Erythropoietin (EPO) is recognized for neuroprotective and angiogenic effects and has been associated with aging and neovascular age-related macular degeneration (AMD). We hypothesized that systemic EPO facilitates the development of choroidal neovascularization (CNV). Wild type mice expressed murine EPOR (mWtEPOR) in RPE/choroids at baseline and had significantly increased serum EPO after laser treatment. To test the role of EPO signaling, we used human EPOR knock-in mice with the mWtEPOR gene replaced by either the human EPOR gene (hWtEPOR) or a mutated human EPOR gene (hMtEPOR) in a laser-induced choroidal neovascularization (LCNV) model. Loss-of-function hWtEPOR mice have reduced downstream activation, whereas gain-of-function hMtEPOR mice have increased EPOR signaling. Compared to littermate controls (mWtEPOR), hMtEPOR with increased EPOR signaling developed larger CNV lesions. At baseline, hMtEPOR mice had increased numbers of macrophages, greater expression of macrophage markers F4/80 and CD206, and following laser injury, had greater expression of cytokines CCL2, CXCL10, CCL22, IL-6, and IL-10 than mWtEPOR controls. These data support a hypothesis that injury from age- and AMD-related changes in the RPE/choroid leads to choroidal neovascularization through EPOR-mediated cytokine production.

Department of Biology Palacky University Olomouc Czech Republic

Department of Hematology University of Utah Salt Lake City UT USA

John A Moran Eye Center University of Utah Salt Lake City UT USA

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Shirley Ding SL, et al. Revisiting the role of erythropoietin for treatment of ocular disorders. Eye (London, England) 2016;30:1293–1309. doi: 10.1038/eye.2016.94. PubMed DOI PMC

Wang ZY, Zhao KK, Song ZM, Shen LJ, Qu J. Erythropoietin as a novel therapeutic agent for atrophic age-related macular degeneration. Medical hypotheses. 2009;72:448–450. doi: 10.1016/j.mehy.2008.09.055. PubMed DOI

Brines M, Cerami A. Discovering erythropoietin’s extra-hematopoietic functions: Biology and clinical promise. Kidney Int. 2006;70:246–250. doi: 10.1038/sj.ki.5001546. PubMed DOI

Grimm C, et al. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration. Nature medicine. 2002;8:718–724. doi: 10.1038/nm723. PubMed DOI

Shah SS, Tsang SH, Mahajan VB. Erythropoetin receptor expression in the human diabetic retina. BMC research notes. 2009;2:234. doi: 10.1186/1756-0500-2-234. PubMed DOI PMC

Lifshitz L, Tabak G, Gassmann M, Mittelman M, Neumann D. Macrophages as novel target cells for erythropoietin. Haematologica. 2010;95:1823–1831. doi: 10.3324/haematol.2010.025015. PubMed DOI PMC

Yodoi Y, Sasahara M, Kameda T, Yoshimura N, Otani A. Circulating hematopoietic stem cells in patients with neovascular age-related macular degeneration. Investigative ophthalmology & visual science. 2007;48:5464–5472. doi: 10.1167/iovs.07-0093. PubMed DOI

Cherepanoff S, McMenamin P, Gillies MC, Kettle E, Sarks SH. Bruch’s membrane and choroidal macrophages in early and advanced age-related macular degeneration. The British journal of ophthalmology. 2010;94:918–925. doi: 10.1136/bjo.2009.165563. PubMed DOI

Bhutto IA, et al. Increased choroidal mast cells and their degranulation in age-related macular degeneration. The British journal of ophthalmology. 2016;100:720–726. doi: 10.1136/bjophthalmol-2015-308290. PubMed DOI PMC

Yang Y, et al. Macrophage polarization in experimental and clinical choroidal neovascularization. Scientific reports. 2016;6:30933. doi: 10.1038/srep30933. PubMed DOI PMC

Kirkeby A, van Beek J, Nielsen J, Leist M, Helboe L. Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor. Journal of neuroscience methods. 2007;164:50–58. doi: 10.1016/j.jneumeth.2007.03.026. PubMed DOI

Elliott S, et al. Anti-Epo receptor antibodies do not predict Epo receptor expression. Blood. 2006;107:1892–1895. doi: 10.1182/blood-2005-10-4066. PubMed DOI

Divoky V, et al. Mouse model of congenital polycythemia: Homologous replacement of murine gene by mutant human erythropoietin receptor gene. Proc Natl Acad Sci USA. 2001;98:986–991. doi: 10.1073/pnas.98.3.986. PubMed DOI PMC

