epo meeting lübeck 2012 supplement 2012 focus uni lübeck · hemopoietic growth factors" will...

Supplement 2012EPO Meeting Lübeck 2012

Zeitschrift für Wissenschaft, Forschung und Lehre an der Universität zu Lübeck

(focus)uni lübeck

9th InternationalLübeck Conference

Pathophysiology andPharmacology ofErythropoietinand other HemopoieticGrowth Factors

University of Lübeck, GermanyJuly 13 - July 15, 2012

Impressumfocus uni lübeck

Zeitschrift für Wissenschaft, Forschung und Lehre an der Universität zu Lübeck

Herausgeber: Das Präsidium der Universität zu Lübeck

Präsidiumsbeauftragter: C. Borck

Schriftleitung: H.‑P. Bruch, W. Kühnel, Th. Martinetz, P. Schmucker

Wissenschaftlicher Beirat: T. Buzug, J. Dunst, A. Ch. Feller, G. Gillessen‑Kaesbach, S. Grisanti, W. Gross, E. Hartmann, M. Herczeg, E. Herting, R. Hilgenfeld, F. Hohagen, C. Hübner, W. Jelkmann, D. Jocham, J. Köhl, H. Lehnert, M. Leucker, V. Linnemann, E. Maehle, P. Mailänder A. Mertins, Th. Münte, D. O. Nutzinger, Th. Peters, D. Petersen, J. Prestin, H.‑H. Raspe, K. R. Reischuk, F. Schmielau, H. Schunkert, A. Schweikard, G. Sczakiel, H. H. Sievers, W. Solbach, N. Tautz, V. Tronnier, A. Vogel, J. Westermann, B. Wollenberg, P. Zabel, D. Zillikens

Redaktion: Rüdiger Labahn (Leitung), Dr. Thorsten Biet (Schwerpunkt Wissenschaft und Technik), Dr. Solveig Simowitsch (Schwerpunkt Chancengleichheit und Familie)Telefon (04 51) 500 3004 ‑ E‑mail: [email protected]‑luebeck.de

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EPO Meeting Luebeck 2012 (focus) uni lübeck Supplement 2012

General Information

The 9th International Luebeck Conference on the "Pathophysiology and Pharmacology of Erythropoietin and otherHemopoietic Growth Factors" will be held at the University of Luebeck, Germany, from July 13 - July 15, 2012. Similar tothe meetings before, the purpose of the conference will be to stimulate the exchange of ideas between basic scientistsand clinicians who are active in this field. About 150 participants are expected to attend the meeting. The majorscientific topics will be:

- Hypoxia sensing

- EPO production in vitro

- EPO production in vivo

- Recombinant erythropoietins

- Treatment of renal anemia

- Treatment of cancer anemia

- Sports medicine

- Colony-stimulating factors

- Non-hemopoietic actions of erythropoietin

Date and Placeof the Meeting

July 13 - July 15, 2012

Lecture Hall Audimax AM1University of LuebeckRatzeburger Allee 160D-23562 LuebeckGermany

Organization Horst Pagel and Wolfgang JelkmannInstitute of PhysiologyMember of theCenter for Structural and Cell Biology in Medicineof the University of Luebeck ( CSCM )

Permanent Office Evelyn KimpelInstitute of PhysiologyUniversity of LuebeckRatzeburger Allee 160D-23562 LuebeckGermany

Phone +49-451-500-4152+49-451-500-5751 ( during the meeting )

Fax +49-451-500-4151

E-mail [email protected]

Presentations Oral presentations: in English only (no translation), 15 min plus 5 min for discussion.

Poster presentations: All posters will be up during the entire meeting in the foyer ofthe lecture hall. Poster presenters are requested to present their main results duringa poster session.

For the presentations of the posters a board will be available (115 cm width, 145 cmheight). Fixing material will be provided by the organizers.

Abstracts The abstracts will be published in a special issue of the Luebeck University Journal"FOCUS uni-luebeck" and electronically in the "American Journal of Hematology".

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Scientific Program EPO-Meeting Luebeck 2012

Saturday, July 14, 2012

8:00 OPENING ADDRESSESW. JelkmannR. Köhling, Secretary of the German Physiological Society

8:30 - 10:00 Session 1 (Oral Presentations)"Hypoxia inducible factors"(Chairpersons: J. Griffiths, V. H. Haase)

8:30 - 8:50 A. Loboda, U. Florczyk, S. Czauderna, A. Stachurska, M. Tertil, M. Kozakowska,L. Poellinger, A. Jozkowicz, J. DulakTHE EFFECT OF HIF-1 AND HIF-2 ON THE REGULATION OF ANGIOGENIC GENES IN ENDOTHELIALCELLS

8:50 - 9:10 T. Otto, S. Lauterbach, S. Frede, J. FandreyINFLAMMATION IN RENAL EPO PRODUCING CELLS

9:10 - 9:30 K. Franke, J. Kalucka , R. Pal Singh, S. Mamlouk, A. Muschter, A. Weidemann,V. Iyengar, J. Fandrey, M. Gassmann, B. WielockxCONDITIONAL PHD2 DEFICIENCY LEADS TO NON-LETHAL EXCESSIVE ERYTHROCYTOSIS ANDDELAYED WOUND HEALING IN MICE

9:30 - 9:50 M. Miró-Murillo, A. Elorza, I. Soro-Arnáiz, L. Albacete-Albacete, A. Ordoñez, E. Balsa,A. Vara-Vega, S. Vázquez, E. Fuertes, C. Fernández-Criado, M. O. Landázuri, J. AragonésACUTE VHL GENE INACTIVATION INDUCES CARDIAC HIF-DEPENDENT ERYTHROPOIETIN GENEEXPRESSION

10:00 - 10:30 Coffee break

10:30 - 12:00 Session 2 (Oral Presentations)"Hypoxia sensing and erythropoiesis"(Chairpersons: R. Depping, N. Suzuki)

10:30 - 10:50 M. Mastrogiannaki, P. Matak, J. R. Mathieu, S. Delga, P. Mayeux, S. Vaulont,C. PeyssonnauxROLE OF HIF-2ALPHA IN IRON METABOLISM

10:50 - 11:10 Q. Liu, V. H. HaaseHYPOXIA-INDUCIBLE FACTOR REGULATION OF HEPCIDIN REQUIRES EPO

11:10 - 11:30 E. Gammella, V. Díaz, S. Recalcati, P. Santambrogio, A. Monge Naldi, J. Vogel, M. Gassmann, G. CairoREGULATION OF IRON HOMEOSTASIS IN MICE OVEREXPRESSING HUMAN ERYTHROPOIETIN

11:30 - 11:50 K. J. Nytko, N. Maeda, P. Schläfli, P. Spielmann, R. H. Wenger, D. P. StiehlVITAMIN C IS NOT REQUIRED FOR OXYGEN SENSING IN VIVO

12:00 - 13:00 Lunch

13:00 - 14:30 Session 3 (Oral Presentations)"Human pathologies"(Chairpersons: P. Kling, R. Wenger)

13:00 - 13:20 M. J. Percy, P. W. Furlow, Y. J. Chung, T. R. J. Lappin, M. F. McMullin, F. S. LeeCRITICAL IMPORTANCE OF THE REGION C TERMINAL TO THE HYDROXYACCEPTOR PROLINE FORTHE REGULATION OF HIF-2ALPHA

13:20 - 13:40 N. Petousi, Q. C. Croft, H. Y. Cheng, F. Formenti, K. Ishida, N. P. Talbot, P. J. Ratcliffe,P. A. RobbinsA PHYSIOLOGICAL STUDY OF TIBETAN NATIVES AT SEA LEVEL

13:40 - 14:00 C. Dame, C. Rau, A. Thorwarth, P. Koehne, M. Klar, C. BührerTHE ERYTHROPOIETIN (EPO) GENE POLYMORPHISM (RS16171640) IN PRETERM INFANTS WITHRETINOPATHY

14:00 - 14:30 Invited Lecture: G. Garibotto, A. Bonanni, D. VerzolaEFFECT OF CHRONIC KIDNEY DISEASE (CKD) AND HEMODIALYSIS ON PROTEIN AND AMINO ACIDMETABOLISM


14:00 - 14:30 Invited Lecture: G. Garibotto, A. Bonanni, D. VerzolaEFFECT OF CHRONIC KIDNEY DISEASE (CKD) AND HEMODIALYSIS ON PROTEIN AND AMINO ACIDMETABOLISM


15:00 - 16:30 Session 4 (Poster Viewing)

1 N. Suzuki, X. Pan, Y. Tojo, T. Dan, T. Miyata, L. Poellinger, M. YamamotoEPIGENETIC REGULATION OF HYPOXIA-DEPENDENT ERYTHROPOIETIN GENE EXPRESSION

2 G. Zhang, V. Emelianov, A. Kromminga, N. van Beek, E. Bodo, U. Duske, R. PausNOVEL POINTERS TO REGULATION AND TARGETS OF ERYTHROPOIETIN IN HUMAN SKIN: AN EXPLORATORYPILOT STUDY

3 I. Hirano, N. Suzuki, X. Pan, S. Yamazaki, N. Minegishi, M. YamamotoERYTHROPOIETIN PRODUCTION IN NEURAL CREST CELLS

4 F. Wenzel, T. Gettmann, N. Zimmermann, G. GiersPERIOPERATIVE SERUM ERYTHROPOIETIN AND THROMBOPOIETIN LEVELS IN CORONARY ARTERY BYPASSGRAFTING (CABG)

5 B. B. Beleslin Cokic, V. P. Cokic, C. T. NoguchiNITRIC OXIDE AND HYPOXIA STIMULATE AND REGULATE ERYTHROPOIETIN RECEPTOR IN ENDOTHELIALCELLS

6 N. Dehne, S. Essler, M. Tausendschön, T. Schmid, B. BrüneIL-4 ATTENUATES THE PROANGIOGENIC CAPACITY OF MACROPHAGES BY DOWNREGULATING HIF-1α PROTEIN

7 K. Flück, G. Breves, S. Schäfer, J. Fandrey, S. WinningTHE ROLE OF HYPOXIA-INDUCIBLE FACTORS IN INFLAMMATORY BOWEL DISEASE

8 R. Lupica, C. Zinnarello, V. Donato, S. Luicisano, A. Lacquaniti, V. Cernaro, M. R. Fazio, P. Ruggeri, M. BuemiERYTHROPOIETIN AS A LINK BETWEEN SKIN AND KIDNEY

9 F. K. Pientka, M. Ströfer, J. Dunst, W. Jelkmann, R. DeppingEXPRESSION OF THE CELLULAR OXYGEN SENSOR PHD2 AFFECTS RADIORESISTANCE IN SQUAMOUS CELLCANCER OF THE HEAD AND NECK

10 K. M. Kirschner, J. Braun, H. ScholzEXPRESSION OF THE AMILORIDE BINDING PROTEIN 1 (ABP1) IS STIMULATED IN RESPONSE TO HYPOXIA ANDMYOCARDIAL INFARCTION

11 W. Sun, T. Hellwig-Bürgel, R. Depping, W. JelkmannDISTINCT IMPACTS OF INTERLEUKIN-1BETA ON HIF-1ALPHA AND HIF-2ALPHA IN TUMOR CELLS

12 A. Bernardini, U. Brockmeier, J. FandreyANALYSIS OF THE ERYTHROPOIETIN RECEPTOR DIMER IN DIFFERENT CANCER CELL LINES BY MEANS OFFLUORESCENCE RESONANCE ENERGY TRANSFER

13 L. Dahm, I. Hassouna, N. Offen, C. Ott, R. Neher, S. Sperling, N. Hagemeyer, K. Hannke, M. Mitkovski, L. Schlee,M. Dittrich, S. Boretius, A. Zeug, T. Dandekar, E. Neher, A.-L. Sirén, H. EhrenreichERYTHROPOIETIN DRIVES NEURODIFFERENTIATION

14 J. A. Griffiths, T. Gallego-Martin, V. Telezhkin, J. Soliz, C. Gonzalez, P. J. KempEPO REGULATES HYPOXIC SIGNAL TRANSDUCTION IN RAT CAROTID BODY GLOMUS CELLS VIA POTASSIUMCHANNEL REMODELLING

15 P. J. Kling, A. T. Quilling, M. Y. Sun, S. E. BlohowiakENTERAL ERYTHROPOIETIN & IRON TRANSPORTER EXPRESSION IN NEWBORN RATS

16 N. Miljus, D. Ostrowski, H. Ehrenreich, R. HeinrichERYTHROPOIETIN PROMOTES SURVIVAL AND REGENERATION OF INSECT NEURONS IN VIVO AND IN VITRO

17 F. Moriconi , P. Ramadori, M. Blaschke, F. C. Schultze, A. Amanzada, G. RamadoriCHARACTERIZATION OF THE ERYTHROPOIETIN/ ERYTHROPOIETIN RECEPTOR AXIS IN A RAT MODEL OF LIVERDAMAGE AND CHOLANGIOCARCINOMA DEVELOPMENT

18 N. Offen, J. Flemming, R. Ahmad, W. Wolber, C. Geis, H. Zaehres, H. Schöler, A. Müller, A.-L. SirénINFLUENCE OF ERYTHROPOIETIN ON SURVIVAL AND NEURONAL DIFFERENTIATION OF MOUSE INDUCEDPLURIPOTENT STEM CELLS


19 N. Hagemeyer, C. Ott, D. Winkler, N. Jensen, S. Boretius, A. von Streitberg, H. Welpinghus, S. Sperling, J. Frahm,M. Simons, P. Ghezzi, H. EhrenreichERYTHROPOIETIN ATTENUATES NEUROLOGICAL AND HISTOLOGICAL CONSEQUENCES OF TOXICDEMYELINATION IN MICE

20 O. Dmytriyeva, T. Novikova, C. R. Gøtzsche, E. Bock, V. Berezin, S. PankratovaA NOVEL MIMETIC OF ERYTHROPOIETIN FOR THE TREATMENT OF ALZHEIMER’S DISEASE-LIKE PATHOLOGY

21 N. Trošt, P. Juvan, G. Serša, N. Hevir, T. Lanišnik Rižner, N. DebeljakERYTHROPOIETIN MODULATES BREAST CANCER CELL RESPONSE TO CISPLATIN IN A TIME-DEPENDENTMANNER

22 U. Brockmeier, A. Wrobeln, E. MetzenSCREENING FOR ER PROTEINS THAT INFLUENCE FOLDING AND CELL SURFACE EXPOSURE OF THEERYTHROPOIETIN RECEPTOR

23 Y. Hüsecken, J. Fandrey, S. WinningCHARACTERIZATION OF DENDRITIC CELLS UNDER INFLAMMATORY HYPOXIA

24 A. Lubkowska, B. Dołęgowska, G. BanfiGROWTH FACTORS IN PRP - GENERAL APPROACHES - A REVIEW OF RECENT DEVELOPMENTS

25 N. Torres-Nagel, D. Ziegler-Landesberger, R. Sircar, S. de Schepper, G. Walker, N. van Acker, M. Schräml, J. Bachmann,U. Klingmüller, M. Jarsch, O. MundiglSPECIFIC AND SENSITIVE IMMUNOHISTOCHEMISTRY DETECTION OF THE HUMAN ERYTHROPOIETINRECEPTOR IN SITU

26 T. V. Parkhomenko, V. V. Tomson, O. A. KlitsenkoERYTHROPOIETIN (EPO) STIMULATES AEROBIC AND ANAEROBIC PROCESSES IN RAT CARDIOMYOCYTES

27 M. Schwartz, T. Otto, U. Brockmeier, S. Lauterbach, E. Metzen, E. Gulbins, A. Carpinteiro, N. Scheerer, J. FandreyEFFECTS OF NITRIC OXIDE ON HEMATOGENOUS METASTASIS

28 A. Thiel, A. Neugebauer, C. Stockmann, U. Berchner-Pfannschmidt, N. Scheerer, J. FandreyMODULATION OF OXYGEN SENSING BY NITRIC OXIDE IN AN AUTOCHTHONUS MOUSE MODEL OF BREASTCANCER

29 V. G. Demikhov, E. V. Demikhova, E.N. Zinovyeva, A.D. Pavlov, E.F. MorshchakovaWHY RECOMBINANT ERYTHROPOIETIN THERAPY IS VERY EFFECTIVE FOR THE TREATMENT OF IRONDEFICIENCY ANEMIA IN PREGNANCY?

30 K. Gottwald, V. Ullmann, A. Reif, M. Rädisch, C. UnverzagtSEMISYNTHESIS OF HUMAN ERYTHROPOIETIN GLYCOSYLATED AT ASN24

31 S. Hiram-Bab, T. Liron, M. C. Souroujon, M. Eisenstein, D. NeumannNOVEL ERYTHROPOIETIN-RECEPTOR PEPTIDE ANTAGONISTS

32 N. V. Inyakova, V. G. Demikhov, E. N. Dolzhenko, O. A. Golovitsyna, E. F. MorshchakovaRECOMBINANT HUMAN ERYTHROPOIETIN THERAPY OF ANEMIC PATIENTS WITH LUNG TUBERCULOSIS

33 S. Schiebel, A.-M. Larsson, M. Vaapil, J. Sun, A. Jögi, L. Rönnstrand, S. PåhlmanEPO-INDEPENDENT FUNCTIONAL EPO RECEPTOR IN BREAST CANCER ENHANCES ESTROGEN RECEPTORACTIVITY AND PROMOTES CELL PROLIFERATION

34 A. N. Nikolaev, V. G. Demikhov, V. L. Dobin, N. V. Inyakova, V. B. Skobin, E. F. MorshchakovaPREOPERATIVE USE OF RECOMBINANT HUMAN ERYTHROPOIETIN FOR PREVENTION OF BLOODTRANSFUSIONS IN PULMONARY TUBERCULOSIS PATIENTS

35 Z. Szygula, M. Wiecek, M. Maciejczyk, W. Pilch, A. Zembron-Lacny, K. de Meirleir, J. SmitzSERUM ERYTHROPOIETIN, PLASMA VOLUME REGULATING HORMONES, AND PLASMA VOLUME CHANGESINDUCED BY EXERCISE AND THERMAL DEHYDRATION

36 A. Rodriguez, M. Schelker, M. Schilling, J. Bachmann, A. Raue, F. Salopiata, S. Depner, L. Adlung, R. Merkle, A. Franke,M. Jarsch, J. Timmer, U. KlingmüllerEPO AND EPOR SIGNALING DYNAMIC IN HEMATOPOIETIC AND TUMOR CONTEXT

37 H. Pagel, E. Metzen, W. JelkmannEFFECTS OF HIF STABILIZERS IN ISOLATED PERFUSED RAT KIDNEYS: IMPLICATIONS FOR BLOOD DOPING

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16:30 - 18:00 Session 5 (Oral Presentations)"Non-hemopoietic actions of erythropoietin"(Chairpersons: J. Fandrey, M. Schwaninger)

16:30 - 16:50 R. HeinrichPRE-VERTEBRATE EVOLUTION OF ERYTHROPOIETIN SIGNALLING SERVING NEUROPROTECTION ANDNEUROREGENERATION

16:50 - 17:10 A. Kästner, S. Grube, A. El-Kordi, B. Stepniak, H. Friedrichs, D. Sargin, J. Schwitulla, M. Begemann, I. Giegling,K. W. Miskowiak, S. Sperling, K. Hannke, A. Ramin, R. Heinrich, O. Gefeller, K.-A. Nave, D. Rujescu,H. EhrenreichCOMMON VARIANTS OF THE GENES ENCODING ERYTHROPOIETIN AND ITS RECEPTOR MODULATECOGNITIVE PERFORMANCE

17:10 - 17:30 A.-L. Sirén, N. Offen, L. Dahme, J. Flemming, H. Kamawal, N. Hagemeyer, S. Sperling, R. Ahmad, C. Geis,A. Müller, H. EhrenreichERYTHROPOIETIN INDUCES NEURAL DIFFERENTIATION IN STEM CELLS

17:30 - 17:50 S. Pankratova, D. Kiryushko, K. Sonn, V. Soroka, L. B. Køhler, M. Rathje, I. Korshunova, B. Gu, K. Gotfryd,O. Clausen, A. Zharkovsky, E. Bock, V. BerezinNEUROTROPHIC AND NEUROPROTECTIVE PROPERTIES OF NON-HEMATOPOIETIC EPO-DERIVEDPEPTIDES

Sunday, Juli 15, 2012

8:30 - 10:00 Session 6 (Oral Presentations)"Non-hemopoietic actions of erythropoietin"(Chairpersons: M. Mittelman, A. Sytkowski)

8:30 - 8:50 C. T. Noguchi, R. Teng, M. AlNaeeliMETABOLIC ACTIVITY ASSOCIATED WITH ERYTHROPOIETIN

8:50 - 9:10 E. Fibach, E. A. RachmilewitzTHE ANTIOXIDANT EFFECT OF ERYTHROPOIETIN ON THALASSEMIC BLOOD CELLS

9:10 - 9:30 S. Yakushev, D. Mihov, M. Gassmann, A. BogdanovaMYOCARDIAL NA,K-ATPASE AND ITS ROLE IN EPO-INDUCED CARDIOPROTECTION

9:30 - 9:50 N. Deshet-Unger, Y. Ohana, S. Hiram-Bab, M. Gassmann, M. Mittelman, M. Souroujon, D. NeumannERYTHROPOIETIN (EPO) EXERTS AN ANTI-NEOPLASTIC ACTIVITY: CHARACTERIZING EPO-DRIVENRESPONSES IN THE 5T33 MULTIPLE MYELOMA MOUSE MODEL.


