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Table of Contents   
ORIGINAL RESEARCH  
Year : 2011  |  Volume : 22  |  Issue : 1  |  Page : 116-121
Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral submucous fibrosis


Department of Oral and Maxillo-facial Pathology, Meenakshi Ammal Dental College and Hospital, Maduravoyal, Chennai, India

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Date of Submission24-Sep-2010
Date of Decision07-Oct-2010
Date of Acceptance15-Oct-2010
Date of Web Publication25-Apr-2011
 

   Abstract 

Introduction: Oral submucous fibrosis (OSMF) is a chronic, progressive, scarring disease that predominantly affects the people of south-east Asian origin.
Aim: The present study is aimed at comparing and correlating the mast cell density (MCD) and micro vascular density (MVD) in normal mucosa and different grades of OSMF and to analyze their role in the disease progression.
Materials and Methods: MCD was assessed immunohistochemically using anti mast cell tryptase and MVD was assessed using anti-factor VIII related von Willebrand factor.
Results: The one way comparison of MVD and MCD in normal mucosa and among different grades of OSMF showed a significant increase in MCD and MVD among OSMF cases. Correlation analysis using Pearson correlation coefficient, showed positive correlation between MCD and MVD i.e. as MCD increases there is an exponential increase in MVD.
Conclusion: The increase in MVD and MCD reveals their role in the pathogenesis of OSMF, a lesion characterized by progressive fibrosis in early stages and failure of degradation or remodeling in the advanced stages.

Keywords: Endothelial proliferation, immunohistochemistry, mast cells, oral submucous fibrosis

How to cite this article:
Sabarinath B, Sriram G, Saraswathi T R, Sivapathasundharam B. Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral submucous fibrosis. Indian J Dent Res 2011;22:116-21

How to cite this URL:
Sabarinath B, Sriram G, Saraswathi T R, Sivapathasundharam B. Immunohistochemical evaluation of mast cells and vascular endothelial proliferation in oral submucous fibrosis. Indian J Dent Res [serial online] 2011 [cited 2019 Jul 17];22:116-21. Available from: http://www.ijdr.in/text.asp?2011/22/1/116/80009
Oral submucous fibrosis (OSMF) is a chronic, progressive, scarring disease that predominantly affects the people of Southeast Asian origin. [1] Overall prevalence of OSMF in India is about 0.5% with a range of 0.2-1.2% in different regions of the country. [2] Excessive fibrosis in the mucosa seems to be the primary pathology in OSMF and the atrophic changes in the epithelium are secondary. Epithelial dysplasia occurs in 10-15% and carcinoma occurs at least in 5% of cases submitted for biopsy. [3]

Mast cells are highly engineered cells which may be activated by a number of stimuli. [4] Following activation by immunological or non-immunological stimuli, mast cells release via their granules a range of preformed mediators. Mast cell products degrade connective tissue matrix to provide space for neovascular sprouts. [5],[6]

As there is paucity of information in the literature correlating mast cell and angiogenic activity in OSMF, this immunological study is designed to evaluate their role in pathogenesis of OSMF.

Aims

  • To quantitate mast cell density (MCD) and microvessel density (MVD) in normal buccal mucosa and in different histological grades of OSMF by immunohistochemistry using anti-factor VIII and anti-mast cell tryptase, respectively.
  • To quantitate immunohistochemically the number of intact and degranulated mast cells using anti-mast cell tryptase.
  • To compare and correlate the MCD and MVD in NM and different grades of OSMF and to analyze their role in the disease progression.

   Materials and Methods Top


This study included 40 formalin-fixed, paraffin-embedded sections retrieved from the Department of Oral and Maxillo-facial Pathology, Meenakshi Ammal Dental College and Hospital, Chennai. Among these, 30 cases were histologically diagnosed OSMF and 10 cases were clinically normal oral buccal mucosa of individuals without any habit, forming the control group. Clinical data and informed consent were taken from these patients.

