|Year : 2015 | Volume
| Issue : 3 | Page : 635-640
Comparative analysis of the immunohistochemical expression of vascular endothelial growth factor and matrix metalloproteinase-9 in keratocystic odontogenic tumor, dentigerous cyst and radicular cyst
Komal Khot, Siddharth B Deshmukh, Sheeba Alex
Department of Oral Pathology, YMT's Dental College and P.G Institute, Kharghar, Navi Mumbai, Maharashtra, India
|Date of Web Publication||9-Oct-2015|
Siddharth B Deshmukh
Rebellow Enclave, 402/B, Seepz, Subhash Nagar, Andheri (E), Mumbai - 400 093, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of cysts. Both these factors seem to be interrelated to each other. The importance of the MMPs in the induction of the angiogenic process has recently been described. MMPs, which are produced by microvascular endothelial cells, break down the extracellular matrix. This is one of the earliest and sustained events in the process of new capillary formation. Thus, we studied the expression of VEGF and MMP-9 in Keratocystic odontogenic tumors (KCOTs), dentigerous cysts (DCs) and radicular cysts (RCs).
Materials and Methods: Ten cases each of KCOTs, DCs and RCs and were included in the study and immunohistochemistry was performed using anti-VEGF and anti-MMP-9 antibody using standard protocol.
Result: When the data of positive cells in the epithelium of KCOTs was compared with DCs and RCs, it showed highly significant results (P < 0.05). Furthermore, the expression of VEGF and MMP-9 in the stroma of KCOTs showed a significant result when compared to DCs and RCs. The expression of VEGF in inflammatory cells was more in RCs when compared to DCs. Also, the expression of MMP-9 was more in RCs and DCs as compared to KCOTs.
Conclusion: Higher expression of VEGF and MMP-9 in KCOTs could be responsible for the aggressive behavior of this cyst that is currently considered a cystic tumor rather than a developmental cyst.
Keywords: Dentigerous cyst, keratocystic odontogenic tumor, matrix metalloproteinase-9, radicular cyst, vascular endothelial growth factor
|How to cite this article:|
Khot K, Deshmukh SB, Alex S. Comparative analysis of the immunohistochemical expression of vascular endothelial growth factor and matrix metalloproteinase-9 in keratocystic odontogenic tumor, dentigerous cyst and radicular cyst. J Can Res Ther 2015;11:635-40
|How to cite this URL:|
Khot K, Deshmukh SB, Alex S. Comparative analysis of the immunohistochemical expression of vascular endothelial growth factor and matrix metalloproteinase-9 in keratocystic odontogenic tumor, dentigerous cyst and radicular cyst. J Can Res Ther [serial online] 2015 [cited 2019 Nov 19];11:635-40. Available from: http://www.cancerjournal.net/text.asp?2015/11/3/635/144591
| > Introduction|| |
The jaws are unique in the skeleton in the variety of epithelial residues contained within them and in the range of cystic lesions to which they give rise.  Cysts are relatively common and may be encountered virtually in any organ or tissue in the body. The head and neck region and the jaws, in particular, collectively comprise one of the more common sites for the occurrence of cysts. Cystic lesions play a key role in jaw destruction.  There is considerable literature on possible mechanisms of the cyst initiation and subsequent enlargement. Theories have ranged from the osmotic/hydrostatic pressure concept to differential growth and inflammatory mediators. More recently, various angiogenic factors and secretory proteins have also been implicated in the growth and expansion of cysts. Expression of these molecules in the epithelium and connective tissue is related to different clinical and biological behavior of various odontogenic cysts.
The Keratocystic odontogenic tumor (KCOT) is noteworthy because of its ability to attain a large size before any clinical signs and symptoms develop, its high recurrence rate and its association with nevoid basal cell carcinoma syndrome.  There is general agreement that the KCOT arises from the cell rests of the dental lamina and from the extensions of basal cells from the overlying oral epithelium. ,
Dentigerous cyst (DC) is defined as a cyst that originates by the separation of the follicle from around the crown of an unerupted tooth.  This is the most common type of developmental odontogenic cyst.
Radicular cyst (RC) are by far the most common cysts of the jaws.  These inflammatory cysts derive their epithelial lining from the proliferation of small odontogenic epithelial residues within the periodontal ligament.
