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Effects of angiotensin II type I receptor blocker losartan on orthodontic tooth movement
ORIGINAL ARTICLE
Effects of angiotensin II type I receptor blocker losartan on orthodontic tooth movement Adriana Pedrosa Moura,a Carina Cristina Montalvany-Antonucci,a Silvana Rodrigues de Albuquerque Taddei,b udia Cristina Biguetti,d Gustavo Pompermayer Garlet,e Celso Martins Queiroz-Junior,c Cla f Anderson Jose Ferreira, Mauro Martins Teixeira,g Tarcılia Aparecida Silva,h and Ildeu Andrade, Jri Belo Horizonte, Minas Gerais, Salvador, Bahia, and Bauru, S~ ao Paulo, Brazil
Introduction: Drugs that block the renin-angiotensin system (RAS) are widely used for treating hypertension, heart and kidney failure, and the harmful effects of diabetes. Components of the RAS have been identified in various organs, but little is known of their effects on bone remodeling. The aim of this study was to evaluate whether the blockage of the RAS influences strain-induced bone remodeling in a model of orthodontic tooth movement. Methods: An orthodontic appliance was placed in C57BL6/J mice that were randomly divided into 2 groups: vehicle-treated mice (VH) and mice treated with losartan (an angiotensin II receptor blocker). Orthodontic tooth movement and the number of tartrate-resistant acid phosphatase-positive cells were determined by histopathologic analysis. The expression of mediators involved in bone remodeling was evaluated by quantitative real-time polymerase chain reaction. Blood pressure was measured before and during the experimental period. Results: Orthodontic tooth movement and tartrate-resistant acid phosphatase-positive cells were significantly reduced in the losartan group compared with the VH group. mRNA levels of osteoclast markers (RANK, RANKL, cathepsin K, and metalloproteinase 13) were lower in the losartan mice than in the VH group, whereas the expressions of osteoblast markers and negative regulators of bone resorption (periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, metalloproteinase 2, and osteoprotegerin) were higher in the VH group. Conclusions: Blockage of the RAS system decreases osteoclast differentiation and activity and, consequently, results in decreased strain-induced bone remodeling in orthodontic tooth movement. (Am J Orthod Dentofacial Orthop 2016;149:358-65)
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he renin-angiotensin system (RAS) is classically known as a circulating endocrine system regulating blood pressure and electrolyte homeostasis.1,2 The main effector peptide in this system is angiotensin II (ANG II), which is formed from angiotensin I by an angiotensin-converting enzyme, a
key molecule in this system that interferes in prostaglandin and nitric oxide production.3 This precursor, angiotensin I, is cleaved from angiotensinogen by renin, a highly selective protease secreted from the juxtaglomerular cells in the kidneys. ANG II exerts its biologic effects through binding to 2 specific
a Resident, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. b Assistant professor, Department of Cellular Biology, Institute of Biological Sciences, Federal University of Bahia, Salvador, Bahia, Brazil. c Postdoctoral resident, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. d Resident, Department of Biological Science, Faculty of Dentistry, University of S~ao Paulo, Bauru, S~ao Paulo, Brazil. e Associate professor, Department of Biological Science, Faculty of Dentistry, University of S~ao Paulo, Bauru, S~ao Paulo, Brazil. f Associate professor, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. g Full professor, Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. h Associate professor, Department of Oral Pathology, Faculty of Dentistry, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
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Associate professor, Department of Orthodontics, School of Dentistry, Pontifıcal Catholic University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Adriana Pedrosa Moura and Carina Cristina Montalvany-Antonucci are joint first authors and contributed equally to this work. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Supported by the Fundac¸~ao de Amparo a Pesquisas do Estado de Minas Gerais and the Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico. Address correspondence to: Ildeu Andrade, Jr, Department of Orthodontics, School of Dentistry, Pontifıcal Catholic University of Minas Gerais, Av. Dom Jose Gaspar 500, CEP 31.270-901, Belo Horizonte, Minas Gerais, Brazil; e-mail, [email protected]. Submitted, August 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.2015.09.019
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angiotensin receptors—angiotensin type 1 receptors (AT1) and angiotensin type 2 receptor (AT2)—that are expressed in many cell types, including osteoclasts and osteoblasts.4 Drugs that block the RAS, such as losartan (LOS), act by blocking the AT1 receptors5 and are a main way to control high blood pressure (hypertension), heart failure, kidney failure, and the harmful effects of diabetes.1,5,6 The relationship between the RAS and bone metabolism is mainly based on the regulation of ANG II on bone metabolism.7-9 Different components of the RAS have been found to be synthesized and active in osteoblasts and in osteoclasts,10-12 and ANG II has been shown to stimulate bone resorption in osteoblast and osteoclast cocultures.9,10 A previous study has also demonstrated expression of the RAS components in osteoblasts and osteoclasts in vivo.12 Further evidence for a potential role of the RAS in bone metabolism comes from clinical studies. Patients treated with an angiotensin-converting enzyme inhibitor showed increased bone mineral density and reduced fracture risk.8,13,14 Moreover, previous animal studies have shown that excessive activation of RAS causes osteoporosis, mainly by elevation of osteoclastic bone resorption.15,16 Along with other factors, ANG II can upregulate the receptor activator of nuclear factor kappa-B ligand (RANKL) expression and downregulate the expression of runt-related transcription factor 2 (RUNX2). These key mediators are responsible for controlling osteoblast and osteoclast differentiation and regulating bone cell activity, leading to reduced bone formation and enhancing bone reabsorption.17 As described above, the bone does express components of the RAS, but little is known about the role of the RAS in the context of orthodontic tooth movement (OTM). The aim of this study was to evaluate the effects of blockers of the RAS on osteoclast recruitment and activity using a well-established mouse model of OTM. We hypothesized that the inhibition of the RAS pathway would impair the expression of proinflammatory cytokines, decrease osteoclast recruitment, and consequently reduce OTM. MATERIAL AND METHODS
Forty 10-week-old male C57BL6/J mice were used in this experiment. They were randomly divided into 2 groups: (1) treated with tap water (VH) as the control group, and (2) treated with 10 mg per kilogram per day of LOS (Sigma-Aldrich, St Louis, Mo). The sample size calculation was based on a previous study from our group.18 No significant weight loss was observed. All treatments followed the ethical regulations of the