Controls

Control sections which included neurofibroma for mast cells and pyogenic granuloma for angiogenesis formed the positive controls and were treated in the same manner as the test groups, and control sections which included one NM and one OSMF formed the negative controls and they were also treated in the same manner as the test groups, except that the primary antibody was omitted and substituted with phosphate-buffered saline (PBS) and a non-immune antibody (normal rabbit serum) at same concentration.

Staining

Hematoxylin and eosin staining

Formalin-fixed, paraffin-embedded tissues were sectioned at 4 μm thickness and taken for routine hemotoxylin and eosin staining. These sections were used for grading of OSMF.

Immunohistochemical staining

The serial sections of the same were stained by immunohistochemical reagents (anti-Mc tryptase for mast cells and anti-factor VIII related von Willebrand factor for endothelial cells).

Methodology

Four-micrometer thin sections were taken on to slides which were kept in 4% chromic acid and coated with amino-propyl ethoxy silane (APES) and were dried in a microwave oven at 60°C overnight. Sections were deparaffinized and were subjected to antigen retrieval in 10 M citrate acid buffer solution and boiled thrice in microwave oven for 5 minutes. The solution was allowed to cool to room temperature and washed in Tris buffer saline (TBS).

Sections were further treated with methanemic hydrogen peroxide for 10 minutes at room temperature to block the endogenous peroxidase activity. Power block was performed for 15 minutes to block background staining. Incubate primary monoclonal antibody anti-mast cell tryptase for 1 hour for slides to be stained for mast cells, and anti-factor VIII related von Willebrand factor for 2 hours followed by secondary antibody-Super enhancer and Poly HRP (horse raddish peroxidase) for 30 minutes. Excess of stain was wiped off and washed with phosphate buffer solution (PBS) for two changes. The sections were incubated with diethylamine benzene (DAB) substrate chromogen for 15-20 minutes and counterstained with one dip of Harris hematoxylin followed by bluing in running tap water. Slides were air dried and mounted with DPX.

Evaluation

Mast cells and microvessels were counted in the systematic field using ×400 using oculometer grid which had 100 subdivisions. The criteria for counting the mast cells were that any cluster of mast cell granule positive for anti-Mc tryptase and clearly separated from an adjacent cluster was considered to be a single mast cell and appearing reddish brown in color; cellular boundary was not necessary for a structure to be defined as mast cell [Figure 1], [Figure 2], [Figure 3] and [Figure 4].
Figure 1: Immunohistochemical demonstration of mast cells in normal mucosa using mouse monoclonal anti-mast cell tryptase antibody (×400)

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Figure 2: Immunohistochemical demonstration of mast cells in VEOSMF using mouse monoclonal anti-mast cell tryptase antibody (×400)

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Figure 3: Immunohistochemical demonstration of mast cells in EOSMF using mouse monoclonal anti-mast cell tryptase antibody (×400)

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Figure 4: Immunohistochemical demonstration of mast cells in MAOSMF using mouse monoclonal anti-mast cell tryptase antibody (×400)

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Similarly, for the blood vessel, any endothelial lined vessel lumen or endothelial cell cluster positive for factor VIII-related antigen, i.e., stained reddish brown and clearly separated from an adjacent cluster was considered to be a single, countable microvessel. Vessel lumen was not necessary for a structure to be defined as a microvessel. Red blood cells were not used to define a vessel lumen [Figure 5], [Figure 6], [Figure 7] and [Figure 8].
Figure 5: Immunohistochemical demonstration of micro vessels in normal mucosa using mouse monoclonal anti-factor VIII related von Willebrand factor antibody (×400)

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Figure 6: Immunohistochemical demonstration of micro vessels in VEOSMF using mouse monoclonal anti-factor VIII related von Willebrand factor antibody (×400)

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Figure 7: Immunohistochemical demonstration of micro vessels in EOSMF using mouse monoclonal anti-factor VIII related von Willebrand factor antibody (×400)

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Figure 8: Immunohistochemical demonstration of micro vessels in MAOSMF using mouse monoclonal anti-factor VIII related von Willebrand factor antibody (×400)

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The mean number of mast cells and microvessels were calculated using the formula

No. of mast cells/microvessels per mm 2



Further, the mean values were subjected to statistical analysis using independent "t" test, analysis of variance (ANOVA), Tukey's Honestly Significant Difference (HSD), and correlation of MCD with MVD was done using Pearson's correlation test.


