Year : 2007 | Volume
: 18 | Issue : 3 | Page : 94--100
Inducible nitric oxide synthase expression is upregulated in oral submucous fibrosis
R Rajendran, Shirley Varkey
Department of Oral Pathology and Microbiology, Govt. Dental College, Trivandrum - 695 011, India
Department of Oral Pathology and Microbiology, Govt. Dental College, Trivandrum - 695 011
Objective: We tested the hypothesis that inducible nitric oxide synthase (iNOS) modulates angiogenesis in human models and this information could be extrapolated in elucidating the pathophysiology of oral submucous fibrosis (OSF). A hypothesis which looks inadequate, but is deep rooted in literature is the epithelial alteration («DQ»atrophy«DQ») seen in OSF and the events that lead to its causation. This aspect was tried to be addressed and an alternative pathogenetic pathway for the disease is proposed.
Materials and Methods: This immunohistochemical study sought to investigate the expression of iNOS in OSF samples (n= 30) a using monospecific antibody (SC- 2050, Santa Cruz Biotechnology, Inc) to the protein and also to correlate it with different grades of epithelial dysplasia associated with the disease. Twenty (20) healthy adults acted as controls.
Results: iNOS staining was not demonstrated in normal oral epithelium. In oral epithelial dysplasia, staining was seen in all cases (100%) in the basal layers of the epithelium and in 30% of cases it extended into the parabasal compartments as well. iNOS staining was uniformly positive in moderate dysplasia with an increase in intensity and distribution noted as the severity of dysplasia progressed. There were highly significant differences in overall positivity for iNOS in epithelium between cases and controls (Mann-Whitney U = 11.000, Wilcoxon W = 221.00, P = 0.000). Significant comparisons were made of mild Vs moderate dysplasia (Mann-Whitney U = 48.000, P = 0.014)
Conclusions: This study supports our earlier morphological assessment (image analysis) of the nature of vascularity in OSF mucosa. The significant vasodilation noticed in these cases argues against the concept of ischemic atrophy of the epithelium. This observation of vascularity and iNOS expression helped to explain the vasodilation noticed (sinusoids) in this disease; NO being a net vasodilator. The mechanism of activation of iNOS in dysplasia is difficult to explain. The role of contingent paracrine-activating factors on keratinocytes and macrophages is discussed.
|How to cite this article:|
Rajendran R, Varkey S. Inducible nitric oxide synthase expression is upregulated in oral submucous fibrosis.Indian J Dent Res 2007;18:94-100
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Rajendran R, Varkey S. Inducible nitric oxide synthase expression is upregulated in oral submucous fibrosis. Indian J Dent Res [serial online] 2007 [cited 2019 Dec 9 ];18:94-100
Available from: http://www.ijdr.in/text.asp?2007/18/3/94/33783
The pathogenesis of oral submucous fibrosis (OSF) and the circumstances leading to its proven pre-cancerous outcome have always aroused curiosity but remained enigmatic till date. What needs to be elucidated is mainly its molecular pathways pathogenic to the events and also those that predispose to its ultimate malignant transformation. Different hypotheses have been put forward in fully elucidating the pathogenic sequence of this disease.  A hypothesis which looks rather naοve but is deep rooted in the literature is the epithelial alteration seen in OSF and the events that lead to its causation.  The epithelium is described to be "atrophied" and therefore, vulnerable to the effects of oral carcinogens. This "atrophy" is explained to be the aftermath of the stromal changes, which undergoes progressive hyalinization, decrease in vascularity and cellularity with resultant tissue ischemia. Thus, the "atrophy" of the overlying epithelium was described as "ischemic atrophy".
This rather simplistic explanation was disputed recently by us on the ground that there was no morphologic evidence of increased cell death (apoptosis / necrosis) associated with atrophy failed to be noticed in OSF epithelium, when compared to site equivalent healthy oral mucosa.  This finding was further strengthened with additional data based on studies of mucosal vasculature aimed at segmentation, characterization and quantification of subepithelial microvasculature in this disease.  The vascularity of mucosa was assessed by estimating the mean vascular density, the mean vascular area percentage and the mean vascular luminal diameter. The mean vascular percentage area and luminal diameter were found to increase with the progress of the disease progresses (p  It was observed that histamine and bradykinin along with prostaglandins (PGI 2 , -E 2 , -E 1 and -D 2 ) seemed to exert their effects on the vasculature. With the advance of the disease and the stroma becomes more and more hyalinised, these mediators are believed to diminish in concentration and its role is assumed to be taken over by other agents perhaps from the endothelial cells along with fibroblasts and fibrocytes. The possible role (s) of basic fibroblast growth factor β -FGF, platelet-derived growth factor PDGF, transforming growth factor-β TGF-β, interleukin-1 IL-1, tumor necrosis factor-α TNF-α, vascular endothelial growth factor VEGF and nitric oxide NO in this regard is worthy of further investigations.
