- Email: [email protected]
Overexpression of Cyclophilin A in Human Periapical Lesions
BASIC RESEARCH – BIOLOGY
Overexpression of Cyclophilin A in Human Periapical Lesions ABSTRACT Introduction: Cyclophilin A (CypA) is a cytosolic protein involved in multiple biological functions, such as inflammation, tissue remodeling, tumorigenesis, and vascular diseases. Human periapical lesions are induced by bacterial infections. However, the expression of CypA in human periapical lesions remains unclear. This study aimed to investigate the presence of CypA in human periapical lesions and the possible association of CypA with angiogenesis, inflammatory cell infiltration, and alveolar bone degradation during inflammatory development. Methods: Fifty-eight human periapical tissues, including periapical granulomas (PGs, n 5 28), radicular cysts (RCs, n 5 24), and healthy control tissues (control group, n 5 6) were collected. Samples were fixed and analyzed. CypA expression was detected and analyzed by immunohistochemistry in different cross sections. Double immunofluorescence was assessed to colocalize CypA with CD34, CypA with matrix metalloproteinase 9 (MMP-9), and CD147 with MMP-9. Results: CypA was significantly overexpressed in the RC and PG groups compared with the control group (P , .05), but the difference between the RC and PG groups was insignificant (P . .05). CypA-positive cells were mainly lymphocytes, endothelial cells, epithelial cells, and plasma cells. The doublelabeling analysis of CypA with CD34 suggested that CypA expression was associated with angiogenesis during periapical lesions. MMP-9 colocalized with both CypA and CD147 indicated that CypA may colocalize with CD147 and may be associated with the degradation of soft and hard tissues around human periapical lesions. Conclusions: CypA may be involved in the development of periapical lesions with an increase in inflammatory cell infiltration, angiogenesis acceleration, and alveolar bone degradation. (J Endod 2019;-:1–8.)
Yanqing Wang, DDS,*† Guojing Liu, DDS,* Jie Zhang, DDS,* Lingxin Zhu, DDS,* Jingjing Yu, DDS,* Shenting Zhu, DDS,* Fengyuan Lv, DDS,* and Bin Peng, DDS, PhD*
SIGNIFICANCE We suggest that CypA may be involved in the development of periapical lesions with an increase in inflammatory cell infiltration, angiogenesis acceleration, and alveolar bone degradation. Therefore, targeting CypA may serve as a new therapy for human periapical lesion treatment.
KEY WORDS Angiogenesis; CD34; CD147; cyclophilin A; matrix metalloproteinase 9; periapical lesions
A periapical lesion is a continuation of a bacterial infection in root canals, which can result in dental pulp necrosis and develop into apical periodontitis with degradation of the periradicular periodontal ligament and destruction of alveolar bone1,2. Periapical granulomas (PGs) and radicular cysts (RCs) are chronic periapical lesions that represent different stages of the same inflammatory process3. Considerable studies have been conducted to improve understanding of the inflammatory mechanisms involved in the granulation tissue formation and jaw cyst enlargement in PG and RC pathogenesis4–6. To date, a series of proinflammatory cytokines, such as matrix metalloproteinases, interferon gamma, and transforming growth factor beta, are synthesized during granulation tissue formation, jaw cyst enlargement, and alveolar bone resorption, respectively5,7. The detailed mechanisms underlying the progression of proinflammatory cytokines on granulation and cyst tissue formation, such as angiogenesis, inflammatory cell infiltration, epithelial proliferation, and alveolar bone degradation, have not been fully elucidated. Cyclophilin A (CypA) is a ubiquitously distributed protein that is first known as the intracellular receptor for the potent immunosuppressive drug cyclosporine A8. To be the most abundant cyclophilin, increasing studies have shown that CypA can be actively released into the extracellular tissue spaces by multiple cells, such as endothelial cells, monocytes/macrophages, and vascular smooth muscle cells (VSMCs), in response to inflammatory stimuli and exhibit potent chemotactic capacity on inflammatory cells9–11. Notably, CypA exerts indispensable roles in VSMC remodeling by promoting inflammation and VSMC proliferation in the vascular system12. CypA also accelerates cartilage and bone degradation by activating the macrophage matrix metalloproteinase 9 (MMP-9) expression in rheumatoid arthritis (RA)13. Recently, many studies have identified the roles of CypA in dental diseases, such as chronic periodontitis and oral submucous fibrosis14,15. Thus, we hypothesized that CypA may be associated with the inflammatory progression of human periapical lesions.
