内皮细胞力学敏感蛋白S1Pr1在长骨缺损修复中参与成血管-成骨偶联的研究

骨骼的发育、重塑和再生均对力学刺激高度敏感。近期研究发现力学刺激可通过调控骨骼组织中与成骨偶联的H型血管调控骨修复，使其有望成为治疗骨损伤的新策略。然而，力学刺激调控骨修复的关键蛋白尚不明确；成血管-成骨偶联的机制也尚未阐明。血管内皮细胞表面的鞘氨醇-1-磷酸受体1（sphingosine-1-phosphate receptor 1, S1Pr1）是调控血管新生、稳定性维持及重构的关键蛋白之一，也是一种力学敏感蛋白。因此，S1Pr1可能是力学刺激调控骨修复、H型血管行为及成血管-成骨偶联的重要机制。探索血管内皮细胞S1Pr1信号在骨修复中的力学响应有望揭示骨修复中的新力学生物学机制，丰富对成血管-成骨偶联的了解，为应用力学刺激促进骨修复新疗法提供科学依据。

首先，本研究基于深层组织成像建立了一种新的单细胞三维空间分析方法（single cell 3D spatial correlation，sc3DSC），克服了在骨修复中原位定量细胞间相互作用受限的问题。本研究利用该方法结合干细胞植入、荧光探针追踪研究了骨修复过程中，植入干细胞的行为及其与H型血管的偶联。本研究发现在骨修复过程中，大部分植入的干细胞定位于H型血管周围并显著提高了缺损组织中成骨细胞的数量。只有少量植入的干细胞进行了成骨分化。部分植入细胞在骨修复过程中持续表达干细胞标记物，并且通过分泌成血管因子VEGFA和PDGF-BB提高了缺损部位H型血管体积，并且招募了大量自体来源干细胞。这一结果显示植入干细胞促进骨缺损修复的作用可能通过招募本体干细胞及调控成血管-成骨偶联而实现的。

随后，本研究利用S1Pr1特异性拮抗剂、活体力学刺激及sc3DSC分析了缺损中S1Pr1信号通路在力学刺激促进长骨修复中的作用。结果发现缺损中高表达S1Pr1的细胞中40-50%是H型血管内皮细胞。拮抗S1Pr1减少了H型血管体积及血管周围成骨系细胞数量，抑制了力学刺激诱导的骨修复，显示S1Pr1是骨修复过程中力学响应及成血管-成骨偶联的重要组成部分。

最后，本研究构建了在血管内皮细胞中条件性敲除S1Pr1的小鼠模型，结合活体力学刺激、sc3DSC分析及体外细胞实验，探索了血管内皮细胞S1Pr1在力学刺激诱导骨修复中的机制。结果显示力学刺激所激活的血管内皮细胞S1Pr1受体，一方面通过下游YAP-VE-cadherin通路抑制新生的血管重构，提高H型血管体积；另一方面促进了内皮细胞分泌成骨相关因子MMP13，提高了血管周围干细胞的成骨分化，促进了骨修复。本研究提出了一种新的骨组织再生中成血管-成骨偶联的力学生物学机制，为推进应用力学刺激治疗骨修复的发展提供了临床前模型动物中的科学依据。

[1] BURGE R, DAWSON-HUGHES B, SOLOMON D H, et al. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025 [J]. Journal of Bone and Mineral Research, 2007, 22(3): 465-75.
[2] REICHERT J C, CIPITRIA A, EPARI D R, et al. A tissue engineering solution for segmental defect regeneration in load-bearing long bones [J]. Science Translational Medicine, 2012, 4(141): 141ra93-ra93.
[3] WILLIAM JR G, EINHORN T A, KOVAL K, et al. Bone grafts and bone graft substitutes in orthopaedic trauma surgery: a critical analysis [J]. The Journal of Bone and Joint Surgery, 2007, 89(3): 649-58.
[4] TANNOURY C A, AN H S J T S J. Complications with the use of bone morphogenetic protein 2 (BMP-2) in spine surgery [J]. Spine Journal, 2014, 14(3): 552-9.
[5] SI L, WINZENBERG T, JIANG Q, et al. Projection of osteoporosis-related fractures and costs in China: 2010–2050 [J]. Osteoporosis International, 2015, 26(7): 1929-37.
[6] DECOSTER T A, GEHLERT R J, MIKOLA E A, et al. Management of posttraumatic segmental bone defects [J]. JAAOS-Journal of the American Academy of Orthopaedic Surgeons, 2004, 12(1): 28-38.
