Influence Of Genetic Polymorphisms In Genes Of Bone Remodeling And Angiogenesis Process In The Apical Periodontitis

Jardel Francisco Mazzi-Chaves Igor Bassi Ferreira Petean Isadora Mello Vilarinho Soares Alessandro Guimarães Salles Lívia Azeredo Alves Antunes Raquel Assed Bezerra Segato Léa Assed Bezerra da Silva Erika Calvano Küchler Leonardo Santos Antunes Manoel Damião Sousa-Neto About the authors

Abstract

Persistent apical periodontitis (AP) is a situation involving an inflammatory and immune response caused mainly by anaerobic polymicrobial infection of the root canal system and the outcome and follow-up of the root canal treatment has been reported as intimately related to host response. The apical periodontitis repair might be associated with genetic polymorphisms. This study aimed to evaluate the association between HIF1A genetic polymorphisms (rs2301113 and rs2057482) with PAP in Brazilian patients. Subjects with at least 1 year of follow-up after root canal therapy (RCT) were recalled. Sixty-four subjects with signs/symptoms of PAP and 84 subjects with root canal-treated teeth exhibiting healthy perirradicular tissues (healed) were included. Genomic DNA was extracted from saliva and used for HIF1A genotyping by real-time PCR. Genotype and allele frequencies were compared by c2 or Fisher’s exact tests and odds ratio was implemented, using Epi Info 3.5.2. All tests were performed with an established alpha of 0.05. There was no association between allele and genotype distribution for HIF1As polymorphisms and PAP (p>0.05). The genetic polymorphisms in HIF1A were not associated with persistent apical periodontitis.

Key words:
HIF1A; genetic polymorphisms; apical periodontitis; angiogenesis

Resumo

A periodontite apical persistente (PAP) é uma condição que envolve uma resposta inflamatória e imunológica causada principalmente por infecções polimicrobianas de origem anaeróbia no sistema de canais radiculares, tornando o resultado e o acompanhamento do tratamento do canal radicular intimamente relacionados à resposta do hospedeiro. O reparo da periodontite apical pode estar associado a polimorfismos genéticos. Este estudo teve como objetivo avaliar a associação entre os polimorfismos genéticos do HIF1A (rs2301113 e rs2057482) com a PAP em pacientes brasileiros. Indivíduos com pelo menos 1 ano de acompanhamento após o tratamento do canal radicular (TCR) foram agendados para consulta de acompanhamento. Sessenta e quatro indivíduos com sinais/sintomas de PAP e 84 indivíduos com dentes tratados endodonticamente e tecidos perirradiculares saudáveis (cicatrizados) foram incluídos no presente estudo. O DNA genômico foi extraído da saliva e utilizado para a genotipagem do HIF1A por PCR em tempo real. O genótipo e as frequências alélicas foram comparados por teste c2 ou exato de Fisher e odds-ratio foi implementado por meio do software Epi Info 3.5.2. Todos os testes realizados foram estabelecidos com a=0,05. Não houve associação entre alelo e distribuição genotípica para polimorfismos do HIF1A e PAP (p> 0,05). Os polimorfismos genéticos em HIF1A não foram associados à periodontite apical persistente.

Introduction

Apical periodontitis (AP) is an inflammatory and immune response condition, which the main cause, is the anaerobic polymicrobial infection of the dental pulp and root canals 11 Siqueira, JF; Rôças, IN. Diversity of endodontic microbiota revisited. J Dent Res 2009; 88:969-981.,22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,33 Liu, B; Liu, Q; Song, Y; Li, X; Wang, Y; Wan, S; et al. Polymorphisms of HIF1A gene are associated with prognosis of early stage non-small-cell lung cancer patients after surgery. Med Oncol 2014;31:877.,44 Müller, AS; Janjic, K; Lilaj, B; Edelmayer, M; Agis, H. Hypoxia-based strategies for regenerative dentistry-Views from the different dental fields. Arch Oral Biol 2017;81:121-130.,55 He, M; Bian, Z. Expression of hypoxia-induced semaphorin 7A correlates with the severity of inflammation and osteoclastogenesis in experimentally induced periapical lesions. Arch Oral Biol2017;75:114-119.,66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145.. This inflammation damages tissues and results in bone destruction around the root apex 11 Siqueira, JF; Rôças, IN. Diversity of endodontic microbiota revisited. J Dent Res 2009; 88:969-981.,22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,33 Liu, B; Liu, Q; Song, Y; Li, X; Wang, Y; Wan, S; et al. Polymorphisms of HIF1A gene are associated with prognosis of early stage non-small-cell lung cancer patients after surgery. Med Oncol 2014;31:877.,66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145..