   Results Top


On evaluating the control sections, the mean MCD and MVD using independent "t" test were 125.60 and 80.40, respectively. All OSMF cases were divided into three groups as very early OSMF (VEOSMF), early OSMF (EOSMF), and moderately advanced OSMF (MADOSMF). The mean MCD and MVD for VEOSMF were 223.11 and 260.88, respectively. Similarly, for EOSMF, they were 238.57 and 272.00, respectively, and for MADOSMF, they were 201.71 and 283.42, respectively [Figure 9].
Figure 9: Comparison of MCD and MVD

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A significant increase was found in the MCD and MVD among VEOSMF, EOSMF, and MADOSMF, when compared to normal cases (P < 0.05), using independent "t" test, ANOVA and Tukey's HSD. But the increase in MCD and MVD between the VEOSMF, EOSMF, MADOSMF groups was not statistically significant (P > 0.05). We observed statistically significant increase of intact mast cells from normal mucosa to different grades of OSMF but degranulated mast cells were significantly increased only in the very early and early stages of OSMF. On the contrary, they were decreased in moderately advanced stage. Correlation analysis revealed a positive correlation in MADOSMF between MCD and MVD and was highly significant (P< 0.01). As MCD increases, there is an exponential increase in MVD [Figure 10].
Figure 10: Correlation analysis

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Similarly, correlation analysis revealed a positive correlation between MCD and MVD but was not statistically significant (P > 0.01) in normal mucosa, VEOSMF and EOSMF.


   Discussion Top


OSMF is a chronic disease of the oral cavity, characterized by inflammation and progressive mucosal fibrosis. [2] Epithelial changes include hyperplasia in the early stage and atrophy in the later stage. The connective tissue changes vary from fibrosis to hyalinization. Normal tissue growth and tissue remodeling depends upon the nutritional status (blood supply). When the demand increases, it is reflected by the presence of formation of new blood vessels - angiogenesis. [7]

Different methods of histopathological classification are available for OSMF. The recent one given by Utsunomiya et al. (2005) [8] is simplified into three grades as early, intermediate and advanced. As it does not define the tissue changes clearly, we adopted the earliest classification of Pindborg et al. (1966) [9] for the present study. Our samples comprised 9 very early, 14 early, and 7 moderately advanced OSMF cases.

Mast cells frequently accumulate at the site of fibrosis, such as the skin involved in scleroderma [10] and fibrotic lung tissue, [11] and are suggestive of the contribution of mast cells to the pathogenesis of various fibrotic conditions. [12]

In the present study, the average number of mast cells/mm 2 was increased from normal mucosa to the different grades of OSMF. Comparison of MCD among the three groups showed highly significant increase in MCD from normal buccal mucosa to very early, early and significant increase in moderately advanced OSMF. But, intergroup comparison of the increase in MCD between very early, early and moderately advanced OSMF was not statistically significant.

This was consistent with the studies carried out by Bhatt and Dholakia (1977) [13] and Ankle (2007) [12] in which they noted abundant mast cells in Grade I and Grade II of OSMF in comparison with normal mucosa. [12]

Hawkins et al. (1985) [10] proposed that an initial, unknown stimulus results in lymphocyte activation (both peripheral blood lymphocytes and tissue mononuclear cell infiltrates) leading to local mast cell proliferation.

Betel quid components may induce the production of various inflammatory mediators such as prostanoids, interleukin (IL)-6, tumor necrosis factor (TNF)-α, IL-8, and granulocyte-macrophage colony stimulating factor (GMCSF) (unpublished observations), which subsequently stimulate infiltration of inflammatory cells like mast cells[14] and their differentiation and activation in the oral mucosa.