[7] KRONENBERG H M. Developmental regulation of the growth plate [J]. Nature, 2003, 423(6937): 332-6.
[8] LONG F. Building strong bones: molecular regulation of the osteoblast lineage [J]. Nature reviews Molecular Cell Biology, 2011, 13(1): 27-38.
[9] RAMASAMY S K, KUSUMBE A P, SCHILLER M, et al. Blood flow controls bone vascular function and osteogenesis [J]. Nature Communications, 2016, 7(1): 13601.
[10] DIOMEDE F, MARCONI G D, FONTICOLI L, et al. Functional Relationship between Osteogenesis and Angiogenesis in Tissue Regeneration [J]. International Journal of Molecular Sciences, 2020, 21(9):3242.
[11] MAES C, KOBAYASHI T, SELIG M K, et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels [J]. Developmental Cell, 2010, 19(2): 329-44.
[12] HAUSMAN M R, SCHAFFLER M B, MAJESKA R J. Prevention of fracture healing in rats by an inhibitor of angiogenesis [J]. Bone, 2001, 29(6): 560-4.
[13] SIVARAJ K K, ADAMS R H. Blood vessel formation and function in bone [J]. Development (Cambridge, England), 2016, 143(15): 2706-15.
[14] RISAU W, FLAMME I, BIOLOGY D. Vasculogenesis [J]. Mechanisms of Development, 1995, 11(1): 73-91.
[15] GEUDENS I, GERHARDT H. Coordinating cell behaviour during blood vessel formation [J]. Nature Reviews Molecular Cell Biology, 2011, 138(21): 4569-83.
[16] ADAMS R H, ALITALO K. Molecular regulation of angiogenesis and lymphangiogenesis [J]. Nature Reviews Molecular Cell Biology, 2007, 8(6): 464-78.
[17] CARMELIET P, JAIN R K. Molecular mechanisms and clinical applications of angiogenesis [J]. Nature, 2011, 473(7347): 298-307.
[18] KUHNERT F, MANCUSO M R, SHAMLOO A, et al. Essential regulation of CNS angiogenesis by the orphan G protein–coupled receptor GPR124 [J]. Science, 2010, 330(6006): 985-9.
[19] NOLAN D J, GINSBERG M, ISRAELY E, et al. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration [J]. Developmental Cell, 2013, 26(2): 204-19.
[20] RAFII S, BUTLER J M, DING B-S. Angiocrine functions of organ-specific endothelial cells [J]. Nature, 2016, 529(7586): 316-25.
[21] CROCK H J. A revision of the anatomy of the arteries supplying the upper end of the human femur [J]. Journal of Anatomy, 1965, 99(Pt 1): 77.
[22] TRUETA J, MORGAN J D. The vascular contribution to osteogenesis. I. Studies by the injection method [J]. The Journal of Bone and Joint Surgery, 1960, 42: 97-109.
[23] CARMELIET P, DE SMET F, LOGES S, et al. Branching morphogenesis and antiangiogenesis candidates: tip cells lead the way [J]. Nature Reviews Clinical Oncology, 2009, 6(6): 315-26.
[24] CARMELIET P. Mechanisms of angiogenesis and arteriogenesis [J]. Nature Medicine, 2000, 6(4): 389-95.
[25] RAMASAMY S K, KUSUMBE A P, WANG L, et al. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone [J]. Nature, 2014, 507(7492): 376-80.
[26] PENG Y, WU S, LI Y, et al. Type H blood vessels in bone modeling and remodeling [J]. Theranostics, 2020, 10(1): 426.
[27] TIEMEIJER L, FRIMAT J, STASSEN O, et al. Spatial patterning of the Notch ligand Dll4 controls endothelial sprouting in vitro [J]. Scientific Reports, 2018, 8(1): 6392.
[28] BOOPATHY G T, HONG W J F I C, BIOLOGY D. Role of hippo pathway-YAP/TAZ signaling in angiogenesis [J]. Frontiers in Cell and Developmental Biology, 2019, 7: 49.
[29] SIVARAJ K K, DHARMALINGAM B, MOHANAKRISHNAN V, et al. YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells [J]. ELife, 2020, 9: e50770.
[30] HENDRIKS M, RAMASAMY S K. Blood Vessels and Vascular Niches in Bone Development and Physiological Remodeling [J]. Frontiers in Cell and Developmental Biology, 2020, 8: 602278.