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From the *The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key, Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; and †Division of Life Sciences and Medicine, The First Affiliated Hospital of University of Science and Technology of China, Hefei, Anhui, China Address requests for reprints to Prof Bin Peng, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2019 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2019.09.006
Cyclophilin A in Human Periapical Lesions
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CypA is able to mediate inflammatory responses by interacting with its cellular receptor, CD147, which plays an important role in neutrophil adhesion, chemotaxis, matrix metalloproteinase production, and oxidative burst16. Previous studies indicated that the abundant CypA increases MMP-9 expression by directly binding to CD147, and the CypACD147 interactions lead to the destruction of cartilage and bone; CypA is also a crucial proinflammatory signaling pathway in response to inflammatory stimulation by accelerating angiogenesis in atherosclerosis9,17. CD34, which is known as the vascular marker related to angiogenesis, has been frequently used in many diseases18,19. Therefore, in the present study, we aimed to investigate the expression of CypA in human periapical lesions. We hypothesized that CypA may be involved in the inflammatory development of human periapical lesions and had a close relationship with angiogenesis and alveolar bone degradation.
MATERIALS AND METHODS Patients and Tissue Sample Collection A total of 58 human apical periodontitis lesions were consecutively obtained from the Department of Pathology after periapical surgery in the School and Hospital of Stomatology, Wuhan University, Wuhan, China. Six healthy dental pulp and gingival samples were obtained from a group of orthodontic teeth. Patients without periodontitis and systematic diseases were enrolled in the study. There were no significant age or sex differences between the healthy donors and the patients with periapical lesions. The Ethics and Investigation Committee of Wuhan University approved this study, and all subjects provided written informed consent.
Histologic Analysis Sections were sequentially fixed in 4% phosphate-buffered formalin (pH 5 7.4) for 48 hours and processed for paraffin embedding. After the sections were deparaffinized and rehydrated, they were subjected to hematoxylin-eosin staining, immunohistochemistry, and double immunofluorescence labeling for histologic examination. One in every 4 sections was observed under a light microscope.
Immunohistochemical Analysis According to previous experiments, pepsin (Maixin, Fuzhou, China) was used to retrieve antigen for 20 minutes. Then, the sections were washed and stained using a streptavidin peroxidase kit (Maixin) according to the
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manufacturer’s instructions. Briefly, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes at 37 C. After washing with phosphate-buffered saline (PBS), the sections were incubated with normal serum for 40 minutes at 37 C and then incubated with rabbit polyclonal antibodies against CypA (1:200; Abcam Biotechnology Inc, Cambridge, UK) overnight at 4 C. After washing with PBS, the sections were incubated with a biotinylated secondary antibody for 40 minutes at 37 C and then with streptavidin peroxidase for another 30 minutes at 37 C. Subsequently, the sections were developed with 3,30-diaminobenzidine (Zhongshan Biotechnology Co, Beijing, China) and counterstained with hematoxylin. Finally, the sections were observed under a microscope. Nonimmune serum was used as the negative control. Sections that were substituted with nonimmune rabbit or goat serum instead of primary antibodies served as negative controls. In each specimen, the CypA-positive cells in 3 randomly selected areas were counted under 400! magnification by 2 independent observers. Three sections from each sample were analyzed for cell measurement, and the average of these observations was calculated. All measurements were performed in a doubleblind manner.
Double Immunofluorescence Labeling This experiment was performed as previously described20. After deparaffinization and rehydration, the slices were treated with 0.1% Triton X-100 (Shenggong Biotechnology Co, Shanghai, China) for 15 minutes (for CypA only) and then incubated with 1% bovine serum albumin for 1 hour at 37 C to eliminate nonspecific staining. Without washing, the slices were incubated with CypA rabbit polyclonal antibody (1:100) and either CD34 mouse monoclonal (1:100; Proteintech, Inc, Wuhan, China) or MMP-9 goat polyclonal antibody (1:100; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and slices of another experimental group were incubated with MMP9 goat polyclonal antibody and rabbit polyclonal antibodies against CD147 (1:100, Abcam Biotechnology Inc) at 4 C overnight. The sections were washed and incubated with the secondary fluorescein donkey antirabbit CY3 (1:200; EarthOx, San Francisco, CA) and either donkey antigoat Dylight 488 (1:200; Proteintech Group, Wuhan, China) or donkey antimouse Dylight 488 antibodies (1:200, Proteintech Group) at 37 C for 1 hour in the dark; 40 ,6-diamidino-2-phenylindole (ZSGBBIO, Beijing, China) was used to stain the nuclei
at 37 C for 5 minutes. Finally, the sections were washed, incubated with AutoFluo Quencher (Applygen, Beijing, China) for 30 minutes, and observed immediately under a fluorescence microscope (Leica, Nussloch, Germany) in each experiment. A single primary antibody (eg, CypA, CD34, MMP-9, or CD147) or PBS was used as the control.
Morphometric Analysis Immunopositive cells were quantified using Image-Pro Plus version 6.0 (Media Cybernetics, Inc, Bethesda, MD) with histologic section images captured by fluorescent microscopy (Leica, Nussloch, Germany). The overlap of the red and green and/or blue fluorescence in the same field were observed as yellow or white. Each specimen was randomly divided into 3 fields and analyzed at 400! magnification (0.0714 mm2/ field), and double-labeling positive cells in human periapical tissues were counted in each field. The density of positive cells was expressed as the number of cells/mm2.