Ebie AZ, Fleming KG. Dimerization of the erythropoietin receptor transmembrane domain in micelles. Journal of molecular biology. 2007;366:517–524. doi: 10.1016/j.jmb.2006.11.035. PubMed DOI

Divoky, V. & Prchal, J. T. Mouse surviving solely on human erythropoietin receptor (EpoR): model of human EpoR-linked disease. Blood99, 3873–3874; author reply 3874–3875 (2002). PubMed

Divoky V, et al. Delayed hemoglobin switching and perinatal neocytolysis in mice with gain-of-function erythropoietin receptor. Journal of molecular medicine (Berlin, Germany) 2016;94:597–608. doi: 10.1007/s00109-015-1375-y. PubMed DOI PMC

Tsutsumi C, et al. The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization. Journal of leukocyte biology. 2003;74:25–32. doi: 10.1189/jlb.0902436. PubMed DOI

Killingsworth MC, Sarks JP, Sarks SH. Macrophages related to Bruch’s membrane in age-related macular degeneration. Eye (London, England) 1990;4(Pt 4):613–621. doi: 10.1038/eye.1990.86. PubMed DOI

McLeod DS, et al. Distribution and Quantification of Choroidal Macrophages in Human Eyes With Age-Related Macular Degeneration. Investigative ophthalmology & visual science. 2016;57:5843–5855. doi: 10.1167/iovs.16-20049. PubMed DOI PMC

Kang J, et al. Erythropoietin priming improves the vasculogenic potential of G-CSF mobilized human peripheral blood mononuclear cells. Cardiovascular research. 2014;104:171–182. doi: 10.1093/cvr/cvu180. PubMed DOI

Liu J, et al. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice. PloS one. 2013;8:e72935. doi: 10.1371/journal.pone.0072935. PubMed DOI PMC

He L, Marneros AG. Macrophages are essential for the early wound healing response and the formation of a fibrovascular scar. The American journal of pathology. 2013;182:2407–2417. doi: 10.1016/j.ajpath.2013.02.032. PubMed DOI PMC

Wang S, et al. Erythropoietin protects against rhabdomyolysis-induced acute kidney injury by modulating macrophage polarization. Cell death & disease. 2017;8:e2725. doi: 10.1038/cddis.2017.104. PubMed DOI PMC

Cao X, et al. Macrophage polarization in the maculae of age-related macular degeneration: a pilot study. Pathology international. 2011;61:528–535. doi: 10.1111/j.1440-1827.2011.02695.x. PubMed DOI PMC

Sakurada Y, et al. Aqueous humor cytokine levels in patients with polypoidal choroidal vasculopathy and neovascular age-related macular degeneration. Ophthalmic research. 2015;53:2–7. doi: 10.1159/000365487. PubMed DOI

Mo FM, Proia AD, Johnson WH, Cyr D, Lashkari K. Interferon gamma-inducible protein-10 (IP-10) and eotaxin as biomarkers in age-related macular degeneration. Investigative ophthalmology & visual science. 2010;51:4226–4236. doi: 10.1167/iovs.09-3910. PubMed DOI

Seddon JM, George S, Rosner B, Rifai N. Progression of age-related macular degeneration: prospective assessment of C-reactive protein, interleukin 6, and other cardiovascular biomarkers. Archives of ophthalmology (Chicago, Ill.: 1960) 2005;123:774–782. doi: 10.1001/archopht.123.6.774. PubMed DOI

Rezar-Dreindl S, et al. The Intraocular Cytokine Profile and Therapeutic Response in Persistent Neovascular Age-Related Macular Degeneration. Investigative ophthalmology & visual science. 2016;57:4144–4150. doi: 10.1167/iovs.16-19772. PubMed DOI

Chan KK, et al. Erythropoietin drives breast cancer progression by activation of its receptor EPOR. Oncotarget. 2017;8:38251–38263. PubMed PMC

Tankiewicz-Kwedlo A, et al. Erythropoietin accelerates tumor growth through increase of erythropoietin receptor (EpoR) as well as by the stimulation of angiogenesis in DLD-1 and Ht-29 xenografts. Molecular and cellular biochemistry. 2016;421:1–18. doi: 10.1007/s11010-016-2779-x. PubMed DOI PMC

Apte RS, Richter J, Herndon J, Ferguson TA. Macrophages inhibit neovascularization in a murine model of age-related macular degeneration. PLoS medicine. 2006;3:e310. doi: 10.1371/journal.pmed.0030310. PubMed DOI PMC

Sakurai E, Anand A, Ambati BK, van Rooijen N, Ambati J. Macrophage depletion inhibits experimental choroidal neovascularization. Investigative ophthalmology & visual science. 2003;44:3578–3585. doi: 10.1167/iovs.03-0097. PubMed DOI

