Immunoexpression of vascular endothelial growth factor in
periapical granulomas, radicular cysts, and residual
radicular cysts
Cassiano Francisco Weege Nonaka, DDS, MSc,aAlexandre Pinto Maia, DDS, MSc,a
George João Ferreira do Nascimento, DDS, MSc,aRoseana de Almeida Freitas, DDS, PhD,b
Lélia Batista de Souza, DDS, PhD,band Hébel Cavalcanti Galvão, DDS, PhD,bNatal, Brazil ORAL PATHOLOGY DEPARTMENT, FEDERAL UNIVERSITY OF RIO GRANDE DO NORTE
Objective. Our aim was to assess and compare the immunoexpression of vascular endothelial growth factor (VEGF) in
periapical granulomas (PGs), radicular cysts (RCs), and residual radicular cysts (RRCs), relating it to the angiogenic index and the intensity of the inflammatory infiltrate.
Study design. Twenty PGs, 20 RCs, and 10 RRCs were evaluated by immunohistochemistry using anti-VEGF antibody.
Angiogenic index was determined by microvessel count (MVC) using anti–von Willebrand factor antibody.
Results. The PGs and RCs showed higher expression of VEGF than the RRCs. Lesions presenting few inflammatory
infiltrate revealed the lowest immunoexpression of VEGF (P⬍ .05). Irrespective of the intensity of the inflammatory infiltrate, most of the RCs and RRCs showed moderate to strong epithelial expression of VEGF. Lesions showing dense inflammatory infiltrate presented higher MVC indices (P⬍ .05). VEGF expression and MVC did not reveal a significant correlation (P⬎ .05).
Conclusions. VEGF is present in periapical inflammatory lesions but at a lower level in RRCs. The expression of this
proangiogenic factor is closely related to the intensity of the inflammatory infiltrate in these lesions. (Oral Surg Oral
Med Oral Pathol Oral Radiol Endod 2008;106:896-902)
Periapical lesions, which include periapical granulomas (PGs) and radicular cysts (RCs), occur as a result of the immunologic response to continuous antigenic stimu-lation from root canals.1,2Persistence of the inflamma-tion is associated with resorpinflamma-tion of adjacent bone, which is replaced by a granulation tissue to form a PG.2
As a consequence of inflammatory and immunologic responses, the epithelial remnants of Malassez are stim-ulated to proliferate, which may result in the develop-ment of an RC.3By definition, residual radicular cysts (RRCs) represent RCs that are inadvertently left behind after the extraction of the involved tooth.4,5
Vascular endothelial growth factor (VEGF) is a po-tent proangiogenic cytokine secreted by many cell types which presents several pivotal functions in phys-iologic and pathologic angiogenesis.6-8VEGF acts on the vasculature by inducing the proliferation, differen-tiation, and migration of vascular endothelial cells.8-10
In addition, VEGF can induce microvascular perme-ability, leading to extravasation of plasma proteins, fluid accumulation, and edema.7,9,10Therefore, VEGF has been implicated as an important factor in granula-tion tissue development.8,11,12
Recent studies suggest a role for VEGF in enlarge-ment of cysts. It has been speculated that the presence of VEGF in cystic lesions is capable of increasing vascular permeability, leading to accumulation of cys-tic fluid.13-17Leonardi et al.18proposed a similar func-tion for VEGF in periapical lesions. According to those authors, the expression of VEGF in PGs and RCs has bioactivity to increase vascular permeability, being at
aDoctoral student. bProfessor.
Received for publication Apr 26, 2008; returned for revision Jun 28, 2008; accepted for publication Jun 30, 2008.
1079-2104/$ - see front matter © 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2008.06.028
896
least partially involved in the accumulation of inflam-matory cells and cyst fluid.