Several evidences demonstrate that inflammatory lesions are characterized by cystic formation, nutrient depletion and ischaemia hypoxia in the central areas of the inflamed AP. The episodes of tissue hypoxia occur as a result of increased metabolism and reduced oxygen supply 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,44 Müller, AS; Janjic, K; Lilaj, B; Edelmayer, M; Agis, H. Hypoxia-based strategies for regenerative dentistry-Views from the different dental fields. Arch Oral Biol 2017;81:121-130.,77 Janjic, K; Lilaj, B; Moritz, A; Agis, H. Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents. Int Endod J 2018;51:e146-156.. The hypoxia and inflammatory environments may induce angiogenic processes 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,88 Konisti, S; Kiriakidis, S; Paleolog, EM. Hypoxia-a key regulator of angiogenesis and inflammation in rheumatoid arthritis. Nat Rev Rheumatol 2012;8:153-162., cell proliferation or cell protection through several mechanisms such as autophagy to aid cells overcome this situation 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.. In fact, one of the most important factor that affects acute and chronic inflammation is the oxygen 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.. Hypoxia is a key feature of inflammatory tissues due to elevated oxygen consumption by infiltrated inflammatory cells, which must adapt to hypoxic environments and maintain the function of the innate immune system against infectious microorganisms 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145..

Inside the root canals and surround periapical tissues, hypoxia plays pathogenic roles in the development of AP, which involve hypoxia-inducible factor 1 (HIF-1) 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145..The HIF-1 is a local regulator especially related to angiogenesis but is also receiving prominence by modulating osteoclastogenesis and osteoclastic activity 99 Gomes-Filho, JE; Wayama, MT; Dornelles, RCM; Ervolino, E; Yamanari, GH; Lodi, CS; et al. Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model. Int Endod J 2015;48:1059-1068.,1010 Palazon, A; Goldrath, AW; Nizet, V; Johnson, RS. HIF Transcription Factors, Inflammation, and Immunity. Immunity2014;41:518-528.. HIF1A gene encodes the alpha subunit of transcription factor HIF-1a, which regulates oxygen dependent gene transcription 44 Müller, AS; Janjic, K; Lilaj, B; Edelmayer, M; Agis, H. Hypoxia-based strategies for regenerative dentistry-Views from the different dental fields. Arch Oral Biol 2017;81:121-130.,77 Janjic, K; Lilaj, B; Moritz, A; Agis, H. Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents. Int Endod J 2018;51:e146-156.. In the hypoxia signaling pathway HIF-1a plays a major role. This factor is O2-sensitive, and in the presence of O2, HIF-1a is hydrolysed and degraded. During hypoxia, HIF1A is stabilized and translocated to the nucleus where it induces transcription of hypoxia-regulated genes, particularly associated with increased angiogenesis 1010 Palazon, A; Goldrath, AW; Nizet, V; Johnson, RS. HIF Transcription Factors, Inflammation, and Immunity. Immunity2014;41:518-528.. However, it has been reported that HIF-1a is also involved in the regulation of the osteoclastogenesis and osteoclast activation 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145.,99 Gomes-Filho, JE; Wayama, MT; Dornelles, RCM; Ervolino, E; Yamanari, GH; Lodi, CS; et al. Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model. Int Endod J 2015;48:1059-1068.,1010 Palazon, A; Goldrath, AW; Nizet, V; Johnson, RS. HIF Transcription Factors, Inflammation, and Immunity. Immunity2014;41:518-528..

HIF1A is expressed in innate and adaptive immune cells, including macrophages 1111 Cramer, T; Yamanishi, Y; Clausen, BE; Förster, I; Pawlinsk,i R; Mackman, N; et al. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell2003;112:645-657., neutrophils 1212 Walmsley, SR; Print, C; Farahi, N; Peyssonnaux, C; Johnson, RS; Cramer ,T; et al. Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity. J Exp Med2005;201:105-115., dendritic cells 1313 Jantsch, J; Chakravortty, D; Turza, N; Prechtel, AT; Buchholz, B; Gerlach, RG; et al. Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function. J Immunol 2008;180:4697-4705., and lymphocytes 1414 McNamee, EN; Korns Johnson, D; Homann, D; Clambey, ET. Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function.Immunol Res2013;55(1-3):58-70.. Recent studies have been demonstrating that HIF-1a plays an important role in the process of AP wound healing 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.,66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145., playing a protective role downregulating of NF-kB, proinflammatory cytokines, M1 macrophages and osteoclastogenesis 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.. Although recent studies have suggested that HIF-1a are a protective factor for AP, the connection between the gene HIF1A and AP have not been evaluated yet. Thus, based on these evidences, this study proposed to evaluate the association between persistent AP and the genetic polymorphisms rs2301113 and rs2057482 in HIF1A.

