metalloproteinase 9, and endoglin (CD105) in odontogenic
keratocysts, dentigerous cysts, and radicular cysts
Pedro Paulo de Andrade Santos, DDS, MSc, Ana Rafaela Luz de Aquino, DDS, MSc, Alessandra Oliveira Barreto, DDS, MSc, Roseana de Almeida Freitas, DDS, MSc, PhD, Hébel Cavalcanti Galvão, DDS, MSc, PhD, and Lélia Batista de Souza, DDS, MSc, PhD, Natal, Brazil
ORAL PATHOLOGY POST GRADUATE PROGRAM, DEPARTMENT OF DENTISTRY, FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE (UFRN)
Objective. The aim of this study was to compare the immunohistochemical expression of nuclear factorB (NF-B), matrix metalloproteinase 9 (MMP-9), and CD105 in odontogenic keratocysts (OKCs), dentigerous cysts (DCs), and radicular cysts (RCs).
Study design. Twenty cases of OKCs, 20 DCs, and 20 RCs were analyzed. A labeling index (LI), which expresses the percentage of NF-B–stained nuclei, was calculated for the analysis of NF-B expression. Expression of MMP-9 in the epithelium and in the capsule of each lesion was scored as 0 (⬍10% stained cells), 1 (10%-50% stained cells), or 2 (⬎50% stained cells). In addition, MMP-9 immunostaining was analyzed in endothelial cells of vessels with a conspicuous lumen. The angiogenic index was determined based on the number of anti-CD105 antibody–stained microvessels.
Results. In the epithelial component, the NF-B LI was higher in OKCs than in DCs and RCs (P⬍.001). Analysis of
MMP-9 expression in the epithelial component showed a predominance of score 2 in OKCs (90%), DCs (70%), and RCs (65%;P⫽.159). Evaluation of the NF-B LI according to the expression of MMP-9 in the epithelial lining
revealed no significant difference between lesions (P⫽.282). In the fibrous capsule, the highest percentage of
MMP-9 –stained cells (score 2) was observed in OKCs (P⫽.100). Analysis of the expression of MMP-9 in the vessels of
odontogenic cysts showed a predominance of score 2 in OKCs (80%) and RCs (50%) and of score 1 in DCs (75%;
P⫽.002). Mean microvessel count was high in RCs (16.9), followed by DCs (12.1) and OKCs (10.0;P⫽.163). No
significant difference in microvessel count according to the expression of MMP-9 was observed between groups (P⫽
.689).
Conclusions. The results suggest that the more aggressive biologic behavior of OKCs is related to the higher expression of MMP-9 and NF-B in those lesions. The differences in the biologic behavior of the lesions studied do not seem to be associated with the angiogenic index.(Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112: 476-483)
Odontogenic cysts comprise a group of osseodestruc-tive lesions that frequently affect the jaws. These cysts can arise from epithelial components of the tooth germ, the reduced epithelium of the enamel organ, epithelial rests of Malassez, remnants of dental lamina, or the basal layer of the oral epithelium.1,2
The odontogenic keratocyst (OKC), recently re-classified by the World Health Organization as a keratocystic odontogenic tumor,3is a developmental
cyst characterized by an aggressive nature and high rate of recurrence, especially compared with other odontogenic cysts.4The dentigerous cyst (DC) is the most common developmental odontogenic cyst and usually associated with the crowns of impacted per-manent teeth. This cyst shows an indolent behavior, and recurrence is rare after removal.2,4,5 The radic-ular cyst (RC) is an odontogenic cyst of inflamma-tory origin that arises from the inflammainflamma-tory stimu-lation of the epithelial rests of Malassez.6,7 RCs, DCs, and OKCs show distinct growth patterns and biologic behaviors. Previous studies have investi-gated transcription factors and factors related to tis-sue remodeling and angiogenesis which might be associated with the development of these lesions in an attempt to determine which factors that may be responsible for the specific biologic behavior of each type of lesion.4
Supported by the Brazilian Research Council for Science and Tech-nology Development (CNPq) and Brazilian Coordination Develop-ment of Senior Staff (CAPES).
Received for publication Mar 16, 2011; accepted for publication Apr 14, 2011.
