ORIGINAL ARTICLE
Effects of vascular endothelial growth factor on osteoblasts and osteoclasts Hechang Huang,a Li Ma,b and Stephanos Kyrkanidesc Stony Brook, NY
Introduction: Bone remodeling is crucial to the success of many dental procedures and is tightly regulated. Vascular endothelial growth factor (VEGF), a key cytokine for angiogenesis, is also an important regulator of bone remodeling. We aimed to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on cultured osteoblasts and osteoclasts. Methods: Preosteoblastic MC3T3-E1 cells were treated with vehicle or VEGF-A165. Cell proliferation, migration, and invasion potentials were assessed. Preosteoclastic RAW264.7 cells were treated with vehicle or VEGF with or without the receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoclast formation was measured with tartrate-resistant acid phosphatase staining. Conditioned media from vehicle-treated or VEGF-treated MC3T3-E1 cells were tested for the levels of RANKL and osteoprotegerin (OPG) and were used to treat RAW264.7 cells to observe osteoclast formation. Results: VEGF significantly induced MC3T3-E1 cell proliferation, migration, and invasion. VEGF did not directly induce osteoclastogenesis but significantly increased the RANKL/OPG ratio in the conditioned media from the MC3T3-E1 cultures; this significantly increased osteoclast formation. Conclusions: VEGF stimulates osteoclast differentiation by increasing the osteoblastic RANKL/OPG ratio but has no direct effect on osteoclast precursor cells, and it induces osteoblast proliferation, migration, and invasion potentials in vitro. (Am J Orthod Dentofacial Orthop 2016;149:366-73)
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one remodeling is the lifelong continuous replacement of old bone tissue with new bone1,2 and is crucial to the success of a variety of dental procedures, including dental implants,3 periodontal disease treatment,4 and orthodontic tooth movement.5 It is a tightly coupled local process starting with bone resorption, followed by reversal and bone formation phases. Osteoclasts and osteoblasts are organized as basic multicellular units and cooperate to perform the resorption-reversalformation sequence of the bone remodeling process.6 The level of bone remodeling is closely regulated by hormones, cytokines, prostaglandins, and mechanical From the Department of Orthodontics and Pediatric Dentistry, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, NY. a Assistant professor. b Research assistant professor. c Professor and chair. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Address correspondence to: Hechang Huang, Department of Orthodontics and Pediatric Dentistry, School of Dental Medicine, State University of New York at Stony Brook, Rockland Hall, Stony Brook, NY 11794-8700; e-mail, hechang. [email protected]. Submitted, September 2014; revised and accepted, September 2015. 0889-5406/$36.00 Copyright Ó 2016 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2015.09.021
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loading.2 Receptor activator of nuclear factor-kappa B ligand (RANKL), an osteoblastic cell-derived factor, is crucial for osteoclast differentiation, survival, and function by binding to osteoclast cell-surface receptor, its receptor on preosteoclasts.7 Osteoprotegerin (OPG), another osteoblastic cell-derived factor, interrupts the RANKL/osteoclast cell-surface receptor binding as a decoy receptor of RANKL, inhibiting osteoclastogenesis.8 Therefore, the RANKL/OPG ratio largely determines the formation of functional osteoclasts and the activation of the resorption phase of bone remodeling.9 The expression of RANKL and OPG by osteoblastic cells is regulated by various systemic hormones and local factors.2 Orthodontic treatment duration is a major concern for patients and is closely associated with various side effects, including external root resorption,10 carious lesions,11 and open gingival embrasures.12 According to the American Association of Orthodontists, about 4 million people in this country receive orthodontic treatment each year. Accelerating tooth movement can therefore benefit millions of patients by reducing their treatment durations and side effects. The rate of orthodontic tooth movement is largely determined by bone remodeling.5 A full-thickness mucoperiosteal flap alone was sufficient to induce de novo regional
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acceleratory phenomenon, a significantly increased bone remodeling activity as part of the trauma healing process.13 Vascular endothelial growth factor (VEGF) was shown to play an essential role in the postflap healing procedure that led to activated bone remodeling.14 Local injections of VEGF in mice significantly increased the numbers of osteoclasts and the rate of orthodontic tooth movement,15 whereas neutralizing the antiVEGF antibody had the opposite effect.16 A previous study showed that VEGF promotes bone remodeling primarily by reducing OPG, but it had no effect on RANKL in osteoblastic cell cultures up to 72 hours. The long-term effects of VEGF on osteoclast and osteoblast formation and activities, and on bone remodeling, are yet to be elucidated.17 The aim of this study was to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on osteoblasts and osteoclasts in vitro. MATERIAL AND METHODS
MC3T3-E1 subclone 4 cells, subcloned by Dr Renny Franceschi at the University of Michigan from the original immortalized neonatal murine calvarial cells, were purchased (CRL-2593; American Type Culture Collection, Manassas, Va).18 The cells were grown in alpha minimum essential medium (A1049001; Gibco BRL, Gaithersburg, Md); 10% heat-inactivated fetal calf serum (Gibco BRL), penicillin (100 U/mL), and streptomycin (100 mg/mL) were added to the media. Cells (5000/cm2) were plated in 6-well dishes in a humidified atmosphere of 5% carbon dioxide at 37 C and grown until confluent. RAW264.7 cells, a tumor cell line induced by Abelson murine leukemia virus, were purchased (TIB-71; American Type Culture Collection). Cells were grown in Dulbecco's Modified Eagle's Medium (30-2002; American Type Culture Collection). We added 10% heatinactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) to the media. The cells were plated in 96-well plates in a humidified atmosphere of 5% carbon dioxide at 37 C. Formation of tartrateresistant acid phosphatase positive multinucleated cells (TRAP1 MNC) was assessed with a commercial kit (F4523; Sigma-Aldrich, St Louis, Mo). For the western blot analysis, protein from MC3T3E1 cell cultures was extracted with radioimmunoprecipitation assay buffer following the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, Calif). Protein concentrations were measured by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, Mass). Conditioned media from MC3T3-E1 cells were filtered with a 0.44-mm filter under sterile conditions and stored at –80 C. The same experimental procedures