10:30 - 12:00 Session 7 (Oral Presentations)"Therapeutic aspects"(Chairpersons: M. Jarsch, T. Lappin)

10:30 - 10:50 Y. Sasaki, M. Noguchi-Sasaki, Y. Matsumoto-Omori, K. Yorozu, Y. ShimonakaIRON DYNAMICS AND ERYTHROBLAST MATURATION KINETICS AFTER C.E.R.A. TREATMENT

10:50 - 11:10 C. I. Günter, U. Dornseifer, F. Siemers, P. Mailänder, M. Ninkovic, O. Thamm, G. Spilker, T. Wolter, S. Dunda,G. Grieb, N. Pallua, C. Ernert, M. Steen, A. Kestel, R. Sievers, D. Fuge, B. Reichert, B. Hartmann,A. Rahmanian-Schwarz, H. E. Schaller, A. Daigeler, M. Otte, H. Menke, S. M. Ryu, Pierson, A. Bader, S. Ebert,W. Jelkmann, A. M. Raem, C. Ohmann, V. Kehl, H. G. MachensFIRST RESULTS ON REGENERATIVE EFFECTS OF ERYTHROPOIETIN IN BURN AND SCALD INJURES(CLINICAL TRIAL: “EPO IN BURNS”)

11:10 - 11:30 E. Pérès, S. Valable, A. Calipel, A. Corroyer-Dumont, L. Durand, E. Lechapt-Zalcman, M. Bernaudin, E. PetitEPOR DEFICIENCY ON GLIOMA CELLS INDUCES SENSITIVITY TO IONIZING RADIATION ANDTEMOZOLOMIDE

11:30 - 11:50 K. W. Woodburn, C. P. Holmes, K.-L. Fong, Y. Moriya, Y. TagawaPHARMACOKINETICS, BIODISTRIBUTION, METABOLISM AND EXCRETION OF PEGINESATIDE, ANERYTHROPOIESIS-STIMULATING AGENT, IN RATS


16:50 - 17:10 A. Kästner, S. Grube, A. El-Kordi, B. Stepniak, H. Friedrichs, D. Sargin, J. Schwitulla,M. Begemann, I. Giegling, K. W. Miskowiak, S. Sperling, K. Hannke, A. Ramin,R. Heinrich, O. Gefeller, K.-A. Nave, D. Rujescu, H. EhrenreichCOMMON VARIANTS OF THE GENES ENCODING ERYTHROPOIETIN AND ITS RECEPTOR MODULATECOGNITIVE PERFORMANCE

17:10 - 17:30 A.-L. Sirén, N. Offen, L. Dahme, J. Flemming, H. Kamawal, N. Hagemeyer, S. Sperling,R. Ahmad, C. Geis, A. Müller, H. EhrenreichERYTHROPOIETIN INDUCES NEURAL DIFFERENTIATION IN STEM CELLS

17:30 - 17:50 S. Pankratova, D. Kiryushko, K. Sonn, V. Soroka, L. B. Køhler, M. Rathje, I. Korshunova,B. Gu, K. Gotfryd, O. Clausen, A. Zharkovsky, E. Bock, V. BerezinNEUROTROPHIC AND NEUROPROTECTIVE PROPERTIES OF NON-HEMATOPOIETIC EPO-DERIVEDPEPTIDES






9:30 - 9:50 N. Deshet-Unger, Y. Ohana, S. Hiram-Bab, M. Gassmann, M. Mittelman, M. Souroujon,D. NeumannERYTHROPOIETIN (EPO) EXERTS AN ANTI-NEOPLASTIC ACTIVITY: CHARACTERIZING EPO-DRIVENRESPONSES IN THE 5T33 MULTIPLE MYELOMA MOUSE MODEL.




10:50 - 11:10 C. I. Günter, U. Dornseifer, F. Siemers, P. Mailänder, M. Ninkovic, O. Thamm, G. Spilker,T. Wolter, S. Dunda, G. Grieb, N. Pallua, C. Ernert, M. Steen, A. Kestel, R. Sievers,D. Fuge, B. Reichert, B. Hartmann, A. Rahmanian-Schwarz, H. E. Schaller, A. Daigeler,M. Otte, H. Menke, S. M. Ryu, Pierson, A. Bader, S. Ebert, W. Jelkmann, A. M. Raem,C. Ohmann, V. Kehl, H. G. MachensFIRST RESULTS ON REGENERATIVE EFFECTS OF ERYTHROPOIETIN IN BURN AND SCALD INJURES(CLINICAL TRIAL: “EPO IN BURNS”)

11:10 - 11:30 E. Pérès, S. Valable, A. Calipel, A. Corroyer-Dumont, L. Durand, E. Lechapt-Zalcman,M. Bernaudin, E. PetitEPOR DEFICIENCY ON GLIOMA CELLS INDUCES SENSITIVITY TO IONIZING RADIATION ANDTEMOZOLOMIDE



12:15 - 13:35 Session 8 (Oral Presentations)"Applied physiology"(Chairpersons: C. Bauer, Z. Szygula)

12:15 - 12:35 K. M. Oliver, C. R. Lenihan, U. Bruning, A. Cheong, J. G. Laffey, P. McLoughlin,S. F. Fitzpatrick, C. C. Scholz, C. Slattery, M. O. Leonard, C. T. Taylor, E. P. CumminsCARBON DIOXIDE (CO2) SENSING AT THE AXIS OF IMMUNITY AND INFLAMMATION.



12:15 - 12:35 K. M. Oliver, C. R. Lenihan, U. Bruning, A. Cheong, J. G. Laffey, P. McLoughlin, S. F. Fitzpatrick, C. C. Scholz,C. Slattery, M. O. Leonard, C. T. Taylor, E. P. CumminsCARBON DIOXIDE (CO2) SENSING AT THE AXIS OF IMMUNITY AND INFLAMMATION.

12:35 - 12:55 B. Christensen, B. Nellemann, M. Larsen, L. Thams, P. Sieljacks, M. H. Vendelbo, T. Krusenstjerna-Hafstrøm, M.Madsen, S. B. Pedersen, K. Vissing, S. Nielsen, N. Jessen, N. Møller, J. O. Lunde JørgensenMETABOLIC EFFECTS OF ACUTE AND PROLONGED TREATMENT WITH RHUEPO TO HEALTHY YOUNGMEN

12:55 - 13:15 B. Schuler, M. Alvarez Sanchez, M. GassmannCEREBRAL EPO AND EXCERCISE PERFORMANCE

13:15 - 13:35 E. Neuberger, D. Moser, P. SimonDETECTION OF EPO GENE DOPING IN BLOOD

13:40 CONCLUDING REMARKSH. Pagel

16:50 - 17:10 A. Kästner, S. Grube, A. El-Kordi, B. Stepniak, H. Friedrichs, D. Sargin, J. Schwitulla,M. Begemann, I. Giegling, K. W. Miskowiak, S. Sperling, K. Hannke, A. Ramin,R. Heinrich, O. Gefeller, K.-A. Nave, D. Rujescu, H. EhrenreichCOMMON VARIANTS OF THE GENES ENCODING ERYTHROPOIETIN AND ITS RECEPTOR MODULATECOGNITIVE PERFORMANCE

17:10 - 17:30 A.-L. Sirén, N. Offen, L. Dahme, J. Flemming, H. Kamawal, N. Hagemeyer, S. Sperling,R. Ahmad, C. Geis, A. Müller, H. EhrenreichERYTHROPOIETIN INDUCES NEURAL DIFFERENTIATION IN STEM CELLS

17:30 - 17:50 S. Pankratova, D. Kiryushko, K. Sonn, V. Soroka, L. B. Køhler, M. Rathje, I. Korshunova,B. Gu, K. Gotfryd, O. Clausen, A. Zharkovsky, E. Bock, V. BerezinNEUROTROPHIC AND NEUROPROTECTIVE PROPERTIES OF NON-HEMATOPOIETIC EPO-DERIVEDPEPTIDES






9:30 - 9:50 N. Deshet-Unger, Y. Ohana, S. Hiram-Bab, M. Gassmann, M. Mittelman, M. Souroujon,D. NeumannERYTHROPOIETIN (EPO) EXERTS AN ANTI-NEOPLASTIC ACTIVITY: CHARACTERIZING EPO-DRIVENRESPONSES IN THE 5T33 MULTIPLE MYELOMA MOUSE MODEL.




10:50 - 11:10 C. I. Günter, U. Dornseifer, F. Siemers, P. Mailänder, M. Ninkovic, O. Thamm, G. Spilker,T. Wolter, S. Dunda, G. Grieb, N. Pallua, C. Ernert, M. Steen, A. Kestel, R. Sievers,D. Fuge, B. Reichert, B. Hartmann, A. Rahmanian-Schwarz, H. E. Schaller, A. Daigeler,M. Otte, H. Menke, S. M. Ryu, Pierson, A. Bader, S. Ebert, W. Jelkmann, A. M. Raem,C. Ohmann, V. Kehl, H. G. MachensFIRST RESULTS ON REGENERATIVE EFFECTS OF ERYTHROPOIETIN IN BURN AND SCALD INJURES(CLINICAL TRIAL: “EPO IN BURNS”)

11:10 - 11:30 E. Pérès, S. Valable, A. Calipel, A. Corroyer-Dumont, L. Durand, E. Lechapt-Zalcman,M. Bernaudin, E. PetitEPOR DEFICIENCY ON GLIOMA CELLS INDUCES SENSITIVITY TO IONIZING RADIATION ANDTEMOZOLOMIDE




12:15 - 12:35 K. M. Oliver, C. R. Lenihan, U. Bruning, A. Cheong, J. G. Laffey, P. McLoughlin,S. F. Fitzpatrick, C. C. Scholz, C. Slattery, M. O. Leonard, C. T. Taylor, E. P. CumminsCARBON DIOXIDE (CO2) SENSING AT THE AXIS OF IMMUNITY AND INFLAMMATION.


Social ProgramThe events are free of charge for participants of the conference.

Friday, July 13

19:00 Get-together Party

Audience Hall ("Audienzsaal") of the Town Hall ("Rathaus") of Lübeck, Breite Straße

Welcome by Mr. Bernd Saxe, Mayor of the City of Lübeck

The Town Hall of Lübeck was commenced by the citizens in 1230. Since it was modified during the following centuries, it incorporates now a variety of styles from the Gothic to the Renaissance. The Town Hall still meets its purpose as domicile of the municipal parliament, the Senate, and the top-departments of the administration. In former times the Audience Hall used to be the convention room of the Hanseatic High Court. Its present Rococo style originates from the years between 1754 and 1761. The paintings by the Italian artist Stefano Torelli show allegorical motives: the liberal arts, trade, freedom, mercy, harmony, industry and abundance, vigilance, intelligence, moderation, and discretion. The interior portal of the Audience Hall dates to 1573 and shows in reliefs the judgement of Solomon, the justice and affection. The portal had/has two openings: Those convicted had to stoop to pass through the lower door, but the Conference guests may walk upright through the high one.

Saturday, July 14

19:00 Snacks and Music (Martina Tegtmeyer, accordion, and Jan Baruschke, violin)

“KulturRösterei” and Restaurant “Remise” Wahmstraße 43-45 (Downtown Lübeck)

Participants of the Conference are invited to meet at this former coffee processing plant that has been transformed into a stage for cabaret, theatre, literature readings and music performances.

Sunday, July 15

16:00 Guided City Tour

Please sign up at the Conference Secretary´s desk for a special city walk (max. 25 participants, due to space restrictions).

Lübeck was founded in 1143 as the first German city at the Baltic Sea. The historic Old Town which is surrounded by water is a significant symbol of brick stone architecture from the Gothic period. The cultural sights symbolize the great past of Lübeck as the "Queen of the Hanse”. Parts of the historic Old Town were added to the list of World Heritage by UNESCO in December 1987. Apart from the Town Hall, the most important buildings include the convent "Burgkloster", the “Holsten Gate”, a unique quarter of the late 13th century (Koberg) with St. Jacob's church, the “Hospital of the Holy Spirit” and all the buildings between "Glockengießerstraße" and "Aegidiensstraße", and a prestigious historic quarter with patrician houses of the 15th and 16th century between St. Peter's church and Lübeck's Cathedral.

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Report Abstracts


The effect of HIF-1 and HIF-2 on the regulation of angiogenic genes in endothelial cells

Agnieszka Loboda1, Urszula Florczyk1, Szymon Czauderna1, Anna Stachurska1, Magdalena Tertil1, Magdalena Kozakowska1, Lorenz Poellinger2, Alicja Jozkowicz1, Jozef Dulak1

1Department of Medical Biotechnology,Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; 2Karolinska Institute, Department of Cell and Molecular Biology, Stockholm, Sweden,

Hypoxia-inducible factor (HIF)-1α and HIF-2α are known to be the major mediators of hypoxic response and regulate the expression of genes involved in angiogenesis, cell proliferation and viability. Although highly related, owing to possess diffe-rent transactivation domains and binding distinct cofactors, they may regulate not only shared but also unique target genes. Among HIF isoforms-interacting proteins c-Myc/Max proteins and Sp-1 transcription factor were found. Interestingly, we have shown that interleukin-8 (IL-8), known mediator of angiogenesis, is downregulated by HIF-1α (Lo-boda et al., 2009). Then we aimed to examine the effect of HIF-2α on IL-8 expression and the involvement of c-Myc/Mxi1/Sp-1 proteins in such regulation.Human microvascular endothelial cells (HMEC-1) were transduced with adenoviral vectors to overexpress HIF-1α or HIF-2α. In contrast to HIF-1α, overexpression of HIF-2α resulted in significantly increased expression of IL-8. Accordingly, HIF-2α was found to augment the activity of Sp-1. Importantly, inhibition of the latter with mithramycin A decreased, while its overexpression with pCMV-Sp-1 vector increased IL-8 expression. Mutation of putative Sp-1-binding site within IL-8 promoter revealed however no direct interaction of Sp-1 and IL-8. From the other hand, we confirmed that in contrast to HIF-1α, overexpression of HIF-2α isoform results in increased expression of c-Myc. We checked the effect of c-Myc silen-cing and revealed diminishment of HIF-2α-dependent induction of IL-8 in such conditions. Conversely, inhibition of Mxi1 by siRNA reversed the effect of HIF-1α on IL-8. Moreover, we showed that severe hypoxia (0.5% oxygen), simultaneously with decrease in IL-8 expression, augments Mxi1, but reduces c-Myc mRNA level. In accordance with the latter, transduction of cells with AdHIF-1α caused decrease in c-Myc level suggesting that effect of hypoxia is primarily mediated by HIF-1α.Current study revealed for the first time opposite role of HIF-1α and HIF-2α in regulation of IL-8 expression in endothelial cells and the involvement of Sp-1 and c-Myc in the enhancement of IL-8 by HIF-2α.

Inflammation in renal EPO producing cells

Otto, Teresa¹; Lauterbach, Silke¹; Frede, Stilla²; Fandrey, Joachim¹

¹Institute of Physiology, University of Duisburg-Essen, Essen, Germany²Department of Anaesthesiology and Intensive Care Medicine, University Hospital Bonn, Bonn, Germany

Anemia of chronic disease (ACD) represents one of the most prevalent anemias and is associated with infections, can-cer, autoimmune diseases, transplantation and chronic kidney disease. It is known to be immune driven by cytokines and results in impaired erythropoietin (EPO) production. Cytokines like interleukin-1β (IL-1β) and tumour necrosis factor α (TNF-α) are already known to mediate a decrease in the production of EPO and several mechanisms in hepatocellular car-cinoma and neuroblastoma cell lines have been proposed. Since adult kidneys produce 90% of systemic EPO it is impor-tant to examine the mechanisms of cytokine mediated EPO suppression in a renal tissue culture model. With our human renal EPO producing cell line (REPC) we were able to unravel the mechanisms, combining inflammatory effects on TNFα, HIF2α and HNF4α expression as well as the involvement of microRNAs.


Conditional PHD2 Deficiency Leads to Non-Lethal Excessive Erythrocytosis and delayed wound healing in Mice

Kristin Franke, Joanna Kalucka , Rashim Pal Singh, Soulafa Mamlouk, Antje Muschter, Alexander Weidemann, Vasuprada Iyengar, Joachim Fandrey, Max Gassmann and Ben Wielockx

Emmy Noether Research Group. - Institute of Pathology, University of Technology, Dresden, Germany.Department of Nephrology and Hypertension, University Clinic Erlangen, Germany.

Department of Physiology, University of Duisburg, Germany.Institute of Veterinary Physiology, Vetsuisse Facult Human Physiology (ZIHP), University of Zürich, Switzerland.

An abnormal increase in the number of circulating red blood cells (RBCs) or erythrocytosis is dangerous and associated with a high mortality rate if not treated. The control of red cell mass is therefore of utmost importance and in many cases reliant on the precise regulation of erythropoietin (EPO) and its transcription factor hypoxia inducible factor (HIF). This entire system is directly controlled by oxygen-sensitive prolyl hydroxylases (PHDs), although their detailed role is far from understood. Here, we present a new conditional PHD2 deficient mouse line (cKO) displaying over-production of EPO in kidney and brain, lea-ding to excessive erythrocytosis, splenomegaly but normal lifespan. Using double deficient mice we were able to demonstra-te that the EPO-induced phenotypes are exclusively driven by HIF2α whilst HIF1α stabilization served as a protective factor ensuring survival. Additionally, in a complex model of skin wound healing we observed in cKO as well as EPO transgenic mice a drastic delay in one of the last stages of wound healing; the resolving phase. Taken together, we have demonstrated that extreme hematocrits in PHD2 conditional deficient mice are not per se lethal due to the distinct regulation of HIF1α and HIF2α.

Acute Vhl Gene Inactivation Induces Cardiac HIF-Dependent Erythropoietin Gene Expression

Marta Miró-Murillo#, Ainara Elorza1, Inés Soro-Arnáiz1, Lucas Albacete-Albacete1, Angel Ordoñez1, Eduardo Balsa1, Alicia Vara-Vega1, Silvia Vázquez1, Esther Fuertes1, Carmen Fernández-Criado2, Manuel O. Landázuri1 and Julián Aragonés1

(1) Servicio de Inmunología, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria Princesa (IP),Universidad Autónoma de Madrid, Madrid, Spain.

(2) Gabinete Veterinario, Universidad Autónoma de Madrid, Madrid, Spain.

Von Hippel Lindau (Vhl) gene inactivation results in embryonic lethality. Therefore the consequences of its inactivation in adult mice, and of the ensuing activation of the hypoxia-inducible factors (HIFs), have been explored mainly in a tissue-specific manner. This mid-gestation lethality can be also circumvented by using a floxed Vhl allele in combination with an ubiquous tamoxifen-inducible recombinase Cre-ERT2. Here, we characterize a widespread reduction in Vhl gene expressi-on in Vhlfloxed-UBC-Cre-ERT2 adult mice after dietary tamoxifen administration. Vhl gene inactivation rapidly resulted in a marked splenomegaly and skin erythema, accompanied by renal and hepatic induction of the erythropoietin (Epo) gene, in-dicative of the in vivo activation of the oxygen sensing HIF pathway. We show that acute Vhl gene inactivation also induced Epo gene expression in the heart, revealing cardiac tissue to be an extra-renal source of EPO. Indeed, primary cardiomyo-cytes and HL-1 cardiac cells both induce Epo gene expression in a HIF-dependent manner when exposed to low oxygen tension. Moreover, we have recently generated Vhlfloxed-HIF1afloxedUBC-Cre-ERT2 and Vhlfloxed-HIF2afloxedUBC-Cre-ERT2 adult mice in which Vhl and HIF1a - or VHL and HIF2a - can be simultaneously inactivated in adult mice. These mice are providing information about (i) additional extra-renal sources of EPO production as well as (ii) the relative contribution of HIF1a or HIF2a isoforms. Thus, this data provides evidence of a cardiac oxygen-sensing VHL/HIF/EPO pathway in adult mice and establish novel genetic tools to evaluate EPO production in vivo associated to oxygen fluctuations.

Acknowledgments: This work was supported by grants from European Comission (Epocan, Grant agreement no.: 282551), Ministerio de Educación y Ciencia (BFU2008-03407/BMC), RECAVA (RD06/0014/0031), CICYT (SAF2007-60592) and CAM (SAL0311/2006).