Material and Methods

Sample Selection and Patients

After the approval of the Ethics Committees (CAAE: 74708517.7.0000.5419 and 37717414.0.0000.5243), all subjects were informed about the research and a consent term was signed for each one.

Initially, subjects who received endodontic treatment at the School of Dentistry of Ribeirão Preto, University of São Paulo, SP, Brazil and at the Fluminense Federal University, RJ, Brazil were screened. Patients that presented pulp necrosis and apical periodontitis at the time of endodontic treatment 1515 Siqueira, JF; Rôças, IN; Provenzano, JC; Daibert, FK; Silva, MG; Lima, KC. Relationship between Fcgamma receptor and interleukin-1 gene polymorphisms and post-treatment apical periodontitis. J Endod2009;35:1186-1192.,1616 Morsani, JM; Aminoshariae, A; Han, YW; Montagnese, TA; Mickel, A. Genetic Predisposition to Persistent Apical Periodontitis. J Endod2011;37:455-459.,1717 Siqueira, JF; Rôças, IN; Provenzano, JC; Guilherme, BPS. Polymorphism of the FcgRIIIa gene and post-treatment apical periodontitis. J Endod2011;37:1345-1348.,1818 Rôças, IN; Siqueira, JF; Del Aguila, CA; Provenzano, JC; Guilherme, BPS; Gonçalves, LS. Polymorphism of the CD14 and TLR4 genes and post-treatment apical periodontitis. J Endod2014;40:168-172. and at least 1 year follow-up were included.

As an inclusion criterion, only subjects with at least one root canal-treated tooth, no specific reason for root canal treatment failure and good or regular health condition, were included. Patients with unsatisfactory endodontic treatment (obturation more than 2 mm shorter from the radiographic apex or overfilled from the root apex and/or with voids, inadequate density, unfilled canals, or poor condensation), medical conditions, immunologically compromised; vertical root fracture; presence of microleakage and absence of final restoration were excluded.

Phenotype Determination

During follow-up visits, the phenotype was determined based on radiographic and clinical aspects. Immediate postoperative radiographs were compared to radiographs obtained during the follow-up session. All radiographs were taken through the bisector technique to allow the comparison between sessions and evaluated by an experienced and calibrated endodontist. If the examiner was not able to clearly determine the phenotype, a second observer was consulted until an agreement was reached between the examiners. In patients who had endodontic treatment in multirooted teeth, the phenotype was determined from the worst result found in the roots.

The data from this study were classified according to criteria used in a previous study 1616 Morsani, JM; Aminoshariae, A; Han, YW; Montagnese, TA; Mickel, A. Genetic Predisposition to Persistent Apical Periodontitis. J Endod2011;37:455-459. as follow: 1) persistent apical periodontitis (PAP) group, which was defined as a lack of healing with apparently well obturated root canal system(s) as determined by a radiographic examination, the preexisting radiographic lesion remained the same size or increased in size, and the presence of a clinical sign or symptom of periapical disease (ie, sinus tract, pain, and swelling), constituting. 2) Healed group was assigned by subjects who had endodontic treatment accomplished with no swelling and pain absence, sinus tract disappearance, no function loss, and no tissue destruction.

DNA Extraction and Genotyping

During the follow-up visit, after the radiographic examination, saliva samples were collected from all included patients. The genotyping analysis was performed from genomic DNA extracted from buccal cells isolated from saliva as previously described 1919 Küchler, EC; Tannure, PN; Falagan-Lotsch, P; Lopes, TS; Granjeiro,, JM; Amorim, LMF. Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR. J Appl Oral Sci;20:467-471.. A spectrophotometer (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA) determined the quantity and purity of the DNA. Two genetic polymorphisms in HIF1A genes were selected and are described in the Table 1.

Table 1
Genes and markers studied in the present study

Bold form indicates ancestral allele; MAF means minor allele frequency. Data obtained from databases: http://www.ncbi.nlm.nih.gov/snp/;http://genome.ucsc.edu/;http://www.thermofisher.com/. *Multivariation model using time of follow-up as a covariant. Bold forms indicated statistical significance; OR means Odds Ratio; CI means Confidence Interval.

Real-time polymerase chain reaction (PCR) using the TaqMan assay 2020 Ranade, K; Chang, MS; Ting, CT; Pei, D; Hsiao, CF; Olivier, M; et al. High-throughput genotyping with single nucleotide polymorphisms. Genome Res2001;11:1262-1268. was used to perform genotyping. Water was used as a non-model control (negative control) to ensure the quality control of genotyping reactions.

