|Year : 2014 | Volume
| Issue : 4 | Page : 243-247
Immunohistochemical evaluation of myofibroblasts using alpha-smooth muscle actin in oral submucous fibrosis
Teena Philip, T Dinesh Kumar, K Rajkumar, K Raghavendhar Karthik, N Priyadharsini, A Ramesh Kumar
Department of Oral Pathology, SRM Dental College, Ramapuram, Chennai, Tamil Nadu, India
|Date of Web Publication||20-Nov-2014|
T Dinesh Kumar
Department of Oral and Maxillofacial Pathology, SRM Dental College, Bharathi Salai, Ramapuram, Chennai - 600 089
Introduction: Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a premalignant condition of the oral cavity characterized by generalized submucosal fibrosis with a multifactorial etiology. Myofibroblasts are a unique group of cells phenotypically intermediate between smooth muscle cells and fibroblast exhibiting contractile properties, expressing α-smooth muscle actin (α-SMA) and are considered primary producers of extracellular matrix after injury. Their accumulation has been established as a marker of progressive fibrosis in various organs. The aim of the present study is to evaluate and compare the myofibroblasts in various histological grades of OSMF. Materials and Method: Fifteen cases of OSMF, which were further categorized histologically into early (5 cases), moderately advanced (5 cases) and advanced (5 cases), were subjected to immunohistochemical evaluation using α-SMA antibody for detection of myofibroblasts. Fifteen benign mucosal proliferation specimens were also stained for comparison. Results: The number of myofibroblasts in OSMF was significantly increased when compared to that of benign mucosal proliferations (P < 0.05). Additionally, a statistically significant increase in the myofibroblasts population between early and advanced stages was observed (P < 0.05). Conclusions: The results of the present study showed that expression of myofibroblasts within the OSMF group showed a progressive increase from the early OSMF through moderate OSMF and the advanced OSMF group indicating that myofibroblasts could serve as effective prognostic marker for disease progression in oral submucous fibrosis.
Keywords: Myofibroblasts, oral submucous fibrosis, α-SMA
|How to cite this article:|
Philip T, Kumar T D, Rajkumar K, Karthik K R, Priyadharsini N, Kumar A R. Immunohistochemical evaluation of myofibroblasts using alpha-smooth muscle actin in oral submucous fibrosis. SRM J Res Dent Sci 2014;5:243-7
|How to cite this URL:|
Philip T, Kumar T D, Rajkumar K, Karthik K R, Priyadharsini N, Kumar A R. Immunohistochemical evaluation of myofibroblasts using alpha-smooth muscle actin in oral submucous fibrosis. SRM J Res Dent Sci [serial online] 2014 [cited 2018 Feb 19];5:243-7. Available from: http://www.srmjrds.in/text.asp?2014/5/4/243/145126
| Introduction|| |
Oral submucous fibrosis (OSMF) is a chronic debilitating disease and a premalignant condition of the oral cavity first described by Pindborg and Sirsat in 1966, which is characterized by inflammation and progressive generalized submucosal fibrosis, leading to limitation of mouth opening.  The pathogenesis of this disease is not well-established and is considered to be multifactorial; however, epidemiological evidence strongly suggests an association between areca nut and OSMF. Other etiological factors suggested are chillies, lime, tobacco, nutritional deficiencies such as iron and zinc, immunological disorders, and collagen disorders. 
Worldwide estimates in 1996 indicated that 2.5 million people were affected by the disease. In 2002, the statistics for OSMF from the Indian continent alone was about 5 million people (0.5% of the population of India).  Recent epidemiological data indicate that the number of cases of OSMF have risen rapidly in India from an estimated 250,000 cases in 1980 to 14 million cases in 2010. This has led to the fact that OSMF to be now globally accepted as an Indian disease. The reasons for the rapid increase of the disease are reported to be due to an upsurge in the popularity of commercially prepared areca nut preparations (pan masala) in India and an increased uptake of this habit by young people due to easy access, effective price changes, and marketing strategies.
