barbieri et al, 2008; Dejana et al, 2008) both of which are critical during the pathological changes in the
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periodontal tissues. Indeed, there is evidence that a major pro-inflammatory peptide, the C-reactive protein mediates the cross-talk between VEGF and VE-cadherin (Verma et al, 2004). This interaction has been speculated to provide a therapeutic mechanism in inflammatory bowel disease and experimental colitis (Chidlow et al, 2007). While limited, these studies provide evidence that the interaction between VEGF and VE-cadherin may have a critical role in the inflammatory process due to the cross-talk between VEGF and VE-cadherin. Such a phenomenon has not been tested as a plausible mechanism for the vascularization during inflammatory periodontal diseases. A major disruption to the periodontal vasculature would be due to smoking (Bergstr€ om et al, 1988; Rezavandi et al, 2002), smoking has already been shown to be an important risk factor for periodontal diseases (Morozumi et al, 2004; Sakallıo glu et al, 2008). One consistent finding in smoking-related diseases is the existence of endothelial dysfunction leading to damage of the endothelium (Koide et al, 2005; Michaud et al, 2006; Lu et al, 2011; Schweitzer et al, 2011). Recent data have demonstrated that smoking may impair vessel health and induce vasoconstriction in gingiva (Mavropoulos et al, 2003), decrease gingival blood flow rate (Morozumi et al, 2004; Mavropoulos et al, 2007), cause higher percent of small blood vessels but similar vascular density in gingiva (Mirbod et al, 2001; S€ onmez et al, 2003), and contribute to the disruption of immune response due to repeated vasoconstrictions (Morozumi et al, 2004). The hypothesis of this study was that smoking-induced disruption of the periodontal vascularization results in changes in VEGF and VE-cadherin expression, which will explain the mechanism of endothelial damage in periodontium. Therefore, the present study aimed: (i) to evaluate GCF levels of VE-cadherin and VEGF in patients with periodontitis and (ii) to investigate the impact of smoking on VE-cadherin and VEGF production in GCF.
Materials and methods Study group characterization Participants referred to the Periodontology Department of Ondokuz Mayıs University Dental Faculty in Samsun, Turkey, were recruited in this cross-sectional study. The study protocol was confirmed by the relevant Human Ethics Committee of Ondokuz Mayıs University-Samsun, and it was carried out according to the ‘Helsinki Declaration of 1975’, as revised in 2008. The experiments were undertaken with the understanding and written consent of each participant. A study population of male smokers and nonsmokers was selected regarding the criteria: (i) to be ≥ 35 years of age and to have localized chronic periodontitis, (ii) not to have previous periodontal therapy in the last 6 months, and (iii) not to have any systemic problems and regular/current (at least 3 weeks prior to data collection) chemotherapeutic regimens. Smoking status was determined as ≥ 5 years of duration and as ≥ 15 per day of consumption in all smokers, whereas non-smoking status included individuals who never smoked before. It has already been demonstrated in many studies that the cotinine levels in serum and urine correlate with the cigarette Oral Diseases
consumption (Wewers et al, 2000; Benowitz et al, 2003; Underner et al, 2004; Johnstone et al, 2006). Therefore, we have decided not to measure the cotinine levels in patients in this study and preferred to use the extensive references. Periodontal statuses of these participants were assessed by clinical examination, and the teeth with periodontitis were matched to the contralateral teeth with no signs of inflammation and destruction. Such a split-mouth study design was carried out to perform evaluations in similar environmental and systemic conditions. Thus, four study groups were arranged according to the smoking habits of the participants and periodontal statuses of their teeth: Group 1 = the teeth with periodontitis in smokers, Group 2 = the teeth without periodontitis in smokers, Group 3 = the teeth with periodontitis in non-smokers, and Group 4 = the teeth without periodontitis in non-smokers. Fifteen smoker and 15 non-smoker participants with healthy and diseased sites were appropriate to determine the results at a = 0.05 with a 90% power. Clinical procedures Clinical records and gingival crevicular fluid (GCF) samples were collected from all participants. The clinical examination procedure to determine periodontal status included: (i) Silness-L€oe plaque index (PI) (L€oe and Silness, 1963), (ii) L€oe-Silness gingival index (GI) (Silness and L€oe, 1964), (iii) probing pocket depth (PPD), and (iv) clinical attachment level (CAL) and radiographical evaluation of alveolar bone loss (ABL). Radiographs taken by full-mouth parallel technique were evaluated for each participant. The distance between enamel-cement line and crestal bone was evaluated, and the distance > 2 mm was accepted as ABL. PI, GI, PPD, and CAL measurements were taken at six sites (mesio-, mid-, disto-buccal/labial and palatal/lingual surfaces) per tooth using a Williams’ periodontal probe. The sites demonstrating > 4 mm PPD and CAL with radiographical evidence of ABL (by parallel technique) were regarded as sites with ‘periodontitis’ and allocated for groups 1 and 3. The tooth site