Steve Stegen

Expansion of Murine Periosteal Progenitor Cells with Fibroblast Growth Factor 2 Reveals an Intrinsic Endochondral Ossification Program Mediated by Bone Morphogenetic Protein 2

The preservation of the bone‐forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell‐based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2‐primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2‐expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2‐primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell‐based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects. Stem Cells 2014;32:2407–2418

HIF-1α Promotes Glutamine-Mediated Redox Homeostasis and Glycogen-Dependent Bioenergetics to Support Postimplantation Bone Cell Survival.

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.

Highly proliferative primitive fetal liver hematopoietic stem cells are fueled by oxidative metabolic pathways

Hematopoietic stem cells (HSCs) in the fetal liver (FL) unlike adult bone marrow (BM) proliferate extensively, posing different metabolic demands. However, metabolic pathways responsible for the production of energy and cellular building blocks in FL HSCs have not been described. Here, we report that FL HSCs use oxygen dependent energy generating pathways significantly more than their BM counterparts. RNA-Seq analysis of E14.5 FL versus BM derived HSCs identified increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the citric acid cycle (TCA). We demonstrated that FL HSCs contain more mitochondria than BM HSCs, which resulted in increased levels of oxygen consumption and reactive oxygen species (ROS) production. Higher levels of DNA repair and antioxidant pathway gene expression may prevent ROS-mediated (geno)toxicity in FL HSCs. Thus, we here for the first time highlight the underestimated importance of oxygen dependent pathways for generating energy and building blocks in FL HSCs.

Biological Activity of CXCL8 Forms Generated by Alternative Cleavage of the Signal Peptide or by Aminopeptidase-Mediated Truncation

Background Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH2-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others. Methodology/Principal Findings Aminopeptidase-cleaved CXCL8(2-77) and CXCL8(3-77), the product of alternative cleavage of the signal peptide CXCL8(-2-77) and the previously studied forms containing 77 and 72 amino acids, CXCL8(1-77) and CXCL8(6-77), were prepared by solid-phase peptide synthesis, purified and folded into active proteins. No differences in binding and calcium signaling potency were detected between CXCL8(1-77), CXCL8(-2-77), CXCL8(2-77) and CXCL8(3-77) on cells transfected with one of the human CXCL8 receptors, i.e. CXCR1 and CXCR2. However, CXCL8(-2-77) was more potent compared to CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) in signaling and in vitro chemotaxis of peripheral blood-derived human neutrophils. Moreover, CXCL8(-2-77) was less efficiently processed by plasmin into the more potent CXCL8(6-77). The truncated forms CXCL8(2-77) and CXCL8(3-77) had higher affinity for heparin than CXCL8(1-77), a property important for the presentation of CXCL8 on endothelial layers. Upon intraperitoneal injection in mice, elongated, truncated and intact CXCL8 were equally potent to recruit neutrophils to the peritoneal cavity. Conclusions In terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH2-terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.

The vasculature: a vessel for bone metastasis

Emerging evidence indicates that the interactions between tumor cells and the bone microenvironment have a crucial role in the pathogenesis of bone metastasis and that they can influence tumor cell dissemination, quiescence and tumor growth in the bone. The vasculature is known to be critical for primary tumor growth, and anti-angiogenesis drugs are approved for the treatment of certain tumor types. The role of the vasculature in bone metastasis is less well known, but recent evidence shows that blood vessels in the bone are a key component of the local microenvironment for the tumor cells and contribute to the different consecutive phases of bone metastasis. A better insight in the importance of the vasculature for bone metastasis may help develop novel treatment modalities that either slow down tumor growth or, preferably, prevent or cure bone metastasis.

Adequate hypoxia inducible factor 1α signaling is indispensable for bone regeneration

Engineered cell-based constructs are an appealing strategy to treat large skeletal defects. However, transplanted cells are often confronted with an environment that is deprived of oxygen and nutrients. Upon hypoxia, most cell types activate hypoxia-inducible factor 1α (HIF-1α) signaling, but its importance for implanted osteoprogenitor cells during bone regeneration is not elucidated. To this end, we specifically deleted the HIF--1α isoform in periosteal progenitor cells and show that activation of HIF-1α signaling in these cells is critical for bone repair by modulating angiogenic and metabolic processes. Activation of HIF-1α 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. HIF-1α signaling limits oxygen consumption to avoid accumulation of harmful ROS and preserve redox balance, and additionally induces a switch to glycolysis to prevent energetic distress. Altogether, our results indicate that the proangiogenic capacity of implanted periosteal cells is HIF-1α regulated and that metabolic adaptations mediate post-implantation cell survival.

The skeletal vascular system – Breathing life into bone tissue

During bone development, homeostasis and repair, a dense vascular system provides oxygen and nutrients to highly anabolic skeletal cells. Characteristic for the vascular system in bone is the serial organization of two capillary systems, each typified by specific morphological and physiological features. Especially the arterial capillaries mediate the growth of the bone vascular system, serve as a niche for skeletal and hematopoietic progenitors and couple angiogenesis to osteogenesis. Endothelial cells and osteoprogenitor cells interact not only physically, but also communicate to each other by secretion of growth factors. A vital angiogenic growth factor is vascular endothelial growth factor and its expression in skeletal cells is controlled by osteogenic transcription factors and hypoxia signaling, whereas the secretion of angiocrine factors by endothelial cells is regulated by Notch signaling, blood flow and possibly hypoxia. Bone loss and impaired fracture repair are often associated with reduced and disorganized blood vessel network and therapeutic targeting of the angiogenic response may contribute to enhanced bone regeneration.

Osteocytic oxygen sensing controls bone mass through epigenetic regulation of sclerostin

Preservation of bone mass is crucial for healthy ageing and largely depends on adequate responses of matrix-embedded osteocytes. These cells control bone formation and resorption concurrently by secreting the WNT/β-catenin antagonist sclerostin (SOST). Osteocytes reside within a low oxygen microenvironment, but whether and how oxygen sensing regulates their function remains elusive. Here, we show that conditional deletion of the oxygen sensor prolyl hydroxylase (PHD) 2 in osteocytes results in a high bone mass phenotype, which is caused by increased bone formation and decreased resorption. Mechanistically, enhanced HIF-1α signalling increases Sirtuin 1-dependent deacetylation of the Sost promoter, resulting in decreased sclerostin expression and enhanced WNT/β-catenin signalling. Additionally, genetic ablation of PHD2 in osteocytes blunts osteoporotic bone loss induced by oestrogen deficiency or mechanical unloading. Thus, oxygen sensing by PHD2 in osteocytes negatively regulates bone mass through epigenetic regulation of sclerostin and targeting PHD2 elicits an osteo-anabolic response in osteoporotic models.Osteocytes reside in a low oxygen environment, but it is not clear if oxygen sensing regulates their function. Here, the authors show that deletion of the oxygen sensor prolyl hydroxylase 2 in osteocytes leads to increased bone mass via regulation of sclerostin, and reduces bone loss in mouse models of osteoporosis.