Most authors believe that DC and RC enlarge as a result of increased osmotic pressure within the lumen of the cyst. This mechanism does not appear to hold true for KCOT. Their growth may be related to unknown factors inherent in the epithelium itself or enzymatic activity in the fibrous wall. The inflammatory exudate plays a negligible role in KCOT enlargement.  The aggressive behavior and high recurrence rate of the KCOT suggests a true neoplastic potential and has prompted the World Health Organization (WHO) Working Group to classify it as a benign tumor. ,, The most recent classification of the WHO reallocated keratocyst within the classification of tumors under the term "Keratocystic Odontogenic Tumor". ,,,,
World Health Organization has selected to rename this cystic lesion as KCOTs, but some authors like Marx and Stern prefer to retain the old terminology. 
Recent studies suggest an important role of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in the pathogenesis of cysts. ,,, A dimeric glycoprotein with a selective mitogenic effect on vascular endothelial cells, VEGF is an extremely potent and specific angiogenic factor.  MMPs are important proteases that cause structural and functional changes in extracellular matrix components. Under physiologic conditions, MMPs are poorly expressed in the tissue. However, over-expression of these proteins is observed under pathologic conditions, which is the result of an imbalance between the activity of MMPs and their inhibitors. 
This study was carried out to assess and compare the expression of VEGF and MMP-9 in the lining epithelium and connective tissue of KCOTs, DCs and RCs.
| > Materials and methods|| |
Paraffin embedded tissue specimens of the desired lesions were selected from archives of our institute. Ten cases each of KCOTs, DCs and RCs, were included in the study. Samples were selected based on microscopic examination of slides stained with haematoxylin and eosin and correlated with clinical and radiographic details. Sections (4.5-5 μm) were cut from paraffin blocks and mounted on super frost silane coated slides (Fisher brand size 25 mm × 75 mm × 1 mm). On dewaxed slides, antigen retrieval procedure was carried out in EZ retriever system (V.2.1, Biogenex, USA).The slides were then flooded with peroxidase block for 15 min and treated with the power block for 5 min. Primary antibody (anti-VEGF - Biogenex) was applied for 1 h. Polymer horseradish peroxidase (HRP - Biogenex) was applied for 40 min.
Slides were cleaned with phosphate buffered saline (PBS) and cleaned properly. Working solution of diaminobenzidene (1 μl DAB buffer + 1 drop of chromogen + 5 μH 2 O 2 ) was applied as chromogen in H 2 O 2 for 10 min. Sections were counter stained and mounted with DPX. Similar procedure was carried out for MMP-9 antibody on another section of the same tissue.
For positive control, sections of the placenta for VEGF and sections of bone marrow for MMP-9 were taken and treated in a similar manner as the test sample. Negative control samples were treated as above, except that the primary antibody was replaced by a solution of bovine serum albumin in PBS.
Immunohistochemical expression of VEGF and MMP-9 was studied in both the epithelium as well as the connective tissue of the three cysts. As VEGF and MMP-9 are cytoplasmic markers, brown staining of the cell membrane or cytoplasm was considered positive.
Using light microscopy under ×10 magnification five fields with the largest number of immunostained cells were identified. Further, using ×40 magnification, the counting of the immunopositive cells was done in of these fields.
A grid (Motic, Image Plus, Version 2.0, Motic Asia, Hong Kong) consisting of 100 2 each measuring 1 μm × 1 μm in size was used for counting the total number of immunopositive cells. The fields containing artefactual changes such as chatter, tears, etc., were omitted from the study.
| > Results|| |
Vascular endothelial growth factor and matrix metalloproteinase-9 in the epithelium
Since P value for the analysis of variance (ANOVA) test was <0.05, this indicated a significant difference between the mean values of VEGF (not for MMP-9) in the cysts [Table 1]. To find out the exact difference for multiple comparisons Tukey's tests was performed. This showed that there was no significant difference between the mean values in DCs and RCs, but when compared with KCOTs the results were highly significant [Table 2].
|Table 1: Difference between the mean values of VEGF and MMP-9 in the KCOTs, DCs and RCs|
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Vascular endothelial growth factor and matrix metalloproteinase-9 in the stroma
Statistical analysis by ANOVA test showed a significant difference between the mean values of these markers in the stroma of the cysts studied [Table 1]. This was indicated by a P < 0.05. To find out the exact difference for multiple comparisons Tukey's test was performed [Table 2]. The results showed that there was a significant difference between the mean values only between RC, DC and KCOT.