with the highest PPD and CAL values in groups 1 and 3 was selected. GCF was collected from this selected sites. The matching sites with < 3 mm PPD and CAL and without radiographical evidence of ABL were regarded as ‘nonperiodontitis/control’ sites and allocated for groups 2 and 4. In groups 2 and 4, GCF was collected from the nonperiodontitis/control sites. All clinical parameters were measured 1 day before GCF sampling to eliminate possible quantitative and/or qualitative alterations in GCF due to the examination methods. For GCF sampling, the teeth were isolated with cotton rolls and air-dried for 5 s. A standard paper strip (Periopaper; Ora Flow Inc., Amityville, NY, USA) was gently inserted into the gingival crevice (i.e., standard depth of 1 mm) and left there for 30 s. Volumetric analysis of the fluid collected in the strip was performed immediately by a volume quantifying device (Periotronâ 8000; Pro Flow Inc., Amityville, NY, USA), and the strips were stored at 80°C for the laboratory
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procedures. Two investigators calibrated for the measurements of the clinical examination and GCF collection. Laboratory procedures GCF levels of VE-cadherin and VEGF were determined and compared among the study groups. GCFs was first eluded from the paper strip by a protocol described elsewhere (Curtis et al, 1998), and the obtained GCF samples were utilized in the biochemical analyses of VE-cadherin and VEGF. VE-cadherin and VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercially available VE-cadherin (Human VE-cadherin ELISA; Bender MedSystems GmbH, Vienna, Austria) and VEGF (Human VEGF ELISA; Bender MedSystems GmbH) kits. These laboratory procedures were performed by a single investigator blinded to the study. The measured marker concentrations were corrected for GCF volume, and VE-cadherin concentrations were expressed in nanogram per milliliter (ng ml 1), whereas VEGF concentrations were expressed in picogram per milliliter (pg ml 1). The limits of detection were 0.10 ng ml 1 for VE-cadherin and 7.9 pg ml 1 for VEGF. Statistical analysis Calculation of sample size was performed regarding the difference between VEGF levels of GCF in healthy and periodontitis sites (Prapulla et al, 2007) as the study outcome, as no similar data were available for VE-cadherin. Normality of the data was analyzed by Shapiro–Wilk test and, multiple comparison of the groups was performed by Kruskal–Wallis and Friedman tests. Wilcoxon and Mann– Whitney-U tests (with Bonferroni correction) were utilized for the comparison between 2 groups. All results were expressed as median (min.-max.), and the differences were determined as significant at P = 0.05 (independent groups) and P = 0.05 (dependent groups) levels. Spearman’s correlation test was utilized to evaluate the correlation between VE-cadherin and VEGF in each study group. These calculations were performed using a statistical software package program (SPSS 12.0v; IBMCo., Chicago, IL, USA).
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Results Thirty smokers and non-smokers with an age range of 35– 58 years were allocated as the study population. Two teeth in each participant, thus 15 teeth in each group were included in the study. Data on clinical parameters and GCF volumes of the groups are presented in Table 1. Study groups of teeth with periodontal disease (groups 1 and 3) showed significantly higher levels of plaque regardless of the smoking status (Table 1). Gingival tissues around teeth with periodontal disease (groups 1 and 3) showed clinical signs of inflammation compared with healthy sites (Table 1). Smoking was not associated with PPD or CAL (Table 1). GCF volume significantly increased at sites with periodontitis (groups 1 and 3) (Table 1). VE-cadherin concentration was higher in the periodontitis sites of smokers and non-smokers (groups 1 and 3) than in the non-periodontitis sites of smokers and nonsmokers (groups 2 and 4) (P < 0.05) with similar levels between the periodontitis sites of smokers and non-smokers (groups 1 and 3) (P > 0.05) (Figure 1a). It was also found to be higher in the non-periodontitis sites of smokers than in the non-periodontitis sites of non-smokers (groups 2 and 4) (P < 0.05) (Figure 1a). Total amount of VE-cadherin was higher in the periodontitis sites of smokers and non-smokers (groups 1 and 3) than in the non-periodontitis sites of smokers and non-smokers (groups 2 and 4) (P < 0.05) (Figure 1b). However, it was found to be similar in non-periodontitis sites of smokers and non-smokers (groups 2 and 4) (P > 0.05) (Figure 1b). VEGF concentration was higher in the periodontitis sites of smokers and non-smokers (groups 1 and 3) than in the non-periodontitis sites of smokers and non-smokers (groups 2 and 4) (P < 0.05), and it was similar between the periodontitis and non-periodontitis sites of both smokers and non-smokers (groups 1 and 3; groups 2 and 4) (P > 0.05) (Figure 2a). Total VEGF amount was higher in the periodontitis sites of smokers and non-smokers (groups 2 and 4) (P < 0.05), and it was similar between the
Table 1 Distribution and comparison of the clinical examination parameters in the study groups. Groups (n = 15) Group 1 (smoker + periodontitis) median (min.–max.) PI Pa GI Pa PPD (mm) Pa PAL (mm) Pa GCF volume (µl) Pa
2 (0–2) 1 (1–2) 5 (5–7) 6 (5–9) 0.82 (0.28–1.27)
Group 2 (smoker + non-periodontitis) median (min. –max.)