NO, a gaseous free radical, was reported to be functionally equivalent to endothelium derived relaxing factor (EDRF), which causes net vasodilatation.  This radical is generated by the action of nitric oxide synthase (NOS), which can be categorized into two functional classes; constitutive (cNOS) and inducible(iNOS), based on their sensitivity to calcium.  It has been reported that iNOS is expressed in various cell types, including oral keratinocytes and macrophages.  There are few reports, which correlate the expression of NOS to the severity of epithelial dysplasia are quite few. The staining for and hence, expression of eNOS (endothelial) was reported to be decrease as the severity of dysplasia increased.  This was interpreted to be the result of enhanced NO production as a result of iNOS upregulation. NO is known to reversibly inhibit eNOS enzymes,  as part of a negative feedback loop
This immunohistochemical study tried to investigate the expression of iNOS in OSF samples using monospecific antibody to the protein to determine the degree of expression of this net vasodilator molecule (NO) and also to correlate it with different grades of epithelial dysplasia associated with the disease.
Materials and Methods
Immuno Cruz TM staining system (SC- 2050, Santa Cruz Biotechnology, Inc. California) utilizing a horseradish peroxidase (HRP)- streptavidin complex for staining of formalin-fixed, paraffin-embedded tissue sections was employed for the immunolocalization of the iNOS proteins. Parallel haematoxylin and eosin (H and E) staining of tissue sections (serial) was performed both to confirm the immunohistochemistry results and to grade the severity of dysplasia according to the criteria suggested by Burkhardt.  Multiple sections of 4μm thickness were obtained from each paraffin block and stained by using a monoclonal antibody against inducible nitric oxide synthase (iNOS). The procedure was carried out as follows: the glass slides were cleaned with 95% ethanol, treated with subbing solution and air-dried. 4-6 μm thick sections were cut using microtome and applied to PLL coated slides. Sections were dewaxed in xylene for ten minutes, rehydrated in alcohol solution and placed in 5% hydrogen peroxide in absolute alcohol for ten minutes to block endogenous peroxidase activity. To unmask antigen by heat treatment, the slides were placed in a container and covered with 10mM sodium citrate buffer, pH 6 and heated at 95 o C for 5 minutes. Topped off with fresh buffer and heated at 95 O C for 5 minutes and allowed slides to cool in buffer for 20 minutes. Washed in deionized water three times for 2 minutes each on stir slate. All subsequent steps were carried out at room temperature in a humidified chamber. Tissue sections were not allowed to dry out at any time during the procedure.
The primary antibody was diluted to 0.5 - 5 μgm/ ml (1:50 dilution) in 5% normal serum in PBS using goat serum (cat# SC-2043) for rabbit and mouse primary antibody (SC-2050). The diluted primary antibody was added in sufficient volume to cover this tissue, incubated for 2 hours. Rinsed with PBS, washed in PBS twice for 2 minutes each on stir plate. The sections were incubated for 30 minutes in 1-3 drops of biotinylated secondary antibody; antimouse immunoglobulin (Dako) at a dilution of 1:200. Rinse with PBS, then wash with PBS twice for 2 minutes each on stir plate. Incubate specimen for 30 minutes in 1-3 drops of HRP-streptavidin complex (Dako) for a further 20 minutes before washing again with PBS. The immuno staining was visualized by developing in diaminobenzidine (DAB) before couter stained with Mayer's haematoxylline.
Negative controls were treated in the same manner, but discarding the primary antibody. Normal oral mucosa from identical site collected from healthy volunteers were also ran in parallel with the test cases.
Scoring of staining intensity
A semi quantitative scoring pattern of the stained sections was employed for the objective assessment of the protein expression 
The scoring begins with the progenitor compartment and the percentage score was depicted as:
1 = 75% of cells stained positively.
For overall assessment, the epithelial staining was divided grossly into lower ¹/³, middle ¹/³ and superficial ¹/³. The stromal reaction was scored per se and a subjective score of 1-3 was given to assess the staining intensity.
Comparison between cases and controls was done using χ2 test or Fisher's exact test depending on the cell count values. Wherever the expected number was less than 5, Fisher's exact test was used. The non-parametric test, Mann-Whitney was used for the comparison of the number of cells stained positively. A 'P' value of 0.05 was required for statistical significance.
