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Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

ABSTRACT: Rationale: Angiogenesis and osteogenesis are highly coupled processes which are indispensable to bone repair. However, the underlying mechanism(s) remain elusive. To bridge the gap in understanding the coupling process is crucial to develop corresponding solutions to abnormal bone healing. Epidermal growth factor-like protein 6 (EGFL6) is an angiogenic factor specifically and distinctively up-regulated during osteoblast differentiation. In contrast with most currently known osteoblast-derived coupling factors, EGFL6 is highlighted with little or low expression in other cells and tissues. Methods: In this study, primary bone marrow mesenchymal stem cells (MSCs) and osteoblastic cell line (MC3T3-E1) were transduced with lentiviral silencing or overexpression constructs targeting EGFL6. Cells were induced by osteogenic medium, followed by the evaluation of mineralization as well as related gene and protein expression. Global and conditional knockout mice were established to examine the bone phenotype under physiological condition. Furthermore, bone defect models were created to investigate the outcome of bone repair in mice lacking EGFL6 expression. Results: We show that overexpression of EGFL6 markedly enhances osteogenic capacity in vitro by augmenting bone morphogenic protein (BMP)-Smad and MAPK signaling, whereas downregulation of EGFL6 diminishes osteoblastic mineralization. Interestingly, osteoblast differentiation was not affected by the exogenous addition of EGFL6 protein, thereby indicating that EGFL6 may regulate osteoblastic function in an intracrine manner. Mice with osteoblast-specific and global knockout of EGFL6 surprisingly exhibit a normal bone phenotype under physiological conditions. However, EGFL6-deficiency leads to compromised bone repair in a bone defect model which is characterized by decreased formation of type H vessels as well as osteoblast lineage cells. Conclusions: Together, these data demonstrate that EGFL6 serves as an essential regulator to couple osteogenesis to angiogenesis during bone repair.

SUBMITTER: Chen K

PROVIDER: S-EPMC8581413 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Osteogenesis and angiogenesis are two integrated components in bone repair and regeneration. A deeper understanding of osteogenesis and angiogenesis has been hampered by technical difficulties of analyzing bone and neovasculature simultaneously in spatiotemporal scales and in 3D formats. To overcome these barriers, a cranial defect window chamber model was established that enabled high-resolution, longitudinal, and real-time tracking of angiogenesis and bone defect healing via multiphoton laser scanning microscopy (MPLSM). By simultaneously probing new bone matrix via second harmonic generation (SHG), neovascular networks via intravenous perfusion of fluorophore, and osteoblast differentiation via 2.3-kb collagen type I promoter-driven GFP (Col2.3GFP), we examined the morphogenetic sequence of cranial bone defect healing and further established the spatiotemporal analyses of osteogenesis and angiogenesis coupling in repair and regeneration. We showed that bone defect closure was initiated in the residual bone around the edge of the defect. The expansion and migration of osteoprogenitors into the bone defect occurred during the first 3 weeks of healing, coupled with vigorous microvessel angiogenesis at the leading edge of the defect. Subsequent bone repair was marked by matrix deposition and active vascular network remodeling within new bone. Implantation of bone marrow stromal cells (BMSCs) isolated from Col2.3GFP mice further showed that donor-dependent bone formation occurred rapidly within the first 3 weeks of implantation, in concert with early angiogenesis. The subsequent bone wound closure was largely host-dependent, associated with localized modest induction of angiogenesis. The establishment of a live imaging platform via cranial window provides a unique tool to understand osteogenesis and angiogenesis in repair and regeneration, enabling further elucidation of the spatiotemporal regulatory mechanisms of osteoprogenitor cell interactions with host bone healing microenvironment.

Project description:Osteoblast-derived extracellular vesicles (EV) are a collection of secreted (sEVs) and matrix-bound nanoparticles that function as foci for mineral nucleation and accumulation. Due to the fact sEVs can be isolated directly from the culture medium of mineralizing osteoblasts, there is growing interest their application regenerative medicine. However, at present therapeutic advancements are hindered by a lack of understanding of their precise temporal contribution to matrix mineralization. This study advances current knowledge by temporally aligning sEV profile and protein content with mineralization status. sEVs were isolated from mineralizing primary osteoblasts over a period of 1, 2, and 3 weeks. Bimodal particle distributions were observed (weeks 1 and 3: 44 and 164 nm; week 2: 59 and 220 nm), indicating a heterogeneous population with dimensions characteristic of exosome- (44 and 59 nm) and microvesicle-like (164 and 220 nm) particles. Proteomic characterization by liquid chromatography tandem-mass spectrometry (LC-MS/MS) revealed a declining correlation in EV-localized proteins as mineralization advanced, with Pearson correlation-coefficients of 0.79 (week 1 vs. 2), 0.6 (2 vs. 3) and 0.46 (1 vs. 3), respectively. Principal component analysis (PCA) further highlighted a time-dependent divergence in protein content as mineralization advanced. The most significant variations were observed at week 3, with a significant (p < 0.05) decline in particle concentration, visual evidence of EV rupture and enhanced mineralization. A total of 116 vesicle-localized proteins were significantly upregulated at week 3 (56% non-specifically, 19% relative to week 1, 25% relative to week 2). Gene ontology enrichment analysis of these proteins highlighted overrepresentation of genes associated with matrix organization. Of note, increased presence of phospholipid-binding and calcium channeling annexin proteins (A2, A5, and A6) indicative of progressive variations in the nucleational capacity of vesicles, as well as interaction with the surrounding ECM. We demonstrate sEV-mediated mineralization is dynamic process with variations in vesicle morphology and protein content having a potential influence on developmental changes matrix organization. These findings have implications for the selection and application of EVs for regenerative applications.

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Osteoblast-derived EGFL6 couples angiogenesis to osteogenesis during bone repair

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