Statistical Analysis

The Epi Info 3.5.2 software was used to analyze the data obtained. Test-t was performed to calculate the age differences between groups. Fisher’s exact and chi-square tests were used to analyze the demographic difference (age, sex, ethnicity, healthy condition and habits) between groups.

Chi-square or Fisher’s exact tests and odds ratio were used to compare allele and genotype distributions between PAP and healed groups. A logistic regression analysis was also implemented using time of follow-up as co-variate. All tests were performed with an established alpha of 0.05. Hardy-Weinberg equilibrium was evaluated using the chi-square test.

Results

The studied group was composed by 150 patients, in which 109 (72.67%) were females and 41 (27.33%) were males. Eight-two (54.67%) patients were Caucasians and 68 (45.33%) African-descendants with age ranging from 16 to 83 (Table 2).

Table 2
Demographic characteristics distribution of studied patients between groups: Persistent Apical Periodontitis (PAP) x healed

Therefore, a total of 42.7% (n=64) of the evaluated patients showed PAP and 57.3% (n=86) were classified as healed, and the follow-up time ranged between 12 and 84 months after the endodontic treatment.

Table 3 demonstrated genotype and allele distributions between PAP and healed groups. The genetic polymorphisms in HIF1A were not associated with PAP (p>0.05).

Table 3
HIF1A Genotypes and Alleles distribution in patient between Persistent Apical Periodontitis (PAP) and healed groups

Table 4 shows the results of the multivariate analysis adjusting by the follow-up time, which was not associated with the persistent PAP (p>0.05).

Table 4
HIF1A multivariation model

Discussion

In the past decades, the understanding of the genetic contributions to the risk of developing AP and the risk to present PAP was explored in some studies 11 Siqueira, JF; Rôças, IN. Diversity of endodontic microbiota revisited. J Dent Res 2009; 88:969-981.,1818 Rôças, IN; Siqueira, JF; Del Aguila, CA; Provenzano, JC; Guilherme, BPS; Gonçalves, LS. Polymorphism of the CD14 and TLR4 genes and post-treatment apical periodontitis. J Endod2014;40:168-172.,2121 Morsani, JM; Aminoshariae, A; Han, YW; Montagnese, TA; Mickel, A. Genetic Predisposition to Persistent Apical Periodontitis. J Endod2011;37:455-459.,2222 Siqueira, JF; Rôças, IN; Provenzano, JC; Guilherme, BPS. Polymorphism of the FcgRIIIa gene and post-treatment apical periodontitis. J Endod2011;37:1345-1348.,2323 Menezes-Silva, R; Khaliq, S; Deeley, K; Letra, A; Vieira, AR. Genetic susceptibility to periapical disease: conditional contribution of MMP2 and MMP3 genes to the development of periapical lesions and healing response. J Endod2012;38:604-607.,2424 Amaya, MP; Criado, L; Blanco, B; Gómez, M; Torres, O; Flórez, L; et al. Polymorphisms of pro-inflammatory cytokine genes and the risk for acute suppurative or chronic nonsuppurative apical periodontitis in a Colombian population. Int Endod J;46:71-78.,2525 Dill, A; Letra, A; Chaves de Souza, L; Yadlapati, M; Biguetti, CC; Garlet GP; et al. Analysis of multiple cytokine polymorphisms in individuals with untreated deep carious lesions reveals IL1B (rs1143643) as a susceptibility factor for periapical lesion development. J Endod2015;41:197-200.,2626 Maheshwari, K; Silva, RM; Guajardo-Morales, L; Garlet, GP; Vieira, AR; Letra, A. Heat Shock 70 Protein Genes and Genetic Susceptibility to Apical Periodontitis. J Endod2016;42:1467-1471.. In a recent review, Aminoshariae and Kulild 2727 Aminoshariae, A; Kulild, JC. Association of Functional Gene Polymorphism with Apical Periodontitis. J Endod2015;41:999-1007. showed that polymorphisms could be biological modifiers of some individual susceptibility. In fact, many of these previous studies proposed that some genetic polymorphisms could be a genetic marker for PAP 11 Siqueira, JF; Rôças, IN. Diversity of endodontic microbiota revisited. J Dent Res 2009; 88:969-981.,2828 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen, YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J2018;51:e125-145.,1818 Rôças, IN; Siqueira, JF; Del Aguila, CA; Provenzano, JC; Guilherme, BPS; Gonçalves, LS. Polymorphism of the CD14 and TLR4 genes and post-treatment apical periodontitis. J Endod2014;40:168-172.,2121 Morsani, JM; Aminoshariae, A; Han, YW; Montagnese, TA; Mickel, A. Genetic Predisposition to Persistent Apical Periodontitis. J Endod2011;37:455-459.,2222 Siqueira, JF; Rôças, IN; Provenzano, JC; Guilherme, BPS. Polymorphism of the FcgRIIIa gene and post-treatment apical periodontitis. J Endod2011;37:1345-1348.,2323 Menezes-Silva, R; Khaliq, S; Deeley, K; Letra, A; Vieira, AR. Genetic susceptibility to periapical disease: conditional contribution of MMP2 and MMP3 genes to the development of periapical lesions and healing response. J Endod2012;38:604-607.,2424 Amaya, MP; Criado, L; Blanco, B; Gómez, M; Torres, O; Flórez, L; et al. Polymorphisms of pro-inflammatory cytokine genes and the risk for acute suppurative or chronic nonsuppurative apical periodontitis in a Colombian population. Int Endod J;46:71-78.,2525 Dill, A; Letra, A; Chaves de Souza, L; Yadlapati, M; Biguetti, CC; Garlet GP; et al. Analysis of multiple cytokine polymorphisms in individuals with untreated deep carious lesions reveals IL1B (rs1143643) as a susceptibility factor for periapical lesion development. J Endod2015;41:197-200.,2727 Aminoshariae, A; Kulild, JC. Association of Functional Gene Polymorphism with Apical Periodontitis. J Endod2015;41:999-1007.,2929 Gomes, MS; Blattner, TC; Sant’Ana Filho, M; Grecca, FS; Hugo, FN; Fouad, AF; et al. Can apical periodontitis modify systemic levels of inflammatory markers? A systematic review and meta-analysis. J Endod 2013;39:1205-1217.,3030 Paradowska-Gorycka, A; Stypinska, B; Pawlik, A; Haladyj, E; Romanowska-Próchnicka, K; Olesinska, M. HIF-1A gene polymorphisms and its protein level in patients with rheumatoid arthritis: a case-control study. Inflamm Res 2018;67:423-433.,3131 Salles, AG; Antunes, LAA; Küchler, EC; Antunes, LS. Association between Apical Periodontitis and Interleukin Gene Polymorphisms: A Systematic Review and Meta-analysis. J Endod 2018;44:355-362..

