Regulation of the Bone Vascular Network is Sexually Dimorphic

Abstract

Osteoblast (OB) lineage cells are an important source of vascular endothelial growth factor (VEGF), which is critical for bone growth and repair. During bone development, pubertal differences in males and females exist, but little is known about whether VEGF signaling contributes to skeletal sexual dimorphism. We have found that in mice, conditional disruption of VEGF in osteocalcin‐expressing cells (OcnVEGFKO) exerts a divergent influence on morphological, cellular, and whole bone properties between sexes. Furthermore, we describe an underlying sexual divergence in VEGF signaling in OB cultures in vitro independent of circulating sex hormones. High‐resolution synchrotron computed tomography and backscattered scanning electron microscopy revealed, in males, extensive unmineralized osteoid encasing enlarged blood vessel canals and osteocyte lacunae in cortical bone after VEGF deletion, which contributed to increased porosity. VEGF was deleted in male and female long bone–derived OBs (OBVEGKO) in vitro and Raman spectroscopic analyses of mineral and matrix repertoires highlighted differences between male and female OBVEGFKO cells, with increased immature phosphate species prevalent in male OBVEGFKO cultures versus wild type (WT). Further sexual dimorphism was observed in bone marrow endothelial cell gene expression in vitro after VEGF deletion and in sclerostin protein expression, which was increased in male OcnVEGFKO bones versus WT. The impact of altered OB matrix composition after VEGF deletion on whole bone geometry was assessed between sexes, although significant differences between OcnVEGFKO and WT were identified only in females. Our results suggest that bone‐derived VEGF regulates matrix mineralization and vascularization distinctly in males and females, which results in divergent physical bone traits.

Multiscale molecular profiling of pathological bone resolves sexually dimorphic control of extracellular matrix composition

“…Both Lama1 and Mmp9 expression exhibited greater upregulation in male than in female OBVEGFKO cells. In particular, elevated levels of MMP9 correlate with osteoporotic bone loss ( Haibo et al, 1997 ), and, given the function of MMP9 in matrix remodelling, these findings indicate possible pathological remodelling effects that could, in part, be responsible for the aberrations in matrix microarchitecture and vascular anomalies in the male OcnVEGFKO bones ( Goring et al, 2019 ). Contrasting with these effects, differential changes in the expression of Hc and Itgae were detected following OBVEGFKO in male and female OBs.…”

“…Uniquely, these techniques have enabled the in-depth assessment and characterisation of nanoscale sex differences in matrix composition following the loss of VEGF. Previous studies linked the loss of VEGF to bone pathology characterised by a profound osteoporotic phenotype present following the deletion of Vegf in early and late-stage OBs ( Goring et al, 2019 ; Liu et al, 2012 ). Furthermore, clinical reports have also identified that the loss of VEGF is associated with the degenerative bone loss seen in osteoporosis ( Senel et al, 2013 ).…”

“…In the present study, successful in vitro culture of male and female OBs was achieved in the absence of circulating hormones. In addition, the expression of both oestrogen and androgen receptors in OBs derived from male and female mice were comparable ( Goring et al, 2019 ). Deletion of Vegf in male and female OBs influenced the expression of genes associated with ECM formation and arrangement, confirming a potential intracrine and/or autocrine role of VEGF signalling in bone.…”

“…Tibiae from 16-week-old male and female Vegf fl/fl (WT) and Vegf fl/fl ; Ocn-Cre (OcnVEGFKO) littermates were derived as previously described ( Goring et al, 2019 ) in accordance with the UK Animals (Scientific Procedures) Act 1986 and regulations set by the UK Home Office. Briefly, tibiae were fixed for 48 h on rotation in 4% formaldehyde, pH 7.4 (from paraformaldehyde; Merck, UK) and dehydrated in ethanol ahead of embedding in poly-methyl methacrylate (PMMA).…”

“…Vascularisation of the collagen matrix is a requirement for mineralisation and is driven, in part, by angiogenic factors produced by matrix-forming OBs, including vascular endothelial growth factor (VEGF; also known as VEGFA) ( Hu and Olsen, 2016 ; Liu et al, 2012 ). We recently reported that loss of Vegf in osteocalcin-expressing OBs (OcnVEGFKO) promotes a poorly mineralised, yet highly vascularised, bone patho-phenotype that is pronounced in males ( Goring et al, 2019 ). In vitro studies confirmed that this role of VEGF was, in part, mediated via autocrine signalling in male OBs, which resulted in compromised bone maturation.…”