1079-2104/$ - see front matter © 2011 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2011.04.022
Nuclear factor B (NF-B) belongs to a family of transcription factors8 that regulates the expression of various genes, including matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor genes whose products are involved in tumorigenesis.9 How-ever, little is known about the role of NF-B in odon-togenic lesions.10
MMPs comprise a family of calcium- and zinc-de-pendent enzymes that are able to degrade extracellular matrix components under both physiologic and patho-logic conditions.11-13 Studies suggest that enzymatic degradation of the bone matrix and basement mem-brane by MMP-9 and other metalloproteinases is in-volved in the expansion of odontogenic cysts.14-16
Endoglin, also known as CD105, is a transmembrane protein that is highly expressed on human vascular endothelial cells.17 Unlike panendothelial antibodies, such as CD31, CD34, and factor VIII, endoglin is able to distinguish endothelial cells proliferating from pre-existing blood vessels. Several studies emphasize the involvement of CD105 in blood vessel formation and that its utility as a powerful marker for the quantifica-tion of microvessels in many types of tumors.18
In view of the distinct clinical behaviors of OKCs, RCs, and DCs the objective of the present investiga-tion was to compare the immunohistochemical ex-pression of NF-B, MMP-9, and endoglin between these lesions.
MATERIALS AND METHODS
Twenty cases each of OKC, DC, and RC were stud-ied. Formalin (10%)–fixed and paraffin-embedded specimens were selected among cases diagnosed and archived at the Pathologic Anatomy Service of the Discipline of Oral Pathology, Department of Dentistry, Federal University of Rio Grande do Norte (UFRN), Natal, Brazil. The study was approved by the Ethics Committee of UFRN (protocol no. 094/09).
Immunohistochemical method
Paraffin-embedded material was cut into 3-m-thick sections and the specimens mounted on glass slides prepared with organosilane coupling agent (3-amino-propyltriethoxysilane; Sigma Chemical Co., St. Louis, MO, USA). The tissue sections were deparaffinized, submitted to antigen retrieval with citrate (pH 6.0) in a Pascal pressure cooker (Dako, Carpinteria, CA, USA), and then immersed in 10 volumes of hydrogen peroxide to block endogenous peroxidase activity. Next, the sec-tions were washed in phosphate-buffered saline solu-tion (PBS). After treatment with normal serum, the sections were incubated with the following primary antibodies in a moist chamber: anti–NF-B (clone sc-109; Santa Cruz Biotechnology, Santa Cruz, CA, USA;
biotin complex (Dako) at room temperature. Peroxidase activity was developed by immersion of the sections in diaminobenzidine (D5637; Sigma Chemical Co.). Fi-nally, the sections were counterstained with Mayer hematoxylin, covered with a glass coverslip, and mounted in Permount (SP15-500; Fisher Scientific, Fair Lawn, NJ, USA).
Prostate carcinoma and kidney and liver fragments were used as positive control samples for NF-B, MMP-9, and CD105, respectively. The negative control involved substitution of the primary antibody with 1% bovine serum albumin in PBS.
Analysis of the immunohistochemical profile The slides were examined under a light microscope (Olympus Cx31; Tokyo, Japan) at⫻400 magnification. NF-B staining was analyzed by an adaptation of the method proposed by Nakayama et al.19 For this pur-pose, 1,000 epithelial cells were counted in 5 different representative fields of the cystic lesions. A labeling index (LI), which expresses the percentage of NF- B-immunostained nuclei, was calculated based on the number of immunopositive nuclei.
MMP-9 staining was evaluated according to Gong et al.,10 with some modifications. Ten histologic fields each were selected in the epithelial component and in the connective tissue capsule. Immunoexpres-sion of MMP-9 was scored in each case as 0 (⬍10% immunostained cells), 1 (10%-50% immunostained cells), or 2 (⬎50% immunostained cells). In addi-tion, expression of MMP-9 was analyzed in endothelial cells of vessels with a conspicuous lumen at⫻400 mag-nification, selecting 5 fields that showed the highest im-munoreactivity.
The angiogenic index of OKCs, DCs, and RCs was determined by the quantification of anti–CD105-im-munoreactive vessels by an adaptation of the method of Maeda et al.20 Ten areas of highest anti-CD105 immunoreactivity were identified at 40⫻ magnifica-tion and the microvessel count (MVC) was obtained
by counting immunostained vessels in these areas at 200⫻ magnification.
Statistical analysis
The results were analyzed with the use of the Statis-tical Package for the Social Sciences (version 17.0; SPSS, Chicago, IL, USA). NF-B LIs in the epithelium were compared between the different cysts by the non-parametric Kruskal-Wallis test. For the analysis of MMP-9 expression, scores 0 and 1 were combined into one group and score 2 remained as another group. Differences in MMP-9 expression in the cystic epithe-lium and in the connective tissue capsule were evalu-ated by the 2 test. Differences between NF-B LIs according to MMP-9 expression in the cystic epithe-lium were evaluated by the nonparametric Mann-Whit-ney test. The Kruskal-Wallis test was used for the comparison of the number of CD105-immunostained blood vessels. Possible differences in MVC according to the expression of MMP-9 in the vessels of odonto-genic cysts were evaluated by the nonparametric Mann-Whitney test. A level of significance of 5% (P ⬍.05) was established for all tests.