Statistical Analysis Data were analyzed with SPSS 17.0 software (SPSS Inc, Chicago, IL). All measurement results were presented as the mean 6 standard deviation. The average value was determined for each group. Statistical differences in each group were subjected to the Tukey multiple comparison test (a 5 0.05). P , .05 was considered significant.
RESULTS CypA Overexpressed in Human Periapical Lesions Immunohistochemical observation showed that CypA-positive cells were detected in all periapical lesion tissues (52 cases), and no evident positive immunostaining of CypA was observed in healthy gingiva and dental pulp tissues (6 cases, P , .05; Fig. 1A–H). Comparing the PG (28 cases) and RC groups (24 cases) showed that CypA expression was stronger in the PG group than in the RC group, but the difference between the RC and PG groups was insignificant (Fig. 1I). CypA was cytoplasmic expression, and CypA-positive cells were mainly lymphocytes, plasma cells, epithelial cells, and endothelial cells, especially on cells that were highly expressed along the vascular wall (Fig. 2A–F).
CypA Colocalized with CD34 in Human Periapical lesions Previous studies reported that CD34 is a vascular marker related to angiogenesis18,19. We evaluated whether CypA is colocalized
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FIGURE 1 – Hematoxylin-eosin staining and immunohistochemical evaluation of CypA. Histologic analysis showed inflammatory infiltrates in (A ) healthy gingiva, (B ) healthy pulp, (C ) PGs, and (D ) RCs and in situ CypA expression in (E ) healthy gingiva, (F ) healthy pulp, (G ) PGs, and (H ) RCs (scale bar 5 50 mm). (I ) Statistical analysis of the number of CypA-positive cells in every group. Significant differences were observed between the healthy control group with PGs and RC groups (P , .05). No significant difference was observed between the PG and RC groups. The horizontal lines in the box represent the median, the inferior and superior lines of the box indicate the 25% and 75% percentile distributions, and the whiskers indicate the minimum and maximum values. The P value from the Kruskal-Wallis test was statistically significant when P , .05. ***P , .001. JOE Volume -, Number -, - 2019
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FIGURE 2 – The immunohistochemical staining of CypA. (A–C ) CypA was cytoplasmic expression, and CypA-positive cells were mainly lymphocytes (green arrows ), plasma cells (red arrows ), epithelial cells (yellow arrows ), and endothelial cells (black arrows ). Further detection of CypA expression on endothelial cells in (D ) oblique plane, (E ) cross, and (F ) sagittal directions. CypA was highly expressed along the vascular wall.
with CD34 in vascular tissues in human periapical lesions. The immunofluorescence staining of human samples showed that a large number of CypA-positive cells overlapped with CD34-positive cells in periapical lesions compared with the healthy control group (P , .05, Fig. 3A–G). CypA and CD34 were mainly colocalized along the vascular wall. Therefore, the association of CypA with CD34 may be involved in the process of angiogenesis in human periapical lesions.
CypA Colocalized with MMP-9 in Human Periapical Lesions Considering that MMP-9 expression can be enhanced by the interaction between CypA and CD147 in accelerating histolysis9,17, we further detected the colocalization among CypA, CD147, and MMP-9 in human periapical lesions. Double immunofluorescence staining showed that a large number of CypA-positive cells overlapped with MMP-9–positive cells, and a large number of CD147-positive cells also overlapped with MMP-9–positive cells in periapical lesions (Fig. 4A–F). These results indirectly indicated that CypA was colocalized with CD147 and may participate in the process of histolysis in human periapical lesions.
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DISCUSSION A periapical lesion is an inflammatory disease caused by root canal bacterial infection characteristic of root apex ligament degradation and bone resorption21. Various inflammatory cells and their by-products are involved during inflammation development22. CypA is a member of the immunophilin family that regulates multiple cell functions in multiple inflammatory diseases, such as cardiovascular disease, RA, and viral infection11,12,23. Recently, CypA has been studied in the field of oral diseases, such as periodontitis14,24, oral submucous fibrosis15, and human oral squamous cell carcinoma25,26. In a mice model of the experimental periapical lesions, we detected a positive relationship between the increased osteoclastic CypA expression and enhanced MMP-9 expression. In the present study, CypA expression in human periapical lesions was investigated for the first time. In this study, CypA was overexpressed in human inflammatory periapical lesions compared with normal tissues. This result suggested that CypA may be involved in the pathogenesis of human periapical lesions. Previous studies have proved that CypA expressed on neutrophils and mononuclear cells and could induce the chemotaxis of neutrophils and mononuclear cells in
periodontitis. In the present study, CypA was strongly expressed on lymphocytes and plasma cells in human periapical lesions, which supported the results of previous studies showing that CypA can be a potent chemotaxis mediator and promoter in the pathogenesis of inflammation-mediated diseases14,17. In RA, CypA mediated neutrophil migration into the joints and enhanced MMP secretion and cell invasion of synoviocytes, all of which may contribute to the cartilage invasion and bone destruction of RA. Our findings complement these results, indicating that CypA may be involved in inflammatory responses and alveolar bone resorption in human periapical lesions24,27. PGs and RCs are 2 different periapical lesion phenotypes consisting of various inflammatory cells and structures. Granulomas are histologically identified by fibrous and granular tissues formed with abundant newborn capillaries, fibroblasts, and infiltrated inflammatory cells. RCs contain cavities completely enclosed by epithelial lining and capsule content28. Endothelial cells, which are the main cells forming new blood vessels in the vascular system, were observed in most human periapical lesions. Currently, CypA was not only localized in lymphocytes but was also highly expressed in vascular endothelial cells. In periodontitis, endothelial cells were
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FIGURE 3 – Double immunolabeling of CypA and CD34 detected by immunofluorescence. Red, CypA; green, CD34; blue, 40 ,6-diamidino-2-phenylindole; and orange or yellow, overlap of the green and red fluorescence. CypA-CD34 double labeling in the (A–C ) healthy control and (D–F ) periapical lesion groups (scale bar 5 50 mm). (G ) The boxplot shows the number of CypA-CD34 double-positive cells in every group. The results are expressed as the number of positive cells/mm2. The horizontal lines in the box represent the median, the inferior and superior lines of the box indicate the 25% and 75% percentile distributions, and the whiskers indicate the minimum and maximum values. The P value from the KruskalWallis test was statistically significant when P , .05. ***P , .001.
