Biological marker for the establishment of periodontal disease: Cross-seccional study in gingival tissue

Biological marker for the establishment of periodontal disease: Cross-seccional study in gingival tissue | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biological marker for the establishment of periodontal disease: Cross-seccional study in gingival tissue Déborah-Pitta-Paraíso Iglesias, Weslay-Rodrigues da-Silva, Glória-Maria de-França, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1946247/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background The present study investigated the expression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the gingival tissue, to verify if there is a correlation between the immunoexpression of these proteins and the changes caused by the inflamed infiltrate present in the gingival tissues. Material and methods A morphological analysis of epithelial changes (hyperplasia, exocytosis, spongiosis, and hydropic degeneration) was performed, as well as a semiquantitative analysis of the immunoexpression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the epithelium and connective tissue of 60 specimens of gingival tissue. Results Epithelial immunoexpression to COX-2 was observed in three cases, while EMMPRIN, HIF-1α, and GLUT-1 were strongly expressed in the basal layer of the epithelium and gradually decreasing until the upper layers. In the connective tissue, COX-2 immunoexpression showed a statistical association (p < 0.001) with the gingival inflammatory infiltrate. In connective tissue, EMMPRIN, and HIF-1α exhibited intense immunopositivity, while GLUT-1 was negative in most cases. Conclusion COX-2 expression may constitute a biological marker of gingival tissues since its epithelial immunoexpression may indicate a greater propensity for the establishment of periodontal disease. Gingiva Periodontal diseases Inflammation Figures Figure 1 Figure 2 Introduction Periodontal disease is a condition of infectious etiology that can develop in all age groups and is the most frequent inflammatory disease of the oral cavity ( 1 , 2 ). The inflammatory response triggered by the microbial accumulation in the gingival sulcus promotes the release of cytokines and the increase in local vascular permeability, clinically characterized by gingival erythema and edema, and the production of matrix metalloproteinases (MMPs) that can contribute to the alteration and/or destruction of the periodontium. This clinical panorama is the result of the secretion of cyclooxygenase-2 (COX-2), a proinflammatory enzyme that participates in the biosynthesis of prostaglandin E2, an inflammatory mediator that is involved in periodontal pathology ( 3 – 5 ). The persistence of the inflammatory condition generates the accumulation of cytokines and contributes to a greater release of MMPs, which are related to the degree of tissue destruction and disorganization ( 3 ). EMMPRIN is a glycoprotein involved in the pathogenesis of various diseases and, its activity is mainly related to the inducing effect on MMPs and its chemotactic capacity. Changes in its expression have been reported during the pathogenesis of periodontal disease with a relationship between increased expression and clinical disease progression ( 6 – 8 ). The chemotactic activity of EMMPRIN requires high energy consumption, also the arrival of inflammatory cells in the microenvironment is accompanied by an increase in the consumption of nutrients and oxygen, which can generate hypoxia conditions ( 7 , 8 ). The local hypoxia generated in the inflamed microenvironment can activate the hypoxia-inducible factor 1 (HIF-1), which represents, in most human cells, the main path of adaptive cellular response to changes in oxygen concentration. The local hypoxia induces a change in energy activity in cells where glycolytic metabolism will prevail. At this time, the activity of proteins such as the glucose transporter 1 (GLUT-1) is essential, which, in hypoxia conditions, facilitates the entry of glucose into cells to maintain metabolic activity ( 9 – 11 ). Thus, the present study investigated the expression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the gingival tissue, in order to verify if there is a correlation between the immunoexpression of these proteins and the changes caused by the inflamed infiltrate in the periodontium and contribute to a better understanding of the action of the inflammatory infiltrate in the pathogenesis of the periodontal disease. Material And Methods Sample This research was cross-sectional study previously approved by the Research Ethics Committee (Approval number 164/2012, C.A.A.E. 0279.0.051.051-11). All samples included in this research were obtained at the Dental Office of the 14th Motorized Infantry Battalion and in the Oral Pathology Service at the UFRN (Natal, RN) between July and December 2011. A total of 60 formalin-fixed paraffin-embedded gingival tissue samples were obtained through a periodontal surgical procedure, for aesthetic reasons or the indication of increasing clinical crown height to restorative treatments. Were excluded from the research gingival tissues of pregnant or lactating patients as well as patients with previously known systemic disease, presence of infections in extraoral sites, smoking history in the last five years, and patients undergoing treatment with drugs that modulate the inflammatory response. Morphological analysis Five-µm thick histological sections were cut from the formalin-fixed paraffin-embedded specimens and stained with hematoxylin-eosin. Slides were independently assessed under light microscopy (Olympus CX31, Olympus Japan Co., Tokyo, Japan). Semi-quantitative morphological analysis was carried out to verify changes due to inflammation (hyperplasia, exocytosis, spongiosis and hydropic degeneration) throughout the epithelial extension, which was categorized as normal epithelium (with no changes or changes present in less than 10% of the epithelial extension); mild changes (11% − 25%); moderate changes (26% − 50%); and severe changes (in more than 50% of the epithelial extension). Connective tissue was evaluated according to its density and organization of collagen fibers by adapting the criteria proposed by Liu et al. (2010) ( 12 ), being categorized into collagen fibers densely organized in more than 50% of the connective tissue; collagen fibers loosely organized in more than 50% of the connective tissue; and collagen fibers, sometimes loose, sometimes densely organized. The inflammatory infiltrate was categorized as absent (no inflammatory cells were observed); scarce inflammatory infiltrate (up to 25% of connective tissue occupied by inflammatory cells); moderate (26–50% of the connective tissue occupied by inflammatory cells); and intense (more than 50% of the connective tissue occupied by inflammatory cells). Immunohistochemical staining For the immunohistochemical study, 3-µm thick sections were obtained from paraffin-embedded tissue blocks and mounted on organosilane-coated slides (3-aminopropyltriethoxysilane; Sigma Chemical Co., St. Louis, MO, USA). The slides were incubated with one the following primary antibodies: anti-COX-2 (SP21; Spring Bioscience; 1:200; 60’), anti-EMMPRIN (Policlonal; Invitrogen; 1:400; 60’), anti-HIF-1α (H1α67; Santa Cruz, 1:200; Overnight), anti-GLUT-1 (Policlonal; Gene Tex; 1:400; 60’). Sections were then washed twice in phosphate-buffered saline and incubated in the HiDef visualization system (HiDef Detection™ HRP Polymer System, Cell-Marque, USA) at room temperature. Finally, tissue sections were counterstained with Mayer's hematoxylin and coverslipped. For all primary antibodies, invasive ductal carcinoma sections were used as positive control. For negative control, the primary antibody was replaced with 1% bovine serum albumin in TBS. Immunostaining assessment The analysis of the immunohistochemical expression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the gingival tissue was performed by a single investigator, who was blinded to the clinical and morphological data, under a light microscope (Olympus CX31, Olympus Japan Co., Tokyo, Japan) with 200x magnification. According the methodology proposed by Morton and Dagnari-Bagtzoglou (2001) ( 13 ), Xiang et al. (2009) ( 14 ), Ng et al. (2011) ( 15 ) and Ito et al. (2002) ( 16 ), cells that showed brownish cytoplasmic immunostaining for COX-2, membrane and cytoplasmic for EMMPRIN, cytoplasmic and nuclear for HIF-1α, and membrane for GLUT-1 were considered positive. The immunohistochemical assessment considered the presence or absence of immunostaining. The immunostaining of epithelial cells was categorized according to the basal, parabasal and superficial (intermediate and superficial) layers of the epithelium. In the connective tissue, the analysis was performed by adapting the methodology proposed by Liu et al. (2010) ( 12 ), where the immunostaining of cells present in connective tissue was categorized as absent; scarce (1% − 25% of immunostained cells); moderate (26% − 50% of immunostained cells); and intense (above 50% of immunostained cells). Statistical analysis Absolute and relative frequency were performed using the IBM Statistical Package for the Social Sciences (SPSS) Statistics 20.0 program (IBM Corp., Armonk, USA). Immunopositivity percentages were submitted to distribution analysis by the Kolmogorov-Smirnov test, which revealed a non-normal data distribution. Pearson’s Chi-square test was performed considering a 5% significance level (p ≤ 0.05). Results Clinicopathological data The sample consisted of 60 cases of gingival tissue. Of these, 41 cases (68.3%) corresponded