Abstract
Gorham-Stout disease (GSD) is a sporadic chronic disease characterized by progressive bone dissolution, absorption, and disappearance along with lymphatic vessel infiltration in bone-marrow cavities. Although the osteolytic mechanism of GSD has been widely studied, the cause of lymphatic hyperplasia in GSD is rarely investigated. In this study, by comparing the RNA expression profile of osteoclasts (OCs) with that of OC precursors (OCPs) by RNA sequencing, we identified a new factor, semaphorin 3A (Sema3A), which is an osteoprotective factor involved in the lymphatic expansion of GSD. Compared to OCPs, OCs enhanced the growth, migration, and tube formation of lymphatic endothelial cells (LECs), in which the expression of Sema3A is low compared to that in OCPs. In the presence of recombinant Sema3A, the growth, migration, and tube formation of LECs were inhibited, further confirming the inhibitory effect of Sema3A on LECs in vitro. Using an LEC-induced GSD mouse model, the effect of Sema3A was examined by injecting lentivirus-expressing Sema3A into the tibiae in vivo. We found that the overexpression of Sema3A in tibiae suppressed the expansion of LECs and alleviated bone loss, whereas the injection of lentivirus expressing Sema3A short hairpin RNA (shRNA) into the tibiae caused GSD-like phenotypes. Histological staining further demonstrated that OCs decreased and osteocalcin increased after Sema3A lentiviral treatment, compared with the control. Based on the above results, we propose that reduced Sema3A in OCs is one of the mechanisms contributing to the pathogeneses of GSD and that expressing Sema3A represents a new approach for the treatment of GSD.
摘要
戈勒姆综合征(Gorham-Stout disease, GSD)是一种罕见的散发性慢性骨科疾病, 以进行性骨溶解、 吸收和消失为特征, 伴骨髓腔淋巴管浸润. 虽然GSD的溶骨机制已被广泛研究, 但其骨中淋巴管增生的原因却很少被触及. 本研究通过RNA测序, 比较破骨细胞(OCs)和破骨细胞前体(OCPs)的RNA表达谱, 发现了具有骨保护作用的因子信号素3A(Sema3A)在OCs中的表达显著降低了. 并且与OCPs相比, OCs促进了淋巴管内皮细胞(LECs)的生长、 迁移和体外淋巴管管状形成. 同时体外研究发现, 重组Sema3A能抑制LECs的生长、 迁移和体外淋巴管管状形成, 证实了Sema3A对LECs的抑制作用. 采用LECs诱导的GSD小鼠模型, 通过在胫骨内注射表达Sema3A的慢病毒, 我们进一步检测Sema3A在体内的作用. 结果表明, 在胫骨中过表达Sema3A可抑制LECs的扩张, 减少骨丢失. 而在胫骨中注射表达Sema3A shRNA的慢病毒以敲低Sema3A的表达可引发小鼠胫骨GSD样表型. 组织学染色分析表明, 与对照组相比, Sema3A慢病毒治疗后OCs减少, 骨钙素增加. 基于以上结果, 我们认为OCs中Sema3A的减少是GSD的发病机制之一, 表达Sema3A代表了一种治疗GSD的新方案.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability statement
All data generated or analyzed during this study are included in this published article and its supplementary information files.
