{"paper_id":"02593c74-7278-4a96-9e4a-cdccbff277cb","body_text":"Activation of PPARγ redirects fibro-adipogenic progenitors to replace ectopic \nbone with fat in models of fibrodysplasia ossificans progressiva and trauma-\ninduced heterotopic ossification \n \nAuthors:  Pratik Koirala1, Ziyu Chen1,2, Samerender Nagam Hanumantharao1, Ashley E \nSiegel1, Chang Liu 1, Zachary Williams 1, Harshaan Sekhon 1, David Maridas 3, Yuji \nMishina4, Vicki Rosen3, Shailesh Agarwal1* \n \nAffiliations: \n1Department of Surgery, Brigham and Women’s Hospital and Harvard Medical School, \nBoston, Massachusetts, USA. \n2Department of Bone and Joint Surgery, Peking University Shenzhen Hospital, Shenzhen, \nGuangdong, China. \n3 Harvard School of Dental Medicine, Boston, Massachusetts, USA. \n4 University of Michigan Dental School, Ann Arbor, Michigan, USA. \n \nCorresponding Author:  \nShailesh Agarwal, MD \nAssistant Professor of Surgery \nDivision of Plastic Surgery \nDepartment of Surgery \nBrigham and Women’s Hospital  \nHarvard Medical School \n \n75 Francis St.,  \nBoston, MA 02115 \nPhone: 248-854-7084 \nEmail: sagarwal15@bwh.harvard.edu \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nABSTRACT \nThe pathologic, osteogenic differentiation of fibroadipogenic progenitor cells (FAPs) is the primary \nrecognized contributor to ectopic bone formation in fibrodysplasia ossificans progressiva (FOP) \nand trauma -induced heterotopic ossification (HO). Both cond itions are characterized by up -\nregulated BMP signaling – the former by a gene mutation rendering the BMP receptor ACVR1 \nsusceptible to activation by inflammatory ligands (Activin A), and the latter by up -regulated \npresence of BMP2 ligand in the setting of unmutated BMP receptor. We performed an unbiased \nassessment of FDA -approved therapies which would optimally target  the transcriptional \naberrations observed in developing FOP and HO lesions based on publicly -available datasets. \nThis analysis uncovered rosiglitazone, a peroxisome proliferator -activated receptor gamma \n(PPARγ) agonist as the highest scoring therapeutic op tion across three data sets for both \nconditions. Rosiglitazone treatment eliminated ectopic bone lesions in a mouse model of FOP, \nand replaced these lesions with ectopic adipose tissue; similarly, systemic and local rosiglitazone \ntreatment eliminated ectopic bone lesions in a mouse model of trauma-induced HO and replaced \nthese lesions with ectopic adipose tissue. Our findings were corroborated by a single case report \nfrom 2010 showing positive results with rosiglitazone in a non-diabetic patient with FOP, with no \nsubsequent studies. Overall, our findings suggest that a previously FDA-approved therapeutic is \nlikely to be a successful therapeutic agent for both FOP and trauma-induced HO, both conditions \nfor which current therapeutic options remain inadequate. \n \nOne Sentence Summary: We show that a previously FDA-approved therapeutic known to induce \nadipogenesis reduces ectopic bone and induces ectopic fat formation in diseases of heterotopic \nossification. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nINTRODUCTION \nHeterotopic ossification (HO) is a pathologic condition in which extra -skeletal bone forms within \nsoft tissues  1,2. A genetic form of HO known as fibrodysplasia ossificans progressiva (FOP), \ndevelops in patients with a hyperactivating mutation of the bone morphogenetic protein (BMP) \nreceptor ACVR1 (ACVR1 R206H) 2. Patients with FOP develop painful , ectopic bony lesions at \nsites of inflammation such as may occur after even minor bruises  or local injections. HO is also \nobserved in patients with wild type ACVR1 who have experienced severe traumatic injury such \nas blast injuries, burns, and even surgical procedures  3-5. Like FOP, trauma-induced HO is \nmediated by excessive BMP signaling albeit due to elevated levels of BMP ligand rather than the \npresence of a hyperactivating mutation in the receptor itself 4,5.  \n \nPrevious studies have established fibro -adipogenic progenitor cells (FAPs) as precursor cells \nresponsible for bone formation in FOP and trauma-induced HO 6,7. These FAPs are also capable \nof undergoing fibrogenic differentiation, as observed after muscle injury in individuals without \nhyperactivating BMP receptor mutations. A less understood differentiation pathway for FAPs is \ntowards the adipogenic fate  8; however, this process is observed with ectopic fat infiltration in \natrophying muscle 8 9.  \n \nMuch of the morbidity which ensues after HO formation is due to the rigidity and firmness of the \nbone, which leads to impaired mobility, open wounds due to pressure, and pain due to nerve \nimpingement. Given the multi-potent capacity of FAPs, we hypothesized that these cells could be \n\"nudged” towards adopting an adipogenic fate, rather than undergoing osteogenic differentiation.  \n \nPeroxisome proliferator-activated receptor gamma (PPARγ) is a master transcriptional regulator \nof adipogenesis  10. Although in some contexts, elevated bone morphogenetic protein (BMP) \nsignaling can up -regulate PPARγ expression 11, adipogenesis is not typically observed in FOP \nlesions or trauma-induced HO. In the clinical setting, the formation of soft, adipose tissue, would \nbe preferable to rigid bone.  Our objective in this study was to determine whether  PPARγ could \nserve as a target for the developing lesions present in FOP and trauma-induced HO, and whether \ndelivery of a PPARγ agonist could instead induce formation of ectopic fat. \n \nRESULTS  \nIn silico drug discovery identifies rosiglitazone as a top candidate to reverse aberrant gene \nexpression in FOP and trauma-induced HO \nTo identify compounds capable of reversing pathological gene expression in fibrodysplasia \nossificans progressiva (FOP) and trauma-induced heterotopic ossification (HO), we analyzed bulk \nRNA-sequencing (RNA -seq) datasets from mouse injury models (GSE220725, GSE218699, \nGSE126118) 12 13 14. Differential expression between FOP/HO -injured sites and controls was \nscored against drug -gene interactions from the Drug –Gene Interaction Database (DGIdb)  to \nprioritize agents predicted to counteract these signatures. For FOP (GSE220725), injured \ngastrocnemius from mutant ACVR1 -expressing mice showed distinct profiles versus normal \ncontrols, with pairwise correlations, confirming clustering (Fig 1A). Volcan o plot highlighted \ndysregulated genes (|log ₂FC| ≥ 3, P < 0.01; Fig 1B). Drugs were scored by opposing effects \n(agonist/antagonist) to inverse differential patterns, filtering for non -immunotherapeutic, non -\nantineoplastic agents (Fig 1C). Rosiglitazone, a PPAR γ agonist, ranked amongst the highest. \nRosiglitazone also led scores in HO datasets: early post -injury in dexamethasone -cardiotoxin \nmodel (GSE218699; Fig 1D) and chronic burn/tenotomy (GSE126118; Fig 1E). Thus, we \nadvanced rosiglitazone therapy in mouse models of FOP and trauma-induced HO.  \n \nAbsence of adipocytes or adipogenic signaling in FOP lesions \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nWe examined FOP lesions in a validated genetic mouse model (Ub.CreERT/ACVR1 R206H) three \nweeks after cardiotoxin-induced muscle injury (Fig 2A). Based on our examination of histologic \nimages, we found no evidence of adipocytes in the injury site using H&E, Picrosirius Red, and \nSafranin O/Fast green with no treatment (Fig 2B). Perilipin-1 immunostaining verified adipocytes \ninside the bone marrow as expected, but not within the surrounding soft tissue (Fig 2C). \n \nTreatment with a PPARγ agonist suppresses osteogenesis and induces adipogenesis in \nbone marrow-derived mesenchymal cells in vitro \nNext, we sought to determine the fate of mesenchymal cells derived from FOP mice, when \ncultured in media with a pro -adipogenic PPARγ agonist. FOP-derived cells cultured in Activin A \nand rosiglitazone exhibited reduced expression of osteogenic genes Col2a1, Aggrecan, and Sox9 \nrelative to FOP-derived cells cultured in Activin A alone. Conversely, FOP-derived cells cultured \nin Activin A and rosiglitazone exhibited increased expression of adipogenic genes such as Lpl and \nFabp4 relative to FOP-derived cells cultured in Activin A alone (Fig 3A) (* is p<0.05). \n \nTo simulate trauma-induced HO, which is dependent on BMP signaling in the setting of wild type \nACVR1, a similar series of experiments were performed with mesenchymal cells obtained from \nwild type mice, and treated with BMP2 and rosiglitazone or vehicle control. WT cells cultured in \nBMP2 and rosiglitazone exhibited reduced expression of osteoge nic genes Col2a1, Aggrecan, \nand Sox9 relative to WT cells cultured in BMP2 alone. Conversely, WT cells cultured in BMP2 \nand rosiglitazone exhibited increased expressio n of adipogenic genes such as Lpl and Fabp4 \nrelative to WT cells cultured in BMP2 alone (Fig 3B) (* is p<0.05).  \n  \nTreatment with rosiglitazone significantly reduces formation of post-injury FOP lesions \nFOP mice which had received intramuscular cardiotoxin were treated with rosiglitazone (10 \nmg/kg) or vehicle control initiated on day of cardiotoxin injection and delivered twice weekly until \neuthanization after 3 weeks . Both MicroCT and x-ray verified a significant reduction in ectopic \nbone formation at the site of injury (Fig 4A, B) (* is p< 0.05). Instead, we observed new adipose \ntissue and decreased cartilage in the affected region using H&E, Picrosirius Red, and Safranin \nO/Fast green (Fig 4C). However, immunostaining of the affected site verified the presence of \nelevated BMP signaling (anti -pSMAD 1/5), coincident with increased expression of adipogenic \nmarkers (anti-perilipin-1 and anti-Pparg). Soft tissue resident adipocytes also displayed PDGFRα \nsignaling (anti-PDGFRα) suggesting that FAPs served as the source of ectopic adipocytes (Fig \n4D).   \n \nSystemic treatment with rosiglitazone reduces formation of heterotopic bone in a trauma \nmodel \nNext, we sought to examine whether rosiglitazone could similarly reduce heterotopic bone in a \nmodel of trauma -induced H O. MicroCT verified ectopic bone formation in soft tissue and \ncalcaneus in the absence of treatment  (Fig 5 A,B). Mice which received tenotomy exhibited \nsignificantly reduced formation of ectopic cartilage production after systemic rosiglitazone \ntreatment relative to control treatment (Safranin O, H&E) (Fig 5C). Immunostaining verified the \npresence of increased pe rilipin and PPAR γ, indicative of adipocytes , in mice treated with \nrosiglitazone (Fig 5D).  \n \nLocal treatment with rosiglitazone reduces formation of heterotopic bone in a trauma \nmodel \nNext, we sought to determine whether local rosiglitazone treatment would sufficiently reduce HO \nformation in a trauma model. Given the association between rosiglitazone and \nosteopenia/osteoporosis, such an approach would avoid systemic exposure in patients who may \nrequire normal skeletal healing to occur following trauma. Mice underwent tendon transection to \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\ninduce local HO formation; additionally, they were treated with local injection of rosiglitazone (10 \nmg/kg; once a week for 3 weeks) (Fig 6A). As with systemic delivery, mice which received \ntenotomy exhibited significantly reduced formation of ectopic cart ilage production after local \nrosiglitazone treatment relative to local control treatment (Safranin O, H&E) (Fig 6B). There was \nno evidence of increased adipogenesis in the contralateral hindlimb. Immunostaining verified that \nperilipin and PPARγ expression was elevated in the affected area, indicative of adipocytes in mice \nlocally treated with rosiglitazone (Fig 6C). \n \nDISCUSSION  \nIn this study, we used an unbiased approach to identify the top -scoring FDA -approved drug \npredicted to intercede on transcriptional changes observed in developing FOP and HO lesions; \nthis unbiased approach uncovered rosiglitazone as a top -scoring candidate  and validated \ndiminished PPARγ signaling as a modifiable aberration in FOP lesions. We found that delivery of \nrosiglitazone induces ectopic fat formation and suppresses ectopic bone formation in models of \nFOP and trauma-induced HO, thereby verifying that this drug has the desired effect in vivo. In \nmice with the genetic mutation responsible for FOP (ACVR1 R206H), rosiglitazone delivery led to \nthe discernible presence of ectopic fat at the injury site. These fat cells expressed traditional \nadipocytes markers including perilipin and adiponectin, while also expressing PDGFRa, a marker \nfor FAPs. Conversely, ectopic bone was significantly and substantially reduced with rosiglitazone \ntreatment. Similarly, rosiglitazone, delivered either systemically or locally, induced ectopic fat \nformation and significantly reduced ectopic bone formation at the tendon transection site in a well-\nstudied model of trauma-induced HO.  \n \nOur findings of diminished ectopic bone and de novo ectopic adipogenesis with rosiglitazone \ntreatment in FOP are consequential to the potential for this drug as a therapeutic option for FOP. \nGatti et al have previously reported on an improvement observed in a patient with FOP treated \nwith rosiglitazone 15. In that case report, the patient had substantial improvement relative to the \nyear prior while on corticosteroid therapy during which time five episodes of flares were noted. In \nthat patient, rosiglitazone was administered at a dose of 4-8 mg daily, which is consistent with the \ndosing used for patients with type 2 diabetes. However, beyond this case report, no further studies \nwere pursued or published to further interrogate