0041-1337/03/7508-1356/0 TRANSPLANTATION Vol. 75, 1356–1360, No. 8, April 27, 2003 Copyright © 2003 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A. HUMAN, NONHUMAN PRIMATE, AND RAT PANCREATIC ISLETS EXPRESS ERYTHROPOIETIN RECEPTORS 1 ELIZABETH S. FENJVES, 2,3 M. SOFIA OCHOA, 2 OVER CABRERA, 2 ARMANDO J. MENDEZ, 2 NORMA S. KENYON, 2 LUCA INVERARDI, 2 AND CAMILLO RICORDI 2 Background. Erythropoietin (EPO) promotes sur- vival in a variety of cells by mediating antiapoptotic signals through the EPO receptor (R). The authors examined pancreatic islets for the presence of EPO-R to determine whether these cells are protected by EPO from cytokine-induced apoptosis. Methods. Reverse-transcriptase polymerase chain reaction, immunohistology, and Western blots were used to establish the presence and localization of EPO-R on rat, nonhuman primate, and human islets. Islets were exposed to cytokines in the presence and absence of recombinant EPO and apoptosis was mea- sured using a terminal deoxynucleotide transferase- mediated dUTP nick-end labeling assay followed by fluorescence-activated cell sorter analysis. Glucose stimulation indices were measured to assess the effect of EPO on islet function. Results. The presence of EPO-R was demonstrated on islets regardless of species. Recombinant EPO pro- tected islets in culture from cytokine-induced apopto- sis in a dose-dependent manner. Furthermore, the presence of EPO in the media does not adversely affect islet function. Conclusions. This is the first demonstration that pancreatic islets express EPO-R and that EPO may prevent islet-cell apoptosis in culture. In vivo trials to evaluate the potential of long-term expression of EPO to augment islet survival in transplantation are underway. Erythropoietin (EPO) is a kidney cytokine that regulates hematopoiesis by promoting the survival, proliferation, and differentiation of erythroid progenitor cells (1). Specifically, EPO blocks the normal apoptotic cycle in erythroid progeni- tors as they progress through maturation (2, 3). The effect of EPO is mediated exclusively by its binding to the EPO re- ceptor (R), through which antiapoptotic signals are mediated (4). Expression of the EPO-R was thought to be exclusive of precursor erythroid cells (5) but has recently been found on a variety of cells including small bowel (6), myoblasts (7), neu- ronal (8), and kidney (9). The observation that the EPO-R is widely expressed extends its putative role beyond hemato- poiesis. Recently, the EPO-R has been linked to the develop- ment of a variety of nonerythroid systems, implying that EPO may shield a variety of tissues against hypoxic and ischemic stress (6, 7, 10, 11). Transplantation of pancreatic islets offers a potential cur- ative treatment for type 1 diabetes, and this clinical approach has shown great promise (12). However, for islet transplan- tation to become a viable treatment option for a significant number of patients, it will be necessary to protect these cells from apoptotic signals triggered by isolation, culture, and transplantation. Islet mass is often lost to apoptosis (13), which accounts for up to 60% of transplanted -cell loss (14). One approach to promote islet survival during transplanta- tion is to take advantage of molecules that would render islets less sensitive to cytokine-mediated apoptosis in the transplant setting. The authors hypothesized that pancreatic islets may express EPO-R and thus be protected from apo- ptosis by recombinant EPO. To this end, the authors exam- ined pancreatic islets derived from rat, nonhuman primates, and humans for EPO-R expression and for protection from apoptosis by recombinant EPO. To determine whether rat, nonhuman primate, and human islets express the EPO-R, molecular and histologic methods were used. The authors demonstrate that pancreatic islets express EPO-R and established the protective effect of EPO on islet survival in the presence of proapoptotic stimuli. Preconditioning the culture media with EPO protects islets from cytokine-induced death in a dose-dependent manner. These results indicate that EPO is useful in protecting cul- tured pancreatic islets from apoptosis after isolation. MATERIALS AND METHODS Animals Experiments using animals were performed in compliance with National Institutes of Health and Institutional Animal Care and Use Committee guidelines. Islet Isolation Rat islets (Lewis; Harlan Sprague Dawley, Indianapolis, IN) were isolated by intraductal injection of the enzyme Liberase (Boehringer Mannheim, Indianapolis, IN), followed by incubation at 37°C (98.6°F), mechanical disruption, and purification by centrifugation on discontinuous gradients. Islets obtained from nonhuman primate pancreata and cadaveric pancreata were isolated by enzymatic di- gestion (Liberase) using the automated method and purification on discontinuous gradients as previously described (15). Functional Assessment of Islets Islet purity was assessed by dithizone (Sigma, St. Louis, MO) staining, and islets were counted and scored for size. An algorithm was used for the calculation of 150-m-diameter islet equivalent number (IEN) (24). Glucose-stimulated insulin secretion assay was used to assess -cell function. Triplicates of 50 IEN were incubated at 37°C in 12-well plates (Nalgene-Nunc, Rochester, NY) in 1 mL Krebs-Ringer bicarbonate buffer with or without EPO at concentra- 1 This work was supported by the Diabetes Research Institute Foundation. 2 Diabetes Research Institute, University Of Miami, School of Medicine, Miami, FL. 3 Address correspondence to: Elizabeth S. Fenjves, Ph.D., Diabe- tes Research Institute, University of Miami, School of Medicine, 1450 N.W. 10th Avenue, R-134, Miami, FL 33136. E-mail: efenjves@miami.edu. Received 10 October 2002; revision requested 19 December 2002. Accepted 5 February 2003. 1356 DOI: 10.1097/01.TP.0000062862.88375.BD