Role of HIF-2α in iron metabolism

Maria Mastrogiannaki, Pavle Matak, Jacques R Mathieu, Stéphanie Delga, Patrick Mayeux, Vaulont S, Peyssonnaux C.

Inserm U1016, Institut Cochin, CNRS UMR 8104, Université Paris Descartes, Sorbonne Paris Cité, Paris, France

The 200 billion new mature red blood cells produced daily are the major consumers of body iron, which is indispensable for the enormous production of heme for hemoglobin synthesis, to carry oxygen from the lungs to every cell in the human body. The number of circulating red blood cells is the major determinant of tissue oxygenation. As a consequence, hypoxia triggers induction of erythropoietin (EPO) to increase the production of erythrocytes. However, the control of erythropoiesis depends not only on EPO but also on the availability of plasma iron. Therefore, the expression of genes involved in erythro-poiesis and iron availability need to be coordinately regulated in response to hypoxia. Hypoxia Inducible Factors, HIF-1 and HIF-2, are iron- and hypoxia-regulated heterodimeric transcription factors, and the central regulators of mammalian oxygen homeostasis. We hypothesized that the HIF factors could play a role in the regulation of iron metabolism in vivo. Hepcidin, a liver expressed hormone, is the key iron hormone regulating iron absorption by the intestine and iron recycling by the mac-rophages of the spleen, by binding and degrading ferroportin (FPN), the only known iron exporter gene. Hepcidin expression is decreased in response to hypoxia and iron deficiency. We generated intestinal-specific and liver-specific HIF knockout mouse models in order to investigate in physiopathological conditions the links between HIF signaling and i) the regulation of hepatic hepcidin expression, and ii) the regulation of intes-tinal iron absorption. We showed that hepatic HIF-2 repress hepcidin expression, indirectly through epo-mediated increase in erythropoiesis. In the intestine, we found that HIF-2, but not HIF-1, controls iron absorption by regulating the expression of DMT1 (Divalent Metal Transporter-1) and DCYTB (Duodenal cytochrome b) proteins which import iron in the enterocytes. Finally, we demonstrated that HIF-2α is involved in the regulation of iron hyper-absorption in a genetic mouse model of here-ditary hemochromatosis (HH). HH is a genetic disorder characterized by abnormally low hepcidin expression and excessive iron accumulation in the liver and parenchyma. These latest findings suggest a prominent role of HIF-2 in the physiopatho-logical regulation of intestinal iron absorption and may provide new therapeutical perspectives for the treatment of anemias and iron overload-associated disorders.

Hypoxia-inducible factor regulation of hepcidin requires EPO

Qingdu Liu and Volker H. Haase

Departments of Medicine, Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA

The increased production of red blood cells (RBC) and thus increased O2-carrying capacity of blood represents a major adaptation to systemic hypoxia. This important physiologic response consists of cell type-specific changes that include increased erythropoietin (EPO) production in kidney and liver and enhanced iron uptake and utilization. Hepcidin, a small polypetide produced by hepatocytes, plays a central role in regulating iron uptake and utilization. It promotes internalization and degradation of ferroportin, the only known cellular iron exporting channel. Hypoxia suppresses hepcidin, thereby en-hancing intestinal iron uptake and release from internal stores. Experimental studies in cell culture and in animals, as well as clinical data from patients with Chuvash polycythemia, who are homozygous for the von Hippel-Lindau (VHL) R200W mutation, demonstrated that regulation of hepcidin synthesis involves the VHL/hypoxia-inducible factor (HIF) pathway. While HIF, a central mediator of cellular adaptation to hypoxia, directly regulates renal and hepatic EPO synthesis under hypoxia, the molecular basis of hypoxia/HIF-mediated hepcidin suppression in the liver remains unclear. To specifically dissect the role of the VHL/HIF axis and EPO in the hypoxic suppression of hepcidin in vivo, we have used a genetic approach to disengage Hif activation from Epo synthesis in mice. We utilized tamoxifen-inducible Cre/loxP-mediated recombination to activate Hif-1 and Hif-2 via ablation of Vhl while simultaneously inactivating Epo. We found that the hypo-xia/Hif-mediated suppression of hepcidin required Epo inducibility. Furthermore, we show that HIF-mediated hepcidin sup-pression requires erythropoietic activity irrespective of serum EPO levels. Taken together, we provide genetic evidence that hepcidin regulation by the HIF pathway occurs indirectly through the stimulation of EPO-induced erythropoiesis.


Regulation of iron homeostasis in mice overexpressing human erythropoietin

Elena Gammella # 1, Víctor Díaz # 2, Stefania Recalcati 1, Paolo Santambrogio 3, Arianne Monge Naldi 2, 4, Johannes Vogel 2, Max Gassmann 2, Gaetano Cairo 1

# These authors contributed equally to the work1 Department of Biomedical Science for Health, University of Milan, Italy; 2 Institute of Veterinary Physiology, Vetsuisse Faculty, Zurich Center for Integrative Human Physiology (ZIHP) and University of Zurich, Switzerland; 3 Proteomics of Iron

Metabolism Unit, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy; 4 Clinic for Otolaryngology, Head and Neck Surgery, University Hospital Zurich, Switzerland

Body iron balance is maintained through the coordination between the erythroid regulator (erythroid precursors are the major sites of iron utilization) and the store regulator (iron accumulation, primarily in liver and reticuloendothelial system). Systemic iron homeostasis is maintained through the hepcidin-ferroportin axis. Hepcidin (Hpc), a liver-derived peptide, negatively regu-lates the iron exporter ferroportin (Fpn) to reduce iron absorption and mobilization, balancing iron demands. Hpc expression, which is stimulated by iron and inflammation, is repressed in response to hypoxia, anemia, and erythropoiesis, in order to increase iron availability for the bone marrow. However, the signal(s) at the basis of this response are not fully understood. To elucidate the control of Hpc expression and body iron balance under conditions of stress erythropoiesis, we investigated iron homeostasis in wild type (Wt) animals and in a transgenic mouse line (Tg6) chronically overexpressing human eryth-ropoietin (Epo) (12-fold compared to Wt) and presenting a hematocrit ~80%. In addition, Wt (Wt_DXT) and Tg6 (Tg6_DXT) animals were treated with iron dextran, and Tg6 mice were splenectomized to reduce erythropoiesis (Tg6_SPL). Hpc mRNA levels were strongly repressed in Tg6. Iron treatment, which increased Hpc expression in Wt_DXT mice, also enhanced Hpc in Tg6_DXT animals to levels comparable to Wt mice. Moreover, Hpc expression was elevated in Tg6_SPL compared to Tg6. The variations in Hpc expression were paralleled by changes in the expression of BMP6, an iron-regulated inducer of Hpc transcription, and the iron storage protein ferritin. As opposed to animal models of β-thalassemia in which the erythroid regulator predominates over the storage regulator to set Hpc levels, these data suggest that body iron overcomes the Epo signal in Hpc regulation and thus is the primary signal affecting iron homeostasis during chronically elevated erythropoiesis. Evaluation of duodenal iron uptake and analysis of proteins involved in intestinal iron absorption, as well as iron deposition, acquisition and export from storage sites (liver, spleen, muscle) indicated that changes in duodenal Fpn, together with iron mobilization, were the major variations in iron homeostasis under these conditions.

Vitamin C is not required for oxygen sensing in vivo

Katarzyna J. Nytko, Nobuyo Maeda, Philipp Schläfli, Patrick Spielmann, Roland H. Wenger and Daniel P. Stiehl

Institute of Physiology, University of Zürich, SwitzerlandDepartment of Pathology and Laboratory of Medicine, University of North Carolina at Chapel Hill, USA

Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as co-substrates with iron(II) and vitamin C serving as co-factors. While vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. We found that vitamin C deprived Gulo-/- knock-out mice show normal HIF-dependent gene expression. The systemic re-sponse of Gulo-/- animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to animals sup-plemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all three PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, tran-sactivation activity and endogenous target gene expression in cells exposed to CoCl2. A C201S mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.


Critical importance of the region C terminal to the hydroxyacceptor proline for the regulation of HIF-2alpha

MJ Percy¹ , PW Furlow ², YJ Chung ², TRJ Lappin³, MF McMullin¹³ and FS Lee ²

¹ Haematology, Belfast City Hospital, Belfast, UK; ²Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, USA; ³Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, UK.

The oxygen sensing pathway regulates the synthesis of erythropoietin (EPO) in response to hypoxia at the level of transcripti-on by the hypoxia inducible factor (HIF). The HIF transcription complex is a heterodimer composed of one alpha and one beta subunit. Although both subunits are constitutively expressed the alpha subunit, of which there are 3 isoforms, is degraded in the presence of oxygen by the proteasome. HIF-alpha is tagged with ubiquitin by the von Hippel Lindau (VHL) protein in response to prolyl hydroxylation. The proline (Pro531 in the case of HIF-2alpha) present in the LXXLAP motif in the oxygen dependent degradation (ODD) domain of HIF-alpha is modified by the PHD family of enzymes. There are 3 family members and they require both oxygen and iron to function. Dysregulation of EPO production can be an underlying cause of red cell hyperplasia, known as erythrocytosis. Defects in the key players of the oxygen sensing pathway have been identified in ery-throcytosis patients and consequently the axis controlling EPO production has been defined as PHD2-HIF-2alpha-VHL. We have described a series of HIF-2alpha mutations located C-terminal to the LXXLAP motif in the ODD domain. The residues N-terminal to the hydroxylacceptor proline (Pro531) are highly conserved compared to those C-terminal to Pro531, but in-terestingly mutations have not been identified in the former region. The presence of erythrocytosis mutations from residues Pro534 to Phe540 highlights the importance of this region for association with VHL and PHD2. All mutations studied thus far led to decreased degradation and increased stabilization of HIF-2alpha. Furthermore, increased expression of HIF-2alpha target genes was observed, thus confirming all mutations resulted in a gain of function. However, not all mutations demons-trated the same affinity towards PHD2 and VHL. One mutation, M535V, retained the ability to bind VHL similar to wild type, but was weakened in its ability to bind PHD2. This suggests that impairment of PHD2’s ability to hydroxylate HIF-2alpha is sufficient to cause an erythrocytosis phenotype. This is consistent with heterozygous PHD2 mutations causing erythrocytosis. Characterisation of HIF-2alpha confirmed the critical importance of the Pro531C-terminal region for the regulation of HIF-2alpha.

A physiological study of Tibetan natives at sea level

Petousi N, Croft QC, Cheng H-Y, Formenti F, Ishida K, Talbot NP, Ratcliffe PJ and Robbins PA

Department of Physiology, Anatomy and Genetics, University of Oxford

The Hypoxia-Inducible Factor (HIF) pathway plays a key role in the regulation of erythropoiesis and generally coordinates human responses to hypoxia at both the cellular and integrative-physiology levels. Humans with genetic disorders of the HIF pathway (e.g. mutations in VHL, HIF2A and PHD2) have polycythaemia and abnormal cardiopulmonary physiology – exag-gerated ventilation and pulmonary vascular response to hypoxia. Recent genomic studies comparing Tibetan highlanders with Han Chinese suggest natural selection on EPAS1 (HIF2A) and PHD2 genes in Tibetans, associated with a phenotype of low haemoglobin at high altitude.In this study we characterise the physiological responses to hypoxia of Tibetan volunteers at sea level. Volunteers are ex-posed to acute (10 min) and sustained (8 hours) of isocapnic hypoxia. Ventilation, pulmonary vascular and cardiac output responses and plasma erythropoietin are measured in response to hypoxia. Results suggest that, compared to Han Chinese, Tibetan volunteers at sea level: have lower haemoglobin (and haematocrit) and higher ventilation at rest; exhibit similar ven-tilatory, cardiac output and erythropoietic responses to hypoxia but have a significantly blunted pulmonary vascular response to hypoxia. There is also evidence that within Tibetans there is variation in the plasma erythropoietin rise in response to hypoxia that might be related to genetic variation in HIF2A and PHD2.


The Erythropoietin (Epo) Gene Polymorphism (rs16171640) in Preterm Infants with Retinopathy

Christof Dame, Carolin Rau, Anne Thorwarth, Petra Koehne, Martin Klar, Christoph Bührer

Department of Neonatology, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany.

Introduction: The homozygous polymorphism (rs16171640; nt-1125G>T) in the 5’ Epo promoter introduces a novel binding motif for different transcription factors, resulting in increased Epo promoter activity. In adults with type II diabetes, who exhibit the homozygous rs16171640 polymorphism, significantly higher intravitreal Epo concentrations and significantly increased incidence of proliferative retinopathy have been reported. Our study aimed to evaluate whether the rs16171640 Epo polymor-phism is also associated with severe retinopathy (ROP ≥3°) in very low birth weight (VLBW) infants. Such association would be important for decision making on the use of recombinant Epo (rEpo) for treatment of the anemia of prematurity, because rEpo has been accused to aggravate ROP in VLBW infants.Methods: Sequencing of the Epo promoter in genomic DNA of 72 VLBW infants with ROP ≥3° and in 70 case-controls with ROP 0/I°. Cohort analysis, considering rEpo treatment (750 U/kg/week iv/sc, initiation on day 7 after birth). Results: The frequency of the homozygous (TT) rs16171640 Epo polymorphism did not significantly differ between VLBW infants with ROP≥3° (39.2%) and ROP 0/I° (35.7%; p=0.86). In VLBW infants with ROP ≥3° and rEpo treatment the frequency of the TT risk genotype was only slightly, but not significantly higher compared to rEpo treated case-controls with ROP 0/I° (45.7% vs 35.1%; p=0.4). Independently from the presence of the rs16171640 Epo polymorphism, the incidence of ROP ≥3° was associated with an earlier initial red blood cell transfusion (p=0.004) as well as a higher number and total volume of red blood cell transfusions than in case-controls (both p<0.001). The cumulative rEpo dose, however, was not associated with the severity of ROP (p=0.5). Discussion: In VLBW infants the homozygous rs16171640 Epo polymorphism is not associated with a higher frequency of ROP≥3°. Considering experimental, cell-protective effects of rEpo on retinal cells and the negative association of red blood cells transfusions with severe ROP in VLBW infants justifies to (re-)evaluate optimized strategies for rEpo treatment in infants with anemia of prematurity.

EFFECT OF CHRONIC KIDNEY DISEASE (CKD) AND HEMODIALYSIS ON PROTEIN AND AMINO ACID METABOLISM

Giacomo Garibotto, Alice Bonanni, Daniela Verzola

Division of Nephrology, Dialysis and Transplatation, University of Genoa and IRCCS San Martino, Genoa, Italy

Despite current innovations in CKD therapy, mortality is still high in patients with end-stage renal disease (ESRD). This in-crease in mortality is not only limited to dialysis patients, but includes all stages of CKD and is mainly due to cardiovascular disease. Protein-energy wasting becomes clinically manifest at an advanced CKD stage, early before or during the dialytic stage and increases the morbidity and mortality in this patient’s population. The purpose of this presentation is to review re-cent observations on alterations of amino acid and protein metabolism which cause wasting and increase cardiovascular risk.

Recent studies have consistently increased our understanding, and new mechanisms have been proposed, to explain was-ting and vascular disease in CKD patients. These include anorexia and endocrine dysfunction, altered muscle intracellular signaling through the insulin receptor substrate/phosphatidylinositol 3-kinase/Akt pathway, myostatin uptrgulation and defec-tive miocyte regeneration. These factors may trigger wasting through an increase in protein degradation and/or acceleration of apoptotic processes in skeletal muscle. These mechanisms may be accelerated by hemodialysis, leading to progression of vascular disease and wasting.This new understanding hold promise for new treatments which can prevent/treat vascular diseases and wasting in CKD patients.


Pre-vertebrate evolution of erythropoietin signalling serving neuroprotection and neuroregeneration

Ralf Heinrich

Department of Cellular Neurobiology, Georg-August-University Göttingen, Germany

Erythropoietin (EPO) initiates adaptive cellular responses to both moderate environmental challenges and tissue damaging insults in various non-hematopoietic mammalian tissues. Homologs of the human EPO gene have been identified in various species including other mammals, amphibia and fish suggesting that EPO signalling is common to all vertebrates. Our studies on insects revealed similar neuroprotective and regenerative functions of EPO as described by numerous studies on mammalian nervous tissues. Recombinant human EPO (rhEPO) increased the survival of primary cultured grasshopper brain neurons in a concentration dependend manner, accelerated neurite regeneration of these neurons and increased neuronal survival under hypoxia. EPO's protective effects under hypoxic conditions were abolished by the janus kinase inhi-bitor AG490, suggesting that the initiation of intracellular signalling cascades is similar in insects and mammals. In addition to these in vitro effects, rhEPO promoted the regeneration of grasshopper auditory receptor axons after tympanal nerve crush, leading to a faster and more complete regeneration of sound localization. Immunoblots of central nervous tissues from mouse, grasshopper, crayfish and leech labeled protein bands of ≈38 kDa, fitting to the molecular weight of EPO reported in earlier studies.Our results indicate that a ligand/receptor system with high structural and functional similarity to the mammalian EPO/EPO receptor system mediates neuroprotective and neuroregenerative effects in insects and probably also in other invertebrates. EPO-like signalling involved in tissue protection appears to be an ancient beneficial function shared by vertebrates and in-vertebrates.

Common variants of the genes encoding erythropoietin and its receptor modulate cognitive performance

Anne Kästner, Sabrina Grube, Ahmed El-Kordi, Beata Stepniak, Heidi Friedrichs, Derya Sargin, Judith Schwitulla, Martin Begemann, Ina Giegling, Kamilla W. Miskowiak, Swetlana Sperling, Kathrin Hannke, Anna Ramin, Ralf Heinrich,

Olaf Gefeller, Klaus-Armin Nave, Dan Rujescu, and Hannelore EhrenreichDivision of Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany, and

DFG Research Center for Molecular Physiology of the Brain (CMPB), Göttingen, Germany

Erythropoietin (EPO) improves cognitive performance in clinical studies and rodent experiments. We hypothesized that an intrinsic role of EPO for cognition exists, with particular relevance in situations of cognitive decline, which is reflected by associations of EPO and EPO receptor (EPOR) genotypes with cognitive functions. To prove this hypothesis, schizophrenic patients (N>1000) were genotyped for 5'upstream-located gene variants, EPO SNP rs1617640 (T/G) and EPOR STR(GA)n. Associations of these variants were obtained for cognitive processing speed, fine motor skills and short-term memory rea-douts, with one particular combination of genotypes superior to all others (p<.0001). In an independent healthy control sam-ple (N>800), these associations were confirmed. A matching preclinical study with mice demonstrated cognitive processing speed and memory enhanced upon transgenic expression of constitutively active EPOR in pyramidal neurons of cortex and hippocampus. We thus predicted that the human genotypes, associated with better cognition, would reflect gain-of-function effects. Indeed, reporter gene assays and quantitative transcriptional analysis of peripheral blood mononuclear cells showed genotype-dependent EPO/EPOR expression differences. Together, these findings reveal a role of endogenous EPO/EPOR for cognition.


Erythropoietin induced neural differentiation in stem cells

Sirén, Anna-Leena1, Offen, Nils1, Dahme, Liane2, Flemming, Johannes1, Kamawal, Hares1, Hagemeyer, Nora2, Sperling, Swetlana2, Ahmad, Ruhel3, Geis, Christian4, Müller, Albrecht3, Ehrenreich H2

1Department of Neurosurgery, University of Würzburg, Würzburg, 2Max-Planck Institute for Experimental Medicine, Göttin-gen, 3Center for Experimental Molecular Medicine (ZEMM), University of Würzburg, 4 Department of Neurology, University of Würzburg, Würzburg, 5Department Cell and Developmental Biology , Max-Planck Institute for Molecular Biomedicine,

Münster, Germany

Neural stem cells express erythropoietin (EPO) receptors and respond to exogenous EPO treatment with increased survival and differentiation but the mechanisms of these events remain elusive. Using stem cells derived from mouse embryonal forebrain or from mouse induced pluripotent stem (iPS) cells, we examined the cellular and molecular mechanisms of EPO induced neural differentiation. Analysis of E14 forebrain neurosphere cultures exposed to EPO showed significant shifts in the expression of several differentiation markers such as Sox9, ND1 or in the Dcx/MAP2 ratio; together with a decrease in sphere growth and proliferation. Similarly, proliferation of iPS-derived pan neural progenitor cells (pNPCs) was inhibited while their survival and efficacy of differentiation into a neuronal phenotype were improved. The effects of EPO on stem cell proliferation and differentiation could be in part abrogated by inhibition of neuronal microRNAs.

Our results show that EPO improves survival and accelerates neuronal differentiation from iPS cell-derived neural progenitor cells and in brain neural stem cells.