Group 3 (non-smoker + periodontitis) median (min. –max.)
1 (0–2) 2 (2–3) 0.001 for groups 1–2, 1–4, 2–3 and 3–4 1 (0–1) 2 (2–3) 0.001 for groups 1–2, 1–4, 2–3, and 3–4 2 (1–3) 5 (5–8) 0.001 for groups 1–2, 1–4, 2–3 and 3–4 2 (1–3) 6 (5–9) 0.001 for groups 1–2, 1–4, 2–3 and 3–4 0.08 (0.01–0.25) 1.12 (0.55–1.60) 0.001 for groups 1–4 and 2–3; 0.002 for groups 1–2 and 3–4
Group 4 (non-smoker + non-periodontitis) median (min. –max.) 1 (0–2) 1 (0–2) 2 (1–3) 2 (1–3) 0.09 (0.03–0.28)
PI, plaque index; GI, gingival index; PPD, probing pocket depth; GCF, Gingival crevicular fluid. a Statistically significant values. Oral Diseases
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0.35 0.3
(a) 30 0.29 (0.20-0.57)
0.28 (0.20-0.51) 0.2 (0.19-0.31)
0.25 0.2 0.15 0.1
0.19 (0.01-0.21)
0.05 0
VEGF CONCENTRATION (pg ml–1))
(a) VE-CADHERIN CONCENTRATION (pg ml–1)
266
22.66 (2.63-43.91)
27.58 (14.-81.54)
25 20 15
0.55 (0.03-2.32)
9.23
(0.01-15.38)
10 5 0
0.35 0.3 0.25
(b) 30
0.29 (0.13-0.66) 0.23 (0.10-0.44)
0.2 0.15 0.1 0.05
0.02 (0.001-0.05)
0.02 (0.01-0.06)
0
TOTAL VEGF AMOUNT (pg)
TOTAL VE-CADHERIN AMOUNT (pg)
(b)
25
periodontitis and non-periodontitis sites of both smokers and non-smokers (groups 1 and 3; groups 2 and 4) (P > 0.05) (Figure 2b). Spearman’s correlation test demonstrated no correlations between the VE-cadherin and VEGF levels of periOral Diseases
22.66 (2.63-43.91)
20 15 10 5 0
Figure 1 (a) Distribution and comparison of VE-cadherin concentrations [median (min.-max.)] in the study groups. P = 0.004 for periodontitis sites and non-periodontitis sites of smokers (groups 1–2). P = 0.001 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of non-smokers (groups 2–3), non-periodontitis sites of smokers and nonsmokers (groups 2–4), and periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.851 for periodontitis sites of smokers and non-smokers (groups 1–3). (b) Distribution and comparison of VEcadherin total amounts [median (min.-max.)] in the study groups. P = 0.002 for periodontitis sites and non-periodontitis sites of smokers (groups 1–2). P = 0.000 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of non-smokers (groups 2–3). P = 0.003 for periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.110 for periodontitis sites of smokers and non-smokers (groups 1– 3). P = 0.340 for non-periodontitis sites of smokers and non-smokers (groups 2–4).
