Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression

Abstract

Due to increasing morbidity worldwide, fractures are becoming an emerging public health concern. This study aimed to investigate the effect of metformin on the healing of osteoporotic as well as normal fractures. Type H vessels have recently been identified as a bone-specific vascular subtype that supports osteogenesis. Here, we show that metformin accelerated fracture healing in both osteoporotic and normal mice. Moreover, metformin promoted angiogenesis in vitro under hypoxia as well as type H vessel formation throughout fracture healing. Mechanistically, metformin increased the expression of HIF-1α, an important positive regulator of type H vessel formation, by inhibiting the expression of YAP1/TAZ in calluses and hypoxia-cultured human microvascular endothelial cells (HMECs). The results of HIF-1α or YAP1/TAZ interference in hypoxia-cultured HMECs using siRNA further suggested that the enhancement of HIF-1α and its target genes by metformin is primarily through YAP1/TAZ inhibition. Finally, overexpression of YAP1/TAZ partially counteracted the effect of metformin in promoting type H vessel-induced angiogenesis-osteogenesis coupling during fracture repair. In summary, our findings suggest that metformin has the potential to be a therapeutic agent for fractures by promoting type H vessel formation through YAP1/TAZ inhibition.

References

[1] Brandi ML. Microarchitecture, the key to bone quality. Rheumatology (Oxford), 2009, 48: iv3-iv8

[2] Wang L et al. Prevalence of osteoporosis and fracture in China: The China osteoporosis prevalence study. JAMA Netw. Open, 2021, 4: e2121106

[3] Burge R et al. Incidence and economic burden of osteoporosis‐related fractures in the United States, 2005–2025. J. Bone Min. Res., 2007, 22: 465-475

[4] Einhorn TA, Gerstenfeld LC. Fracture healing: mechanisms and interventions. Nat. Rev. Rheumatol, 2015, 11: 45-54

[5] Cheung WH, Miclau T, Chow SK, Yang FF, Alt V. Fracture healing in osteoporotic bone. Injury, 2016, 47: S21-S26

[6] Garrison KR et al. Bone morphogenetic protein (BMP) for fracture healing in adults. Cochrane Database Syst. Rev., 2010, 2010: Cd006950

[7] Hak DJ. The biology of fracture healing in osteoporosis and in the presence of anti-osteoporotic drugs. Injury, 2018, 49: 1461-1465

[8] Sivaraj KK, Adams RH. Blood vessel formation and function in bone. Development, 2016, 143: 2706-2715

[9] Kusumbe AP, Ramasamy SK, Adams RH. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature, 2014, 507: 323-328

[10] Ramasamy SK, Kusumbe AP, Wang L, Adams RH. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature, 2014, 507: 376-380

[11] Xie H et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nat. Med., 2014, 20: 1270-1278

[12] Xu R et al. Targeting skeletal endothelium to ameliorate bone loss. Nat. Med., 2018, 24: 823-833

[13] Liu, J.-H. et al. Akkermansia muciniphila promotes type H vessel formation and bone fracture healing by reducing gut permeability and inflammation. Dis. Models Mech. 13, dmm043620 (2020).

[14] Fu V, Plouffe SW, Guan KL. The Hippo pathway in organ development, homeostasis, and regeneration. Curr. Opin. Cell Biol., 2017, 49: 99-107

[15] Dey A, Varelas X, Guan KL. Targeting the Hippo pathway in cancer, fibrosis, wound healing and regenerative medicine. Nat. Rev. Drug Discov, 2020, 19: 480-494

[16] Kim J et al. YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation. J. Clin. Invest, 2017, 127: 3441-3461

[17] Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med., 2003, 9: 677-684

[18] Sivaraj KK et al. YAP1 and TAZ negatively control bone angiogenesis by limiting hypoxia-inducible factor signaling in endothelial cells. Elife, 2020, 9: e50770

[19] Flory J, Lipska K. Metformin in 2019. JAMA, 2019, 321: 1926-1927

[20] Melton LJ 3rd, Leibson CL, Achenbach SJ, Therneau TM, Khosla S. Fracture risk in type 2 diabetes: update of a population-based study. J. Bone Miner. Res, 2008, 23: 1334-1342

[21] Molinuevo MS et al. Effect of metformin on bone marrow progenitor cell differentiation: In vivo and in vitro studies. J. Bone Miner. Res., 2010, 25: 211-221

[22] Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Sugimoto T. Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression. Biochem. Biophys. Res. Commun., 2008, 375: 414-419

[23] Sedlinsky C et al. Metformin prevents anti-osteogenic in vivo and ex vivo effects of rosiglitazone in rats. Eur. J. Pharmacol, 2011, 668: 477-485

[24] Jeyabalan J et al. The anti-diabetic drug metformin does not affect bone mass in vivo or fracture healing. Osteoporos Int, 2013, 24: 2659-2670

[25] Pryor R, Cabreiro F. Repurposing metformin: an old drug with new tricks in its binding pockets. Biochem. J., 2015, 471: 307-322

[26] Bahrambeigi S, Yousefi B, Rahimi M, Shafiei-Irannejad V. Metformin; an old antidiabetic drug with new potentials in bone disorders. Biomed. Pharmacother., 2019, 109: 1593-1601

[27] Hidayat K, Du X, Wu MJ, Shi BM. The use of metformin, insulin, sulphonylureas, and thiazolidinediones and the risk of fracture: Systematic review and meta-analysis of observational studies. Obes. Rev., 2019, 20: 1494-1503