The importance of vascular networks in the devel-opment and maintenance of tissues have been demon-strated in many physiologic and pathologic processes, such as embryogenesis, wound healing, inflammation, and tumor progression.6-8,10,11Angiogenesis is the pro-cess by which new blood vessels are produced by sprouting from preexisting vasculature,7,8,10 and the measurement of its role may be important for a better understanding of the pathogenesis of neoproliferative lesions.19 Although angiogenesis cannot be measured directly, it can be assessed by quantification of the vessels immunolabeled by specific antibodies against endothelial cell epitopes, such as von Willebrand factor (vWF).20 In this manner, it is possible to indirectly establish the angiogenic activity of a tissue.20,21
To our knowledge, there are no studies analyzing the expression of VEGF in PGs, RCs, and RRCs. There-fore, the aim of the present study was to assess and compare the immunoexpression of VEGF in these le-sions, relating it to the angiogenic index and the inten-sity of the inflammatory infiltrate.
MATERIALS AND METHODS
Fifty tissue specimens, 20 PGs, 20 RCs and 10 RRCs, archived in the files of the Oral Pathology De-partment of the Federal University of Rio Grande do Norte (UFRN) were randomly selected for this study. All PGs and RCs were obtained from teeth without endodontic treatment. Only PGs without odontogenic epithelium were selected. Moreover, all RCs and RRCs presented unequivocal cystic cavity lined by odonto-genic epithelium. The cases were not matched for age, gender, or anatomic location. Serial sections, 3-5m thick, were taken from tissue blocks and processed for morphologic and immunohistochemical studies. The study was approved by the Research Ethics Committee of the UFRN, Natal, Brazil.
Morphologic analysis
For the morphologic analysis, tissue sections were stained with hematoxylin and eosin. The intensity of the inflammatory infiltrate was evaluated according to the method proposed by Tsai et al.4Briefly, each spec-imen was graded at ⫻200 magnification as: grade I,
inflammatory cells less than one-third per field; grade II, inflammatory cells between one-third and two-thirds per field; and grade III, inflammatory cells more than two-thirds per field. Grading of each specimen was recorded on the average inflammatory condition in 3 consecutive microscopic fields, starting from the inner portion of the specimen and proceeding deeper into connective tissue. Epithelial thickness was defined as atrophic (2-10 cell layers) or hyperplastic (⬎10 cell layers), according to Moreira et al.22
Immunohistochemical methods
For the immunohistochemical study, tissue sections were deparaffinized and immersed in methanol with 0.3% hydrogen peroxide to block endogenous peroxi-dase activity. The tissue sections were then washed in phosphate-buffered saline (PBS). The antigen retrieval, antibody dilution, and clone type for VEGF and vWF are shown in Table I. After treatment with normal serum, the tissue sections were incubated in a moist chamber with primary antibodies. The tissue sections were then washed twice in PBS and treated with strep-toavidin-biotin-peroxidase complex (SABC; Dako, Glostrup, Denmark) at room temperature to bind the primary antibodies. Peroxidase activity was visualized by immersing tissue sections in diaminobenzidine (D5637; Sigma Chemical, St. Louis, MO), resulting in a brown reaction product. Finally, tissue sections were counterstained with Mayer hematoxylin and cover-slipped. Positive control samples for VEGF and vWF were sections of normal human kidney. As negative control, samples were treated as above, except that the primary antibody was replaced by a solution of bovine serum albumin in PBS.
Immunostaining assessment and statistical analysis
After the immunohistochemical treatment, the tissue sections were examined by light microscopy. Immuno-histochemical expression of VEGF was evaluated both in the connective tissue of PGs, RCs, and RRCs and in the epithelial lining of RCs and RRCs. In the connec-tive tissue, a quantitaconnec-tive assessment of the immunopo-sitive cells, irrespective of the color intensity, was performed according to the method proposed by Freitas et al.20Tissue sections were examined by light
micros-Table I. Specificity, dilution, antigen retrieval, and incubation of the antibody (Ab) clones
Ab clone Specificity Dilution Antigen retrieval Incubation
C-1* VEGF 1:400 Trypsin pH 7.9, oven 37°C, 60 min Overnight (18 h)
F8/86† vWF 1:50 Citrate pH 6.0, steamer, 30 min 60 min
*Santa Cruz Biotechnology. †Dako Cytomation.
copy using⫻100 magnification to identify 5 fields with the largest number of immunostained cells. Using ⫻400 magnification, the counting of the immunoposi-tive cells was performed in each one of these fields.
