Osteocytic oxygen sensing: Distinct impacts of VHL and HIF-2alpha on bone integrity.

Skeletal fracture resistance emerges from multiple components of bone structure like microarchitecture, matrix mineralization, and organization. These characteristics are engendered via mechanisms like the hypoxia-inducible factors (HIF) pathway, involving two paralogs, HIF-1α and HIF-2α. Under normoxia, HIF-α is targeted for degradation via von-Hippel Lindau (VHL); hypoxia enables HIF-α stabilization and induction of target genes.

Lipolysis products from triglyceride-rich lipoproteins induce stress protein ATF3 in osteoblasts.

High fat diets overwhelm the physiological mechanisms for absorption, storage, and utilization of triglycerides (TG); consequently TG, TG-rich lipoproteins (TGRL), and TGRL remnants accumulate, circulate systemically, producing dyslipidemia. This associates with, or is causative for increased atherosclerotic cardiovascular risk, ischemic stroke, fatty liver disease, and pancreatitis. TGRL hydrolysis by endothelial surface-bound lipoprotein lipase (LPL) generates metabolites like free fatty acids which have proinflammatory properties.

Degradation-Resistant Hypoxia Inducible Factor-2α in Murine Osteocytes Promotes a High Bone Mass Phenotype.

Molecular oxygen levels vary during development and disease. Adaptations to decreased oxygen bioavailability (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are composed of an oxygen-dependent α subunit (HIF-α), of which there are two transcriptionally active isoforms (HIF-1α and HIF-2α), and a constitutively expressed β subunit (HIFβ).

Hypoxia-Inducible Factor-2α Signaling in the Skeletal System.

Hypoxia-inducible factors (HIFs) are oxygen-dependent heterodimeric transcription factors that mediate molecular responses to reductions in cellular oxygen (hypoxia). HIF signaling involves stable HIF-β subunits and labile, oxygen-sensitive HIF-α subunits. Under hypoxic conditions, the HIF-α subunit is stabilized, complexes with nucleus-confined HIF-β subunit, and transcriptionally regulates hypoxia-adaptive genes.

Expression of Sex Hormone Receptors in Canine Osteosarcoma.

Sex steroids regulate bone metabolism directly and indirectly through receptors on bone. Estrogen receptors (ER-∝, ER-β), progesterone receptor (PR), and androgen receptor (AR), have been previously identified on human osteosarcoma (OSA) cells, and are considered to influence tumor growth, but their expression and role in canine OSA is unknown. The aim of this study was to characterize sex hormone receptor expression levels in naturally occurring OSA tissue and in three canine OSA cell lines.

Circulating progenitor cells and the expression of Cxcl12, Cxcr4 and angiopoietin-like 4 during wound healing in the murine ear.

Migration of cells from both local and systemic sources is essential for the inflammatory and regenerative processes that occur during normal wound healing. CXCL12 is considered a critical regulator of CXCR4-positive cell migration during tissue regeneration. In this study, we investigated the expression of Cxcl12 and Cxcr4 during healing of a murine full thickness ear wound.

Hypoxia Signaling in the Skeleton: Implications for Bone Health.

Purpose Of Review: We reviewed recent literature on oxygen sensing in osteogenic cells and its contribution to development of a skeletal phenotype, the coupling of osteogenesis with angiogenesis and integration of hypoxia into canonical Wnt signaling, and opportunities to manipulate oxygen sensing to promote skeletal repair.

Recent Findings: Oxygen sensing in osteocytes can confer a high bone mass phenotype in murine models; common and unique targets of HIF-1α and HIF-2α and lineage-specific deletion of oxygen sensing machinery suggest differentia utilization and requirement of HIF-α proteins in the differentiation from mesenchymal stem cell to osteoblast to osteocyte; oxygen-dependent but HIF-α-independent signaling may contribute to observed skeletal phenotypes. Manipulating oxygen sensing machinery in osteogenic cells influences skeletal phenotype through angiogenesis-dependent and angiogenesis-independent pathways and involves HIF-1α, HIF-2α, or both proteins.

Age Dependence of Systemic Bone Loss and Recovery Following Femur Fracture in Mice.

The most reliable predictor of future fracture risk is a previous fracture of any kind. The etiology of this increased fracture risk is not fully known, but it is possible that fracture initiates systemic bone loss, leading to greater fracture risk at all skeletal sites. In this study, we investigated systemic bone loss and recovery after femoral fracture in young (3-month-old) and middle-aged (12-month-old) mice.

Vhl deficiency in osteocytes produces high bone mass and hematopoietic defects.

Tissue oxygen (O) levels vary during development and disease; adaptations to decreased O (hypoxia) are mediated by hypoxia-inducible factor (HIF) transcription factors. HIFs are active in the skeleton, and stabilizing HIF-α isoforms cause high bone mass (HBM) phenotypes. A fundamental limitation of previous studies examining the obligate role for HIF-α isoforms in the skeleton involves the persistence of gene deletion as osteolineage cells differentiate into osteocytes.

Parathyroid hormone regulation of hypoxia-inducible factor signaling in osteoblastic cells.

Osteoblasts perceive and respond to changes in their pericellular environment, including biophysical signals and oxygen availability, to elicit an anabolic or catabolic response. Parathyroid hormone (PTH) affects each arm of skeletal remodeling, with net anabolic or catabolic effects dependent upon duration of exposure. Similarly, the capacity of osteoblastic cells to perceive pericellular oxygen has a profound effect on skeletal mass and architecture, as mice expressing stable hypoxia-inducible factor (HIF)-1α and -2α demonstrate age-dependent increases in bone volume per tissue volume and osteoblast number.