Neurotrophic and neuroprotective properties of non-hematopoietic EPO-derived peptides

Stanislava Pankratova, Darya Kiryushko, Katrin Sonn, Vladislav Soroka, Lene B. Køhler, Mette Rathje, Irina Korshunova, Bing Gu, Kamil Gotfryd, Ole Clausen, Alexander Zharkovsky, Elisabeth Bock and Vladimir Berezin

Protein Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen

Apart from its hematopoietic activity, erythropoietin (EPO) is also known as a tissue protective cytokine. In the brain, EPO and its receptor (EPOR) are upregulated in response to insult and exert pro-survival effects. EPO binds to EPOR via high- and low-affinity binding sites (Sites 1 and 2, respectively), inducing conformational changes in the receptor, followed by the activation of downstream signalling cascades. Based on the crystal structure of the EPO:EPOR2 complex, we designed two peptides, termed Epobis and Epotris, whose sequences encompassed amino acids of binding Site 1 and Site 2, respectively. The present study shows that both peptides specifically bind to EPOR and induce neurite outgrowth from primary neurons in an EPOR-expression dependent manner. Epobis and Epotris promoted the survival of cerebellar and hippocampal neuro-nal cultures. We also demonstrated that systemically administered Epotris penetrated the blood-brain barrier and had both anti-epileptic and neuroprotective effects in an animal model of kainic acid-induced neurotoxicity. Neuroprotective doses of Epotris and Epobis do not stimulate hematopoiesis in vivo which makes these peptides attractive drug candidates for the treatment of neurodegenerative disorders.


Metabolic activity associated with erythropoietin

Constance Tom Noguchi, Ruifeng Teng and Mawadda AlNaeeli

Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA 20892

Erythropoietin (EPO) receptor (EpoR) expression extends beyond erythroid progenitor cells and is found in other select cell/tissue types including brain during development and adulthood. Selective rescue of EpoR in erythroid tissue results in viable mice with no abnormal organogenesis or gross morphological defects. Unexpectedly, these mice exhibit glucose intolerance, become obese and insulin resistance. In white adipose tissue of these mice, adipocyte cell number is markedly increased and cell size is disproportionately decreased. Conversely, diet induced obese mice and ob/ob mice treated with high dose EPO exhibit improved glucose tolerance, decreased serum insulin level and decreased body fat accumulation. EPO treatment in mice with EpoR restricted to erythroid tissue show the expected increase in hematocrit, but no change in fat mass compared with saline treatment. A tissue survey of EpoR expression reveals high EpoR in white fat of wild type mice. EPO treatment of preadipocytes decreases activity of PPARγ, a transcription factor required for adipogenesis, via increased PPARγ phospho-rylation and decreases adipocyte differentiation, consistent with decreased PPARγ phosphorylation and increased adipocyte number in white fat with loss of EpoR. Interestingly, high dose EPO treatment in wild type mice decreases food intake and increases energy expenditure while mice with EpoR restricted to erythroid tissue exhibit decreased energy expenditure but no change in food intake. In addition to high EpoR expression in white adipose tissue we also observe high EpoR expressi-on in the hypothalamus where EpoR specifically localized to proopiomelanocortin neurons. Epo treatment in wild-type mice induces the expression of the polypeptide hormone precursor gene, proopiomelanocortin in the hypothalamus. Furthermore, primary neural cell cultures from the hypothalamus show that EPO treatment preferentially stimulates proopiomelanocortin expression compared with other hypothalamic neuropeptides. These data suggest that in addition to systemic effects of EPO relating to erythrocyte production, EPO can affect adipocyte differentiation, fat mass accumulation and directly regulate hy-pothalamic neuropeptide expression to modify central regulation of energy homeostasis and food intake.

The antioxidant effect of erythropoietin on thalassemic blood cells

Eitan Fibach and Eliezer A. Rachmilewitz

Dept. of Hematology, Hadassah - Hebrew University Medical Center, Jerusalem, IsraelDept. of Hematology, E. Wolfson Medical Center, Holon, Israel

Erythropoietin (Epo) has a stimulating effect on RBC production and is commonly used for treatment of anemia in a variety of disorders, e.g., in patients on dialysis and following chemo-therapy. In β-thalassemia, where Epo levels are low relative to the degree of anemia, several studies demonstrated improvement of the anemia following treatment with Epo. The RBC and platelets in thalassemia are under oxidative stress, which result in accelerated senescence and clearance of RBC and activation of platelets, leading to anemia and thromboembolic complications.We investigated Epo as an antioxidant. Using flow-cytometry technology, we found that in-vitro treatment with Epo of blood cells from these patients increased their glutathione content and reduced their reactive oxygen species, membrane lipid per-oxides and phosphatidylserine exposure which consequently resulted in reduced susceptibility of the RBC to hemolysis and erythrophagocytosis. Injection of Epo to heterozygous (Hbbth3/+) β-thalassemic mice reduced the oxidative markers within 3 hrs. Our results suggest that, in addition to stimulating RBC production and fetal hemoglobin synthesis, Epo has features of an antioxidant which may alleviate the severity of thalassemia.


Myocardial Na,K-ATPase and its role in Epo-induced cardioprotection

Yakushev S.¹, Mihov D.², Gassmann M.¹, Bogdanova A.¹

¹ University of Zurich, Institute of Veterinary Physiology, ² Biozentrum, University of Basel

Our studies revealed that suppression of oxidative stress and oedema progression during the first minutes of reperfusion were decisive in acute cardioprotective action of Epo in the post-ischemic heart. These cardioprotective effects were associ-ated with induction of IP3K-Akt signalling pathway and stimulation of NO production. Furthermore, we have recently shown that acute reversible alterations in activity of the Na,K-ATPase (NAK) in response to the changes in oxygenation are caused by a switch between S-nitrosylation and S-glutathionylation of cysteine residues within the ATP binding site. This study was designed to assess the action of Epo on the function of NAK in post-ischemic heart and cardioprotective effect of the cytoki-ne. Changes in redox state and NO production during the first 10 min of reperfusion following 40 min of cold global ischemia were monitored in ex vivo blood-perfused rat heart in the presence or absence of 130U/ml Epo and/or 3mM L-NAME added at the onset of reperfusion. Thiol modifications in the α-subunit of the NAK were detected and its activity was measured in tissue homogenate. The obtained data indicate that Epo application causes an increase in NO production and therefore shifts thiol redox state of the α-subunit of NAK towards S-nitrosylated form. In untreated hearts ischemia-reperfusion is associated with oxidative stress and S-glutathionylation of the α-subunit. The NAK activity is thereby suppressed and oedema persists due to the accumulation of Na+ in ischemic and even more so in reperfused heart tissue. As a consequence, microcirculation of ventricular capillaries is compromised and restoration of transmembrane ion gradients delayed. Inhibition of NO production by L-NAME abolishes antioxidative effect of Epo and Epo-induced facilitation of capillary perfusion in post-ischemic heart. The obtained results highlight NAK as an important target in Epo-induced cardioprotection. Recent observations suggest that reversible thiol modifications coordinate activity of several ion transporters (NAK, SERCA, RyR, Na/Ca exchanger). Re-versible thiol modifications may therefore represent a universal regulatory mechanism tuning the function of ion transporters involved in control of heart contractility in accordance with oxygen availability. Epo-stimulated NO production may therefore have an effect on numerous targets altering heart function and facilitating post-ischemic recovery.

Erythropoietin (Epo) exerts an anti-neoplastic activity: characterizing Epo-driven responses in the 5T33 multiple myeloma mouse model.

Naamit Deshet-Unger*, Yasmin Ohana*, Sahar Hiram-Bab*, Max Gassmann#, Moshe Mittelman§, Miriam Souroujon† and Drorit Neumann*

*Department of Cell and Developmental Biology, §Department of Medicine, Tel Aviv Sourasky Medical Center, Tel Aviv, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel, †Department of Natural Sciences, The Open

University of Israel, Raanana 43107, Israel, #University of Zurich, Institute of Veterinary Physiology, Zurich, Switzerland

Erythropoietin (Epo), mainly produced by the adult kidney, is the major hormone that promotes erythropoiesis. As such, intro-duction of recombinant human Epo (rHuEpo) and its derivatives (Epoetins), has been a breakthrough in treating patients with anemia, mainly chronic kidney failure patients and cancer patients on chemotherapy. In contrast to 'red flags' which point to possible detrimental effects of Epo in certain cases of cancer, the hormone was found by numerous studies to have bene-ficial neuroprotective, cardio-protective and immune-mediated anti-cancer effects as exemplified in multiple myeloma (MM) patients and mouse models. These effects, at least in part, are mediated by the Epo receptors (EpoRs) present on cells other than the erythroid lineage. We have recently identified functional EpoRs in bone marrow-derived macrophages isolated from C57BL/6 mice. Moreover, we have demonstrated an increase in the levels of splenic macrophages in mice that were treated with Epo as well as in transgenic mice constitutively overexpressing human Epo (tg6 mice). Our recent data reflect the effect of Epo administration on the immune system of mice inoculated with 5T33 cells. MM is characterized by clonal proliferation of malignant plasma cells that produce a pathological paraprotein. Western blot analysis show that Epo treatment was associa-ted with a 50% decrease in the levels of the pathological κ light chain, 28 days after the injection of 5T33 MM cells. Moreover, FACS analysis demonstrated that Epo-treated 5T33 MM mice show a tenfold increase in the levels of splenic macrophages (F4/80+,CD11b+) and a two fold increase in natural killer cells in blood (NK1.1,+CD3-). These findings were associated with an increase in the transcript levels of the TH1 cytokine IFN-γ in the bone marrow of Epo-treated 5T33 MM mice as compared to controls. As MM is typically associated with a TH2 response, Epo may have a beneficial effect in MM, possibly by shifting the balance towards TH1 responses.Taken together, our data demonstrate that Epo’s impact extends beyond erythropoiesis. Moreover, our data are in favor of the concept that the usage of rHuEpo may be considered as an adjunct treatment of pati-ents in whom enhancement of the immune system is warranted. Supported by the Multiple Myeloma Research Foundation (DN). DN and MG are members of the HypoxiaNet COST ActionTD0901.


Iron dynamics and erythroblast maturation kinetics after C.E.R.A. treatment

Yusuke Sasaki, Mariko Noguchi-Sasaki, Yuki Matsumoto-Omori, Keigo Yorozu, and Yasushi Shimonaka

Product Research Dept., Chugai Pharmaceutical Co., Ltd.

Aims: We evaluated the mechanism of iron mobilization by erythropoiesis stimulating agents (ESAs) and the effects of continuous erythropoietin receptor activator (C.E.R.A.), a methoxy polyethylene glycol-epoetin beta, on iron dynamics and erythroblast maturation kinetics. Methods: C57BL/6N (B6) mice with total body irradiation (TBI) followed by bone marrow transplantation (BMT) were treated with epoetin beta (EPO) or vehicle intravenously. EPO or carbamylated EPO (C-EPO) was intravenously administrated into B6 mice. Hematological and iron parameters including serum hepcidin were analyzed in these mice. B6 mice were treated with C.E.R.A. or vehicle intravenously. We analyzed hematological and iron parameters, as well as the maturation status of bone marrow erythroblast by flow cytometry stained with TER119 and CD71. Results: Alt-hough TBI abolished the reduction of serum hepcidin by EPO injection which was observed in control mice, BMT recovered the reduction of serum hepcidin by EPO. C-EPO, which preserves in vitro direct and slight inhibitory effect on hepatic hep-cidin production but no erythropoietic activity, did not reduce serum hepcidin in B6 mice. C.E.R.A.-treated mice showed sig-nificantly higher hemoglobin levels than in vehicle-treated mice for 14 days after treatment, while serum hepcidin was conti-nuously suppressed in C.E.R.A.-treated mice and nevertheless serum iron was markedly decreased at day 5. Rapid increase followed by gradual decrease of TER119(+)CD71(high) immature erythroblast and gradual increase of TER119(+)CD71(low) mature erythroblast were observed in C.E.R.A.-treated mice. Conclusions: These results indicate that erythropoietic activity of ESA is essential for iron mobilization through hepcidin down-regulation. C.E.R.A. stimulates effective erythropoiesis and causes marked iron consumption and hepcidin down-regulation, leads to effective iron recruitment for erythroblasts. The gra-dual maturation of erythroblast after C.E.R.A. treatment suggests the process is controlled by iron supplementation. These unique features of C.E.R.A. might contribute to prevent rapid increase of hemoglobin after C.E.R.A. treatment.

First results on regenerative effects of erythropoietin in burn and scald injures (clinical trial: “EPO in Burns”)

Günter CI (1), Dornseifer U, Siemers F, Mailänder P, Ninkovic M, Thamm O Spilker G, Wolter T, Dunda S, Grieb G, Pallua N, Ernert C, Steen M, Kestel A, Sievers R, Fuge D, Reichert B, Hartmann B, Rahmanian-Schwarz A, Schaller HE, Daigeler A,

Otte M, Menke H, Ryu SM, Pierson, Bader A, Ebert S, Jelkmann W, Raem AM, Ohmann C, Kehl V, Machens HG

(1) Klinik für Plastische und Handchirurgie, Klinikum Rechts der Isar, Technische Universität München, Ismaninger Str. 22, 81675 München, [email protected]; in charge of the "EPO in Burns" Study Group

Introduction: Large 3° and deep 2° thermal injuries are life threatening and wound surfaces need quick coverage by split skin grafts following tangential or epifascial necrectomy. Initially, it was assumed that EPO was only a hormone affecting erythro-poiesis, it has now been demonstrated, that EPO plays a key role in its reaction to acute and chronic tissue damage. Further-more, EPO has a significant anti-inflammatory effect, thus EPO enhances healing and „restitutio ad integrum“ after trauma. To address the clinical need for regenerative tools after thermal injury we stared to examine the effects of systemic EPO application in severely burned patients. Methods:This trial is a randomized, controlled, double-blinded, national multicenter trial. Patients of both sexes, aged 18 – 75 years can be included. Primer efficiency point is the complete reepithelialisation of a defined split skin graft donor area. We will examine cellular and molecular regenerative effects, stem cell recruitment, DNA and mRNA expression, [EPO] receptor up-regulation, protein expression, quality of scar formation, quality of life, gender differences, and organ dysfunction parameters, number of transfused packed red cells units, adverse events and serious adverse events.Preliminary results: The trial is still in its recruitment phase. Total number of includes patients is 105. So far only 15% of the patients developed SAE`s, which is a low number regarding the severity of the injuries (up to ABSI 12). During the pre-clinical phase of the trial we examined cell cultures and animal models, these showed: After thermal trauma, mice receiving systemic or local EPO, exhibited clearly improved and faster reepithelialization and faster wound healing as compared to control groups. In culture, under hypoxic conditions the addition of IL-6 decreases cell proliferation, if EPO is adjoined the proliferation increases significantly. Conclusion: The trial is still blinded; definite results can only be presented after unblinding. The results from the cell cultures and animal models and the low number of SAE`s in the trial leads to the assumption, that EPO might represent a new, effec-tive therapeutic opportunity in the treatment of dermal thermal injuries.


EPOR deficiency on glioma cells induces sensitivity to ionizing radiation and temozolomide

Elodie Pérès¹ ² ³, Samuel Valable¹ ² ³, Armelle Calipel¹ ² ³, Aurélien Corroyer-Dumont¹ ² ³, Lucile Durand¹ ² ³,Emmanuèle Lechapt-Zalcman¹ ² ³4, Myriam Bernaudin¹ ² ³, Edwige Petit¹ ² ³

¹ CNRS, UMR 6301 ISTCT, CERVOxy group. GIP CYCERON, Bd Henri Becquerel, BP5229, F-14074 CAEN cedex, France. ² Université de Caen Basse-Normandie, UMR 6301 ISTCT, CERVOxy group. GIP CYCERON, Bd Henri Becquerel, BP5229, F-14074 CAEN cedex, France. ³CEA, DSV/I2BM, UMR 6301 ISTCT, CERVOxy group. GIP CYCERON, Bd Henri Becquerel,

BP5229, F-14074 CAEN cedex, France. 4CHU de Caen, anatomopathology unit, Caen, F-14000, France

The standard of care for glioblastoma (GBM) patients consists primarily of surgical resection followed by radiotherapy and concomitant temozolomide (TMZ) which provides a modest improvement in survival compared to radiation alone. One pro-gnostic factor identified as a reliable biomarker for GBM sensitivity to TMZ is the methylation status of O6-methylguanine-methyl-transferase (MGMT). On the other hand, recombinant EPO has been, until recently, widely used in clinic treatment of anemia associated with cancer, with a potential effect on the increase in tumor oxygenation and thereby an improvement of radiosensitivity. Recently, we and other demonstrated that glioma cells express EPOR and that targeting EPOR on glioma cells reduces tumor growth. Accordingly, the purpose of this study was to evaluate whether silencing EPOR on glioma cells might contribute to modulate sensitivity of these tumor cells to either radiation or chemotherapy. We showed that silencing EPOR on U251 or U87 cells, two human glioma cell lines, led to a reduction in tumor growth both in vitro and in vivo which might be in part due to a G2/M arrest of these cells, an increase in apoptosis and/or senescence. Our results also demons-trated that the inhibition of EPOR of these cells (shEPOR-U87 or shEPOR-U251) increased their radiosensitivity to X-Rays and, more importantly, counteracted the hypoxia-induced radioresistance. On the other hand, when U87 or U251 cells, which exhibit a methylation of MGMT promoter, were exposed to TMZ, EPOR inhibition reinforced the efficacy of TMZ treatment in vitro. In vivo, we showed that animals which received an orthotopic implantation of shEPOR-U251 cells in the striatum, and treated with a TMZ regimen closed to that of glioma patients, displayed a reduction in tumor volume as compared to scrambled-U251 tumor-bearing animals (scrambled-U251 + TMZ group: 57%; shEPOR-U251 + TMZ group: 77%, p<0.01) accompanied with an increase in survival median of 34% versus scrambled-U251 animals treated with TMZ (p<0.005). In conclusion, our results suggest that EPOR on glioma cells might be one of the candidate targets for radiotherapy combined with TMZ for patients with TMZ-resistant GBM and/or radiotherapy-resistant GBM.

Pharmacokinetics, biodistribution, metabolism and excretion of peginesatide, an erythropoiesis-stimulating agent, in rats

Kathryn W. Woodburn¹, Christopher P. Holmes¹, Kei-Lai Fong², Yuu Moriya³, and Yoshihiko Tagawa³

¹Affymax Inc., Palo Alto, CA, USA, ²Accellient Partners LLC, Waltham, MA, USA, and ³Takeda Pharmaceutical Company Ltd, Osaka, Japan

The pharmacokinetic (PK), distribution, metabolism, and excretion profiles of peginesatide, a synthetic, PEGylated peptide-based erythropoiesis stimulating agent (ESA), were evaluated in rats. The PK profile was evaluated at 0.1–5 mg·kgˉ¹ IV using unlabeled or [14C]-labeled peginesatide. Mass balance, tissue dis-tribution andmetabolism were evaluated following IV administration of 5 mg·kgˉ¹ [14C]-peginesatide. Tissue distribution was also evalua-ted by quantitative whole-body autoradiography (QWBA).

Plasma clearance was slow and consistent with the erythropoietic response observed. Elimination of peginesatidefrom the plasma exhibited a biphasic pattern typical of PEGylated compounds characterized by a relatively rapid initial phase and a prolonged terminal phase. The PK parameters of unlabeled peginesatide determined by ELISA were similar to the kinetics derived via quantitative radiometric profiling. Unchanged peginesatide represented 90% of the total radioac-tive exposure. Slow uptake of the radiolabeled compound from the vascular compartment into the tissues was observed. Biodistribution to bone marrow and extramedullary hematopoietic sites, and to highly vascularized lymphatic and excretory tissues was observed. A predominant degradation event to occur in vivo was the loss of one PEG chain from the branched PEG moiety to generate mono-PEG. Renal excretion was the primary mechanism (41%) of elimination, with parent molecule (67%) the major moiety excreted. In conclusion, elimination of [14C]-peginesatide-derived radioactivity was extended, retention preferentially occurred at sites of erythropoiesis (bone marrow). Peginesatide’s pronounced persistence within bone marrow in rats may contribute to the extended pharmacologic action. Urinary excretion was a major elimination route with parent molecule representing the pri-mary moiety excreted.