The immunoexpression of VEGF in the epithelial lining of RCs and RRCs was semiquantitatively eval-uated, using⫻100 magnification. Performing an adap-tation of the method proposed by Leonardi et al.,18the epithelial immunoexpression of VEGF was classified according to the following parameters: no staining in ⬍10% of cells; weak, staining in 11%-25% of cells; moderate, staining in 26%-75% of cells; strong, stain-ing in⬎76% of cells.
Angiogenic index in PGs, RCs, and RRCs was de-termined based on the number of vessels immunoreac-tive to anti-vWF antibody. Adopting the methodology proposed by Freitas et al.,20 a microvessel count (MVC) was performed. Tissue sections were examined by light microscope at⫻40 magnification, and 5 areas showing the highest vascularization were identified subjectively. In these areas, vessels were counted at ⫻200 magnification.
The results obtained were submitted to statistical analysis. Computations were made using the Statistical Package for the Social Sciences (SPSS 13.0). To ana-lyze the immunohistochemical expression of VEGF in the epithelial lining of RCs and RRCs, Fisher exact test was performed. To compare the number of cells immu-noreactive for VEGF in the connective tissue of PGs, RCs, and RRCs, the nonparametric Kruskal-Wallis test was performed. The analysis of the number of vessels in PGs, RCs, and RRCs was evaluated by the Kruskal-Wallis and Mann-Whitney nonparametric tests. Finally, Spearman correlation test was performed to verify pos-sible correlations between the number of immunoposi-tive cells for VEGF and the number of MVC. For all tests, significance level was set at .05 (P⬍ .05).
RESULTS
Morphologic analysis
Analysis of the inflammatory infiltrate in PGs re-vealed 19 cases (95%) with inflammatory infiltrate grade III and only 1 specimen (5%) with inflammatory infiltrate grade II. In RCs, 14 cases (70%) showed inflammatory infiltrate grade III, 5 cases (25%) matory infiltrate grade II, and only 1 case (5%) inflam-matory infiltrate grade I. In the group of RRCs, 4 cases (40%) showed inflammatory infiltrate grade I, 3 cases (30%) inflammatory infiltrate grade II, and 3 cases (30%) inflammatory infiltrate grade III.
Morphologic analysis of the epithelial thickness in RCs revealed the presence of a hyperplastic epithelium in 14 cases (70%) and an atrophic epithelium in only 6 cases (30%). In RRCs, 6 cases (60%) presented an
atrophic epithelium and 4 cases (40%) showed a hy-perplastic epithelium.
Considering all cystic lesions with hyperplastic epi-thelium (n⫽ 18), 13 cases (72.2%) presented inflam-matory infiltrate grade III, 3 cases (16.7%) inflamma-tory infiltrate grade II, and 2 cases (11.1%) inflammatory infiltrate grade I. Considering cystic le-sions with atrophic epithelium (n ⫽ 12), 5 cases (41.7%) presented inflammatory infiltrate grade II, 4 cases (33.3%) inflammatory infiltrate grade III, and 3 cases (25%) inflammatory infiltrate grade I.
Immunohistochemical analysis
The mean number of cells immunostained with anti-VEGF antibody was 564.90 (range 254-916) and 565.05 (range 214-792) in PGs and RCs, respectively. In RRCs, the mean number of immunopositive cells was 443.90 (range 222-660). Grouping all lesions ac-cording to the intensity of the inflammatory infiltrate, the mean number of immunopositive cells for VEGF was 390.40 (range 222-548) in lesions with inflamma-tory infiltrate grade I (n⫽ 5) (Fig. 1). In lesions with inflammatory infiltrate grade II (n ⫽ 9), the mean number of immunoreactive cells was 428.22 (range 214-792). In lesions with inflammatory infiltrate grade III (n⫽ 36), the mean number of immunopositive cells was 589.78 (range 314-916) (Fig. 2).