Expression of angiopoietin-like protein 4 at the fracture site: Regulation by hypoxia and osteoblastic differentiation.

Vascular disruption that occurs as a consequence of bone fracture, leads to hypoxia at the site of damage. Hypoxia regulates the expression of a number of genes that can modulate energy conservation, cell survival, tissue regeneration and angiogenesis. In this study we investigated the expression of Angiopoietin-like 4, an adipocytokine that has additional roles in angiogenesis, at the fracture site.

Impaired osteoblast differentiation in annexin A2- and -A5-deficient cells.

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls.

Nestin expression in mesenchymal stromal cells: regulation by hypoxia and osteogenesis.

Background: The intermediate filament protein nestin is used as a marker for neural stem cells, and its expression is inversely correlated with cellular differentiation. More recently, nestin expression has also been described in other cell types including multipotential mesenchymal stromal cells (MSCs). In this study, we examined the expression of nestin in equine, canine and human bone marrow-derived MSCs undergoing osteogenic differentiation, to determine whether nestin levels were attenuated as the cells acquired a more mature phenotype.

Remineralized bone matrix as a scaffold for bone tissue engineering.

There is a need for improved biomaterials for use in treating non-healing bone defects. A number of natural and synthetic biomaterials have been used for the regeneration of bone tissue with mixed results. One approach is to modify native tissue via decellularization or other treatment for use as natural scaffolding for tissue repair.

Detection of bacterial DNA by PCR in dogs with stifle pathology.

Objective: To determine presence of bacterial DNA in canine stifles with cranial cruciate ligament rupture (CCLR) and medial patellar luxation (MPL) compared to normal canine stifles (control).

Study Design: Prospective clinical study.

Animals: Dogs (n = 44).

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model.

Background Aims: Delivery of bone marrow-derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood.

Long-term administration of AMD3100, an antagonist of SDF-1/CXCR4 signaling, alters fracture repair.

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF-1 (CXCL12) is considered a master regulator of CXCR4-positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF-1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF-1 expression.

Regulation of tenascin expression in bone.

Tenascins regulate cell interaction with the surrounding pericellular matrix. Within bone, tenascins C and W influence osteoblast adhesion and differentiation, although little is known about the regulation of tenascin expression. In this study we examined the effect of osteogenic differentiation, bone morphogenetic protein (BMP) and Wnt growth factors, and mechanical loading on tenascin expression in osteogenic cells.

Hypoxic regulation of mesenchymal stem cell migration: the role of RhoA and HIF-1α.

A variety of pathologies such as skeletal fracture, neoplasia and inflammation compromise tissue perfusion and thereby decrease tissue oxygen tension. We and others have demonstrated that hypoxia is a potent stimulant for MSC (mesenchymal stem cell) recruitment and differentiation, yet to date little research has focused on the effects of oxygen tension on MSC migration. In the present study, we examined the effects of hypoxia and the potential role of the GTPase RhoA and HIF-1α (hypoxia-inducible factor 1α) on MSC migration.

Prostaglandin E2 signals through PTGER2 to regulate sclerostin expression.

The Wnt signaling pathway is a robust regulator of skeletal homeostasis. Gain-of-function mutations promote high bone mass, whereas loss of Lrp5 or Lrp6 co-receptors decrease bone mass. Similarly, mutations in antagonists of Wnt signaling influence skeletal integrity, in an inverse relation to Lrp receptor mutations.

Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia.

The aim of this study was to compare the osteogenic and proliferative potential of canine mesenchymal stromal cells (cMSCs) derived from bone marrow (BM-cMSCs) and adipose tissue (AT-cMSCs). Proliferation potential was determined under varying oxygen tensions (1%, 5%, and 21% O(2)). Effects of reduced oxygen levels on the osteogenic differentiation of AT-cMSCs were also investigated.

Comparison of the osteogenic potential of equine mesenchymal stem cells from bone marrow, adipose tissue, umbilical cord blood, and umbilical cord tissue.

Objective: To determine the optimal osteogenic source of equine mesenchymal stem cells (eMSCs) and optimize collection of and expansion conditions for those cells.

Animals: 10 adult Quarter Horses and 8 newborn Thoroughbred foals.

Procedures: eMSCs were isolated from bone marrow (BM), adipose tissue, and umbilical cord blood and tissue, and the osteogenic potential of each type was assessed.

Hypoxia increases Annexin A2 expression in osteoblastic cells via VEGF and ERK.

Vascular endothelial growth factor (VEGF)-stimulated angiogenesis is critical for endochondral ossification that occurs during bone development and bone repair. Under these circumstances, VEGF production appears to be driven by low oxygen tension, under the control of the hypoxia-inducible factor-α family of transcription factors (HIF-α). Annexin 2 (AnxA2) a calcium-dependent phospholipid binding protein has been implicated in VEGF-mediated retinal neovascularization and is upregulated by VEGF in choroid retinal endothelial cells.

Hypoxia decreases sclerostin expression and increases Wnt signaling in osteoblasts.

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF-alpha isoforms and thereby enables HIF-alpha/beta-driven gene transcription, increases bone mineral content and cross-sectional area compared to wild-type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling.