RESEARCH Open Access

VEGF as a potential molecular target inperiodontitis: a meta-analysis andmicroarray data validationBo Ren†, Que Feng†, Shan He†, Yanfeng Li*, Jiadong Fan*, Guangquan Chai*, Le Liu , Haiyun Liu, Chunhao Yang,Yingdi Wang, Huihui Liu, Huanyue Liu and Yafan Song

Abstract

Background: Anti-vascular endothelial growth factor (VEGF) has been used as a therapeutic drug for the treatmentof some human diseases. However, no systematic evidence is performed for assessing the role of VEGF inperiodontitis. We carried out a comprehensive analysis to explore the role of VEGF in patients with periodontitis.

Methods: Multiple databases were searched for eligible studies. The pooled standardized mean difference (SMD)and odds ratio (OR) with the corresponding 95% confidence interval (CI) were applied to evaluate the effect sizes.Clinical data validation from microarray analysis was used. Pathway and process enrichment analysis were alsoinvestigated.

Results: Finally, 16 studies were included in this analysis. Overall, there was a significantly higher level of VEGFexpression in periodontitis than in healthy control groups (OR = 16.64, 95% CI = 6.01–46.06, P < 0.001; SMD = 2.25,95% CI = 1.25–3.24, P < 0.001). Subgroup analysis of ethnicity showed that VEGF expression was still correlated withperiodontitis in the Asian and European populations. No correlation was observed between VEGF expression andage, gender, and pathological type. A large clinical sample data (427 periodontitis patients and 136 healthycontrols) further validated that VEGF expression was higher in periodontitis than in healthy control groups (P =0.023). VEGF was involved in many functions such as blood vessel development, response to growth factor, cellproliferation, and cell adhesion.

Conclusions: High levels of VEGF were credible implications for the development of periodontitis. Anti-VEGFtherapy may be valuable for the treatment of periodontitis in clinical management.

Keywords: VEGF, Function, Periodontitis, Development, Clinical

© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

* Correspondence: 949427779@qq.com; 359117684@qq.com;Chaiguangquan@126.com†Bo Ren, Shan He and Que Feng contributed equally to this work.Department of Stomatology, the Fourth Medical Center, Chinese PLAGeneral Hospital, No. 51 Fucheng Road, Beijing 100048, Haidian District,China

Ren et al. Journal of Inflammation (2021) 18:18 https://doi.org/10.1186/s12950-021-00281-9

BackgroundPeriodontitis, one of the most common inflammatorydiseases, is caused by the host inflammatory responseand the oral microbiome and affects periodontal con-nective tissue [1, 2]. Periodontitis is recognized as amajor public health problem with the global cost bur-den. It impacts approximately 10–15% of the populationin the world [3, 4]. The diagnosis of periodontitis is cur-rently based on clinical rather than etiologic aspect,which can result in limited therapeutic guidance [5].Hence, it is of great importance to explore potential mo-lecular targets and further conduct effective preventionfor patients with periodontitis.Genetic and epigenetic molecular mechanisms are

shown to be contributed to the development of peri-odontitis [6, 7]. Angiogenesis is closely related to somebiological processes (i.e., embryonic development,reproduction, tissue repair, and wound repair) and mayplay a key role in the pathogenesis of some inflammatorydiseases such as periodontitis [8, 9]. Vascular endothelialgrowth factor (VEGF), mapped to the 6p21.3 chromo-some, is a critical potent pro-angiogenic factor and hasbeen implicated in angiogenesis [10]. VEGF is involvedin many biological functions such as cell proliferation,cell adhesion, and chemotaxis [11, 12]. VEGF expressionhas been reported to be higher in some cancers than inhealthy control groups and to be correlated with poorprognosis [13, 14]. As an anti-angiogenic drug, anti-VEGF by the U.S. FDA approval has been administeredfor the therapy of some cancers [15]. However, the roleof VEGF in the pathogenesis of periodontitis has notbeen fully elucidated. The results on VEGF in periodon-titis were still conflicting. For example, VEGF expressionshows no difference between periodontitis and healthycontrol groups [16]. Balci 2019 et al. reported that VEGFexpression was higher in periodontitis than in healthycontrol groups [17].Therefore, in order to explore a full insight into the

role of VEGF expression in periodontitis, we first per-formed a meta-analysis based on available studies. More-over, independent clinical data were also used to verifythe results and pathway and process analyses were alsoinvestigated in periodontitis, which could provide sys-tematic evidence for a better understanding of periodon-titis etiology and develop potential therapeutic targets inclinical practice.