The analysis of the immunoreactivity of VEGF in the epithelium of RCs revealed weak expression in 2 cases (10%), moderate expression in 7 cases (35%), and strong expression in 11 cases (55%). Only moderate and strong immunoreactivities for VEGF were ob-served in the epithelium of RRCs. Three cases (30%) presented moderate expression and 7 cases (70%) strong expression.
Fig. 1. Immunoexpression of VEGF in specimen of residual radicular cyst presenting inflammatory infiltrate grade I (SABC method, original magnification⫻400).
Grouping RCs and RRCs according to epithelium thickness, in lesions with hyperplastic epithelium (n⫽ 18) 8 cases (44.4%) presented moderate epithelial ex-pression of VEGF and 10 cases (55.6%) strong immu-noreactivity (Fig. 3). In lesions with atrophic epithe-lium (n⫽ 12), 2 cases (16.7%) showed weak epithelial expression of VEGF, 2 cases (16.7%) moderate expres-sion, and 8 cases (66.6%) strong expression (Fig. 4).
The mean number of blood vessels determined by MVC in PGs and RCs was 210.45 (range 124-279) and 250.85 (range 159-350), respectively (Fig. 5). In RRCs, the mean number of blood vessels was 217.00 (range 114-366). Grouping all lesions according to the inten-sity of the inflammatory infiltrate, the mean number of blood vessels was 192.20 (range 152-252) in lesions with inflammatory infiltrate grade I (n⫽ 5). In lesions
with inflammatory infiltrate grade II (n⫽ 9), the mean number of blood vessels was 202.89 (range 114-350). Lesions with inflammatory infiltrate grade III (n⫽ 36) showed a mean number of blood vessels of 239.14 (range 124-409).
Statistical analysis
Analysis of the number of immunopositive cells for VEGF in the connective tissue of PGs, RCs, and RRCs, performed by nonparametric Kruskal-Wallis test, showed no statistically significant difference (P⬎ .05) (Table II). Nevertheless, comparison of the number of immunopositive cells for VEGF according to the inten-sity of the inflammatory infiltrate revealed a statistically significant difference between groups (P⬍ .05) (Table III).
Fig. 2. Immunoexpression of VEGF in specimen of periapi-cal granuloma presenting inflammatory infiltrate grade III (SABC method, original magnification⫻400).
Fig. 3. Strong epithelial expression of VEGF in specimen of residual radicular cyst with hyperplastic epithelial lining (SABC method, original magnification⫻400).
Fig. 4. Strong epithelial expression of VEGF in specimen of radicular cyst with atrophic epithelial lining (SABC method, original magnification⫻400).
Fig. 5. Vessels immunoreactive to anti-vWF antibody in specimen of residual radicular cyst (SABC method, original magnification⫻100).
Fisher exact test was performed to compare the ep-ithelial expression of VEGF in RCs and RRCs. No differences were observed between groups (P⬎ .05). In addition, comparison of the immunoexpression of VEGF according to the epithelial thickness revealed no statistically significant differences between lesions with atrophic epithelium and lesions with hyperplastic epi-thelium (P⬎ .05).
Regarding the number of blood vessels of PGs, RCs, and RRCs, nonparametric Kruskal-Wallis test revealed no difference between groups (P ⬎ .05). In addition, comparison of the number of blood vessels according to the intensity of the inflammatory infiltrate did not re-veal significant differences (P ⬎ .05). Nevertheless, when lesions showing inflammatory infiltrate grades I and II were grouped and compared with those present-ing inflammatory infiltrate grade III, nonparametric Mann-Whitney test revealed a significant difference (P ⬍ .05) (Table IV).
Spearman correlation test disclosed no significant correlation between the number of immunopositive cells for VEGF and the angiogenic index (P⫽ 0.118).
