Bone has the unique capacity to heal without the formation of a fibrous scar, likely because several of the cellular and molecular processes governing bone healing recapitulate the events during skeletal development. A critical component in bone healing is the timely appearance of blood vessels in the fracture callus. Angiogenesis, the formation of new blood vessels from pre-existing ones, is stimulated after fracture by the local production of numerous angiogenic growth factors. The fracture vasculature not only supplies oxygen and nutrients, but also stem cells able to differentiate into osteoblasts and in a later phase also the ions necessary for mineralization. This review provides a concise report of the regulation of angiogenesis by bone cells, its importance during bone healing and its possible therapeutic applications in bone tissue engineering. This article is part of a Special Issue entitled "Stem Cells and Bone".

Highlights d Single-cell RNA-seq reveals EC heterogeneity in choroidal neovascularization d ECs display metabolic transcriptome heterogeneity in the cell cycle and quiescence d Data integration with a genome-scale metabolic model identifies angiogenic targets d SQLE and ALDH18A1 are validated as metabolic angiogenic candidates

Extracellular matrix (ECM) is a major component of the local environment, i.e. the niche, that can determine cell behavior 1 . During metastatic growth, cancer cells shape the ECM of the metastatic niche by hydroxylating collagen to promote their own metastatic growth 2 , 3 . However, only particular nutrients might support the ability of cancer cells to hydroxylate collagen because nutrients dictate which enzymatic reactions are active in cancer cells 4 , 5 . Here, we discovered that breast cancer cells rely on the nutrient pyruvate to drive collagen-based ECM remodeling in the lung metastatic niche. Specifically, we discovered that pyruvate uptake induces the production of α-ketoglutarate. This metabolite in turn activated collagen hydroxylation by increasing the activity of the enzyme collagen prolyl-4-hydroxylase (P4HA). Strikingly, inhibition of pyruvate metabolism was sufficient to impair collagen hydroxylation and consequently the growth of breast cancer-derived lung metastases in different mouse models. In summary, we provide a mechanistic understanding of the link between collagen remodeling and the nutrient environment in the metastatic niche.

Endochondral ossification, an important bone formation process in vertebrates, highly depends on proper functioning of growth plate chondrocytes 1 . Their proliferation determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment 1,2 . In addition, the centre of the expanding growth plate becomes hypoxic and local activation of the hypoxiainducible transcription factor HIF-1α is necessary for chondrocyte survival by still unknown cellintrinsic mechanisms [3][4][5][6] . Whether HIF-1α signalling has to be contained in the other regions of the growth plate and whether chondrocyte metabolism controls cell function remains undefined. We here show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response (UPR) and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate (αKG) levels, which in turn increases collagen proline and lysine hydroxylation. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to proteasemediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other extracellular matrix-related diseases such as cancer and fibrosis.To investigate whether HIF signalling needs to be controlled in growth plate chondrocytes, we conditionally inactivated HIF prolyl hydroxylase 2 (PHD2; Phd2 chonmice), its main negative regulator 7 , resulting in HIF-1α accumulation (Extended Data Fig. 1a-d).This approach caused skeletal dysplasia, characterized by impaired longitudinal bone growth and increased trabecular bone mass (Fig. 1a,b, Extended Data Fig. 1e,f). The growth plate was shorter, but normally organized and, interestingly, the high bone mass was not due to altered bone resorption or formation (Extended Data Fig. 1g-l). Instead, we observed more cartilage remnants in the bony trabeculae, evidenced by more type II collagen (COL2)positive and proteoglycan-rich matrix (Fig. 1c, Extended Data Fig. 1m). The decreased serum CTx-II levels, measuring COL2 degradation, indicated that the cartilage matrix was incompletely resorbed, and the unaltered chondrocyte-to-matrix ratio pointed to a qualitative, rather than quantitative, change in matrix properties (Extended Data Fig. 1j,n). Thus, inactive oxygen sensing in chondrocytes increases trabecular bone mass, caused by abundant cartilage remnants, likely resulting from modifications in the cartilage matrix itself.HIF-1α stabilization in PHD2-deficient chondrocytes resulted, as expected 7,8 , in metabolic reprogramming. Mitochondrial content was reduced, likely because of decreased biogenesis without changing autophagy (Extended D...

Cell-based therapy is a promising strategy in regenerative medicine, but the poor survival rate of the implanted cells remains a major challenge and limits clinical translation. We preconditioned periosteal cells to the hypoxic and ischemic environment of the bone defect site by deleting prolyl hydroxylase domain-containing protein 2 (PHD2), resulting in hypoxia-inducible factor 1 alpha (HIF-1α) stabilization. This strategy increased postimplantation cell survival and improved bone regeneration. The enhanced cell viability was angiogenesis independent but relied on combined changes in glutamine and glycogen metabolism. HIF-1α stabilization stimulated glutaminase-mediated glutathione synthesis, maintaining redox homeostasis at baseline and during oxidative or nutrient stress. Simultaneously, HIF-1α signaling increased glycogen storage, preventing an energy deficit during nutrient or oxygen deprivation. Pharmacological inhibition of PHD2 recapitulated the adaptations in glutamine and glycogen metabolism and, consequently, the beneficial effects on cell survival. Thus, targeting cellular metabolism is an appealing strategy for bone regeneration and cell-based therapy in general.