The most important risk factor in oral cancer development in western countries is the consumption of tobacco and alcohol. However, in Asian countries, the use of smokeless tobacco products such as gutkha, masala, and betel quid is responsible for a considerable percentage of oral cancer.  The development of oral cancer is a tobacco related multistep process involving field cancerization and intraepithelial, clonal spread. Oral squamous cell carcinoma (OSCC) can be preceded by the appearance of lesions which have the potential either to develop into cancer, or portend the development of cancer in the oral cavity. There are several histologically distinct lesions of the oral mucosa which are characterized as having malignant potential and hence are designated as premalignancies.  The most common premalignancies which precede OSCC include leukoplakia, erythroplakia, and OSMF. Given the current scenario of prevalence of OSMF in India, the risk profile of the general population for developing OSCC is alarmingly high with a reported malignant transformation rate of OSMF in the range of 7-13%. 
Persistent chemical irritation from the betel quid constituents and the mechanical irritation of the oral mucosa from the coarse fibers of areca nut results in microtrauma that facilitates the diffusion of areca alkaloids and flavonoids into the subepithelial connective tissue. Any external factor that causes any form of injury to tissue can elicit an inflammatory response. This stress, that is inflammation and wounding in turn leads to fibroblast production and fibrosis. 
Fibroblasts are widely recognized as a critical cell type involved in wound healing and tissue repair. In this regard, well-appreciated is the notion that the transformation of fibroblast to myofibroblasts is a key, perhaps essential, event for the cells to perform these functions. In addition to their normal role in tissue homeostasis and repair, altered number, and function of myofibroblasts have been implicated in diseases with increased extracellular matrix (ECM) deposition and resultant fibrosis. Furthermore, myofibroblasts appear to be the major effector cells in many fibrotic disorders such as scleroderma, hepatic and pancreatic fibrosis and pulmonary fibrosis. 
Myofibroblasts are a unique group of cells phenotypically intermediate between smooth muscle cells and fibroblast or more commonly referred to as phenotypically altered fibroblasts, take part in physiological tissue repair (wound healing) and disappear after healing by apoptosis. But when there is continued insult, these myofibroblasts persist in the tissue and result in dysfunctional repair mechanisms causing excessive secretion of ECM with resultant fibrosis and scarring. This has been established as a marker of progressive fibrosis in organs such as liver, lungs, kidney, and skin. These myofibroblasts can be identified by certain characteristic features of the cytoskeleton, particularly by the expression of α-smooth muscle actin (SMA), and are believed to be primary producers of ECM after injury.
A common finding in many types of solid tumors is that stromal fibroblasts become "activated" and express a number of contractile proteins, particularly α-SMA. Moreover, an altered microenvironment or stroma such as persistent myofibroblasts has been shown to result in tumorigenesis and tumor progression. Historically, most studies of neoplastic transformation and progression have focused on the tumor cell. However, the focus on solely epithelial changes has begun to change, and a recent paradigm shift leads to increasing recognition that the microenvironment makes significant contributions to tumor progression. Although stroma was initially thought to support tumor development passively, there is increasing evidence to suggest that it actively contributes to malignant progression. 
Currently, only limited studies exist to elucidate the presence and role of myofibroblasts in OSMF and OSMF being a chronic scarring disease and a known premalignant condition, this study is aimed to evaluate the presence of myofibroblasts in various histological grades of OSMF using α-SMA antibody and to know if myofibroblasts could be used as a marker to evaluate disease progression.
| Materials and methods|| |
This laboratory-based immunohistochemical study involved the use of buffered formalin fixed, paraffin embedded tissues of previously diagnosed cases of OSMF and oral mucosal proliferations, with each category consisting 15 samples. The samples were reviewed, and findings on hematoxylin and eosin staining were recorded. The OSMF cases were further subdivided histopathologically into early, moderately advanced and advanced according to Pindborg and Sirsat  with each category consisting of five cases. Immunohistochemical procedure was carried out using NovoLink™ Novocastra™ polymer detection system and ready-to-use mouse monoclonal α-SMA (Leica Biosystems Newcastle Ltd, UK. Lot No. 6020842). Stromal spindle cells positive for α-SMA were regarded as myofibroblasts. The immunostaining for α-SMA was performed in a two-step process that involved the demonstration of antigens in tissues and cells first, by the binding of an antibody to the antigen of interest, followed by detection and visualization of the bound antibody by an enzyme chromogenic system. To evaluate the specificity of the immunoreactions, known positive and negative tissue controls were used.
| Immunohistochemical analysis|| |
Immunostaining was assessed by the evaluation of the staining intensity and percentage of α-SMA positive cells, according to the method used by Etemad-Moghadam et al.  The staining percentage was scored as 0 = no positive cells, 1 = 1-33% positive cells, 2 = 34-66% positive cells, 3 = 67-100% positive cells. Staining intensity scoring was recorded as 0 = no staining, 1 = in parts where positivity was observed only at a magnification of ×400, 2 = in cases where the staining was obvious at ×100, but not at ×40, 3 = in fields where immunopositive cells were seen even at ×40. The immunopositive cells in the noninflammatory and nonendothelial stromal cells in the subepithelial connective tissue of OSMF were recorded for the percentage of positive cells and the intensity of staining. Multiplication of the percentage and intensity scores comprised the staining index of each specimen. The staining index was recorded as zero (0), low (1, 2), moderate (3, 4), high (6-9). All the sections were evaluated by two observers to eliminate interobserver bias.
The differences in the presence of myofibroblasts in different groups were analyzed using the Chi-square test, and Fisher's exact test was done to compare staining index of α-SMA between and within the groups. A P < 0.05 was considered statistically significant.
| Results|| |
A positive reaction for α-SMA was observed. Salivary glandular tissue that served as a positive control exhibited intense positivity for α-SMA [Figure 1]. The number of α-SMA-stained myofibroblasts in OSMF was significantly increased when compared to that of the benign mucosal proliferations (P < 0.05) [Table 1] and [Figure 2]. In addition, a statistically significant increase in the myofibroblasts population between early and advanced grades was observed (P < 0.05). However, comparison between the moderate and the advanced OSMF groups, the pattern of expression was not found to be statistically significant [Table 2].
|Figure 1: Intense α -smooth muscle actin expression signifying myofibroblasts in positive control tissue (×40|
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|Figure 2: α -smooth muscle actin expression signifying myofibroblasts in advanced oral submucous fibrosis (×10)|
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|Table 1: Comparison of staining index between OSMF and benign proliferations group|
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| Discussion|| |
Oral submucous fibrosis is a high-risk precancerous condition, characterized by changes in the connective tissue fibers of the lamina propria and deeper parts leading to stiffness of the mucosa and restricted mouth opening. Fibrosis is a progressive disease characterized by accumulation of ECM proteins, which disrupt normal tissue architecture  and OSMF is one such disorder characterized by exuberant deposition of subepithelial collagen in response to chronic areca nut chewing resulting in mucosal rigidity that leads to limitation in mouth opening.
According to studies in various fibrotic disorders, the key cellular mediator of fibrosis is myofibroblasts, which when activated serves as the primary collagen-producing cell.  These cells can be occasionally found in normal tissues, but increased numbers of myofibroblasts appear during wound-healing process. After tissue injury, the myofibroblasts are involved in healing and creating granulation tissue. They produce numerous inflammatory mediators, chemokines, growth factors, and ECM proteins like collagen, fibronectin, etc., which results in ECM reorganization. In addition, they are also important in contraction of ECM because of their component of α-SMA and thus aid in tissue contraction.
Normally, after completion of repair, the myofibroblasts disappear by apoptosis. However, sustained myofibroblast presence stimulates dysfunctional repair mechanisms, leading to excessive contraction, and ECM secretion resulting in fibrosis. It is proposed that in fibrosis, the myofibroblasts acquire an immune-privileged cell phenotype, which helps them to evade apoptosis and allows their uninterrupted accumulation. 
Oral squamous cell carcinoma is the most common oral malignancy and is often preceded by the appearance of lesions which have the potential to develop into invasive carcinoma and are termed as precancerous lesions and conditions. The most common premalignancies include leukoplakia and OSMF. Theoretically, identification and elimination of cancer precursors would lead to the eradication of most human cancers, suggesting that precancers are the most important lesions in modern man. 
The transformation of normal oral mucosa to squamous dysplasia and ultimately OSCC represents a complicated process involving numerous etiologic factors.  Considering that genetic and epigenetic factors are capable of affecting the entire tissue (epithelium and lamina propria), it would be logical to assume that carcinogenesis and tumor progression result from a defective response of both compartments. 