moderately positive for CypA in inflamed gingiva14. Considering CypA overexpressed in endothelial cells, we further examined the localization of CypA and CD34 by immunofluorescence staining to improve our understanding of the role of CypA in angiogenesis in periapical lesions. CD34, which is known as the vascular marker related to angiogenesis, has been studied in many diseases18,19. CypA is a paracrine and autocrine regulator of endothelial cell behavior in vascular diseases29. Under hypoxia, CypA expression is increased and acetylated, and the acetylated CypA is essential for the
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excessive proliferation, migration, and formation of tube networks in the pulmonary arterial endothelial cells, which are directly linked to autophagy activation30. CypA can also enhance neoangiogenesis and restore revascularization in ischemic tissues31. Furthermore, CypA was correlated significantly with vascular endothelial growth factor C and closely related to angiogenesis in oral and maxillofacial tumors, such as tongue squamous cell carcinoma and oral submucous fibrosis15,26. Therefore, our findings were consistent with those of previous studies; the high overlap of CypA and CD34 localization in
the human periapical lesions indicated that CypA may be associated with angiogenesis, which accelerated granuloma formation in human periapical lesions and served as a novel functional therapeutic target in alleviating angiogenesis in human periapical lesions, such as PGs. CD147 was known as the main receptor for extracellular CypA and is involved in many inflammatory diseases24,27. CypA/CD147 signaling serves as an upstream regulator of MMP-9 production17. MMP-9 is an essential factor of periradicular periodontal ligament degradation and alveolar bone destruction32.
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FIGURE 4 – Double immunolabeling of CypA and MMP-9 and CD147 and MMP-9 detected by immunofluorescence. Red, (A ) CypA or (D ) CD147; green, MMP-9; blue, 40 ,6diamidino-2-phenylindole; and orange or yellow, the overlap of the green and red fluorescence. (A–C ) CypA MMP-9 double labeling shown. (D–F ) CD147 MMP-9 double labeling shown (scale bar 5 50 mm). Previous studies have reported that CypA acted as an important mediator in accelerating MMP-9 and CD147 expression11. Under CypA inhibitor treatment, the protein expression of CD147 and MMP-9 down-regulated in myocarditis33, RA34, and cancer35. In the present study, the high overlap of CypA and CD147-positive cells with MMP-9–positive cells in human periapical lesions indicated that CypA/CD147 signaling may exist in human periapical lesions, and MMP-9 expression may be regulated by CypA/CD147 signaling. Combined with the findings reported previously, we suggested that CypA may be an important regulator involved in the
development of human periapical lesions by recruiting inflammatory cells, enhancing vascular permeability, and accelerating MMP9 expression, thereby promoting the periradicular periodontal ligament and alveolar bone destruction. In conclusion, our study is the first to show the immunoexpression of CypA in human periapical lesions. CypA was mainly expressed in lymphocytes, plasma cells, epithelial cells, and endothelial cells. The expression of CypA may be associated with angiogenesis and CD147-mediated MMP-9 secretion. Therefore, CypA may be involved in inflammatory development, lymphocyte
invasion, granulation tissue formation, and MMP-9 secretion in periapical lesions. Targeting CypA may serve as a new therapy for human periapical lesion treatment.
ACKNOWLEDGMENTS Yanqing Wang and Guojing Liu contributed equally to this work. Supported by the National Natural Science Foundation of China (grant nos. 81570973 and 81700966). The authors deny any conflicts of interest related to this study.