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institutional ethics committee of the Pontifıcal Catholic University of Minas Gerais. Daily gavages of 100 mL of 10 mg per kilogram per day of LOS or tap water were administered to the LOS and VH groups, respectively. The drug was administered for 6 or 12 days for the histopathologic assays and for 0 or 12 hours for the molecular analyses. Tooth movement was induced as previously described.19,20 Briefly, the mice were anesthetized with 0.2 mL of a xylazine (0.02 mg/mL) and ketamine (50 mg/mL) solution. An orthodontic appliance consisting of a nickel-titanium 0.25 3 0.76-mm coil spring (Lancer Orthodontics, San Marcos, Calif) was bonded by light-cured resin (Transbond; 3M Unitek, Monrovia, Calif) between the maxillary right first molar and both maxillary incisors (Fig 1). The magnitude of force was calibrated by a tension gauge (Shimpo Instruments, Itasca, Ill) to exert a force of 35 g in the mesial direction. No reactivation was performed during the entire experimental period. For the histopathologic analysis, the left side of the maxilla (without an orthodontic appliance) was used as the control. The mice were killed after 6 or 12 days for the histopathologic analysis or after 0 or 12 hours for the molecular analysis. For each set of experiments, 5 animals were used at each time point. As was previously described, the right and left maxillae halves were fixed in 10% buffered formalin (pH 7.4), decalcified in 14% ethylenediaminetetraacetic acid (pH 7.4) for 20 days, and embedded in paraffin.19,20 The samples were cut into sagittal sections 5 mm thick. The sections were stained with tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich), counterstained with hematoxylin, and used for the histologic examinations. The mesial periodontal site of the distobuccal root of the first molar was used for osteoclast counts on 5 sections per animal. Osteoclasts were identified as TRAP-positive, multinucleated cells on the bone surface. Measurement of OTM was performed as previously described.19,20 Images of the first and second molars were obtained using an optical microscope (Axioskop 40; Carl Zeiss, Gottingen, Germany) and an adapted digital camera (PowerShot A620; Canon, Tokyo, Japan). ImageJ software (National Institutes of Health) was used to quantify the degree of OTM by measuring the distance between the cementoenamel junction of the first and second molars on the right hemimaxilla in relation to the same measurements for the left hemimaxilla. Five vertical sections per animal were evaluated, and 3 measurements were made for each evaluation; the variability was less than 5%. RNA extraction and real-time polymerase chain reaction were performed for each animal separately, as
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Fig 1. A, Occlusal view of the maxillae before placing the orthodontic device; B, a nickel-titanium opencoil spring was placed between the maxillary right first molar and incisor.
previously described.19,20 The periodontal ligament and the surrounding alveolar bone were extracted from the maxillary first molars with a stereomicroscope. All gingival tissues, oral mucosa, and teeth were discarded. The periodontal tissues and the alveolar bone that were extracted from the distal area of the distobuccal root of the maxillary first molar were considered the tension site. The mesial area of the same root was considered the compression site. The bone samples (4-8 mg) were frozen in liquid nitrogen after collection and then stored at –80 C. Previous to mRNA extraction, each sample was mechanically fragmented in liquid nitrogen using a bone chisel and homogenized in sterile Milli-Q water with Ultra Turrax (IKA Laboratory Equipment, Staufen, Germany), and subsequently submitted to RNA extraction using TRIZOL reagent (Life Technologies, Carlsbad, Calif), following the manufacturer's instructions. The mRNA quality and RNA integrity number were assessed with the BioAnalyzer (Agilent, Santa Clara, Calif). The RNA integrity number results ranged between 7.25 and 8.66 (mean, 7.87; SD, 0.36). Complementary DNA was synthesized using 2 mg of RNA through a reverse transcription reaction (Superscript II; Life Technologies). The targets included molecules known to regulate osteoclast functions: RANK (receptor activator of nuclear factor kappa-B), RANKL, osteoprotegerin, cathepsin K, and metalloproteinase 13. We also analyzed the expression of molecules known to regulate osteoblast functions: periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, and metalloproteinase 2. The analyses were
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performed in ViiA7 equipment (Applied Biosystems, Warrington, United Kingdom) with real-time polymerase chain reaction using TaqMan chemistry (Life Technologies) with inventoried optimized primers/probes sets (Life Technologies) as follows: molecules known to regulate osteoclast functions: RANK (catalog number 433118), RANKL (catalog number 4331182), osteoprotegerin (catalog number 433118), cathepsin K (catalog number 4331182), and metalloproteinase 13 (catalog number 4331182); and molecules known to regulate osteoblast functions: periostin (catalog number 4331182), dentin matrix protein (catalog number 4331182), alkaline phosphatase (catalog number 4331182), collagen 1A1 (catalog number 4331182), semaphorin 3A3 (catalog number 4331182), and metalloproteinase 2 (catalog number 4331182). The basic reaction conditions (40 cycles) were 95 C (10 minutes), 94 C (1 minute), 56 C (1 minute), and 72 C (2 minutes). The mean cycle threshold (Ct) values from duplicate measurements were used to calculate the expression of the target gene, with normalization to an internal control (b-actin) using the 2 DDCt formula. Some researchers have used a dosage of antihypertensive drugs that promotes a reduction in blood pressure by 27%.21,22 In our study, we chose to use a dose to keep the blood pressure stable to eliminate bias. The mean arterial pressure measurement was evaluated by the tail-cuff method, which is a noninvasive computerized system for measuring blood pressure (Kent Scientific, Torrington, Conn). This tail-cuff blood pressure system uses volume-pressure recording technology to measure the blood pressure in the mouse's tail. The
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Table. Systolic blood pressure evaluation Systolic blood pressure (mm Hg) Day 1 day before 1 day after 6 days after 12 days after
Vehicle-treated mice 80.7 6 2.7 77.3 6 3.1 78.6 6 2.1 79.8 6 2.6
Losartan-treated mice 81.2 6 1.9 78.3 6 2.1 79.3 6 2.6 80.8 6 2.4
Systolic blood pressure was evaluated (n 5 5 mice per group) by tailcuff plethysmography 24 hours before, and 6 and 12 days after mechanical loading in vehicle-treated and losartan-treated mice (10 mg/kg). The results show the means and standard deviations. Intergroup and intragroup analyses had no significant difference. P .0.05.