to male patients. The patients had a mean age of 37.7 years (range: 18–80 years). Concerning the morphological findings, all epithelial changes (hyperplasia, exocytosis, spongiosis and edematous degeneration) were frequent, and it was observed that in 30 cases (50%) there were changes in a sparse manner, followed by moderate changes (n = 17; 28.3%) and intense (n = 11; 18.3%), while two cases (3.3%) did not show any changes. Also, there was a higher frequency of intense inflammatory infiltrate (n = 25), and 43 cases exhibited collagen fibers densely organized. There was a significant association between the intensity of the inflammatory infiltrate and the density of the connective tissue ( p = 0.009) (Table 01 ). Immunohistochemical findings Immunohistochemical analysis showed COX-2 epithelial positivity in only three cases, which was restricted to the parabasal layer. Of these, sparse immunoexpression was observed in two cases (3.3%) and moderate in one (1.7%). Regarding the immunostaining of COX-2 in connective tissue, positivity was observed in 36 (60%) cases and a higher frequency of sparse expression (n = 18; 30%), followed by moderate expression (n = 9; 15%) and intense (n = 9; 15%) (Fig. 1 A). It was also observed that this immunostaining was present in inflammatory infiltrate mononuclear cells, fibroblasts, and endothelial cells (Fig. 1 B). The results of positivity for COX-2 compared to the intensity of the inflammatory infiltrate revealed a significant association ( p < 0.001), as shown in Table 1 . Table 1 Association between connective tissue organization, the intensity of COX-2 expression, and the intensity of inflammatory infiltrate. Inflammatory Infiltrate p Scarce n (%) Moderate n (%) Intense n (%) Total n (%) Connective tissue organization Densely organized 21 (48.8) 10 (23.3) 12 (27.9) 43 (100) 0.009* Loosely organized 0 (0) 1 (16.7) 5 (83.3) 6 (100) Densely and loosely organized 2 (18.2) 1 (9.1) 8 (72.7) 11 (100) COX-2 immunoexpression intensity Absent 15 (62.5) 7 (29.2) 2 (8.3) 24 (100) < 0.001* Scarce 6 (33.9) 3 (16.7) 9 (50) 18 (100) Moderate 2 (22.2) 2 (22.2) 5 (55.6) 9 (100) Intense 0 (0) 0 (0) 9 (100) 9 (100) *Significant association by Pearson's Chi-square test Concerning EMMPRIN, the epithelial cells exhibited a predominantly membrane immunoexpression pattern in the cells of the basal layer, which reduced gradually until the superficial layer. Positive membrane immunostaining was observed intensely in the basal layer of 42 cases (70%), in the parabasal of 14 cases (23.3%), while it was absent in the superficial layer of 49 cases (81.7%) (Fig. 2 A). The gingival connective tissue showed positive immunostaining, at the cytoplasmic level, for EMMPRIN in 98.3% (n = 59) of the sample, which was intense in 70% (n = 42) of the cases (Fig. 2 B). There was a significant association ( p < 0.001) between the expression of EMMPRIN with the layers of epithelial tissue and connective tissue (Table 2 ). Table 2 Association of immunostaining for EMMPRIN, HIF-1α, and GLUT-1 with gingival epithelium and connective tissue. Immunoexpression Intensity Epithelium Connective Tissue n (%) p Basal Layer n (%) Parabasal Layer n (%) Superficial Layer n (%) EMMPRIN Absent 0 (0) 15 (25) 49 (81.7) 1 (1.7) < 0.001* Scarce 4 (6.7) 16 (26.7) 9 (15) 5 (8.3) Moderate 14 (23.3) 15 (25) 2 (3.3) 12 (20) Intense 42 (70) 14 (23.3) 0 (0) 42 (70) HIF-1α Absent 14 (23.3) 15 (25) 0 (0) 8 (13.3) 0.001* Scarce 4 (6.7) 0 (0) 0 (0) 8 (13.3) Moderate 3 (5) 16 (26.7) 0 (0) 9 (15) Intense 39 (65) 29 (48.3) 0 (0) 35 (58.3) GLUT-1 Absent 1 (1.73) 6 (10) 58 (96.6) 35 (58.3) < 0.001* Scarce 3 (5) 16 (26.7) 2 (3.4) 18 (30) Moderate 12 (20) 20 (33.3) 0 (0) 3 (5) Intense 44 (73.3) 18 (30) 0 (0) 4 (6.7) *Significant association by Pearson's Chi-square test Immunostaining of HIF-1α in epithelial cells demonstrated cytoplasmic, and sometimes nuclear expression in the basal layer (n = 46; 76.7%) (Fig. 2 C). In connective tissue, immunostaining was highly positive (n = 35; 58.3%) in both inflammatory cells and fibroblasts (Fig. 2 C). A significant association was observed between HIF-1α immunoexpression and epithelial and connective tissue immunostaining ( p = 0.001) (Table 2 ). GLUT-1 immunostaining was identified in the cytoplasmic membrane of epithelial cells intensely in the basal layer (n = 44; 73.3%), moderate in the parabasal (n = 20; 33.3%) and absence in the superficial layer (n = 58; 96.6%). In areas where the epithelial tissue had inflammatory changes, such as edematous degeneration, the expression was markedly reduced, showing itself to be weak or absent (Fig. 2 E). The immunostaining of GLUT-1 in connective tissue was positive in 41.7% of the sample (n = 25), showing a membrane pattern in fibroblasts and a cytoplasmic pattern in inflammatory cells. Besides, a greater amount of positive endothelial cells was observed in cases with greater intensity of cell infiltrate (Fig. 2 F). There was significance between GLUT-1 immunoexpression and epithelial and connective tissue immunostaining ( p < 0.001) (Table 2 ). There were no statistically significant results between the immunoexpression of COX-2, EMMPRIN, HIF-1α and GLUT-1. Discussion The present study was carried out on 60 gingival tissue to investigate the changes caused by the inflammatory infiltrate in the periodontium. Our analyzes demonstrate that although the inflammatory infiltrate has no association with the morphological changes of the epithelium, the epithelial immunoexpression of COX-2 can be used as a possible biological marker in investigating the presence of periodontal disease. Histologically, the gingiva presents a variable amount of inflammatory infiltrate, thus obtaining a gingival connective tissue that histologically does not present inflammatory infiltrate is quite unusual ( 17 , 18 ). This inflammatory infiltrate is a basic defense mechanism against microbial persistence in the gingival sulcus. However, it will also contribute to the destruction and increase of gingival tissues through the secretion of lysosomal enzymes and cytokines that stimulate the release of MMPs, vasodilation, increased permeability vascular and local blood flow, and the accumulation of exudate in the extracellular environment ( 2 , 19 ). This reduction in the amount of collagen is microscopically visualized as a looser and more irregular organization of collagen fibers and a large amount of extracellular matrix, characteristics observed in the present study, especially in cases that exhibited intense inflammatory infiltrate. Vasodilation and increased vascular permeability that develops in gingival tissue is partly the result of prostaglandin biosynthesis. The inflammatory infiltrate induces the metabolism of arachidonic acid in epithelial, endothelial cells, macrophages, and fibroblasts and, consequently, the production of PGE2 through the cyclooxygenase pathway and action of COX-2. COX-2 is an enzyme that mediates inflammatory effects and has been identified as a key mediator in the pathogenesis of periodontal disease, with an absent or low expression pattern in healthy tissues and an increase in inflamed tissues ( 20 – 23 ). In the present study, there was a significant increase in the immunoexpression of this protein in cases with intense inflammatory infiltrate. Also, COX-2 immunoexpression in epithelial tissue was observed in only three cases. In previous studies, a high immunoexpression of COX-2 was observed, with no absence of immunostaining in the analyzed cases ( 20 – 23 ). The present research diverges from these results since, in some cases, there was no immunostaining for COX-2. We believe that the lower intensity or absence of this immunostaining is due to the lower intensity of the inflammatory infiltrate observed in some cases. In the study performed by Mesa et al. , (2014) ( 22 ), positive epithelial immunostaining for COX-2 was found only in gingival tissues obtained in sites with periodontal disease and, consequently, bone resorption. Thus, we believe that the cases, with COX-2 epithelial immunoexpression, would be in more advanced stages of periodontal disease, possibly indicating possible gingivitis. In periodontal tissues, the integrity of the extracellular matrix is ​​important for maintaining tissue stability. This integrity is maintained through the activation of MMPs and their inhibitors, which results in the balance between degradation and production of the extracellular matrix. In periodontal disease, progressive destruction of gingival tissues is observed due to increased activity of MMPs, such as collagenase, both due to the action of fibroblasts and mononuclear inflammatory cells ( 14 ). The participation of EMMPRIN in the balance and maintenance of the extracellular matrix in the gingival tissue is demonstrated, in healthy periodontal sites, by its intense expression in the epithelial cells of the basal layer, at the cytoplasmic level, with gradual decrease as the cells move away from the germ layer of the epithelium ( 7 , 8 , 12 , 14 , 24 ). The literature also reports that this pattern of immunostaining confirms the participation of EMMPRIN in maintaining tissue integrity and cell adhesion ( 7 , 25 , 26 ). The present study observed a similar immunostaining pattern. Besides, it was noted that the expression of EMMPRIN was lower in areas where epithelial tissue shows intense spongiosis, findings that corroborate those reported in the literature. In inflammatory gingival conditions, increased cellular inflow increases EMMPRIN expression. In this way, both fibroblasts and the lymphoplasmocytic cells themselves, which overexpress EMMPRIN, can activate and recruit more