Erythropoietin and Erythropoietin-Like
Agents As Pharmacological
Countermeasures During Space
Exploration
Arthur J. Sytkowski, MD
Laboratory for Cell and Molecular Biology
Department of Medicine
Beth Israel Deaconess Medical Center
Harvard Medical School
Center for Advanced Space Studies, Houston , 28 June 2005
Outline of the Presentation
• Physiology and cell biology of erythropoietin,
a.k.a. “Epo”.
• Hematopoietic and non-hematopoietic antiapoptotic actions of Epo.
• Epo as a pharmacologic countermeasure in
spaceflight.
• Long-acting Epo-like agents.
Epo Physiology in the Adult Human
Ebert & Bunn, 1999
Importantly, Epo also acts on other organs and tissues and is produced
and acts locally (autocrine/paracrine action).
Peritubular Interstitial Cells Produce Epo: mRNA
Detected By In Situ Hybridization and
Autoradiography
Koury et al., 1988
Primary Structure of Human Epo
Oligosaccharides are critical for in vivo activity.
Cytokine Receptor Superfamily
Type I,
Homodimer
Watowich, 1996
EpoR Dimerization and Conformational
Change Upon Epo Binding Initiates Signal
Transduction
There is also evidence of pre-formed EpoR dimers.
dimers
Structure of the Epo Receptor
Phosphorylated
tyrosines and
other special
domains are
docking sites for
signal
transduction
molecules.
Signaling Proteins That
Associate With the EpoR
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Jak2, STAT5, Cis1.
PTKs: Lyn, Syk, Tec
PTPs: SHP1 & SHP2
Phospholipid modifying
enzymes: PI3-K, PLCγ
& SHIP
• Adaptor proteins:
GRB2, Shc, Cbl,
CrkL, APS, IRS-2,
Gab1 & Gab2.
• Nucleotide exchange
factors: Vav, C3G &
mSOS
Principal EpoR Signaling Pathways
• JAK2/STAT5…Phospho-STAT 5b is a hallmark
of Epo’s anti-apoptotic signal.
• PI3-Kinase…regulates c-myc transcription and
phosphorylates Akt, another anti-apoptotic signal.
• Raf/MEK/MAP kinase …also regulates c-myc
transcription by a different mechanism.
• PKC…the epsilon isoform is especially important.
EpoR Is Found on Other Tissues: Epo
Also Has Non-Hematopoietic Actions
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Endothelium: angiogenesis and wound healing.
Central nervous system: neuroprotection.
Heart: tissue protection.
Gastrointestinal tract: tissue protection.
Other cell types: actions unknown.
Epo Stimulates Endothelial Cell Growth
and Binds to Cell Surface Receptors
Anagnostou et al. 1990
Epo and Endothelial Cells
• Increases angiogenesis in rat aortic ring. Carlini et al.
1995.
• Up-regulates immediate/early genes (growth).
Fodinger et al. 2000.
• Induces phosphorylation of JAK2 and STAT5 antiapoptosis. Fuste et al. 2002.
• Protects against hypoxia-induced apoptosis by
activation of AKT-1. Chong et al. 2002.
Epo Promotes Wound Healing
• Increases granulation tissue an in vivo woundhealing assay. Haroon 2003.
• Stimulates angiogenesis and healing of
experimental ischemic skin wounds. Buemi 2004.
• Stimulates angiogenesis and wound healing in the
genetically diabetic mouse. Galeano 2004.
Epo and EpoR in the CNS
• Hypoxia induces appearance of endogenous Epo mRNA in rat
brain. Tan et al. 1992.
•
125I-Epo
binds to mouse brain slices. Endogenous Epo and
EpoR mRNA detected by RT-PCR. Digicaylioglu et al. 1995.
• Epo and EpoR detected in monkey and human brain by RTPCR. Marti et al. 1996.
• Epo and EpoR detected in developing human brain by
immunohistochemistry. Juul et al. 1999.
Affinity Cross-Linking of 125I-Epo to
EpoR of Erythroid and Neuronal Cells
Difference in size of cross-linked complexes implies
fundamental difference between EpoRs of the two cell types.
