|Year : 2021 | Volume
| Issue : 3 | Page : 158-164
Vascular endothelial growth factor levels in the gingival crevicular fluid of Type II diabetes mellitus patients with and without chronic periodontitis – A clinico-biochemical study
Patil Rujuta1, AR Pradeep2, Purva Chougule1, S Swathika1
1 Department of Periodontology, The Oxford Dental College, Bengaluru, Karnataka, India
2 Department of Periodontology, Government Dental College, Bengaluru, Karnataka, India
|Date of Submission||14-Jun-2021|
|Date of Decision||20-Jul-2021|
|Date of Acceptance||20-Jul-2021|
|Date of Web Publication||30-Nov-2021|
Dr. Patil Rujuta
Department of Periodontology, The Oxford Dental College, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic cytokine that plays a central role in inflammation and wound healing. The role of this angiogenic factor in periodontal destruction could be significant, and VEGF could act as a potent marker of periodontal disease progression. Its role in diabetes mellitus-related complications has been evaluated, and diabetes mellitus may be a potent modulator of VEGF in periodontal disease. Objective: The objective of this study is to evaluate and compare gingival crevicular fluid (GCF) VEGF levels of systemically healthy, chronic periodontitis, and Type II diabetes mellitus participants with and without chronic periodontitis and to investigate the role of VEGF in periodontal disease progression. Materials and Methods: Eighty participants were divided into four groups based on the gingival index, plaque index, probing pocket depth, and clinical attachment level: (Group 1) healthy, (Group 2) chronic periodontitis, (Group 3) Type II diabetes mellitus without chronic periodontitis, and (Group 4) Type II diabetes mellitus with chronic periodontitis. GCF samples collected from each subject were quantified for VEGF levels using an enzyme-linked immune sorbent assay. Further, the correlation between VEGF levels within groups and with the clinical parameters was analyzed in all groups. The statistical analysis was performed using the SPSS software. Results: The mean concentration of VEGF in GCF was the highest in Group 4 (2179.24 pg/ml) followed by Group 3 (1948.32 pg/ml), Group 2 (1776.83 pg/ml), and the least in Group 1 (1266.80 pg/ml). Further, GCF VEGF levels showed a positive correlation with all of the clinical parameters. Conclusions: VEGF concentrations increased from health to disease. Type II diabetes mellitus may affect VEGF concentrations in periodontal disease. These data indicate that VEGF plays a key role in periodontal disease progression and can be considered a biomarker of periodontal disease progression.
Keywords: Chronic periodontitis, diabetes mellitus, gingival crevicular fluid, vascular endothelial growth factor
|How to cite this article:|
Rujuta P, Pradeep A R, Chougule P, Swathika S. Vascular endothelial growth factor levels in the gingival crevicular fluid of Type II diabetes mellitus patients with and without chronic periodontitis – A clinico-biochemical study. Niger J Exp Clin Biosci 2021;9:158-64
|How to cite this URL:|
Rujuta P, Pradeep A R, Chougule P, Swathika S. Vascular endothelial growth factor levels in the gingival crevicular fluid of Type II diabetes mellitus patients with and without chronic periodontitis – A clinico-biochemical study. Niger J Exp Clin Biosci [serial online] 2021 [cited 2022 Nov 29];9:158-64. Available from: https://www.njecbonline.org/text.asp?2021/9/3/158/331553
| Introduction|| |
Periodontitis corresponds to the inflammation and destruction of highly vascularized tissues that support teeth, caused due to microbial dysbiosis and a susceptible host., Disease expression involves complex interactions of the biofilm with host immunity leading to alterations in the bone and connective tissue homeostasis. Diabetes mellitus, being a disease of metabolic dysfunction, characterized by hyperglycemia has a bidirectional relationship with periodontal disease, wherein progression of any of the diseases affects the severity of both diseases.
Vascular endothelial growth, which is known to be an angiogenic factor, is a highly conserved homodimeric 45-kDa glycoprotein that has endothelial cell-selective mitogenic activity and plays a crucial role in vasculogenesis. VEGF is 50,000 times more potent than histamine in inducing permeability and hence was prior recognized as a vascular permeability factor. Studies have shown link between hyperglycemia, inflammation, and VEGF with Type II diabetes-mediated microvascular diseases.