Carbon dioxide (CO2) sensing at the axis of immunity and inflammation

Oliver KM1, Lenihan CR1, Bruning U1, Cheong A1, Laffey JG2, McLoughlin P1, Fitzpatrick SF1, Scholz CC1, Slattery C1, Leonard MO1, Taylor CT1 & Cummins EP1

University College Dublin1 National University of Ireland Galway 2

Molecular oxygen (O2) and carbon dioxide (CO2) are the primary substrate and product of aerobic metabolism, respectively. Thus, the levels of these gases in the cellular microenvironment can vary significantly under normal and pathophysiologi-cal conditions. The Hypoxia-Inducible Factor (HIF)-dependent transcriptional response to low oxygen levels has been well described, however the impact of CO2 on gene expression remains relatively unexplored. CO2 is increasingly being appre-ciated as an intracellular signaling molecule that affects inflammatory and immune responses through the regulation of gene expression. Elevated CO2 (hypercapnia) is encountered in a range of clinical conditions including chronic obstructive pulmo-nary disease (COPD) and cystic fibrosis (CF) where it is associated with susceptibility to infection and worse patient outcome. Interestingly, elevated CO2 as a consequence of therapeutic ventilation strategies to treat patients with acute respiratory distress syndrome (ARDS) has been associated with improved patient survival. The molecular mechanisms underpinning these clinical outcomes are not well understood. Thus we have used in-vitro and in-vivo approaches to examine the sensi-tivity of the key immune/ inflammatory pathway NF-κB to elevated CO2. We found that CO2 can influence NF-κB signaling on multiple levels. The non-canonical NF-κB family members IKKα and RelB reversibly translocated to the nucleus of cells in response to elevated CO2 in a manner which was independent of cellular O2 levels and the HIF system or changes in intracellular and extracellular pH. The changes in response to CO2 were associated with a suppression of NF-κB-dependent transcriptional activity and expression of key pro-inflammatory genes. Taken together, we provide novel molecular insight into the cellular response to CO2 that suggests the existence of a molecular CO2 sensor that can direct transcriptional events and influence immune signaling in a CO2-dependent manner.

Metabolic effects of acute and prolonged treatment with rHuEpo to healthy young men

Britt Christensen1,3, Birgitte Nellemann1,3, Mads Larsen2, Line Thams2, Peter Sieljacks2, Mikkel H. Vendelbo1,3, Thomas Krusenstjerna-Hafstrøm1, Michael Madsen1, Steen B. Pedersen1, Kristian Vissing2, Søren Nielsen1, Niels Jessen4,

Niels Møller3, Jens Otto Lunde Jørgensen11Department of Endocrinology and Internal Medicine, NBG/THG, Aarhus University Hospital, Aarhus, Denmark; 2Depart-ment of Sports Science, Aarhus Univeisity, Aarhus, Denmark; 3Medical Research Laboratories, Institute for Clinical Medici-

ne, Aarhus University Hospital, Aarhus, Denmark; 4Research Laboratory for Biochemical Pathology, Institute for Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark

Treatment with recombinant human erythropoietin (rHuEpo) improves insulin sensitivity in patients with end-stage renal disease, and animal studies indicate that Epo increases fat oxidation. However, the metabolic effects of rHuEpo have never been experimentally studied in healthy humans. The aim of these studies were to investigate the effects of 1) an acute rHuE-po bolus and 2) prolonged administration (10 weeks) of rHuEpo, on substrate metabolism and insulin sensitivity in health young men. In the acute study, ten healthy young men were studied in a single-blinded, randomized cross-over design with a 2-wk wash-out period receiving 400 IU/kg rHuEpo (Eprex) or placebo. In the prolonged study, 18 healthy young men were di-vided into two groups and received either placebo (n=9) or rHuEpo (n=9) (Aranesp) s.c. once weekly at a dose of 40 μg (week 0-3) / 20 μg (week 4-10). Substrate metabolism was evaluated by indirect calorimetry and insulin sensitivity by a hyperinsuli-nemic euglycemic clamp. Acute exposure to rHuEpo resulted in increased resting energy expenditure (REE) [Basal; 1863 ± 67 (kcal/day) (placebo) vs. 2042 ± 81 (rHuEpo) p<0.001, Clamp; 1904 ± 68 (placebo) vs. 2016 ± 114 (rHuEpo), p<0.05]. Fat oxidation in the basal state tended to be higher after rHuEpo, but insulin stimulated glucose disposal, glucose metabolism, and protein metabolism did not change significantly in response to acute rHuEpo administration. Prolonged administration of rHuEpo for 10 weeks also lead to a significant increase in REE [Week 0; 1713 ± 48 (kcal/day) (placebo) and 1762 ± 39 (rHuEpo), Week 10; 1700± 62 (placebo) and 1910 ± 59 (rHuEpo), p<0.05]. Fat oxidation and insulin sensitivity increased, but this did not reach statistical significance, otherwise, rHuEpo did not affect substrate metabolism.

In conclusion, a single injection and prolonged administration of rHuEpo both increases REE in healthy human subjects. This calorigenic effect is not accompanied by distinct alterations in the pattern of substrate metabolism or insulin sensitivity.


Cerebral Epo and excercise performance

Beat Schuler, Maria Alvarez Sanchez & Max Gassmann

Institute of Veterinary Physiology, Vetsuisse Faculty, and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Switzerland

To define as whether an optimal hematocrit in acutely and chronically Epo-treated mice exists, (i.e. a given hematocrit al-lowing the highest oxygen transport during maximal exercise), hematocrit levels of wild type (wt) mice were acutely elevated by injecting novel erythropoiesis stimulating protein (wtNESP). Moreover, in the transgenic mouse line termed Tg6 that reaches hematocrit levels of up to 90% due to hypoxia-independent expression of human Epo, we reduced the hematocrit gradually by injecting phenylhydrazine (tg6PHZ). Maximal cardiovascular performance was measured in exercising mice by telemetry. Highest maximal oxygen uptake (VO2max) and maximal time to exhaustion (TTE) at sub-maximal exercise in-tensities were reached at hematocrit values of 0.58 and 0.57 for wtNESP, and 0.68 and 0.66 for tg6PHZ, respectively. While maximal working capacity of the heart increased with elevated hematocrit values, blood viscosity correlated with VO2max. These observations suggest that increasing blood viscosity is the crucial factor limiting the blood’s oxygen transport capacity.In a next set of experiments we aimed to test as whether Epo present in the brain has an enhancing effect on exercise per-formance. To test this notion we used either another transgenic mouse line termed Tg21 that constitutively overexpresses human Epo solely in the brain, or wt mice treated with a single high dose of rhEpo (wt+rhEpo). As expected, cerebral Epo concentrations in Tg21 and wt+rhEpo animals were elevated compared to wt mice. Exercise performace tests showed that both Epo mouse models (e.g. Tg21 mice and wt+rhEpo 6h after injection of Epo i.p.) exhibit a significant improvement in maximal exercise performance (VO2max) and TTE that - importantly - was independent of changes in blood and cardiovas-cular parameters. We conclude that increased levels of cerebral Epo do improve exercise performance without altering either hematological or cardiovascular parameters.

Detection of EPO gene doping in blood

Elmo Neuberger, Dirk Moser and Perikles Simon

Department of Sports Medicine, Rehabilitation and Disease Prevention, Johannes Gutenberg-University Mainz, Mainz, Germany

Over a decade ago the fear emerged that somatic gene transfer technology could be misused by athletes or trainers with the intention to enhance athletic performance. Erythropoietin (EPO) is seen as one of the top candidates for prohibited gene transfer. Recently used vector systems, including viral or non-viral vector systems, which can be used for in vivo or ex vivo gene transfer, do not offer both, efficiency and safety, and EPO gene transfer did not reach clinical significance. Although EPO gene doping does not offer a beneficial alternative to conventional EPO doping or blood doping yet, the detectability is prerequisite to deter uncontrolled abuse of gene transfer technology for doping purposes. We and others suggested the detection of transgenic DNA (tDNA) out of whole blood samples. Based on the specific PCR amplification of intronless EPO DNA fragments, this approach enables the direct detection of EPO gene doping. Following in vivo gene transfer of recombinant adeno-associated virus vector systems in animal models, tDNA can be detected over an extended period of time in a reliable, specific and sensitive way. Next to the detection EPO tDNA, the PCR based detection approach has been established for the detection of a number of other potential gene doping candidates.


notes


Poster Abstracts


Epigenetic regulation of hypoxia-dependent erythropoietin gene expression

Norio Suzuki, Xiaoqing Pan, Yutaka Tojo, Takashi Dan, Toshio Miyata, Lorenz Poellinger, Masayuki Yamamoto

United Centers for Advanced Research and Translational Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan; Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden

Erythropoietin (Epo) production is strictly regulated at gene transcription level with a tissue (kidney and liver)-specific and hypoxia-dependent manner. We have identified cis-regulatory elements essential for the Epo gene expression by generation of GFP reporter transgenic mouse lines. Recently, we analyzed mice conditionally lacking hypoxia-inducible transcription factors (HIFs) and HIF-prolyl hydroxylases (PHDs), and determined trans-regulatory factors and signaling pathways required for hypoxic induction of the Epo gene. Next, we demonstrated by chromatin immunoprecipitation (ChIP) assays that histones at the Epo promoter were highly acetylated under normal conditions, and the acetylation level was not increased in hypoxic/anemic mouse livers. This indicates that the Epo promoter region is epigenetically prepared for immediate transcriptional in-duction by hypoxic stresses, because histone acetylation is very predictive for gene expression. Additionally, the acetylation at the promoter was blunted in the liver of the mice lacking the enhancer located 3’ to the Epo gene, in which a HIF-binding site is contained, suggesting important roles of HIFs in establishment of the promoter competence. Moreover, we found that HIFs were essential for hypoxia-dependent formation of nucleosome-free naked DNA regions in the promoters of some hypoxia-responsive genes. These results indicate that hypoxia alters epigenetic status of the promoter/enhancer regions of hypoxia-responsive genes including the Epo gene through PHD-HIF pathway

Novel pointers to regulation and targets of erythropoietin in human skin: An exploratory pilot study

Guoyou Zhang, 1, 2 Vladimir Emelianov, 1 Arno Kromminga, 3 Nina van Beek, 1 Eniko Bodo, 1 Ute Duske, 3 Ralf Paus 1, 4

1Dept. of Dermatology, University of Luebeck, Luebeck, Germany; 2Dept. of Hand and Plastic Surgery, the Second Affiliated Hospital of Wenzhou Medical College, Wenzhou, China; 3IPM Biotech, Hamburg, Germany;

4Institute of Inflammation and Repair, University of Manchester, Manchester, UK

The chief hormonal regulator of erythropoiesis, erythropoietin (EPO), is also expressed in human skin, namely in human scalp hair follicles (HFs), where its expression is up-regulated by hypoxia. However, our understanding of the role of EPO in skin biology, and of how intracutaneous EPO expression in human skin may be up-regulated, is still very poor. Therefore, the current pilot study attempted to generate pointers to novel EPO target genes in EPO-treated, organ-cultured human skin. This identified CREG2: cellular repressor of E1A-stimulated genes 2 (CREG2), MAPK1, Fc fragment of IgE high affinity I receptor for alpha polypeptide (FCE1A), inositol monophosphatase domain containing 1 (IMPA3), T-cell leukemia/lymphoma 1A (TCL1) and corneodesmosin (CDSN) as (direct or indicrect) candidate EPO target genes in normal human skin. Next, we asked whether agents reported to modulate EPO expression/production in selected cell culture systems, i.e. thyroid hormones (T4, T3), the antifungal agent ciclopiroxolamine (CPX), and cobalt chloride (CoCl2), also do so in cultured human keratinocytes, skin and/or HFs. Our preliminary results indicate that T4 and CoCl2 increase EPO transcription in HaCaT keratinocytes, while CPX up-regulates the novel EPO target genes, CREG2 and CDSN, and CoCl2 up-regulates IMPA3, in human HFs. T4 also significantly increases EPO transcription in human skin. In addition, both T4 and T3 up-regulate EPO immunoreactivity in human HFs and organ-cultured human epidermis. CoCl2 also stimulates intraepithelial EPO expression in human skin organ culture, while topically applied CoCl2 up-regulates CREG2 transcription in organ-cultured human skin. However, by ELISA, the EPO protein concentration in the supernatant was below the limit of detection. This exploratory pilot study has identified novel candidate target genes for EPO bioregulation in human skin and shows that clinically interesting EPO regulators can indeed up-regulate EPO expression in human skin. It remains to be clarified whether these can lead to any significant release of EPO from human skin and HFs.


Erythropoietin production in neural crest cells

Ikuo Hirano, Norio Suzuki, Xiaoqing Pan, Shun Yamazaki, Naoko Minegishi and Masayuki Yamamoto

Tohoku University School of Medicine

Erythropoietin (Epo) is necessary for both embryonic (primitive) and adult-type (definitive) erythropoiesis. Epo-producing cells located in the kidney and liver are well known to be important for definitive erythropoiesis, however the cellular source of Epo for primitive one are less understood. To identify Epo-producing cells in mid-gestational stage embryos, we analyzed Epo-GFP transgenic mouse lines (22k-Epo-GFP, 17k-Epo-GFP) expressing green fluorescent protein (GFP) under the regu-lation of Epo gene. The GFP expression was detected in the neural tube, neural crest and its derivatives between embryonic day 8.5 (E8.5) and E13.5. A part of Epo-GFP expressing cells were labeled with a neural-crest tracer (Wnt1-Cre mouse) at E9.5, and the cells expressed mRNAs for neural crest marker genes (Wnt1, FoxD3 and Sox10). Furthermore, we detected Epo protein in ex vivo culture supernatant of the E8.5 neural tubes. The growth and differentiation of the yolk sac-derived erythroid cells were induced by the neural tube supernatant; this effect was blocked by supplementation of a neutralizing antibody against Epo. All of the results indicate that embryonic neural cells secrete Epo and support the development of primitive erythropoiesis.

Perioperative Serum Erythropoietin and Thrombopoietin levels in Coronary Artery Bypass Grafting (CABG)

F. Wenzel1, Th. Gettmann2, N. Zimmermann3, G. Giers2

1Institut für Transplantationsdiagnostik und Zelltherapeutika, Universitätsklinik Düsseldorf, Germany2Institut für Hämostaseologie und Transfusionsmedizin, Universitätsklinik Düsseldorf, Germany

3Institute of Pharmacology and Clinical Pharmacology, Heinrich Heine University Medical Center, Düsseldorf, Germany

Introduction: The hormones erythropoietin (EPO) and thrombopoietin (TPO) are main regulators of erythro- and thrombopoi-esis. Cell loss caused by operative procedures may alter serum levels of the hormones, resulting in well known phenome-nons like reactice thrombocytosis.Material and Methods: Blood samples from 10 patients (mean age 63 ± 9 years) were obtained before and on days 1, 5 and 10 after CABG. sEPO and sTPO levels were determined by commercially available ELISA-Kits (R&D Systems, Germany). In addition, platelet count (PC) and hemoglobin concentration (Hb) were determined.Results: Prior to CABG, sEPO (12.6 ± 8.3 mU/ml), sTPO (191.2 ± 64..0 pg/ml), PC (250.0 ± 124.6/nl) and Hb (9.3 ± 1 mmol/l) were within a normal range. At day 1 after surgery, Hb and PC were significantly decreased to 6.7 ± 0.8 mmol/l and 146.5 ± 92.8/nl. In contrast, sEPO and sTPO were significantly elevated to 39.7 ± 28.5 mU/ml and 346.7 ± 107.2 pg/ml, respectively, in spite of hemodilution. In particular, sTPO elevation was followed by a significant increase in PC (344.9 ± 165.4/nl) at day 10 after surgery compared to preoperative values.Conclusions: Appropriate to the decrease in hemoglobin concentration and platelet count, clear alterations of serum erythro-poietin and thrombopoietin levels could postoperatively be observed. Whereas hemoglobin concentration returned nearly in a normal range, the alterations of the hormones resulted in the phenomenon of reactive thrombocytosis at day 10 after surgery.


Nitric oxide and hypoxia stimulate and regulate erythropoietin receptor in endothelial cells

Bojana B Beleslin Cokic, Vladan P Cokic, Constance T Noguchi

Clinic for Endocrinology, Diabetes and Metabolic Diseases, Genetic Laboratory Clinical Center of Serbia, Dr Subotica 13 11000 Belgrade, Serbia

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. EPOR is ex-pressed on endothelial cells and expression is increased at low oxygen tension. With EPO stimulation of endothelial cells during hypoxia, we observed a corresponding increase in endothelial nitric oxide (NO) synthase (eNOS) expression and acti-vation as well as NO production. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethy-lenetriamine NONOate (DETANONOate) at different oxygen tension for 24 hours showed statistically significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANONOate demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 hours particularly at low oxygen tension. In TrHBMEC and HUVEC EPOR protein was sig-nificantly induced by DETANONOate at 2% oxygen. Different signaling pathways were examined after NO donor stimulation. We found that DETANONOate activated MAPK kinase in TrHBMEC both in normoxia and hypoxia and activity was blocked with MAPK inhibitor PD98059. In contrast, DETANONOate stimulated Akt anti-apoptotic activity after 30 minutes in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In TrHBMEC, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation during first 30 minutes in normoxia and 15 minutes in hypoxia. The cytoprotective effect of EPO in endothelial cells, particularly at low oxygen, was demonstrated by induction of the antiapoptotic protein Bcl-xL. We used an EPOR promoter/luciferase reporter gene assay to examine if NO could regulate EPOR expression at the transcripti-onal level. In reporter gene assays, DETANONOate treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 50% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 70% increase in activity with a 410 bp EPOR promoter fragment. There was no induction of EPOR promoter activity after DETANONOate stimulation at 21% of oxygen, suggesting that NO regulated EPOR expression at the transcriptional level by promoter activation only at low oxygen tension. These data provide a new effect of NO on EPOR expression and regulation in endothelial cells under normal and low oxygen tensions.

IL-4 attenuates the proangiogenic capacity of macrophages by downregulating HIF-1α protein

Nathalie Dehne, Silke Essler, Michaela Tausendschön, Tobias Schmid and Bernhard Brüne.

Institute of Biochemistry I/ZAFES, Goethe-University, Frankfurt, Germany

Macrophages (MΦ) are extremely versatile cells showing many different activation profiles in vivo and in vitro. They are categorized in three extreme phenotypes one being classically activated MΦ after stimulation with LPS or INFγ and two alternative activated forms, known as wound healing MΦ (IL-4/IL-13) and regulatory MΦ (IL-10/TGFβ). We analyzed HIF-1α and HIF-2α expression in mouse and human MΦ polarized towards these phenotypes and noticed distinct expression pat-tern. Classically activated MΦ are characterized by induction of HIF-1α and reduction of HIF-2α mRNA, while wound healing MΦ decreased HIF-1α and slightly increased HIF-2α protein expression. In regulatory MΦ both HIF isoforms are present or slightly induced.Analysis of several HIF target genes in wound healing or regulatory MΦ did not reveal any specific HIF-2 target gene, alt-hough HIF-2 seems to be activated in these phenotypes. Target genes consistently showed reduced hypoxic induction in MΦ stimulated with IL-4, thereby recapitulating HIF-1α modulation. Following angiogenic-sprouting using embryonic stem cells exposed to supernatants of MΦ incubated with IL-4 under hypoxia revealed shorter sprouts compared to supernatants of hypoxic MΦ. Conclusively, IL-4 reduces HIF-1 activity in MΦ and concomitantly attenuates their ability to promote angio-genesis.

Supported by DFG (SFB 815)


The Role of Hypoxia-Inducible Factors in Inflammatory Bowel Disease

Katharina Flück¹,², Gerhard Breves², Simon Schäfer¹,³, Joachim Fandrey¹, Sandra Winning¹

1 Institut für Physiologie, Universitätsklinikum Essen, Universität Duisburg-Essen2 Physiologisches Institut, Tierärztliche Hochschule Hannover

3 Klinik für Anästhesiologie und Intensivmedizin, Universitätsklinikum Essen

Inflammatory bowel diseases are widespread and severe, with Crohn´s disease and ulcerative colitis representing the most prevalent types. It is increasingly recognized that inflammatory processes are always associated with low oxygen tension (hypoxia). Tissue hypoxia requires adaptation of the cells which is mainly controlled by transcription factors designated hypoxia-inducible factors (HIFs), composed of oxygen-regulated α-subunits and a constitutive HIF-1β.Herein, we analyzed how the activation of HIF-1α and HIF-2α subunits in myeloid cells (neutrophil granulocytes, monocytes/macrophages and dendritic cells) influences the progression of an acute colitis.To this aim, mice with a conditional knockout of HIF-1α or HIF-2α either in myeloid cells or in dendritic cells were treated with dextran sodium sulfate to induce experimental colitis.The severity of disease was determined by general outcome, weight loss, and measurement of colon weight and length. We determined the number and distribution of mononuclear cells in colon sections from control and knockout mice using immu-nohistologic analysis. Expression of hypoxia-inducible genes in the inflamed colon was studied at the mRNA level.First results show that mice lacking HIF-2α in dendritic cells started to lose weight at an earlier time point than controls. Colon samples of these animals showed increased expression of inflammatory genes.We conclude that loss of hypoxia-inducible factors in immune cells may lead to augmented inflammation in an experimental model of acute colitis.

Erythropoietin as a link between skin and kidney

Lupica R.1,Zinnarello Clara 2,Donato V.1, Luicisano S.1,,Lacquaniti A.1,Cernaro V1.,Fazio MR.1, Ruggeri Paolo 2, Buemi M.1.

1 University of Messina, Policlinic G. Martino Departement of Internal Medicine UOC of UTSI eTD.2 University of Messina, Policlinic G. Martino Departement of Toraco-Cardio-Vascolare, UOC of Pneumology.