inflammatory cells, thus constituting a pattern of positive self-regulation ( 7 , 8 , 12 , 24 ). In the present study, a similar immunostaining pattern of EMMPRIN was observed in lymphoplasmacytic cells and fibroblasts. However, although we do not find statistical significance, we cannot rule out the participation of this molecule and MMPs in the pathogenesis process of periodontal disease. It is also important to note that the chemotactic activity triggered by EMMPRIN generates a high energy consumption and that the arrival of more cells to the inflammatory site is accompanied by an increase in the consumption of nutrients and oxygen. Thus, the infectious process establishes tissue hypoxia through high oxygen consumption by both microorganisms and host defense cells recruited to the gingival tissues ( 27 ). Endothelial damage and edema resulting from inflammation are also factors that cause tissue hypoxia, as they result in microcirculatory failure and thus reduced oxygen supply. However, the response of gingival tissue to oxygen depletion is coordinated by HIF − 1α ( 28 , 29 ). The present study observed a high immunoexpression of HIF-1α both in the epithelium and in the connective tissue. We believe that the injury to the gingival epithelium, caused by dental biofilm, may justify the intense expression of HIF-1α in the analyzed specimens. The pattern of high nuclear and cytoplasmic immunoexpression, observed by us, corroborating the findings ( 29 , 30 ), which denoted that cytoplasmic expression may represent the activation of the HIF-1α factor. Besides, HIF-1α is associated with the survival of activated T lymphocytes. Once evading the programmed cell death pathway, these cells remain for a longer time in the inflammatory sites, delaying the resolution of the inflammatory condition ( 27 , 29 ). In the present study, many lymphocytes were observed in the connective tissue. Thus, this fact could be attributed to the high immunoexpression of HIF-1α in the gingival connective tissue of our sample. The high expression of HIF-1α modifies the expression of hypoxia-related genes, thus increasing the synthesis of VEGF, a proangiogenic cytokine, and the glucose transporter GLUT-1 ( 9 – 11 ). Agandi and Agandi (2015) ( 31 ) found in their study that the strong immunoexpression of GLUT-1 in the basal and parabasal layer of the oral epithelium occurs due to the high proliferative activity in this layer, while the absence of GLUT-1 in the most superficial layers reflects the maturation of epithelial cells. Our results revealed similar immunostaining of GLUT-1, and it suggests that the expression of this protein in the gingival epithelium have not alterations due to the action of the inflammatory infiltrate. Also, the low immunoexpression of this protein in connective tissue suggests a possible balance of hypoxia control in the inflammatory microenvironment. Thus, we suggest that GLUT-1 participates in the physiology of gingival tissue, mainly contributing to the maintenance of epithelial tissue. In conclusion, the results of the present study demonstrate that the expression of COX-2 may constitute a biological marker of gingival tissues since its epithelial immunoexpression may indicate a greater propensity to establish periodontal disease. Besides that, the results indicated that our immunoexpression patterns of EMMPRIN, HIF-1α, and GLUT-1 in the gingival epithelium and connective tissue confirm the physiological role played by these markers in maintaining the integrity and homeostasis of the gingival tissue. Declarations Ethics approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All procedures performed in this retrospective study involving human participants were approved by the Institutional Ethics Comitee Protocol number 164/2012, C.A.A.E. 0279.0.051.051-11 (UFRN). Acknowledgements We thank the Pathological Anatomy Service of the Discipline of Oral Pathology, Department of Dentistry, Federal University of Rio Grande do Norte, for providing the material and laboratory resources necessary for the study. Funding None Consent to participate All patients involved in this study signed the informed consent form. Conflict of interest The authors declare no competing interests. Author contributions DéborahPitta-Paraíso Iglesias: Participation in conducting the research and preparation of the manuscrip; Weslay Rodrigues da Silva: Participation in conducting the research and preparation of the manuscript Glória Maria de França and Caio César da Silva Barros: wrote the main manuscript text Roseana de Almeida Freitas and Hébel-Cavalcanti Galvão: prepared figures. All authors reviewed the manuscript. Acknowledgements The authors declare no conflict of interest. This study was supported by the National Council for Scientific and Technological Development (CNPq, Brazil), Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil), and Postgraduate Program in Dental Sciences (UFRN, Natal, RN, Brazil). References Holmstrup P, Plemons J and Meyle J. Non–plaque-induced gingival disease. Journal of Periodontology 2018; 89: S28–S45. Kumar S. Evidence-Based Update on Diagnosis and Management of Gingivitis and Periodontitis. Dental Clinics of North America 2019; 63: 69-81. Lazăr L, Loghin A, Bud E, Cerghizan D, Horváth E and Nagy EE. Cyclooxygenase-2 and matrix metalloproteinase-9 expressions correlate with tissue inflammation degree in periodontal disease. Romanian Journal of Morphology and Embryology 2015; 56: 1441–1446. Arweiler NB and Netuschil L. The Oral Microbiota. Advances in Experimental Medicine and Biology 2016; 902: 45-60. Stone SJ, Kumar PS, Offenbacher S, Heasman PA and McCracken GI. Exploring a temporal relationship between biofilm microbiota and inflammatory mediators during resolution of naturally-occurring gingivitis. Journal of Periodontology 2019; 90: 627-636. de Oliveira Nóbrega FJ, de Oliveira DHIP, Vasconcelos RG, Nonaka CFW, Queiroz LMG. Study of the participation of MMP-7, EMMPRIN and cyclophilin A in the pathogenesis of periodontal disease. Archives of Oral Biology 2016; 72: 172–178. Liu X, Zhang Z, Pan S, Shang S and Li C. Interaction between the Wnt/β-catenin signaling pathway and the EMMPRIN/MMP-2, 9 route in periodontitis. Journal of Periodontal Research 2018; 53: 842-852. Zhang Z, Yang X, Zhang H, Liu X, Pan S and Li C. The Role of extracellular Matrix Metalloproteinase Inducer Glycosylation in Regulating Matrix Metalloproteinases in Periodontitis. Journal of Periodontal Research 2018; 53: 391-402. Zhu P, Ding J, Zhou J, Dong W-J, Fan C-M and Chen Z-N. Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production. Arthritis Research and Therapy 2005; 7: R1023-R1033. Huang N and Gibson III FC. Immuno-pathogenesis of Periodontal Disease: Current and Emerging Paradigms. Current Oral Health Report 2014; 1: 124–132. Afacan B, Öztürk VO, Paşalı Ç, Bozkurt E, Köse T and Emingil G. Gingival crevicular fluid and salivary HIF-1𝜶, VEGF, and TNF-𝜶 levels in periodontal health and disease. Journal of Periodontology 2019; 90: 788-797. Liu L, Li C, Cai X, Xiang J, Cao Z and Dong W. The temporal expression and localization of extracellular matrix metalloproteinase inducer (EMMPRIN) during the development of periodontitis in an animal model. Journal of Periodontal Research 2010; 45: 541-549. Morton RS and Dongari-Bagtzoglou AI. Cyclooxygenase-2 is upregulated in inflamed gingival tissues. Journal of Periodontology 2001; 72: 461-469. Xiang J, Cao Z, Dong W and Li C. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in healthy and inflamed human gingival. Quintessence International 2009; 44: 125-132. Ng KT, Li JP, Ng KM, Tipoe GL, Leung WK and Fung ML. Expression of hypoxia-inducible factor-1α in human periodontal tissue. Journal of Periodontology 2011; 82:1 36-141. Ito H, Duxbury M, Zinner MJ, Ashley SW and Whang EE. Glucose transporter-1 gene expression is associated with pancreatic cancer invasiveness and MMP-2 activity. Surgery 2004; 136: 548-556. Idrees MM, Azzeghaiby SN, Hammad MM and Kujan OB. Prevalence and severity of plaque-induced gingivitis in a Saudi Adult population. Saudi Medical Journal 2014; 25: 1373-1377. Soanca A, Lupse M, Moldovan M, et al. Application of inflammation-derived Gingival Stem Cells for Testing the Biocompatibility of Dental Restorative Biomaterials. Annals of Anatomy – Anatomischer Anzeiger 2018; 218: 28-39. Magán-Fernández A, O’Valle F, Abadía-Molina F, Muñoz R, Puga-Guil P and Mesa F Characterization and comparison of neutrophil extracellular traps in gingival samples of periodontitis and gingivitis: a pilot study. Journal of Periodontal Research 2019; 54: 218-224. Bage T, Lindeberg J, Lundeberg J, Modeer T and Yucel-Lindeberg T. Signal pathways JNK and NF-κB, identified by global gene expression profiling, are involved in regulation of TNFα-induced mPGES-1 and COX- 2 expression in gingival fibroblasts. BMC Genomics 2010; 11: 241-268. Bage T, Kats A, Lopez BS, et al. Expression of prostaglandin e synthases in periodontitis immunolocalization and cellular regulation. The American Journal of Pathology 2011; 178: 1676-1688 Mesa F, O’Valle F, Rizzo M, et al. Association between COX-2 rs 6681231 Genotype and Interleukin-6 in Periodontal Connective Tissue. A Pilot Study. Plos One 2014; 9: e87023. Moro MG, Oliveira MDS, Oliveira LR, et al. Effects of selective Versus non-selective COX-2 inhibition on Experimental periodontitis. Brazilian Dental Journal 2019; 30: 133-138. Xue L, Su L, Xie J, Du Y and Yu X. EMMPRIN-Cypa contributes to the inflammatory process in human periodontitis through infiltrating CD68 + inflammatory cells. International Journal of Clinical and Experimental Pathology 2018; 11: 3828-3832. Iacono KT, Brown A, Greene MI and Saouaf SJ. CD147 Immunoglobulin Superfamily Receptor Function and Role in Pathology. Experimental and Molecular Pathology 2007; 83: 283-295. Agrawal SM and Yong VW. The many faces of EMMPRIN - roles in neuroinflammation. Biochimica et Biophysica Acta (BBA) – Molecular Basis of Disease 2011; 1812: 213-219. Koinsky D, Campbell EL and Colgan SP. Metabolic Shift in immunity and inflammation. The Journal of Immunology 2010; 184: 4062-4069. Imtyaz HZ and Simon MC. Hypoxia-inducible factors as essential regulators of inflammation. Current Topics in Microbiology and Immunology 2010; 345: 105-120. Vasconcelos RC, Costa ALL, Freitas RA, et al. Immunoexpression of HIF-1α and VEGF in periodontal disease and healthy gingival tissues. Brazilian Dental Journal 2016; 27: 117-122. Yan K, Lin O, Tang K, et al. Substance P participates in periodontitis by upregulating HIF-1 α and RANKL/OPG ratio. BMC Oral Health 2020; 20: 27. Angadi VC, Angadi PV. GLUT-1 immunoexpression in oral epithelial dysplasia, oral squamous cell carcinoma, and verrucous carcinoma . J Oral Sci. 2015; 57 :115-22. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 21 Nov, 2022 Reviewers agreed at journal 28 Sep, 2022 Reviews received at journal 29 Aug, 2022 Reviewers agreed at journal 22 Aug, 2022 Reviewers invited by journal 14 Aug, 2022 Editor assigned by journal 12 Aug, 2022 Submission checks completed at journal 12 Aug, 2022 First submitted to journal 09 Aug, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1946247","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":128401660,"identity":"9eb8c7dd-5512-4f43-87c4-7e12dd9582cf","order_by":0,"name":"Déborah-Pitta-Paraíso Iglesias","email":"","orcid":"","institution":"Federal University of Pernambuco","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Déborah-Pitta-Paraíso","middleName":"","lastName":"Iglesias","suffix":""},{"id":128401661,"identity":"f415be08-2455-4e7f-b3f6-c5e8b01990e0","order_by":1,"name":"Weslay-Rodrigues da-Silva","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABLUlEQVRIiWNgGAWjYHACxgNg6gBjA4ji4QOKPIAycAJULWwMDMwGUAYhLVAeUCWbBJSBFRgcb39w4GfbPTm+24ebX/NU1MmwSSQ/q/yaYyfDxsD88NENLFrOnDE42NtWbCx5LrHNmufMYR42iTSz27LbkoEOYzM2zsHUYnYjh+EAb1tC4oYzjG3GvG0HeNh4DpjdltzGDNTCwyaNTcv95w8O/oVr+VcH1HL8W7HktnrcWm4wGByG2tL8mLcBaDh7jxnjx22HcWqxP5NjcFjmXIKxJNAWxjnHDoO0FEszbjvOw8aM3S+S7ccfPnxTliDHd4b98Yc3NXX2/MzsGz/+3FZtz8/e/PAxFi1gwAiJAkh0gAAzD5jEoRwM/kAUfoCb8QOf6lEwCkbBKBhpAADLtmUpIgLOiwAAAABJRU5ErkJggg==","orcid":"","institution":"Real Hospital Português","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Weslay-Rodrigues","middleName":"","lastName":"da-Silva","suffix":""},{"id":128401662,"identity":"8940e390-9650-45c4-a9eb-1eda68b88958","order_by":2,"name":"Glória-Maria de-França","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Glória-Maria","middleName":"","lastName":"de-França","suffix":""},{"id":128401663,"identity":"a494ccec-11b0-4f5d-b705-beaf16edb016","order_by":3,"name":"Caio-César-da-Silva Barros","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Caio-César-da-Silva","middleName":"","lastName":"Barros","suffix":""},{"id":128401664,"identity":"e3b7d051-eba7-4371-8cb3-b8d2a8155ca2","order_by":4,"name":"Roseana de Almeida Freitas","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Roseana","middleName":"de Almeida","lastName":"Freitas","suffix":""},{"id":128401665,"identity":"b77a3ad7-7973-4c45-9b16-9467eba7b4d0","order_by":5,"name":"Hébel Cavalcanti Galvão","email":"","orcid":"","institution":"Federal University of Rio Grande do Norte","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hébel","middleName":"Cavalcanti","lastName":"Galvão","suffix":""}],"badges":[],"createdAt":"2022-08-09 17:29:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1946247/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1946247/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25321138,"identity":"00343fa8-cc34-4041-8235-32d42ceda869","added_by":"auto","created_at":"2022-08-17 14:26:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1755903,"visible":true,"origin":"","legend":"\u003cp\u003eScarce immunohistochemical expression of COX-2 in connective tissue \u003cstrong\u003e(A)\u003c/strong\u003e and immunostaining positivity in inflammatory infiltrate cells, fibroblasts, and endothelial cells \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1946247/v1/d5fb18b08f518601e0887829.png"},{"id":25321139,"identity":"d788003b-a445-4b68-b9a2-57b20bf41667","added_by":"auto","created_at":"2022-08-17 14:26:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1771508,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical expression of EMMPRIN, HIF-1α, GLUT-1 - \u003cstrong\u003e(A)\u003c/strong\u003e Immunostaining of EMMPRIN in the epithelial cell membrane, which gradually reduced from the basal to the superficial layer. \u003cstrong\u003e(B)\u003c/strong\u003e Positive cytoplasmic immunostaining of EMMPRIN in cells of the inflammatory infiltrate in the gingival connective tissue. \u003cstrong\u003e(C)\u003c/strong\u003e In epithelial cells, HIF-1α immunostaining occurred in the cytoplasm and nucleus, showing greater intensity in the basal layer. \u003cstrong\u003e(D)\u003c/strong\u003e Intense immunoexpression of HIF-1α in inflammatory cells and fibroblasts. \u003cstrong\u003e(E)\u003c/strong\u003e GLUT-1 immunostaining in the gingival epithelium was similar to the pattern observed in EMMPRIN. \u003cstrong\u003e(F)\u003c/strong\u003e In connective tissue, immunostaining GLUT-1 in the fibroblast membrane and the cytoplasm of inflammatory cells.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1946247/v1/0a1232edcdf3e2dc4dadc6f2.png"},{"id":25321140,"identity":"8106fe8c-f375-4aed-89a8-ad8f74433598","added_by":"auto","created_at":"2022-08-17 14:26:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":357222,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1946247/v1/684d2a71-c5ce-4991-82ba-5f6b46da751c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological marker for the establishment of periodontal disease: Cross-seccional study in gingival tissue","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePeriodontal disease is a condition of infectious etiology that can develop in all age groups and is the most frequent inflammatory disease of the oral cavity (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The inflammatory response triggered by the microbial accumulation in the gingival sulcus promotes the release of cytokines and the increase in local vascular permeability, clinically characterized by gingival erythema and edema, and the production of matrix metalloproteinases (MMPs) that can contribute to the alteration and/or destruction of the periodontium. This clinical panorama is the result of the secretion of cyclooxygenase-2 (COX-2), a proinflammatory enzyme that participates in the biosynthesis of prostaglandin E2, an inflammatory mediator that is involved in periodontal pathology (\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe persistence of the inflammatory condition generates the accumulation of cytokines and contributes to a greater release of MMPs, which are related to the degree of tissue destruction and disorganization (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). EMMPRIN is a glycoprotein involved in the pathogenesis of various diseases and, its activity is mainly related to the inducing effect on MMPs and its chemotactic capacity. Changes in its expression have been reported during the pathogenesis of periodontal disease with a relationship between increased expression and clinical disease progression (\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe chemotactic activity of EMMPRIN requires high energy consumption, also the arrival of inflammatory cells in the microenvironment is accompanied by an increase in the consumption of nutrients and oxygen, which can generate hypoxia conditions (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The local hypoxia generated in the inflamed microenvironment can activate the hypoxia-inducible factor 1 (HIF-1), which represents, in most human cells, the main path of adaptive cellular response to changes in oxygen concentration. The local hypoxia induces a change in energy activity in cells where glycolytic metabolism will prevail. At this time, the activity of proteins such as the glucose transporter 1 (GLUT-1) is essential, which, in hypoxia conditions, facilitates the entry of glucose into cells to maintain metabolic activity (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Thus, the present study investigated the expression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the gingival tissue, in order to verify if there is a correlation between the immunoexpression of these proteins and the changes caused by the inflamed infiltrate in the periodontium and contribute to a better understanding of the action of the inflammatory infiltrate in the pathogenesis of the periodontal disease.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cp\u003eSample\u003c/p\u003e \u003cp\u003e This research was cross-sectional study previously approved by the Research Ethics Committee (Approval number 164/2012, C.A.A.E. 0279.0.051.051-11). All samples included in this research were obtained at the Dental Office of the 14th Motorized Infantry Battalion and in the Oral Pathology Service at the UFRN (Natal, RN) between July and December 2011. A total of 60 formalin-fixed paraffin-embedded gingival tissue samples were obtained through a periodontal surgical procedure, for aesthetic reasons or the indication of increasing clinical crown height to restorative treatments. Were excluded from the research gingival tissues of pregnant or lactating patients as well as patients with previously known systemic disease, presence of infections in extraoral sites, smoking history in the last five years, and patients undergoing treatment with drugs that modulate the inflammatory response.