Studies have ascertained the presence of VEGF in periodontal tissues within endothelial and plasma cells and junctional, sulcular, and gingival epithelium along with its increased levels in gingival crevicular fluid (GCF) of periodontitis individuals. Increased prostaglandin E2, interleukin-1, and tumor necrosis-α in GCF have been associated with active destruction of periodontal structures, and these mediators are the potent stimulators of VEGF. Therefore, VEGF may promote periodontal disease progression by increasing vascular permeability and angiogenesis. Further work into the role of VEGF in the pathogenesis of periodontal disease and diabetes mellitus would be beneficial.
Thus, the aim of this clinic-biochemical study was to estimate and compare VEGF levels in GCF of systemically healthy, chronic periodontitis, and Type II diabetes mellitus participants with and without chronic periodontitis.
| Materials and Methods|| |
The study participants were recruited from the outpatient section, Department of Periodontology, The Oxford Dental College, Bangalore, India. The study population consisted of 80 participants (both males and females) age ranging from ≥18 to ≤60 years, having body mass index in the range of 18.5–24.9 kg/and waist circumference <90 cm (men) and <80 cm (women) (WHO 2008) (Source: NHLBI Obesity education initiative 2000).
The exclusion criteria included alcoholics, individuals who smoke and chew tobacco, individuals with a history of periodontal therapy during the previous 6 months, patients on either long-term or current administration of anti-inflammatory, antibiotic, and immune suppressant medication, pregnant and lactating women, and Type II diabetes mellitus patients with insulin replacement therapy and with additional systemic disorders. Each subject underwent full mouth clinical and radiographic evaluation, and the parameters assessed were following: Plaque index, gingival index (GI), gingival bleeding index, probing pocket depth (PPD), and clinical attachment level (CAL). Clinical measurements were performed on four sites per tooth (mesial, buccal, distal, and lingual/palatal) using William's calibrated periodontal probe by the same investigator.
All the participants were divided into four groups – Group 1 – 20 healthy individuals; Group 2 – 20 chronic periodontitis individuals; Group 3 – 20 Type II diabetes mellitus participants without chronic periodontitis; and Group 4 – 20 Type II diabetes mellitus participants with chronic periodontitis.
Based on the criteria of the American Academy of Periodontology, Classification of Periodontal diseases (1999) chronic periodontitis was defined, where GI score was >1, PPD ≥5 mm, CAL ≥3 mm with radiographic evidence of moderate bone loss and Type II diabetes mellitus was diagnosed based on the criteria defined by American Diabetes Association, classification of diabetes mellitus in 2017 and glycated hemoglobin levels values >7%. The written informed consent was obtained from all the study participants. The sample size was determined based on the power analysis at a confidence interval of 90% (P ≤ 0.05). The ethical clearance was obtained from the Institutional Committee and Review Board of The Oxford Dental College, Bangalore (Ref No: 258/2017-2018). The study was conducted in accordance with Helsinki Declaration, 1975.
Site selection and collection of gingival crevicular fluid
A single examiner performed clinical and radiographic evaluation, group allocation, and sampling-site selection. The GCF collection procedure was carried out by another examiner. The collection of the GCF samples was carried out following the day after recording clinical parameters to avoid contamination with blood. The selected site isolation was done using cotton rolls, and it was air-dried. Without touching the marginal gingiva, the supragingival plaque was removed to avoid contamination with blood and saliva and microcapillary pipette blockage. GCF was collected by placing the tip of the microcapillary pipette extracrevicularly (unstimulated) until it gently touched the marginal gingiva, and a standard volume of 3 μl was obtained from each site. The test sites which did not yield a standard volume of 3 μl of GCF were excluded, and contaminated micropipettes were discarded. Only one site per subject was selected as a sampling site in chronic periodontitis, Type II diabetes mellitus with and without chronic periodontitis groups (Group 2, 3, and 4), whereas in the healthy group (Group 1), to ensure collection of an adequate amount of GCF, multiple sites were sampled. The collected GCF samples were stored in a freezer (temperature maintained at − 80°C) till the assay procedure was carried out.