Thirty cases of OSF, clinically advanced and biopsy proven, constituted the test sample and twenty healthy adults acted as controls. Demographic details of test cases are shown in [Table 1],[Table 2]. From all the individuals, biopsy was performed at the same oral site (left buccal mucosa, close to the occlusal plane) in all the individuals (case and control) and the epithelial dysplasia was graded using established criteria  as mild, moderate and severe. iNOS staining was not demonstrated in normal oral epithelium while in mild dysplasia, it was seen in all cases (100%) in the basal layers of the epithelium. In the parabasal compartment staining was positive in all cases at different intensities and in 6 (30%) cases it was extended to the surface layers as well [Table 3]. iNOS staining was seen in the majority of moderate dysplasia cases [Table 4] with an increase in both the intensity and distribution being noted in the epithelium as the severity of dysplasia increased. Mild, n=20. Moderate, n=10. Severe, n=0.
The enhanced expression of iNOS in OSF mucosa may have a bearing on its pathophysiology. This, rather well-characterized signaling molecule is proved to have cytotoxic and possible genotoxic effects. It is recognized that NO and related compounds have a play complex role in cancer biology, being implicated in both tumor progression and inhibition.  NOS expression and activity have been studied in head and neck squamous cell carcinoma (HNSCC).  These studies demonstrated significantly increased expression and activity of iNOS in HNSCC with no activity in normal oral mucosa. It was theorized that high NO production may lead to increased angiogenesis and facilitate tumor dissemination.  Extrapolating these background information in elucidating the pathogenesis of OSF sound reasonable. We had addressed the issue of epithelial "atrophy" in OSF earlier  and had discussed an alternative hypothesis explaining the epithelial "thinness" noticed with the condition. It was proved using reliable criteria that the epithelium in OSF failed to demonstrate an increased Absolute Cell death Index (ACI) often seen in tissue atrophy. The ACI was computed as the cumulative figure of both apoptopic and necrotic indices (AI and NI) judged here by morphologic criteria at ultrastructural level. This prompted us to consider an alternative hypothesis in favour of a reduced proliferation index of the adult stem cell compartment of the oral epithelium. Therefore we proposed a hypothesis of epithelial hypoproliferation, rather than atrophy, which causes thinning of surface epithelium in clinically advanced OSF. The role of NO, being a powerful cytotoxic and genotoxic agent, probably exerting its effects on epithelial keratinocytes and stromal cell needs redressal against the backdrop of our earlier findings. A failure to notice an increased ACI in OSF epithelium again points to the its effect of NO on the stem cell compartments, with the associated possible cell damage. The progressive cellular hypoplasia with lack of tissue cellularity noticed in the stroma in cases of advanced OSF (hyalinization) may again be a reflection of its (NO) effect on the stromal stem cell compartments. The susceptibility of the stem cells to the deleterious effects of NO cannot be explained fully but we theorized that being a labile cell, constantly under turnover and therefore under steady genetic instability make it more vulnerable to the effects of NO than could be expected from more stable and genetically stubborn differentiated cellular compartments (somatic pool). The demonstration of an augmented iNOS expression in advanced OSF mucosa, therefore lends more credence to our earlier hypothesis in favour of epithelial 'hypoproliferation' over that of "atrophy", since ischemia seemed to have a limited role in the course of disease progression.
NO is a critical regulatory molecule for physiological angiogenesis, that constitutes naturally occurring, compensatory response to ischemia. Mucosal inflammation is associated with induction of iNOS in epithelial cells and to a much lesser extent in inflammatory cells of the lamina propria.  What could be the role of NO produced by the abundantly expressed iNOS in mucosal epithelial cells? Is it harmful or protective? The result of the study do not allow solving the intriguing questions, especially the still unsettled issue of the status of mucosal vasculature in OSF and the consequent "ischemic atrophy". NO has diverse physiological and pathological role as a vasodilator and a pivotal role in angiogenesis, vascular permeability and it reduces leucocyte endothelial (L/E) adhesiveness.  It seems interesting to pursue the role of this vascular mediator in OSF in the special context of its putative role in modulating mucosal vascularity. While good case-control studies on the integrity and viability of mucosal microvasculature in OSF are still lacking, histological evidence goes against the concept of decreased vascularity in this disease.  The concept of epithelial "atrophy" in OSF is based on the assumption of an ischemic epithelium resultant to poorly vascularised stroma. The concept of ischemia and therefore the resultant "atrophy" was disputed recently based on the data generated in our laboratory. The mean vascular density in OSF samples was found to be more or less same when compared to normal, healthy mucosa harvested from healthy volunteers. The mean percentage area showed an increasing trend as the disease progresses (F = 8.63, p 
The present data on iNOS overactivity and expression in OSF samples support our earlier contention of lack of tissue ischemia in these cases and also provide laboratory evidence to the reported vascular dilatation and engorgement. The properties of angiogenesis, vascular dilatation, increased permeability of vessels are all contrary to the concept of tissue hypoxia in OSF and therefore the proposed "ischemic atrophy" of the overlying epithelium. This being an aftermath of this stromal reaction seems rather naοve a hypothesis. This augments our earlier contention of an alternative explanation for the "thinning" of the epithelium often noticed in clinically advanced cases. We attribute it again to the defective replenishment of the desquamated epithelial cell pool probably due to decreased proliferation index (P.I.) of the adult stem cell compartment. In this context, hypoplasia being a more plausible concept explaining epithelial "thinning" than that of "atrophy". The possible cytotoxic and genotoxic effects of NO on adult stem cell pools of epithelium and supporting stroma (fibroblast) lends further impetus to this assumption.