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were followed as published previously.19 Briefly, equal amounts (100 mg) of protein (for VEGF receptor-1 [VEGRF-1] and VEGF receptor-2 [VEGFR-2]) or equal volumes (25 mL) of media (for RANKL and OPG) were used for 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif). The total protein on the nitrocellulose membrane was stained with a protein stain kit (24580; Thermo Scientific, Rockford, Ill), which has been used in previous studies.20,21 Membranes were washed with trisbuffered saline solution (pH 7.6) before blocking with 5% weight per volume of nonfat dry milk in 1 3 trisbuffered saline solution containing 0.05% Tween 20 (Sigma-Aldrich) for 60 minutes. Then the membranes were incubated with primary antibody of VEGFR-1 and VEGFR-2 for cell extracts and RANKL or OPG for media samples, washed with tris-buffered saline solution containing 0.05% Tween 20, and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000). The signal was detected with LumiGLO chemiluminescent reagent (Cell Signaling Technology, Danvers, Mass). Primary antibodies, including VEGFR-1 (AF471; R&D Systems, Minneapolis, Minn), VEGFR-2 (AF644; R&D Systems), RANKL (AF462; R&D Systems), or OPG (AF459; R&D Systems), were incubated at 0.1 mg per milliliter overnight at 4 C. Cell proliferation was tested with the 5-bromo-20 deoxyuridine (BrdU) assay. MC3T3-E1 cells (1 3 103 cells per well) were plated in 96-well plates in the media described above. Vehicle or VEGF (10, 20, or 40 ng/mL) was added at the beginning of cell plating and treated for 24 hours. The BrdU assay followed the same procedures as published previously.22 Briefly, the cells were fixed, and the DNA was denatured after removing the labeling medium. Then anti-BrdUperoxidase antibody was added. Substrate reactions to detect the immune complexes were quantified by measuring the absorbance with a spectrophotometer. For cell migration and invasion assessment, we used 8.0-mm-pore BD BioCoat Control Insert 24-well plates (354578; BD Bioscience, San Jose, Calif) and Matrigel Invasion Chamber 24-well plates (354480; BD Biosciences) to measure migration and invasion of MC3T3-E1 cells, respectively. We plated 2 3 105 cells in 0.4% fetal bovine serum medium in the top chamber after it was collected and washed. Vehicle or VEGF (20 ng/mL) was added to the bottom chamber. Cell migration and invasion assessment followed the same experimental procedures as published before.23 Briefly, cells on the top surface were removed after 22 hours. Cells on the lower surface were stained with 0.5% crystal violet after being fixed.
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Then the stained cells were photographed under bright field microscopy. Ten random high-power fields per insert were used to count migrated or invaded cells, and averages were calculated. Fold changes of the numbers of migrated or invaded cells to the untreated controls in triplicate experiments were determined. RESULTS
For VEGF stimulated osteoblastic MC3T3-E1 cell proliferation, migration, and invasion, the expression of VEGF receptors by MC3T3-E1 cells was assessed by western immunoblotting. These cells express VEGFR-1 and VEGFR-2 (data not shown). Proliferation analysis by the BrdU assay showed that VEGF at 10 ng per milliliter did not affect MC3T3-E1 cell proliferation (Fig 1, A; P .0.05), whereas VEGF at 20 or 40 ng per milliliter significantly stimulated MC3T3-E1 cell proliferation (Fig 1, A; 3.60- and 3.98-fold, respectively; P \0.01). In addition, VEGF (20 ng/mL) significantly increased MC3T3-E1 cell migration (Fig 1, B; 2.29-fold; P \0.01) and invasion (Fig 1, C; 15.74-fold; P \0.001) compared with the vehicle-treated cells. VEGF did not directly affect osteoclast formation in RAW264.7 cell cultures. To test the direct effects of VEGF on osteoclast formation, we first treated osteoclastic precursor RAW264.7 cells directly with VEGF (20 ng/mL). RAW264.7 cells can differentiate into mature osteoclasts by RANKL without macrophage colony-stimulating factor. As expected, RANKL induced TRAP1 MNC formation used here as the control. VEGF treatment alone did not stimulate any osteoclast formation, nor did VEGF increase osteoclast formation induced by RANKL (Fig 2). TRAP1 MNC counts are shown in the Table. Conditioned media from VEGF-treated MC3T3-E1 cells stimulated osteoclast formation in RAW264.7 cell cultures. Osteoclast formation in RAW264.7 cells was also assessed with conditioned media from osteoblastic MC3T3-E1 cells previously treated with VEGF (20 ng/ mL) or vehicle for 4, 7, 14, and 21 days (Fig 3, A). Conditioned media from MC3T3-E1 cells treated with VEGF at all time points significantly increased TRAP1 MNC formation, whereas those from the control group had a minimum effect (Fig 3, B; P \0.001) at all time points. Among the RAW264.7 cultures treated with conditioned media from VEGF-treated MC3T3-E1 cells, osteoclast formation was significantly higher at the earlier times of days 4 and 7 compared with the later times of days 14 and 21 (Fig 3, B; P \0.01). VEGF induced RANKL and reduced OPG expression in MC3T3-E1 cells. We measured RANKL and OPG
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protein levels in the conditioned media from osteoblastic MC3T3-E1 cultures with western blot. We found that VEGF treatment for 4, 7, 14, and 21 days significantly increased RANKL expression by MC3T3-E1 cells (Fig 4, A; 1.36 6 0.19, 1.94 6 0.25, 1.28 6 0.22, and 1.03 6 0.04-fold, respectively), while potently inhibiting OPG expression (Fig 4, B; 0.15 6 0.04, 0.37 6 0.01, 0.41 6 0.02, 0.43 6 0.06-fold, respectively), resulting in a significantly increased RANKL/OPG ratio in the media (Fig 4, C; 9.46 6 2.04, 5.21 6 0.75, 3.12 6 0.64, 2.42 6 0.40-fold, respectively). The effects of VEGF to increase the RANKL/OPG ratio were more significant at the earlier times of days 4 and 7 than at the later times of days 14 and 21 (P\0.01). Since RANKL and OPG proteins measured in this study are extracellular proteins in the culture media, intracellular proteins such as action or glyceraldehyde 3-phosphate dehydrogenase are not suitable to be used as loading controls. Therefore, staining of total proteins on the stripped nitrocellulose membrane was performed with a protein stain kit that has been used previously.20,21 Loading control of total protein is shown in Figure 4, D. Visual assessment of protein loading was variable, possibly due to different background staining. The effects of VEGF on RANKL and OPG expressions in MC3T3-E1 cells are consistent with osteoclast formation induced by the conditioned media from MC3T3-E1 cells treated with VEGF or vehicle. DISCUSSION