Vascular endothelial growth factor assay procedure
The VEGF concentration was determined by a quantitative sandwich enzyme immunoassay kit. The samples were incubated in the wells of a divided microplate that had been precoated with the Human VEGF monoclonal antibody. For the assay, samples were added to antibody precoated wells followed by incubation. To blank wells, 100 μl/well of standard diluent was added. To standard wells, 50 μl of standard solution and 50 μl of streptavidin- Horseraddish Peroxidase (HRP) were added. To sample wells, 40 μl undiluted sample, 10 μl of VEGF antibody, and 50 μl of streptavidin-HRP were added. The assay plate was sealed using a plate sealer, mixed gently, and finally incubated in the plate at 37°C for 60 min. After incubation, the solution was completely discarded, and the plate was tapped on a clean paper towel. 1X washing buffer solution in each well was added, incubated the plate for 25–30 s at the room temperature, the solution was completely discarded and the plate was tapped on a clean paper towel to drain the reagent out of wells. This washing procedure was repeated five times. For color development, 50 μl of chromogen reagent A was added to each well followed by the addition of 50 μl of chromogen reagent B to each well. The plate was shaken gently to mix the A and B reagents and incubated for 10 min at 37°C in dark. Fifty microliter stop solution was added to each well to stop the reaction (color changed from blue to yellow immediately at that moment). The optical density (OD) of each well was determined within 10 min after adding stop solution using a microplate reader set to 450 nm. The concentration of VEGF in the tested samples was estimated using the standard curve plotted using OD values with the standards. Samples and standards were assayed in duplicate as suggested by the manufacturer.
Data were analyzed using the SPSS (Statistical Package for Social Sciences for Windows, Version 22.0. Released 2013. Armonk, NY: IBM Corp). One-way ANOVA test was performed to compare the mean values of clinical parameters between the groups. Tukey's post hoc analysis was used for the multiple pairwise comparisons between the groups for different parameters. Kruskal–Wallis test was used for the comparison of mean GCF VEGF levels. Mann–Whitney post hoc analysis was used for multiple pair-wise comparisons of GCF VEGF levels between groups. Spearman's correlation test was used to establish the relationship between GCF-VEGF biomarker and periodontal parameters. Stepwise multiple linear regression analysis for GCF VEGF in different groups was done.
| Results|| |
The VEGF was detected in every sample assessed from each group. The mean GCF VEGF levels were the highest in Group 4 (2179.24 ± 1125.63 pg/ml) followed by Group 3 (1948.32 ± 820.0 pg/ml), Group 2 (1776.83 ± 475.18 pg/ml), and lowest in Group 1 (1266.80 ± 97.21 pg/ml) at P < 0.001 [Graph 1]. One-way ANOVA was done to compare the clinical parameters between all the groups, revealed the highest score of all parameters in Group 4 and least in Group 1 [Table 1]. Kruskal–Wallis test was done to compare GCF VEGF levels concerning CAL in Group 2 and Group 4 and the test demonstrated direct proportionality between CAL and GCF VEGF concentration [Table 2].
|Table 1: Comparison of mean values of different study parameters using the one-way ANOVA test|
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|Table 2: Comparison of mean gingival crevicular fluid-vascular endothelial growth factor levels (pg/mL) with respect to clinical attachment level in Group 2 and Group 4 using Kruskal–Wallis test|
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Spearman's correlation test used to establish the relationship between GCF VEGF biomarker and periodontal parameters showed a positive correlation of all clinical parameters with VEGF levels in GCF of Groups 2, 3, and 4 which was statistically significant at P < 0.001. The results suggested that there was a progressive increase in GCF VEGF levels from health to disease [Table 3]. Stepwise multiple regression analysis was done for GCF VEGF levels for different groups. The regression analysis demonstrates that for all the groups, as GI score increased by one score, the GCF VEGF levels increased by 1199.38 pg/ml, which was statistically significant at P < 0.001, as CAL increased by 1 mm, the GCF VEGF levels increased by 418.53 pg/ml, which was statistically significant at P = 0.004, as PPD increased by 1 mm, the GCF VEGF levels increased by 364.90 pg/ml, which was statistically significant at P = 0.04. The results suggest that all the clinical parameters have direct proportionality with GCF VEGF levels [Table 4].