Huang et al. 66 Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145. demonstrated that hypoxia is presented in inflammatory AP by immunoblotting and potentially interacts with the immune and inflammatory responses. In our present study, we did not find an association between genetic polymorphisms in HIF1A and PAP, although HIF1A was a promising candidate gene. Hirai et al. 22 Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12. demonstrated that the activation of HIF-1a exhibited an anti-inflammatory effect in AP, and they proposed that HIF-1a might be related to infiltration of myeloid cells in AP inflammation. Their gene expression analysis also indicated a possible mechanism of the less inflammatory state mediated by activation of HIF-1a through downregulation of NF-kB activation and subsequent proinflammatory gene expressions.

The genetic polymorphism rs2301113 in HIF1A was associated with the lung cancer outcome but not for colorectal cancer 33 Liu, B; Liu, Q; Song, Y; Li, X; Wang, Y; Wan, S; et al. Polymorphisms of HIF1A gene are associated with prognosis of early stage non-small-cell lung cancer patients after surgery. Med Oncol 2014;31:877.,3232 Zhang, Y; Wang, P; Zhou, X-C; Bao, G-Q; Lyu, Z-M; Liu, X-N; et al. Genetic variations in the HIF1A gene modulate response to adjuvant chemotherapy after surgery in patients with colorectal cancer. Asian Pac J Cancer Prev 2014;15:4637-4642.. On the other hand, the genetic polymorphism rs2057482 was associated with the efficiency of the chemotherapy in colorectal cancer patients 3232 Zhang, Y; Wang, P; Zhou, X-C; Bao, G-Q; Lyu, Z-M; Liu, X-N; et al. Genetic variations in the HIF1A gene modulate response to adjuvant chemotherapy after surgery in patients with colorectal cancer. Asian Pac J Cancer Prev 2014;15:4637-4642. and with the prognosis of perimenopause coronary artery disease 3333 Guo, N; Zhang, N; Yan, L; Cao, X; Wang, J; Wang, Y. Correlation between genetic polymorphisms within the MAPK1/HIF-1/HO-1 signaling pathway and risk or prognosis of perimenopausal coronary artery disease. Clin Cardiol 2017;40:597-604.. In our study, we were not able to identify a polymorphism in HIF1A as a marker to the endodontic treatment outcome prognosis. This could be related with the fact that these polymorphisms are not associated with PAP or with the fact that this gene has a small effect in the prognosis of the endodontic treatment, and this effect could only be detect in a larger sample size. It is also possible that other genetic polymorphisms in this same gene are involved in PAP, since HIF-1a plays an important role in angiogenesis and osteogenesis.