This study shows that VEGF stimulated osteoblast proliferation, migration, and invasion potentials, and stimulated osteoclast formation from precursor cells indirectly through an increased RANKL/OPG ratio produced by osteoblasts. Since it was initially identified in 1989,24 VEGF has been found to play an important role in both angiogenesis and bone remodeling, including bone resorption and formation.25 In dentistry, VEGF has been shown to be a crucial factor in the pathogenesis of periodontal disease26 and orthodontic tooth movement.15,16 Several studies have shown that the expression of VEGF and its receptor VEGFR-1 are intensified by mechanical forces during orthodontic tooth movement,27-30 and VEGF mediates the induction of RANKL from osteoblastic cells by mechanical stress.30 VEGF also plays a key role in accelerated orthodontic tooth movement. Alveolar corticotomy, a procedure used clinically to accelerate orthodontic tooth movement in periodontally accelerated osteogenic orthodontics, significantly increased the expression of VEGF.31,32 Local injections of VEGF significantly increased the rate of orthodontic tooth movement,33
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Fig 1. VEGF promoted osteoblastic MC3T3-E1 cell proliferation, migration, and invasion. A, MC3T3-E1 cells in 96-well plates were cultured for 24 hours before incubation with BrdU for 2 hours. Cell proliferation was measured with ELISA for BrdU incorporation. VEGF at different concentrations significantly increased MC3T3-E1 proliferation compared with vehicle-treated cultures (n 5 3). B, MC3T3-E1 cells were plated in migration chambers with vehicle or VEGF (20 ng/mL) for 24 hours. Representative images and numbers of cells migrating through the chamber membranes are shown (n 5 3). C, MC3T3-E1 cells were plated in invasion chambers with Matrigel matrix with vehicle or VEGF (20 ng/mL) for 24 hours. Percentage of invasion 5 mean number of cells through Matrigel insert membrane/mean number of cells through control membrane. Relative invasion index 5 percentage of invasion of experimental (VEGF) cell/percentage of invasion of control (vehicle) cell (n 5 3). a, Significant effect of VEGF; P \0.01.
whereas neutralizing the antibody to VEGF had the opposite effect.16 VEGF accelerates orthodontic tooth movement by stimulating osteoclast formation and enhancing alveolar bone remodeling as shown by a previous study.33 Alveolar bone remodeling can be
activated with a full-thickness mucogingival flap,13 and VEGF was shown to play an essential role in the postflap healing procedure that led to activated bone remodeling.34 The bone remodeling level is the determining factor for the rate of orthodontic tooth
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Fig 2. VEGF had no direct effect on osteoclast differentiation in monocytic RAW264.7 cell cultures. RAW264.7 cells were cultured in 96-well plates for 3 to 5 days with vehicle, RANKL (25 ng/mL), VEGF (20 ng/mL), or RANKL (25 ng/mL) plus VEGF (20 ng/mL). Representative images of TRAP staining in the culture wells at each time point are shown. Counts of TRAP1 MNC in the cultures are shown in the Table.
Table. Osteoclast formation in RAW264.7 cell cul-
tures treated with RANKL (25 ng/mL), VEGF (20 ng/ mL), or RANKL (25 ng/mL) plus VEGF (20 ng/mL) Vehicle RANKL VEGF RANKL 1 VEGF
Day 3 0 61.3 6 7.8* 0 54.7 6 4.2*
Day 4 0 71.7 6 7.2* 0 79.3 6 4.7*
Day 5 0 23.6 6 4.0* 0 20.4 6 6.1*
Data are presented as means and standard deviations. No statistically significant difference was found between the RANKL and the RANKL 1 VEGF groups. *Significant difference from vehicle.
movement.5 Previous studies have demonstrated that VEGF increases the rate of orthodontic tooth movement by enhancing bone remodeling.15 Our study further elucidates the cellular and molecular mechanisms of VEGF's effects on bone remodeling. The osteoblastic reaction to VEGF stimulus plays a pivotal role in increasing bone remodeling. First, our study showed that VEGF significantly increased the RANKL/OPG ratio produced by the osteoblastic MC3T3-E1 cells by increasing RANKL expression and decreasing OPG expression. Consistently, conditioned media from MC3T3-E1 cells treated with VEGF significantly induced differentiation of osteoclasts from precursor RAW264.7 cells. RANKL is the chief factor determining the differentiation, function, and survival of osteoclasts,7 whereas OPG, a decoy receptor of RANKL, antagonizes the effects of RANKL.8 The RANKL/OPG ratio has been shown to determine osteoclast differentiation.9 Bone resorption by osteoclasts is the initial phase
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of the bone remodeling cycle. During this phase, bone mineral density decreases, causing transient osteopenia, which is believed to be the major factor facilitating accelerated orthodontic tooth movement.31 An important finding in this study was that the effects of VEGF to increase the RANKL/OPG ratio and osteoclast formation were more potent at earlier time points. This temporal pattern of the effects of VEGF is favorable for more bone resorption in the early phase of bone remodeling to accelerate orthodontic tooth movement. These effects become less significant during the bone remodeling process when the balance gradually shifts to more bone formation to replace the resorbed bone, indicating that accelerating orthodontic tooth movement by VEGF is time sensitive. An early time window is important to achieve accelerated tooth movement after VEGF administration, and repeated use of VEGF may be needed if prolonged acceleration is desired. On the other hand, VEGF does not directly induce osteoclast differentiation, nor does it increase RANKL-induced osteoclast formation, indicating that osteoblasts are needed for VEGF to induce osteoclast formation. A previous study on the effects of VEGF on MC3T3-E1 cells showed that VEGF significantly increased OPG at 72 hours, but it had no effect on RANKL.17 However, we did not examine the effects of VEGF on RANKL and OPG beyond 72 hours. Our investigation provides a clearer view of the longer effects of VEGF on osteoblastic RANKL and OPG expression and the effects on osteoclast formation, which is more clinically relevant. Second, we found that VEGF directly stimulated osteoblast proliferation, migration, and invasion potentials. As shown by earlier studies, VEGF induced
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Vehicle Or VEGF
CondiƟoned media at 4, 7, 14 or 21 Days TRAP staining at day 4
Raw264.7 Cells
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b b b
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a
250
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Fig 3. Conditioned media from VEGF-treated osteoblastic MC3T3-E1 cultures significantly induced osteoclast formation in monocytic RAW264.7 cultures. Conditioned media from MC3T3-E1 cultures in the presence (EXP) or absence (CTR) of VEGF (20 ng/mL) on days 4, 7, 14, and 21 were used to treat RAW264.7 cells for 4 days in 96-well plates. A, Experimental design; B, number of TRAP1 MNC in the cultures. Osteoclast formation was significantly increased in RAW265.7 cultures treated with conditioned media from MC3T3-E1 cells treated with VEGF (n 5 3). a, Significant effect of conditioned media from VEGF-treated MC3T3-E1 cultures (P \0.001); b, significant effect of time among the EXP groups (P \0.01); D, day.
