Hypoxia signaling in the regulation of bone homeostasis, angiogenesis and cell metabolism: Implications for bone tissue engineering

It is well established that oxygen and nutrients are crucial for cellular functioning, including survival, proliferation and differentiation. As simple oxygen diffusion is limited to about 200 µm, cells will become hypoxic in tissues beyond a certain size. Cells have however developed mechanisms to resist tissue hypoxia and these are mediated by hypoxia inducible factors (HIFs) that are considered as the executors of the cellular response to low oxygen together with HIF prolyl hydroxylases (PHDs) that regulate HIFs in an oxygen-dependent manner. In general, activation of the HIF pathway will ultimately result in (i) stimulation of angiogenesis to resupply oxygen and (ii) adaptations in cell metabolism to ensure adequate energy generation and redox balance. Despite the remarkable healing potential of bone tissue, about 10% of all fractures results in delayed healing or non-union. Bone tissue engineering combines biological basic research and the principles of engineering in order to repair damaged bone using skeletal progenitor or stem cells and/or appropriate signaling molecules seeded on a biocompatible scaffold. The clinical impact of cell-based constructs is however limited, which urges for a better understanding of the cellular and molecular mechanisms of bone regeneration. Upon transplantation, grafted cells encounter an injured tissue that is poorly perfused, turning them in a hypoxic state. In a first part of this PhD project, we investigated the importance of endogenous HIF-1a signaling for bone regeneration. To this end, we specifically deleted the HIF­1a isoform in periosteal progenitor cells and show that activation of HIF-1a signaling in these cells is critical for bone repair by modulating angiogenic and metabolic processes. Activation of HIF‑1a is not only crucial for blood vessel invasion, by enhancing angiogenic growth factor production, but also for periosteal cell survival early after implantation, when blood vessels have not yet invaded the construct. Mechanistically, HIF-1a signaling limits oxygen consumption in hypoxia to avoid accumulation of harmful reactive oxygen species and thereby preserves redox balance, and additionally induces a switch to glycolysis to prevent energetic distress. Although activation of endogenous HIF-1a signaling prevents massive apoptosis, the majority of the grafted cells do not survive. Therefore, we preconditioned periosteal cells to the hypoxic environment of the bone defect site through inhibition of PHD2, the most important PHD isoform in skeletal cells. This strategy increased post-implantation cell survival and improved bone regeneration, an effect that was mediated by HIF-1a. The enhanced cell viability was angiogenesis-independent, but relied on simultaneous and combined changes in glutamine and glycogen metabolism. HIF­1a stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. At the same time, HIF­1a signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Lastly, we showed that pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and thus the beneficial effects on cell survival, supporting further investigations to develop novel treatment modalities for bone regeneration. Throughout life, bone is being constantly remodeled through the coupled action of bone-forming osteoblasts and bone-resorbing osteoclasts, which is controlled among others by matrix-embedded osteocytes. This observation suggests that an adequate supply of oxygen and nutrients is needed to meet the high metabolic demand of these processes. Intriguingly, the bone microenvironment during development, adult life and several pathologies is characterized by low oxygen tensions and several reports have demonstrated a functional role for HIF signaling in osteoprogenitors and osteoblasts. However, the role of the hypoxia signaling pathway in osteocytes, the key regulators of bone mass during homeostasis and pathology, remains elusive. The aim of the last part of this PhD study was therefore to investigate the role of PHD2 in osteocytes, which are considered to be crucial regulators of postnatal bone mass. Transgenic mice lacking PHD2 in osteocytes displayed a high bone mass phenotype, caused by increased bone formation and decreased resorption, processes that were associated with enhanced angiogenesis. Mechanistically, stabilization of HIF-1a in PHD2-deficient osteocytes resulted in a Sirtuin 1-dependent decrease in the WNT/b-catenin inhibitor sclerostin, which stimulates osteogenesis while limiting osteoclast activity. The enhanced angiogenic response was mediated by a HIF-1a-dependent increase in vascular endothelial growth factor. Lastly, we show that genetic ablation of PHD2 was sufficient to protect mice from osteoporotic bone loss, which was potentially mediated by sustained WNT/b-catenin signaling. In this PhD thesis, we have determined the pleiotropic role of PHD2/HIF signaling during bone regeneration and homeostasis. Moreover, our data suggest that targeting PHD2 might be an interesting strategy to improve bone regeneration and, potentially, to limit bone loss during osteoporosis.status: publishe