DISCUSSION
Despite intense investigation,2,3,22-24 the molecular mechanisms involved in the formation of granulation tissue and enlargement of jaw cysts have not been fully clarified. Recent studies suggest an important role for VEGF in the pathogenesis of PGs and RCs.18,19 A dimeric glycoprotein with a selective mitogenic effect on vascular endothelial cells, VEGF is an extremely potent and specific angiogenic factor.6-10 Moreover, VEGF is capable of inducing microvascular permeabil-ity with a potency some 50,000 times that of hista-mine, leading to extravasation of plasma proteins and a predictable sequence of proangiogenic stromal changes.6,18Therefore, VEGF has been implicated as an important factor in granulation tissue develop-ment7,8,11,12and cyst enlargement.13-17
In the present study, we evaluated the expression of VEGF in PGs, RCs, and RRCs. The results revealed
that VEGF is expressed in the connective tissue of all of these lesions and in the epithelial lining of RCs and RRCs, corroborating earlier studies.18,19 According to
Leonardi et al.,18the expression of VEGF in periapical lesions has bioactivity to increase vascular permeabil-ity, being partially involved in the accumulation of inflammatory cells and cyst fluid. Therefore, in accor-dance with the biologic functions described for VEGF, such as inducing microvascular permeability and being a potent mitogenic factor for endothelial cells,6-8 the present findings suggest the participation of this proan-giogenic factor in the development and enlargement of PGs, RCs, and RRCs.
Despite the absence of statistical significance (P⬎ .05), PGs and RCs presented a higher mean number of immunopositive cells for VEGF compared with RRCs. PGs and RCs present a constant source of stimulation by bacterial toxins released from the infected root canal which are not present in RRCs.3In addition, Muglali et al.3verified higher levels of interleukin-1␣, monocyte chemotactic protein 1, and regulated upon activation normal T-cell expressed and secreted (RANTES) in cystic fluids of RCs compared with cystic fluids of RRCs. Interleukin-1␣ is a cytokine that has been involved in up-regulation of the expression of VEGF.7,25,26 Monocyte chemotactic protein 1 and RANTES are important chemokines that regulate mac-rophage infiltration in tissues.3,27 Besides their func-tions as antigen-presenting cells and phagocytes during wound repair, macrophages present a proangiogenic potential,28 suggesting a functional contribution of these cells in wound angiogenesis.27,29 Therefore, the low mean number of immunopositive cells for VEGF in RRCs observed in the present study might be related to a decreased level of cytokines in these lesions, as a consequence of the reduction of the inflammatory stim-uli.
Leonardi et al.18 verified marked differences in the expression of VEGF between PGs and RCs. In their study, many inflammatory cells in PGs were immu-nopositive for VEGF, but there was a progressive de-crease in the number of such immunolabeled cells to almost none in RCs. Therefore, Leonardi et al.18 sug-gested that inflammatory cells and fibroblasts would be responsible for synthesis of VEGF in the early phases of periapical lesion development and epithelial cells in the later ones.
In the present study, we could not verify such pro-gressive decrease in the immunoexpression of VEGF. The differences between our results and those reported by Leonardi et al.18might be due to the small sample of
RCs used in their research. Moreover, Leonardi et al.18
did not report if the intensity of the inflammatory in-filtrate was evaluated in their study. In the present
Table II. Parameters used for the calculation of the
Kruskal-Wallis (KW) test for the evaluation of the number of immunopositive cells for VEGF in PGs, RCs, and RRCs Lesion n Median Q25-Q75 Mean of the ranks KW P value PG 20 586.00 406.25–690.25 27.55 3,315 .191 RC 20 590.50 505.75–688.75 27.20 RRC 10 460.00 256.00–618.50 18.00
PG, Periapical granuloma; RC, radicular cyst; RRC, residual radicular cyst; Q, quartile.
research, lesions with inflammatory infiltrate grade I revealed a lower number of immunopositive cells for VEGF than lesions showing inflammatory infiltrate grade III (P⬍ .05). Similarly, Graziani et al.19verified a positive correlation between the intensity of the in-flammatory infiltrate and the expression of VEGF in periapical lesions.