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Yang Y, Lu N, Zhou J, et al. Cyclophilin A up-regulates MMP-9 expression and adhesion of monocytes/macrophages via CD147 signalling pathway in rheumatoid arthritis. Rheumatology (Oxford) 2008;47:1299–310.
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Overexpression of Cyclophilin A in Human Periapical Lesions ABSTRACT Introduction: Cyclophilin A (CypA) is a cytosolic protein involved in multiple biological functions, such as inflammation, tissue remodeling, tumorigenesis, and vascular diseases. Human periapical lesions are induced by bacterial infections. However, the expression of CypA in human periapical lesions remains unclear. This study aimed to investigate the presence of CypA in human periapical lesions and the possible association of CypA with angiogenesis, inflammatory cell infiltration, and alveolar bone degradation during inflammatory development. Methods: Fifty-eight human periapical tissues, including periapical granulomas (PGs, n 5 28), radicular cysts (RCs, n 5 24), and healthy control tissues (control group, n 5 6) were collected. Samples were fixed and analyzed. CypA expression was detected and analyzed by immunohistochemistry in different cross sections. Double immunofluorescence was assessed to colocalize CypA with CD34, CypA with matrix metalloproteinase 9 (MMP-9), and CD147 with MMP-9. Results: CypA was significantly overexpressed in the RC and PG groups compared with the control group (P , .05), but the difference between the RC and PG groups was insignificant (P . .05). CypA-positive cells were mainly lymphocytes, endothelial cells, epithelial cells, and plasma cells. The doublelabeling analysis of CypA with CD34 suggested that CypA expression was associated with angiogenesis during periapical lesions. MMP-9 colocalized with both CypA and CD147 indicated that CypA may colocalize with CD147 and may be associated with the degradation of soft and hard tissues around human periapical lesions. Conclusions: CypA may be involved in the development of periapical lesions with an increase in inflammatory cell infiltration, angiogenesis acceleration, and alveolar bone degradation. (J Endod 2019;-:1–8.)
Yanqing Wang, DDS,*† Guojing Liu, DDS,* Jie Zhang, DDS,* Lingxin Zhu, DDS,* Jingjing Yu, DDS,* Shenting Zhu, DDS,* Fengyuan Lv, DDS,* and Bin Peng, DDS, PhD*
SIGNIFICANCE We suggest that CypA may be involved in the development of periapical lesions with an increase in inflammatory cell infiltration, angiogenesis acceleration, and alveolar bone degradation. Therefore, targeting CypA may serve as a new therapy for human periapical lesion treatment.
KEY WORDS Angiogenesis; CD34; CD147; cyclophilin A; matrix metalloproteinase 9; periapical lesions
A periapical lesion is a continuation of a bacterial infection in root canals, which can result in dental pulp necrosis and develop into apical periodontitis with degradation of the periradicular periodontal ligament and destruction of alveolar bone1,2. Periapical granulomas (PGs) and radicular cysts (RCs) are chronic periapical lesions that represent different stages of the same inflammatory process3. Considerable studies have been conducted to improve understanding of the inflammatory mechanisms involved in the granulation tissue formation and jaw cyst enlargement in PG and RC pathogenesis4–6. To date, a series of proinflammatory cytokines, such as matrix metalloproteinases, interferon gamma, and transforming growth factor beta, are synthesized during granulation tissue formation, jaw cyst enlargement, and alveolar bone resorption, respectively5,7. The detailed mechanisms underlying the progression of proinflammatory cytokines on granulation and cyst tissue formation, such as angiogenesis, inflammatory cell infiltration, epithelial proliferation, and alveolar bone degradation, have not been fully elucidated. Cyclophilin A (CypA) is a ubiquitously distributed protein that is first known as the intracellular receptor for the potent immunosuppressive drug cyclosporine A8. To be the most abundant cyclophilin, increasing studies have shown that CypA can be actively released into the extracellular tissue spaces by multiple cells, such as endothelial cells, monocytes/macrophages, and vascular smooth muscle cells (VSMCs), in response to inflammatory stimuli and exhibit potent chemotactic capacity on inflammatory cells9–11. Notably, CypA exerts indispensable roles in VSMC remodeling by promoting inflammation and VSMC proliferation in the vascular system12. CypA also accelerates cartilage and bone degradation by activating the macrophage matrix metalloproteinase 9 (MMP-9) expression in rheumatoid arthritis (RA)13. Recently, many studies have identified the roles of CypA in dental diseases, such as chronic periodontitis and oral submucous fibrosis14,15. Thus, we hypothesized that CypA may be associated with the inflammatory progression of human periapical lesions.
