Temporal and Spatial Expression of Erythropoietin, Erythropoietin Receptor, and Common β Receptor in Wound Fluid and Granulation
- 0 Comments
- 6647 reads
Abstract: 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 βreceptor (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 βcR 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 βcR, may be an effective therapeutic approach to improve wound healing.
Address correspondence to:
Thomas R. Howdieshell, MD
Trauma/Surgical Critical Care
Department of Surgery
University of New Mexico HSC
Albuquerque, NM 87131
Erythropoietin (Epo) is a 30.4 kDa glycoprotein produced primarily in the adult kidney under the control of an oxygen-sensing mechanism. It regulates the daily production of 2 x 1011 red blood cells in adult bone marrow to maintain the oxygen-carrying capacity of blood under physiologic conditions. Epo has long been known to be the principle hematopoietic growth factor that regulates cellular proliferation and differentiation along the erythroid lineage.1 A recent study has shown that Epo is a pleiotropic cytokine that is proangiogenic and exerts broad tissue-protective effects in diverse nonhematopoietic organs.2
The biological effects of Epo in hematopoietic cells are mediated through its binding to its specific cell surface receptor—the erythropoietin receptor (EpoR). EpoR is a member of the type I cytokine receptor family that includes cellular transmembrane receptors for factors such as granulocyte-colony stimulating factor, many of the interleukins, prolactin, as well as growth hormone.2 EpoR expression and signaling in hematopoietic tissues is essential for normal mammalian erythropoiesis during development.
1. Bunn HF. New agents that stimulate erythropoiesis. Blood. 2007;109(3):868–873.
2. Parganas E, Wang D, Stravopodis D, et al. Jak2 is essential for signaling through a variety of cytokine receptors. Cell. 1998;93(3):385–395.
3. Kertesz N, Wu J, Chen TH, Sucov HM, Wu H. The role of erythropoietin in regulating angiogenesis. Dev Biol. 2004;276(1):101–110.
4. Wu H, Lee SH, Gao J, Liu X, Iruela-Arispe ML. Inactivation of erythropoietin leads to defects in cardiac morphogenesis. Development. 1999;126(16):3597–3605.
5. Parsa CJ, Kim J, Riel RU, et al. Cardioprotective effects of erythropoietin in the reperfused ischemic heart: a potential role for cardiac fibroblasts. J Biol Chem. 2004;279(20):20655–20662.
6. Nagai A, Nakagawa E, Choi HB, Hatori K, Kobayashi S, Kim SU. Erythropoietin and erythropoietin receptors in human CNS neurons, astrocytes, microglia, and oligodendrocytes grown in culture. J Neuropathol Exp Neurol. 2001;60(4):386–392.
7. Westenfelder C, Biddle DL, Baranowski RL. Human, rat, and mouse kidney cells express functional erythropoietin receptors. Kidney Int. 1999;55(3):808–820.
8. Fiordaliso F, Chimenti S, Staszewsky L, et al. A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc Natl Acad Sci U S A. 2005;102(6):2046–2051.
9. Haroon ZA, Amin K, Jiang X, Arcasoy MO. A novel role for erythropoietin during fibrin-induced wound-healing response. Am J Pathol. 2003;163(3):993–1000.
10. Buemi M, Galeano M, Sturiale A, et al. Recombinant human erythropoietin stimulates angiogenesis and healing of ischemic skin wounds. Shock. 2004;22(2):169–173.
11. Buemi M, Vaccaro M, Sturiale A, et al. Recombinant human erythropoietin influences revascularization and healing in a rat model of random ischaemic flaps. Acta Derm Venereol. 2002;82(6):411–417.
12. Galeano M, Altavilla D, Bitto A, et al. Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds. Crit Care Med. 2006;34(4):1139–1146.
13. Howdieshell TR, Riegner C, Gupta V, et al. Normoxic wound fluid contains high levels of vascular endothelial growth factor. Ann Surg. 1998;228(5):707–715.
14. Howdieshell TR, Webb WL, Sathyanarayana, McNeil PL. Inhibition of inducible nitric oxide synthase results in reductions in wound vascular endothelial growth factor expression, granulation tissue formation, and local perfusion. Surgery. 2003;133(5):528–537.
15. Clark RA. Cutaneous tissue repair: basic biologic consideration. I. J Am Acad Dermatol. 1985;13(5 Pt 1):701–725.
16. Stadelmann WK, Digenis AG, Tobin GR. Impediments to wound healing. Am J Surg. 1998;176(2A Suppl):39S–47S.
17. Brem H, Balledux J, Bloom T, Kerstein MD, Hollier L. Healing of diabetic foot ulcers and pressure ulcers with human skin equivalent, a new paradigm in wound healing. Arch Surg. 2000;135(6):627–634.
18. Höckel M, Schlenger K, Doctrow S, Kissel T, Vaupel P. Therapeutic angiogenesis. Arch Surg. 1993;128(4):423–429.
19. Eckardt KU. The potential of erythropoietin and related strategies to stimulate erythropoiesis. Curr Opin Investig Drugs. 2001;2(8):1081–1085.
20. Grasso G, Sfacteria A, Cerami A, Brines M. Erythropoietin as a tissue-protective cytokine in brain injury: what do we know and where do we go? Neuroscientist. 2004;10(2):93–98.
21. Hanlon PR, Fu P, Wright GL, Steenbergen C, Arcasoy MO, Murphy E. Mechanisms of erythropoietin-mediated cardioprotection during ischemia-reperfusion injury: role of protein kinase C and phosphatidylinositol 3-kinase signaling. FASEB J. 2005;19(10):1323–1325.
22. Vesey DA, Cheung C, Pat B, Endre Z, Gobé G, Johnson DW. Erythropoietin protects against ischemic acute renal injury. Nephrol Dial Transplant. 2004;19(2):348–355.
23. Hayashida K, Kitamura T, Gorman DM, Arai K, Yokota T, Miyajima A. Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci U S A. 1990;87(24):9655–9660.
24. Jubinsky PT, Krijanovski OI, Nathan DG, Tavernier J, Sieff CA. The beta chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor. Blood. 1997;90(5):1867–1873.
25. Brines M, Grasso G, Fiordaliso F, et al. Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc Natl Acad Sci U S A. 2004;101(41):14907–14912.
26. Coleman TR, Westernfelder C, Tögel FE, et al. Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities. Proc Natl Acad Sci U S A. 2006;103(15):5965–5970.