animals were acclimated to the restraint and tail-cuff inflation for 1 day before the experiments. The restraint platform was maintained at approximately 32 C to 34 C. For each section, the mouse was placed in an acrylic box restraint, and the tail was inserted into a compression cuff that measured the blood pressure 15 times. The average of these values was calculated (Table). Statistical analysis
The data are expressed as the means and standard deviations. The comparisons between the groups were performed using 1-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test. P #0.05 was considered statistically significant. RESULTS
Strain-induced alveolar bone remodeling and osteoclast recruitment are negatively modulated when the RAS is inhibited. The number of osteoclasts was significantly reduced in the LOS group compared with the VH mice (Fig 2, A, D, and F). The quantification of the TRAP-positive cells steadily increased until day 12 in the VH mice subjected to orthodontic force, and the numbers of osteoclasts were greater in the VH than in the LOS mice (Fig 2, A). At 6 days, the amount of movement of the teeth after application of force was similar in the control group and LOS-treated animals. However, the amount of OTM was significantly diminished in LOS-treated animals compared with the VH mice after 12 days of mechanical loading (Fig 2, B). The inhibition of the RAS affects osteoclast and osteoblast markers during bone remodeling induced by orthodontic force. In view of the altered phenotype of alveolar bone microscopy observed in experimental mice subjected
to mechanical force, we investigated whether the blockage of the RAS by LOS could interfere with the expression of markers involved in bone resorption. The orthodontic force significantly increased the mRNA levels of RANK (Fig 3, A), RANKL (Fig 3, B), cathepsin K (Fig 3, C), and metalloproteinase 13 (Fig 3, D) in both the control and experimental groups. However, the expression of these molecules was significantly lower in the LOS group, mainly in the compression sites (Fig 3). In addition, we characterized the expression of osteoblast markers and negative regulators of bone resorption. Our results showed that the expressions of periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, and metalloproteinase 2 (Fig 3, E-J) were augmented in the compression and tension sides of the periodontia of both groups. However, this increase was significantly more pronounced in the experimental mice than in the VH mice after 12 hours of mechanical loading. In parallel, the same scenario happened with the levels of osteoprotegerin, which were significantly elevated in mice when the RAS system was blocked by LOS (Fig 3, K). DISCUSSION
The inhibition of the RAS pathway by blocking the AT1 receptors (LOS) has been widely studied for their hemodynamic and antihypertensive activity, but there is little information about the role of this drug on bone remodeling, and no data are available on whether it affects OTM.2,3 In this study, the blood pressure in the VH group remained stable throughout the experimental period, and treatment with LOS had little effect on blood pressure as assessed at the end of the experimental period. More significantly, this study showed for the first time that the inhibition of the RAS by LOS significantly diminished bone remodeling induced by mechanical force. Associated with the diminished remodeling, the expression of regulatory markers of bone function was modified, so that there were less expression of markers associated with resorption and increases in markers associated with osteoblast function. Our results agree with previous studies that showed the effects of ANG II in osteoclast activation16 and alveolar bone resorption.23 Osteoclasts originate from monocyte/macrophage lineage multinucleated cells, which can also be the target of ANG II. Osteoblasts and stromal cells express RANKL in response to several bone-resorbing factors to support osteoclast differentiation from their precursors.17 Osteoclast precursors, which express RANK, recognize RANKL through cellto-cell interactions with osteoblasts and stromal cells and differentiate into mature osteoclasts.17,24 Targeted
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Fig 2. A, Numbers of TRAP-positive osteoclasts; B, time course of changes in the amount of OTM between the VH and LOS animals; C-F, histologic changes related to OTM, with sections of the periodontium around the distobuccal root of the first molar stained with TRAP; C and E, VH- and LOStreated groups (12 days of mechanical loading), respectively; D and F, higher views of the identified areas in C and E. Small yellow arrows, TRAP-positive osteoclasts; MB, mesial alveolar bone; PL, periodontal ligament; r, root; black arrows indicate the direction of tooth movement. Data are expressed as means and standard deviations. *P #0.05 comparing the results between 6 and 12 days of mechanical loading in the same group. #P #0.05 comparing the results between the VH and LOS groups (1-way ANOVA and Newman-Keuls multiple comparison test). Bar 5 100 mm.