[31] KUSUMBE A P, RAMASAMY S K, ADAMS R H. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone [J]. Nature, 2014, 507(7492): 323-8.
[32] PENG Y, WU S, LI Y, et al. Type H blood vessels in bone modeling and remodeling [J]. Theranostics, 2020, 10(1): 426-36.
[33] KUSUMBE A P, RAMASAMY S K, ITKIN T, et al. Age-dependent modulation of vascular niches for haematopoietic stem cells [J]. Nature, 2016, 532(7599): 380-4.
[34] ZHOU B O, YUE R, MURPHY M M, et al. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow [J]. Cell Stem Cell, 2014, 15(2): 154-68.
[35] RAMASAMY S K J S C I. Structure and functions of blood vessels and vascular niches in bone [J]. Stem Cells International, 2017, 2017.
[36] ONO N, ONO W, MIZOGUCHI T, et al. Vasculature-associated cells expressing nestin in developing bones encompass early cells in the osteoblast and endothelial lineage [J]. Developmental Cell, 2014, 29(3): 330-9.
[37] JEFFERY E C, MANN T L, POOL J A, et al. Bone marrow and periosteal skeletal stem/progenitor cells make distinct contributions to bone maintenance and repair [J]. Cell Stem Cell, 2022, 29(11): 1547-61. e6.
[38] MIZOGUCHI T, PINHO S, AHMED J, et al. Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development [J]. Developmental Cell, 2014, 29(3): 340-9.
[39] CHEN C, ULUDAĞ H, WANG Z, et al. Noggin suppression decreases BMP-2-induced osteogenesis of human bone marrow-derived mesenchymal stem cells in vitro [J]. Journal of Cellular Biochemistry, 2012, 113(12): 3672-80.
[40] ROMEO S G, ALAWI K M, RODRIGUES J, et al. Endothelial proteolytic activity and interaction with non-resorbing osteoclasts mediate bone elongation [J]. Nature Cell Biology, 2019, 21(4): 430-41.
[41] XU R, YALLOWITZ A, QIN A, et al. Targeting skeletal endothelium to ameliorate bone loss [J]. Nature Medicine, 2018, 24(6): 823-33.
[42] GERBER H P, VU T H, RYAN A M, et al. VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation [J]. Nature Medicine, 1999, 5(6): 623-8.
[43] TOMBRAN-TINK J, BARNSTABLE C J. Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone [J]. Biochemical and Biophysical Research Communications, 2004, 316(2): 573-9.
[44] JAMES A W, PéAULT B. Perivascular Mesenchymal Progenitors for Bone Regeneration [J]. Journal of Orthopaedic Research, 2019, 37(6): 1221-8.
[45] ZHANG X, PéAULT B, CHEN W, et al. The Nell-1 growth factor stimulates bone formation by purified human perivascular cells [J]. Tissue Engineering Part A, 2011, 17(19-20): 2497-509.
[46] SACCHETTI B, FUNARI A, REMOLI C, et al. No Identical "Mesenchymal Stem Cells" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels [J]. Stem Cell Reports, 2016, 6(6): 897-913.
[47] ASKARINAM A, JAMES A W, ZARA J N, et al. Human perivascular stem cells show enhanced osteogenesis and vasculogenesis with Nel-like molecule I protein [J]. Tissue Engineering Part A, 2013, 19(11-12): 1386-97.
[48] JAMES A W, ZHANG X, CRISAN M, et al. Isolation and characterization of canine perivascular stem/stromal cells for bone tissue engineering [J]. PloS one, 2017, 12(5): e0177308.
[49] GöKçINAR-YAGCI B, YERSAL N, KORKUSUZ P, et al. Generation of human umbilical cord vein CD146+ perivascular cell origined three-dimensional vascular construct [J]. Microvascular Research, 2018, 118: 101-12.
[50] WOLFF J. The law of bone remodelling [M]. Springer Science & Business Media, 2012.
[51] FROST D E, GREGG J M, TERRY B C, et al. Mandibular interpositional and onlay bone grafting for treatment of mandibular bony deficiency in the edentulous patient [J]. Journal of Oral and Maxillofacial Surgery, 1982, 40(6): 353-60.
[52] SUN L-W, LI S, YANG X, et al. Contribution of bone micromechanical behavior beyond lamellar length scale to the macroscopic bone quality of hind limb unloading rats [J]. Acta Astronautica, 2018, 152: 468-73.