|Table 3: Spearman's correlation test to establish the relationship between gingival crevicular fluid-vascular endothelial growth factor biomarker and periodontal parameters|
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|Table 4: Stepwise multiple linear regression analysis for gingival crevicular fluid-vascular endothelial growth factor in over-all samples and different groups|
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Although there was no statistically significant difference seen in mean VEGF concentration in GCF between Groups 2, 3, and 4, suggesting that diabetes mellitus may have possible influences on the progression of periodontal disease. Further, the concentration of VEGF in GCF increased with the progression of periodontal disease.
| Discussion|| |
Periodontitis is an infection of highly vascularized supporting tissues of the teeth characterized by active and quiescent periods. Dysbiotic microbiota and a susceptible host are required to develop periodontitis which is associated with an increased risk for certain systemic disorders. Angiogenesis is a prominent feature of inflammation and healing, but the role of angiogenesis in promoting the progression or healing of periodontal lesions is not clear.
VEGF is a multifunctional angiogenic cytokine of importance in inflammation and wound healing. It is a principal mediator of diabetic retinopathy, capable of inducing the changes observed in proliferative retinopathy, macular edema, and possibly nonproliferative diabetic retinopathy. VEGF is found to contribute in the pathogenesis of Type II diabetes-mediated microangiopathy.
Prapulla et al. assessed VEGF levels in GCF in participants with clinically healthy gingiva, gingivitis, and chronic periodontitis and after initial therapy (scaling and root planing [SRP]) in the chronic periodontitis participants. Jayaraj et al. assessed GCF VEGF levels in chronic periodontitis patients with and without type II diabetes mellitus before and after SRP. However, till today, no studies have been reported, that compare GCF VEGF levels in Type II diabetes mellitus individuals with and without chronic periodontitis. Thus, the present study is the first study designed to correlate VEGF concentrations in GCF of Type II diabetes mellitus individuals with and without chronic periodontitis and assess the influence of Type II diabetes mellitus on periodontal disease progression.
In this study, the use of a commercially available, sensitive, sandwich enzyme-linked immunosorbent assay kit has been done to quantify VEGF. The GCF was collected using microcapillary pipettes to avoid nonspecific attachment of the analyte to filter paper fibers ensuing in a false reduction in the detectable VEGF levels that in turn can underestimate the correlation of VEGF levels to disease severity/progression.
The results of the present study indicated that the concentration of VEGF in GCF was the highest in type II diabetes mellitus patients with chronic periodontitis (2179.24 pg/ml) followed by type II diabetes mellitus patients without chronic periodontitis (1948.32 pg/ml), systemically healthy chronic periodontitis patients (1776.83 pg/ml), and the least in healthy individuals (1266.80 pg/ml), which is in accordance with studies done by Prapulla et al. where they saw highest GCF VEGF in chronic periodontitis followed by gingivitis and the least in healthy individuals and also in accordance with study done by Jayaraj et al. who found an increase in GCF VEGF concentration in chronic periodontitis participants with and without type II diabetes mellitus.
In the present study, the clinical parameters correlated positively with GCF VEGF concentrations in Type II diabetes mellitus patients with and without chronic periodontitis and also in systemically healthy Type II diabetes mellitus patients. Prapulla et al. also found an increase in the concentration of VEGF as CAL increased. The positive correlation could be due to the angiogenic potential of VEGF. The vasoconstriction caused during inflammation causes hypoxia which in turn stimulates the production of VEGF and thereby plays a pivotal role during inflammation by mediating neovascularization. These findings suggest the primitive role of VEGF in inflammation, thus mediating disease progression. The variability of VEGF concentration within-subjects of each group can be attributed to the role of VEGF in different stages of the disease process at the time of collection of GCF samples.