[28] Salari-Moghaddam A, Sadeghi O, Keshteli AH, Larijani B, Esmaillzadeh A. Metformin use and risk of fracture: a systematic review and meta-analysis of observational studies. Osteoporos Int., 2019, 30: 1167-1173

[29] Jang WG et al. Metformin induces osteoblast differentiation via orphan nuclear receptor SHP-mediated transactivation of Runx2. Bone, 2011, 48: 885-893

[30] Fu LJ, Tang TT, Hao YQ, Dai KR. Long-term effects of alendronate on fracture healing and bone remodeling of femoral shaft in ovariectomized rats. Acta Pharmacol. Sin., 2013, 34: 387-392

[31] Aspenberg P et al. Teriparatide for acceleration of fracture repair in humans: a prospective, randomized, double-blind study of 102 postmenopausal women with distal radial fractures. J. Bone Miner. Res, 2010, 25: 404-414

[32] Peichl P, Holzer LA, Maier R, Holzer G. Parathyroid hormone 1-84 accelerates fracture-healing in pubic bones of elderly osteoporotic women. J. Bone Joint Surg. Am., 2011, 93: 1583-1587

[33] Fang TD et al. Angiogenesis is required for successful bone induction during distraction osteogenesis. J. Bone Miner. Res., 2005, 20: 1114-1124

[34] Liu Y, Tang G, Zhang Z, Wang Y, Yang GY. Metformin promotes focal angiogenesis and neurogenesis in mice following middle cerebral artery occlusion. Neurosci. Lett., 2014, 579: 46-51

[35] Takahashi N et al. Metformin stimulates ischemia-induced revascularization through an eNOS dependent pathway in the ischemic hindlimb mice model. J. Vasc. Surg., 2015, 61: 489-496

[36] Yu JW et al. Metformin improves the angiogenic functions of endothelial progenitor cells via activating AMPK/eNOS pathway in diabetic mice. Cardiovasc. Diabetol, 2016, 15: 88

[37] Di Pietro M et al. Metformin regulates ovarian angiogenesis and follicular development in a female polycystic ovary syndrome rat model. Endocrinology, 2015, 156: 1453-1463

[38] Kannarkatt J, Alkharabsheh O, Tokala H, Dimitrov NV. Metformin and angiogenesis in cancer—revisited. Oncology, 2016, 91: 179-184

[39] Baker CE et al. Bone fracture acute phase response-a unifying theory of fracture repair: Clinical and scientific implications. Clin. Rev. Bone Miner. Metab., 2018, 16: 142-158

[40] Chen M et al. Skeleton-vasculature chain reaction: a novel insight into the mystery of homeostasis. Bone Res., 2021, 9: 21

[41] Yang M et al. Ophiopogonin D promotes bone regeneration by stimulating CD31(hi) EMCN(hi) vessel formation. Cell Prolif, 2020, 53: e12784

[42] Marenzana M, Arnett TR. The key role of the blood supply to bone. Bone Res., 2013, 1: 203-215

[43] Hooglugt A, van der Stoel MM, Boon RA, Huveneers S. Endothelial YAP/TAZ signaling in angiogenesis and tumor vasculature. Front. Oncol, 2020, 10: 612802

[44] DeRan M et al. Energy stress regulates hippo-YAP signaling involving AMPK-mediated regulation of angiomotin-like 1 protein. Cell Rep., 2014, 9: 495-503

[45] Tian Y et al. Metformin mediates resensitivity to 5-fluorouracil in hepatocellular carcinoma via the suppression of YAP. Oncotarget, 2016, 7: 46230-46241

[46] Ramasamy SK et al. Blood flow controls bone vascular function and osteogenesis. Nat. Commun, 2016, 7

[47] Caire R et al. Parathyroid hormone remodels bone transitional vessels and the leptin receptor-positive pericyte network in mice. J. Bone Miner. Res., 2019, 34: 1487-1501

[48] Chen Q et al. Fate decision of mesenchymal stem cells: Adipocytes or osteoblasts? Cell Death Differ, 2016, 23: 1128-1139

[49] Gao Y, Xue J, Li X, Jia Y, Hu J. Metformin regulates osteoblast and adipocyte differentiation of rat mesenchymal stem cells. J. Pharm. Pharmacol, 2008, 60: 1695-1700

[50] Zhao B, Li L, Lei Q, Guan KL. The Hippo-YAP pathway in organ size control and tumorigenesis: an updated version. Genes Dev., 2010, 24: 862-874

[51] Dupont S et al. Role of YAP/TAZ in mechanotransduction. Nature, 2011, 474: 179-183

[52] Wang X et al. YAP/TAZ orchestrate VEGF signaling during developmental angiogenesis. Dev. Cell, 2017, 42: 462-478.e467

[53] Zaidi SK et al. Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. EMBO J., 2004, 23: 790-799

[54] Kegelman CD et al. Skeletal cell YAP and TAZ combinatorially promote bone development. FASEB J., 2018, 32: 2706-2721

Funding

National Natural Science Foundation of China (National Science Foundation of China)(82072504)

Natural Science Foundation of Hunan Province (Hunan Provincial Natural Science Foundation)(2021JJ40492)

Independent Exploration and Innovation Project of Central South University (Grant Nos. 2020zzts255)