Erythropoietin

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ErythropoietinNeuronal Recovery

• Matthew Barber Michael Mienaltowski
• VIPS V Presentation
• October 11, 2002

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What is EPO?- hematopoiesis -
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What is EPO?- recombinant human EPO -

• The cloning and expression of human
  erythropoietin (EPO) was first described by Lin
  et al. in 1985.

• Recombinant erythopoietin (r-hEPO) is used to
  treat patients with EPO-dependent nonregenerative
  anemia.

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What is EPO?- recombinant human EPO -

• Recombinant human EPO therapy has been successful
  with humans and of limited success in animals.
• A large proportion of treated animals develop an
  anti-hEPO immune response.

Is there a solution to the immunogenicity dilemma
for dogs and cats?
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What is EPO?- recombinant cEPO fEPO -

• Dog and cat therapies are being developed.
  Results of early clinical trials are favorable.

• Cloning and expression of canine and feline
  erythropoietin genes were first described by the
  MacLeod Lab in 1998.
• Right Data from MacLeod Lab at Cornell University

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What is EPO?- An alternative therapy? -

• Erythropoietin has also been shown to
• Reduce inflammation
• Inhibit apoptosis
• Promote growth and development in neural stem
  cells.

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Nerve Damage Apoptosis
Two Phases of Neuronal Death 1) Immediate
necrosis of most severely affected neurons (due
to glutamate-mediated excitotoxicity, free
radical production, inflammatory mediators) 2)
Apoptosis of neurons outside of
ground zero due to prolonged
hypoxia
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Nerve Damage Apoptosis

• Proapoptotic
• TR3
• Caspase-1 3
• bax, bak, bid, bad
• p53
• Antiapoptotic
• NF-?B
• TRAF 1 2
• c-IAP 1 2
• Bcl-2 Bcl-w

TUNEL assay for apoptosis
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Nerve Damage Apoptosis

• Proposed Process of Ischemia Induced Apoptosis
• Buildup of nuclease in the nucleus
• Synthesis of transglutaminase
• Release of transcription factor TR3
• Increase in mitochondrial membrane permeability
• Release of caspase enzymes and cytochrome C
• Activation of caspase cascade and nuclease
• Apoptosis

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Nerve Damage Apoptosis
TR3
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Nerve Damage Apoptosis

• Cleanup of Cellular Debris After Neuronal Insult
• Removal of debris from immediate necrosis is
  mediated primarily by PMNs due to the release of
  proinflammatory cytokines released at initial
  insult
• Removal of apoptotic debris is mediated almost
  exclusively by macrophages, so inflammation is
  greatly reduced

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Cellular EPO Recovery

• Treatments to Date for CNS Injury
• Primarily supportive
• High-dose glucocorticoid therapy has been shown
  to be effective, although mechanisms are unclear
• Ex Methylprednisolone

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Cellular EPO Recovery

• Possible treatments in the future include IL-1ra
  and especially EPO, the topic of todays
  discussion

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Cellular EPO Recovery

• EPO induced neuronal Recovery
• Phosphorylation of I-?B
• Nuclear translocation of NF-?B
• Initiation of transcription of neuroprotective
  genes and their subsequent translation-thus
  resulting in ischemic tolerance
• Also, EPO receptors on endothelial cells
  initiates vascular actions to preserve blood
  supply to ischemic areas, thus lessening ischemic
  damage

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EPOs Role InNeuronal Recovery
Goldman Nedergaard. Nature (2002) 8 785-787.
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EPONeuronal Recovery
Recombinant human erythropoietin counteracts
secondary injury and markedly enhances
neurological recovery from experimental spinal
cord trauma Alfredo Gorio, Necati Gokmen, Serhat
Erbayraktar, Osman Yilmaz, Laura Madaschi, Cinzia
Cichetti, Anna Maria Di Guilo, Enver Vardar,
Anthony Cerami, and Michael Brines. Proc. Natl.
Acad. Sci., USA (2002) 99 9450-9455.

• Researchers experimentally injured rats to model
• Aneurysm
• Traumatic Spinal Cord Injury

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EPONeuronal Recovery
Aneurysm Clip Model (48 animals)
Sham (n 6) No aneurysm (just incision)
Control (n 14) Aneurysm Immediate Saline
i.p.
Exp 1a (n 14) Aneurysm Immediate 1000
U/kg r-hEPO, i.p.
Exp 2a (n 14) Aneurysm 1000 U/kg r-hEPO,
i.p. SID for 3 days
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PNAS fig 1-- Treatment after removing aneurysm
clip --

• Good short-term neuro response to EPO at doses of
  1000U/kg (Exp 1a) and 3000U/ml (Exp 2a).
• Likewise, a favorable neurological recovery (18
  out of 21) beginning at days 20-28.

