The role of EGF-like protein family members in angiogenesis and bone homeostasis

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    [Truncated] Bone is a highly vascularised living tissue that forms our skeletal system. It is continuously being shaped, moulded and repaired to maintain structural and mechanical integrity in a process known as bone remodelling. The vasculature is critical for the transportation of oxygen, nutrients, various growth factors and differentiation factors in the bone to facilitate skeletal development, postnatal growth and bone remodelling. Bone remodelling takes place in a specialised structure known as the bone remodelling compartment (BRC) and involves two key processes, bone resorption by osteoclasts followed by new bone matrix formation by osteoblasts. Importantly, the BRC is closely associated with blood vessels formed by angiogenesis and play a pivotal role in supporting the bone remodelling process.

    The regulation of angiogenesis in the bone microenvironment is highly complex and orchestrated by intercellular crosstalk between bone cells and endothelial cells. Osteoclasts, osteoblasts and osteocytes are capable of producing angiogenic factors to mediate angiogenesis and osteogenesis. In recent years, many epidermal growth factor (EGF)-like family members, which are characterised by differing EGF repeat domains and vary in amino acid lengths, have been implicated to have a role in bone biology and angiogenesis. In addition, our group has identified several new EGF-like factors that are expressed in the bone microenvironment, including EGFL6, EGFL7 and nephronectin (NPNT). As many EGF-like proteins are known to regulate angiogenesis and bone homeostasis, it was hypothesised that EGFL6, EGFL7 and NPNT may regulate angiogenesis and homeostasis via autocrine/paracrine mechanisms.

    In this study, EGFL7 was found to be expressed in both osteoclast and osteoblast lineages. By using conditioned medium containing EGFL7, it was found that EGFL7 promoted SVEC (simian virus 40-transformed mouse microvascular endothelial cell line) cell migration and tube-like structure formation in vitro. Moreover, recombinant EGFL7 promoted angiogenesis, characterised by the formation of web-like structures, in ex vivo foetal mouse metatarsal angiogenesis assay. By using Western blot analysis, it was revealed that EGFL7 induced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), and focal adhesion kinase (FAK) in SVEC cells. Furthermore, inhibition of ERK1/2 and STAT3 signalling impaired EGFL7-induced endothelial cell migration and angiogenesis. Bioinformatics analyses indicated that EGFL7 contains a conserved integrin-binding region, RGD/QGD motif. It was found that EGFL7-induced endothelial cell migration was significantly reduced in the presence of RGD peptides, suggesting its potential to interact with integrin. Finally, EGFL7 gene expression was significantly upregulated during rat growth plate injury repair. Together, these results show that EGFL7 expressed by bone cells regulates endothelial cell activities through integrin-mediated signalling.
    Original languageEnglish
    QualificationDoctor of Philosophy
    Publication statusUnpublished - 2015


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