Vascular Endothelial Growth Factor Levels in Gingival Crevicular Fluid Before and after Periodontal Therapy

Abstract

Introduction: Of the various cytokines and growth factors regulating angiogenesis, the most potent agent acting on vascular endothelium is vascular endothelial growth factor(VEGF). The present study aims to access VEGF concentration in periodontal diseases and the effect of periodontal therapy on its concentration in gingival crevicular fluid (GCF).

Materials and Methods: Ninety five subjects (47 females and 48 males) 25- 47 y of age were divided into healthy (group-1), gingivitis (group-2), and periodontitis (group-3). GCF samples were collected using micro-capillary pipettes & were transferred immediately to plastic vials and stored at -70˚C until the time of the assay. The concentration of VEGF was determined using commercially available ELISA kit.

Results: The mean VEGF concentration was highest in periodontitis patients (Group 3) (88.08±8.04pg/ml), with lowest in healthy patients (Group 1). VEGF levels reduced significantly after therapy in Groups 2 and 3.

Conclusion: VEGF levels in GCF had a significant correlation with both periodontal disease progression and healing after therapy.

Introduction

Angiogenesis is the formation of new capillaries by the budding of endothelial cells residing in the surrounding pre-existing vessels. There is considerable evidence to indicate that vascular endothelial growth factor (VEGF) also known as vascular permeability factor or vasculotropin plays a critical role in neo-vascularization [1]. Increase in vessel profiles along the periodontal pocket wall could be a result of angiogenesis [2]. VEGF induces the permeability of fluids and proteins 50,000 times more than histamine [3]. VEGF potently increases micro-vascular permeability, stimulates endothelial cell (EC) proliferation, induces proteolytic enzymes and migration of endothelial cell and monocytes, all of which are essential for angiogenesis [4-11].

Several members of VEGF family have been described during the past few years including VEGF-A, PIGF, VEGF-B, VEGF-C, VEGF-D and VEGF-E [12]. VEGF A is a dimeric glycoprotein which has atleast nine subtypes due to the alternative splicing of the single gene; VEGF 121, VEGF 145, VEGF148, VEGF165b, VEGF183, VEGF189, VEGF206 [13,14]. VEGF121 does not bind heparin and is freely released from the cell. VEGF165 readily binds to heparin and it remains bound to the cell surface or to the extracellular matrix following secretion [15]. VEGF 189 and VEGF 206 bind to heparin with greater affinity than VEGF165 and are completely sequestrated within the extracellular matrix [15,16].

VEGF has a role in physiological (menstrual cycle, pregnancy and wound healing) and pathological conditions (diabetes, rheumatoid arthritis, psoriasis, cancer and chronic inflammation) [17-21]. VEGF is also shown to be important for memory and learning [22]. Exaggerated levels of VEGF A have been detected in tissues and biologic samples from subjects of asthma where these levels correlate directly with the disease [23] and inversely with airway function [24]. Increased levels of circulating soluble fms -like tyrosine kinase 1 (sFlT-1) and reduced levels of free placental growth factor (PlGF) and VEGF predict the subsequent development of pre-eclampsia [25].

In gingival crevicular fluid (GCF) the levels of bacterial antigens, inflammatory mediators and tissue breakdown products have been assessed as risk markers of active periodontal destruction [26]. PGE2 in GCF is associated with destruction of periodontal attachment [27]. PGE2 is a potent stimulant of VEGF synthesis. In addition to PGE2, interleukin-1 (IL1) and tumour necrosis factor α (TNF α) are also implicated in the induction of VEGF [28]. Both these cytokines are released at higher levels by monocytes and macrophages from patients with periodontitis than from healthy controls particularly in response to bacterial risk factor [29,30]. VEGF expression is also reported to be regulated by the oxygen concentration of the tissues with hypoxia inducing its expression [31].

In view of the above mentioned findings this clinico-biochemical study was undertaken to investigate the VEGF levels detectable in GCF in subjects with clinically healthy gingiva, gingivitis and chronic periodontitis and after initial therapy i.e. scaling in the gingivitis subjects and scaling root planning (SRP) in periodontitis subjects.

Materials and Methods

The study population consisted of 95 subjects (47 females and 48 males) 25-47 y of age that were selected from the out-patient section of the Dept. of Periodontics, St. Josephs Dental College, Eluru, Andhra Pradesh, India.

Exclusion criteria were: Pregnancy, lactation, Smoking, alcoholism, diabetes, hypertension, asthma, tumours, rheumatoid arthritis, cardiac diseases, long term administration of anti-inflammatory and antibiotic medication, aggressive periodontitis and previous periodontal therapy within a period of twelve months. Each subject underwent a full mouth periodontal probing and charting along with intra oral peri-apical radiographs for crestal bone loss.

The subjects were then categorised into three groups based on the clinical examination and radiographic evidence of bone loss. Forty five subjects with clinically healthy periodontium, Gingival Index (GI<1), Probing pocket depth less than or equal to 3mm (PPD ≤ 3), no clinical attachment loss (CAL=0) and no radiographic evidence of crestal bone loss were included in group 1.

Group 2 (Gingivitis group) consisted of 25 subjects with GI≥2, PPD≤3mm and CAL=0.

Group3 (Chronic periodontitis) consisted of 25 subjects with PPD ≥5mm, CAL ≥2mm and/or radiographic evidence of bone loss.

Subjects satisfying the above criteria were enrolled in the study after obtaining the ethical clearance from the institutional review board. All the recordings were performed by a single examiner. Single site was selected from each subject for GCF sampling. In the healthy subjects sampling was predetermined to be from mesio-buccal region of maxillary right first molar in the absence of which the left molar was considered. In the gingivitis patients, sites with most severe gingival inflammation were considered and in periodontitis group sites showing maximum probing pocket depth or sites with CAL ≥2mm along with radiographic confirmation of crestal bone loss was sampled. GCF samples were collected using calibrated volumetric micro capillary pipettes (TT3 Top Tech, Biomedicals, Mumbai, India). Sites selected for sampling were isolated with cotton wool rolls, supragingival plaque gently removed with a curette and the site dried with a pellet of cotton roll. GCF was collected in micropipette with 5μl capacity and colour coded markings at each μl. The pipette was kept at the entrance of gingival sulcus until the pipette is filled with 3μl of gingival fluid. Pipettes being contaminated with blood or saliva were discarded and fresh samples were again collected. The GCF samples were transferred immediately to plastic vials containing 5ml of phosphate buffered solution (PBS) and stored at -70˚ until the time of the assay. The concentration of VEGF was determined by using commercially available enzyme linked immunosorbent assay (ELISA) kit {R &D system Minneapolis, USA}, as instructed by the manufacturer.

Group 2 patients were treated with single sitting ultrasonic scaling.

Group 3 patients were treated with a non-surgical approach which included single sitting ultrasonic scaling and root planing (SRP) and GCF samples were collected after 12 wk.

The study was performed in accordance with the Declaration of Helsinki. All study participants gave their signed, informed consent before inclusion in the project.

Statistical Analysis

The mean and standard deviation was calculated for all the parameters, then these values were subjected for analysis of variance (ANOVA) and paired t-test for further analysis using SPSS software for Microsoft excel.

Results

All of the samples in each group tested positive for the presence of VEGF. [Table/Fig-1] shows the mean and the standard deviation of VEGF in the three groups. The mean VEGF levels in GCF shows modest increase from 31.91± 3.06 (control/group-1) to 48.71 ± 3.61(gingivitis/group-2) while, a sharp increase is noticed in the periodontitis group tantamounting to a level of 88.08±8.04 (periodontitis/group-3). When subjected for analysis of variance test (ANOVA) the results showed a statistically significant difference in VEGF values in each group (p-value 0.00) [Table/Fig-2]. When compared between control (Group-1) and gingivitis (Group-2) group the mean difference was 16.79 and between control (group-1) and periodontitis (Group-3) the mean difference was 56.16. Similarly, when compared between gingivitis (Group-2) and periodontitis (Group-3) the mean difference was 39.36. These differences were found to be statistically significant with p-value 0.00 [Table/Fig-3]. This indicated that as the disease progressed from health to gingivitis and then to periodontitis the VEGF levels increased significantly in the GCF.

[Table/Fig-1]:

GCF VEGF levels (pg/ml) of the three groups

	N	Mean	Std. Deviation	Std. Error	95% confidence interval for mean		Minimum	Maximum
					Lower bound	Upper bound
1	45	31.9144	3.06842	0.45741	30.9926	32.8363	27.15	38.75
2	25	48.7120	3.61021	0.72204	47.2218	50.2022	42.15	55.85
3	25	88.0888	8.04723	1.60945	84.7583	91.4017	76.20	105.35
Total	95	51.1153	23.78442	2.44023	46.2701	55.9604	27.15	105.35

[Table/Fig-2]:

Analysis of variance for VEGF levels in the three groups

ANOVA
	Sum of squares	df	Mean square	F	Sig.
Between groups	50894.401	9	25447.200	1026.248	.000
Within groups	2281.265	92	24.796

[Table/Fig-3]:

Intergroup comparison of VEGF levels (pg/ml) before treatment

(I)VAVAR00006 (J)VAVAR00006	Mean differences (I-J)	Std. error	Sig.	95% confidence interval
				Lower bound	Upper bound
Tukey HSD 1.00 2.00 3.00	-16.79756 -56.16556	1.24213 1.24213	0.00 0.00	-19.7566 -59.1246	-13.8385 -56.2065
2.00 1.00 3.00	16.79756 -39.36800	1.24213 1.40844	0.00 0.00	13.8385 -42.7232	19.7566 -36.0128

The GCF levels of VEGF were re-evaluated after 3 months of intervention. In group-2 the mean VEGF levels reduced from 48.71±3.61 to 36.72±4.27 with a mean difference of 11.98±3.74. This difference was statistically significant with p-value 0.0002 [Table/Fig-4]. Similarly in Goup-3 the mean VEGF values reduced from 88.08±8.04 to 66.39±9.07 after three months with a difference of 21.68±4.66 with a statistically significant p-value 0.00038 [Table/Fig-4].

[Table/Fig-4]:

Intra group comparison of VEGF levels (pg/ml) before and after therapy

Paired samples test
	Mean	SD	Std Error	95% confidence intervel of the	t	Diff.	Sig.
Pair 1 gr2 before - gr2 after	11.98800	3.74417	.74883	10.44248	13.53352	16.009	24	0.0002
Pair 2 gr3 before - gr3 after	21.68920	4.66712	.93342	19.76271	23.61569	23.2356	24	0.00038

Upon subjecting the group-2 patients for scaling and root planning, it was seen that the mean gingival index (GI) decreased from 2.24±0.18 to 0.82±0.27. When subjected to paired sample test the mean difference of GI before & after therapy was 1.41± 0.32 which is statistically significant (p-value 0.00) [Table/Fig-5] The group-3 patients upon completion of therapy showed a reduction in GI from 2.59±0.17 to 1.36±0.20. Similarly, the probing pocket depth reduced from 6.04±0.67 to 4.28±0.89. The results of the paired sample test showed the mean difference of 1.23±0.25, 1.76±0.77 and 0.36±0.48 for GI, PPD and CAL respectively. These differences were found to be statistically significant with p-value 0.00 [Table/Fig-6].This decrease in the clinical parameters suggests the efficacy of the scaling and root planing on periodontitis.

[Table/Fig-5]:

Paired sample statistics of Group 2

Mean	N	Std. deviation	Std. error of mean	Differences		p-value
				Mean	S.D.
Pair 1 GIb GIa	2.2440 0.8280	25 25	0.18502 0.27917	0.3700 0.5583	1.41600	0.32104	0.00

[Table/Fig-6]:

Paired sample statistics of Group 3

Mean	N	Std. deviation	Std. error of mean	Differences		p-value
				Mean	S.D.
Pair1 GIb GIa	2.5960 1.3600	25 25	0.17673 0.20817	0.03535 0.04163	1.23600	0.25311	0.00
Pair 2 PDb PDa	6.0400 4.2800	25 25	0.67577 0.89069	0.13515 0.17814	1.76000	0.77889	0.00
Pair 3 CALb CALa	2.3200 1.9600	25 25	0.47610 0.35119	0.9522 0.7024	0.3600	0.48990	0.001

Discussion

Aberrant angiogenesis is associated with lesion formation in chronic periodontitis but the mediators that contribute to angiogenesis or therapeutic agents that control the action of mediators have not been well described [32].

Periodontitis is a chronic inflammatory disease with episodes of active destruction and periods of quiescence [33]. However, individuals vary in their response to chronic gingival inflammation. VEGF one of the most potent angiogenic mediators was detectable in periodontal tissues within the vascular endothelial cells, plasma cells, macrophages and in junctional and sulcular gingival epithelium. This extensive cellular distribution shows that VEGF may play a role in the maintenance of periodontal physiology and the progression of periodontal diseases [34]. In recent years the possible role of VEGF in the pathogenesis of periodontal diseases has been investigated in clinical studies [32,35-38]. To the readers knowledge the present study is one of the few studies designed to investigate the concentration of VEGF in GCF of periodontal healthy subjects and to compare with disease subjects (gingivitis and periodontitis) also, before and after therapy in disease subjects. The patients were carefully selected to eliminate the possible alterations of hormonal conditions seen women such as in menstruation cycle and menopause, because of the possible effects of smoking on gingival microcirculation and increased inflammatory cytokine production may aggravate periodontal disease therefore, smokers were excluded from the study. The patients with psoriasis and hypersensitivity have also shown high levels of VEGF [39] levels so, they were also excluded from this study.