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Fig 3. mRNA expression of osteoclast differentiation and activity markers: A, RANK; B, RANKL; C, cathepsin K (CATK); D, metalloproteinase 13 (MMP13); K, osteoprotegerin (OPG). mRNA expression of osteoblast markers: E, periostin; F, dentin matrix protein (DMP); G, alkaline phosphatase (ALP); H, collagen 1A1 (COL1A1); I, semaphorin 3A3 (SEM3A3); J, metalloproteinase 2 (MMP2) in VH- and LOS-treated animals at compression (cs) and tension (ts) sites. Data are expressed as means and standard deviations. *P #0.05 comparing 0 and 12 hours of mechanical loading in the same group; #P #0.05 comparing the compression and tension sites in the same experimental group; 1P #0.05 comparing the VH and LOS groups for compression and tension sites (1-way ANOVA and Newman-Keuls multiple comparison test).
disruption of either RANKL or RANK in mice causes a lack of osteoclasts and decreased bone resorption. The levels of proresorptive markers, including RANK, RANKL, cathepsin K, and metalloproteinase 13, decreased in the LOS mice after mechanical loading; this is consistent with reduced bone resorption. Previous studies have demonstrated that ANG II significantly upregulated the expression of RANKL in osteoblasts, inducing osteoclast-osteoblast interactions, increasing osteoclast differentiation, and consequently bone resorption.16,17,25 In parallel, cathepsin K and
metalloproteinase 13 are widely known proteases that degrade the bone matrix. They are a critical determinant of resorptive activity by osteoclasts.26 Our findings showed that inhibition of the RAS by LOS significantly decreased the levels of these proteases, pointing to a possible mechanism by which ANG II contributes to strain-induced bone resorption. Because differentiation and function of osteoblasts are essential to bone remodeling, we investigated the expression of osteoblast markers, such as periostin, dentin matrix protein, alkaline phosphatase, collagen
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1A1, semaphorin 3A3, and metalloproteinase 2 (late markers of osteoblast differentiation and activity).27 We observed increases in the levels of these molecules in the compression and tension sides of the periodontia of the LOS-treated mice. Therefore, our findings suggest that the inhibition of the RAS by blocking the ANG II receptors affects the expression of osteoblast differentiation markers. Moreover, our results also showed a greater increase in the expression of osteoprotegerin in the LOS mice than in the VH mice. These findings indicate that when the RAS is blocked, bone resorbing inhibitors (such as osteoprotegerin) are increased, and proresorptive markers are decreased.28 Taken all together, these data indicate an antiresorptive scenario after the inhibition of the RAS by LOS. Since bone remodeling is regulated by a variety of hormones, local factors, and inflammatory cytokines, the RAS is a novel component of the osteoclast differentiation system.8-10 Because ANG II can cause osteoclast activation, leading to increased bone resorption, blockage of the RAS pathway could inhibit this resorptive process, lessen the risk of bone diseases in elderly people, and modulate OTM.10,15,16 Our experimental system showed a decreased balance of marker expression toward bone maintenance and decreased OTM. Whether these experimental findings will translate into the clinical situation is not known, but considering the widespread availability of drugs that modify the RAS, clinical trials with ANG II receptor blockers could be performed to evaluate this boneprotective role of RAS blockers in humans. CONCLUSIONS
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2. 3.
Blockage of the RAS pathway decreases osteoclast differentiation and activity, leading to reduced tooth movement. This was the first demonstration that the RAS plays an important role in bone remodeling induced by mechanical force, as during OTM. The RAS plays a role in osteoclast differentiation and activation and, consequently, in OTM. AT1 antagonists provide potentially relevant clinical strategies for future therapeutic interventions, including modulation of the extent of OTM and potentially in other conditions associated with bone loss, such as osteoporosis and rheumatoid arthritis. Accordingly, it is reasonable to assume that the chronic use of RAS blockers by orthodontic patients might suppress tooth movement and therefore prolong treatment times. This should be considered by orthodontists during treatment planning.
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REFERENCES 1. Paul M, Poyan Mehr A, Kreutz R. Physiology of local reninangiotensin systems. Physiol Rev 2006;86:747-803. 2. Peach MJ. Renin-angiotensin system: biochemistry and mechanism of action. Physiol Rev 1977;57:313-70. 3. Garcia P, Schwenzer S, Slotta JE, Scheuer C, Tami AE, Holstein JH, et al. Inhibition of angiotensin-converting enzyme stimulates fracture healing and periosteal callus formation—role of a local renin-angiotensin system. Br J Pharmacol 2010;159: 1672-80. 4. Akazawa H, Yano M, Yabumoto C, Kudo-Sakamoto Y, Komuro I. Angiotensin II type 1 and type 2 receptor-induced cell signaling. Curr Pharm Des 2013;19:2988-95. 5. Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, et al. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 diabetes and nephropathy. N Engl J Med 2001;345:861-9. 6. So WY, Ma RC, Ozaki R, Tong PC, Ng MC, Ho CS, et al. Angiotensin-converting enzyme (ACE) inhibition in type 2, diabetic patients—interactions with ACE insertion/deletion polymorphism. Kidney Int 2006;69:1438-43. 7. Nakagami H, Osako MK, Shimizu H, Hanayama R, Morishita R. Potential contribution of action of renin angiotensin system to bone metabolism. Curr Hypertens Rev 2007;3:129-32. 8. Sernia C, Huang H, Nguyuen K, Li YH, Hsu S, Chen M, et al. Bone homeostasis: an emerging role for the renin-angiotensin system. In: Leung PS, editor. Frontiers in research of the renin-angiotensin system on human disease. Proteases in biology and disease. Dordrecht, The Netherlands: Springer; 2007. p. 179-95. 9. Nakagami H, Morishita R. Hormones and osteoporosis update. Effect of angiotensin II on bone metabolism. Clin Calcium 2009;19: 997-1002. 