[53] ACEVEDO C, STADELMANN V A, PIOLETTI D P, et al. Fatigue as the missing link between bone fragility and fracture [J]. Nature Biomedical Engineering, 2018: 1.
[54] HUISKES R, RUIMERMAN R, VAN LENTHE G H, et al. Effects of mechanical forces on maintenance and adaptation of form in trabecular bone [J]. Nature, 2000, 405(6787): 704.
[55] LI H, LI R-X, WAN Z-M, et al. Counter-effect of constrained dynamic loading on osteoporosis in ovariectomized mice [J]. Journal of Biomechanics, 2013, 46(7): 1242-7.
[56] LIU C, CARRERA R, FLAMINI V, et al. Effects of mechanical loading on cortical defect repair using a novel mechanobiological model of bone healing [J]. Bone, 2018, 108: 145-55.
[57] RIDDLE R C, DONAHUE H J. From streaming potentials to shear stress: 25 Years of bone cell mechanotransduction [J]. Journal of Orthopaedic Research, 2009, 27(2): 143-9.
[58] CLAES L E, HEIGELE C A. Magnitudes of local stress and strain along bony surfaces predict the course and type of fracture healing [J]. Journal of Biomechanics, 1999, 32(3): 255-66.
[59] ANANI T, CASTILLO A B. Mechanically-regulated bone repair [J]. Bone, 2022, 154: 116223.
[60] ESTES B T, GIMBLE J M, GUILAK F. Mechanical signals as regulators of stem cell fate [J]. Current Topics in Developmental Biology, 2004, 60: 91-126.
[61] YUAN L, SAKAMOTO N, SONG G, et al. Low-level shear stress induces human mesenchymal stem cell migration through the SDF-1/CXCR4 axis via MAPK signaling pathways [J]. Stem Cells and Development, 2013, 22(17): 2384-93.
[62] PALOMARES K T, GLEASON R E, MASON Z D, et al. Mechanical stimulation alters tissue differentiation and molecular expression during bone healing [J]. Journal of Orthopaedic Research, 2009, 27(9): 1123-32.
[63] WANG L, YOU X, LOTINUN S, et al. Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk [J]. Nature Communications, 2020, 11(1): 282.
[64] UDA Y, AZAB E, SUN N, et al. Osteocyte Mechanobiology [J]. Current Osteoporosis Reports, 2017, 15(4): 318-25.
[65] GHAFFARI S, LEASK R L, JONES E A. Flow dynamics control the location of sprouting and direct elongation during developmental angiogenesis [J]. Development (Cambridge, England), 2015, 142(23): 4151-7.
[66] ROSENFELD D, LANDAU S, SHANDALOV Y, et al. Morphogenesis of 3D vascular networks is regulated by tensile forces [J]. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(12): 3215-20.
[67] RUEHLE M A, EASTBURN E A, LABELLE S A, et al. Extracellular matrix compression temporally regulates microvascular angiogenesis [J]. Science Advances, 2020, 6(34): eabb6351.
[68] GARDNER M J, VAN DER MEULEN M C, DEMETRAKOPOULOS D, et al. In vivo cyclic axial compression affects bone healing in the mouse tibia [J]. Journal of Orthopaedic Research, 2006, 24(8): 1679-86.
[69] LIU C, CABAHUG-ZUCKERMAN P, STUBBS C, et al. Mechanical Loading Promotes the Expansion of Primitive Osteoprogenitors and Organizes Matrix and Vascular Morphology in Long Bone Defects [J]. Journal of Bone and Mineral Research, 2019, 34(5): 896-910.
[70] FRANCO C A, JONES M L, BERNABEU M O, et al. Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling [J]. ELife, 2016, 5: e07727.
[71] CHOI D, PARK E, JUNG E, et al. ORAI1 Activates Proliferation of Lymphatic Endothelial Cells in Response to Laminar Flow Through Krüppel-Like Factors 2 and 4 [J]. Circulation Research, 2017, 120(9): 1426-39.
[72] CHOI D, PARK E, JUNG E, et al. Laminar flow downregulates Notch activity to promote lymphatic sprouting [J]. The Journal of Clinical Investigation, 2017, 127(4): 1225-40.
[73] MORROW D, CULLEN J P, CAHILL P A, et al. Cyclic strain regulates the Notch/CBF-1 signaling pathway in endothelial cells: role in angiogenic activity [J]. Arteriosclerosis, Thrombosis, and Vascular biology, 2007, 27(6): 1289-96.