The mean VEGF concentration in GCF was highest in group 3 and lowest in group 1. And intermediate in group 2. The results of this study suggested that VEGF levels in GCF increased progressively from healthy to gingivitis and periodontitis subjects. The results of the present study are in accordance with those of Guneri et al., who also reported increased VEGF levels in GCF in periodontitis sites compared to healthy sites [37]. This suggests that in periodontitis patients the production of VEGF is generally upregulated. The reported increase in the release of IL-1β [40] & TNF-α from peripheral blood mono-nuclear cells of periodontitis patients compared with healthy subjects. This might explain the higher concentration of VEGF found in patients since these cytokines can induce the expression of VEGF. Johnson et al., [35] from their study suggested that VEGF is likely to be a factor in the etiology of gingivitis and its progression to periodontitis, possibly by initiating expansion of the vascular network which in turn could be initiated by Interleukin 1β concentration at sites of active periodontal inflammation. Further, Fusen et al., investigated the association between VEGF, Diabetes mellitus and periodontitis and concluded that VEGF is increased in gingival tissues of diabetic patients especially those with periodontal diseases [40].

Contrary to the above findings Booth et al., have reported that VEGF is up-regulated in relatively healthy sites. They explained this finding to be due to the following possible mechanisms; the presence of sub-clinical levels of inflammation, healing after the microbial assault, revealing the presence of VEGF as a component of physiological angiogenesis in the gingival or periodontal environment. Similarly, Cetinkaya BO et al., investigated the association between VEGF expression and vascularisation with regard to the number and diameter of blood vessels and concluded that VEGF may be related more to the healing stage of periodontal disease than to the destruction stage [41]. In another study by Keles GC et al., on VEGF expression levels of gingiva in gingivitis and periodontitis patients with/without diabetes mellitus it was stated that VEGF expression is probably related to both maintenance of periodontal health and periodontal tissue destruction [42]. Therefore, it can be stated that studies regarding the role of VEGF in the pathogenesis of periodontal diseases have had conflicting results.

The gingivitis and periodontitis subjects were treated by non-surgical periodontal therapy (SRP) and strict oral hygiene measures were instituted. Post-treatment both the gingivitis and periodontitis group showed significant reduction in VEGF concentration of GCF [Table/Fig-3]. These results are in accordance with those of Devi V Prapulla [43]. The variability of the VEGF concentration in each group can be attributed to the different stages of the disease process at the time of collection of GCF samples. The results of our study indicate a direct correlation of VEGF values in GCF and the inflammatory condition of the periodontal tissues.

In the present study the GI scores of gingivitis group reduced significantly after therapy. The mean GI and PPD scores of periodontitis subjects also reduced significantly post therapy. According to Becker et al., [44], the decreases in probing pocket depths have however proved to be smaller following non-surgical treatment although these differences have not always been sustained over longer periods of time. At deeper sites gain in attachment have been reported by some but not other investigators following non-surgical therapy [45]. However, according to Goodson et al., and Page and De Rouen any treatment would appear successful when the experimental sites are not actively loosing attachment [46,47]. The mean decrease in the clinical parameters (GI & PD) in the periodontitis subjects post therapy in our study suggests the efficacy of treatment procedures, however our study did not show any CAL gain.

Further, the work into the role of VEGF in the pathogenesis and healing of periodontal disease should be carried out. Longitudinal clinical research would provide further information and should monitor the levels of VEGF both during development and resolution of experimental gingivitis and periodontitis. Further emphasis should also be given to quantify the VEGF levels during treatment intervention.

Conclusion

In this study VEGF concentration in GCF increased proportionally with the progression of periodontal disease. Further treatment aimed at arresting periodontal disease progression resulted in improvement in the clinical parameters (GI & PD) and also, a statistically significant reduction in GCF VEGF levels proportionally confirming to its active pathological role. However, to reach a more precise conclusion longitudinal clinical studies with a larger study population and longer duration on those patients who are on maintenance program is suggested.

Financial or Other Competing Interests

None.