Penfold PL, Killingsworth MC, Sarks SH. Senile macular degeneration: the involvement of immunocompetent cells. Graefe’s archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie. 1985;223:69–76. doi: 10.1007/BF02150948. PubMed DOI

Combadiere C, et al. CX3CR1-dependent subretinal microglia cell accumulation is associated with cardinal features of age-related macular degeneration. The Journal of clinical investigation. 2007;117:2920–2928. doi: 10.1172/JCI31692. PubMed DOI PMC

Yu X, Lin CS, Costantini F, Noguchi CT. The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse. Blood. 2001;98:475–477. doi: 10.1182/blood.V98.2.475. PubMed DOI

Wen D, Boissel JP, Showers M, Ruch BC, Bunn HF. Erythropoietin structure-function relationships. Identification of functionally important domains. J Biol Chem. 1994;269:22839–22846. PubMed

Robbie SJ, et al. Enhanced Ccl2-Ccr2 signaling drives more severe choroidal neovascularization with aging. Neurobiology of aging. 2016;40:110–119. doi: 10.1016/j.neurobiolaging.2015.12.019. PubMed DOI

Nakamura R, et al. IL10-driven STAT3 signalling in senescent macrophages promotes pathological eye angiogenesis. Nature communications. 2015;6:7847. doi: 10.1038/ncomms8847. PubMed DOI PMC

Kelly J, Ali Khan A, Yin J, Ferguson TA, Apte RS. Senescence regulates macrophage activation and angiogenic fate at sites of tissue injury in mice. The Journal of clinical investigation. 2007;117:3421–3426. doi: 10.1172/JCI32430. PubMed DOI PMC

Luhmann UF, et al. The drusenlike phenotype in aging Ccl2-knockout mice is caused by an accelerated accumulation of swollen autofluorescent subretinal macrophages. Investigative ophthalmology & visual science. 2009;50:5934–5943. doi: 10.1167/iovs.09-3462. PubMed DOI PMC

Hoch M, et al. Erythropoietin preserves the endothelial differentiation capacity of cardiac progenitor cells and reduces heart failure during anticancer therapies. Cell stem cell. 2011;9:131–143. doi: 10.1016/j.stem.2011.07.001. PubMed DOI

Yang Z, Wang H, Jiang Y, Hartnett ME. VEGFA Activates Erythropoietin Receptor and Enhances VEGFR2-Mediated Pathological Angiogenesis. Am J Pathol. 2014;184:1230–1239. doi: 10.1016/j.ajpath.2013.12.023. PubMed DOI PMC

McCloskey M, et al. Anti-VEGF Antibody Leads to Later Atypical Intravitreous Neovascularization and Activation of Angiogenic Pathways in a Rat Model of Retinopathy of Prematurity. Invest Ophthalmol Vis Sci. 2013;54:2020–2026. doi: 10.1167/iovs.13-11625. PubMed DOI PMC

Wang H, et al. VEGF-mediated STAT3 activation inhibits retinal vascularization by down-regulating local erythropoietin expression. Am J Pathol. 2012;180:1243–1253. doi: 10.1016/j.ajpath.2011.11.031. PubMed DOI PMC

Wang H, et al. Retinal Inhibition of CCR3 Induces Retinal Cell Death in a Murine Model of Choroidal Neovascularization. PLoS One. 2016;11:e0157748. doi: 10.1371/journal.pone.0157748. PubMed DOI PMC

Wang H, et al. Upregulation of CCR3 by Age-Related Stresses Promotes Choroidal Endothelial Cell Migration via VEGF-Dependent and -Independent Signaling. Investigative Ophthalmology & Visual Science. 2011;52:8271–8277. doi: 10.1167/iovs.11-8230. PubMed DOI PMC

Zajac E, et al. Angiogenic capacity of M1- and M2-polarized macrophages is determined by the levels of TIMP-1 complexed with their secreted proMMP-9. Blood. 2013;122:4054–4067. doi: 10.1182/blood-2013-05-501494. PubMed DOI PMC

Ershler WB, et al. Serum erythropoietin and aging: a longitudinal analysis. Journal of the American Geriatrics Society. 2005;53:1360–1365. doi: 10.1111/j.1532-5415.2005.53416.x. PubMed DOI

Ghaem Maralani H, et al. Metabolic syndrome and risk of age-related macular degeneration. Retina (Philadelphia, Pa.) 2015;35:459–466. doi: 10.1097/IAE.0000000000000338. PubMed DOI