Vascular endothelial growth factor and matrix metalloproteinase-9 in the inflammatory cells
The expression of VEGF in inflammatory cells was more in RCs as compared to DCs. Also, the expression of MMP-9 was more in RCs and DCs when compared to KCOTs, although the results were not statistically significant as shown in [Table 1].
| > Discussion|| |
Despite intense investigations, the molecular mechanisms involved in the formation of cystic lesions and enlargement of jaw cysts have not been fully understood. It is now well established that the KCOT is an aggressive cystic lesion that has a propensity for recurrence if not adequately removed and may undergo malignant changes.
Studies investigating the expression of MMP-9 and VEGF in periapical lesions are scarce. In view of KCOTs aggressive biologic behavior, studies have tried to identify the molecular basis underlying the distinct behavior of KCOTs compared with other odontogenic cysts. The present study has evaluated the expression of VEGF and MMP-9 in the epithelium, stroma and inflammatory infiltrate of KCOTs, DCs and RCs.
When the data of positive cells for VEGF in the epithelium of cysts was compared, it was found that the expression of KCOTs as compared with RCs and DCs, showed highly significant results [P < 0.05, [Table 1]]. However when the expression of VEGF was compared in DCs and RCs, the difference was not significant. This suggested that the expression of VEGF in KCOT was more [[Figure 1]a,c and d]. VEGF is capable of inducing microvascular permeability leading to extravasation of plasma protein and a predictable sequence of proangiogenic stromal changes. VEGF is a sensitive measure of the angiogenic potential of a lesion, and it is well known that tumors need a rich vascular supply.
|Figure 1: Expression of vascular endothelial growth factor and matrix metalloproteinase-9 in components of keratocystic odontogenic tumor, dentigerous cyst and radicular cyst|
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Accumulation in the cystic fluid of serum proteins from the vasculature has been thought to elevate the hydrostatic pressure and maintain their expansion. , Smith et al., considered VEGF as a potent inducer of vascular permeability. On the other hand, mechanical forces have been shown to up-regulate VEGF and its inducing growth factors (transforming growth factor, platelet-derived growth factor, and fibroblast growth factor) in endothelial cells.  Thus, it would be reasonable to suggest that there is a positive feedback mechanism, where VEGF induces an increase in cystic pressure, and this is induced by the pressure exerted on the lining epithelium from the cystic fluid. In the present study VEGF expression was more intense in KCOT, which is currently considered as a cystic tumor rather than a developmental cyst.
The expression of MMP-9 was noted in the epithelium of 2 KCOT cases and was not seen in RCs and DCs [Table 3] and [Figure 1]b. MMP-9 helps in degradation of basement membrane and invasion of dysplastic cells into the underlying connective tissue. , Some investigators suggest that it is caused by metalloproteinase mediated degradation of collagen in juxta epithelial regions. Although others suggest that it is due to collagenolytic activity of collagenase. It is important to note that if epithelium gets separated from the connective tissue with ease, fragments could easily be left behind during the removal, resulting in a recurrence. , In the present study, only two cases were positive for MMP-9, so it is difficult to predict the actual role of MMP-9 in KCOTs.
|Table 3: Percentage of cases positive for VEGF and MMP-9 in the epithelium, stroma and inflammatory infiltrate of KCOTs, DCs and RCs|
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It was found that 70.0% of cases showed VEGF expression in the stroma of KCOTs, 44.0% of DCs and 40% cases of RCs [Figure 1]c and d and [Figure 2]. The expression of VEGF in KCOTs when compared with DCs and RCs was significantly more. But when compared with DCs and RCs though there is considerable expression the difference was not significant. This is in accordance with previous studies, and it may be inferred that angiogenesis may be important in the progression and enlargement of odontogenic cysts similarly to what occurs in neoplastic conditions. ,
|Figure 2: Expression of vascular endothelial growth factor in connective tissue of keratocystic odontogenic tumor (×40)|
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Data of the present study reveals that 84.0% of cases showed MMP-9 expression in the stroma of KCOTs. This was significantly more as compared to 38.0% in DCs and 54.0% in RCs [P < 0.05, [Table 4]] which is much higher as compared to DCs and RCs [Figure 1]e and f but it did not show a significant difference between DCs and RCs. Silveira et al. observed the expression of MMP-9 in the fibrous capsule of odontogenic lesions and noted the tendency towards higher expression of this protein in the fibrous capsule of KCOTs as compared to DCs and RCs.  Kumamato et al., analyzing the expression of MMP-9 in ameloblastomas, also detected strong reactivity to this metalloproteinase in the stroma suggesting that an increased production of this protein by neoplastic cells is related to the neoplastic transformation of odontogenic tissues and aggressiveness of these tumors.  Taken together, these findings and the results of the present study suggest that the higher expression of MMP-9 in the mesenchymal component of KCOTs contributes to more aggressive behavior of these cysts by promoting the degradation of extracellular matrix.
