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

doi:10.21203/rs.3.rs-1946247/v1

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

https://doi.org/10.21203/rs.3.rs-1946247/v1

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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

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.

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).

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. 1A). It was also observed that this immunostaining was present in inflammatory infiltrate mononuclear cells, fibroblasts, and endothelial cells (Fig. 1B). 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 (%)	p
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. 2A). 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. 2B). 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
Immunoexpression Intensity	Basal Layer n (%)	Parabasal Layer n (%)	Superficial Layer n (%)	Connective Tissue n (%)	p
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. 2C). In connective tissue, immunostaining was highly positive (n = 35; 58.3%) in both inflammatory cells and fibroblasts (Fig. 2C). 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. 2E). 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. 2F). 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.

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.

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).

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

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