10. Hatton R, Stimpel M, Chambers TJ. Angiotensin II is generated from angiotensin I by bone cells and stimulates osteoclastic bone resorption in vitro. J Endocrinol 1997;152:5-10. 11. Hiruma Y, Inoue A, Hirose S, Hagiwara H. Angiotensin II stimulates the proliferation of osteoblast-rich populations of cells from rat calvariae. Biochem Biophys Res Commun 1997;230:176-8. 12. Izu Y, Mizoguchi F, Kawamata A, Hayata T, Nakamoto T, Nakashima K, et al. Angiotensin II type 2 receptor blockade increases bone mass. J Biol Chem 2009;284:4857-64. 13. Perez-Castrillon JL, Silva J, Justo I, Sanz A, Martin-Luquero M, Igea R, et al. Effect of quinapril, quinapril-hydrochlorothiazide, and enalapril on the bone mass of hypertensive subjects: relationship with angiotensin converting enzyme polymorphisms. Am J Hypertens 2003;16:453-9. 14. Rejnmark L, Vestergaard P, Mosekilde L. Treatment with betablockers, ACE inhibitors, and calcium-channel blockers is associated with a reduced fracture risk: a nationwide case-control study. J Hypertens 2006;24:581-9. 15. Asaba Y, Ito M, Fumoto T, Watanabe K, Fukuhara R, Takeshita S, et al. Activation of renin-angiotensin system induces osteoporosis independently of hypertension. J Bone Miner Res 2009;24: 241-50. 16. Shimizu H, Nakagami H, Osako MK, Hanayama R, Kunugiza Y, Kizawa T, et al. Angiotensin II accelerates osteoporosis by activating osteoclasts. FASEB J 2008;22:2465-75. 17. Teitelbaum SL. Bone resoption by osteoclasts. Science 2000;289: 1504-8. 18. Taddei SR, Queiroz-Junior CM, Moura AP, Andrade I Jr, Garlet GP, Proudfoot AE, et al. The effect of CCL3 and CCR1 in bone
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23. Suda N, Moriyama K, Ganburged G. Effect of angiotensin II receptor blocker on experimental periodontitis in a mouse model of Marfan syndrome. Infect Immun 2013;81:182-8. 24. Brooks PJ, Nilforoushan D, Manolson MF, Simmons CA, Gong SG. Molecular markers of early orthodontic tooth movement. Angle Orthod 2009;79:1108-13. 25. Tintut Y. Inhibition of osteoblast-specific transcription factor Cbfa1 by the cAMP pathway in osteoblastic cells. J Biol Chem 1999;247:28875-9. 26. Rachner TD, Khosla S, Hofbauer LC. Osteoporosis: now and the future. Lancet 2011;377:1276-87. 27. Liu W, Toyosawa S, Furuichi T, Kanatani N, Yoshida C, Liu Y, et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J Cell Biol 2001;155:157-66. 28. Kanzaki H, Chiba M, Takahashi I, Haruyama N, Nishimura M, Mitani H. Local OPG gene transfer to periodontal tissue inhibits orthodontic tooth movement. J Dent Res 2004;83: 920-5.
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Effects of angiotensin II type I receptor blocker losartan on orthodontic tooth movement Adriana Pedrosa Moura,a Carina Cristina Montalvany-Antonucci,a Silvana Rodrigues de Albuquerque Taddei,b udia Cristina Biguetti,d Gustavo Pompermayer Garlet,e Celso Martins Queiroz-Junior,c Cla f Anderson Jose Ferreira, Mauro Martins Teixeira,g Tarcılia Aparecida Silva,h and Ildeu Andrade, Jri Belo Horizonte, Minas Gerais, Salvador, Bahia, and Bauru, S~ ao Paulo, Brazil
Introduction: Drugs that block the renin-angiotensin system (RAS) are widely used for treating hypertension, heart and kidney failure, and the harmful effects of diabetes. Components of the RAS have been identified in various organs, but little is known of their effects on bone remodeling. The aim of this study was to evaluate whether the blockage of the RAS influences strain-induced bone remodeling in a model of orthodontic tooth movement. Methods: An orthodontic appliance was placed in C57BL6/J mice that were randomly divided into 2 groups: vehicle-treated mice (VH) and mice treated with losartan (an angiotensin II receptor blocker). Orthodontic tooth movement and the number of tartrate-resistant acid phosphatase-positive cells were determined by histopathologic analysis. The expression of mediators involved in bone remodeling was evaluated by quantitative real-time polymerase chain reaction. Blood pressure was measured before and during the experimental period. Results: Orthodontic tooth movement and tartrate-resistant acid phosphatase-positive cells were significantly reduced in the losartan group compared with the VH group. mRNA levels of osteoclast markers (RANK, RANKL, cathepsin K, and metalloproteinase 13) were lower in the losartan mice than in the VH group, whereas the expressions of osteoblast markers and negative regulators of bone resorption (periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, metalloproteinase 2, and osteoprotegerin) were higher in the VH group. Conclusions: Blockage of the RAS system decreases osteoclast differentiation and activity and, consequently, results in decreased strain-induced bone remodeling in orthodontic tooth movement. (Am J Orthod Dentofacial Orthop 2016;149:358-65)
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he renin-angiotensin system (RAS) is classically known as a circulating endocrine system regulating blood pressure and electrolyte homeostasis.1,2 The main effector peptide in this system is angiotensin II (ANG II), which is formed from angiotensin I by an angiotensin-converting enzyme, a
key molecule in this system that interferes in prostaglandin and nitric oxide production.3 This precursor, angiotensin I, is cleaved from angiotensinogen by renin, a highly selective protease secreted from the juxtaglomerular cells in the kidneys. ANG II exerts its biologic effects through binding to 2 specific
a Resident, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. b Assistant professor, Department of Cellular Biology, Institute of Biological Sciences, Federal University of Bahia, Salvador, Bahia, Brazil. c Postdoctoral resident, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. d Resident, Department of Biological Science, Faculty of Dentistry, University of S~ao Paulo, Bauru, S~ao Paulo, Brazil. e Associate professor, Department of Biological Science, Faculty of Dentistry, University of S~ao Paulo, Bauru, S~ao Paulo, Brazil. f Associate professor, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. g Full professor, Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. h Associate professor, Department of Oral Pathology, Faculty of Dentistry, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