[74] MOKRES L M, PARAI K, HILGENDORFF A, et al. Prolonged mechanical ventilation with air induces apoptosis and causes failure of alveolar septation and angiogenesis in lungs of newborn mice [J]. American Journal of Physiology Lung Cellular and Molecular Physiology, 2010, 298(1): L23-35.
[75] TAKUWA Y, DU W, QI X, et al. Roles of sphingosine-1-phosphate signaling in angiogenesis [J]. Pharmacological Research, 2010, 1(10): 298.
[76] JOSIPOVIC I, PFLüGER B, FORK C, et al. Long noncoding RNA LISPR1 is required for S1P signaling and endothelial cell function [J]. Journal of Molecular and Cellular Cardiology, 2018, 116: 57-68.
[77] XIN Q, CHENG G, KONG F, et al. STAT1 transcriptionally regulates the expression of S1PR1 by binding its promoter region [J]. Gene, 2020, 736: 144417.
[78] JUETTNER V V, KRUSE K, DAN A, et al. VE-PTP stabilizes VE-cadherin junctions and the endothelial barrier via a phosphatase-independent mechanism [J]. The Journal of Cell Biology, 2019, 218(5): 1725-42.
[79] LIU Y, WADA R, YAMASHITA T, et al. Edg-1, the G protein-coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation [J]. The Journal of Clinical Investigation, 2000, 106(8): 951-61.
[80] LIU S, NI C, ZHANG D, et al. S1PR1 regulates the switch of two angiogenic modes by VE-cadherin phosphorylation in breast cancer [J]. Cell Death & Disease, 2019, 10(3): 200.
[81] JUNG B, OBINATA H, GALVANI S, et al. Flow-regulated endothelial S1P receptor-1 signaling sustains vascular development [J]. Developmental Cell, 2012, 23(3): 600-10.
[82] CAO J, EHLING M, MäRZ S, et al. Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis [J]. Nature Communications, 2017, 8(1): 2210.
[83] BALAJI RAGUNATHRAO V A, ANWAR M, AKHTER M Z, et al. Sphingosine-1-Phosphate Receptor 1 Activity Promotes Tumor Growth by Amplifying VEGF-VEGFR2 Angiogenic Signaling [J]. Cell Reports, 2019, 29(11): 3472-87.e4.
[84] ANWAR M, MEHTA D. Post-translational modifications of S1PR1 and endothelial barrier regulation [J]. Biochimica et Biophysica Acta Molecular and Cell Biology of Lipids, 2020, 1865(9): 158760.
[85] HSIEH H-J, LIU C-A, HUANG B, et al. Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications [J]. Journal of Biomedical Science, 2014, 21: 1-15.
[86] CAMPINHO P, VILFAN A, VERMOT J. Blood flow forces in shaping the vascular system: a focus on endothelial cell behavior [J]. Frontiers in Physiology, 2020, 11: 552.
[87] KWAK B R, BäCK M, BOCHATON-PIALLAT M-L, et al. Biomechanical factors in atherosclerosis: mechanisms and clinical implications [J]. European Heart Journal, 2014, 35(43): 3013-20.
[88] CHACHISVILIS M, ZHANG Y-L, FRANGOS J A. G protein-coupled receptors sense fluid shear stress in endothelial cells [J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(42): 15463-8.
[89] YU F X, ZHAO B, PANUPINTHU N, et al. Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling [J]. Cell, 2012, 150(4): 780-91.
[90] DELA PAZ N G, MELCHIOR B, FRANGOS J A. Shear stress induces Gαq/11 activation independently of G protein-coupled receptor activation in endothelial cells [J]. The Journal of Biological Chemistry, 2017, 312(4): C428-C37.
[92] MEDEROS Y SCHNITZLER M, STORCH U, MEIBERS S, et al. Gq‐coupled receptors as mechanosensors mediating myogenic vasoconstriction [J]. EMBO Journal, 2008, 27(23): 3092-103.
[93] ZOU Y, AKAZAWA H, QIN Y, et al. Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II [J]. Naunyn Schmiedebergs Arch Pharmacol, 2004, 6(6): 499-506.
[94] SILTARI A, KORPELA R, VAPAATALO H. Bradykinin–induced vasodilatation: Role of age, ACE1-inhibitory peptide, mas-and bradykinin receptors [J]. Peptides, 2016, 85: 46-55.
[95] ERDOGMUS S, STORCH U, DANNER L, et al. Helix 8 is the essential structural motif of mechanosensitive GPCRs [J]. Nature Communications, 2019, 10(1): 5784.