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From the *The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key, Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; and †Division of Life Sciences and Medicine, The First Affiliated Hospital of University of Science and Technology of China, Hefei, Anhui, China Address requests for reprints to Prof Bin Peng, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright © 2019 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2019.09.006
Cyclophilin A in Human Periapical Lesions
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CypA is able to mediate inflammatory responses by interacting with its cellular receptor, CD147, which plays an important role in neutrophil adhesion, chemotaxis, matrix metalloproteinase production, and oxidative burst16. Previous studies indicated that the abundant CypA increases MMP-9 expression by directly binding to CD147, and the CypACD147 interactions lead to the destruction of cartilage and bone; CypA is also a crucial proinflammatory signaling pathway in response to inflammatory stimulation by accelerating angiogenesis in atherosclerosis9,17. CD34, which is known as the vascular marker related to angiogenesis, has been frequently used in many diseases18,19. Therefore, in the present study, we aimed to investigate the expression of CypA in human periapical lesions. We hypothesized that CypA may be involved in the inflammatory development of human periapical lesions and had a close relationship with angiogenesis and alveolar bone degradation.
MATERIALS AND METHODS Patients and Tissue Sample Collection A total of 58 human apical periodontitis lesions were consecutively obtained from the Department of Pathology after periapical surgery in the School and Hospital of Stomatology, Wuhan University, Wuhan, China. Six healthy dental pulp and gingival samples were obtained from a group of orthodontic teeth. Patients without periodontitis and systematic diseases were enrolled in the study. There were no significant age or sex differences between the healthy donors and the patients with periapical lesions. The Ethics and Investigation Committee of Wuhan University approved this study, and all subjects provided written informed consent.
Histologic Analysis Sections were sequentially fixed in 4% phosphate-buffered formalin (pH 5 7.4) for 48 hours and processed for paraffin embedding. After the sections were deparaffinized and rehydrated, they were subjected to hematoxylin-eosin staining, immunohistochemistry, and double immunofluorescence labeling for histologic examination. One in every 4 sections was observed under a light microscope.
Immunohistochemical Analysis According to previous experiments, pepsin (Maixin, Fuzhou, China) was used to retrieve antigen for 20 minutes. Then, the sections were washed and stained using a streptavidin peroxidase kit (Maixin) according to the
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manufacturer’s instructions. Briefly, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes at 37 C. After washing with phosphate-buffered saline (PBS), the sections were incubated with normal serum for 40 minutes at 37 C and then incubated with rabbit polyclonal antibodies against CypA (1:200; Abcam Biotechnology Inc, Cambridge, UK) overnight at 4 C. After washing with PBS, the sections were incubated with a biotinylated secondary antibody for 40 minutes at 37 C and then with streptavidin peroxidase for another 30 minutes at 37 C. Subsequently, the sections were developed with 3,30-diaminobenzidine (Zhongshan Biotechnology Co, Beijing, China) and counterstained with hematoxylin. Finally, the sections were observed under a microscope. Nonimmune serum was used as the negative control. Sections that were substituted with nonimmune rabbit or goat serum instead of primary antibodies served as negative controls. In each specimen, the CypA-positive cells in 3 randomly selected areas were counted under 400! magnification by 2 independent observers. Three sections from each sample were analyzed for cell measurement, and the average of these observations was calculated. All measurements were performed in a doubleblind manner.
Double Immunofluorescence Labeling This experiment was performed as previously described20. After deparaffinization and rehydration, the slices were treated with 0.1% Triton X-100 (Shenggong Biotechnology Co, Shanghai, China) for 15 minutes (for CypA only) and then incubated with 1% bovine serum albumin for 1 hour at 37 C to eliminate nonspecific staining. Without washing, the slices were incubated with CypA rabbit polyclonal antibody (1:100) and either CD34 mouse monoclonal (1:100; Proteintech, Inc, Wuhan, China) or MMP-9 goat polyclonal antibody (1:100; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), and slices of another experimental group were incubated with MMP9 goat polyclonal antibody and rabbit polyclonal antibodies against CD147 (1:100, Abcam Biotechnology Inc) at 4 C overnight. The sections were washed and incubated with the secondary fluorescein donkey antirabbit CY3 (1:200; EarthOx, San Francisco, CA) and either donkey antigoat Dylight 488 (1:200; Proteintech Group, Wuhan, China) or donkey antimouse Dylight 488 antibodies (1:200, Proteintech Group) at 37 C for 1 hour in the dark; 40 ,6-diamidino-2-phenylindole (ZSGBBIO, Beijing, China) was used to stain the nuclei
at 37 C for 5 minutes. Finally, the sections were washed, incubated with AutoFluo Quencher (Applygen, Beijing, China) for 30 minutes, and observed immediately under a fluorescence microscope (Leica, Nussloch, Germany) in each experiment. A single primary antibody (eg, CypA, CD34, MMP-9, or CD147) or PBS was used as the control.
Morphometric Analysis Immunopositive cells were quantified using Image-Pro Plus version 6.0 (Media Cybernetics, Inc, Bethesda, MD) with histologic section images captured by fluorescent microscopy (Leica, Nussloch, Germany). The overlap of the red and green and/or blue fluorescence in the same field were observed as yellow or white. Each specimen was randomly divided into 3 fields and analyzed at 400! magnification (0.0714 mm2/ field), and double-labeling positive cells in human periapical tissues were counted in each field. The density of positive cells was expressed as the number of cells/mm2.