\u003c/p\u003e \u003cp\u003eMorphological analysis\u003c/p\u003e \u003cp\u003eFive-\u0026micro;m thick histological sections were cut from the formalin-fixed paraffin-embedded specimens and stained with hematoxylin-eosin. Slides were independently assessed under light microscopy (Olympus CX31, Olympus Japan Co., Tokyo, Japan). Semi-quantitative morphological analysis was carried out to verify changes due to inflammation (hyperplasia, exocytosis, spongiosis and hydropic degeneration) throughout the epithelial extension, which was categorized as normal epithelium (with no changes or changes present in less than 10% of the epithelial extension); mild changes (11% \u0026minus;\u0026thinsp;25%); moderate changes (26% \u0026minus;\u0026thinsp;50%); and severe changes (in more than 50% of the epithelial extension).\u003c/p\u003e \u003cp\u003eConnective tissue was evaluated according to its density and organization of collagen fibers by adapting the criteria proposed by Liu \u003cem\u003eet al.\u003c/em\u003e (2010) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), being categorized into collagen fibers densely organized in more than 50% of the connective tissue; collagen fibers loosely organized in more than 50% of the connective tissue; and collagen fibers, sometimes loose, sometimes densely organized. The inflammatory infiltrate was categorized as absent (no inflammatory cells were observed); scarce inflammatory infiltrate (up to 25% of connective tissue occupied by inflammatory cells); moderate (26\u0026ndash;50% of the connective tissue occupied by inflammatory cells); and intense (more than 50% of the connective tissue occupied by inflammatory cells).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eFor the immunohistochemical study, 3-\u0026micro;m thick sections were obtained from paraffin-embedded tissue blocks and mounted on organosilane-coated slides (3-aminopropyltriethoxysilane; Sigma Chemical Co., St. Louis, MO, USA). The slides were incubated with one the following primary antibodies: anti-COX-2 (SP21; Spring Bioscience; 1:200; 60\u0026rsquo;), anti-EMMPRIN (Policlonal; Invitrogen; 1:400; 60\u0026rsquo;), anti-HIF-1α (H1α67; Santa Cruz, 1:200; Overnight), anti-GLUT-1 (Policlonal; Gene Tex; 1:400; 60\u0026rsquo;). Sections were then washed twice in phosphate-buffered saline and incubated in the HiDef visualization system (HiDef Detection\u0026trade; HRP Polymer System, Cell-Marque, USA) at room temperature. Finally, tissue sections were counterstained with Mayer's hematoxylin and coverslipped. For all primary antibodies, invasive ductal carcinoma sections were used as positive control. For negative control, the primary antibody was replaced with 1% bovine serum albumin in TBS.\u003c/p\u003e \u003cp\u003eImmunostaining assessment\u003c/p\u003e \u003cp\u003eThe analysis of the immunohistochemical expression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the gingival tissue was performed by a single investigator, who was blinded to the clinical and morphological data, under a light microscope (Olympus CX31, Olympus Japan Co., Tokyo, Japan) with 200x magnification. According the methodology proposed by Morton and Dagnari-Bagtzoglou (2001) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e), Xiang \u003cem\u003eet al.\u003c/em\u003e (2009) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), Ng \u003cem\u003eet al.\u003c/em\u003e (2011) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and Ito \u003cem\u003eet al.\u003c/em\u003e (2002) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), cells that showed brownish cytoplasmic immunostaining for COX-2, membrane and cytoplasmic for EMMPRIN, cytoplasmic and nuclear for HIF-1α, and membrane for GLUT-1 were considered positive. The immunohistochemical assessment considered the presence or absence of immunostaining. The immunostaining of epithelial cells was categorized according to the basal, parabasal and superficial (intermediate and superficial) layers of the epithelium. In the connective tissue, the analysis was performed by adapting the methodology proposed by Liu \u003cem\u003eet al.\u003c/em\u003e (2010) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), where the immunostaining of cells present in connective tissue was categorized as absent; scarce (1% \u0026minus;\u0026thinsp;25% of immunostained cells); moderate (26% \u0026minus;\u0026thinsp;50% of immunostained cells); and intense (above 50% of immunostained cells).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAbsolute and relative frequency were performed using the IBM Statistical Package for the Social Sciences (SPSS) Statistics 20.0 program (IBM Corp., Armonk, USA). Immunopositivity percentages were submitted to distribution analysis by the Kolmogorov-Smirnov test, which revealed a non-normal data distribution. Pearson\u0026rsquo;s Chi-square test was performed considering a 5% significance level (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eClinicopathological data\u003c/p\u003e \u003cp\u003eThe sample consisted of 60 cases of gingival tissue. Of these, 41 cases (68.3%) corresponded to male patients. The patients had a mean age of 37.7 years (range: 18\u0026ndash;80 years). Concerning the morphological findings, all epithelial changes (hyperplasia, exocytosis, spongiosis and edematous degeneration) were frequent, and it was observed that in 30 cases (50%) there were changes in a sparse manner, followed by moderate changes (n\u0026thinsp;=\u0026thinsp;17; 28.3%) and intense (n\u0026thinsp;=\u0026thinsp;11; 18.3%), while two cases (3.3%) did not show any changes. Also, there was a higher frequency of intense inflammatory infiltrate (n\u0026thinsp;=\u0026thinsp;25), and 43 cases exhibited collagen fibers densely organized. There was a significant association between the intensity of the inflammatory infiltrate and the density of the connective tissue (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e01\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImmunohistochemical findings\u003c/p\u003e \u003cp\u003eImmunohistochemical analysis showed COX-2 epithelial positivity in only three cases, which was restricted to the parabasal layer. Of these, sparse immunoexpression was observed in two cases (3.3%) and moderate in one (1.7%). Regarding the immunostaining of COX-2 in connective tissue, positivity was observed in 36 (60%) cases and a higher frequency of sparse expression (n\u0026thinsp;=\u0026thinsp;18; 30%), followed by moderate expression (n\u0026thinsp;=\u0026thinsp;9; 15%) and intense (n\u0026thinsp;=\u0026thinsp;9; 15%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). It was also observed that this immunostaining was present in inflammatory infiltrate mononuclear cells, fibroblasts, and endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The results of positivity for COX-2 compared to the intensity of the inflammatory infiltrate revealed a significant association (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation between connective tissue organization, the intensity of COX-2 expression, and the intensity of inflammatory infiltrate.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eInflammatory Infiltrate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eScarce\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eModerate\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eIntense\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eConnective tissue organization\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDensely organized\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21 (48.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12 (27.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.009*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLoosely organized\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (16.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (83.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eDensely and loosely organized\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (18.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (9.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 (72.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eCOX-2 immunoexpression intensity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAbsent\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15 (62.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (29.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (8.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScarce\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 (33.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (16.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eModerate\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2 (22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (55.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIntense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*Significant association by Pearson's Chi-square test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eConcerning EMMPRIN, the epithelial cells exhibited a predominantly membrane immunoexpression pattern in the cells of the basal layer, which reduced gradually until the superficial layer. Positive membrane immunostaining was observed intensely in the basal layer of 42 cases (70%), in the parabasal of 14 cases (23.3%), while it was absent in the superficial layer of 49 cases (81.