In fact, activation of the HIF-1a pathway accelerates bone regeneration, reduces inflammatory cell infiltration and promotes wound healing 3434 Wan, C; Gilbert, SR; Wang, Y; Cao, X; Shen, X; Ramaswamy, G; et al. Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration. Proc Natl Acad Sci U S A 2008;105:686-691.,3535 Cummins, EP; Seeballuck, F; Keely, SJ; Mangan, NE; Callanan, JJ; Fallon, PG; et al. The hydroxylase inhibitor dimethyloxalylglycine is protective in a murine model of colitis. Gastroenterology2008;134:156-165.,3636 Botusan, IR; Sunkari, VG; Savu, O; Catrina, AI; Grünler, J; Lindberg, S; et al. Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice. Proc Natl Acad Sci U S A2008;105:19426-19431.. An association between HIF-1a, RANKL and angiogenesis has also been showed by some studies 3737 Trebec-Reynolds, DP; Voronov, I; Heersche, JNM; Manolson, MF. VEGF-A expression in osteoclasts is regulated by NF-kappaB induction of HIF-1alpha. J Cell Biochem2010;110:343-351.,3838 Dandajena, TC; Ihnat, MA; Disch, B; Thorpe, J; Currier, GF. Hypoxia triggers a HIF-mediated differentiation of peripheral blood mononuclear cells into osteoclasts. Orthod Craniofac Res2012;15:1-9.. In addition, studies demonstrated that HIF-1a promotes increased vascular endothelial growth factor, which in turn recruit osteoclast to the remodeling area, to stimulate osteoclast differentiation and to promote osteoclast resorption 99 Gomes-Filho, JE; Wayama, MT; Dornelles, RCM; Ervolino, E; Yamanari, GH; Lodi, CS; et al. Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model. Int Endod J 2015;48:1059-1068.,3939 Niida, S; Kaku, M; Amano, H; Yoshida, H; Kataoka, H; Nishikawa, S; et al. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med1999;190:293-298.,4040 Maes, C; Carmeliet, P; Moermans, K; Stockmans, I; Smets, N; Collen, D; et al. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev2002;111:61-73.,4141 Sipola, A; Nelo, K; Hautala, T; Ilvesaro, J; Tuukkanen, J. Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro. BMC Musculoskelet Disord 2006;7:56.,4242 Knowles, HJ; Athanasou, NA. Hypoxia-inducible factor is expressed in giant cell tumour of bone and mediates paracrine effects of hypoxia on monocyte-osteoclast differentiation via induction of VEGF. J Pathol2008;215:56-66..

Briefly, hypoxia-based strategies in dentistry are emerging in all dental specialties 44 Müller, AS; Janjic, K; Lilaj, B; Edelmayer, M; Agis, H. Hypoxia-based strategies for regenerative dentistry-Views from the different dental fields. Arch Oral Biol 2017;81:121-130.. Further research on this topic is necessary to harvest the benefits of these approaches in the future and identify the most applicable screening strategy in the endodontic research. According to the results of the present study, it can be concluded that the genetic polymorphisms rs2301113 and rs2057482 in HIF1A were not associated with persistent apical periodontitis.