Most of the RCs and RRCs evaluated in the present study presented a moderate to strong epithelial expression of VEGF, irrespective of the intensity of the inflammatory infiltrate or the thickness of the epithelial lining. The epithelial expression of VEGF in RCs and RRCs might constitute an additional mechanism for the enlargement of these lesions, maintaining the stimulus for angiogenesis and vascular permeability, a role also proposed by Leo-nardi et al.18 Results from studies of the expression of VEGF in brain tumor cysts13,15,17and cyst fluid of thyroid nodules14,16 also corroborate this suggestion. According to Vaquero et al.,15VEGF might enhance accumulation of cyst fluid, together with an increase in oxygen supply boosting the development of the cyst.
The epithelium status of the RCs (hyperplastic or atro-phic) has been suggested as a reliable histologic parameter of biologic activity and/or inactivity of cystic growth.22,30 On quiescent lesions (atrophic epithelium), despite the presence of antigens and enzymes able to induce immu-nologic responses, epithelium proliferation, and bone re-sorption, cyst enlargement does not occur.22 In such phases, immunosuppressor effectors22 or apoptotic events31 may operate to regulate cyst enlargement. De-spite those reports, the present results revealed that most of the RCs and RRCs with atrophic epithelial lining present strong epithelial expression of VEGF. These find-ings might suggest the existence of a potential for expan-sion in cystic leexpan-sions with atrophic epithelium.
The present results showed that lesions with a dense inflammatory infiltrate (grade III) present both a high
number of immunopostive cells for VEGF (P ⬍ .05) and a high MVC (P ⬍ .05). These findings are in accordance with other studies and highlight the impor-tance of inflammatory cells, particularly neutrophils and macrophages,27 on VEGF expression.19,32 In this way, the low level of inflammation, probably due to reduced antigenic stimulation,3could be suggested as a possible explanation for the low expression of VEGF, and consequently low MVC, in RRCs.
Graziani et al.19 observed a positive correlation be-tween the microvessel density and the expression of VEGF. However, in our research, we could not find a statistically significant correlation between MVC and the number of immunopositive cells for VEGF. The differences between our results and those reported by Graziani et al.19 could be related to differences in the methods selected for evaluation of the immunoreactiv-ity. In addition, differing from our study, Graziani et al.19 only evaluated RCs.
Despite the absence of statistical significance, RCs presented a higher number of blood vessels compared with PGs and RRCs. To our knowledge, there are no previous studies in the literature that compared the angiogenic index between PGs, RCs, and RRCs. The low MVC observed in RRCs could be related to the lower number of cells immunopositive for VEGF in these lesions compared with RCs. However, PGs and RCs showed a similar number of cells immunopositive for VEGF. It could be hypothesized that the difference in the number of blood vessels might be related to a possible conversion of granulation tissue to a cyst. In this way, it can be speculated that the limits of neovas-cularization are reached and, as a result, the center of the granuloma necroses. Additional studies are neces-sary to clarify these observations.
In conclusion, our results revealed that VEGF is present in periapical inflammatory lesions, but at a
Table III. Parameters used for the calculation of the Kruskal-Wallis (KW) test for the evaluation of the number of
immunopositive cells for vascular endothelial growth factor according to the grade of the inflammatory infiltrate
Inflammatory infiltrate n Median Q25-Q75 Mean of the ranks KW P value
Grade I 5 373.00 242.00–547.50 11.70 9.736 .008
Grade II 9 298.00 246.00–633.00 17.56
Grade III 36 611.00 542.75–690.25 29.40
Q, Quartile.
Table IV. Parameters used for the calculation of the Mann-Whitney U test for the evaluation of the number of vessels
immunostained by anti–von Willebrand factor antibody according to the grade of the inflammatory infiltrate.
Inflammatory infiltrate n Median Q25-Q75 Mean of the ranks U P value
Grade I/II 14 191.00 155.00–231.50 18.57 155.00 .036
Grade III 36 232.50 198.75–259.00 28.19
lower level in RRCs. The expression of this pro-angio-genic factor is closely related to the intensity of the inflammatory infiltrate in these lesions. Further studies need to be performed to clarify the role of VEGF and other angiogenic factors in the pathogenesis of PGs, RCs, and RRCs.
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