Statistical Analysis Data were analyzed with SPSS 17.0 software (SPSS Inc, Chicago, IL). All measurement results were presented as the mean 6 standard deviation. The average value was determined for each group. Statistical differences in each group were subjected to the Tukey multiple comparison test (a 5 0.05). P , .05 was considered significant.
RESULTS CypA Overexpressed in Human Periapical Lesions Immunohistochemical observation showed that CypA-positive cells were detected in all periapical lesion tissues (52 cases), and no evident positive immunostaining of CypA was observed in healthy gingiva and dental pulp tissues (6 cases, P , .05; Fig. 1A–H). Comparing the PG (28 cases) and RC groups (24 cases) showed that CypA expression was stronger in the PG group than in the RC group, but the difference between the RC and PG groups was insignificant (Fig. 1I). CypA was cytoplasmic expression, and CypA-positive cells were mainly lymphocytes, plasma cells, epithelial cells, and endothelial cells, especially on cells that were highly expressed along the vascular wall (Fig. 2A–F).
CypA Colocalized with CD34 in Human Periapical lesions Previous studies reported that CD34 is a vascular marker related to angiogenesis18,19. We evaluated whether CypA is colocalized
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FIGURE 1 – Hematoxylin-eosin staining and immunohistochemical evaluation of CypA. Histologic analysis showed inflammatory infiltrates in (A ) healthy gingiva, (B ) healthy pulp, (C ) PGs, and (D ) RCs and in situ CypA expression in (E ) healthy gingiva, (F ) healthy pulp, (G ) PGs, and (H ) RCs (scale bar 5 50 mm). (I ) Statistical analysis of the number of CypA-positive cells in every group. Significant differences were observed between the healthy control group with PGs and RC groups (P , .05). No significant difference was observed between the PG and RC groups. The horizontal lines in the box represent the median, the inferior and superior lines of the box indicate the 25% and 75% percentile distributions, and the whiskers indicate the minimum and maximum values. The P value from the Kruskal-Wallis test was statistically significant when P , .05. ***P , .001. JOE Volume -, Number -, - 2019
Cyclophilin A in Human Periapical Lesions
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FIGURE 2 – The immunohistochemical staining of CypA. (A–C ) CypA was cytoplasmic expression, and CypA-positive cells were mainly lymphocytes (green arrows ), plasma cells (red arrows ), epithelial cells (yellow arrows ), and endothelial cells (black arrows ). Further detection of CypA expression on endothelial cells in (D ) oblique plane, (E ) cross, and (F ) sagittal directions. CypA was highly expressed along the vascular wall.
with CD34 in vascular tissues in human periapical lesions. The immunofluorescence staining of human samples showed that a large number of CypA-positive cells overlapped with CD34-positive cells in periapical lesions compared with the healthy control group (P , .05, Fig. 3A–G). CypA and CD34 were mainly colocalized along the vascular wall. Therefore, the association of CypA with CD34 may be involved in the process of angiogenesis in human periapical lesions.
CypA Colocalized with MMP-9 in Human Periapical Lesions Considering that MMP-9 expression can be enhanced by the interaction between CypA and CD147 in accelerating histolysis9,17, we further detected the colocalization among CypA, CD147, and MMP-9 in human periapical lesions. Double immunofluorescence staining showed that a large number of CypA-positive cells overlapped with MMP-9–positive cells, and a large number of CD147-positive cells also overlapped with MMP-9–positive cells in periapical lesions (Fig. 4A–F). These results indirectly indicated that CypA was colocalized with CD147 and may participate in the process of histolysis in human periapical lesions.