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The gingival connective tissue showed positive immunostaining, at the cytoplasmic level, for EMMPRIN in 98.3% (n\u0026thinsp;=\u0026thinsp;59) of the sample, which was intense in 70% (n\u0026thinsp;=\u0026thinsp;42) of the cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). There was a significant association (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between the expression of EMMPRIN with the layers of epithelial tissue and connective tissue (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAssociation of immunostaining for EMMPRIN, HIF-1α, and GLUT-1 with gingival epithelium and connective tissue.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eImmunoexpression Intensity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eEpithelium\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConnective Tissue\u003c/p\u003e \u003cp\u003en (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eBasal Layer\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eParabasal Layer\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eSuperficial Layer\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003en (%)\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEMMPRIN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAbsent\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49 (81.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (1.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScarce\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (6.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9 (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (8.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eModerate\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12 (20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIntense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42 (70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42 (70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHIF-1α\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAbsent\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (23.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (13.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScarce\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (6.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 (13.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eModerate\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9 (15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIntense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39 (65)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29 (48.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35 (58.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGLUT-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAbsent\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 (1.73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 (10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58 (96.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35 (58.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eScarce\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eModerate\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12 (20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 (33.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIntense\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44 (73.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (6.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e*Significant association by Pearson's Chi-square test\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eImmunostaining of HIF-1α in epithelial cells demonstrated cytoplasmic, and sometimes nuclear expression in the basal layer (n\u0026thinsp;=\u0026thinsp;46; 76.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). In connective tissue, immunostaining was highly positive (n\u0026thinsp;=\u0026thinsp;35; 58.3%) in both inflammatory cells and fibroblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). A significant association was observed between HIF-1α immunoexpression and epithelial and connective tissue immunostaining (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GLUT-1 immunostaining was identified in the cytoplasmic membrane of epithelial cells intensely in the basal layer (n\u0026thinsp;=\u0026thinsp;44; 73.3%), moderate in the parabasal (n\u0026thinsp;=\u0026thinsp;20; 33.3%) and absence in the superficial layer (n\u0026thinsp;=\u0026thinsp;58; 96.6%). In areas where the epithelial tissue had inflammatory changes, such as edematous degeneration, the expression was markedly reduced, showing itself to be weak or absent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). The immunostaining of GLUT-1 in connective tissue was positive in 41.7% of the sample (n\u0026thinsp;=\u0026thinsp;25), showing a membrane pattern in fibroblasts and a cytoplasmic pattern in inflammatory cells. Besides, a greater amount of positive endothelial cells was observed in cases with greater intensity of cell infiltrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). There was significance between GLUT-1 immunoexpression and epithelial and connective tissue immunostaining (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). There were no statistically significant results between the immunoexpression of COX-2, EMMPRIN, HIF-1α and GLUT-1.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study was carried out on 60 gingival tissue to investigate the changes caused by the inflammatory infiltrate in the periodontium. Our analyzes demonstrate that although the inflammatory infiltrate has no association with the morphological changes of the epithelium, the epithelial immunoexpression of COX-2 can be used as a possible biological marker in investigating the presence of periodontal disease.\u003c/p\u003e \u003cp\u003eHistologically, the gingiva presents a variable amount of inflammatory infiltrate, thus obtaining a gingival connective tissue that histologically does not present inflammatory infiltrate is quite unusual (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). This inflammatory infiltrate is a basic defense mechanism against microbial persistence in the gingival sulcus. However, it will also contribute to the destruction and increase of gingival tissues through the secretion of lysosomal enzymes and cytokines that stimulate the release of MMPs, vasodilation, increased permeability vascular and local blood flow, and the accumulation of exudate in the extracellular environment (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). This reduction in the amount of collagen is microscopically visualized as a looser and more irregular organization of collagen fibers and a large amount of extracellular matrix, characteristics observed in the present study, especially in cases that exhibited intense inflammatory infiltrate.\u003c/p\u003e \u003cp\u003eVasodilation and increased vascular permeability that develops in gingival tissue is partly the result of prostaglandin biosynthesis. The inflammatory infiltrate induces the metabolism of arachidonic acid in epithelial, endothelial cells, macrophages, and fibroblasts and, consequently, the production of PGE2 through the cyclooxygenase pathway and action of COX-2. COX-2 is an enzyme that mediates inflammatory effects and has been identified as a key mediator in the pathogenesis of periodontal disease, with an absent or low expression pattern in healthy tissues and an increase in inflamed tissues (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In the present study, there was a significant increase in the immunoexpression of this protein in cases with intense inflammatory infiltrate. Also, COX-2 immunoexpression in epithelial tissue was observed in only three cases.\u003c/p\u003e \u003cp\u003eIn previous studies, a high immunoexpression of COX-2 was observed, with no absence of immunostaining in the analyzed cases (\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The present research diverges from these results since, in some cases, there was no immunostaining for COX-2. We believe that the lower intensity or absence of this immunostaining is due to the lower intensity of the inflammatory infiltrate observed in some cases. In the study performed by Mesa \u003cem\u003eet al.\u003c/em\u003e, (2014) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), positive epithelial immunostaining for COX-2 was found only in gingival tissues obtained in sites with periodontal disease and, consequently, bone resorption. Thus, we believe that the cases, with COX-2 epithelial immunoexpression, would be in more advanced stages of periodontal disease, possibly indicating possible gingivitis.\u003c/p\u003e \u003cp\u003eIn periodontal tissues, the integrity of the extracellular matrix is ​​important for maintaining tissue stability. This integrity is maintained through the activation of MMPs and their inhibitors, which results in the balance between degradation and production of the extracellular matrix. In periodontal disease, progressive destruction of gingival tissues is observed due to increased activity of MMPs, such as collagenase, both due to the action of fibroblasts and mononuclear inflammatory cells (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The participation of EMMPRIN in the balance and maintenance of the extracellular matrix in the gingival tissue is demonstrated, in healthy periodontal sites, by its intense expression in the epithelial cells of the basal layer, at the cytoplasmic level, with gradual decrease as the cells move away from the germ layer of the epithelium (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The literature also reports that this pattern of immunostaining confirms the participation of EMMPRIN in maintaining tissue integrity and cell adhesion (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The present study observed a similar immunostaining pattern. Besides, it was noted that the expression of EMMPRIN was lower in areas where epithelial tissue shows intense spongiosis, findings that corroborate those reported in the literature.