References
al Baroudi S, Jabre NA, Dunn E, et al., 2020. A 15-year-old boy with dyspnea and vanishing bones. Am J Respir Crit Care Med, 202(3):451–452. https://doi.org/10.1164/rccm.201907-1339IM
Bernatchez PN, Rollin S, Soker S, et al., 2002. Relative effects of VEGF-A and VEGF-C on endothelial cell proliferation, migration and PAF synthesis: role of neuropilin-1. J Cell Biochem, 85(3):629–639. https://doi.org/10.1002/jcb.10155
Biswas L, Chen JY, de Angelis J, et al., 2023. Lymphatic vessels in bone support regeneration after injury. Cell, 186(2):382–397.e24. https://doi.org/10.1016/j.cell.2022.12.031
Bruch-Gerharz D, Gerharz CD, Stege H, et al., 2007. Cutaneous lymphatic malformations in disappearing bone (Gorham-Stout) disease: a novel clue to the pathogenesis of a rare syndrome. J Am Acad Dermatol, 56(S2):S21–S25. https://doi.org/10.1016/j.jaad.2006.01.063
Bussolino F, Giraudo E, Serini G, 2013. Class 3 semaphorin in angiogenesis and lymphangiogenesis. In: S. Karger AG (Ed.), Angiogenesis, Lymphangiogenesis and Clinical Implications. Karger, Basel, p.71-88. https://doi.org/10.1159/000353315
Casazza A, Fu X, Johansson I, et al., 2011. Systemic and targeted delivery of semaphorin 3A inhibits tumor angiogenesis and progression in mouse tumor models. Arterioscler Thromb Vasc Biol, 31(4):741–749. https://doi.org/10.1161/atvbaha.110.211920
Dellinger MT, McCormack FX, 2020. The emergence of targetable MEKanisms in sporadic lymphatic disorders. EMBO Mol Med, 12(10):e12822. https://doi.org/10.15252/emmm.202012822
Dellinger MT, Garg N, Olsen BR, 2014. Viewpoints on vessels and vanishing bones in Gorham-Stout disease. Bone, 63:47–52. https://doi.org/10.1016/j.bone.2014.02.011
Edwards JR, Williams K, Kindblom LG, et al., 2008. Lymphatics and bone. Hum Pathol, 39(1):49–55. https://doi.org/10.1016/j.humpath.2007.04.022
Favier B, Alam A, Barron P, et al., 2006. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration. Blood, 108(4):1243–1250. https://doi.org/10.1182/blood-2005-11-4447
Franco-Barrera MJ, Zavala-Cerna MG, Aguilar-Portillo G, et al., 2017. Gorham-Stout disease: a clinical case report and immunological mechanisms in bone erosion. Clin Rev Allergy Immunol, 52:125–132. https://doi.org/10.1007/s12016-016-8594-z
Fukuda T, Takeda S, Xu R, et al., 2013. Sema3A regulates bone-mass accrual through sensory innervations. Nature, 497(7450):490–493. https://doi.org/10.1038/nature12115
Gorham LW, Stout AP, 1955. Massive osteolysis (acute spontaneous absorption of bone, phantom bone, disappearing bone): its relation to hemangiomatosis. J Bone Joint Surg Am, 37-A(5):985–1004.
Gu CH, Rodriguez ER, Reimert DV, et al., 2003. Neuropilin-1 conveys semaphorin and VEGF signaling during neural and cardiovascular development. Dev Cell, 5(1):45–57. https://doi.org/10.1016/s1534-5807(03)00169-2
Guttmann-Raviv N, Shraga-Heled N, Varshavsky A, et al., 2007. Semaphorin-3A and semaphorin-3F work together to repel endothelial cells and to inhibit their survival by induction of apoptosis. J Biol Chem, 282(36):26294–26305. https://doi.org/10.1074/jbc.M609711200
Hayashi M, Nakashima T, Taniguchi M, et al., 2012. Osteoprotection by semaphorin 3A. Nature, 485(7396):69–74. https://doi.org/10.1038/nature11000
Hayashi M, Nakashima T, Yoshimura N, et al., 2019. Autoregulation of osteocyte Sema3A orchestrates estrogen action and counteracts bone aging. Cell Metab, 29(3):627–637.e5. https://doi.org/10.1016/j.cmet.2018.12.021
Hu LH, Wu W, Zou J, 2022. Circular RNAs: typical biomarkers for bone-related diseases. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 23(12):975–988. https://doi.org/10.1631/jzus.B2200211
Hirayama T, Sabokbar A, Itonaga I, et al., 2001. Cellular and humoral mechanisms of osteoclast formation and bone resorption in Gorham-Stout disease. J Pathol, 195(5):624–630. https://doi.org/10.1002/path.989
Homayun-Sepehr N, Mccarter AL, Helaers R, et al., 2021. KRAS-driven model of Gorham-Stout disease effectively treated with trametinib. JCI Insight, 6(15):e149831. https://doi.org/10.1172/jci.insight.149831
Hominick D, Silva A, Khurana N, et al., 2018. VEGF-C promotes the development of lymphatics in bone and bone loss. Elife, 7:e34323. https://doi.org/10.7554/eLife.34323