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Associate professor, Department of Orthodontics, School of Dentistry, Pontifıcal Catholic University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Adriana Pedrosa Moura and Carina Cristina Montalvany-Antonucci are joint first authors and contributed equally to this work. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Supported by the Fundac¸~ao de Amparo a Pesquisas do Estado de Minas Gerais and the Conselho Nacional de Desenvolvimento Cientıfico e Tecnol ogico. Address correspondence to: Ildeu Andrade, Jr, Department of Orthodontics, School of Dentistry, Pontifıcal Catholic University of Minas Gerais, Av. Dom Jose Gaspar 500, CEP 31.270-901, Belo Horizonte, Minas Gerais, Brazil; e-mail, [email protected]. Submitted, August 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.2015.09.019
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angiotensin receptors—angiotensin type 1 receptors (AT1) and angiotensin type 2 receptor (AT2)—that are expressed in many cell types, including osteoclasts and osteoblasts.4 Drugs that block the RAS, such as losartan (LOS), act by blocking the AT1 receptors5 and are a main way to control high blood pressure (hypertension), heart failure, kidney failure, and the harmful effects of diabetes.1,5,6 The relationship between the RAS and bone metabolism is mainly based on the regulation of ANG II on bone metabolism.7-9 Different components of the RAS have been found to be synthesized and active in osteoblasts and in osteoclasts,10-12 and ANG II has been shown to stimulate bone resorption in osteoblast and osteoclast cocultures.9,10 A previous study has also demonstrated expression of the RAS components in osteoblasts and osteoclasts in vivo.12 Further evidence for a potential role of the RAS in bone metabolism comes from clinical studies. Patients treated with an angiotensin-converting enzyme inhibitor showed increased bone mineral density and reduced fracture risk.8,13,14 Moreover, previous animal studies have shown that excessive activation of RAS causes osteoporosis, mainly by elevation of osteoclastic bone resorption.15,16 Along with other factors, ANG II can upregulate the receptor activator of nuclear factor kappa-B ligand (RANKL) expression and downregulate the expression of runt-related transcription factor 2 (RUNX2). These key mediators are responsible for controlling osteoblast and osteoclast differentiation and regulating bone cell activity, leading to reduced bone formation and enhancing bone reabsorption.17 As described above, the bone does express components of the RAS, but little is known about the role of the RAS in the context of orthodontic tooth movement (OTM). The aim of this study was to evaluate the effects of blockers of the RAS on osteoclast recruitment and activity using a well-established mouse model of OTM. We hypothesized that the inhibition of the RAS pathway would impair the expression of proinflammatory cytokines, decrease osteoclast recruitment, and consequently reduce OTM. MATERIAL AND METHODS
Forty 10-week-old male C57BL6/J mice were used in this experiment. They were randomly divided into 2 groups: (1) treated with tap water (VH) as the control group, and (2) treated with 10 mg per kilogram per day of LOS (Sigma-Aldrich, St Louis, Mo). The sample size calculation was based on a previous study from our group.18 No significant weight loss was observed. All treatments followed the ethical regulations of the
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institutional ethics committee of the Pontifıcal Catholic University of Minas Gerais. Daily gavages of 100 mL of 10 mg per kilogram per day of LOS or tap water were administered to the LOS and VH groups, respectively. The drug was administered for 6 or 12 days for the histopathologic assays and for 0 or 12 hours for the molecular analyses. Tooth movement was induced as previously described.19,20 Briefly, the mice were anesthetized with 0.2 mL of a xylazine (0.02 mg/mL) and ketamine (50 mg/mL) solution. An orthodontic appliance consisting of a nickel-titanium 0.25 3 0.76-mm coil spring (Lancer Orthodontics, San Marcos, Calif) was bonded by light-cured resin (Transbond; 3M Unitek, Monrovia, Calif) between the maxillary right first molar and both maxillary incisors (Fig 1). The magnitude of force was calibrated by a tension gauge (Shimpo Instruments, Itasca, Ill) to exert a force of 35 g in the mesial direction. No reactivation was performed during the entire experimental period. For the histopathologic analysis, the left side of the maxilla (without an orthodontic appliance) was used as the control. The mice were killed after 6 or 12 days for the histopathologic analysis or after 0 or 12 hours for the molecular analysis. For each set of experiments, 5 animals were used at each time point. As was previously described, the right and left maxillae halves were fixed in 10% buffered formalin (pH 7.4), decalcified in 14% ethylenediaminetetraacetic acid (pH 7.4) for 20 days, and embedded in paraffin.19,20 The samples were cut into sagittal sections 5 mm thick. The sections were stained with tartrate-resistant acid phosphatase (TRAP) (Sigma-Aldrich), counterstained with hematoxylin, and used for the histologic examinations. The mesial periodontal site of the distobuccal root of the first molar was used for osteoclast counts on 5 sections per animal. Osteoclasts were identified as TRAP-positive, multinucleated cells on the bone surface. Measurement of OTM was performed as previously described.19,20 Images of the first and second molars were obtained using an optical microscope (Axioskop 40; Carl Zeiss, Gottingen, Germany) and an adapted digital camera (PowerShot A620; Canon, Tokyo, Japan). ImageJ software (National Institutes of Health) was used to quantify the degree of OTM by measuring the distance between the cementoenamel junction of the first and second molars on the right hemimaxilla in relation to the same measurements for the left hemimaxilla. Five vertical sections per animal were evaluated, and 3 measurements were made for each evaluation; the variability was less than 5%. RNA extraction and real-time polymerase chain reaction were performed for each animal separately, as
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Fig 1. A, Occlusal view of the maxillae before placing the orthodontic device; B, a nickel-titanium opencoil spring was placed between the maxillary right first molar and incisor.