27.58 (14.05-59.98)
0.55 (0.03-2.32)
0.66 (0.001-3.21)
Figure 2 (a) Distribution and comparison of VEGF concentrations [median (min.-max.)] in the study groups. P = 0.001 for periodontitis and non-periodontitis sites of smokers (groups 1–2). P = 0.000 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of nonsmokers (groups 2–3). P = 0.001 for periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.663 for periodontitis sites of smokers and non-smokers (group 1–3), and non-periodontitis sites of smokers and non-smokers (groups 2–4). (b) Distribution and comparison of VEGF total amounts [median (min.-max.)] in the study groups. P = 0.002 for periodontitis and non-periodontitis sites of smokers (groups 1–2). P = 0.000 for periodontitis sites of smokers and non-periodontitis sites of non-smokers (groups 1–4), non-periodontitis sites of smokers and periodontitis sites of non-smokers (groups 2–3). P = 0.003 for periodontitis and non-periodontitis sites of non-smokers (groups 3–4). P = 0.176 for periodontitis sites of smokers and non-smokers (group 1–3). P = 0.853 for non-periodontitis sites of smokers and non-smokers (groups 2–4).
odontitis sites in smokers (r = 0.265) and non-smokers (r = 0.338) as well as in the non-periodontitis sites of smokers (r = 0.006) and non-smokers (r = 0.114).
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Discussion The study results suggest that higher GCF levels of VEcadherin and VEGF are associated with periodontal disease. Smoking further increases VE-cadherin expression in the GCF. On the other hand, data also suggest that increased VE-cadherin levels were found in the periodontitis sites compared with the non-periodontitis sites, independent of the smoking status. This finding is within lines of the previous reports demonstrating that the diseased and/or inflammatory conditions that increase the vascular permeability may affect the structure and organization of vascular junctions (Corada et al, 1999). One possible explanation for such an increase would be due to a physical phenomenon through which the permeability of the vessel wall increases cytokines such as histamine, TNF-a, platelet-activating factor. During this process, VEGF may induce the tyrosine phosphorylation of VE-cadherin and its binding partner, b-catenin (Dejana et al, 2008). Consequently, an endothelial cell retraction may occur, therefore reducing the endothelial junctional strength and causing an extraordinary opening in the intercellular gaps (Esser et al, 1998; Andriopoulou et al, 1999). Dramatic increase in permeability and vascular fragility via administration of anti-VE-cadherin in mouse model was observed (Corada et al, 1999). The relation between VE-cadherin and vascular permeability in inflamed endothelial cells in cell cultures (Sheets et al, 2005), and a loss of cell adhesion (Takeichi et al, 2008), the barrier breakdown and increased paracellular permeability (Shen et al, 2011) as a result of altered expression of VE-cadherin were also reported in different tissues. Therefore, alterations in VE-cadherin expression (as VEGF expression) may be an important stage in the initiation and progression of inflammation. Our results demonstrated higher VEGF levels in the periodontitis sites of both smokers and non-smokers compared with the matching non-periodontitis sites. VEGF may regulate the periodontal inflammation by (i) promoting a vascular network, (ii) increasing the extent of inflammatory process, and (iii) inducing angiogenesis into the spaces created by the destruction of periodontal tissues based on the previous reports (Johnson et al, 1999; Sakallıo glu et al, 2007; Pradeep et al, 2011) and the findings from the current study. VEGF may also be effective on the disruption of adherens junctions, as it induces the dissociation of VE-cadherin/catenin complexes in endothelium and causes destabilization in the adherens junction complexes to modify vascular permeability (Harhaj et al, 2006; Barbieri and Weksler, 2007; Barbieri et al, 2008; Dejana et al, 2008; Pannekoek et al, 2011). Indeed, VEGF has been shown to produce significant changes in the VE-cadherin concentration of endothelial cell cultures as well as alterations in actin fibers resulting in increased capillary hyperpermeability (Villasante et al, 2008). VE-cadherin was found to be increased in the non-periodontitis sites of smokers compared with the non-periodontitis sites of non-smokers with similar levels in the periodontitis sites of both smokers and non-smokers. Deterioration of VE-cadherin may be evaluated as a sign of endothelial degeneration (Michaud et al, 2006; Barbieri