\u003c/p\u003e \u003cp\u003eIn inflammatory gingival conditions, increased cellular inflow increases EMMPRIN expression. In this way, both fibroblasts and the lymphoplasmocytic cells themselves, which overexpress EMMPRIN, can activate and recruit more inflammatory cells, thus constituting a pattern of positive self-regulation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In the present study, a similar immunostaining pattern of EMMPRIN was observed in lymphoplasmacytic cells and fibroblasts. However, although we do not find statistical significance, we cannot rule out the participation of this molecule and MMPs in the pathogenesis process of periodontal disease.\u003c/p\u003e \u003cp\u003eIt is also important to note that the chemotactic activity triggered by EMMPRIN generates a high energy consumption and that the arrival of more cells to the inflammatory site is accompanied by an increase in the consumption of nutrients and oxygen. Thus, the infectious process establishes tissue hypoxia through high oxygen consumption by both microorganisms and host defense cells recruited to the gingival tissues (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Endothelial damage and edema resulting from inflammation are also factors that cause tissue hypoxia, as they result in microcirculatory failure and thus reduced oxygen supply. However, the response of gingival tissue to oxygen depletion is coordinated by HIF \u0026minus;\u0026thinsp;1α (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). The present study observed a high immunoexpression of HIF-1α both in the epithelium and in the connective tissue.\u003c/p\u003e \u003cp\u003eWe believe that the injury to the gingival epithelium, caused by dental biofilm, may justify the intense expression of HIF-1α in the analyzed specimens. The pattern of high nuclear and cytoplasmic immunoexpression, observed by us, corroborating the findings (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), which denoted that cytoplasmic expression may represent the activation of the HIF-1α factor. Besides, HIF-1α is associated with the survival of activated T lymphocytes. Once evading the programmed cell death pathway, these cells remain for a longer time in the inflammatory sites, delaying the resolution of the inflammatory condition (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In the present study, many lymphocytes were observed in the connective tissue. Thus, this fact could be attributed to the high immunoexpression of HIF-1α in the gingival connective tissue of our sample.\u003c/p\u003e \u003cp\u003eThe high expression of HIF-1α modifies the expression of hypoxia-related genes, thus increasing the synthesis of VEGF, a proangiogenic cytokine, and the glucose transporter GLUT-1 (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Agandi and Agandi (2015) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) found in their study that the strong immunoexpression of GLUT-1 in the basal and parabasal layer of the oral epithelium occurs due to the high proliferative activity in this layer, while the absence of GLUT-1 in the most superficial layers reflects the maturation of epithelial cells. Our results revealed similar immunostaining of GLUT-1, and it suggests that the expression of this protein in the gingival epithelium have not alterations due to the action of the inflammatory infiltrate. Also, the low immunoexpression of this protein in connective tissue suggests a possible balance of hypoxia control in the inflammatory microenvironment. Thus, we suggest that GLUT-1 participates in the physiology of gingival tissue, mainly contributing to the maintenance of epithelial tissue.\u003c/p\u003e \u003cp\u003eIn conclusion, the results of the present study demonstrate that the expression of COX-2 may constitute a biological marker of gingival tissues since its epithelial immunoexpression may indicate a greater propensity to establish periodontal disease. Besides that, the results indicated that our immunoexpression patterns of EMMPRIN, HIF-1α, and GLUT-1 in the gingival epithelium and connective tissue confirm the physiological role played by these markers in maintaining the integrity and homeostasis of the gingival tissue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. All procedures performed in this retrospective study involving human participants were approved by the Institutional Ethics Comitee Protocol number 164/2012, C.A.A.E. 0279.0.051.051-11 (UFRN).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Pathological Anatomy Service of the Discipline of Oral Pathology, Department of Dentistry, Federal University of Rio Grande do Norte, for providing the material and laboratory resources necessary for the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients involved in this study signed the informed consent form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD\u0026eacute;borahPitta-Para\u0026iacute;so Iglesias: Participation in conducting the research and preparation of the manuscrip;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWeslay Rodrigues da Silva: Participation in conducting the research and preparation of the manuscript\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGl\u0026oacute;ria Maria de Fran\u0026ccedil;a and Caio C\u0026eacute;sar da Silva Barros: \u0026nbsp;wrote the main manuscript text\u003c/p\u003e\n\u003cp\u003eRoseana de Almeida Freitas and H\u0026eacute;bel-Cavalcanti Galv\u0026atilde;o: prepared figures.\u003c/p\u003e\n\u003cp\u003eAll authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest. This study was supported by the National Council for Scientific and Technological Development (CNPq, Brazil), Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil), and Postgraduate Program in Dental Sciences (UFRN, Natal, RN, Brazil).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eHolmstrup P, Plemons J and Meyle J. Non\u0026ndash;plaque-induced gingival disease. \u003cem\u003eJournal of Periodontology\u003c/em\u003e 2018; \u003cstrong\u003e89:\u003c/strong\u003eS28\u0026ndash;S45.\u003c/li\u003e\n \u003cli\u003eKumar S. Evidence-Based Update on Diagnosis and Management of Gingivitis and Periodontitis. \u003cem\u003eDental Clinics of North America\u003c/em\u003e 2019; \u003cstrong\u003e63:\u003c/strong\u003e69-81.\u003c/li\u003e\n \u003cli\u003eLazăr L, Loghin A, Bud E, Cerghizan D, Horv\u0026aacute;th E and Nagy EE. Cyclooxygenase-2 and matrix metalloproteinase-9 expressions correlate with tissue inflammation degree in periodontal disease. \u003cem\u003eRomanian Journal of Morphology and Embryology\u003c/em\u003e 2015; \u003cstrong\u003e56:\u003c/strong\u003e1441\u0026ndash;1446.\u003c/li\u003e\n \u003cli\u003eArweiler NB and Netuschil L. The Oral Microbiota. \u003cem\u003eAdvances in Experimental Medicine and Biology\u003c/em\u003e 2016; \u003cstrong\u003e902:\u003c/strong\u003e45-60.\u003c/li\u003e\n \u003cli\u003eStone SJ, Kumar PS, Offenbacher S, Heasman PA and McCracken GI. Exploring a temporal relationship between biofilm microbiota and inflammatory mediators during resolution of naturally-occurring gingivitis. \u003cem\u003eJournal of Periodontology\u003c/em\u003e 2019; \u003cstrong\u003e90:\u003c/strong\u003e627-636.\u003c/li\u003e\n \u003cli\u003ede Oliveira N\u0026oacute;brega FJ, de Oliveira DHIP, Vasconcelos RG, Nonaka CFW, Queiroz LMG.\u0026nbsp;Study of the participation of MMP-7, EMMPRIN and cyclophilin A in the pathogenesis of periodontal disease. \u003cem\u003eArchives of Oral Biology\u003c/em\u003e 2016; \u003cstrong\u003e72:\u003c/strong\u003e172\u0026ndash;178.\u003c/li\u003e\n \u003cli\u003eLiu X, Zhang Z, Pan S, Shang S and Li C. Interaction between the Wnt/\u0026beta;-catenin signaling pathway and the EMMPRIN/MMP-2, 9 route in periodontitis. \u003cem\u003eJournal of Periodontal Research\u003c/em\u003e 2018; \u003cstrong\u003e53:\u003c/strong\u003e842-852.\u003c/li\u003e\n \u003cli\u003eZhang Z, Yang X, Zhang H, Liu X, Pan S and Li C. The Role of extracellular Matrix Metalloproteinase Inducer Glycosylation in Regulating Matrix Metalloproteinases in Periodontitis. \u003cem\u003eJournal of Periodontal Research\u003c/em\u003e 2018; \u003cstrong\u003e53:\u003c/strong\u003e391-402.\u003c/li\u003e\n \u003cli\u003eZhu P, Ding J, Zhou J, Dong W-J, Fan C-M and Chen Z-N. Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production. Arthritis Research and\u0026nbsp;Therapy 2005; \u003cstrong\u003e7:\u003c/strong\u003eR1023-R1033.\u003c/li\u003e\n \u003cli\u003eHuang N and Gibson III FC. Immuno-pathogenesis of Periodontal Disease: Current and Emerging Paradigms. \u003cem\u003eCurrent Oral Health Report\u003c/em\u003e 2014; \u003cstrong\u003e1:\u003c/strong\u003e124\u0026ndash;132.\u003c/li\u003e\n \u003cli\u003eAfacan B, \u0026Ouml;zt\u0026uuml;rk VO, Paşalı \u0026Ccedil;, Bozkurt E, K\u0026ouml;se T and Emingil G. Gingival crevicular fluid and salivary HIF-1𝜶, VEGF, and TNF-𝜶\u0026nbsp;levels in periodontal health and disease. \u003cem\u003eJournal of Periodontology\u003c/em\u003e 2019; \u003cstrong\u003e90:\u003c/strong\u003e788-797.