previously described.19,20 The periodontal ligament and the surrounding alveolar bone were extracted from the maxillary first molars with a stereomicroscope. All gingival tissues, oral mucosa, and teeth were discarded. The periodontal tissues and the alveolar bone that were extracted from the distal area of the distobuccal root of the maxillary first molar were considered the tension site. The mesial area of the same root was considered the compression site. The bone samples (4-8 mg) were frozen in liquid nitrogen after collection and then stored at –80 C. Previous to mRNA extraction, each sample was mechanically fragmented in liquid nitrogen using a bone chisel and homogenized in sterile Milli-Q water with Ultra Turrax (IKA Laboratory Equipment, Staufen, Germany), and subsequently submitted to RNA extraction using TRIZOL reagent (Life Technologies, Carlsbad, Calif), following the manufacturer's instructions. The mRNA quality and RNA integrity number were assessed with the BioAnalyzer (Agilent, Santa Clara, Calif). The RNA integrity number results ranged between 7.25 and 8.66 (mean, 7.87; SD, 0.36). Complementary DNA was synthesized using 2 mg of RNA through a reverse transcription reaction (Superscript II; Life Technologies). The targets included molecules known to regulate osteoclast functions: RANK (receptor activator of nuclear factor kappa-B), RANKL, osteoprotegerin, cathepsin K, and metalloproteinase 13. We also analyzed the expression of molecules known to regulate osteoblast functions: periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, and metalloproteinase 2. The analyses were
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performed in ViiA7 equipment (Applied Biosystems, Warrington, United Kingdom) with real-time polymerase chain reaction using TaqMan chemistry (Life Technologies) with inventoried optimized primers/probes sets (Life Technologies) as follows: molecules known to regulate osteoclast functions: RANK (catalog number 433118), RANKL (catalog number 4331182), osteoprotegerin (catalog number 433118), cathepsin K (catalog number 4331182), and metalloproteinase 13 (catalog number 4331182); and molecules known to regulate osteoblast functions: periostin (catalog number 4331182), dentin matrix protein (catalog number 4331182), alkaline phosphatase (catalog number 4331182), collagen 1A1 (catalog number 4331182), semaphorin 3A3 (catalog number 4331182), and metalloproteinase 2 (catalog number 4331182). The basic reaction conditions (40 cycles) were 95 C (10 minutes), 94 C (1 minute), 56 C (1 minute), and 72 C (2 minutes). The mean cycle threshold (Ct) values from duplicate measurements were used to calculate the expression of the target gene, with normalization to an internal control (b-actin) using the 2 DDCt formula. Some researchers have used a dosage of antihypertensive drugs that promotes a reduction in blood pressure by 27%.21,22 In our study, we chose to use a dose to keep the blood pressure stable to eliminate bias. The mean arterial pressure measurement was evaluated by the tail-cuff method, which is a noninvasive computerized system for measuring blood pressure (Kent Scientific, Torrington, Conn). This tail-cuff blood pressure system uses volume-pressure recording technology to measure the blood pressure in the mouse's tail. The
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Table. Systolic blood pressure evaluation Systolic blood pressure (mm Hg) Day 1 day before 1 day after 6 days after 12 days after
Vehicle-treated mice 80.7 6 2.7 77.3 6 3.1 78.6 6 2.1 79.8 6 2.6
Losartan-treated mice 81.2 6 1.9 78.3 6 2.1 79.3 6 2.6 80.8 6 2.4
Systolic blood pressure was evaluated (n 5 5 mice per group) by tailcuff plethysmography 24 hours before, and 6 and 12 days after mechanical loading in vehicle-treated and losartan-treated mice (10 mg/kg). The results show the means and standard deviations. Intergroup and intragroup analyses had no significant difference. P .0.05.
animals were acclimated to the restraint and tail-cuff inflation for 1 day before the experiments. The restraint platform was maintained at approximately 32 C to 34 C. For each section, the mouse was placed in an acrylic box restraint, and the tail was inserted into a compression cuff that measured the blood pressure 15 times. The average of these values was calculated (Table). Statistical analysis
The data are expressed as the means and standard deviations. The comparisons between the groups were performed using 1-way analysis of variance (ANOVA) followed by the Newman-Keuls multiple comparison test. P #0.05 was considered statistically significant. RESULTS
Strain-induced alveolar bone remodeling and osteoclast recruitment are negatively modulated when the RAS is inhibited. The number of osteoclasts was significantly reduced in the LOS group compared with the VH mice (Fig 2, A, D, and F). The quantification of the TRAP-positive cells steadily increased until day 12 in the VH mice subjected to orthodontic force, and the numbers of osteoclasts were greater in the VH than in the LOS mice (Fig 2, A). At 6 days, the amount of movement of the teeth after application of force was similar in the control group and LOS-treated animals. However, the amount of OTM was significantly diminished in LOS-treated animals compared with the VH mice after 12 days of mechanical loading (Fig 2, B). The inhibition of the RAS affects osteoclast and osteoblast markers during bone remodeling induced by orthodontic force. In view of the altered phenotype of alveolar bone microscopy observed in experimental mice subjected
to mechanical force, we investigated whether the blockage of the RAS by LOS could interfere with the expression of markers involved in bone resorption. The orthodontic force significantly increased the mRNA levels of RANK (Fig 3, A), RANKL (Fig 3, B), cathepsin K (Fig 3, C), and metalloproteinase 13 (Fig 3, D) in both the control and experimental groups. However, the expression of these molecules was significantly lower in the LOS group, mainly in the compression sites (Fig 3). In addition, we characterized the expression of osteoblast markers and negative regulators of bone resorption. Our results showed that the expressions of periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, and metalloproteinase 2 (Fig 3, E-J) were augmented in the compression and tension sides of the periodontia of both groups. However, this increase was significantly more pronounced in the experimental mice than in the VH mice after 12 hours of mechanical loading. In parallel, the same scenario happened with the levels of osteoprotegerin, which were significantly elevated in mice when the RAS system was blocked by LOS (Fig 3, K). DISCUSSION