more bone formation.35 Thus, increasing osteoblast proliferation is also important during the bone formation phase of the remodeling cycle to restore bone mass lost during the resorption phase, achieving balance between resorption and formation to preserve normal bone mass. Increased migration and invasion potentials of osteoblasts by VEGF may be important for the activated bone remodeling. By traveling to other portions of the bone in addition to the initial VEGF stimulation area through migration and invasion activities, osteoblasts can bring RANKL closer to more osteoclast precursor cells for their differentiation into functional osteoclasts, spreading the effects of VEGF to larger areas. In-vivo animal studies are needed to verify this hypothesis. Previous studies have shown that MC3T3-E1 cells express both VEGFR-1 and VEGFR-1 messenger RNA, and VEGF treatment significantly increased the level of VEGFR-2 messenger RNA in MC3T3-E1 cells for 24
to 72 hours.17 In this study, we also detected both VEGFR-1 and VEGFR-2 proteins with western blot. It is not uncommon that receptor expression is changed by the agonist. In this study, we did not measure the changes in the expression levels of the VEGF receptors in response to VEGF treatment or the intracellular mechanisms leading to the effects of VEGF on the osteoblastic MC3T3-E1 cells. The details of intracellular signaling are worth further investigation to determine the molecular mechanisms for the effects of VEGF on osteoblasts. CONCLUSIONS
This study shows that VEGF stimulates osteoclast formation by increasing the osteoblastic RANKL/OPG ratio and induces osteoblast proliferation, migration, and invasion potentials in vitro. These results provide insight into the mechanisms by which VEGF stimulates
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Fig 4. VEGF significantly increased RANKL and decreased OPG in conditioned media from osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in the presence (EXP) or absence (CTR) of VEGF (20 ng/mL) for 4, 7, 14, and 21 days in 6-well plates. We used 37.5 mL of media from each treatment group for the western blot test to measure A, RANKL; and B, OPG. Relative densities of bands were measured with control media at day 4 set as 1. C, The ratio of band density of RANKL/OPG at each time point (n 5 3). a, Significant effect of VEGF treatment (P \0.001); b, significant effect of time among EXP groups (P \0.01). D, Staining of total protein on the nitrocellulose membrane.
bone remodeling, which governs many physiologic and pathologic processes in dentistry from the speed of orthodontic tooth movement to periodontal disease, for
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example. Future studies are needed to examine the in-vivo effects of VEGF on bone cells and bone remodeling.
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19. Huang H, Chikazu D, Voznesensky OS, Herschman HR, Kream BE, Drissi H, et al. Parathyroid hormone induction of cyclooxygenase2 in murine osteoblasts: role of the calcium-calcineurin-NFAT pathway. J Bone Miner Res 2010;25:819-29. 20. Yabluchanskiy A, Ma Y, Chiao YA, Lopez EF, Voorhees AP, Toba H, et al. Cardiac aging is initiated by matrix metalloproteinase-9mediated endothelial dysfunction. Am J Physiol Heart Circ Physiol 2014;306:H1398-407. 21. McCurdy SM, Dai Q, Zhang J, Zamilpa R, Ramirez TA, Dayah T, et al. SPARC mediates early extracellular matrix remodeling following myocardial infarction. Am J Physiol Heart Circ Physiol 2011;301:H497-505. 22. Huang H, Shank G, Ma L, Tallents RH, Kyrkanides S. Nerve growth factor induced after temporomandibular joint inflammation decelerates chondrocyte differentiation. Oral Dis 2013; 19:604-10. 23. Brouxhon SM, Kyrkanides S, Teng X, O'Banion MK, Clarke R, Byers S, et al. Soluble-E-cadherin activates HER and IAP family members in HER21 and TNBC human breast cancers. Mol Carcinog 2014;53:893-906. 24. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306-9. 25. Clarkin CE, Gerstenfeld LC. VEGF and bone cell signalling: an essential vessel for communication? Cell Biochem Funct 2013; 31:1-11. 26. Matarese G, Isola G, Anastasi GP, Favaloro A, Milardi D, Vermiglio G, et al. Immunohistochemical analysis of TGF-beta1 and VEGF in gingival and periodontal tissues: a role of these biomarkers in the pathogenesis of scleroderma and periodontal disease. Int J Mol Med 2012;30:502-8. 27. Kaku M, Motokawa M, Tohma Y, Tsuka N, Koseki H, Sunagawa H, et al. VEGF and M-CSF levels in periodontal tissue during tooth movement. Biomed Res 2008;29:181-7. 28. Miyagawa A, Chiba M, Hayashi H, Igarashi K. Compressive force induces VEGF production in periodontal tissues. J Dent Res 2009;88: 752-6. 29. Kohno T, Matsumoto Y, Kanno Z, Warita H, Soma K. Experimental tooth movement under light orthodontic forces: rates of tooth movement and changes of the periodontium. J Orthod 2002;29: 129-35. 30. Nakai T, Yoshimura Y, Deyama Y, Suzuki K, Iida J. Mechanical stress up-regulates RANKL expression via the VEGF autocrine pathway in osteoblastic MC3T3-E1 cells. Mol Med Rep 2009;2:229-34. 31. Wang L, Lee W, Lei DL, Liu YP, Yamashita DD, Yen SL. Tisssue responses in corticotomy- and osteotomy-assisted tooth movements in rats: histology and immunostaining. Am J Orthod Dentofacial Orthop 2009;136:770.e1-11:discussion, 70-1. 32. Iino S, Sakoda S, Ito G, Nishimori T, Ikeda T, Miyawaki S. Acceleration of orthodontic tooth movement by alveolar corticotomy in the dog. Am J Orthod Dentofacial Orthop 2007;131:448.e1-8. 33. Kohno S, Kaku M, Tsutsui K, Motokawa M, Ohtani J, Tenjo K, et al. Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement. J Dent Res 2003;82:177-82. 34. Nobuto T, Suwa F, Kono T, Taguchi Y, Takahashi T, Kanemura N, et al. Microvascular response in the periosteum following mucoperiosteal flap surgery in dogs: angiogenesis and bone resorption and formation. J Periodontol 2005;76:1346-53. 35. Street J, Lenehan B. Vascular endothelial growth factor regulates osteoblast survival—evidence for an autocrine feedback mechanism. J Orthop Surg Res 2009;4:19.