Introduction: recently, research has pointed out that numerous tissues such as heart and lung are able to produce Erythropi-etin. In particular, the skin seems to behave as a hypoxia-sensible organ: in fact, the keratinocytes fix the F5 nitro imidazole and express high levels of HIF-1 transcripts. iNOS (inducible oxide nitric synthase) is a target gene of the HIF transcriptional response to hypoxia, and NO is an essential intermediary through which the HIF modification at the skin level modulates the EPO synthesis stimulation. The aim of our study has been to evaluate the role of NO in the EPO synthesis following cutane-ous stimulation.Materials and methods : the study population consisted of 20 patients (10 men, 10 women; aged 60-75 years; GFR 21-54 ml/min; Hb 11.6-15.5 g/dl; Hct 38-45%) affected by chronic renal failure; none of them had been treated with recombinant human EPO before. For each patient we dosed EPO serum concentration at baseline (T0) and 6 and 12 hours after oral administ-ration of isosorbide-5-mononitrate (20 mg, 2 tablets) and, after 3 days, we performed the same measurements at T0 and 6 and 12 hours after administration of transdermal nitroglycerin patch (5 mg). Moreover, we assessed the NO concentrations in exhaled air in fasting conditions and at the same times of EPO dosage. In particular, we measured the Fractional Exhaled Nitric Oxide (FeNO) basing on the electrochemical sensor technology, according to the American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines.Results: we observed a statistically significant increase in EPO serum concentrations after transdermal nitroglycerin admi-nistration (from 12.74 ± 4.5 mUl/ml at T0 to 16.0 ± 4.0 mUI/ml and 16.62 ± 5.37 mUI/ml respectively 6 and 12 hours after ap-plying the patch; T0 vs 12h: p= 0.017). Moreover, changes occurred in the exhaled air NO concentrations, with a statistically significant increase 6 and 12 hours after transdermal nitroglycerin (from 18.6 ± 2.6 at baseline to 24.0 ± 2.35 and 24.7 ± 2.2 ppb respectively; T0 vs 12h: p= 0.001); such modifications did not happen after oral isosorbide-5-mononitrate administration (from 12.93 ± 2.47 at baseline to 12.0 ± 2.38 and 12.68 ± 2.62 ppb respectively; T0 vs 12h: p>0.5). Conclusion, the transdermal administration of nitroglycerin, by acting on the skin receptor, induces an increase in NO con-centration.


Expression of the cellular oxygen sensor PHD2 affects radioresistance in squamous cell cancer of the head and neck

Friederike Katharina Pientka, Mareike Ströfer, Jürgen Dunst, Wolfgang Jelkmann, Reinhard Depping

Institute of Physiology, Center for Structural and Cell Biology in Medicine, University of Lübeck, GermanyDepartment of Radiotherapy, University of Lübeck, Germany

The human prolyl-4-hydroxylase domain (PHD) proteins 1-3 are known as cellular oxygen sensors involved in degradation of hypoxia-inducible factor (HIF) α-subunits. All three PHDs differ in their intracellular expression patterns and show distinct cellular functions. PHD2 is considered to be the key regulator of HIF-1α stability in normoxia and mild hypoxia. Our previous results have demonstrated that PHD2 shuttles between nucleus and cytoplasm. The import and export from the nucleus depend on specific transport signals in PHD2. Recently, it could be shown that nuclear PHD2 expression associates with malignant cancer phenotype and radiation resistance. The aim of our study was to evaluate the relationship between sub-cellular PHD2 expression and radioresistance in squamous cell cancer of the head and neck. Therefore, we treated wildtype and PHD2-deficient cells as well as cells expressing compartment-specific PHD2 mutants with ionising radiation in doses from 2 to 6 Gray and examined clonogenic survival. Our results demonstrate that PHD2 expression decreases resistance to radiation in human cancer cells. However, the subcellular localization of PHD2 does not affect clonogenic survival in our model. These data extend previous observations on PHD2 by showing an inverse correlation between PHD2 expression and the outcome of radiotherapy.

Expression of the amiloride binding protein 1 (Abp1) is stimulated in response to hypoxia and myocardial infarction

Karin M. Kirschner, Julian Braun, Holger Scholz

Institut für Vegetative Physiologie, Charité-Universitätsmedizin Berlin, Hessische Straße 3-4, 10115 Berlin

Putrescine is a polyamine, which is important for normal cell growth and differentiation. Its formation is enhanced in response to myocardial ischemia presumably due to increased activity of ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in putrescine synthesis. Recent findings indicate that tissue levels of putrescine are regulated not only by the rate of synthesis, but also by its degradation through the diamine oxidase, amiloride-binding protein 1 (Abp1).The goal of this study was to assess the role of Abp1 in regulating putrescine degradation in the ischemic rat heart.For this purpose, adult rats were subjected either to left coronary artery ligation or sham-surgery, and Abp1 mRNA was measured by real-time RT-PCR. Twenty-four hours after surgery, a 10-fold increase of Abp1 transcripts was observed in the hearts of rats with myocardial infarction compared to sham-operated animals (Student's t-test, P<0.001, n=5). In contrast, ODC expression was not significantly changed at 24 hours after infarction. Consistently, exposure of mice to 8% inspiratory oxygen for 6 hours caused an 11-fold increase of Abp1 mRNA in the hearts (P<0.05, n=5) with no significant changes of ODC transcripts. Abp1 mRNA levels were also significantly increased in several cell lines grown at 1% oxygen for 24 h. A luciferase reporter construct carrying the promoter of the Abp1 gene was stimulated approx. 3.5-fold in transfected C2C12 myoblasts at 1% oxygen. Abp1 mRNA levels were increased 10-fold upon in vitro differentiation of C2C12 cells to myotubes. Inhibition of Abp1 by amiloride (100 µM) prevented the in vitro differentiation of C2C12 myoblasts.In conclusion, cardiac expression of Abp1 is stimulated in response to hypoxia and myocardial infarction. It is suggested that enhanced Abp1 expression has a role in tissue remodeling after myocardial ischemia through regulating putrescine metabolism.


Distinct impacts of Interleukin-1β on HIF-1α and HIF-2α in tumor cells

Wenwen Sun, Thomas Hellwig-Buergel, Reinhard Depping, Wolfgang Jelkmann

Institute of Physiology, University of Luebeck, Germany

Hypoxia inducible factors (HIFs) are transcription factors which mediate the primary transcriptional responses to hypoxic stress in mammalian cells. HIFs function as heterodimers consisting of an oxygen-labile α-subunit (HIF-1α, HIF-2α and HIF-3α) and a stable β-subunit (ARNT). In the presence of oxygen, HIF-α is hydroxylated at conserved proline residues by HIF-PHD, which labels them for rapid degradation by the proteasome. Hypoxia inhibits PHD activity through various mechanisms, which results in HIF-α stabilization, heterodimerization with HIF-β and increased HIF transcriptional activity. Because of the competence in affecting many key aspects of tumor progression, invasion and metastasis, HIF-1α and HIF-2α represent attractive targets for tumor therapies. Although HIF-1α and HIF-2α share some redundant functions, they exhibit unique and even opposing activities in many processes that affect tumor growth.Interleukin-1β (IL-1β) is a product of tumor-infiltrating macrophages which are usually associated with unfavorable progno-sis. Several in vivo experiments showed that tumor growth and metastasis can be decreased by injection with IL-1receptor antagonist. Therefore, IL-1β is suspected to be a tumor promoter.Recent Studies have shown that IL-1β induces accumulation of HIF-1α in many tumor cell lines. In this study, we focused on the influence of IL-1β on HIF-2α. We found that although IL-1β increases HIF-1α protein content under normoxia and hypoxia in hepatocellular carcinoma cell line HepG2, no change of HIF-2α protein level could be observed. The influence of IL-1β on the HIF transcriptional activity and expression of HIF target genes needs further to be studied. It is also necessary to investigate the exact mechanism by which IL-1β increases HIF-1α, but not HIF-2α in HepG2 cells. Our finding will probably help understand the tumor-promoting effect of IL-1β.

Analysis of the erythropoietin receptor dimer in different cancer cell lines by means of Fluorescence Resonance Energy Transfer

André Bernardini, Ulf Brockmeier, Joachim Fandrey

Institute of Physiology / University of Duisburg-EssenHufelandstr. 55, D-45122 Essen

Anemia is a common complication during chemotherapy. Erythropoietin (Epo) is commonly used to treat anemia. Recent studies found however that Epo treatment of cancer patients is apparently associated with tumor growth and hence increases mortality. While the Epo receptor (EpoR) mRNA is shown to be detectable in tumor cells, the presence of functional EpoR molecules on the surface of tumor cells has not unambiguously been demonstrated. Furthermore there is no valid study that could show increased proliferation of these cell lines under pharmacologically relevant doses of Epo. This raises the question whether the cellular environment of tumor cells is able to correctly assemble the EpoR dimer and integrate it into the cytoplasm membrane where it can bind to Epo. To study this question, we cloned human EpoR cDNA into ECFP and EYFP expression vectors. Both constructs were co-transfected into two different tumor cell lines and analyzed by confocal FRET microscopy. The chimeras localized mostly as described by other working groups. In most cells fluorescence accumulates in the endo-plasmic reticulum while only a very weak signal is detectable in the cytoplasm membrane. There are however also some cells that exhibit an endosome-like staining pattern instead of the ER staining. We could also observe some cells where fluorescence was localized only to the membrane. The cause for this heterogeneity remains unclear.Although our data show excellent co-localization of both chimeras we were not able to detect significant FRET between them. Even upon addition of the ligand no FRET signal was detectable. This does however not necessarily mean that no dimers are formed. Little is known about the three-dimensional structure of the intracellular domain of EpoR and hence both fluorophores (both attached to the C-terminus of the EpoR) may simply be too far apart to allow for energy transfer.Supported by European Commission under the 7th Framework Programme (FP7-HEALTH-2011-single-stage Grant Agree-ment Number 282551EpoCan) to Joachim Fandrey. This report reflects only the authors’ views, and the European Commu-nity is in no way liable for any use that may be made of the information contained herein.


Erythropoietin drives neurodifferentiation

Liane Dahm, Imam Hassouna, Nils Offen, Christoph Ott, Richard Neher, Swetlana Sperling, Nora Hagemeyer, Kathrin Hannke, Miso Mitkovski, Lucia Schlee, Marcus Dittrich, Susann Boretius, Andre Zeug, Thomas Dandekar,

Erwin Neher, Anna-Leena Sirén and Hannelore Ehrenreich

Division of Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany

Erythropoietin (EPO) enhances cognition and hippocampal long-term potentiation in juvenile mice via yet unclear mecha-nisms. Searching for cellular insight, we surprisingly found after 3-week EPO treatment around 15% more neurons and 20% more oligodendrocytes in hippocampal CA1/CA3 regions, without evidence of increased proliferation or decreased apopto-sis. We hypothesized that EPO drives differentiation of local precursors, and indeed saw under EPO decreases of immature neurons and oligodendrocyte precursors in CA1/CA3. In E14 neural stem cell cultures, EPO provoked rapid shifts in differen-tiation markers Sox9 and NeuroD1, stimulated miR124 and neurodifferentiation, and reduced sphere growth. This reduction was inhibited by miR124 antisense oligos. In more mature E17 hippocampal cultures, EPO caused similar marker shifts and stimulated differentiation/maturation, reflected by a larger active area of synapses. To conclude, EPO-induced differentiation of local precursors in CA1/CA3 led to increased neuron numbers and hippocampal size. This capacity sheds new light on adult neurogenesis in general.

Epo regulates hypoxic signal transduction in rat carotid body glomus cells via potassium channel remodelling

Julia A. Griffiths, Teresa Gallego-Martin, Vsevolod Telezhkin, Jorge Soliz, Constancio Gonzalez, Paul J. Kemp

Department of Pathophysiology and Repair,Cardiff School of Biosciences, Cardiff University, UK

Understanding the molecular mechanism involved in the cardioprotective effect of Epo is of significant value when conside-ring targeted therapy for pathologies resulting from severe cases of hypoxia and ischemia. Epo has been proposed to aug-ment the ventilatory response to hypoxia, suggesting that one protective mechanism may involve remodelling of the carotid chemoreflex; a notion investigated here in adult rat carotid body glomus cells. EpoR co-localized with tyrosine hydroxylase, suggesting that EpoR is expressed in glomus cells. Acute treatment (5h) of glomus cells with Epo (500 U/L) had no effect on glomus cell ionic currents, whereas chronic (>24h) exposure resulted in a significant increase in outward potassium current density from 48.9 ± 3.4 to 95.0 ± 11.7 pA/pF (measured at +60 mV, n=22, p<0.05). This augmentation was accompanied by a parallel increase in the oxygen sensitive potassium current density. In addition TEA (20 mM) + 4AP (1mM) insensitive currents were also enhanced by chronic Epo treatment. In a separate set of experiments the potassium current density in the chronic presence of Epo was significantly decreased by co-treatment with 20 µM of the NFkβ inhibitor, SN50 (from 170.05 ± 20.41 to 85.62 ± 18.36 at +60 mV, n=8, p<0.05), suggesting that the Epo response is dependent upon transcription, consistent with the consensus NFkβ binding site in the BK α subunit promoter. To identify the major Epo-regulated, voltage-sensitive current, we employed single channel analysis using inside-out patches. In symmetrical potassium solutions, unitary conductance was unchanged by Epo treatment (control = 177.0 ± 9.0 pS (n=13), Epo = 164.5 ± 11.1 (n=15), p>0.5) and open state probability (Po) was augmented by 100 µM calcium, NS1619 or CORM2, confirming that they were BKCa channels. Importantly, chronic Epo treatment resulted in an increase in the mean maximal number of BKCa channels per patch, as assessed when fully activated by 100 µM calcium (1.54 ± 0.25, n=11 to 2.71 ± 0.45, n=17, p≤0.01). In addition, Po at physio-logical calcium was always higher in the Epo group (e.g. At 300 nM, control Po = 0.13 ± 0.04 (n=11), Epo Po = 0.35 ± 0.06 (n=17), p<0.01). Taken together, these data suggest that potassium channel remodelling may contribute to the Epo-evoked augmentation of the ventilatory response to chronic hypoxia.


Enteral Erythropoietin & Iron Transporter Expression in Newborn Rats

Pamela J. Kling, Alex T. Quilling, Mary Y. Sun, Sharon E. Blohowiak

University of Wisconsin Department of Pediatrics School of Medicine and Public Health, Madison, WI, USA.

Transferrin (Tf) and erythropoietin (Epo) are found in the mammalian milk and transferrin receptor (TfR) in enterocytes. In erythropoietic cells, erythropoietin (Epo) increases TfR expression. In neonates, parenteral Epo has little impact on iron ab-sorption, but our goal was to examine whether enteral Epo improves TfR and iron absorption in newborn rat intestine. Both control (Dam) and iron-deficient (IDA) formula-fed rats were fed enteral Epo (540 U/Kg/d). Blood and tissues were harvested. Dam-fed rats had higher body iron than IDA, with intermediate levels in IDA+Epo. Plasma levels of Tf, iron and Tf saturation were similar between groups, but plasma ferritin levels were higher in Dam vs. IDA, with intermediate results in IDA+Epo. Duodenal TfR expression was highest in IDA+Epo and localized to apical surface of enterocytes. Enteral Epo increased iron absorption and plasma ferritin (reflecting body iron stores), with liver iron higher than anticipated. Epo increased TfR in a fashion similar to erythrocyte precursors, by increasing enterocyte TfR levels. Newborns differ from adults, with TfR playing a major role in enteral iron transport and further study of enteral Epo and newborn iron status is needed.

Erythropoietin promotes survival and regeneration of insect neurons in vivo and in vitro

Natasa Miljus, Daniela Ostrowski, Hannelore Ehrenreich & Ralf Heinrich

Department of Cellular Neurobiology; Georg-August-University Göttingen, Germany (N.M., D.O., R.H.)Division of Clinical Neurosciences; Max Planck Institute for Experimental Medicine, Göttingen, Germany (H.E.)

Both erythropoietin (EPO) and erythropoietin receptor (EPOR) are expressed in mammalian central nervous systems and play important functions during neural development and in protective responses against diverse types of injury. EPO’s be-neficial functions are mediated through janus kinases (JAK) / signal transducers and activators of transcription (STAT) trans-duction pathways.We studied potential neuroprotective and neuroregenerative effects of EPO in the grasshopper, an invertebrate that lacks EPO-stimulated erythropoiesis. Recombinant human EPO (rhEPO; 4 U/ml) increased the survival of primary cultured gras-shopper brain neurons while decreasing survival of brain glial cells. The same concentration of rhEPO both improved in vitro neuronal survival during periods of hypoxia (36 hrs) and promoted neurite regeneration of cell bodies that lost all of their neurites during the dissociation process. rhEPO-mediated improvement of anatomical and functional regeneration of gras-shopper neurons was also seen in vivo after transection of centrally projecting axons of auditory receptor neurons. A single application of rhEPO (4 U in 10µl saline) to the experimentally injured nerve accelerated and generally improved the rege-neration of sound source localization, that was monitored by nerve backfills with a tracer and daily behavioral testing for 20 days following auditory nerve crush. In order to characterize the intracellular pathways that mediate EPO’s neuroprotective effects in insects, primary cultured grasshopper brain neurons were subjected to various combinations of hypoxia-, rhEPO- and “inhibitor”-treatments. In these experiments, the janus kinase inhibitor AG490 completely abolished the protective effect of rhEPO during hypoxia. The involvement of downstream intracellular pathways is currently beeing investigated.The results of our studies on EPO-mediated neuroprotection in insects parallel a large number of similar studies on mam-malian nervous systems. This indicates the existence of a ligand/receptor system in insects that shares high structural and functional similarities with EPO/EPOR signalling in the nervous system and other non-hematopoietic tissues.


CHARACTERIZATION OF THE ERYTHROPOIETIN/ ERYTHROPOIETIN RECEPTOR AXIS IN A RAT MODEL OF LIVER DAMAGE AND CHOLANGIOCARCINOMA DEVELOPMENT

Federico Moriconi(1) , Pierluigi Ramadori(2), Martina Blaschke(1), Frank C. Schultze(1), Ahmad Amanzada(1), Giuliano Ramadori(1)

(1) Department of Gastroenterology and Endocrinology, University Medical Center of Goettingen, Germany; (2) Department of Cell Physiology and Metabolism, University Medical Centre of Geneva, Switzerland.

It has been recently shown that the biological effects of erythropoietin (EPO) are not limited to the hematopoietic compart-ment but, as pleiotropic glycoprotein, this hormone can exert pro-angiogenic and tissue-protective functions also in a wide range of non-hematopoietic organs. The role of EPO and the effective functionality of its receptor in solid tumours are still a controversial point of debate. In the present work we analyzed the gene expression of EPO and its cognate receptor (EpoR) in a rat model of thioacetamide (TAA)-induced damage and tumor. An analysis of the EPO/EpoR axis was also performed on human cholangiocarcinoma (CC) cell lines performing real time RT-PCR, Western blot analysis and immunohistochemical studies. A progressive increase of EPO and EpoR mRNA can already be observed during the fibrotic-cirrhotic development with a peak of expression rising at tumour formation (24.7±9.9 fold increase and 15.5±1.1 fold increase respectively for the two genes). Co-localization studies by immunofluorescence revealed hepatocytes in the regenerative cirrhotic nodules (HEPPAR+) and in the dysplastic bile duct cells (CK19+) as the major EPO producers in this specific condition. The same cell populations, together with endothelial cells, exhibited an increased expression of Epo-R although all the non-parenchymal cell populations in the liver exhibited modest basal mRNA levels. Challenging human CC cells, Mz-Cha-2, with a combination of EPO and SCF resulted in a synergistic effect on the gene expression of EPO, Cyclin-D1 and PCNA. In the liver, a parallel upregulation of the hypoxia-inducible factor-1 (HIF-1) α gene was observed (up to 6-fold increase 18 weeks after TAA admi-nistration), while HIF-2 α gene expression was downregulated reaching the minimum 4 weeks after beginning the treatment. Same results were obtained in rat livers from TAA-induced tumor. This study suggests that the autocrine and paracrine release of endogenous EPO in the microenvironment may contribute to the development and maintenance of the CC and indicates HIF-1α as a major regulator of EPO gene expression in these experimental conditions.