RESULTS
The mean NF-B LI in the epithelial lining was higher in OKCs (21.1%, range 3.7%-44.2%) than in DCs (8.7%, range 0.2%-31.6%) and RCs (1.4%, range 0.2%-5.5%;Fig. 1). Nonparametric tests revealed sig-nificant difference between groups (P⬍.001;Table I). Regarding the epithelial expression of MMP-9, there was a predominance of score 2 (n⫽18; 90%) in OKCs, whereas score 1 was predominantly observed in the other 2 cases (10%). Similarly, score 2 predominated in DCs (n ⫽ 14; 70%), but the distribution of scores 0 (n⫽4; 20%) and score 1 (n⫽2; 10%) was relatively similar. In RCs, most cases were classified as score 2 (n⫽13; 65%), followed by score 1 (n⫽5; 25%) and score 0 (n ⫽ 2; 10%; Fig. 2). Statistical analysis re-Table I. Parameters used for the calculation of the Krukal-Wallis test (KW) for the evaluation of the labeling index (LI) of nuclear factor (NF)B nuclear staining in epithelial tissue and evaluation of the angiogenic index (CD105) of radicular cyst, dentigerous cyst, and odontogenic keratocyst
n Median Q25-Q75 Mean rank KW P value
NF-B (LI) ⬍.001
Odontogenic keratocyst 20 21.7 12.85-28.57 44.55 22.382
Dentigerous cyst 20 4.8 2.25-9.05 28.23
Radicular cyst 20 1 0.67-5.37 18.73
CD-105 (angiogenic index) .163
Odontogenic keratocyst 20 6.1 1.7-39.0 25.55 3.632
Dentigerous cyst 20 7.4 0.0-48.0 29.93
vealed no significant difference between groups (P⫽ 0.159;Table II).
Evaluation of the NF-B LI according to the level of MMP-9 expression in epithelial cells lining the cysts showed a mean index of 8.9% (range 0.2%-44.2%) for
cysts scored as 1. On the other hand, cysts scored as 2 presented a mean LI of 10.9% (range 0.2%-39.2%). Nonparametric Mann-Whitney test revealed no signif-icant difference between groups (P⫽.282). Similarly, separate analysis of OKCs, DCs, and RCs showed no significant differences in NF-B LI according to the level of MMP-9 expression in the epithelial lining (P⫽ .313;P⫽ .650; andP⫽ .721; respectively).
Analysis of the expression of MMP-9 in the capsule of the lesions studied showed a relatively similar dis-tribution of score 1 (n⫽8; 40%) and score 2 (n⫽9; 45%) in OKCs. Score 0 was observed in 15% of cases (n⫽3). In contrast, in DCs there was a similar distri-bution of score 0 (n ⫽ 8; 40%) and score 1 (n ⫽ 9; 45%), whereas score 2 was observed in 15% of cases (n⫽3). In RCs, there was a predominance of score 2 (n⫽11; 55%), followed by score 3 (n⫽5; 25%) and score 0 (n ⫽ 4; 20%; Fig. 2). Statistical analysis showed no significant difference between groups (P⫽ .100;Table II).
Analysis of the expression of MMP-9 in vessels of the odontogenic cysts showed a predominance of score 2 (n⫽16; 80%), followed by score 1 (n⫽4; 20%), in OKCs. DCs presented a higher percentage of cases classified as score 1 (n⫽15; 75%), followed by score 2 (n⫽4; 20%) and score 0 (n⫽1; 5%). In RCs, there was a similar distribution of cases scored as 1 (n⫽9; 45%) and 2 (n ⫽ 10; 50%). Only 1 case (5%) was classified as score 0. The2test revealed a significant difference between groups (P⫽ .002;Table II).
Regarding angiogenesis, the highest mean MVC was obtained for RCs (16.9, range 2.0-44.0), followed by DCs (12.1, range 0.0-48.0) and OKCs (10.0, range 1.7-39.0; Fig. 3). However, the Kruskal-Wallis test revealed no significant difference in MVC between groups (P⫽.163; Table I).
Analysis of MVC according to the level of expres-sion of MMP-9 in endothelial cells revealed a mean count of 13.9 vessels (median 15.4, range 0.0-48.0) for cysts with score 0/1, whereas cysts scored as 2 had a mean count of 12.2 vessels (median of 8.16, range 1.7-39.0). There was no significant difference between groups (P⫽.689; nonparametric Mann-Whitney test). Similarly, separate analysis of OKCs, DCs, and RCs showed no significant differences in MVC according to the level of MMP-9 expression in endothelial cells (P⫽.965; P⫽.965; P⫽ .676; respectively).