Several studies have shown that the microenvironment or stroma of neoplastic tissues plays an active role in tumor progression. Concurrent with the conversion of nondiseased epithelial tissue to precancerous epithelium to carcinoma, the stroma also changes from normal to primed to activated or tumor associated. In this regard Albini and Sporn  have even suggested a revision of the nomenclature of malignant epithelial tumors. Accordingly, the altered epithelial cells of OSCC would not be solely responsible for carcinogenesis, but different stromal factors participate in its development via communication with the epithelial elements. Fibroblasts are considered as one of the most important mesenchymal cells involved in tumor progression. Transdifferentiation of fibroblasts to myofibroblasts is a crucial and early event in tumorigenesis.
According to the results obtained in this study, the number of myofibroblasts were significantly higher in OSMF compared to the benign mucosal proliferations which were relatively devoid of myofibroblasts. These findings were in agreement with those reported by Angadi et al., who also found that there is lack of myofibroblasts in the normal epithelium when compared with OSMF and suggested that possibly OSMF represents an abnormal healing process in response to chronic mechanical and chemical irritation because of areca nut chewing as demonstrated by the increased incidence of myofibroblasts in OSMF. 
In addition, on comparing the different groups of OSMF, the staining index progressively increased from early OSMF through advanced OSMF with significant increase demonstrable in the advanced stages when compared to early OSMF. However, the difference between early and moderate OSMF did not show significant statistical difference. This was in accordance with the study done by Utsunomiya et al., who studied the expression of myofibroblasts and ECM dynamics in various histological stages of OSMF and concluded that the progression of OSMF stages could be regarded as kind of maturation mode of granulation tissue. 
The localization of myofibroblasts among the study group was varied and was present in the stroma only subjacent to the epithelium in few cases, and few cases showed scattered presence all over the stroma. This feature as explained by fact that the myofibroblasts that are present in the stroma are basically present in two dominant patterns, "spindle" and "network," as described by Vered et al., and Kellermann et al., , In the "network" pattern, myofibroblasts are exceptionally abundant and occupy almost the entire tumor stroma, while the "spindle" pattern is characterized by stromal myofibroblasts that have spindle-shaped morphology and are located at the periphery.
An interesting enigma regarding the premalignant potential of OSMF, a predominantly connective tissue disease resulting in an epithelial malignancy has been the subject of long-standing controversy. There has been increasing evidence that indicates that cancer development and progression are facilitated by interactions between epithelium and stroma. The stroma is characterized by marked alteration of fibroblast phenotype into myofibroblasts that express α-SMA which have been implicated in carcinogenesis, tumor progression, and invasion. Moreover, it is found that myofibroblasts derived from fibrotic tissues are more capable of promoting tumorigenesis through their interaction with carcinoma cells compared to normal fibroblasts derived from normal tissues. Several animal models have provided evidence that fibrotic and wounded tissues have the potential to develop into a tumor, suggesting the presence of oncogenic factors in both the afflicted tissues. 
Consequently, it appears that altered tissue microenvironment, elicited by wounding and fibrosis, significantly increases the risk of tumor incidence and progression. Correlating this to OSMF, the possibility of epithelial-mesenchymal interactions resulting in altered keratinocyte phenotype predisposing to the development of malignancy in OSMF has been reported. This possible role of the myofibroblast in malignant transformation of OSMF requires illumination with further research.
A major issue in this study is that there was no way of knowing whether the examined OSMF cases would have transformed into OSCCs or remained as such. Another problem was that we were unable to obtain samples of OSMF cases with co-existing OSCC. This would have been helpful to draw probable conclusions as to where and when myofibroblasts are first observed.
| Conclusion|| |
Regarding the role of myofibroblasts as a prognostic marker, the present results showed that the expression of myofibroblasts showed a progressive and steady increase from early OSMF through moderate and advanced OSMF cases indicating that myofibroblasts could be used as an effective prognostic marker for disease progression in OSMF. Considering the observations of this study, and the role of myofibroblasts resulting in fibrosis and its role in tumorigenesis and tumor progression, therapeutic targets against myofibroblasts could be used to decrease the disease severity and prevent progression to OSCC.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]