MethodsSearch strategyThe current meta-analysis was conducted in accordancewith the Preferred Reporting Items for Systematic Re-view and Meta-analysis (PRISMA) statement [18]. Weperformed a comprehensive literature search from thePubMed, Embase, Web of Science, Wanfang, and CNKI

databases for studies published until March 19, 2020.The keywords and search terms used were shown as fol-lows: “vascular endothelial growth factor OR VEGF ORVEGF-A OR VEGFA”, “periodontitis OR periapical peri-odontitis OR periodontal abscess OR periodontal dis-ease”. We also hand searched the reference lists fromthe included publications to identify additional studies.

Study selectionIf studies fulfilling the following inclusion criteria wereincluded in this meta-analysis: 1) periodontitis patientswere clinically or pathologically diagnosed and healthycontrol group without a history of periodontitis andother diseases; 2) the expression level of VEGF was re-ported from original clinical studies; 3) studies providedavailable data for calculating standardized mean differ-ence (SMD) or odds ratio (OR) with 95% confidenceinterval (CI). If authors published multiple papers usingoverlapping sample study population, we only includedthe study containing most comprehensive informationor the recent study. If data were not directly reported,we would contact the corresponding author via email.Exclusion criteria were mainly listed as follows: 1) peri-odontitis patients with systematic disorders such as dia-betes mellitus, etc.; 2) case reports, reviews, conferenceabstracts, editorial letters, comments, or non-human re-searches; 3) duplicate studies; 4) studies with insufficientavailable data.

Data extractionThe following data were extracted from the eligible pub-lications: first author’s surname, publication year, coun-try, ethnicity, sample size, age of participants (mean ormedian age), VEGF levels (mean and standard devi-ation), VEGF expression levels (frequency), and testmethod, etc. If the data were not directly reported, wewould contact the corresponding author through emailas possible as we can. If there were any disagreements,disagreements were resolved by the discussion of allauthors.

Further clinical data validation from microarray analysisThe normalized gene expression profiles of microarraydatasets for periodontitis were obtained from the GeneExpression Omnibus (GEO). Three datasets of tissuesamples with periodontitis, GSE10334, GSE16134, andGSE23586 were used. For the GSE23586 dataset, weconducted a log2 transformation. To achieve a largesample population, batch effects were adjusted with theComBat function for these three datasets [19]. Finally,563 tissue samples involving in 427 patients with peri-odontitis and 136 healthy controls were identified forVEGF expression analysis.

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Pathway and process enrichment analysisThe Spearman correlation between VEGF and genes wascalculated in periodontitis, and the corresponding geneswith the correlation with > 0.4 were selected. Based onthese significant genes, Metascape, an effective and effi-cient tool for experimental biologists [20], was appliedto investigate the function of VEGF.

Statistical analysisWith regard to data of mean and standard deviation(SD), the SMDs with 95% CIs were used to compare thelevels of VEGF in the patients with periodontitis and thelevels in healthy controls. For categorical data, thepooled ORs and their corresponding 95% CIs were used.The random-effects model was applied to estimate thepooled effect size (SMD and OR) in this meta-analysis.Heterogeneity among the studies was measured usingCochran’s Q statistics test [21], the statistical heterogen-eity was significant for a Q test P-value < 0.1. When sig-nificant heterogeneity was detected, a sensitivity analysisthrough deleting each study was carried out to deter-mine the stability of the pooled results. Publication bias

was conducted by using the Begg’s and Egger’s test [22,23]. This meta-analysis was performed using Stata soft-ware, version 12.0 (Stata Corp., College Station, TX,USA).For bioinformatics validation data, the difference of

VEGF between periodontitis and healthy control groupswas analyzed with the Wilcoxon signed-rank test. Spear-man correlation coefficient between VEGF and geneswas analyzed. The corresponding analyses were con-ducted using the R software version 3.5.1 (Institute forStatistics and Mathematics, Vienna, Austria).