Some Effects of Epo on Neuronal Cells
• Increases intracellular free calcium. Assandri
1999.
• Increases membrane polarization, dopamine
release and tyrosine hydroxylase activity.
Koshimura 1999.
• Promotes differentiation of oligodendrocytes
and growth of astrocytes. Sugawa 2002.
EpoR Is Found Within And Around
Human Brain Capillaries.
Brines 2000
(A) EpoR in capillaries (arrow). (B) High-power view (C) EpoR at capillary (c)
identified as an astrocytic process (a). Astrocytes contain EpoR. (D) EM shows
EpoR within astrocytic foot processes (*), and ECs (arrows).
Epo Crosses the Blood-Brain Barrier
(A) Biotinylated Epo (bEPO) seen around capillaries 5 h after i.p. injection.
(B) bEPO surrounds the lumen of capillaries (arrow). (C) bEpo not observed if
injected along with 100 times excess of unlabeled Epo (bEPO + EPO).
Epo Attenuates Brain Injury
After Blunt Trauma
Injury without
Epo pretreatment
Injury with
Epo pretreatment
Brines et al. PNAS 2000
Anti-Inflammatory Effect on the CNS:
Exptl Autoimmune Encephalomyelitis
H & E stain
Anti-glial
fibrillary acidic
protein
(GFAP) stain
Anti-CD11b stain
Rat lumbar spinal cord sections from unimmunized
(control), EAE rat (immunized with myelin basic
protein), and EAE/Epo rat.
Agnello et al., 2002.
2002
Epo Protects Retinal Neurons From
Acute Ischemia-Reperfusion Injury
Non-ischemic
Ischemic
Ischemic +
Epo Pre-Rx
Ischemic +
Epo Post-Rx
Histological appearances of the non-ischemic (control) and
ischemic (vehicle and Epo-treated) retinas at 7 days of
reperfusion after ischemia. A, control; B, ischemic (45-min)
vehicle-treated; C, ischemic (45-min) and Epo-pretreated;
D, ischemic (45-min), Epo-posttreated. Epo-treated animals have
significantly less retinal thinning compared with vehicle-treated
controls.
Junk et al, 2002
Epo and the Heart
• Epo and retinoic acid are secreted from
epicardium during embryogenesis.
• Blocking of endogenous Epo inhibits
proliferation and survival of cardiomyocytes.
• Epo (both endogenous and administered)
protects against ischemia/reperfusion injury.
Epo and the Gut
• Epo found in human milk.
• EpoR detected in small bowel of human fetuses.
• Enterocytes of post-natal rats migrate faster if exposed
to Epo.
• Reduced incidence of necrotizing enterocolitis in low
birth weight infants receiving rhEpo.
Epo and Other Cell Types
• EpoR in kidneys.
• Epo in myoblasts.
• EpoR on pancreatic islets.
So What Does All Of This Have To
Do With Spaceflight?
• Long-term human spaceflight presents unique
problems in autonomous medical care as well as
identified risks to health for which countermeasures,
including novel pharmaceuticals, especially longacting pharmaceuticals, will be needed.
• Among the risks identified in the Bioastronautics
Roadmap of special relevance to Epo are
• hemorrhage/anemia /blood replacement,
• major trauma and
• pharmacology of space medicine delivery.
We hypothesize that long-acting and ultra-longacting forms of Epo will prove to be valuable
therapeutic agents and pharmacological
countermeasures in support of the
treatment/prevention of trauma and of
hemorrhage/anemia/blood replacement.
Importantly, Epo has several crucial non-hematopoietic
actions including
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increased wound healing
neuroprotection and
cardioprotection.
Thus, Epo can serve as a new countermeasure for these
and, potentially, other diverse risks of human
spaceflight.
Long-acting forms of Epo, such as those that we are
developing, should have even greater impact.
In addition to these risks for which Epo and
derivatives may serve as valuable
countermeasures/treatments, additional
health risks may be counteracted by other
long-acting recombinant protein therapeutic
agents, including, but not limited to, muscle
wasting and bone loss.