The inhibition of the RAS pathway by blocking the AT1 receptors (LOS) has been widely studied for their hemodynamic and antihypertensive activity, but there is little information about the role of this drug on bone remodeling, and no data are available on whether it affects OTM.2,3 In this study, the blood pressure in the VH group remained stable throughout the experimental period, and treatment with LOS had little effect on blood pressure as assessed at the end of the experimental period. More significantly, this study showed for the first time that the inhibition of the RAS by LOS significantly diminished bone remodeling induced by mechanical force. Associated with the diminished remodeling, the expression of regulatory markers of bone function was modified, so that there were less expression of markers associated with resorption and increases in markers associated with osteoblast function. Our results agree with previous studies that showed the effects of ANG II in osteoclast activation16 and alveolar bone resorption.23 Osteoclasts originate from monocyte/macrophage lineage multinucleated cells, which can also be the target of ANG II. Osteoblasts and stromal cells express RANKL in response to several bone-resorbing factors to support osteoclast differentiation from their precursors.17 Osteoclast precursors, which express RANK, recognize RANKL through cellto-cell interactions with osteoblasts and stromal cells and differentiate into mature osteoclasts.17,24 Targeted
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Fig 2. A, Numbers of TRAP-positive osteoclasts; B, time course of changes in the amount of OTM between the VH and LOS animals; C-F, histologic changes related to OTM, with sections of the periodontium around the distobuccal root of the first molar stained with TRAP; C and E, VH- and LOStreated groups (12 days of mechanical loading), respectively; D and F, higher views of the identified areas in C and E. Small yellow arrows, TRAP-positive osteoclasts; MB, mesial alveolar bone; PL, periodontal ligament; r, root; black arrows indicate the direction of tooth movement. Data are expressed as means and standard deviations. *P #0.05 comparing the results between 6 and 12 days of mechanical loading in the same group. #P #0.05 comparing the results between the VH and LOS groups (1-way ANOVA and Newman-Keuls multiple comparison test). Bar 5 100 mm.
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Fig 3. mRNA expression of osteoclast differentiation and activity markers: A, RANK; B, RANKL; C, cathepsin K (CATK); D, metalloproteinase 13 (MMP13); K, osteoprotegerin (OPG). mRNA expression of osteoblast markers: E, periostin; F, dentin matrix protein (DMP); G, alkaline phosphatase (ALP); H, collagen 1A1 (COL1A1); I, semaphorin 3A3 (SEM3A3); J, metalloproteinase 2 (MMP2) in VH- and LOS-treated animals at compression (cs) and tension (ts) sites. Data are expressed as means and standard deviations. *P #0.05 comparing 0 and 12 hours of mechanical loading in the same group; #P #0.05 comparing the compression and tension sites in the same experimental group; 1P #0.05 comparing the VH and LOS groups for compression and tension sites (1-way ANOVA and Newman-Keuls multiple comparison test).
disruption of either RANKL or RANK in mice causes a lack of osteoclasts and decreased bone resorption. The levels of proresorptive markers, including RANK, RANKL, cathepsin K, and metalloproteinase 13, decreased in the LOS mice after mechanical loading; this is consistent with reduced bone resorption. Previous studies have demonstrated that ANG II significantly upregulated the expression of RANKL in osteoblasts, inducing osteoclast-osteoblast interactions, increasing osteoclast differentiation, and consequently bone resorption.16,17,25 In parallel, cathepsin K and
metalloproteinase 13 are widely known proteases that degrade the bone matrix. They are a critical determinant of resorptive activity by osteoclasts.26 Our findings showed that inhibition of the RAS by LOS significantly decreased the levels of these proteases, pointing to a possible mechanism by which ANG II contributes to strain-induced bone resorption. Because differentiation and function of osteoblasts are essential to bone remodeling, we investigated the expression of osteoblast markers, such as periostin, dentin matrix protein, alkaline phosphatase, collagen
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1A1, semaphorin 3A3, and metalloproteinase 2 (late markers of osteoblast differentiation and activity).27 We observed increases in the levels of these molecules in the compression and tension sides of the periodontia of the LOS-treated mice. Therefore, our findings suggest that the inhibition of the RAS by blocking the ANG II receptors affects the expression of osteoblast differentiation markers. Moreover, our results also showed a greater increase in the expression of osteoprotegerin in the LOS mice than in the VH mice. These findings indicate that when the RAS is blocked, bone resorbing inhibitors (such as osteoprotegerin) are increased, and proresorptive markers are decreased.28 Taken all together, these data indicate an antiresorptive scenario after the inhibition of the RAS by LOS. Since bone remodeling is regulated by a variety of hormones, local factors, and inflammatory cytokines, the RAS is a novel component of the osteoclast differentiation system.8-10 Because ANG II can cause osteoclast activation, leading to increased bone resorption, blockage of the RAS pathway could inhibit this resorptive process, lessen the risk of bone diseases in elderly people, and modulate OTM.10,15,16 Our experimental system showed a decreased balance of marker expression toward bone maintenance and decreased OTM. Whether these experimental findings will translate into the clinical situation is not known, but considering the widespread availability of drugs that modify the RAS, clinical trials with ANG II receptor blockers could be performed to evaluate this boneprotective role of RAS blockers in humans. CONCLUSIONS
1.
2. 3.
Blockage of the RAS pathway decreases osteoclast differentiation and activity, leading to reduced tooth movement. This was the first demonstration that the RAS plays an important role in bone remodeling induced by mechanical force, as during OTM. The RAS plays a role in osteoclast differentiation and activation and, consequently, in OTM. AT1 antagonists provide potentially relevant clinical strategies for future therapeutic interventions, including modulation of the extent of OTM and potentially in other conditions associated with bone loss, such as osteoporosis and rheumatoid arthritis. Accordingly, it is reasonable to assume that the chronic use of RAS blockers by orthodontic patients might suppress tooth movement and therefore prolong treatment times. This should be considered by orthodontists during treatment planning.
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