Temporal and spatial expression of erythropoietin, erythropoietin receptor, and common β receptor in wound fluid and granulation tissue

Background. Erythropoietin (Epo) and its cognate receptor (EpoR) have been recently identified in nonhematopoietic cells. Epo structural variants, which possess tissue protective effects while exhibiting no effect on erythropoiesis, appear to require a second distinct receptor component, the common receptor (βcR) of IL-3, IL-5, and GM-CSF for ligand signal transduction. The goal of this work was to determine the temporal and spatial presence of Epo, EpoR, and βcR in porcine wound fluid and granulation tissue. Methods. A ventral hernia, surgically created in the abdominal wall of female swine (n = 8), was repaired with silicone sheeting and skin closure. Over time, a fluid-filled wound compartment formed, bounded by subcutaneous and omental granulation tissue; its thickness was measured by ultrasonography. Serial wound fluid samples were obtained by percutaneous aspiration. On day 14, the animals were sacrificed. Protein isolated from skin, kidney, granulation tissue, and peritoneal and wound fluids was analyzed by Western blotting. Sections of formalin-fixed abdominal wall tissue were stained for immunoreactivity to Epo, EpoR, and βcR. Results. A progressive increase in granulation tissue thickness was measured during the 14-day interval. Western blot analysis of serial wound fluid samples demonstrated an 8-fold increase in local wound fluid Epo concentration. Immunoblotting of day 0 skin and day 14 granulation tissue homogenates demonstrated presence of Epo, EpoR, and β2cR in wound granulation tissue but not in control skin. Immunostaining demonstrated localization of Epo and its receptors in granulation endothelial cells, fibroblasts, macrophages, and smooth muscle cells. Conclusion. Temporal expression of soluble Epo was associated with a progressive increase in porcine granulation tissue formation. Receptor expression, spatially localized to cellular constituents of granulation tissue, increased in the wound environment compared to control tissue. Epo variants, which signal via a heteroreceptor complex including both EpoR and β2cR, may be an effective therapeutic approach to improve wound healing.