Problems With Conventional
Recombinant Epo
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Must be glycosylated- produced in mammalian cells.
Relatively short in vivo half-life: 6-12 hr.
Must be injected.
Delay in physiological response: 2-4 weeks.
There are several approaches to address these problems,
including darbepoetin, an Epo mutant with two additional
glycosylation sites, which increase its half-life..
Our strategy is the production of Epo dimers by
chemical crosslinking or as a fusion protein.
Hypothetical Action of Epo Dimer
Monomer
Epo Dimer
Increased affinity and
EpoR clustering
amplifies signaling.
Linker length is critical
• Also, the increased size and glycosylation
of the Epo-Epo dimer should result in a
markedly prolonged plasma half-life.
• So we predict a double effect of Epo
dimerization, increased action on the cell
and increased survival in the circulation.
Chemical Crosslinking of Epo
• Purpose
• Hypothesize that T1/2 is a function of size
• Create a “large Epo” or oligomer with
increase number of EpoR binding sites
• Approach
• Control degree of crosslinking and size
• Use two chemical modifying agents to
prepare two pools of modified Epo
First Approach: Chemical
Crosslinking of Epo
LC-SPDP
Epo A
SMCC
SH M
Epo B
LC-SPDP = succinimidyl 6-[3-(2-pyridyldithio) propionamido] hexanoate
SMCC = succinimidyl 4-(N-maleimidomethyl
M = maleimido group
SDS-PAGE of Epo-SH, M-Epo and
Crosslinked Epo Dimer and Trimer
Trimer
Dimer
Monomer
SH
M Epo-SH
+
M-Epo
Activity of Epo Oligomers
Separated By SE HPLC
IU/µg
Monomer 160
Dimer
210
Trimer
100
Pharmacokinetics of Epo
Monomer and Dimer in Rabbits
Epo Dimer ( ) Activity Is
Superior to Monomer ( ) In Vivo
A: 300 IU/kg 3X
B: 300 IU/kg 1X
C: 30 IU/kg 3X
D: 30 IU/kg 1X
Groups of 6 mice were injected subcutaneously.
A Second Approach:
Epo-Epo Fusion Protein cDNA
Encodes A “Recombinant Dimer”
Epo
5’ UTR
Leader
Epo
Linker
Peptide
3’ UTR
Stop
SDS-PAGE and Western Blot of
Epo and Epo-Epo Fusion Protein
Specific Activity,
U/µg
U/pmol
Epo
Epo-Epo
350
13
1000
76
Pharmacokinetics of Epo and Epo-Epo
in Mice
Intravenous injection. Each curve represent 1 mouse.
mouse
Epo
Epo-Epo
Note much longer half-life of Epo-Epo
Epo-Epo is Active In Vivo And is
Superior To Epo
Single subcutaneous dose of 300 U/kg. Post-Hct done after 7 days.
Each line represent 1 mouse.
New Epo Fusion Proteins Under
Study In Our Laboratory
Hexamer
Pentamer
M.W.
Tetramer
Trimer
Dimer = Epo-Epo
SDS PAGE
and
Western Blot
Summary
• Long-term human spaceflight is associated with
novel risks that require countermeasures,
including new pharmacological agents, and with
unique problems in autonomous medical care.
• Long-acting and ultra-long-acting protein
therapeutic agents can be designed to address
some of these risks.
• The Epo dimer and fusion protein concept can
serve as a paradigm for the development of these
new agents.
For more information
about
Erythropoietin
www.wiley.com
Acknowledgment
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Julia Sue
James Fisher
Laurie Feldman
Yijuang Chern
Jennifer Grodberg
Elizabeth Lunn
Mary Risinger
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Kelly Donahue
Kerry Wellenstein
Rudy Spangler
Stephen Bailey
Hiren Patel
Yuqui Li
Changmin Chen
Funding from NIH, DOD and NASA
Greetings from the Laboratory for
Cell and Molecular Biology
Thank you for your kind attention
Arthur J. Sytkowski, MD 617 632 9980
asytkows@bidmc.harvard.edu