and Weksler, 2007; Barbieri et al, 2008), as it is known as the main component of adherens junctions in endothelial cells. This has been demonstrated as a barrier dysfunction with the disruption of VE-cadherin in mouse cardiac endothelium due to tobacco smoke (Barbieri et al, 2008). An in vitro study of endothelial progenitor cells obtained from the peripheral blood of smokers further reported significant downregulation of VE-cadherin expression (Michaud et al, 2006). It has also been revealed that vascular damage caused by the disassembly of VE-cadherin complex induced endothelial dysfunction (Harris and Nelson, 2010; Schulte et al, 2011; Yakovlev et al, 2011). Because VE-cadherin is an essential protein in the maintenance of intact vasculature by providing the integrity of endothelial lining cells (Harris and Nelson, 2010), it may only be detected in serum or body fluids whenever a destructive process leads to its release. The VE-cadherin increase in GCF may accordingly be a consequence of the adherens junction degradation on endothelium secondary to smoking. However, the similarity between the periodontitis sites suggests that this phenomenon may be masked within an established inflammatory process, and thus, an additive effect of smoking on the VE-cadherin increase may not be noted in the chronic inflammatory diseases or lesions such as periodontitis. This assumption, however, requires further testing in larger clinical and in vivo experimental models. Similar VEGF concentration in the periodontitis and non-periodontitis sites of both smokers and non-smokers was an another interesting finding in this study. Although insufficient data are available on the effect of smoking on the VEGF profile of healthy periodontium, increased (Kuwano et al, 1993; Hiroshima et al, 2002; Hashimoto et al, 2005) and/or decreased (Blomberg et al, 1997; Bocchino et al, 1997; Liu et al, 2003) VEGF levels have been detected on the pulmonary tissues and secretions of smokers and, tobacco-related diseases causing pathological angiogenesis such as neoplasies and angiogenic squamose dysplasia have been described before in pulmonary tissues (Keith et al, 2000; Heeschen et al, 2001). In the present study, smoking was not found to be directly associated with GCF levels of VEGF, and the results suggest that VEGF is likely to increase only in inflammatory conditions. Increased VEGF levels in both GCF and periodontal tissues were showed at the periodontal disease (Pradeep et al, 2011; Bletsa et al, 2012; Tian et al, 2013) and diabetes (Guneri et al, 2004; Sakallıoglu et al, 2007; Aspriello et al, 2009; Keles et al, 2010). In this respect, further larger scale studies are clearly required to investigate the role of VEGF in pathogenesis of periodontal disease at especially smoking status because of its regulator role in vascular integrity, dysfunction, and hyperpermeability. Contrary to our hypothesis, we could not identify any correlation between VEGF and VE-cadherin both in the periodontitis and non-periodontitis sites of smokers and non-smokers. This finding does not preclude the fact that there may be a mechanistic regulation due to the limitations of the current study. Even if the power analysis was performed prior to the study and intended patient number was recruited, the variation between the groups still suggests that a larger study needs to be designed and
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conducted. On the other hand, statistical analyses demonstrate significant differences, which suggest that the results are reliable as a basis for future studies. One limitation of the current study is obviously the tissue level expression of the studied markers and their correlations to the GCF levels. This shortcoming requires an immunohistochemical analysis and testing of additional markers to elaborate the mechanistic link between the analytes studied. Therefore, we have refrained from making any mechanistic suggestions and present the data as associative findings. Future studies are needed to identify a possible link at various stages of the inflammatory process, because inflammatory markers may be found altered at smoking status than those in non-smoking status. This has been becoming an important area of research in periodontology and many milieu such as saliva, GCF, and serum have been tested for potential samples for diagnostic and prognostic markers of disease. On the other hand, this field is at its infancy especially due to the need for indepth understanding of high-throughput technologies. Another limitation to this end is the difficulty in designing longitudinal studies that will demonstrate the progression of gingivitis to periodontitis in humans. Due to obvious ethical concerns, only reversible gingivitis can be induced experimentally in humans while non-reversible periodontitis can only be tested in animals in vivo. Therefore, studies on human periodontitis are associative in nature, and biomarkers of inflammation can only be inferred upon based on the cross-sectional study designs and/or longitudinal treatment protocols. Our study is a cross-sectional evaluation of the existing periodontitis in humans and cannot address if and how the analytes studied are critical for the progression of disease. Therefore, longitudinal testing of these molecules (and others) in response to periodontal therapy is required for validation of their ‘biomarker’ status. Within these limitations, it can be concluded that (i) VE-cadherin and VEGF may increase in GCF due to the periodontal inflammatory process and (ii) VE-cadherin and VEGF may be utilized to confirm periodontal inflammation or active process. Acknowledgement The authors declare that they have no conflict of interest and that there is no source of funding for this research.
Author contributions Dr. E.E. Sakallioglu: Creation of the study hypothesis and coordination of the experimental design. Dr. M. Lutfioglu: Patient selection and statistical analyses. Dr. U. Sakallıoglu: Coordination of the study phases and manuscript editing. Dr. F. Pamuk: Laboratory procedures and ELISA. Dr. A. Kantarcı: Control of the experimental procedures and manuscript editing.
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