Intraosseous Endothelial MTORC1 Affects Skeletal Development Via Regulating Type H Vessels

BACKGROUND:Intraosseous blood vessels participate in the regulation of bone formation and metabolism.Angiogenesis and vascular maintenance is vital to bone development and keeping the balance of bone homeostasis.Various bone injuries and diseases(such as bone fractures,osteoporosis and femoral head necrosis,etc.)is closely related to bone vascular abnormalities.Bone vascular endothelial cells,as the most important components and functional cells of intraosseous blood vessels,regulate the process of skeletal development and metabolism by the secretion of various cytokines.Various growth factors or cytokines like VEGF,BMPs,Endothelin,Noggin have been reported to promote bone formation,but there aren't so many factors,of which mechanism are not so clear as well,that can entirely explain the complexity of angiogenesis and osteogenesis coupling.A special subtype of capillaries in the metaphysis with a predominantly columnar organization and in the endosteum show high expression of CD31 and EMCN.This kind of capillaries were defined as type H vessels.However,how type H vessels form are unrevealed.mTORC1 is a key factor in regulation of cell metabolism,proliferation,differentiation and survival.That makes it very likely to play an important role in type H vessel formation and maintenance.Type H vessels have significant positive effect on osteogenesis and bone metabolism while the mechanism in them is quite unclear.Several theories have been brought up to explain the phenomenon especially that type H vessels secret different levels and kinds of various and enormous growth factors and cytokines acting on perivascular cells in bone microenvironment.There are still many unsolved mysteries in this field.Therefore,it's really important and meaningful to confirm and further explore the functions of type H vessels in bone and their effects on skeletal development and metabolism,and to discover and examine novel key factors that matters.METHODS:The activity levels of intraosseous endothelial mTORC1 signaling in aged and postmenopausal osteoporosis C57 mice are compared to normal adult ones.How different levels of endothelial mTORC1 activity affect the formation and maintenance of type H vessels is analyzed.CCK-8 cell proliferation assay,wound healing assay and endothelial cell tube formation assay are performed.Endothelial cell-specific Raptor(an mTORC1 component and promotor)conditional gene knock out(CKO)mice model is constructed,to help proceed the experiment in vivo.Bone volume and bone mineral density(BMD)and bone structure of CKO mice and their littermate controls are evaluated by micro-CT scan and reconstruction.Osteoblastic biomarker Runx2 immunohistochemical staining,Osteoclastic TRAP staining and TUNEL staining for chondrocytes are performed.mTORC1 signaling in bone marrow-derived endothelial cells(BMECs)is activated or inactivated via siRNA transfection.Cells are collected for extracting mRNAs and proteins.Conditional medium supernatent is collected carefully to re-incubate osteoblasts(OBs),osteoclasts(OCs)and chondrocytes(CCs)in cell culture imitating endothelial cell paracrine in bone microenvironment as much as possible in vitro.OB,OC,CC's proliferation,differentiation and apoptosis abilities are tested.Cytokine bio-microarray contains more than 200 vital candidate cytokines related to cell proliferation,differentiation,chemotaxis and apoptosis is performed to test the distribution and abundance of factors in conditional medium supernatent previously mentioned.A novel target named MFG-E8 is found and this protein has great potential on regulating osteogenesis and BMECs phagocytosis to the clearance of apoptotic chondrocytes.The function of MFG-E8 in bone is explored and confirmed in vitro vitro.After all,to verify our hypothesis and seeking reasonable explanation to the interesting phenomenon we observe,we complete our research experiments in molecular,cellular and animal levels.RESULTS:The activity of intraosseous endothelial mTORC1 signaling in aged and postmenopausal osteoporosis C57 mice is significantly decreased compared to normal adult ones.Activation of mTORC1 signaling in intraosseous endothelial cells promoted the formation and maintenance of type H vessels.Osteopenia is observed in CKO mice.It's been shown that the decrease of osteoblasts,increase of osteoclasts and the accumulation of chondrocytes in metaphysis in CKO mice.Type H vessels secret a cytokine named MFG-E8 which has multiple functions.Type H endothelial mTORC1 signaling promotes the secretion of MFG-E8.MFG-E8 is of great value because it promotes osteoblasts differentiation and bone formation,reduces TRAP+osteoclasts numbers and bone resorption,helps BMEC itself's phagocytosis to the clearance of hypertrophic apoptotic chondrocytes.CONCLUSION:mTORC1 is a key signal regulating the formation and the maintenance of bone type H vessels.Type H endothelial cells secret MFG-E8 to regulate skeletal development and metabolism.MFG-E8 may become a novel target for clinical therapies of bone related injuries and metabolic diseases such as bone fractures and osteoporosis.