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Effects of vascular endothelial growth factor on osteoblasts and osteoclasts Hechang Huang,a Li Ma,b and Stephanos Kyrkanidesc Stony Brook, NY
Introduction: Bone remodeling is crucial to the success of many dental procedures and is tightly regulated. Vascular endothelial growth factor (VEGF), a key cytokine for angiogenesis, is also an important regulator of bone remodeling. We aimed to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on cultured osteoblasts and osteoclasts. Methods: Preosteoblastic MC3T3-E1 cells were treated with vehicle or VEGF-A165. Cell proliferation, migration, and invasion potentials were assessed. Preosteoclastic RAW264.7 cells were treated with vehicle or VEGF with or without the receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoclast formation was measured with tartrate-resistant acid phosphatase staining. Conditioned media from vehicle-treated or VEGF-treated MC3T3-E1 cells were tested for the levels of RANKL and osteoprotegerin (OPG) and were used to treat RAW264.7 cells to observe osteoclast formation. Results: VEGF significantly induced MC3T3-E1 cell proliferation, migration, and invasion. VEGF did not directly induce osteoclastogenesis but significantly increased the RANKL/OPG ratio in the conditioned media from the MC3T3-E1 cultures; this significantly increased osteoclast formation. Conclusions: VEGF stimulates osteoclast differentiation by increasing the osteoblastic RANKL/OPG ratio but has no direct effect on osteoclast precursor cells, and it induces osteoblast proliferation, migration, and invasion potentials in vitro. (Am J Orthod Dentofacial Orthop 2016;149:366-73)
B
one remodeling is the lifelong continuous replacement of old bone tissue with new bone1,2 and is crucial to the success of a variety of dental procedures, including dental implants,3 periodontal disease treatment,4 and orthodontic tooth movement.5 It is a tightly coupled local process starting with bone resorption, followed by reversal and bone formation phases. Osteoclasts and osteoblasts are organized as basic multicellular units and cooperate to perform the resorption-reversalformation sequence of the bone remodeling process.6 The level of bone remodeling is closely regulated by hormones, cytokines, prostaglandins, and mechanical From the Department of Orthodontics and Pediatric Dentistry, School of Dental Medicine, State University of New York at Stony Brook, Stony Brook, NY. a Assistant professor. b Research assistant professor. c Professor and chair. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Address correspondence to: Hechang Huang, Department of Orthodontics and Pediatric Dentistry, School of Dental Medicine, State University of New York at Stony Brook, Rockland Hall, Stony Brook, NY 11794-8700; e-mail, hechang. [email protected]. Submitted, September 2014; revised and accepted, September 2015. 0889-5406/$36.00 Copyright Ó 2016 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2015.09.021
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loading.2 Receptor activator of nuclear factor-kappa B ligand (RANKL), an osteoblastic cell-derived factor, is crucial for osteoclast differentiation, survival, and function by binding to osteoclast cell-surface receptor, its receptor on preosteoclasts.7 Osteoprotegerin (OPG), another osteoblastic cell-derived factor, interrupts the RANKL/osteoclast cell-surface receptor binding as a decoy receptor of RANKL, inhibiting osteoclastogenesis.8 Therefore, the RANKL/OPG ratio largely determines the formation of functional osteoclasts and the activation of the resorption phase of bone remodeling.9 The expression of RANKL and OPG by osteoblastic cells is regulated by various systemic hormones and local factors.2 Orthodontic treatment duration is a major concern for patients and is closely associated with various side effects, including external root resorption,10 carious lesions,11 and open gingival embrasures.12 According to the American Association of Orthodontists, about 4 million people in this country receive orthodontic treatment each year. Accelerating tooth movement can therefore benefit millions of patients by reducing their treatment durations and side effects. The rate of orthodontic tooth movement is largely determined by bone remodeling.5 A full-thickness mucoperiosteal flap alone was sufficient to induce de novo regional