|Table 4: Mean values for VEGF and MMP-9 in the epithelium, stroma and inflammatory components|
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The inflammatory infiltrate was found to be mild in the selected cases of KCOTs as compared to the other cysts. When the data of positive cells for inflammatory infiltrate of KCOT was compared with RCs and DCs, significant results were not obtained [P > 0.05, [Table 1]]. The expression of VEGF and MMP-9 in the inflammatory infiltrate was more in DCs and RCs as compared to KCOTs. Inflammatory infiltrate of various grades was studied to understand the effects of inflammation on the lining of KCOTs, DCs and RCs.
The epithelium in the three cases of KCOT showing inflammation was proliferative and had lost its characteristic appearance. These findings were correlated with a previous study which has stated that in the presence of inflammation, the transformation of a classical parakeratinized epithelium to a nonkeratinizing squamous epithelium may occur. In the present study too this kind of transformation was noted, thus indicating that there may be an association between inflammation and morphology of the epithelial lining in KCOTs. 
Previous studies have found that VEGF expression was higher in cysts showing more inflammation. Inflammation causes up-regulation of cytokines that induce VEGF expression that causes the expansion of cysts.  Even though the inflammatory infiltrate was less in KCOTs as compared to DCs and RCs, the expression of VEGF in the epithelium was much higher.
In RCs moderate to severe inflammation was seen in most of the cases. VEGF and MMP-9 expression in the inflammatory cells was higher in RCs and DCs as compared to KCOTs. The expression of VEGF in inflammatory cells was more in RCs as compared to DCs. Also, the expression of MMP-9 was more in RCs and DCs as compared to KCOTs.
In contrast, the KCOTs showed higher expression of VEGF and MMP-9 in the epithelium and stroma. Thus taking these observations into consideration it may be inferred from results of the present study that the expansion and growth of KCOTs could be a result of VEGF and MMP-9 expression in the epithelium and stroma. This may not be true for RCs and to a certain extent for DCs where intense inflammation and expression of markers by inflammatory cells could play an important role in the expansion of these cysts.
According to Li et al. (2006), angiogenesis is a process that not only needs several vascular growth factors, but also requires degradation of vascular basement membrane and remodeling of the extracellular matrix to enable endothelial cells to migrate into surrounding tissues. In one of the previous study results suggest that MMP-9 might be an important molecule for angiogenesis in odontogenic cysts, probably promoting degradation of components of vascular basement membrane, and thereby allowing proteolysis-associated migration of endothelial cells. 
The importance of the MMPs in the induction of the angiogenic process has recently been described in various studies. Results from studies in several pathologic processes have demonstrated the existence of an important relationship between VEGF and MMP-9. Hollborn et al., verified that hypoxic expression of MMP-9 may stimulate the production and secretion of VEGF under pathologic conditions.  In addition, Lee et al. (2007) observed that intracerebral hemorrhage induced by VEGF is associated with increased expression of MMP-9.  In the present study also a positive correlation between these two markers was noted in a few cases which are in accordance with the above mentioned study.
| > Conclusion|| |
The results of the present study can support the hypothesis that angiogenesis could be an active mechanism in the invasive growth of the KCOT. The role of VEGF in the pathogenesis of KCOTs and odontogenic cysts should be further evaluated as angiogenesis could be a potential target for the therapeutic management of these lesions. The expression of VEGF and MMP-9 in KCOTs, DCs and RCs should be further evaluated with larger sample size for better understanding of the role of these markers in pathogenesis of cysts.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]