Such an increase in VEGF concentrations has been seen in other pathologic conditions as well. Kucukardali et al. concluded that increased plasma VEGF levels in were noted in patients with coronary artery disease. Kim et al. (2020) reported the high levels of pro-angiogenic cytokine VEGF in the synovial fluid and sera of rheumatoid arthritis patients. The authors also stated the use of serum VEGF as an alternative biomarker to C-reactive protein and erythrocyte sedimentation rate to asses rheumatoid arthritis activity and treatment response. Gomułka et al. assessed the possible association between VEGF serum concentration and irreversible bronchoconstriction in adult patients with a diagnosis of asthma and concluded that increased VEGF serum concentration is characteristic of patients with asthma, especially those with irreversible bronchoconstriction The findings also suggest the influence of VEGF on the hyperactivity of airways and reduction of bronchial diameter by thickening the bronchial wall during the remodeling process. Brown et al. showed upregulation of VEGF mRNA expression in three bullous disorders with subepidermal blister formation: Bullous pemphigoid, erythema multiforme, and dermatitis herpetiformis. Tandon et al. stated that higher serum VEGF levels were seen in pregnancy-induced hypertension compared to healthy pregnant women.
In the present study, increased VEGF levels in GCF in chronic periodontitis patients with and without Type II diabetes mellitus indicate the progression of periodontal disease by an amplified inflammatory response, as both chronic periodontitis and diabetes mellitus are the chronic inflammatory disease. The presence of VEGF in the GCF of healthy individuals indicates the role of VEGF in homeostasis by angiogenesis.
The evidence from systematic review and meta-analysis substantiates the results obtained in our study. Ren et al. and team conducted a meta-analysis and microarray data validation for the use of VEGF as a potential molecular target in periodontitis. They concluded that high levels of VEGF were credible implications for the development of periodontitis and anti–VEGF therapy could prove to be a valuable therapeutic modality in treating periodontitis. Nardi et al. performed a systematic review and found increased expression of VEGF in the gingival tissue and GCF of diabetic patients with periodontitis. They also stated the possibility of major role of VEGF in neovascularization physiology and pathophysiology in microvasculature of the periodontium.
Thus, our study shows that VEGF concentration in GCF increases in both chronic periodontitis and type II diabetes mellitus. However, VEGF concentration increases with the increasing severity of the periodontal disease. Thus, VEGF in GCF can be considered as a marker of angiogenic activity in periodontal disease as well as diabetes mellitus. This study confirms the critical role of VEGF in periodontal disease progression employing increased angiogenesis as a result of hypoxia caused to tissue destruction and these findings also propose the putative effect of diabetes mellitus on VEGF levels, which may be considered as one of the critical factors causing the severity of periodontal disease with its inflammatory features.
The possible limitations of the present study could be that it was not gender matched with more males compared to females, which can act as a confounding factor. The study consists of a small sample size. The effect of nonsurgical periodontal therapy on GCF VEGF should have been assessed for all the groups.
| Conclusions|| |
The present study showed GCF VEGF concentrations to be the highest in chronic periodontitis participants with Type II diabetes mellitus, which denotes the pro-inflammatory role of VEGF, as both chronic periodontitis and Type II diabetes mellitus are the chronic inflammatory disease. The results also state that Type II diabetes mellitus may be an important modifying factor for VEGF production in periodontal disease and this modifying effect during inflammation may not be altered even under good control of diabetes mellitus. However, there are still not enough data about the destructive and progressive role of VEGF in periodontal disease of diabetic patients, particularly in large populations and in different metabolic control levels.
Thus, within the limits of the present study, it could be proposed that VEGF plays a crucial role in periodontal disease progression and could be used as a biochemical marker. However, long-term, prospective, multi-centric studies involving a larger sample size need to be carried out to confirm the above findings. The role of VEGF in periodontal disease of Type II diabetes mellitus patients with different metabolic control should be assessed, along with the duration of diabetes mellitus. Further studies that will investigate the relationship between VEGF, inflammation, and diabetes mellitus in periodontal disease should also match with vascularization, vascular permeability, and fluid dynamics of periodontal tissues. In addition, chair-side diagnostic tests and VEGF-specific therapeutic strategies could be developed to arrest the periodontitis-associated alveolar bone destruction.
The authors are greatful to Dr. R Kavyashree, Principal of The Oxford Science College who has provided facility for the storage of samples and to Mr. Venkatswamy, Department of Microbiology, Kempegowda Institute of Medical Sciences, Bangalore, for his help in carrying out ELISA procedure.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]