Jiao B, Liu SY, Tan X, et al., 2021. Class-3 semaphorins: potent multifunctional modulators for angiogenesis-associated diseases. Biomed Pharmacother, 137:111329. https://doi.org/10.1016/j.biopha.2021.111329
Joyal JS, Sitaras N, Binet F, et al., 2011. Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A. Blood, 117(22):6024–6035. https://doi.org/10.1182/blood-2010-10-311589
Jurisic G, Maby-El Hajjami H, Karaman S, et al., 2012. An unexpected role of semaphorin3A–neuropilin-1 signaling in lymphatic vessel maturation and valve formation. Circ Res, 111(4):426–436. https://doi.org/10.1161/circresaha.112.269399
Kim JM, Lin CJ, Stavre Z, et al., 2020. Osteoblast-osteoclast communication and bone homeostasis. Cells, 9(9):2073. https://doi.org/10.3390/cells9092073
Lee H, Macpherson LJ, Parada CA, et al., 2017. Rewiring the taste system. Nature, 548(7667):330–333. https://doi.org/10.1038/nature23299
Li TT, Zhang SH, Yang YX, et al., 2022. Co-regulation of circadian clock genes and microRNAs in bone metabolism. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 23(7):529–546. https://doi.org/10.1631/jzus.B2100958
Liu SZ, Zhou X, Song A, et al., 2018a. A rare case of Gorham-Stout syndrome of femur treated with cement augmentation. Chin Med J (Engl), 131(13):1628–1629. https://doi.org/10.4103/0366-6999.235121
Liu SZ, Zhou X, Song A, et al., 2018b. Successful treatment of Gorham-Stout syndrome in the spine by vertebroplasty with cement augmentation: a case report and literature review. Medicine (Baltimore), 97(29):e11555. https://doi.org/10.1097/md.0000000000011555
Maione F, Molla F, Meda C, et al., 2009. Semaphorin 3A is an endogenous angiogenesis inhibitor that blocks tumor growth and normalizes tumor vasculature in transgenic mouse models. J Clin Invest, 119(11):3356–3372. https://doi.org/10.1172/jci36308
Mao TJ, Xu L, Yu YM, et al., 2018. Use of zoledronic acid combined with thalidomide in the treatment of Gorham-Stout disease. Rheumatology (Oxford), 57(6):1122–1124. https://doi.org/10.1093/rheumatology/key055
Möller G, Priemel M, Amling M, et al., 1999. The Gorham-Stout syndrome (Gorham’s massive osteolysis). A report of six cases with histopathological findings. J Bone Joint Surg Br, 81(3):501–506. https://doi.org/10.1302/0301-620x.81b3.9468
Monroy M, Mccarter AL, Hominick D, et al., 2020. Lymphatics in bone arise from pre-existing lymphatics. Development, 147(21):dev184291. https://doi.org/10.1242/dev.184291
Nozawa A, Ozeki M, Niihori T, et al., 2020. A somatic activating KRAS variant identified in an affected lesion of a patient with Gorham-Stout disease. J Hum Genet, 65(11):995–1001. https://doi.org/10.1038/s10038-020-0794-y
Ochsenbein AM, Karaman S, Jurisic G, et al., 2014. The role of neuropilin-1/semaphorin 3A signaling in lymphatic vessel development and maturation. In: Kiefer F, Schulte-Merker S (Eds.), Developmental Aspects of the Lymphatic Vascular System. Springer, Vienna, p.143–152. https://doi.org/10.1007/978-3-7091-1646-3_11
Ochsenbein AM, Karaman S, Proulx ST, et al., 2016. Endothelial cell-derived semaphorin 3A inhibits filopodia formation by blood vascular tip cells. Development, 143(4):589–594. https://doi.org/10.1242/dev.127670
Páez Codeso FM, Morillo Domínguez MC, Dorado Galindo A, 2017. A rare case of chylothorax. Gorham-Stout syndrome. Arch Bronconeumol, 53(11):640. https://doi.org/10.1016/j.arbres.2017.04.010
Qu LY, Cai XY, Wang BL, 2018. Diagnosis and treatment of Gorham-Stout disease in maxillofacial regions. J Craniofac Surg, 29(2):460–461. https://doi.org/10.1097/scs.0000000000004188
Raimondi C, Ruhrberg C, 2013. Neuropilin signalling in vessels, neurons and tumours. Semin Cell Dev Biol, 24(3):172–178. https://doi.org/10.1016/j.semcdb.2013.01.001
Ricci KW, Hammill AM, Mobberley-Schuman P, et al., 2019. Efficacy of systemic sirolimus in the treatment of generalized lymphatic anomaly and Gorham-Stout disease. Pediatr Blood Cancer, 66(5):e27614. https://doi.org/10.1002/pbc.27614
Sakurai A, Doçi CL, Gutkind JS, 2012. Semaphorin signaling in angiogenesis, lymphangiogenesis and cancer. Cell Res, 22:23–32. https://doi.org/10.1038/cr.2011.198
Serini G, Valdembri D, Zanivan S, et al., 2003. Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function. Nature, 424(6947):391–397. https://doi.org/10.1038/nature01784