ResultsStudy characteristicsThe flow diagram of the initial search strategy is shownin Fig. 1. According to the inclusion criteria, 16 studiesfrom 13 publications containing 1532 participants (566patients with periodontitis and 966 healthy controls)were identified [17, 24–35]. These studies were pub-lished between 2000 and 2019 and were conducted inTurkey, India, China, Japan, Italy, and Poland. Of thesestudies, three studies reporting categorical data evaluated

Fig. 1 Flowchart of the study selection process

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the correlation of VEGF between periodontitis andhealthy control groups [29, 32, 34]; the remaining stud-ies reporting mean and standard deviation (SD) evalu-ated the difference between the VEGF expression levelsin periodontitis. The main characteristics of the includedpublications are summarized in Table S1, Table 1.

VEGF expression in periodontitis and normal controlsFor categorical data, the results from three studiesshowed that VEGF expression was higher in the peri-odontitis group than the healthy control group (OR =16.64, 95% CI = 6.01–46.06, P < 0.001), including 67 peri-odontitis patients and 77 healthy controls (Fig. 2).For continuous data, the pooled results from 13 stud-

ies also demonstrated a significant association betweenVEGF expression and periodontitis (SMD = 2.25, 95%

CI = 1.25–3.24, P < 0.001), including 566 periodontitispatients and 889 healthy controls (Fig. 3).

Association of VEGF expression with the clinical andpathological characteristics of periodontitisData from two studies with 27 periodontitis patientsshowed no association between VEGF expression andage (≥ 50 vs. < 50 years: OR = 0.23, 95% CI = 0.02–2.72, P = 0.241) and gender (male vs. female: OR =0.38, 95% CI = 0.05–2.87, P = 0.35) (Fig. 4a). Addition-ally, data from two studies involving 32 patients withperiodontitis showed no correlation between VEGFexpression and disease type (aggressive vs. chronicperiodontitis: SMD = 2.85, 95% CI = − 2.22-7.92, P =0.27) (Fig. 4b).

Table 1 Study characteristics of the included studies on VEGF levels

Firstauthor

Country Ethnicity Age Method Histology Source of Ab Staining Case(N)

Control(N)

Case Healthycontrol

Mean SD Mean SD

Yuan2000

China Asian 32 IHC Periodontitis NA Cytoplasm 15 15 0.6 0.27 0.38 0.21

Kubota2001

Japan Asian 28–42

RT-PCR Aggressiveperiodontitis

NA NA 6 8 5.34 1.36 3 1.27

Kubota2001

Japan Asian 55–71

RT-PCR Chronicperiodontitis

NA NA 6 4.95 0.91

Lucarini2009

Italy European 47 IHC Periodontitis dil. 1:200; SantaCruzBiotechnology,Santa Cruz, CA,USA

Epithelial cells 16 16 40 14.14 10 2.6

Artese2010

Italy European < 35 IHC Aggressiveperiodontitis

diluted at 1:100 Oral epithelium 6 7 74 2.28 27.85 1.95

Artese2010

Italy European > 35 IHC Chronicperiodontitis

diluted at 1:100 Oral epithelium 14 56.28 3.51

Loo2011

China Asian 48 ELISA Chronicperiodontitis

Diaclone, France NA 280 250 126.3 56.086 42.76 14.17

Kasprzak2012

Poland European 43 IHC Chronicperiodontitis

clone VG1, 1:50,DAKO

HEVs andordinary/inflammatoryinfiltrate cells

40 15 3.57 5.02 0.02 0.04

Tian2013

China Asian 54 ELISA Chronicperiodontitis

PhoenixPharmaceuticals,Inc., USA

NA 122 532 127.88 52.13 40.13 13.63

Balci2019

Turkey European 47.25 IHC Chronicperiodontitis

diluted 1:100,Abcam

Inflammatorycells

16 16 219.72 30.48 93.29 51.07

Taskan2019

Turkey European 40.37 IHC Periodontitis(D-GradeB)

diluted 1:250 Inflammatorycells

15 15 98.74 3.73 122.7 17.37

Taskan2019

Turkey European 35.25 IHC Periodontitis(D-GradeC)

diluted 1:250 Inflammatorycells

15 98.25 4.09

Li 2014 China Asian 35–76

IHC Chronicperiodontitis

NA Endothelialcells, stromalcells/inflammatorycells

15 15 83.53 7 41.87 5.97

RT-PCR reverse transcription-polymerase chain reaction, ELISA enzyme-linked immunosorbent assay, IHC immunohistochemistry, Ab antibody, N the number of thestudy population, SD standard deviation