Influence of erythropoietin on survival and neuronal differentiation of mouse induced pluripotent stem cells

Offen, Nils¹, Flemming, Johannes¹, Ahmad, Ruhel², Wolber, Wanja¹, Geis, Christian³, Zaehres, Holm4, Schöler Hans R4, Müller, Albrecht², Sirén, Anna-Leena¹

1 Department of Neurosurgery, 2 Center for Experimental Molecular Medicine (ZEMM), 3 Department of Neurology, University of Wuerzburg, 4 Department of Cell and Developmental Biology ,

Max-Planck Institute for Molecular Biomedicine, Muenster, Germany

Genetic reprogramming of somatic cells represents a novel strategy to produce autologous stem cells so-called induced pluripotent stem cells (iPS). The potential of iPS cells to differentiate into various cell types from the endo-, ecto- and meso-dermal lineage have been extensively studied, however the efficiency to produce functional neurons from iPS cells remains elusive. Here, we generated pan-neural progenitor cells (pNPCs) from mouse four factor iPS cells (Kim et al., Nature 2008, 454:646-654) and characterized their capacity to generate different neural subtypes. Furthermore, we investigated the effect of erythropoietin (EPO) on pNPC survival, growth and differentiation. We found that most iPS cell-derived pNPCs gave rise to cells that express markers for neurons (ß-III-tubulin, MAP2, synapsin, homer-1) and oligodendrocytes (platelet-derived-growth-factor-receptor, O4) while only a few cells expressed astrocytic (glial fibrillary acidic protein, vimentin) markers. Electrophysiological analyses of pNPC-derived neuron-like cells revealed their ability to generate action potentials and in-ward and outward currents. Moreover, labeling of synaptic vesicles with an anti-synaptotagmin antibody demonstrated the formation of active synapses. EPO (0.1-3U/ml) treatment of pNPCs increased cell survival and reduced pNPC sphere growth by inhibiting cell proliferation. Moreover EPO treatment enhanced the number of ß-III-tubulin positive cells and increased neurite numbers. Our results show that EPO improves survival and accelerates neuronal differentiation from iPS cell-derived neural progenitor cells.


Erythropoietin attenuates neurological and histological consequences of toxic demyelination in mice

Nora Hagemeyer, Christoph Ott, Daniela Winkler, Niels Jensen, Susann Boretius, Axel von Streitberg, Henrike Welpinghus, Swetlana Sperling, Jens Frahm,

Mikael Simons, Pietro Ghezzi, and Hannelore EhrenreichMax Planck Institute of Experimental Medicine

Erythropoietin (EPO) reduces symptoms of experimental autoimmune encephalomyelitis (EAE) in rodents and shows neuro-regenerative effects in chronic progressive multiple sclerosis. EPO's mechanisms of action in these conditions with shared immunological etiology are still unclear. Therefore, we employed a model of toxic demyelination allowing exclusion of T-cell mediated inflammation. In a 'double-blind' (for food/injections), placebo-controlled, longitudinal 4-arm design, 8-week old C57BL/6 mice (n=26/group) were assigned to cuprizone-containing (0.2%) or regular food (ground chow) for 6 weeks. After 3 weeks, mice were injected every other day with placebo or EPO (5,000IU/kg intraperitoneally) until the end of cuprizone feeding. Half of the mice were exposed to behavioral testing, magnetic resonance imaging (MRI) and histology immediately after treatment cessation, whereas the other half were allowed a 3-week treatment-free recovery. Immediately after termi-nation of cuprizone feeding, all toxin-exposed mice were compromised regarding vestibulomotor function/coordination, with EPO treated animals performing better than placebo. Likewise, ventricular enlargement after cuprizone as documented by MRI was less pronounced upon EPO. After 3-week recovery, remarkable spontaneous improvement was observed in all mice with no measurable further benefit in the EPO group (‘ceiling effect’). Histological analysis of the corpus callosum revealed attenuation by EPO of the cuprizone-induced increase in microglial numbers and amyloid precursor protein accumulations as readout of inflammation and axonal degeneration. To conclude, EPO ameliorates neurological symptoms in the cuprizone model of demyelination possibly by reduction of inflammation-associated axonal degeneration in white matter tracts. These findings underscore the value of future therapeutic strategies for multiple sclerosis based on EPO or EPO variants.

A novel mimetic of erythropoietin for the treatment of Alzheimer’s disease-like pathology

Oksana Dmytriyeva, Tatjana Novikova, Casper R. Gøtzsche, Elisabeth Bock, Vladimir Berezin, Stanislava Pankratova

Protein Laboratory, Department of Neuroscience and Pharmacology, University of Copenhagen

Alzheimer’s disease (AD) is the most common cause of dementia and is characterized by a progressive decline in cognitive function, neuronal degeneration, and the cerebral accumulation of extracellular amyloid. The hematopoietic cytokine Eryth-ropoietin (EPO) is now well known to have beneficial effects in a variety of nervous system disorders. Treatment with EPO improves neurologic function and reduces brain damage in acute and chronic neurodegenerative disorders including AD. EPO treatment has been demonstrated to improve memory in healthy mice and humans volunteers, and improve cognitive functions in schizophrenia patients. However, given the potential adverse effects of EPO treatment (increase in blood pres-sure, hemoglobin contents, and risk of thrombosis) the development of non-erythropoietic EPO mimetics is of importance. Here we show that a neuritogenic non-erythropioetic peptide, NL100, improves memory in intact rats as measured by a social recognition test. In the rat animal model of AD, NL100 treatment restored social memory to normal levels. The histological analysis of brain samples showed the decreased number of damaged neurons and lower astrogliosis in the NL100-treated group. The results of in vitro experiments on survival of hippocampal neurons suggest an anti-oxidative mechanism of the NL100-induced neuronal protection in the employed AD model.


Erythropoietin modulates breast cancer cell response to cisplatin in a time-dependent manner

Nina Trošt¹ , Peter Juvan¹, Gregor Serša², Neli Hevir¹, Tea Lanišnik Rižner¹, Nataša Debeljak¹

¹Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia²Institute of Oncology, Department of Experimental Oncology, Ljubljana, Slovenia

BACKGROUND: The erythropoietin receptor (EpoR) expression in breast cancer cells was shown to correlate with the ex-pression of steroid receptors and was associated with response to tamoxifen in ER(+)/PR(+) but not in ER(-) tumors. The aim of this study was to elucidate whether long and short-term rHuEpo administration influences breast cancer cell response to ci-splatin (cDDP) and whether this influence depends on steroid receptor and p53 status. METHODS: In the MCF-7, MCF-10A, MDA-MB-231, MDA-MB-361, T-47D, SKBR3, Hs578T and Hs578Bst cell lines we analyzed correlations between expression of steroid receptors genes (ESR1-2, PGR, and GPER) and EPHB4, CSF2RB and EPOR. According to the differences in ER/PR and p53 status, MCF-7 and MDA-MB-231 cells were selected for furtherer analysis. Cells were cultured with or without rHuEpo for 24h and 9 weeks and their growth characteristics before/after exposure to cDDP were determined. Expression of p53-dependent genes and bcl2-gene family members and activation of MAPK and PI-3K signaling pathways were analyzed. RESULTS and CONCLUSION: Variations in the expression of estrogen and progesterone receptors genes were seen in the analyzed cell lines. Expression of ESR1 was positively correlated with the expression of PGR, GPER, ESR2 and EPO, while ESR2 positively correlated with PGR and EPOR. ER/PR protein expression and p53 status were then further paralleled with cell response to rHuEpo and cDDP. Short-term exposure to rHuEpo reduces proliferation and protects breast cancer cells from CDDP cytotoxicity. Long-term exposure of MCF-7 cells that are ER(+)/PR(+) and express wild-type p53 increases their proliferation and induces sensitivity to cDDP. The MCF-7 cells proliferation and viability seem to be reversely influenced by the length of rHuEpo treatment. The MDA-MB-231 cells with ER(+)/PR(-) status and mutated p53 are almost irresponsive to long-term rHuEpo treatment which was further confirmed in the lowered level of ERK phosphorylation when compared to short-term treated cells. The expression analysis of p53-dependent genes and bcl-2 gene family members confirmed diffe-rences between long and short-term rHuEpo treatment, indicating the most prominent changes in BCL2 and BAD expression. Speculatively, ER/PR and p53 genetic signature may be used to predict response to rHuEpo supportive therapy in breast cancer patients.

Screening for ER proteins that influence folding and cell surface exposure of the erythropoietin receptor

Ulf Brockmeier, Anna Wrobeln and Eric Metzen

Institute of Physiology, Faculty of Medicine, University Duisburg-Essen, 45122 Essen, Germany

The Erythropoietin receptor (EPO-R) and its ligand (EPO) are exoproteins which become glycosylated and form disulfide bonds in the endoplasmic reticulum (ER) before traversing the Golgi to their final destinations, the cell membrane and ext-racellular space, respectively. Although extensive research over the last decades revealed many important insights into the EPO/EPO-R axis, little is known about the individual set of chaperones required for the cell surface exposure of the EPO-R. Of particular interest is the finding that, even after overexpression, most of the EPO-R protein remains in an intracellular compartment and less than 5 % of the total protein reach the cell surface. To investigate this process we used an inducible lentiviral knockdown system in an EPO-R expressing cell line to perform loss of function experiments for various ER chape-rones. Surprisingly, neither the knockdown of the ER protein OS-9, which is involved in ER associated protein degradation, nor the knockdown of one of the key members of the PDI family, ERp57, that promotes disulfide bond formation and isome-rization, did influence the cell surface exposure of EPO-R. However, downregulation of the ER lectin-chaperone calreticulin dramatically decreased the amount of cell surface exposed EPO-R. Additional co-immunoprecipitation experiments confir-med a direct protein-protein interaction between EPO-R and calreticulin.


Characterization of dendritic cells under inflammatory hypoxia

Yvonne Hüsecken, Joachim Fandrey, Sandra Winning

Institut für Physiologie, Universitätsklinikum Essen, Universität Duisburg-Essen

The transcription factor complex hypoxia-inducible factor (HIF)-1 regulates the adaptation of macrophages and dendritic cells to hypoxic and inflammatory conditions. Bone marrow derived dendritic cells (BmDC) are always contaminated with macro-phages, which are known to react to inflammatory stimuli with a dramatic induction of HIF-1 activity. This could mislead to the assumption that the transcription factor HIF-1 is important for the adaption of dendritic cells to inflammatory hypoxia. To analyze if the observed effects in our bone marrow derived cell cultures were caused by DCs we differentiated cells with and without Interleukin-4 (IL-4). IL-4 is the major cytokine driving DC differentiation. The lack of IL-4 during differentiation led to increased numbers of macrophages while the number of mature dendritic cells was decreased. Then, DC cultures from con-trol and HIF-1α knockout mice were cultivated with and without IL-4. The cells were compared in their response to bacterial lipopolysaccharides under normoxia and hypoxia.No differences in HIF-1α protein or HIF-1 target gene expression were detected between cell cultures with and without IL-4. LPS-stimulation led to similar activation of HIF-1 and was independent of DC purity. Furthermore, no significant differences in gene expression of the inflammatory cytokines IL-1β or TNF-α were obvious after differentiation under the distinct conditions.Thus, contaminating macrophages in DC cultures have a negligible influence on hypoxia and inflammation induced HIF-1 activation in dendritic cells.

Growth factor in PRP - general approaches - a review of recent developments

Lubkowska A. , Dołęgowska B., Banfi G.

1 Department of Physiology, Faculty of Biology, Szczecin University, ul. Felczaka 3c, 71-412 Szczecin, Poland2 Chair and Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, al. Powstańców Wlkp. 72,

70-111, Szczecin, Poland

Autogenous platelet-rich plasma (PRP) was first used in the 1990s and is raising interest and controversy among researchers and clinicians. Investigations relating to the use of autogenous material in medicine focus on inter-relationships between factors released during the activation of platelets, plasma factors, potential additive mechanisms, and the search for optimal preparation procedures and dosing of blood products, including PRP. Platelet-rich plasma concentrate is applied in many fields of medicine. The general concept of useful PRP is to concentrate blood platelets through centrifugation of whole blood and to apply them with or without direct activation where their healing potential is needed . Platelet α-granules have been shown to contain mitogenic and chemotactic growth factors (GF) and associated healing molecules in an inactive form, im-portant in wound healing, such as platelet-derived growth factor (PDGF), transforming growth factors β1, β2, β3 (TGF-β1, TGF-β2, TGF-β3), platelet-derived angiogenesis factor (PDAF), insulin-like growth factor 1 (IGF-1), platelet factor 4 (PF-4), epidermal growth factor (EGF), epithelial cell grow factor (ECGF), vascular endothelial cell growth factor (VEGF), basic fib-roblast growth factor (bFGF) and others cytokines. They mediate many of the cellular functions including cell migration, pro-liferation, differentiation, metabolism and apoptosis, as well as cellular cycle. Platelet concentrates containing growth factors are applied in clinical practice in various ways. A lot of studies have been performed to investigate the effect of PRP upon tissue regeneration, however, the results are controversial, often due to differing methods of platelet concentrate preparation. Biology and clinical potential of preparations are dependent on their composition and the way the growth factors, matrix and cells are assembled together. These technologies have been popularized particularly in cardiac surgery, in many areas of dentistry such as periodontics, oral implantology and oral and maxillofacial surgery, orthopedics, repositioning skin flaps in plastic surgery, against small diffuse bleedings, a mal perforant ulcer in the foot of a person with diabetes, to speed up the healing process of burns, to promote retinal neurogenesis, and in the treatment of musculoskeletal injuries and disorders but also in sports medicine.


Specific and sensitive immunohistochemistry detection of the human erythropoietin receptor in situ

Nora Torres-Nagel1, Doris Ziegler-Landesberger1, Ranjan Sircar1, Stefanie De Schepper3, Gaby Walker2, Nathalie Van Acker3, Michael Schräml1, Julie Bachmann4, Ursula Klingmüller4, Michael Jarsch1, Olaf Mundigl1

1 Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, Penzberg, Germany 2 Hoffmann La Roche AG, Basel, Switzerland

3 HistoGeneX N.V., Campus Middelheim, B-2020 Antwerp, Belgium4 German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany

A crucial limitation of currently available antibodies directed against the human Epo-receptor (EpoR) is that these antibodies produce inconsistent and artifactual results when used on tissue sections. Here we provide a novel anti-EpoR antibody that enables specific and sensitive detection of EpoR via immunohistochemistry (IHC) and western blotting (WB). Antibody GBb detected the different glycosylation forms of EpoR 62-66 kDa by WB that could specifically be knocked down by shRNA. Moreover, GBb immunoprecipitated a 66 kDa protein that became phosphorylated upon Epo stimulation of UT-7 cells. Immu-nofluorescence microscopy revealed that GBb exclusively stained EpoR clusters in cells that overexpressed an EpoR-GFP fusion protein. BIACORE analysis further indicated that this antibody displays favorable binding kinetics with a KD = 0.63 nM and a dissociation constant of 23 min. (t/2 diss).Finally, using immunohistochemistry analysis, we were able to show EpoR expression in erythroid progenitor cells in bone marrow and xenograft tissues from EpoR overexpressing HELA cells .This novel antibody will allow researchers to thoroughly reevaluate EpoR expression in clinical tissue samples.

Erythropoietin (EPO) stimulates aerobic and anaerobic processes in ratcardiomyocytes

T.V. Parkhomenko, V.V Tomson, O.A. Klitsenko

SRC of St-Petersburg Pavlov State Medical University

This study was designed to investigate the influence of EPO on cardiomyocytes of young male Wistar rats (150-210 g) in vitro. We determined histoenzyme activity of one of the key Krebs cycle enzyme – suczinatdehydrogenase(SDG) and of gly-colytic marker enzyme – lactatdehydrogenase(LDG) in rat cardiomyocytes before(contr.) and after(exp.) 30 min incubation with EPO(Cepo=2,0 U/ml) at 37º C since the activity of this enzymes reflect the processes of energetic vital functions of cells. Additionally we registered the fluorescence intensity DSM (fluorescent cationic probe) (Fdsm) in rat cardiomyocytes before and after incubation with EPO as far as at present moment it is established that changes in FDSM depend on the physiological status of cells.Methods. Rat myocardium specimens were frozen directly after removal in two steps: by immersing in intermediate medium and then – in liquid nitrogen; the sections were sliced using a microtome at -20º C; the sections were placed on a glass microscope slides; the Fdsm was analyzed by fluorescent microscope; enzyme activity was analyzed on cytophotometer by point counting method in 10 fields of vision.Results. Mean values of SDG and LDG activity in cardiomyocytes were increased from contr. to exp. data by 31,6+1,1%(P≤0,05) and by 38,5+1,5%(P≤0,05) respectively . The incubation of cardiomyocytes with EPO resulted in significant increase of average fluorescence intensity of DSM(Δ Fdsm) by 53,1+2,5%(P≤0,01). Moreover positive correlation between the incre-ase of Δ Fdsm and mean values of SDG activity(0,005≤P≤0,01); between increase of Δ Fdsm and mean values of LDG activity(P≤0,005) was found. Conclusions. 1.The incubation with EPO activated aerobic and anaerobic pathways of energy support of cardiomyocytes. 2. Changes in Δ Fdsm allow to suggest about biochemical changes in energetic activity of cardiomyocytes.


Effects of nitric oxide on hematogenous metastasis

M. Schwartz¹, T. Otto¹, U. Brockmeier¹, S. Lauterbach¹, E. Metzen¹, E. Gulbins², A. Carpinteiro², N. Scheerer¹, J. Fandrey¹

¹Institut für Physiologie²Institut für Molekularbiologie, Universität Duisburg-Essen

The appearance of metastases is a characteristic feature of malignant tumor disease. Tumor hypoxia thereby tends to result in a significant increase of metastasis. The adaption of tumor cells to hypoxic conditions is basically driven by hypoxia-indu-cible transcription factors such as the hypoxia-inducible factor 1 (HIF-1). In vitro hypoxic HIF-1 activity can be modulated by nitric oxide. To investigate the effects of this modulation on hematogenous metastasis B16F10-melanoma cells were injected into the tail vein of C57Bl/6J-mice. Nodules visible on lung surface were counted fifteen days after injection. Twelve hours of hypoxic pre-incubation of B16F10-melanoma cells led to increased bulk of lung nodules. In contrast simultaneous pre-incubation with 100 µM DETA-NO completely eliminated this hypoxic effect. Parallel performed in vitro experiments showed a reduced hypoxic induction of the HIF-1 target gene carbonic anhydrase IX after the application of DETA-NO. Further in vitro and in vivo experiments with stable HIF-1 knock down B16F10-melanoma cells will provide evidence for the role of HIF-1 in hematogenous metastasis after NO exposure.

Modulation of oxygen sensing by nitric oxide in an autochthonous mouse model of breast cancer

Anika Thiel¹, Agnes Neugebauer¹, Christian Stockmann¹, Utta Berchner-Pfannschmidt² , Nina Scheerer¹, Joachim Fandrey¹

¹Institut für Physiologie, Universitätsklinikum Essen, Universität Duisburg-Essen²Zentrum für Augenheilkunde, Universitätsklinikum Essen

Tumor cells respond to hypoxia by adapting gene expression through the activation of the transcription factor hypoxia-induci-ble factor 1 (HIF-1). Hydroxylation of the HIF-1α subunit by prolyl hydroxylase (PHD) oxygen sensors leads to its degradation and inactivation. HIF-1 has been recognized as an accelerating factor in mammary tumor progression and metastasis. In vitro data provide evidence that accumulation of HIF-1 can be modulated by nitric oxide (NO)-releasing substances. Thus the aim of this study was to examine the influence of the NO donor DETA-NO on tumor growth and metastasis in vivo using an autochthonous mouse model of breast carcinoma. We studied mice that develop mammary tumors over a period of 20 weeks due to expression of the polyoma middle T (PyMT) oncoprotein in mammary epithelial cells. Mice carrying the PyMT transgene were injected every third day intra-peritoneally with DETA-NO or saline at an age of eight weeks over a period of 12 weeks. Our data suggest that treatment with DETA-NO results in an accelerated tumor onset, higher tumor burden and increased pulmonary metastasis. Further studies including mice with a deletion of HIF-1α in mammary epithelial cells will elucidate the role of HIF-1 in NO-mediated tumor progression and metastasis.


WHY RECOMBINANT ERYTHROPOIETIN THERAPY IS VERY EFFECTIVE FOR THE TREATMENT OF IRON DEFICIENCY ANEMIA IN PREGNANCY?

1Demikhov V.G., 1Demikhova E.V., 2Zinovyeva E.N., 1Pavlov A.D., 1Morshchakova E.F.