DISCUSSION
DCs are the most common developmental cysts. In the past, it was believed that the epithelial lining of these cysts has a higher proliferative potential than the epithelial lining of inflammatory cysts, such as RC.7 OKCs are one of the most frequent odontogenic lesions Fig. 2. Immunohistochemical reactivity for MMP-9 in
and require special attention because of their aggressive biologic behavior and tendency toward recurrence.21,22 Studies have demonstrated a high proliferative activity of these lesions and the expression of proteins associ-ated with the inhibition of apoptosis,23,24in addition to the observation of allelic losses, especially in theP16, P53,PTCH,MCC,TSLC1,LTAS2, andFHITgenes.25 In view of OKC’s aggressive biologic behavior, studies have tried to identify the molecular basis un-derlying the distinct behavior of OKCs compared with other odontogenic cysts. The present study evaluated the immunohistochemical expression of NF-B, MMP-9, and CD105 in DCs, RCs, and OKCs and compared the expression of these markers between lesions.
In quiescent cells, the nuclear transcription factor NF-B is sequestered in the cytoplasm by IB pro-teins.26 Its activation by cytokines requires the phos-phorylation and degradation of the cytoplasmic inhibi-tor IB␣.27 Once activated, NF-B is translocated to the nucleus, where it induces the expression of a set of target genes, including those involved in tumor me-tastasis, cell-cycle regulatory genes, and growth factor genes, in addition to regulating MMP genes, such as MMP-9.8Therefore, activated NF-B plays an impor-tant role in cell differentiation, proliferation, and apo-ptosis in response to a variety of physiologic and patho-logic stimuli. Studies have shown that NF-B activity can be detected in cells of different tumors, but little is known about its role in odontogenic lesions.10
In a comparative study of ameloblastomas and tooth germs, Kumamoto and Ooya28observed scarce nuclear NF-B staining in both tumors and normal odontogenic epithelium. Similarly, Gong et al.,10 analyzing NF-B staining in calcifying odontogenic cysts and classic
ameloblastomas, found scarce nuclear staining (⬍1%) in the 2 groups. Despite these findings, an association between higher NF-B expression and greater aggres-siveness has been demonstrated for different malignant tumors.27,29,30
In the present study, a significant difference in the NF-B LI was observed among OKCs, DCs, and RCs, with a higher mean index (21.1%) in OKCs. Therefore, the higher nuclear expression of this factor in OKCs might be related to the greater aggressiveness of those tumors compared with DCs and RCs.
NF-B regulates the expression of a large number of proinflammatory cytokines, such as tumor necrosis fac-tor ␣, interleukin-1, and interleukin-6, as well as MMPs.31 MMPs are important proteases that cause structural and functional changes in extracellular matrix components. Under physiologic conditions, MMPs are poorly expressed in tissues. However, overex-pression of these proteins is observed under patho-logic conditions, which is the result of an imbalance between the activity of MMPs and their inhibitors (TIMPs).12
Among MMPs, gelatinase B (MMP-9) plays an im-portant role in angiogenesis as well as in tumor inva-sion and metastasis, mainly because of its capacity to cleave collagen IV present in the basement mem-brane.32Despite the small number of studies involving odontogenic lesions, the expression of MMP-9 was demonstrated in ameloblastoma, calcifying cystic odontogenic tumor, dentinogenic ghost cell tumor, odontogenic ghost cell carcinoma, OKC, DC, and RC.10,12,16 Kubota et al.,33 investigating whether the latent (92 kDa) or active (83 kDa) form of MMP-9 is present in fluids of OKCs, DCs, and RCs, observed the presence of the active form in 75% of OKCs and Table II. Distribution of cases of radicular cysts, dentigerous cysts, and odontogenic keratocysts according to the percentage of cells stained by MMP-9 in epithelium, fibrous capsule, and blood vessels, n (%)
Odontogenic keratocyst Dentigerous cyst Radicular cyst 2 P value
Epithelial tissue 6.933 .159
⬍10% (score 0) 0 (0) 4 (20) 2 (10)
10%-50% (score 1) 2 (10) 2 (10) 5 (25)
⬎50% (score 2) 18 (90) 14 (70) 13 (65)
Total 20 (100) 20 (100) 20 (100)
Fibrous capsule 6.594 .100
⬍10% (score 0) 3 (15) 8 (40) 4 (20)
10%-50% (Score 1) 8 (40) 9 (45) 11 (55)
⬎50% (Score 2) 9 (45) 3 (15) 5 (25)
Total 20 (100) 20 (100) 20 (100)
Blood vessels 0.116 .002
⬍10% (score 0) 0 (0) 1 (5) 1 (5)
10%-50% (score 1) 4 (20) 15 (75) 9 (45)
⬎50% (score 2) 16 (80) 4 (20) 10 (50)
in only 30% of DCs and RCs. In agreement with these findings, in the present study MMP-9 expression tended to be higher in epithelial cells of OKCs com-pared with DCs and RCs (P⬎.05). Immunohistochem-istry is unable to differentiate between the latent and
active forms of proteins. However, in a study by Ikebe et al.,34MMP activity analyzed by zymography showed a significant correlation with immunohistochemical staining.