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Subgroup analysisSubgroup analyses were carried out based on the avail-able information (Table 1). The results by ethnicityshowed that VEGF expression was correlated with peri-odontitis in Asians (SMD = 2.53, P < 0.001) and Euro-peans (SMD = 2.53, P = 0.02).In the subgroup analysis of the testing method, we

found that VEGF expression was sensitive to reversetranscription-polymerase chain reaction (RT-PCR),enzyme-linked immunosorbent assay (ELISA), andimmunohistochemistry (IHC) methods (all P values< 0.01).

Heterogeneity analysisAs shown in Table 1, because all P values of heterogen-eity were not more than 0.1, subgroup analyses based onethnicity, testing method, and sample size demonstratedthat these factors could not explain the potential sourcesof heterogeneity. In the sensitivity analysis, we removedeach individual study to assess the influence and changeof the re-calculated results. The results showed that noindividual study significantly change heterogeneity andthe pooled results (Table S2).

Publication biasUsing the Begg’s and Egger’s test, no publication biaswas observed between VEGF and periodontitis (all Pvalues > 0.05) (Fig. S1).

Further clinical data validation and pathway and processenrichment analysisA large study population with 427 patients with peri-odontitis and 136 healthy controls was used to validateVEGF expression in periodontitis and control groups.The result revealed that VEGF expression was stillhigher in periodontitis than in healthy controls (P =0.023) (Fig. 5a). To determine the functions of VEGF,pathway and process enrichment analysis showed thatVEGF was predominantly involved in blood vessel devel-opment, response to growth factor, cell proliferation,and cell adhesion, etc. (Fig. 5b).

DiscussionPeriodontitis is widely prevalent in many countries andcauses a major global social and economic impact. It isurgent and crucial to prevent and control periodontitis[4, 36]. Microbiome and environmental factors havebeen considered as potential risk factors for periodontitis

Fig. 2 Comparison of the difference of VEGF expression between periodontitis and control groups based on the pooled odds ratio (OR) with95% confidence interval (CI)

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[37]. In recent years, increasing evidence suggests thatgenomic programming is closely implicated in thepathogenesis of periodontitis [38–40]. VEGF, located onchromosome 6p21.3, is reported to be involved in manybiological roles such as cell proliferation, cell adhesion,chemotaxis, regulation of blood vessel development,hematopoietic stem cell development, extracellularmatrix remodeling, and inflammatory cytokine regener-ation, etc. [10–12, 41, 42]. The role of anti-VEGF couldserve as anti-vascular, anti-angiogenic, anti-permeability,or immunomodulator factor. In the era of precisionmedicine, the VEGF blockade could become a promisingand optimal strategy for therapeutic intervention [43].VEGF has been implicated in many diseases [44–46].For example, VEGF -634G > C polymorphism is corre-lated with diabetic retinopathy risk [47]. VEGF expres-sion can predict poor survival in esophageal carcinoma[46]. The expression of VEGF is associated with meta-bolic syndrome or its components [44]. Recently, somestudies have reported that VEGF expression can be

detected in periodontitis [17, 30, 31]. However, the re-sults of VEGF expression in periodontitis and controlswere still inconsistent [16, 17]. Here, we performed asystematic analysis to investigate whether VEGF couldbe a promising therapeutic method for periodontitis.We determined whether VEGF expression was associ-

ated with periodontitis. The results of the current meta-analysis via pooling available publications showed thatVEGF expression was significantly higher in patientswith periodontitis than in healthy controls, which was inagreement with the previous studies on the correlationbetween periodontitis and healthy control groups [17,24, 27, 29, 31]. An extensive clinical sample data (427periodontitis patients and 136 healthy controls) was alsoused to validate the finding of our meta-analysis, and weobserved that VEGF expression remained higher in peri-odontitis than healthy control groups. Further pathwayand process enrichment analysis suggested that VEGFwas also involved in blood vessel development, responseto growth factor, cell proliferation, and cell adhesion,