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acceleratory phenomenon, a significantly increased bone remodeling activity as part of the trauma healing process.13 Vascular endothelial growth factor (VEGF) was shown to play an essential role in the postflap healing procedure that led to activated bone remodeling.14 Local injections of VEGF in mice significantly increased the numbers of osteoclasts and the rate of orthodontic tooth movement,15 whereas neutralizing the antiVEGF antibody had the opposite effect.16 A previous study showed that VEGF promotes bone remodeling primarily by reducing OPG, but it had no effect on RANKL in osteoblastic cell cultures up to 72 hours. The long-term effects of VEGF on osteoclast and osteoblast formation and activities, and on bone remodeling, are yet to be elucidated.17 The aim of this study was to examine the mechanisms by which VEGF induces bone remodeling by studying its effects on osteoblasts and osteoclasts in vitro. MATERIAL AND METHODS
MC3T3-E1 subclone 4 cells, subcloned by Dr Renny Franceschi at the University of Michigan from the original immortalized neonatal murine calvarial cells, were purchased (CRL-2593; American Type Culture Collection, Manassas, Va).18 The cells were grown in alpha minimum essential medium (A1049001; Gibco BRL, Gaithersburg, Md); 10% heat-inactivated fetal calf serum (Gibco BRL), penicillin (100 U/mL), and streptomycin (100 mg/mL) were added to the media. Cells (5000/cm2) were plated in 6-well dishes in a humidified atmosphere of 5% carbon dioxide at 37 C and grown until confluent. RAW264.7 cells, a tumor cell line induced by Abelson murine leukemia virus, were purchased (TIB-71; American Type Culture Collection). Cells were grown in Dulbecco's Modified Eagle's Medium (30-2002; American Type Culture Collection). We added 10% heatinactivated fetal calf serum, penicillin (100 U/mL), and streptomycin (100 mg/mL) to the media. The cells were plated in 96-well plates in a humidified atmosphere of 5% carbon dioxide at 37 C. Formation of tartrateresistant acid phosphatase positive multinucleated cells (TRAP1 MNC) was assessed with a commercial kit (F4523; Sigma-Aldrich, St Louis, Mo). For the western blot analysis, protein from MC3T3E1 cell cultures was extracted with radioimmunoprecipitation assay buffer following the manufacturer's instructions (Santa Cruz Biotechnology, Santa Cruz, Calif). Protein concentrations were measured by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, Mass). Conditioned media from MC3T3-E1 cells were filtered with a 0.44-mm filter under sterile conditions and stored at –80 C. The same experimental procedures
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were followed as published previously.19 Briefly, equal amounts (100 mg) of protein (for VEGF receptor-1 [VEGRF-1] and VEGF receptor-2 [VEGFR-2]) or equal volumes (25 mL) of media (for RANKL and OPG) were used for 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif). The total protein on the nitrocellulose membrane was stained with a protein stain kit (24580; Thermo Scientific, Rockford, Ill), which has been used in previous studies.20,21 Membranes were washed with trisbuffered saline solution (pH 7.6) before blocking with 5% weight per volume of nonfat dry milk in 1 3 trisbuffered saline solution containing 0.05% Tween 20 (Sigma-Aldrich) for 60 minutes. Then the membranes were incubated with primary antibody of VEGFR-1 and VEGFR-2 for cell extracts and RANKL or OPG for media samples, washed with tris-buffered saline solution containing 0.05% Tween 20, and incubated with horseradish peroxidase-conjugated secondary antibody (1:1000). The signal was detected with LumiGLO chemiluminescent reagent (Cell Signaling Technology, Danvers, Mass). Primary antibodies, including VEGFR-1 (AF471; R&D Systems, Minneapolis, Minn), VEGFR-2 (AF644; R&D Systems), RANKL (AF462; R&D Systems), or OPG (AF459; R&D Systems), were incubated at 0.1 mg per milliliter overnight at 4 C. Cell proliferation was tested with the 5-bromo-20 deoxyuridine (BrdU) assay. MC3T3-E1 cells (1 3 103 cells per well) were plated in 96-well plates in the media described above. Vehicle or VEGF (10, 20, or 40 ng/mL) was added at the beginning of cell plating and treated for 24 hours. The BrdU assay followed the same procedures as published previously.22 Briefly, the cells were fixed, and the DNA was denatured after removing the labeling medium. Then anti-BrdUperoxidase antibody was added. Substrate reactions to detect the immune complexes were quantified by measuring the absorbance with a spectrophotometer. For cell migration and invasion assessment, we used 8.0-mm-pore BD BioCoat Control Insert 24-well plates (354578; BD Bioscience, San Jose, Calif) and Matrigel Invasion Chamber 24-well plates (354480; BD Biosciences) to measure migration and invasion of MC3T3-E1 cells, respectively. We plated 2 3 105 cells in 0.4% fetal bovine serum medium in the top chamber after it was collected and washed. Vehicle or VEGF (20 ng/mL) was added to the bottom chamber. Cell migration and invasion assessment followed the same experimental procedures as published before.23 Briefly, cells on the top surface were removed after 22 hours. Cells on the lower surface were stained with 0.5% crystal violet after being fixed.
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Then the stained cells were photographed under bright field microscopy. Ten random high-power fields per insert were used to count migrated or invaded cells, and averages were calculated. Fold changes of the numbers of migrated or invaded cells to the untreated controls in triplicate experiments were determined. RESULTS
For VEGF stimulated osteoblastic MC3T3-E1 cell proliferation, migration, and invasion, the expression of VEGF receptors by MC3T3-E1 cells was assessed by western immunoblotting. These cells express VEGFR-1 and VEGFR-2 (data not shown). Proliferation analysis by the BrdU assay showed that VEGF at 10 ng per milliliter did not affect MC3T3-E1 cell proliferation (Fig 1, A; P .0.05), whereas VEGF at 20 or 40 ng per milliliter significantly stimulated MC3T3-E1 cell proliferation (Fig 1, A; 3.60- and 3.98-fold, respectively; P \0.01). In addition, VEGF (20 ng/mL) significantly increased MC3T3-E1 cell migration (Fig 1, B; 2.29-fold; P \0.01) and invasion (Fig 1, C; 15.74-fold; P \0.001) compared with the vehicle-treated cells. VEGF did not directly affect osteoclast formation in RAW264.7 cell cultures. To test the direct effects of VEGF on osteoclast formation, we first treated osteoclastic precursor RAW264.7 cells directly with VEGF (20 ng/mL). RAW264.7 cells can differentiate into mature osteoclasts by RANKL without macrophage colony-stimulating factor. As expected, RANKL induced TRAP1 MNC formation used here as the control. VEGF treatment alone did not stimulate any osteoclast formation, nor did VEGF increase osteoclast formation induced by RANKL (Fig 2). TRAP1 MNC counts are shown in the Table. Conditioned media from VEGF-treated MC3T3-E1 cells stimulated osteoclast formation in RAW264.7 cell cultures. Osteoclast formation in RAW264.7 cells was also assessed with conditioned media from osteoblastic MC3T3-E1 cells previously treated with VEGF (20 ng/ mL) or vehicle for 4, 7, 14, and 21 days (Fig 3, A). Conditioned media from MC3T3-E1 cells treated with VEGF at all time points significantly increased TRAP1 MNC formation, whereas those from the control group had a minimum effect (Fig 3, B; P \0.001) at all time points. Among the RAW264.7 cultures treated with conditioned media from VEGF-treated MC3T3-E1 cells, osteoclast formation was significantly higher at the earlier times of days 4 and 7 compared with the later times of days 14 and 21 (Fig 3, B; P \0.01). VEGF induced RANKL and reduced OPG expression in MC3T3-E1 cells. We measured RANKL and OPG
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protein levels in the conditioned media from osteoblastic MC3T3-E1 cultures with western blot. We found that VEGF treatment for 4, 7, 14, and 21 days significantly increased RANKL expression by MC3T3-E1 cells (Fig 4, A; 1.36 6 0.19, 1.94 6 0.25, 1.28 6 0.22, and 1.03 6 0.04-fold, respectively), while potently inhibiting OPG expression (Fig 4, B; 0.15 6 0.04, 0.37 6 0.01, 0.41 6 0.02, 0.43 6 0.06-fold, respectively), resulting in a significantly increased RANKL/OPG ratio in the media (Fig 4, C; 9.46 6 2.04, 5.21 6 0.75, 3.12 6 0.64, 2.42 6 0.40-fold, respectively). The effects of VEGF to increase the RANKL/OPG ratio were more significant at the earlier times of days 4 and 7 than at the later times of days 14 and 21 (P\0.01). Since RANKL and OPG proteins measured in this study are extracellular proteins in the culture media, intracellular proteins such as action or glyceraldehyde 3-phosphate dehydrogenase are not suitable to be used as loading controls. Therefore, staining of total proteins on the stripped nitrocellulose membrane was performed with a protein stain kit that has been used previously.20,21 Loading control of total protein is shown in Figure 4, D. Visual assessment of protein loading was variable, possibly due to different background staining. The effects of VEGF on RANKL and OPG expressions in MC3T3-E1 cells are consistent with osteoclast formation induced by the conditioned media from MC3T3-E1 cells treated with VEGF or vehicle. DISCUSSION
This study shows that VEGF stimulated osteoblast proliferation, migration, and invasion potentials, and stimulated osteoclast formation from precursor cells indirectly through an increased RANKL/OPG ratio produced by osteoblasts. Since it was initially identified in 1989,24 VEGF has been found to play an important role in both angiogenesis and bone remodeling, including bone resorption and formation.25 In dentistry, VEGF has been shown to be a crucial factor in the pathogenesis of periodontal disease26 and orthodontic tooth movement.15,16 Several studies have shown that the expression of VEGF and its receptor VEGFR-1 are intensified by mechanical forces during orthodontic tooth movement,27-30 and VEGF mediates the induction of RANKL from osteoblastic cells by mechanical stress.30 VEGF also plays a key role in accelerated orthodontic tooth movement. Alveolar corticotomy, a procedure used clinically to accelerate orthodontic tooth movement in periodontally accelerated osteogenic orthodontics, significantly increased the expression of VEGF.31,32 Local injections of VEGF significantly increased the rate of orthodontic tooth movement,33
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Fig 1. VEGF promoted osteoblastic MC3T3-E1 cell proliferation, migration, and invasion. A, MC3T3-E1 cells in 96-well plates were cultured for 24 hours before incubation with BrdU for 2 hours. Cell proliferation was measured with ELISA for BrdU incorporation. VEGF at different concentrations significantly increased MC3T3-E1 proliferation compared with vehicle-treated cultures (n 5 3). B, MC3T3-E1 cells were plated in migration chambers with vehicle or VEGF (20 ng/mL) for 24 hours. Representative images and numbers of cells migrating through the chamber membranes are shown (n 5 3). C, MC3T3-E1 cells were plated in invasion chambers with Matrigel matrix with vehicle or VEGF (20 ng/mL) for 24 hours. Percentage of invasion 5 mean number of cells through Matrigel insert membrane/mean number of cells through control membrane. Relative invasion index 5 percentage of invasion of experimental (VEGF) cell/percentage of invasion of control (vehicle) cell (n 5 3). a, Significant effect of VEGF; P \0.01.
whereas neutralizing the antibody to VEGF had the opposite effect.16 VEGF accelerates orthodontic tooth movement by stimulating osteoclast formation and enhancing alveolar bone remodeling as shown by a previous study.33 Alveolar bone remodeling can be
activated with a full-thickness mucogingival flap,13 and VEGF was shown to play an essential role in the postflap healing procedure that led to activated bone remodeling.34 The bone remodeling level is the determining factor for the rate of orthodontic tooth
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Fig 2. VEGF had no direct effect on osteoclast differentiation in monocytic RAW264.7 cell cultures. RAW264.7 cells were cultured in 96-well plates for 3 to 5 days with vehicle, RANKL (25 ng/mL), VEGF (20 ng/mL), or RANKL (25 ng/mL) plus VEGF (20 ng/mL). Representative images of TRAP staining in the culture wells at each time point are shown. Counts of TRAP1 MNC in the cultures are shown in the Table.