It is possible that inflammatory cells releasing cytokines may act as the stimulant for the increase in the number of mast cells in OSMF. Our results showing a proportionate increase in the mast cells from normal to different grades of OSMF confirm this concept.

New vessel formation is a multistep, highly orchestrated process involving not only vessel sprouting but also endothelial cell migration, proliferation, tube formation and survival. [6]

As a measure of angiogenic activity, the number of microvessels in tissue sections is counted in most of the studies, and is expressed as MVD. [15]

In the present study, MVD expressed as microvessels/mm 2 was significantly higher in OSMF as compared to normal buccal mucosa. However, intergroup comparison of the increase in MVD between VEOSMF, EOSMF and MADOSMF groups was not statistically significant. Rajendran et al. (2005) [16] selected early and advanced cases of OSMF based on the clinical criteria in their study and found that mean vascular density was the same in different grades. Rather, they observed an increase in mean vascular dilatation and considered it as a part of adaptive response to compensate tissue ischemia.

Mast cells could have an important effect in promoting angiogenesis by secreting angiogenic factors or enzymes that degrade extracellular matrix. Mast cell products, including histamine, basic fibroblast growth factor (bFGF) (Qu Z et al., 1995), [7] and heparin, [17] might directly affect endothelial cells by stimulating their migration or proliferation or indirectly affect them by degrading the connective tissue matrix, thereby providing space for neovascular sprouts to form. [18] Angiogenic factors including vascular endothelial growth factor (VEGF), bFGF, and platelet derived growth factor-AB stimulate mast cell migration. In the hypoxic areas, they might produce angiogenic products that stimulate the infiltration of even more mast cells. [19]

A chemical mediator, tryptase, released by mast cells, which is a potent pro-angiogenic factor, contributes to extracellular degradation as well as stimulates angiogenesis (Toda et al. 1999, Gruber et al. 1989, Blair 1997). [6],[19]

Functional status of mast cell is represented by mast cell degranulation. Mast cell-derived heparin and TGF-β have both been shown to display chemotactic activity for endothelial cells and to stimulate the growth of fibrotic tissue. [20]

We observed statistically significant increase of intact mast cells from normal mucosa to different grades of OSMF but degranulated mast cells were significantly increased only in the very early and early stages of OSMF. On the contrary, they were decreased in moderately advanced stage in which the functional contribution of fibroblasts reaches the maximum and the demand for nutritional supply is less. However, in this study, we found that MVD was increased in moderately advanced stage. This increase in MVD may be due to the possible role of angiogenic factors such as nitric oxide, VEGF, bFGF, angiopoietin 1 and 2, other than mast cells.

Thus, increase in MCD and MVD observed in this study reveals their role in the pathogenesis of OSMF. We consider that further clinico-pathological research is needed for the possible intervention of fibrosis by controlling the angiogenic and mast cell activity in OSMF.


   Conclusion Top


As MCD increases, there is an exponential increase in MVD. Similarly, Pearson correlation analysis revealed a positive correlation between MCD and MVD but was not statistically significant in normal mucosa, VEOSMF and EOSMF. The increase in MCD and MVD reveals their role in the pathogenesis of OSMF. Our findings suggest that mast cells are involved in angiogenesis via secretion of angiogenic factors. However, other factors may also be involved in modulation of angiogenesis. MCD and MVD may be used as indicators for disease progression in OSMF, which has a clinical significance by helping in delineating a risk population.

We consider that as the fibrosis progress, it requires blood supply for collagen fibres growth and mast cells act as one of the factors involved in modulation of angiogenesis.

 
   References Top

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Correspondence Address:
B Sabarinath
Department of Oral and Maxillo-facial Pathology, Meenakshi Ammal Dental College and Hospital, Maduravoyal, Chennai
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.80009

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]

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