A negative feedback loop seems to exist between NO production and p53 tumor suppressor gene.  The genotoxic and cytotoxic effects of NO produced in excess at local sites due to the upregulation of iNOS is expected to be nullified atleast partly by the activation of the wild type p53 gene. However, for cancer cells that have a mutant p53 gene or lack the gene completely, dysregulation of NO (upregulation?) will subsequently occur, with the potential to lead to a cancerous state. Mutations of the p53 gene are frequent in human oral cancerous tissues,  it seems reasonable to suggest that oral mucosa with a high level of iNOS expressivity, could be implicated in the development of neoplastic transformation.
Our earlier observations on OSF  more so to study the frequency of epithelial cell apoptosis and necrosis in clinically advanced samples seem relevant in this context. We have reported on a lower frequency of apoptosis and necrosis (computed as Absolute Cell death Index) in this disease which could an aftermath of p53 mutation and is reported as rampant in OSF samples. The enhanced expression of iNOS in our cases of OSF samples could be causative to the increased frequency of p53 mutation reported in these cases. When there is a mutation of the p53 gene, it has been postulated that this not only give those dysplastic cells an advantage by their failure to undergo p53 mediated apoptosis, but also allows iNOS expression to go unabated. Wild-type p53 protein has a short half-life and is difficult to detect immunohistochemically in normal oral mucosa or benign mucosal lesions. Immunohistochemical overexpression of p53 (mutated form) has been reported in 54%- 67% of oral carcinomas  and in up to 85% of dysplasia.  . In some instances, stabililization of non-mutant forms of the protein (p53) has been reported to give a positive immunostaining and therefore, is not an absolute marker of mutation. 
P53 gene mutation has widely been reported in OSF samples where the percentage prevalence varies from 60%-75%.  . The degree of p53 staining increased with the morphological transformation of normal - appearing epithelial cells in to dysplastic ones. Observing the dynamics of p53 expression from primary pre-cancer to carcinoma development provide additional information on accumulation of the protein during progression of the lesion. Extrapolating this background information on p53 and its mode of expression in OSF samples was necessitated due to our failure to subject these samples for such an assay. Nevertheless it sounds reasonable to speculate that the over expression of iNOS and the resultant over production of NO in OSF mucosa could have its effect in downregulating p53 action, with its resultant negative impact on the apoptotic pathway operational in the mucosa. Our earlier finding of lack of increased apoptotic activity in OSF epithelium, even during advanced clinical stages of the disease, goes well with this observation.
The positive correlation noticed of the iNOS immonoreactivity and the grades of tissue dysplasia (epithelial) warrant some consideration. To the best of our knowledge, very few reports to date have tried to address the issue of expression of iNOS and oral epithelial dysplasia.  Although there was an increase in eNOS (endothelial) expression in both inflammatory and neoplastic cases relative to normal mucosa, the staining for eNOS reduced as the severity of dysplasia increased. Their explanation for the finding was by the increased expression of iNOS in moderate and severe dysplasias, which results in enhanced NO production and NO is known to be reversibly inhibits constitutive NOS enzymes.  The mechanism of activation of iNOS in dysplasia is difficult to explain. It has been suggested that the interaction of tumor cells and macrophages might cause activation of the enzyme in the former probably caused by direct contact or contingent paracrine-activating factor. 
No studies have been published so far to determine the effects, if any, of areca nut chewing on iNOS expression. All OSF patients in this study were areca nut chewers in some forms (betel quid, 'Gutka' etc). Therefore it would be interesting to know whether areca chewing has any effect on NO/iNOS activity in those OSF patients. Recruiting a balanced and well standardized control population without the habit of areca chewing and those who indulge in the habit but did not develop the disease could draw more valuable conclusions in this regard.
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