[96] IWASAWA E, ISHIBASHI S, SUZUKI M, et al. Sphingosine-1-phosphate receptor 1 activation enhances leptomeningeal collateral development and improves outcome after stroke in mice [J]. Journal of Stroke and Cerebrovascular Diseases, 2018, 27(5): 1237-51.
[97] CANTALUPO A, GARGIULO A, DAUTAJ E, et al. S1PR1 (sphingosine-1-phosphate receptor 1) signaling regulates blood flow and pressure [J]. Hypertension, 2017, 70(2): 426-34.
[98] MEISSNER A J H. S1PR (Sphingosine-1-Phosphate Receptor) signaling in the regulation of vascular tone and blood pressure: is S1PR1 doing the Trick? [Z]. American Heart Association. 2017: 232-4
[99] BEN SHOHAM A, MALKINSON G, KRIEF S, et al. S1P1 inhibits sprouting angiogenesis during vascular development [J]. Development (Cambridge, England), 2012, 139(20): 3859-69.
[100]GENG X, YANAGIDA K, AKWII R G, et al. S1PR1 regulates the quiescence of lymphatic vessels by inhibiting laminar shear stress-dependent VEGF-C signaling [J]. Journal of Clinical Investigation Insight, 2020, 5(14).
[101]XIAO L, ZHOU Y, FRIIS T, et al. S1P-S1PR1 Signaling: the "Sphinx" in Osteoimmunology [J]. Frontiers in Immunology, 2019, 10: 1409.
[102]MATSUZAKI E, HIRATSUKA S, HAMACHI T, et al. Sphingosine-1-phosphate promotes the nuclear translocation of β-catenin and thereby induces osteoprotegerin gene expression in osteoblast-like cell lines [J]. Bone, 2013, 55(2): 315-24.
[103]SINHA R K, PARK C, HWANG I Y, et al. B lymphocytes exit lymph nodes through cortical lymphatic sinusoids by a mechanism independent of sphingosine-1-phosphate-mediated chemotaxis [J]. Immunity, 2009, 30(3): 434-46.
[104]TAKAYANAGI H. Osteoimmunology: shared mechanisms and crosstalk between the immune and bone systems [J]. Nature Reviews Immunology, 2007, 7(4): 292-304.
[105]MCLELLAN M A, ROSENTHAL N A, PINTO A R. Cre‐loxP‐mediated recombination: general principles and experimental considerations [J]. Current Protocols in Mouse Biology, 2017, 7(1): 1-12.
[106]ALLENDE M L, YAMASHITA T, PROIA R L. G-protein-coupled receptor S1P1 acts within endothelial cells to regulate vascular maturation [J]. Blood, 2003, 102(10): 3665-7.
[107]SANNA M G, WANG S-K, GONZALEZ-CABRERA P J, et al. Enhancement of capillary leakage and restoration of lymphocyte egress by a chiral S1P1 antagonist in vivo [J]. Nature Chemical Biology, 2006, 2(8): 434-41.
[108]MULLERSHAUSEN F, ZECRI F, CETIN C, et al. Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors [J]. Nature Chemical Biology, 2009, 5(6): 428-34.
[109]NEWBY J, SCHILLER J L, WESSLER T, et al. A blueprint for robust crosslinking of mobile species in biogels with weakly adhesive molecular anchors [J]. Nature Communications, 2017, 8(1): 833.
[110]THOMPSON M S, SCHELL H, LIENAU J, et al. Digital image correlation: a technique for determining local mechanical conditions within early bone callus [J]. Medical Engineering & Physics, 2007, 29(7): 820-3.
[111]KUTZNER I, HEINLEIN B, GRAICHEN F, et al. Loading of the knee joint during activities of daily living measured in vivo in five subjects [J]. Journal of Biomechanics, 2010, 43(11): 2164-73.
[112]LIEN Y-H, YONG K-C, CHO C, et al. S1P1-selective agonist, SEW2871, ameliorates ischemic acute renal failure [J]. Kidney International, 2006, 69(9): 1601-8.
[113]GE J, GUO L, WANG S, et al. The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke [J]. Stem Cell Reviews and Reports, 2014, 10(2): 295-303.
[114]GREEN A C, RUDOLPH-STRINGER V, STRASZKOWSKI L, et al. Retinoic Acid Receptor γ Activity in Mesenchymal Stem Cells Regulates Endochondral Bone, Angiogenesis, and B Lymphopoiesis [J]. Journal of Bone and Mineral Research, 2018, 33(12): 2202-13.