In an attempt to elucidate the molecular basis under-lying the more aggressive biologic behavior of OKCs compared with DCs and RCs and in view of the higher nuclear NF-B staining in OKCs observed in the pres-ent study, we analyzed NF-B LIs according to the expression of MMP-9 in the epithelial cells lining these lesions. Despite the lack of a significant difference, MMP-9 expression by epithelial cells tended to be higher in OKCs compared with DCs and RCs. This finding suggests a possible role of NF-B as a tran-scription factor of this metalloproteinase, because, like MMP-9 expression, the activated factor was detected in the epithelium of most OKCs. This fact has also been reported in the literature by dell’Agli et al.,8who sug-gested that NF-B acts as a transcription factor, and by Garg and Aggarwal,9who reported a role of this factor as a regulator of MMP-9 gene expression.
Regarding the expression of MMP-9 in the fibrous capsule of the odontogenic lesions studied, the ten-dency toward higher expression of this protein in the fibrous capsule of OKCs compared with DCs and RCs observed in the present study agrees with the findings reported by Silveira et al.16Kumamoto et al.,35 analyz-ing the expression of MMP-9 in ameloblastomas, also detected strong reactivity to this metalloproteinase in the stroma of these tumors, 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 sug-gest that the higher expression of MMP-9 in the mes-enchymal component of OKCs contributes to the more aggressive behavior of these tumors compared with inflammatory cysts and DCs by promoting the degra-dation of extracellular matrix. In contrast, expression of MMP-9 in epithelial cells might be responsible for the degradation of the basement membrane.
Different methods are available for the evaluation of angiogenesis in biologic material, including MVC and the determination of microvessel density and volume.36-38Among these methods, MVC is an easy technique that has prognostic relevance in different tumors.38,39
One widely used angiogenic marker is endoglin, or CD105, which was originally identified as a human endothelial marker although subsequent studies dem-onstrated that this cell surface antigen is also expressed by macrophages, erythrocyte precursors, and stromal cells.40Endoglin binds to various transforming growth factorisoforms and exhibits high affinity for human Fig. 3. Immunohistochemical reactivity for CD-105 in
endothelial cells. This protein plays an important role in angiogenesis and is considered to be a powerful marker of neovascularization.41
In the present study, mean MVC was higher in RCs than in DCs and OKCs (P⫽.163). Despite the lack of reports using MVC for the evaluation of angiogenesis in these lesions, in a comparative study of microvessel density between OKCs and RCs, Tete et al.42observed higher vascularization in RCs. The higher mean MVC in RCs seen in the present study might be related to the presence of an exuberant inflammatory infiltrate in these lesions, because, according to Graziani et al.43 and Nonaka et al.,6inflammatory cells can exert angio-genic activity in these cysts.
In the present study, MVC was higher in DCs than in OKCs. These results differ from those reported by Alaeddini et al.4who observed higher microvessel den-sity in OKCs compared with DCs. One possible expla-nation for this difference is the fact that in the latter study microvessel density was evaluated based on im-munoreactivity to the anti-CD34 antibody, whereas in the present investigation CD105 immunostaining was used for microvessel quantification.
In addition, analysis of MVC according to the ex-pression of MMP-9 in endothelial cells revealed no significant difference between OKCs, DCs, and RCs. In fact, the role of MMP-9 in the development of the lesions studied might be associated with the regulation of other factors unrelated to angiogenesis, such as fac-tors involved in cell proliferation and migration, apo-ptosis, and immune and inflammatory responses.44
In conclusion, the present results suggest that the more aggressive biologic behavior of OKCs compared with RCs and DCs is related to the higher expression of MMP-9 and NF-B in these lesions. In addition, dif-ferences in the biologic behavior of the lesions studied do not seem to be associated with the angiogenic index.
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