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DISCUSSION A periapical lesion is an inflammatory disease caused by root canal bacterial infection characteristic of root apex ligament degradation and bone resorption21. Various inflammatory cells and their by-products are involved during inflammation development22. CypA is a member of the immunophilin family that regulates multiple cell functions in multiple inflammatory diseases, such as cardiovascular disease, RA, and viral infection11,12,23. Recently, CypA has been studied in the field of oral diseases, such as periodontitis14,24, oral submucous fibrosis15, and human oral squamous cell carcinoma25,26. In a mice model of the experimental periapical lesions, we detected a positive relationship between the increased osteoclastic CypA expression and enhanced MMP-9 expression. In the present study, CypA expression in human periapical lesions was investigated for the first time. In this study, CypA was overexpressed in human inflammatory periapical lesions compared with normal tissues. This result suggested that CypA may be involved in the pathogenesis of human periapical lesions. Previous studies have proved that CypA expressed on neutrophils and mononuclear cells and could induce the chemotaxis of neutrophils and mononuclear cells in
periodontitis. In the present study, CypA was strongly expressed on lymphocytes and plasma cells in human periapical lesions, which supported the results of previous studies showing that CypA can be a potent chemotaxis mediator and promoter in the pathogenesis of inflammation-mediated diseases14,17. In RA, CypA mediated neutrophil migration into the joints and enhanced MMP secretion and cell invasion of synoviocytes, all of which may contribute to the cartilage invasion and bone destruction of RA. Our findings complement these results, indicating that CypA may be involved in inflammatory responses and alveolar bone resorption in human periapical lesions24,27. PGs and RCs are 2 different periapical lesion phenotypes consisting of various inflammatory cells and structures. Granulomas are histologically identified by fibrous and granular tissues formed with abundant newborn capillaries, fibroblasts, and infiltrated inflammatory cells. RCs contain cavities completely enclosed by epithelial lining and capsule content28. Endothelial cells, which are the main cells forming new blood vessels in the vascular system, were observed in most human periapical lesions. Currently, CypA was not only localized in lymphocytes but was also highly expressed in vascular endothelial cells. In periodontitis, endothelial cells were
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FIGURE 3 – Double immunolabeling of CypA and CD34 detected by immunofluorescence. Red, CypA; green, CD34; blue, 40 ,6-diamidino-2-phenylindole; and orange or yellow, overlap of the green and red fluorescence. CypA-CD34 double labeling in the (A–C ) healthy control and (D–F ) periapical lesion groups (scale bar 5 50 mm). (G ) The boxplot shows the number of CypA-CD34 double-positive cells in every group. The results are expressed as the number of positive cells/mm2. The horizontal lines in the box represent the median, the inferior and superior lines of the box indicate the 25% and 75% percentile distributions, and the whiskers indicate the minimum and maximum values. The P value from the KruskalWallis test was statistically significant when P , .05. ***P , .001.
moderately positive for CypA in inflamed gingiva14. Considering CypA overexpressed in endothelial cells, we further examined the localization of CypA and CD34 by immunofluorescence staining to improve our understanding of the role of CypA in angiogenesis in periapical lesions. CD34, which is known as the vascular marker related to angiogenesis, has been studied in many diseases18,19. CypA is a paracrine and autocrine regulator of endothelial cell behavior in vascular diseases29. Under hypoxia, CypA expression is increased and acetylated, and the acetylated CypA is essential for the
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excessive proliferation, migration, and formation of tube networks in the pulmonary arterial endothelial cells, which are directly linked to autophagy activation30. CypA can also enhance neoangiogenesis and restore revascularization in ischemic tissues31. Furthermore, CypA was correlated significantly with vascular endothelial growth factor C and closely related to angiogenesis in oral and maxillofacial tumors, such as tongue squamous cell carcinoma and oral submucous fibrosis15,26. Therefore, our findings were consistent with those of previous studies; the high overlap of CypA and CD34 localization in
the human periapical lesions indicated that CypA may be associated with angiogenesis, which accelerated granuloma formation in human periapical lesions and served as a novel functional therapeutic target in alleviating angiogenesis in human periapical lesions, such as PGs. CD147 was known as the main receptor for extracellular CypA and is involved in many inflammatory diseases24,27. CypA/CD147 signaling serves as an upstream regulator of MMP-9 production17. MMP-9 is an essential factor of periradicular periodontal ligament degradation and alveolar bone destruction32.
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FIGURE 4 – Double immunolabeling of CypA and MMP-9 and CD147 and MMP-9 detected by immunofluorescence. Red, (A ) CypA or (D ) CD147; green, MMP-9; blue, 40 ,6diamidino-2-phenylindole; and orange or yellow, the overlap of the green and red fluorescence. (A–C ) CypA MMP-9 double labeling shown. (D–F ) CD147 MMP-9 double labeling shown (scale bar 5 50 mm). Previous studies have reported that CypA acted as an important mediator in accelerating MMP-9 and CD147 expression11. Under CypA inhibitor treatment, the protein expression of CD147 and MMP-9 down-regulated in myocarditis33, RA34, and cancer35. In the present study, the high overlap of CypA and CD147-positive cells with MMP-9–positive cells in human periapical lesions indicated that CypA/CD147 signaling may exist in human periapical lesions, and MMP-9 expression may be regulated by CypA/CD147 signaling. Combined with the findings reported previously, we suggested that CypA may be an important regulator involved in the
development of human periapical lesions by recruiting inflammatory cells, enhancing vascular permeability, and accelerating MMP9 expression, thereby promoting the periradicular periodontal ligament and alveolar bone destruction. In conclusion, our study is the first to show the immunoexpression of CypA in human periapical lesions. CypA was mainly expressed in lymphocytes, plasma cells, epithelial cells, and endothelial cells. The expression of CypA may be associated with angiogenesis and CD147-mediated MMP-9 secretion. Therefore, CypA may be involved in inflammatory development, lymphocyte
invasion, granulation tissue formation, and MMP-9 secretion in periapical lesions. Targeting CypA may serve as a new therapy for human periapical lesion treatment.
ACKNOWLEDGMENTS Yanqing Wang and Guojing Liu contributed equally to this work. Supported by the National Natural Science Foundation of China (grant nos. 81570973 and 81700966). The authors deny any conflicts of interest related to this study.
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