[115]MENEZES K, ROSA B G, FREITAS C, et al. Human mesenchymal stromal/stem cells recruit resident pericytes and induce blood vessels maturation to repair experimental spinal cord injury in rats [J]. Scientific Reports, 2020, 10(1): 19604.
[116]WILK K, YEH S-C A, MORTENSEN L J, et al. Postnatal calvarial skeletal stem cells expressing PRX1 reside exclusively in the calvarial sutures and are required for bone regeneration [J]. Stem Cell Reports, 2017, 8(4): 933-46.
[117]YANG C, LIU Y, WANG Z, et al. Controlled mechanical loading improves bone regeneration by regulating type H vessels in a S1Pr1-dependent manner [J]. FASEB Journal, 2022, 36(10): e22530.
[118]MCDERMOTT A M, HERBERG S, MASON D E, et al. Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration [J]. Science Translational Medicine, 2019, 11(495).
[119]SAWALL S, BECKENDORF J, AMATO C, et al. Coronary micro-computed tomography angiography in mice [J]. Scientific Reports, 2020, 10(1): 16866.
[120]GOMARIZ A, HELBLING P M, ISRINGHAUSEN S, et al. Quantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy [J]. Nature Communications, 2018, 9(1): 2532.
[121]ZHOU Z, SIDDIQUEE M M R, TAJBAKHSH N, et al. UNet++: A Nested U-Net Architecture for Medical Image Segmentation [J]. Deep Learn Med Image Anal Multimodal Learn Clin Decis Support (2018), 2018, 11045: 3-11.
[122]BATTEGAY E J, RUPP J, IRUELA-ARISPE L, et al. PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors [J]. The Journal of Cell Biology, 1994, 125(4): 917-28.
[123]XIE H, CUI Z, WANG L, et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis [J]. Nature Medicine, 2014, 20(11): 1270-8.
[124]DUPONT K M, SHARMA K, STEVENS H Y, et al. Human stem cell delivery for treatment of large segmental bone defects [J]. Proceedings of the National Academy of Sciences of The United States Of America, 2010, 107(8): 3305-10.
[125]LIU S, FENG G, TANG B Z, et al. Recent advances of AIE light-up probes for photodynamic therapy [J].Chemical Science, 2021, 12(19): 6488-506.
[126]GAENGEL K, NIAUDET C, HAGIKURA K, et al. The sphingosine-1-phosphate receptor S1PR1 restricts sprouting angiogenesis by regulating the interplay between VE-cadherin and VEGFR2 [J]. Developmental Cell, 2012, 23(3): 587-99.
[127]EINHORN T A, GERSTENFELD L C. Fracture healing: mechanisms and interventions [J]. Nat Rev Rheumatol, 2015, 11(1): 45-54.
[128]GENETOS D C, GEIST D J, LIU D, et al. Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts [J]. Journal of Bone and Mineral Research, 2005, 20(1): 41-9.
[129]LOTINUN S, KIVIRANTA R, MATSUBARA T, et al. Osteoclast-specific cathepsin K deletion stimulates S1P-dependent bone formation [J]. The Journal of Clinical Investigation, 2013, 123(2): 666-81.
[130]WANG Z, KAWABORI M, HOUKIN K. FTY720 (Fingolimod) ameliorates brain injury through multiple mechanisms and is a strong candidate for stroke treatment [J]. Current Medicinal Chemistry, 2020, 27(18): 2979-93.
[131]WANG X, LI X, LI J, et al. Mechanical loading stimulates bone angiogenesis through enhancing type H vessel formation and downregulating exosomal miR-214-3p from bone marrow-derived mesenchymal stem cells [J]. FASEB Journal, 2021, 35(1): e21150.
[132]YU N, WU J L, XIAO J, et al. HIF-1α regulates angiogenesis via Notch1/STAT3/ETBR pathway in trophoblastic cells [J]. Cell Cycle (Georgetown, Tex), 2019, 18(24): 3502-12.
[133]HOLSTEIN J H, ORTH M, SCHEUER C, et al. Erythropoietin stimulates bone formation, cell proliferation, and angiogenesis in a femoral segmental defect model in mice [J]. Bone, 2011, 49(5): 1037-45.
