Elucidating the role of osteoblastderived vascular endothelial growth factor in the regulation of bone development: Sexually dimorphic effects and cellular mechanisms

Abstract

Bone is highly vascularised and bone forming osteoblast-derived vascular endothelial growth factor (VEGF) has been reported to be a key regulator of bone development and bone repair. Sex differences during skeletal growth are well reported and this early divergence is thought to influence the onset and prevalence of degenerative bone pathologies in men and women with age. Although VEGF is critical during early bone development, little remains understood about the role that the vasculature plays in driving the sexual dimorphism in bone. Therefore, the main objective of my PhD was to improve understanding of the function of osteoblast-derived VEGF signalling in male and female bone development and mineralisation. For VEGF deletion in mature osteoblast cells, male and female mice carrying floxed alleles of VEGF and expressing Cre recombinase under the control of the osteocalcin promotor were compared and termed herein as OcnVEGFKO. The following aims were the focus of this thesis i) Quantification and validation of vascular phenotype within the bone cortex in male versus female adult OcnVEGFKO mice ii) Investigation of whole bone traits following osteoblast-VEGF deletion including bone geometry in males versus females, iii) Elucidation of the role of osteoblast-VEGF in prepubertal development and its direct effects on male and female osteoblast function and iv) Investigation into indirect effects of VEGF deletion in male and female osteoblasts on vascular endothelial cell function. High-resolution synchrotron computed tomography, histology and backscattered scanning electron microscopy at the tibiofibular junction revealed increased intracortical porosity (+2.73 fold increase, p<0.0001), increased vasculature (% blood vessel area; WT 0.54, OcnVEGFKO 3.6, p=0.05) and signs of widespread deficiency in matrix mineralisation in 16 week old male OcnVEGFKOs versus WT. In contrast, in female OcnVEGFKOs there were no notable alterations in intracortical porosity, vasculature or mineralisation, but an increase in lacunar density, volume and diameter versus WT. Comparable to the changes observed in 16 week old mice, analysis of 4 week old male and female tibia using SR CT identified increased intracortical canal volume in male OcnVEGFKOs versus WT only. Medium-resolution micro-CT enabled the quantification of changes in bone geometry along the whole tibial length. In 4 week old mice, bone architecture was significantly altered in male OcnVEGFKO versus WT. In 16 week old mice however, differences in bone geometry were identified in female OcnVEGFKO versus WT, but not in males. Trabecular measures were largely unaltered in 4 and 16 week old male and female OcnVEGFKOs versus WT. To assess the ii directionality of VEGF signalling and its contribution to observed sexual dimorphisms, LOBs were isolated from male and female Vegffl/fl mice in vitro. VEGF was deleted using Adenovirus-Cre (OBVEGFKO) to assess direct effects of VEGF deletion on OB function in males and females. OBVEGFKO had no significant effect on viability or alkaline phosphatase (ALP) elution in males or females versus WT. mRNA was extracted from OBVEGFKO and WT OBs and qPCR was utilised to measure changes in relative expression of mRNA. Negligible levels of Vegfr2 expression was identified in male and female OBs, in comparison to the endothelial cell (EC) lysate positive control. Expression of Ar, Esr1, Esr2, Rankl, Opg and Sost was unchanged in male and female OBs as a result of VEGFKO. Indirect effects of OBVEGFKO on EC functions were investigated and WT and OBVEGFKO conditioned media was collected from OBs and used to treat mouse bone marrow endothelial cells (MBMECs). Additional sexual dimorphism was identified here, with differential expression of genes implicated in angiogenesis and osteogenesis in OBVEGFKO versus WT represented by fold changes, including Igf-1 (female; +3.07, male; -2.33), Hif1-α (female; +1.22, male; -1.32), Runx2 (female; +3.43, male; -1.55) and Sost (female;+6.58, male; +3.21). Correlating with in vitro mechanistic findings of sexual dimorphism in endothelial cells driven by osteoblastderived VEGF, immunohistochemistry of tibia sections revealed high levels of sclerostin, a potent inhibitor of bone mineralisation in male OcnVEGFKO and localised to CD31 positive cells at the tibiofibular junction. My results propose that osteoblast-derived VEGF regulates osteoblast matrix mineralisation directly and vascularisation indirectly, that this regulation is distinct to males and females and results in divergent physical bone traits between sexes. Importantly my cell culture studies have found that these dimorphic bone phenotypes are linked to alterations in endothelial cell gene expression which could be targeted clinically to improve bone mineralisation. Targeting vascular signals to modulate bone formation distinctly in males and females could therefore provide a more effective way to treat degenerative bone disease in our ageing population.

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Identifiers

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

ORCID for Philipp Schneider: orcid.org/0000-0001-7499-3576

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