References

  • 1
    Siqueira, JF; Rôças, IN. Diversity of endodontic microbiota revisited. J Dent Res 2009; 88:969-981.
  • 2
    Hirai, K; Furusho, H; Hirota, K; Sasaki, H. Activation of hypoxia-inducible factor 1 attenuates periapical inflammation and bone loss. Int J Oral Sci 2018;10:12.
  • 3
    Liu, B; Liu, Q; Song, Y; Li, X; Wang, Y; Wan, S; et al. Polymorphisms of HIF1A gene are associated with prognosis of early stage non-small-cell lung cancer patients after surgery. Med Oncol 2014;31:877.
  • 4
    Müller, AS; Janjic, K; Lilaj, B; Edelmayer, M; Agis, H. Hypoxia-based strategies for regenerative dentistry-Views from the different dental fields. Arch Oral Biol 2017;81:121-130.
  • 5
    He, M; Bian, Z. Expression of hypoxia-induced semaphorin 7A correlates with the severity of inflammation and osteoclastogenesis in experimentally induced periapical lesions. Arch Oral Biol2017;75:114-119.
  • 6
    Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen ,YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J 2018;51:e125-145.
  • 7
    Janjic, K; Lilaj, B; Moritz, A; Agis, H. Formation of spheroids by dental pulp cells in the presence of hypoxia and hypoxia mimetic agents. Int Endod J 2018;51:e146-156.
  • 8
    Konisti, S; Kiriakidis, S; Paleolog, EM. Hypoxia-a key regulator of angiogenesis and inflammation in rheumatoid arthritis. Nat Rev Rheumatol 2012;8:153-162.
  • 9
    Gomes-Filho, JE; Wayama, MT; Dornelles, RCM; Ervolino, E; Yamanari, GH; Lodi, CS; et al. Raloxifene modulates regulators of osteoclastogenesis and angiogenesis in an oestrogen deficiency periapical lesion model. Int Endod J 2015;48:1059-1068.
  • 10
    Palazon, A; Goldrath, AW; Nizet, V; Johnson, RS. HIF Transcription Factors, Inflammation, and Immunity. Immunity2014;41:518-528.
  • 11
    Cramer, T; Yamanishi, Y; Clausen, BE; Förster, I; Pawlinsk,i R; Mackman, N; et al. HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell2003;112:645-657.
  • 12
    Walmsley, SR; Print, C; Farahi, N; Peyssonnaux, C; Johnson, RS; Cramer ,T; et al. Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity. J Exp Med2005;201:105-115.
  • 13
    Jantsch, J; Chakravortty, D; Turza, N; Prechtel, AT; Buchholz, B; Gerlach, RG; et al. Hypoxia and hypoxia-inducible factor-1 alpha modulate lipopolysaccharide-induced dendritic cell activation and function. J Immunol 2008;180:4697-4705.
  • 14
    McNamee, EN; Korns Johnson, D; Homann, D; Clambey, ET. Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function.Immunol Res2013;55(1-3):58-70.
  • 15
    Siqueira, JF; Rôças, IN; Provenzano, JC; Daibert, FK; Silva, MG; Lima, KC. Relationship between Fcgamma receptor and interleukin-1 gene polymorphisms and post-treatment apical periodontitis. J Endod2009;35:1186-1192.
  • 16
    Morsani, JM; Aminoshariae, A; Han, YW; Montagnese, TA; Mickel, A. Genetic Predisposition to Persistent Apical Periodontitis. J Endod2011;37:455-459.
  • 17
    Siqueira, JF; Rôças, IN; Provenzano, JC; Guilherme, BPS. Polymorphism of the FcgRIIIa gene and post-treatment apical periodontitis. J Endod2011;37:1345-1348.
  • 18
    Rôças, IN; Siqueira, JF; Del Aguila, CA; Provenzano, JC; Guilherme, BPS; Gonçalves, LS. Polymorphism of the CD14 and TLR4 genes and post-treatment apical periodontitis. J Endod2014;40:168-172.
  • 19
    Küchler, EC; Tannure, PN; Falagan-Lotsch, P; Lopes, TS; Granjeiro,, JM; Amorim, LMF. Buccal cells DNA extraction to obtain high quality human genomic DNA suitable for polymorphism genotyping by PCR-RFLP and Real-Time PCR. J Appl Oral Sci;20:467-471.
  • 20
    Ranade, K; Chang, MS; Ting, CT; Pei, D; Hsiao, CF; Olivier, M; et al. High-throughput genotyping with single nucleotide polymorphisms. Genome Res2001;11:1262-1268.
  • 21
    Morsani, JM; Aminoshariae, A; Han, YW; Montagnese, TA; Mickel, A. Genetic Predisposition to Persistent Apical Periodontitis. J Endod2011;37:455-459.
  • 22
    Siqueira, JF; Rôças, IN; Provenzano, JC; Guilherme, BPS. Polymorphism of the FcgRIIIa gene and post-treatment apical periodontitis. J Endod2011;37:1345-1348.
  • 23
    Menezes-Silva, R; Khaliq, S; Deeley, K; Letra, A; Vieira, AR. Genetic susceptibility to periapical disease: conditional contribution of MMP2 and MMP3 genes to the development of periapical lesions and healing response. J Endod2012;38:604-607.
