EPO Promotes Bone Repair through Enhanced Cartilaginous Callus Formation and Angiogenesis

Abstract

Erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling is involved in the development and regeneration of several non-hematopoietic tissues including the skeleton. EPO is identified as a downstream target of the hypoxia inducible factor-α (HIF-α) pathway. It is shown that EPO exerts a positive role in bone repair, however, the underlying cellular and molecular mechanisms remain unclear. In the present study we show that EPO and EPOR are expressed in the proliferating, pre-hypertrophic and hypertrophic zone of the developing mouse growth plates as well as in the cartilaginous callus of the healing bone. The proliferation rate of chondrocytes is increased under EPO treatment, while this effect is decreased following siRNA mediated knockdown of EPOR in chondrocytes. EPO treatment increases biosynthesis of proteoglycan, accompanied by up-regulation of chondrogenic marker genes including SOX9, SOX5, SOX6, collagen type 2, and aggrecan. The effects are inhibited by knockdown of EPOR. Blockage of the endogenous EPO in chondrocytes also impaired the chondrogenic differentiation. In addition, EPO promotes metatarsal endothelial sprouting in vitro. This coincides with the in vivo data that local delivery of EPO increases vascularity at the mid-stage of bone healing (day 14). In a mouse femoral fracture model, EPO promotes cartilaginous callus formation at days 7 and 14, and enhances bone healing at day 28 indexed by improved X-ray score and micro-CT analysis of microstructure of new bone regenerates, which results in improved biomechanical properties. Our results indicate that EPO enhances chondrogenic and angiogenic responses during bone repair. EPO's function on chondrocyte proliferation and differentiation is at least partially mediated by its receptor EPOR. EPO may serve as a therapeutic agent to facilitate skeletal regeneration.

Osteoblasts: a Novel Source of Erythropoietin

“…Many of the studies described above reported that EPO-mediated bone formation and healing was associated with increased vascular density and angiogenesis. EPO treatment was shown to increase endothelial sprouting from metatarsal bones of E17.5 embryos, induce the vasculature in bone in vivo, as well as stimulate endothelial cell proliferation in vitro [33–36]. These findings are consistent with previous reports that EPO signaling regulates angiogenesis [27].…”

“…Subsequent studies demonstrated that EPO alone is sufficient to increase bone volume and repair. EPO treatment enhanced both bone volume and biomechanical properties in multiple murine femoral fracture repair models [33–35]. Similarly, EPO stimulated BMP2-induced bone formation in cranial defect and scaffold models [36, 37].…”

Extracellular signaling molecules to promote fracture healing and bone regeneration

“…As well, PDGF is highly expressed by platelets, as are other signaling proteins, such as thrombospondin-1, both will be discussed in the next section. While RBC are not signaling cells, it is notable that Erythropoietin (EPO) that is necessary for RBC production has been shown in a number of studies to positively influence bone regeneration (18,19). formation), must be recruited to the provisional inflammatory callus ( Figure 1).…”

Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone

“…LOXL2 was shown to be important in sprouting angiogenesis [15], a process important in endochondral bone formation and bone healing [79]. Hypoxia induces a variety of gene expressions in endothelial cells to induce angiogenesis.…”