rosiglitazone in FOP. Currently, only one therapy \nhas received FDA approval for FOP  – palovarotene, a retinoic acid receptor agonist. However, \nadverse effects associated with this medication, such as premature joint plate fusion in pediatric \npatients have been reported 16. This is of particular interest given that ectopic bone formation in \nFOP occurs during childhood and accumulates throughout the patient’s lifespan; therefore, early \nintervention is paramount. Interestingly, while PPARg and RARg both bind to retinoid X receptor \n(RXR) to form separate heterodimer complexes which activate gene expression, rosiglitazone \ntherapy has been  recently shown to be safe in children without adverse effects  17. Therefore, \nrepurposing rosiglitazone may represent an efficacious and safe therapeutic opportunity; it should \nbe noted as well that palovarotene has similar roots as a drug developed initially developed for \nan alternative indication – emphysema 18.  \n \nPrevious studies have established FAPs as a progenitor cell population responsible for ectopic \nbone formation 6,7. FAPs are multipotent and have been shown to contribute to ectopic adipose \ntissue deposition in muscle atrophy, and to intramuscular fibrosis. The factors which modify the \nfate of FAPs in vivo are likely myriad. Our findings from this study suggest that FAPs responsible \nfor ectopic bone formation may be directed towards an adipogenic fate through drug delivery. It \nshould be noted that ectopic adipogenesis was not readily observed in the contralateral hindlimb \nwhich did not receive an injury, suggesting tha t the initial traumatic event is responsible for the \ninflux of FAPs. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\n \nWe have also demonstrated that rosiglitazone reduces ectopic bone formation in trauma-induced \nHO. Both systemic and local treatment were highly effective in this role. In the setting of severe \nburns or high -impact trauma where the likely location of HO for mation may not be easily \ndetermined for local intervention, systemic delivery may present a viable option for HO prevention. \nFor example, burn patients who are at elevated risk for HO formation (>30% total body surface \narea burns, arm burns, increased number of days on the ventilator or operations) 19 may benefit \nfrom systemic rosiglitazone therapy.  \n \nIn other patients who are elevated risk for HO, such as those with hip arthroplasty, systemic \nrosiglitazone may not be desirable due to the presence of comorbid osteopenia/osteoporosis in \nolder patients. Local rosiglitazone delivery into the tissues surrou nding the surgical joint space \nwhich are susceptible to HO formation may represent a therapeutic option for these patients. \nPreviously, poly(lactic-go-glycolic) acid (PLGA) particles have been used for rosiglitazone delivery \nto mitigate pulmonary arterial hyperplasia 20; separately, hydrogel loaded with rosiglitazone \nreduced joint contractures in a rabbit limb immobilization model 21.  \n \nOther drugs have shown promise in prevention of heterotopic ossification. For example, \nrapamycin, an mTOR inhibitor has been shown to be efficacious in FOP  3 and has led to the \ndevelopment of a clinical trial in Japan  22. Whether a combinatorial treatment including both \nrapamycin and rosiglitazone may provide improved protection from FOP lesions with reduced \ndosing requirements may be of interest in the future. Separately, combinatorial treatment with \npalovarotene may also be an opportunity to reduce palovarotene dosing and adverse effects.  \n \nA major challenge in the development of new drugs for FOP is the small patient population at risk \nfor this condition. While it is certainly desirable for few patients to be struck by this condition, this \nis of little solace to those who do have FOP . Importantly, the small number of patients with FOP \npresents a challenge to clinical trial enrollment and drug development; this is compounded by \nreticence due to unknown risk profiles of newer medications. However the low risk profile of \nrosiglitazone may engender increased trial enrollment, relative to newer drugs without a previous \ntrack record of clinical use. In addition, consideration of studies with multiple treatment arms may \nbe feasible when drugs are no longer on -patent. One strategy which may enable commercial \ninterest in these drugs which are off -patent includes the FDA 505(b)(2) pathway which may \nprovide market exclusivity up to 7 years in the setting of orphan drug designation. Regardless, \nour findings underscore the importance of iteratively and cont inuously examining new data and \nthe literature for potential therapeutic avenues with a history of FDA approval.  \n \nMATERIALS AND METHODS \nSex as a biological variable \nOur study examined male and female animals, and similar findings are reported for both sexes. \n \nEthics statement \nAll experiments on animals were conducted in accordance with the protocol approved by \nInstitutional Animal Care and Use Committee (IACUC) of Brigham and Women’s Hospital \n(#2020N000036).  \nIn silico drug discovery \nBulk RNA -seq data was sourced from GEO accessions GSE220725 (FOP vs. normal injury; \nn=5/group), GSE218699 (cardiotoxin + dexamethasone HO vs. saline; n=4/group), and \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nGSE126118 (burn/tenotomy HO vs. uninjured; n=2/group). Drug-gene interactions were obtained \nfrom DGIdb v5.0 (accessed August 2025; https://dgidb.org)  23. Differential expression involved \ngroup-wise mean calculation, log₂FC with pseudocount (0.01), and Welch's t test. Visualizations \nincluded pairwise Pearson correlations on log₂(TPM + 1) for top 3000 variable genes and volcano \nplots (|log₂FC| ≥ 3, P < 0.01), with top genes labeled by composite rank. Drug effects were signed \n(+1 agonist, -1 antagonist), score -scaled, and aggregated per pair; filtered to non -\nimmunotherapeutic, non-antineoplastic agents. \n \nAnimals \nC57BL/6 mice and Ub.CreERT/ACVR1 R206H mice were housed in the Brigham and Women’s \nHospital (BWH) vivarium. Tamoxifen was dissolved in corn oil and intraperitoneally injected into \nUb.CreERT/ACVR1R206H mice (100 mg/kg) for 4 consecutive days to induce mutation. 8 \nUb.CreERT/ACVR1R206H mice were treated with rosiglitazone and 8 mice were used as control. 5 \nWT C57BL/6 mice were treated with rosiglitazone post tenotomy and 6 mice were used as control. \nFinally, 5 WT mice were treated with local rosiglitazone post tenotomy and 5 mice were used as \ncontrol. \n \nMouse model \nFor FOP model, two weeks after the tamoxifen injection, cardiotoxin (0.1 mg/ml in PBS, 30 μl per \nhindlimb) was injected intramuscularly into the hindlimb of the Ub.CreERT/ACVR1R206H mice to \ninduce muscle injury and promote heterotopic ossification. Hindlimb samples were harvested 3 \nweeks after the cardiotoxin injection. For tenotomy model, the achilles of C57BL/6 mice were \ntransected. Hindlimb samples were harvested 3 weeks after the tenotomy. Sacrifice was \nperformed via CO2 asphyxiation. Hindlimb samples were fixed in 10% formalin for 48h followed \nby 70% ethanol for further assessment. \n \nRosiglitazone treatment \nFor systemic treatment of rosiglitazone, it was dissolved in DMSO at the concentration of 25 \nmg/ml and was diluted in corn oil to the final concentration of 2.5 mg/ml before injection. Mice \nwere intraperitoneally injected with rosiglitazone (10 mg/kg) twice a week started in parallel with \ncardiotoxin injection or surgery. Mice in the control group received coin oil injection at the same \ntime points. For local treatment, 10 mg/kg rosiglitazone was injected locally around the site of \ntendon transection once a week for 3 weeks. Mice in the control group received PBS at the same \ntime points. \n \nMicro-CT and analysis \nHindlimb samples were scanned using high -resolution Micro -Computed Tomography ( μCT40, \nSCANCO Medical AG, Brüttisellen, Switzerland). Scan parameters were 30 μm3 isotropic voxel \nsize, 55 kVp peak X -ray tube intensity, 145 µA X-ray tube current, and 300 ms i ntegration time. \nDICOM images were exported for analysis of ectopic bone volume. \n \nHistology \nFormalin-fixed samples were dehydrated, embedded in paraffin, and sectioned with 5 -micron-\nthickness. Sections were stained with Hematoxylin and Eosin (H&E), Picrosirius Red and \nSafranin-O/Fast green according to standard protocol. The images were taken by Olympus BX53 \nmicroscope. \n \nImmunofluorescent staining \nTissue sections were de-paraffinized, rehydrated and blocked with 10% goat serum, 1% bovine \nserum albumin (BSA), and 0.3% Triton (MilliporeSigma) in PBS for 1 hour and incubated with \nprimary antibodies at 4°C overnight. The sections were probed with antibodies for Perilipin-1 (Cell \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nSignaling Technology, cat# 3467S, 1:100), PPAR γ ( Proteintech, cat 16643 -1-AP, 1:100), \npSMAD1/5 (Cell Signaling Technology, cat# 9516S, 1:100).  After washing, the sections were \nincubated for one hour in the dark using the following secondary antibodies: Alexa Fluor Plus 488 \ngoat anti-rabbit IgG (1:1000) and Alexa Fluor 594 goat anti-rabbit IgG (1:1000). ProLong Diamond \nAntifade Mountant with DAPI (P36971, Invitrogen) was used to stain the nuclei and mount the \nsamples. Fluorescent images were taken by Olympus Fluoview Confocal microscope. \n \nCell culture \nBone marrow-derived mesenchymal cells were isolated from both C57BL/6 mice and tamoxifen \ntreated Ub.CreERT/ACVR1R206H mice. Briefly, femur and tibia were obtained after the euthanation \nof the mice. The proximal and distal end of the femurs and tibia were cut using sharp scissors. \nIce-cold PBS was injected into the femur and tibia to flush out the bone marrow from the proximal \nend onto a 70 µm nylon cell strainer. The cells were centrifuged at 1500 rpm for 5 minutes at 4 \n°C. Cells were cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin. All cells \nwere cultured in a humidified atmosphere with 5% CO2 at 37 °C. \n \nCell treatment \nBone marrow-derived mesenchymal cells from C57BL/6 mice were treated with BMP2 (25 ng/ml). \nBone marrow-derived mesenchymal cells from tamoxifen treated Ub.CreERT/ACVR1R206H mice \nwere treated with Activin A (25 ng/ml). Rosiglitazone (10 µM) was administered at the same time. \nAll cells were harvested on day 7. \n \nRT-qPCR \nTotal mRNA was isolated from cells using Direct -zol RNA Miniprep Kits (Zymo  Research). \nReverse transcription was performed using qMax cDNA Synthesis Kit (Accuris). Real-Time PCR \nwas performed using the following primers for Lpl, Fabp4, Sox9, Col2a1, Aggrecan, and Actin \nrespectively: 5’-GCGTAGCAGGAAGTCTGACCAA-3’, 5’ -AGCGTCATCAGGAGAAAGGCGA-3’; \n5’-TGAAATCAC-CGCAGACGACAGG-3’, 5’ -GCTTGTCACCATCTCGTTTTCTC-3’; 5’ -\nCACACGTCAAGCGACCCA-TGAA-3’, 5’ -TCTTCTCGCTCTCGTTCAGCAG-3’; 5’ \nGCTGGTGAAGAAGGCAAACGAG-3’, 5’ -CCATCTTGACCTGGGAATCCAC-3’; 5’ -\nCAGGCTATGAGCAGTGTGATGC-3’, 5’ -GCTGCTGTCTT-TGTCACCCACA-3’; 5’-\nCATTGCTGACAGGATGCAGAAGG-3’, 5’-TGCTGGAAGGTGGACAGTG-AGG-3’. The PCR \ncondition was: 95 °C for 10 minutes; 40 cycles 95°C for 15 s, 60 °C for 1 minute, 72 °C for 30 s; \n72 °C for 10 min The PCR was performed using the machine QuantStudio 7 Flex Real-Time PCR \nSystemand analyzed using QuantStudio™ Real-Time PCR Software. \n \nStatistics \nAll data are presented as mean ± S.D. Statistical analysis was performed using GraphPad Prism \n9. Comparisons between two groups were performed by using 2 -tailed Student’s t -tests. \nComparisons among multiple groups were performed by using a one-way ANOVA with a Tukey’s \nmultiple-comparisons test. Significant difference was established when P value was smaller than \n0.05. \n \nREFERENCE \n1. Kaplan, F.S., Tabas, J.A., Gannon, F.H., Finkel, G., Hahn, G.V., and Zasloff, M.A. (1993). \nThe histopathology of fibrodysplasia ossificans progressiva. An endochondral process. J \nBone Joint Surg Am 75, 220-230. 10.2106/00004623-199302000-00009. \n2. 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Rosen, E.D., Sarraf, P., Troy, A.E., Bradwin, G., Moore, K., Milstone, D.S., Spiegelman, \nB.M., and Mortensen, R.M. (1999). PPAR gamma is required for the differentiation of \nadipose tissue in vivo and in vitro. Mol Cell 4, 611-617. 10.1016/s1097-2765(00)80211-7. \n11. Takada, I., Yogiashi, Y ., and Kato, S. (2012). Signaling Crosstalk between PPARgamma \nand BMP2 in Mesenchymal Stem Cells. PPAR Res 2012, 607141. \n10.1155/2012/607141. \n12. Sun, L., Jin, Y., Nishio, M., Watanabe, M., Kamakura, T., Nagata, S., Fukuda, M., \nMaekawa, H., Kawai, S., Yamamoto, T., and Toguchida, J. (2024). Oxidative \nphosphorylation is a pivotal therapeutic target of fibrodysplasia ossificans progressiva. \nLife Sci Alliance 7. 10.26508/lsa.202302219. \n13. Alexander, K.A., Tseng, H.W., Lao, H.W., Girard, D., Barbier, V., Ungerer, J.P .J., \nMcWhinney, B.C., Samuel, S.G., Fleming, W., Winkler, I.G., Salga, M., et al. (2024). 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Arsoy, D., Salib, C.G., Trousdale, W.H., Tibbo, M.E., Limberg, A.K., Viste, A., Lewallen, \nE.A., Reina, N., Yaszemski, M.J., Berry, D.J., van Wijnen, A.J., et al. (2018). Joint \ncontracture is reduced by intra-articular implantation of rosiglitazone-loaded hydrogels in \na rabbit model of arthrofibrosis. J Orthop Res 36, 2949-2955. 10.1002/jor.24068. \n22. Hino, K., Zhao, C., Horigome, K., Nishio, M., Okanishi, Y., Nagata, S., Komura, S., \nYamada, Y., Toguchida, J., Ohta, A., and Ikeya, M. (2018). An mTOR Signaling \nModulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans \nProgressiva. Stem Cell Reports 11, 1106-1119. 10.1016/j.stemcr.2018.10.007. \n23. Cannon, M., Stevenson, J., Stahl, K., Basu, R., Coffman, A., Kiwala, S., McMichael, J.F., \nKuzma, K., Morrissey, D., Cotto, K., Mardis, E.R., et al. (2024). DGIdb 5.0: rebuilding the \ndrug-gene interaction database for precision medicine and drug discovery platforms. \nNucleic Acids Res 52, D1227-D1235. 10.1093/nar/gkad1040. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nACKNOWLEDGEMENT:  \nThe authors acknowledge 1K08AR082031 (NIH/NIAMS), Plastic Surgery Foundation National \nEndowment for Plastic Surgery Award, Million Dollar Bike Ride Foundation, International FOP \nAssociation, Stepping Strong Foundation and Beal Fellowship to SA, Stepping Strong Innovator \nAward to PK, National Nature Science Foundation of China (82404113), Basic and Applied \nBasic Research Foundation of Guangdong Province (2023A1515111068) and Shenzhen \nScience and Technology Program (JCYJ20230807095121041) to ZC, Hale fellowship, Coller \nAward and PSF fellowship to AS. This work in part was supported by NIH T35 fellowship to ZM, \nNIH T35 HL110843 fellowship to HS. \n \nAuthor contributions: Conceptualization: PK, ZC, SNH, SA; Methodology: PK, ZC, SNH, AS, \nCL, ZM, HS, DM; Investigation: PK, ZC, SNH, SA; Visualization: PK, ZC, SNH, HS; Funding \nacquisition: ZC, SA, Project administration: PK, ZC, SNH, SA, Supervision: YM, VR, SA, Writing \n– original draft: PK, ZC, HS, SA, Writing – review & editing: PK, ZC, YM, VR, SA. \n \nCompeting interests: Authors declare that they have no competing interests. \n \nData and materials availability: All data are available in the main text. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nFIGURES \nFig. 1. Drug repurposing for fibrodysplasia ossificans progressiva (FOP) and heterotopic \nossification (HO). (A) Pairwise correlation heatmap of RNA-seq profiles. Pearson correlations \nfor log₂(TPM + 1)–normalized expression of top 3000 variable genes in injured gastrocnemius \nfrom normal (n = 5) and FOP (n = 5) mice (GSE220725). (B) Volcano plot of FOP vs. normal \ninjury differential expression. Points show genes with log₂ fold change (x axis) and –log₁₀(P \nvalue) from Welch's t test (y axis). Gray: all genes; red: significant (|log₂FC| ≥ 3, P < 0.01). \nDashed blue lines: thresholds. Top 10 genes per direction labeled by composite rank of fold \nchange and significance. (C) Drug scoring for FOP gene expression reversal. Scatter plot of \nnon-immunotherapeutic, non-antineoplastic drugs. horizontal axis: total score (sum of [drug \neffect × –log₂FC]); vertical axis: SD of contributions. Rosiglitazone highlighted in red/bold. (D) \nDrug scoring for HO reversal (cardiotoxin model). Scoring as in (C), using differential expression \nfrom cardiotoxin + dexamethasone (HO; n = 4) vs. cardiotoxin + saline (control; n = 4) at day 4 \n(GEO: GSE218699). (E) Drug scoring for HO reversal (burn/tenotomy model). Scoring as in (C), \nusing differential expression from burn/tenotomy sites (HO; n = 2) vs. uninjured contralateral \nlimbs (n = 2) at 3 weeks (GEO: GSE126118).  \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\n \nFig. 2. FOP lesions do not contain adipocytes or adipogenic signals. (A) Schematic \nrepresentation of the procedure used to develop the FOP mouse model. Mice were treated with \ntamoxifen to induce the R206H mutation in ACVR1, and cardiotoxin was injected to induce \nheterotopic ossification (HO). Tissue was harvested after 3 weeks of cardiotoxin injection. (B) \nPicrosirius Red, Safranin-O/Fast Green, and H&E staining of hindlimbs from FOP mice treated \nwith cardiotoxin show bone lesions but no adipocytes. (C) Immunostaining of FOP mice treated \nwith cardiotoxin shows Perilipin-1–positive cells in the bone marrow, but not in the soft tissue \nsurrounding the bone. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nFig. 3. Treatment with a PPARγ agonist suppresses osteogenesis and induces \nadipogenesis in bone marrow-derived mesenchymal cells in vitro. (A) Bone marrow–\nderived mesenchymal cells from FOP mice were treated with 10µM rosiglitazone, a PPARγ \nagonist, in the presence or absence of 25ng/ml Activin A. Activin A induces osteogenic gene \nexpression in FOP cell lines. Expression levels of adipogenic genes such as LPL and FABP4 \nincreased with rosiglitazone treatment, whereas expression of osteogenic genes such as Sox9, \nCol2A1, and Aggrecan decreased. (n = 3) Statistical significance between two groups was \ndetermined using Student's t-test (p < 0.05). B-actin was used for normalization. (B) Bone \nmarrow–derived mesenchymal cells from WT mice were treated with 10µM rosiglitazone in the \npresence or absence of 25ng/ml BMP2, which induces osteogenic gene expression. Like FOP \ncells, expression of adipogenic genes (LPL and FABP4) increased, while expression of \nosteogenic genes (Sox9, Col2A1, and Aggrecan) decreased. (n = 3) Statistical significance \nbetween two groups was determined using Student's t-test (p < 0.05). B-actin was used for \nnormalization. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nFig. 4. Treatment with rosiglitazone significantly reduces formation of post-injury FOP \nlesions. (A) X-ray and Micro-CT imaging revealed a greater volume of ectopic bone in the \nhindlimbs of FOP mice treated with the corn oil vehicle control compared to those treated with \nrosiglitazone. (B) Quantification of ectopic bone showed an approximately 10-fold decrease in \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nectopic bone volume following rosiglitazone treatment. (n = 8) Statistical significance between \ntwo groups was determined using Student’s t-test (p < 0.05). (C) H&E, Picrosirius Red, and \nSafranin-O/Fast Green staining of FOP mouse hindlimbs showed a reduction in FOP lesions \nand an increase in soft tissue–resident adipocytes after rosiglitazone treatment. Chondrocytes \nare represented by the red staining of Saffranin-O whereas the Fast Green stain represents the \nbone. (D) Immunofluorescence imaging of rosiglitazone-treated FOP mice showed an increase \nin soft tissue–resident adipocytes, indicated by PPARγ and Perilipin-1–positive cells. In contrast, \nhindlimbs of control mice exhibited adipogenic signals only within the bone marrow, consistent \nwith bone marrow–resident adipocytes. Soft tissue–resident adipocytes were absent in control \nmice. pSMAD1/5 staining revealed abundant positive cells in the bone marrow of control mice \nand in the soft tissue of rosiglitazone-treated mice. Soft tissue resident adipocytes were also \npositive for PDGFRα, suggesting that the adipocytes were derived from FAPs. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nFig. 5. Systemic treatment with rosiglitazone reduces heterotopic bone formation in a \ntrauma-induced HO model. (A) Schematic representation of the procedure used to induce \nheterotopic ossification (HO) in wild-type (WT) mice via Achilles tendon tenotomy. Tissue was \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\nharvested 3 weeks post tenotomy. (B) Micro-CT imaging showed ectopic bone in the soft tissue \nand surrounding the calcaneus of the right hindlimb where tenotomy was performed. (C) H&E \nand Safranin-O/Fast Green staining of tenotomized WT mice showed a reduction in HO lesions \nand an increase in soft tissue–resident adipocytes following rosiglitazone treatment. (D) \nImmunofluorescence imaging of rosiglitazone-treated tenotomy mice revealed an increase in \nsoft tissue–resident adipocytes, indicated by PPARγ and Perilipin-1–positive cells. In contrast, \ncontrol mice displayed adipogenic signals only within the bone marrow, consistent with bone \nmarrow–resident adipocytes. Soft tissue–resident adipocytes were absent in control mice. \npSMAD1/5 staining showed abundant positive cells in the bone marrow of control mice and in \nthe soft tissue of rosiglitazone-treated mice. \n \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\n \nFig. 6. Local treatment with rosiglitazone reduces heterotopic bone formation in a \ntrauma-induced HO model. (A) Schematic representation of the procedure used to induce \nheterotopic ossification (HO) in wild-type (WT) mice via Achilles tendon tenotomy, followed by \nlocal rosiglitazone injection. Tissue was harvested 3 weeks post tenotomy. (B) H&E and \nSafranin-O/Fast Green staining of tenotomized WT mice showed a reduction in HO lesions and \nan increase in soft tissue–resident adipocytes after rosiglitazone treatment. (C) \nImmunofluorescence imaging of rosiglitazone-treated tenotomy mice revealed an increase in \nsoft tissue–resident adipocytes, as indicated by PPARγ and Perilipin-1–positive cells. In \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint \n\ncontrast, control mice showed adipogenic signals only within the bone marrow, consistent with \nbone marrow–resident adipocytes, while soft tissue–resident adipocytes were absent. \npSMAD1/5 staining showed abundant positive cells in the bone marrow of control mice and in \nthe soft tissue of rosiglitazone-treated mice. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 2, 2026. ; https://doi.org/10.64898/2026.02.26.708276doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}