Table. Osteoclast formation in RAW264.7 cell cul-
tures treated with RANKL (25 ng/mL), VEGF (20 ng/ mL), or RANKL (25 ng/mL) plus VEGF (20 ng/mL) Vehicle RANKL VEGF RANKL 1 VEGF
Day 3 0 61.3 6 7.8* 0 54.7 6 4.2*
Day 4 0 71.7 6 7.2* 0 79.3 6 4.7*
Day 5 0 23.6 6 4.0* 0 20.4 6 6.1*
Data are presented as means and standard deviations. No statistically significant difference was found between the RANKL and the RANKL 1 VEGF groups. *Significant difference from vehicle.
movement.5 Previous studies have demonstrated that VEGF increases the rate of orthodontic tooth movement by enhancing bone remodeling.15 Our study further elucidates the cellular and molecular mechanisms of VEGF's effects on bone remodeling. The osteoblastic reaction to VEGF stimulus plays a pivotal role in increasing bone remodeling. First, our study showed that VEGF significantly increased the RANKL/OPG ratio produced by the osteoblastic MC3T3-E1 cells by increasing RANKL expression and decreasing OPG expression. Consistently, conditioned media from MC3T3-E1 cells treated with VEGF significantly induced differentiation of osteoclasts from precursor RAW264.7 cells. RANKL is the chief factor determining the differentiation, function, and survival of osteoclasts,7 whereas OPG, a decoy receptor of RANKL, antagonizes the effects of RANKL.8 The RANKL/OPG ratio has been shown to determine osteoclast differentiation.9 Bone resorption by osteoclasts is the initial phase
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of the bone remodeling cycle. During this phase, bone mineral density decreases, causing transient osteopenia, which is believed to be the major factor facilitating accelerated orthodontic tooth movement.31 An important finding in this study was that the effects of VEGF to increase the RANKL/OPG ratio and osteoclast formation were more potent at earlier time points. This temporal pattern of the effects of VEGF is favorable for more bone resorption in the early phase of bone remodeling to accelerate orthodontic tooth movement. These effects become less significant during the bone remodeling process when the balance gradually shifts to more bone formation to replace the resorbed bone, indicating that accelerating orthodontic tooth movement by VEGF is time sensitive. An early time window is important to achieve accelerated tooth movement after VEGF administration, and repeated use of VEGF may be needed if prolonged acceleration is desired. On the other hand, VEGF does not directly induce osteoclast differentiation, nor does it increase RANKL-induced osteoclast formation, indicating that osteoblasts are needed for VEGF to induce osteoclast formation. A previous study on the effects of VEGF on MC3T3-E1 cells showed that VEGF significantly increased OPG at 72 hours, but it had no effect on RANKL.17 However, we did not examine the effects of VEGF on RANKL and OPG beyond 72 hours. Our investigation provides a clearer view of the longer effects of VEGF on osteoblastic RANKL and OPG expression and the effects on osteoclast formation, which is more clinically relevant. Second, we found that VEGF directly stimulated osteoblast proliferation, migration, and invasion potentials. As shown by earlier studies, VEGF induced
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A
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Vehicle Or VEGF
CondiƟoned media at 4, 7, 14 or 21 Days TRAP staining at day 4
Raw264.7 Cells
MC3T3-E1 Cells
B
b b b
TRAP+ MNC
300
b
a
250
a
CTR
200
EXP
a
150
a
100 50 0
D4 Media
D7 Media
D14 Media D21 Media
Fig 3. Conditioned media from VEGF-treated osteoblastic MC3T3-E1 cultures significantly induced osteoclast formation in monocytic RAW264.7 cultures. Conditioned media from MC3T3-E1 cultures in the presence (EXP) or absence (CTR) of VEGF (20 ng/mL) on days 4, 7, 14, and 21 were used to treat RAW264.7 cells for 4 days in 96-well plates. A, Experimental design; B, number of TRAP1 MNC in the cultures. Osteoclast formation was significantly increased in RAW265.7 cultures treated with conditioned media from MC3T3-E1 cells treated with VEGF (n 5 3). a, Significant effect of conditioned media from VEGF-treated MC3T3-E1 cultures (P \0.001); b, significant effect of time among the EXP groups (P \0.01); D, day.
more bone formation.35 Thus, increasing osteoblast proliferation is also important during the bone formation phase of the remodeling cycle to restore bone mass lost during the resorption phase, achieving balance between resorption and formation to preserve normal bone mass. Increased migration and invasion potentials of osteoblasts by VEGF may be important for the activated bone remodeling. By traveling to other portions of the bone in addition to the initial VEGF stimulation area through migration and invasion activities, osteoblasts can bring RANKL closer to more osteoclast precursor cells for their differentiation into functional osteoclasts, spreading the effects of VEGF to larger areas. In-vivo animal studies are needed to verify this hypothesis. Previous studies have shown that MC3T3-E1 cells express both VEGFR-1 and VEGFR-1 messenger RNA, and VEGF treatment significantly increased the level of VEGFR-2 messenger RNA in MC3T3-E1 cells for 24
to 72 hours.17 In this study, we also detected both VEGFR-1 and VEGFR-2 proteins with western blot. It is not uncommon that receptor expression is changed by the agonist. In this study, we did not measure the changes in the expression levels of the VEGF receptors in response to VEGF treatment or the intracellular mechanisms leading to the effects of VEGF on the osteoblastic MC3T3-E1 cells. The details of intracellular signaling are worth further investigation to determine the molecular mechanisms for the effects of VEGF on osteoblasts. CONCLUSIONS
This study shows that VEGF stimulates osteoclast formation by increasing the osteoblastic RANKL/OPG ratio and induces osteoblast proliferation, migration, and invasion potentials in vitro. These results provide insight into the mechanisms by which VEGF stimulates
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Fig 4. VEGF significantly increased RANKL and decreased OPG in conditioned media from osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in the presence (EXP) or absence (CTR) of VEGF (20 ng/mL) for 4, 7, 14, and 21 days in 6-well plates. We used 37.5 mL of media from each treatment group for the western blot test to measure A, RANKL; and B, OPG. Relative densities of bands were measured with control media at day 4 set as 1. C, The ratio of band density of RANKL/OPG at each time point (n 5 3). a, Significant effect of VEGF treatment (P \0.001); b, significant effect of time among EXP groups (P \0.01). D, Staining of total protein on the nitrocellulose membrane.
bone remodeling, which governs many physiologic and pathologic processes in dentistry from the speed of orthodontic tooth movement to periodontal disease, for
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example. Future studies are needed to examine the in-vivo effects of VEGF on bone cells and bone remodeling.
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