1 Federal Clinical Research Center for Pediatric Hematology, Oncology and Immunology named D. Rogachov, Ryazan Branch, Ryazan, Russian Federation

2 Ryazan Maternity Hospital №1, Ryazan, Russian Federation

It is reputed that pathogenesis of anemia in pregnancy (AP) is relation to simple iron deficiency and iron therapy is the basic treatment of anemic pregnant women all over the world. But own and other researchers data demonstrated that recombinant human erythropoietin (rHuEPO) therapy is very effective for the treatment of AP. We have hypothesized AP has more com-plex pathogenesis than ineffective erythropoiesis, caused by iron or folate deficiency. If AP is iron deficiency anemia then rHuEPO therapy of the anemia has not been such beneficial indeed. To show more complex pathogenesis an anemia in pregnancy we investigated the adequacy of the EPO production for the degree of anemia in pregnant women. A total 268 anemic pregnant women and 24 normal Hb level pregnants were tested. Control group consisted of 22 non-pregnant women with iron-deficiency anemia (IDA). EPO values were measured immunoenzymometrically by using ELISA-EPO kits. Serum levels of some proinflammatory cytokines (IL-8, INF-gamma) and estrogens were determinate in anemic pregnants and in 22 healthy non-pregnant women in addition. Majority anemic pregnant had iron deficiency but showed significantly lower serum EPO levels, than non-pregnant women with IDA. As compared with anemic non pregnants controls, the mean O/E (log EPO) ratio was significantly lower in these anemic pregnant women. But interestingly that inadequately low production of EPO was found in women who had Hb level loss 90 g/dL only. The significant elevated serum levels of IL-8 and INF-γ observed at all pregnant women versus healthy non-pregnant women. Highest INF-γ levels were found in pregnants with IDA as compared with normal iron status anemia. It is very interesting that anemia in pregnancy associated with increased levels of INF-gamma and estrogens only in aggregate, but not separately. We are suppose that development of IDA during second half of pregnancy can lead to hypoxia of placenta which increase production of proinflammatory cytokines (INF-γ). It is known that hyperestrogenemia increase lymphocytic production of INF-γ too. Excess of proinflammatory cytokines (INF-γ) inhibits erythropoiesis in IDA during pregnancy. That is why rHuEPO has high effectiveness in AP.

Semisynthesis of human Erythropoietin glycosylated at Asn24

Karen Gottwald, Vera Ullmann, Andreas Reif, Marisa Rädisch, Carlo Unverzagt

Department of Bioorganic Chemistry, Building NW I, University of Bayreuth, 95440 Bayreuth, Germany

Due to its biomedical relevance, the availability of erythropoietin (EPO) variants with a defined glycosylation pattern should lead towards a new generation of this highly specific glycoprotein therapeutic [1]. Depending on the presence and the struc-ture of EPO's carbohydrate moieties, different biological activities emerge [2]. Full in vivo activity relies on the presence of three tetraantennary N-glycans and the degree of sialylation [3]. Biantennary N-glycan EPO variants show stronger binding to the EPO receptor, but display lower in vivo activity [4]. These seminal data were deduced from mixtures of glycoforms since homogenous glycoproteins are still not available. Thus for more detailed structure-activity-relationships synthetic al-ternatives are required. One approach to accomplish this task is the native chemical ligation (NCL) developed by Kent and co-workers [5]. Having established a semisynthesis of bovine RNase C via NCL [6], we currently try to apply the methodology to more complex glycoproteins, like EPO [7]. For creating an EPO with a defined single glycosylation at Asn24, the protein was divided into two segments. The glycosylated thioester fragment 1-28 was synthesized on the solid phase [6], while the cysteine-fragment 29-166 was expressed in E. coli as SUMO fusion protein [8]. Initially inclusion bodies were obtained, which were purified and refolded prior to enzymatic cleavage with the SUMO protease Ulp1 [9]. With the two EPO building blocks (EPO 1-28 (Asn24GlcNAc) thioester and EPO 29-166) in hand, ligations were carried out leading to full-length EPO 1-166 glycoprotein. [1] E. Higgins, Glycoconj. J. 2010, 27, 211-225. [2] C. Kan, S. J. Danishefsky, Tetrahedron 2009, 65 (45), 9047–9065. [3] W. Jelkmann, Physiol. Rev. 1992, 72 (2), 449-489. [4] T. Misaizu, S. Takasaki et al., Blood 1995, 86, 4097-4104. [5] S. Liu, B. L. Pentelute, S. B. H. Kent, Angew. Chem. Int. Ed. Engl. 2012, 51(4), 993-999. [6] C. Piontek, C. Unverzagt et al., Angew. Chem. Int. Ed. Engl. 2009, 48 (11), 1936-1940. [7] M. Murakami, Y. Kajihara et al., Angew. Chem. Int. Ed. Engl. 2012, 124, doi: 10.1002/anie.201200881. [8] T. Panavas, C. Sanders, T. R. Butt, Methods Mol. Biol. 2009, 497, 303-317. [9] S. J. Li, M. Hochstrasser, Nature 1999, 398, 246-251.


Novel Erythropoietin-Receptor peptide antagonists

Hiram-Bab S.*#, Liron T.*#, Souroujon M.C.†, Eisenstein M.§ and Neumann D.*# These two authors contributed equally to the study

*Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel†Department of Natural Sciences, The Open University of Israel, 1 University Rd, Raanana 43107, Israel.

§ Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, IsraelThis study is supported by the FP7 European commission grant; 282551 EpoCan.

Erythropoietin (EPO) is a cytokine that promotes the proliferation and differentiation of erythroid progenitor cells. It does so by binding to its surface receptor, EPO-R. Recombinant Human EPO (rHuEPO) is therefore extensively used as treatment for anemia in chronic kidney failure patients and in cancer patients suffering from treatment-associated anemia. Recently, EPO-R was identified in non-erythroid cells in the heart, lung, brain, the immune system as well as in several types of cancer cells. EPO signaling in non-erythroid EPO-R expressing cells was shown to have anti-apoptotic and anti-inflammatory ef-fects. Thus, the presence of EPO-R on certain cancer cells raises the concern that activation of the receptor upon treatment with rHuEPO would promote undesirable proliferation of these cells. Currently there are no available EPO-R blocking agents except for anti-EPO neutralizing antibodies and anti-EPO-R antibodies. Here we present a rational design of novel EPO-R antagonists. Based on structure analysis of the murine EPO-EPO-R complex and computational anchoring spots mapping, we have synthesized several short peptides that were able to inhibit the binding of EPO to EPO-R. Furthermore, these pep-tides were able to inhibit EPO-induced EPO-R phosphorylation and cell proliferation. Such novel EPO antagonists may be used as research tools to study EPO-induced functions and to treat patients suffering from an increase in red cell mass (erythrocytosis). Moreover, targeting of such antagonists to malignant cells that express EPO-R could be used to selectively inhibit potential undesirable proliferative effects of EPO on these cells.

RECOMBINANT HUMAN ERYTHROPOIETIN THERAPY OF ANEMIC PATIENTS WITH LUNG TUBERCULOSIS

1Inyakova N.V., 1Demikhov V.G., 2Dolzhenko E.N., 1Golovitsyna O.A., 1Morshchakova E.F.

1 Federal Clinical Research Center for Pediatric Hematology, Oncology and Immunoly named D. Rogachov, Ryazan Branch, Ryazan, Russian Federation

2 Ryazan Regional Tuberculosis Hospital, Ryazan, Russian Federation

Background. Blunted erythropoiesis is one of main factors of TB-associated anemia pathogenesis. These data substantiates recombinant human erythropoietin (rHuEPO) therapy of anemic tuberculosis patients. We could not search out any publica-tions which are relation to the management of the anemia in TB patients with rHuEPO. Patients and methods. To evaluate efficacy of rHu-EPO therapy, sixteen anemic patients with lung tuberculosis the mean age 38.8±3.85 years were enrolled. They met the following criteria for inclusion in the study: Hb concentration < 11.0 g/dl, absence of thrombosis history and diastolic blood pressure less than 100 mm Hg. All patients received rHu-EPO according to protocol 1or 2. Eight patients were treated with 300 IU/kg of rHu-EPO three times per week subcutaneously and 200 mg iron (Fe II) orally daily for a total period of the rHu-EPO therapy (protocol 1). They had a baseline Hb level 81.1±2.74 g/dl. The treatment protocol 2 in other 8 patients comprised a combined therapy with 600 IU/kg rHu-EPO intravenously and 100 mg (5 ml) iron sucrose intravenously twice weekly. In these patients baseline Hb level was 76.9±5.34 g/dl. Target range of Hb was 11.0-12.0 g/dl. Withheld dose of rHu-EPO if Hb>12.0 g/dl. All patients received rHu-EPO during 5-6 weeks. Results. Six (75%) out of 8 patients treated according to protocol 1 showed a complete response to rHuEPO therapy, with Hb reaching target range within the first 4 weeks of the treatment. The overall Hb level after rHuEPO therapy was 11.5±1.19 g/dl. All these 8 anemic TB patients showed complete reticulocyte reactions. Target range of Hb in patients reached in 7 (87.5%) out 8 anemic patients treated according to pro-tocol 2. Mean Hb after rHuEPO therapy was 11.6±5.56 g/dl. Thus, complete response to rHuEPO therapy was found in 13 (81.3%) out of 16 anemic patients with lung tuberculosis. No influence of epoetin therapy on blood pressure, laboratory safety variables, or the frequency of specific adverse events was observed. Conclusions. Results of the pilot study demonstrated high effectiveness of rHuEPO therapy of anemic TB patients. We speculate that increased Hb level isn't the one benefit of rHuEPO therapy in these patients. Other advantage of the rHuEPO therapy seem is an improvement of quality of life them, so far as this effect is typical for all epoetins. We hope that rHuEPO therapy will be to improve outcomes and prognosis in anemic ТB patients too.


EPO-Independent Functional EPO Receptor in Breast Cancer Enhances Estrogen Receptor Activity and Promotes Cell Proliferation

Susann Schiebel (1,2), Anna-Maria Larsson (1,2), Marica Vaapil (1,2), Jianmin Sun (3), Annika Jögi (1,2), Lars Rönnstrand (3) and Sven Påhlman (1,2)

(1) Center for Molecular Pathology, (2) CREATE Health, (3) Experimental Clinical Chemistry, Department of Laboratory Medicine, Malmö University Hospital, Lund University, Sweden

Background The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis in response to low blood oxygen levels. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. Recent clinical trials have indicated that rhEPO treatment can have a negative effect on tumor progression and patient survival. In addition, EPOR expression has been detected in different cancer forms. Until today the presence and function of EPOR in tumors are still controversial, in part due to the lack of specific anti-EPOR antibodies. Former findings of our group have shown that high EPOR levels are related to an impaired tamoxifen response in estrogen receptor-positive (ER+) breast tumors.Aim Based on this former finding the aim of this study is to reveal the function of the EPOR in ER+ breast cancer cells. Results Here we report an in-house polyclonal antibody that reliably detects the full-length isoform of the EPOR and we show that breast cancer cell lines as well as breast cancer tissue express the receptor protein. Interestingly rhEPO treatment failed to stimulate phosphorylation of the EPOR and its classical downstream signaling proteins: JAK2, STAT3, STAT5, AKT and ERK1/2. A function of the receptor was however demonstrated in EPOR knockdown experiments resulting in significant de-creased proliferation of ER+ cancer cells, while these effects were not seen in ER negative cells. EPOR knockdown induced a decrease in ER activity, which could explain the EPOR effect on proliferation. Conclusion In conclusion we show that EPOR protein is expressed in breast cancer cells where it appears to act as a novel EPO-inde-pendent proliferation factor in ER expressing breast cancer cells, possibly via its capacity to modulate ER activity.

PREOPERATIVE USE OF RECOMBINANT HUMAN ERYTHROPOIETIN FOR PREVENTION OF BLOOD TRANSFUSIONS IN PULMONARY TUBERCULOSIS PATIENTS

1Nikolaev, A.N., 2Demikhov V.G., 3Dobin V.L., 2Inyakova N.V., 2Skobin V.B., 2Morshchakova E.F.

1 Ryazan Regional Tuberculosis Hospital, Ryazan, Russian Federation 2 Federal Clinical Research Center for Pediatric Hematology, Oncology and Immunology named D. Rogachov,

Ryazan Branch, Ryazan, Russian Federation3 Ryazan State Medical University named I.P.Pavlov, Ryazan, Russian Federation

Background. Tuberculosis is a chronic, infectious disease that primarily attacks the lungs. In some cases these patients may undergo operative intervention to remove the infected areas. In these cases postoperative anemia are usual. Postoperative anemia is harmful for these patients as it impairs rehabilitation and hospitalization time them. Aim of the study was an evalu-ation of ability of preoperative use of recombinant human erythropoietin (rHuEPO, epoetin alfa) to decrease allogenic blood transfusions and the associated risks in patients undergoing surgical treatment of pulmonary tuberculosis. Patients and me-thods. Ten pulmonary tuberculosis patients aged 24 to 53 years those who had high risk to receive blood transfusions after planned operative intervention were enrolled into the trial after providing informed consent. Mean Hb values before rHuEPO administration were 123.1±11.43 g/L, mean serum ferritin levels – 31.4±18.23 μg/L. The trial medication 600 IU epoetin-alfa/kg body weight per week was administered subcutaneously on preoperative days 14, 7, and day of operation. The exclusion criteria were diastolic blood pressure greater than 100 mm Hg, hematocrit level greater than 0.45, Hb level over 140 g/l, platelet count greater than 500×109/L. All patients received 100 mg (5 ml) iron sucrose intravenously twice weekly during two weeks before operation and one more on operation’s day. After operative intervention we evaluated Hb concentrations and serum ferritin levels during four weeks and blood transfusion’s volume and quantity. Results. Perioperative blood loss were from 165 to 1350 mL (407.3±237.65 mL). None of all patients who received rHuEPO and iron sucrose has not required peri-operative blood transfusions. The mean Hb concentrations in operated patients in 1 day after surgery were 124.2±16,37 g/L. Mean serum ferritin levels in patients to four week of observation composed 91.1±47.35 μg/L. No adverse effects of rHuEPO were noted. Conclusions. This study suggests that in pulmonary tuberculosis patients, who has high risk to receive blood transfusions after planned operative intervention, preoperative use of epoetin alfa combined with iron sucrose treatment can be an efficient method to decrease perioperative transfusion requirements and to increase perioperative Hb concentration. Moreover, we expect that preoperative epoetin alfa treatment might also improve rehabilitation of these patients.


Serum erythropoietin, plasma volume regulating hormones, and plasma volume changes induced by exercise and thermal dehydration

Zbigniew Szygula¹, Magdalena Wiecek¹, Marcin Maciejczyk¹, Wanda Pilch¹, Agnieszka Zembron-Lacny², Kenny De Meirleir³, Johan Smitz³

¹University School of Physical Education, Krakow, Poland²Faculty of Physical Culture, Gorzow Wlkp., Poland

³Free University of Brussels (VUB), Brussels, Belgium

The phenomenon of EPO biosynthesis under conditions of exercise load has not been fully explained yet. It has been hypo-thesized that some of the variability in the EPO response may be a result of post-exercise changes in plasma volume (PV), and that EPO may also act as blood regulating hormone. Therefore, the aim of our study was to investigate changes in serum EPO concentration (s[EPO]), as well as the key PV regulating hormones after changes in PV induced by long lasting exercise and by thermal dehydration. Fifteen healthy men (20-24 yr) participated in the study. Subjects participating in the experiment I (Exp I; n=7) exercised at 55% VO2max on the cycloergometer till exhaustion (80-140 min). Eight other subjects participated in thermal dehydration in Finnish sauna (Exp II). In Exp I PV decreased by 8.8% just after exercise, however after 3 h the PV slightly (+3%) exceeded the resting value, and after 9 h increased by 7.8% (p<0.05). The highest increase in PV was observed on the 2nd day (+9%) and remained elevated on the third day (+5.6%). An increased s[EPO] was noted after 9 h (+71%), on the 2nd (+64%) and 3rd third (+52,5%) day following exercise (p<0.05). Plasma renin activity (PRA) increased 4 times, aldosterone (ALDO) concentration 8.5 times, ANP by 70%, and AVP by +105%. PRA, as well as ALDO, and AVP concentration remained elevated up to 3 hours following exercise, however, ANP concentration decreased. Concentration of the hormones were not correlated with EPO concentration. Exp II: PV decreased by 10% just after sauna (p<0.05), then it increased with the highest value on the 2nd day (+10.7%) and maintained at that level during the two subsequent days (+7% and +8.5%) (p<0.05). It also resulted in the increase in s[EPO] - most intensified after 9 hours (+55%) and maintained at that level during the 2nd (+44.6%), 3rd (+35.5%) and 4th (+35.5%) day (p<0.05). A positive correlation was found between the changes in PV and s[EPO]. Conclusions: The decrease in PV (dehydration) may stimulate EPO synthesis through the incre-ase of RAA activity, the increase in sodium reabsorption in renal tubules, and intensified metabolism in renal tissue. Although EPO is considered to be one of the hormones regulating PV, no constant relationship was observed between the changes in s[EPO] and other volume regulating hormones (renin, ALDO, ANP and AVP).

Epo and EpoR signaling dynamic in hematopoietic and tumor context

Agustin Rodriguez(1,2)*, Max Schelker(3,4)*, Marcel Schilling(1), Julie Bachmann(1), Andreas Raue(3,4), Florian Salopia-ta(1), Sofia Depner(1), Lorenz Adlung(1), Ruth Merkle(1), Andreas Franke(5), Michael Jarsch(5), Jens Timmer(3,4,6,7) and

Ursula Klingmüller(1,2)

1 Division Systems Biology of Signal transduction, DKFZ-ZMBH Alliance, German Cancer Research Center, Heidelberg, Germany.

2 Bioquant, Heidelberg University, Germany.3 Institute of Physics, University of Freiburg, Germany. 4 Freiburg Center for Systems Biology (ZBSA), University of Freiburg, Germany.5 Roche Diagnostics GmbH, Penzberg, Germany.6 Freiburg

Institute for Advanced Studies, University of Freiburg, Germany. 7 Centre for Biological Signaling Studies (BIOSS), University of Freiburg, Germany.

Lung carcinoma is the most frequent cause of death in cancer. The current therapeutic approaches cause anemia, reducing the therapeutic response and the quality of life in patients. In order to correct this, recombinant human erythropoietin (rhEpo) has been widely used to reestablish normal levels of erythrocytes. But, EpoR expression has been found in tumor cell lines and primary cancers. Furthermore, several clinical trials have associated Epo treatments with cancer progression. Due to the referred studies, the administration of Epo to the cancer patients is still under discussion. In order to clarify the potential role of Epo in a tumor context, we combined mathematical modeling with quantitative data from NSCLC (non Small Cell Line Carcinoma) and hematopoietic cells. For this purpose we firstly screened NSCLC cells for significant levels of EpoR mRNA (by RT-PCR) and EpoR protein expression (by quantitative IB and MS). Later on, we cha-racterized the dynamic of EpoR activation, Epo depletion and the subsequent signaling of JAK-STAT pathway in NSCLC cells (by quantitative IB and ELISA). Two ordinary differential equation models have been calibrated with the EpoR-transduced Baf3 (mouse hematopoietic cell line) and with the CFU-E (mouse Colony Forming Unit Erythroid). The first model describes the EpoR dynamic, and the second model describes the JAK2-STAT5 signaling upon Epo stimulation. These two models enable us to compare and define the dynamic of Epo/EpoR interaction and signaling activation, in hematopoietic and tumor context. Based on model predictions, we hypothesize sensitive and safe range of Epo concentrations that induce erythropoi-esis but do not activate the NSCLC cells.


Effects of HIF stabilizers in isolated perfused rat kidneys: implications for blood doping

Horst Pagel¹, Eric Metzen², Wolfgang Jelkmann¹

¹Institute of Physiology, University of Luebeck, and ²Institute of Physiology, University of Duisburg-Essen, Germany

Some athletes apply “blood doping” to improve performance. Since peptidic erythropoiesis stimulating agents can be de-tected by chemical tests, cheating athletes may seek alternatives. Of interest are substances that stabilize the HIFs and induce Epo expression. Herein, effects of cobalt chloride (Co) and the 2-oxoglutarate competitor pro-drug di-tert-butyroyl-oxymethyl-2,4-pyridine-dicarboxylate (tBu-2,4-PDC) were compared in isolated perfused rat kidneys (3 h perfusion periods). The results show that both HIF stabilizers stimulated the production of immunoreactive Epo under normoxic conditions. At 50 μM Co, Epo levels were elevated to the levels on hypoxic perfusion (3% O2), and further increased at 100 μM Co. The addition of Co to the perfusion medium had no influence on renal function, i.e., renal vascular resistance, perfusion flow rate, glomerular filtration rate and urine production rate were unaltered vs. the normoxic controls. Neither were water, sodium, calcium or glucose reabsorption rates altered. Epo production was also stimulated by the addition of tBu-2,4-PDC. At 50 µM tBu-2,4-PDC, Epo levels in the perfusion medium were similar to the level measured on hypoxic perfusion; at 100 µM tBu-2,4-PDC the production of Epo was almost doubled. However, tBu-2,4-PDC increased diuresis, and the ability to produce hypertonic urine was greatly reduced, as indicated by a low urine/perfusate-inulin ratio. tBu-2,4-PDC impaired tubular func-tions (reduced water, sodium and calcium reabsorption rates). Further, it increased the permeability of the glomerular filtration barrier for macromolecules. In conclusion, although cobalt is no longer used as an anti-anemic drug in clinical routine, it may be misused in sports, as it exhibits relatively little nephrotoxicity, is readily available, inexpensive and very potent with respect to stimulating Epo production.


The Meeting is Supported by

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