\u003c/li\u003e\n \u003cli\u003eLiu L, Li C, Cai X, Xiang J, Cao Z and Dong W. The temporal expression and localization of extracellular matrix metalloproteinase inducer (EMMPRIN) during the development of periodontitis in an animal model. \u003cem\u003eJournal of Periodontal Research\u003c/em\u003e 2010; \u003cstrong\u003e45:\u003c/strong\u003e541-549.\u003c/li\u003e\n \u003cli\u003eMorton RS and Dongari-Bagtzoglou AI. Cyclooxygenase-2 is upregulated in inflamed gingival tissues. \u003cem\u003eJournal of Periodontology\u003c/em\u003e 2001; \u003cstrong\u003e72:\u003c/strong\u003e461-469.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eXiang J, Cao Z, Dong W and Li C. Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in healthy and inflamed human gingival. \u003cem\u003eQuintessence International\u003c/em\u003e 2009; \u003cstrong\u003e44:\u003c/strong\u003e125-132.\u003c/li\u003e\n \u003cli\u003eNg KT, Li JP, Ng KM, Tipoe GL, Leung WK and Fung ML. Expression of hypoxia-inducible factor-1\u0026alpha;\u0026nbsp;in human periodontal tissue. \u003cem\u003eJournal of Periodontology\u003c/em\u003e 2011; \u003cstrong\u003e82:1\u003c/strong\u003e36-141.\u003c/li\u003e\n \u003cli\u003eIto H, Duxbury M, Zinner MJ, Ashley SW and Whang EE. Glucose transporter-1 gene expression is associated with pancreatic cancer invasiveness and MMP-2 activity. \u003cem\u003eSurgery\u003c/em\u003e 2004; \u003cstrong\u003e136:\u003c/strong\u003e548-556.\u003c/li\u003e\n \u003cli\u003eIdrees MM, Azzeghaiby SN, Hammad MM and Kujan OB. Prevalence and severity of plaque-induced gingivitis in a Saudi Adult population. \u003cem\u003eSaudi Medical Journal\u003c/em\u003e 2014; \u003cstrong\u003e25:\u003c/strong\u003e1373-1377.\u003c/li\u003e\n \u003cli\u003eSoanca A, Lupse M, Moldovan M, et al. Application of inflammation-derived Gingival Stem Cells for Testing the Biocompatibility of Dental Restorative Biomaterials. \u003cem\u003eAnnals of Anatomy \u0026ndash; Anatomischer Anzeiger\u003c/em\u003e 2018; \u003cstrong\u003e218:\u003c/strong\u003e28-39.\u003c/li\u003e\n \u003cli\u003eMag\u0026aacute;n-Fern\u0026aacute;ndez A, O\u0026rsquo;Valle F, Abad\u0026iacute;a-Molina F, Mu\u0026ntilde;oz R, Puga-Guil P and Mesa F Characterization and comparison of neutrophil extracellular traps in gingival samples of periodontitis and gingivitis: a pilot study.\u0026nbsp;\u003cem\u003eJournal of Periodontal Research\u003c/em\u003e 2019; \u003cstrong\u003e54:\u003c/strong\u003e218-224.\u003c/li\u003e\n \u003cli\u003eBage T, Lindeberg J, Lundeberg J, Modeer T and Yucel-Lindeberg T. Signal pathways JNK and NF-\u0026kappa;B, identified by global gene expression profiling, are involved in regulation of TNF\u0026alpha;-induced mPGES-1 and COX- 2 expression in gingival fibroblasts. \u003cem\u003eBMC Genomics\u003c/em\u003e 2010; \u003cstrong\u003e11:\u003c/strong\u003e241-268.\u003c/li\u003e\n \u003cli\u003eBage T, Kats A, Lopez BS, et al. Expression of prostaglandin e synthases in periodontitis immunolocalization and cellular regulation.\u0026nbsp;\u003cem\u003eThe American Journal of Pathology\u003c/em\u003e 2011; \u003cstrong\u003e178:\u003c/strong\u003e1676-1688\u003c/li\u003e\n \u003cli\u003eMesa F, O\u0026rsquo;Valle F, Rizzo M, et al. Association between COX-2 rs 6681231 Genotype and Interleukin-6 in Periodontal Connective Tissue. A Pilot Study. \u003cem\u003ePlos One\u003c/em\u003e 2014; \u003cstrong\u003e9:\u003c/strong\u003ee87023.\u003c/li\u003e\n \u003cli\u003eMoro MG, Oliveira MDS, Oliveira LR, et al. Effects of selective Versus non-selective COX-2 inhibition on Experimental periodontitis. \u003cem\u003eBrazilian Dental Journal\u003c/em\u003e 2019; \u003cstrong\u003e30:\u003c/strong\u003e133-138.\u003c/li\u003e\n \u003cli\u003eXue L, Su L, Xie J, Du Y and Yu X. EMMPRIN-Cypa contributes to the inflammatory process in human periodontitis through infiltrating CD68\u003csup\u003e+\u003c/sup\u003e inflammatory cells. \u003cem\u003eInternational Journal of Clinical and Experimental Pathology\u003c/em\u003e 2018; \u003cstrong\u003e11:\u003c/strong\u003e3828-3832.\u003c/li\u003e\n \u003cli\u003eIacono KT, Brown A, Greene MI and Saouaf SJ.\u0026nbsp;CD147 Immunoglobulin Superfamily Receptor Function and Role in Pathology. Experimental and Molecular Pathology 2007;\u0026nbsp;\u003cstrong\u003e83:\u003c/strong\u003e283-295.\u003c/li\u003e\n \u003cli\u003eAgrawal SM and Yong VW. The many faces of EMMPRIN - roles in neuroinflammation. \u003cem\u003eBiochimica et Biophysica Acta (BBA) \u0026ndash; Molecular Basis of Disease\u003c/em\u003e 2011;\u0026nbsp;\u003cstrong\u003e1812:\u003c/strong\u003e213-219.\u003c/li\u003e\n \u003cli\u003eKoinsky D, Campbell EL and Colgan SP. Metabolic Shift in immunity and inflammation. \u003cem\u003eThe Journal of Immunology\u003c/em\u003e 2010; \u003cstrong\u003e184:\u003c/strong\u003e4062-4069.\u003c/li\u003e\n \u003cli\u003eImtyaz HZ and Simon MC. Hypoxia-inducible factors as essential regulators of inflammation. \u003cem\u003eCurrent Topics in Microbiology and Immunology\u003c/em\u003e 2010; \u003cstrong\u003e345:\u003c/strong\u003e105-120.\u003c/li\u003e\n \u003cli\u003eVasconcelos RC, Costa ALL, Freitas RA, et al. Immunoexpression of HIF-1\u0026alpha;\u0026nbsp;and VEGF in periodontal disease and healthy gingival tissues. \u003cem\u003eBrazilian Dental Journal\u003c/em\u003e 2016; \u003cstrong\u003e27:\u003c/strong\u003e117-122.\u003c/li\u003e\n \u003cli\u003eYan K, Lin O, Tang K, et al. Substance P participates in periodontitis by upregulating HIF-1\u0026nbsp;\u0026alpha;\u0026nbsp;and RANKL/OPG ratio. \u003cem\u003eBMC Oral Health\u003c/em\u003e 2020; \u003cstrong\u003e20:\u003c/strong\u003e27.\u003c/li\u003e\n \u003cli\u003eAngadi VC, Angadi PV. GLUT-1 immunoexpression in oral epithelial dysplasia, oral squamous cell carcinoma, and verrucous carcinoma\u003cem\u003e. J Oral Sci.\u003c/em\u003e 2015; \u003cstrong\u003e57\u003c/strong\u003e:115-22.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oral-and-maxillofacial-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"omfs","sideBox":"Learn more about [Oral and Maxillofacial Surgery](http://link.springer.com/journal/10006)","snPcode":"10006","submissionUrl":"https://submission.nature.com/new-submission/10006/3","title":"Oral and Maxillofacial Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Gingiva, Periodontal diseases, Inflammation","lastPublishedDoi":"10.21203/rs.3.rs-1946247/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1946247/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe present study investigated the expression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the gingival tissue, to verify if there is a correlation between the immunoexpression of these proteins and the changes caused by the inflamed infiltrate present in the gingival tissues.\u003c/p\u003e\u003ch2\u003eMaterial and methods\u003c/h2\u003e \u003cp\u003eA morphological analysis of epithelial changes (hyperplasia, exocytosis, spongiosis, and hydropic degeneration) was performed, as well as a semiquantitative analysis of the immunoexpression of COX-2, EMMPRIN, HIF-1α, and GLUT-1 in the epithelium and connective tissue of 60 specimens of gingival tissue.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eEpithelial immunoexpression to COX-2 was observed in three cases, while EMMPRIN, HIF-1α, and GLUT-1 were strongly expressed in the basal layer of the epithelium and gradually decreasing until the upper layers. In the connective tissue, COX-2 immunoexpression showed a statistical association (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with the gingival inflammatory infiltrate. In connective tissue, EMMPRIN, and HIF-1α exhibited intense immunopositivity, while GLUT-1 was negative in most cases.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eCOX-2 expression may constitute a biological marker of gingival tissues since its epithelial immunoexpression may indicate a greater propensity for the establishment of periodontal disease.\u003c/p\u003e","manuscriptTitle":"Biological marker for the establishment of periodontal disease: Cross-seccional study in gingival tissue","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-17 14:26:15","doi":"10.21203/rs.3.rs-1946247/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-11-21T09:27:40+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"9a33f2b6-afcd-4f3b-b71b-6fef3856638a","date":"2022-09-28T09:58:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-08-29T15:55:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"c5ece892-f0bf-412e-8826-de3001faa8e4","date":"2022-08-22T13:23:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-08-14T06:05:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-08-12T06:59:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-08-12T06:59:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oral and Maxillofacial Surgery","date":"2022-08-09T17:25:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oral-and-maxillofacial-surgery","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"omfs","sideBox":"Learn more about [Oral and Maxillofacial Surgery](http://link.springer.com/journal/10006)","snPcode":"10006","submissionUrl":"https://submission.nature.com/new-submission/10006/3","title":"Oral and Maxillofacial Surgery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b49b2e51-f9c2-4acc-94ad-9dc531a24b7a","owner":[],"postedDate":"August 17th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-12-04T15:14:11+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-17 14:26:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1946247","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1946247","identity":"rs-1946247","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}