Fig. 3 Comparison of the difference of VEGF levels between periodontitis and control groups based on the pooled standardized mean difference(SMD) with 95% confidence interval (CI)

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Fig. 4 Association between VEGF expression and the clinical and pathological characteristics of periodontitis. a age (≥ 50 vs. < 50 years) andgender, (male vs. female); b pathological type (aggressive vs. chronic type). OR: odds ratio; CI: confidence interval; SMD: standardizedmean difference

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etc. Moreover, subgroup analysis of ethnicity demon-strated that VEGF expression was still related to peri-odontitis in the Asian and European populations. These

suggested that VEGF was closely involved in periodon-titis pathogenesis and had the potential to become atherapeutic strategy in the treatment of periodontitis.

Fig. 5 clinical data validation and pathway and process enrichment analysis. a VEGF expression in periodontitis and healthy control groups; bVEGF involved in the pathway and process enrichment analysis

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Interestingly, subgroup analysis of the testing methodshowed that RT-PCR, ELISA, and IHC methods weresensitive to the detection of VEGF expression.We also evaluated whether VEGF expression was cor-

related with the clinical and pathological characteristicsof periodontitis and found no correlation between VEGFexpression and age, gender, and pathological type. Thesefindings were consistent with the previous studies onage [29, 32] and gender [29, 32]. However, the resultsshould be carefully considered with caution, only twostudies with small sample sizes were induced in thismeta-analysis. In the future, more studies with a largestudy population are needed to confirm the findings be-tween VEGF expression and age, gender, and patho-logical type of periodontitis.This meta-analysis had several limitations that should

be included. First, this meta-analysis consisted of Asiansand Europeans, other ethnic groups, such as Africanswere insufficient. Second, although subgroup and sensi-tivity analyses were conducted, ethnicity, testing method,sample size, and individual study could not explain thepotential sources of heterogeneity. The potential reasonsfor heterogeneity were not very clear. Perhaps the differ-ent range of mean and SD values of VEGF levels fromthe original articles, which may result in the potentialheterogeneity. Third, analyses between VEGF expressionand age, gender, and pathological type were based ononly two studies.

ConclusionIn conclusion, our results suggested that VEGF ex-pression had a significantly higher level in peri-odontitis than in healthy controls. VEGF wasinvolved in some functions such as blood vessel de-velopment, response to growth factor, cell prolifera-tion, and cell adhesion, etc. Targeting VEGF hasthe potential to serve as an efficient therapeutic ap-proach for periodontitis in clinical management. Inthe future, more studies with larger sample sizesand multi-center design are required to gain defini-tive conclusions.

AbbreviationsVEGF: Anti-vascular endothelial growth factor; SMD: Standardized meandifference; OR: Odds ratio; CI: Confidence interval; SD: Standard deviation; RT-PCR: Reverse transcription-polymerase chain reaction; ELISA: Enzyme-linkedimmunosorbent assay; IHC: Immunohistochemistry

Supplementary InformationThe online version contains supplementary material available at https://doi.org/10.1186/s12950-021-00281-9.

Additional file 1:.

Additional file 2: Table S1. Study characteristics of the included studieson VEGF positive expression.

Additional file 3: Table S2. Sensitivity analysis.

AcknowledgementsNone.

Authors’ contributionsYL, GC, and LL contributed to the conception and design of this research. Allauthors contributed to the completion of articles and data extraction. BR, SH,and QF contributed to data calculated and the design of figures and tables.All authors approved the final manuscript.

FundingThe study supported by the Clinical Research Support Fund of PLA GeneralHospital (2018FC-304 M-TSYS-05); the Capital Characteristic Clinical Applica-tion Research and Achievement Promotion Project of China(Z171100001017154); Key Project of Transformation Medicine of General Hos-pital of PLA (2017TM-030) and Capital’s Funds for Health Improvement andResearch (No.CFH2020-2-5021). The funder didn’t make any substantial con-tributions to the research of the article.

Availability of data and materialsThe datasets used or/and analyzed during the current study are availablefrom the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participateAll procedures performed in studies involving human participants were inaccordance with the ethical standards of the Gene Expression OmnibusHuman Subjects Protection and Data Access Policies. Informed consent isnot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no conflict of interest.

Received: 6 September 2020 Accepted: 22 March 2021

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