  • 24
    Amaya, MP; Criado, L; Blanco, B; Gómez, M; Torres, O; Flórez, L; et al. Polymorphisms of pro-inflammatory cytokine genes and the risk for acute suppurative or chronic nonsuppurative apical periodontitis in a Colombian population. Int Endod J;46:71-78.
  • 25
    Dill, A; Letra, A; Chaves de Souza, L; Yadlapati, M; Biguetti, CC; Garlet GP; et al. Analysis of multiple cytokine polymorphisms in individuals with untreated deep carious lesions reveals IL1B (rs1143643) as a susceptibility factor for periapical lesion development. J Endod2015;41:197-200.
  • 26
    Maheshwari, K; Silva, RM; Guajardo-Morales, L; Garlet, GP; Vieira, AR; Letra, A. Heat Shock 70 Protein Genes and Genetic Susceptibility to Apical Periodontitis. J Endod2016;42:1467-1471.
  • 27
    Aminoshariae, A; Kulild, JC. Association of Functional Gene Polymorphism with Apical Periodontitis. J Endod2015;41:999-1007.
  • 28
    Huang, HY; Wang, WC; Lin, PY; Huang, CP; Chen, CY; Chen, YK. The roles of autophagy and hypoxia in human inflammatory periapical lesions. Int Endod J2018;51:e125-145.
  • 29
    Gomes, MS; Blattner, TC; Sant’Ana Filho, M; Grecca, FS; Hugo, FN; Fouad, AF; et al. Can apical periodontitis modify systemic levels of inflammatory markers? A systematic review and meta-analysis. J Endod 2013;39:1205-1217.
  • 30
    Paradowska-Gorycka, A; Stypinska, B; Pawlik, A; Haladyj, E; Romanowska-Próchnicka, K; Olesinska, M. HIF-1A gene polymorphisms and its protein level in patients with rheumatoid arthritis: a case-control study. Inflamm Res 2018;67:423-433.
  • 31
    Salles, AG; Antunes, LAA; Küchler, EC; Antunes, LS. Association between Apical Periodontitis and Interleukin Gene Polymorphisms: A Systematic Review and Meta-analysis. J Endod 2018;44:355-362.
  • 32
    Zhang, Y; Wang, P; Zhou, X-C; Bao, G-Q; Lyu, Z-M; Liu, X-N; et al. Genetic variations in the HIF1A gene modulate response to adjuvant chemotherapy after surgery in patients with colorectal cancer. Asian Pac J Cancer Prev 2014;15:4637-4642.
  • 33
    Guo, N; Zhang, N; Yan, L; Cao, X; Wang, J; Wang, Y. Correlation between genetic polymorphisms within the MAPK1/HIF-1/HO-1 signaling pathway and risk or prognosis of perimenopausal coronary artery disease. Clin Cardiol 2017;40:597-604.
  • 34
    Wan, C; Gilbert, SR; Wang, Y; Cao, X; Shen, X; Ramaswamy, G; et al. Activation of the hypoxia-inducible factor-1alpha pathway accelerates bone regeneration. Proc Natl Acad Sci U S A 2008;105:686-691.
  • 35
    Cummins, EP; Seeballuck, F; Keely, SJ; Mangan, NE; Callanan, JJ; Fallon, PG; et al. The hydroxylase inhibitor dimethyloxalylglycine is protective in a murine model of colitis. Gastroenterology2008;134:156-165.
  • 36
    Botusan, IR; Sunkari, VG; Savu, O; Catrina, AI; Grünler, J; Lindberg, S; et al. Stabilization of HIF-1alpha is critical to improve wound healing in diabetic mice. Proc Natl Acad Sci U S A2008;105:19426-19431.
  • 37
    Trebec-Reynolds, DP; Voronov, I; Heersche, JNM; Manolson, MF. VEGF-A expression in osteoclasts is regulated by NF-kappaB induction of HIF-1alpha. J Cell Biochem2010;110:343-351.
  • 38
    Dandajena, TC; Ihnat, MA; Disch, B; Thorpe, J; Currier, GF. Hypoxia triggers a HIF-mediated differentiation of peripheral blood mononuclear cells into osteoclasts. Orthod Craniofac Res2012;15:1-9.
  • 39
    Niida, S; Kaku, M; Amano, H; Yoshida, H; Kataoka, H; Nishikawa, S; et al. Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption. J Exp Med1999;190:293-298.
  • 40
    Maes, C; Carmeliet, P; Moermans, K; Stockmans, I; Smets, N; Collen, D; et al. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech Dev2002;111:61-73.
  • 41
    Sipola, A; Nelo, K; Hautala, T; Ilvesaro, J; Tuukkanen, J. Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro. BMC Musculoskelet Disord 2006;7:56.
  • 42
    Knowles, HJ; Athanasou, NA. Hypoxia-inducible factor is expressed in giant cell tumour of bone and mediates paracrine effects of hypoxia on monocyte-osteoclast differentiation via induction of VEGF. J Pathol2008;215:56-66.

Publication Dates

  • Publication in this collection
    Mar-Apr 2018

History

  • Received
    13 Oct 2017
  • Accepted
    04 Feb 2018
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