[134]BAKER C E, MOORE-LOTRIDGE S N, HYSONG A A, et al. Bone Fracture Acute Phase Response-A Unifying Theory of Fracture Repair: Clinical and Scientific Implications [J]. Clinical Reviews in Bone and Mineral Metabolism, 2018, 16(4): 142-58.
[135]KORN C, AUGUSTIN H G. Mechanisms of Vessel Pruning and Regression [J]. Developmental Cell, 2015, 34(1): 5-17.
[136]SOUILHOL C, SERBANOVIC-CANIC J, FRAGIADAKI M, et al. Endothelial responses to shear stress in atherosclerosis: a novel role for developmental genes [J]. Nature Reviews Cardiology, 2020, 17(1): 52-63.
[137]WEN L, ZHANG T, WANG J, et al. The blood flow-klf6a-tagln2 axis drives vessel pruning in zebrafish by regulating endothelial cell rearrangement and actin cytoskeleton dynamics [J]. PLoS Genetics, 2021, 17(7): e1009690.
[138]DUPONT S, MORSUT L, ARAGONA M, et al. Role of YAP/TAZ in mechanotransduction [J]. Nature, 2011, 474(7350): 179-83.
[139]LIN C, YAO E, ZHANG K, et al. YAP is essential for mechanical force production and epithelial cell proliferation during lung branching morphogenesis [J]. Elife, 2017, 6.
[140]NETO F, KLAUS-BERGMANN A, ONG Y T, et al. YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development [J]. ELife, 2018, 7: e31037.
[141]KOHARA Y, KITAZAWA R, HARAGUCHI R, et al. Macrophages are requisite for angiogenesis of type H vessels during bone regeneration in mice [J]. Bone, 2022, 154: 116200.
[142]JIANG M, SHEN Q, ZHOU Y, et al. Fluid shear stress and endothelial cells synergistically promote osteogenesis of mesenchymal stem cells via integrin β1-FAK-ERK1/2 pathway [J]. Turkish Journal of Biology, 2021, 45(6): 683-94.
[143]OZEKI N, MOGI M, HASE N, et al. Polyphosphate-induced matrix metalloproteinase-13 is required for osteoblast-like cell differentiation in human adipose tissue derived mesenchymal stem cells [J]. Bioscience Trends, 2016, 10(5): 365-71.
[144]CHOI J B, LEE J, KANG M, et al. Dysregulated ECM remodeling proteins lead to aberrant osteogenesis of Costello syndrome iPSCs [J]. Stem Cell Reports, 2021, 16(8): 1985-98.
[145]NAKAJIMA H, YAMAMOTO K, AGARWALA S, et al. Flow-Dependent Endothelial YAP Regulation Contributes to Vessel Maintenance [J]. Developmental Cell, 2017, 40(6): 523-36 e6.
[146]WANG X, FREIRE VALLS A, SCHERMANN G, et al. YAP/TAZ Orchestrate VEGF Signaling during Developmental Angiogenesis [J]. Developmental Cell, 2017, 42(5): 462-78 e7.
[147]BECHTEL T J, REYES-ROBLES T, FADEYI O O, et al. Strategies for monitoring cell–cell interactions [J]. Nature Chemical Biology, 2021, 17(6): 641-52.
[148]DIMITROV D, TüREI D, GARRIDO-RODRIGUEZ M, et al. Comparison of methods and resources for cell-cell communication inference from single-cell RNA-Seq data [J]. Nature Communications, 2022, 13(1): 3224.
[149]ISHII M, EGEN J G, KLAUSCHEN F, et al. Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis [J]. Nature, 2009, 458(7237): 524-8.
[150]WESKE S, VAIDYA M, REESE A, et al. Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss [J]. Nature Medicine, 2018, 24(5): 667-78.
[151]TANTIKANLAYAPORN D, TOURKOVA I L, LARROUTURE Q, et al. Sphingosine-1-Phosphate Modulates the Effect of Estrogen in Human Osteoblasts [J]. Journal of Bone and Mineral Research Plus, 2018, 2(4): 217-26.
[152]PRASAD J, WIATER B P, NORK S E, et al. Characterizing gait induced normal strains in a murine tibia cortical bone defect model [J]. Journal of Biomechanics, 2010, 43(14): 2765-70.
[153]KEGELMAN C D, NIJSURE M P, MOHARRER Y, et al. YAP and TAZ Promote Periosteal Osteoblast Precursor Expansion and Differentiation for Fracture Repair [J]. Journal of Bone and Mineral Research, 2021, 36(1): 143-57.