Staton CA, 2011. Class 3 semaphorins and their receptors in physiological and pathological angiogenesis. Biochem Soc Trans, 39(6):1565–1570. https://doi.org/10.1042/bst20110654
Toledano S, Nir-Zvi I, Engelman R, et al., 2019. Class-3 semaphorins and their receptors: potent multifunctional modulators of tumor progression. Int J Mol Sci, 20(3):556. https://doi.org/10.3390/ijms20030556
van der Klaauw AA, Croizier S, de Oliveira EM, et al., 2019. Human semaphorin 3 variants link melanocortin circuit development and energy balance. Cell, 176(4):729–742.e18. https://doi.org/10.1016/j.cell.2018.12.009
Wang WS, Wang H, Zhou XC, et al., 2017. Lymphatic endothelial cells produce M-CSF, causing massive bone loss in mice. J Bone Miner Res, 32(5):939–950. https://doi.org/10.1002/jbmr.3077
Wilkinson MF, 2019. Genetic paradox explained by nonsense. Nature, 568(7751):179–180. https://doi.org/10.1038/d41586-019-00823-5
Wu JH, Zhou YF, Hong CD, et al., 2019. Semaphorin-3A protects against neointimal hyperplasia after vascular injury. EBioMedicine, 39:95–108. https://doi.org/10.1016/j.ebiom.2018.12.023
Yamashita Y, Hayashi M, Saito M, et al., 2022. Osteoblast lineage cell-derived Sema3A regulates bone homeostasis independently of androgens. Endocrinology, 163(10):bqac126. https://doi.org/10.1210/endocr/bqac126
Yang K, Miron RJ, Bian Z, et al., 2018. A bone-targeting drug-delivery system based on semaphorin 3A gene therapy ameliorates bone loss in osteoporotic ovariectomized mice. Bone, 114:40–49. https://doi.org/10.1016/j.bone.2018.06.003
Zhang Q, Guo RL, Lu Y, et al., 2008. VEGF-C, a lymphatic growth factor, is a RANKL target gene in osteoclasts that enhances osteoclastic bone resorption through an autocrine mechanism. J Biol Chem, 283(19):13491–13499. https://doi.org/10.1074/jbc.M708055200
Acknowledgments
This study was supported by the National Natural Science Foundation of China (No. 81974321), the Discipline Innovation and Talent Introduction Program to Universities from Ministry of Education of China (Project 111), the Tackling Project for Science and Technology of Xinxiang City (No. GG2019003), the Natural Science Foundation of Henan Province of China (No. 212300410173), the State Administration of Traditional Chinese Medicine Young Qi Huang Scholar, and the Innovation Team Project of Scientific Research of Traditional Chinese Medicine of Shanghai Health Committee (No. 2022CX001), China. We thank Dr. Peipei ZHANG (Henan Normal University, Xinxiang, China) and Mr. Yongjian ZHAO (Shanghai University of Traditional Chinese Medicine, Shanghai, China) for micro-CT scanning.
Author information
Authors and Affiliations
Contributions
Wensheng WANG: conceptualization, supervision, funding acquisition, methodology, project administration, and writing - review and editing. Qianqian LIANG: conceptualization, supervision, project administration, writing - original draft, and writing - review and editing. Dongfang ZHANG: writing - original draft, formal analysis, investigation, and visualization. Hao XU, Chi QIN, Kangming CAI, Jing ZHANG, and Xinqiu XIA: formal analysis, investigation, and visualization. Jingwen BI: visualization. Li ZHANG: funding acquisition, writing–original draft, and writing–review and editing. Lianping XING: methodology and writing–review and editing. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
Corresponding authors
Ethics declarations
Dongfang ZHANG, Hao XU, Chi QIN, Kangming CAI, Jing ZHANG, Xinqiu XIA, Jingwen BI, Li ZHANG, Lianping XING, Qianqian LIANG, and Wensheng WANG declare that they have no conflicts of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed. The animal study protocol was approved by the Ethics Committee of Henan Normal University (No. HNSD-2019-12-02).
Additional information
Materials and methods
Detailed methods are provided in the electronic supplementary materials of this paper.
Supplementary information
Materials and methods; Table S1; Figs. S1–S4
Supplementary information
Rights and permissions
About this article
Cite this article
Zhang, D., Xu, H., Qin, C. et al. Reduced expression of semaphorin 3A in osteoclasts causes lymphatic expansion in a Gorham-Stout disease (GSD) mouse model. J. Zhejiang Univ. Sci. B 25, 38–50 (2024). https://doi.org/10.1631/jzus.B2300180
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1631/jzus.B2300180