0 no observable hindlimb movements 21
normal gait
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EPONeuronal Recovery
Gorio et al. (2002), Fig 5
Traumatic Spinal Cord Injury Model (56 animals)
Control (n 14) Impaction Saline i.p. 1 hr
post-injury
Exp 1t (n 14) Impaction 5000 U/kg r-hEPO,
i.p., 1 hr post-injury
Exp 2t (n 14) Impaction 5000 U/kg r-hEPO,
i.p. SID for 7 days
Exp 3t (n 14) Impaction 500 U/kg r-hEPO,
i.p. SID for 7 days
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PNAS fig 2-- TSCI treatment studies --

• Favorable short-term neuro response seen with EPO
  treatment (intermediate to significant)
• Favorable neurological recovery after one month
  with HI EPO dose given once.

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PNAS fig 3-- TSCI treatment studies --

• Swimming studies show significance of 5000 U/kg
  r-hEPO dose.
• 500 U/kg r-hEPO SID for 3 days produces a
  response comparable to saline treatment (control)
• BOTTOM LINE
• Dose-dependence suggested

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PNAS fig 4-- EPO is a neuroprotectant --

• Upper 5000 U/kg r-hEPO i.p. for 7 days. White
  matter is preserved.
• Lower Saline treatment. Cavitation with
  widespread degnereration and swollen myelin
  sheaths.

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ArticleSummary
These observations suggest that r-hEPO provides
early recovery of function, especially after
spinal cord compression, as well as
longer-latency neuroprotective, antiinflammatory
and antiapoptotic functions. - Gorio et al.
Proc. Natl. Acad. Sci., USA (2002) 99 9450-9455.
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Just A Few Possible Future Indications For EPO

• EPO-dependent anemia, of course
• Spinal Cord Injury
• HBC
• Vessel nerve damage due to prolonged surgery
• retinopathies

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EPONeuronal Recovery
Beneficial effects of systemic administration of
recombinant human erythropoietin in rabbits
subjected to subarachnoid hemorrhage Giovanni
Grasso et al. Proc. Natl. Acad. Sci., USA (2002)
99 5627-5631.
After an aneurysmal subarachnoid hemorrhage,
cerebral vasoconstrictions leads to ischemia,
impairment of neurological function, and then
death
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PNASSAH fig 2
A control B control placebo C control
r-hEPO
D SAH E SAH placebo F SAH r-hEPO
EPO eases vasoconstriction
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PNAS SAH BBB access

• Also Increased CSF concentrations of EPO in
  rabbits with SAH
• Possible mechanism

SAH
Administration of r-hEPO
BBB allows increased EPO access to CSF
Endothelial EPO receptor activation
Neuroprotection
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EPO Nerve Regeneration

• Erythropoietin and VEGF Promote Neural Outgrowth
  from Retinal Explants in Postnatal Rats
• Simone Böcker-Meffert, Philip Rosenstiel, Claudia
  Röhl, Nils Warneke, Janka Held-Feindt, Jobst
  Sievers and Ralph Lucius
• From the Institute of Anatomy,
  Christian-Albrechts-University of Kiel, Kiel,
  Germany.

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EPO Nerve Regeneration
Addition of EPO to retinal explants in vitro
increased the number of neurites up to 169
19, a hitherto unknown function of EPO in the
CNS
Bocker-Meffert et al. (2002) Fig. 1
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Summary

• Erythropoietin is a hormone produced by the
  kidney which was commercially developed for
  treating EPO-dependent anemia.
• Basic research into apoptosis and the regulatory
  role of EPO has sparked interest into
  erythropoietins potential roles in
  neuroprotective mechanisms.

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Indications for Further Research

• Proper dosage?
• Timing of administration?
• Will EPO regenerate neurons in the spinal cord?
• Can EPO be used to protect retinas in animals w/
  glaucoma, central artery occlusion, and diabetic
  retinopathy?

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References

• MacLeod JN, Tetrault JW, Lorschy KAS, Gu DN.
  Expression and bioactivity of recombinant canine
  erythropoietin. American Journal of Veterinary
  Research (1998) 59 1144-1148.
• Piercy RJ, Swardon CJ, Hinchcliff KW. Erythroid
  hypoplasia and anemia following administration of
  recombinant human erythropoietin to two horses.
  JAVMA (1998) 212 244-247.
• Randolph JF, Stokol T, Scarlett JM, MacLeod JN.
  Comparison of biological activity and safety of
  recombinant canine erythropoietin in clinically
  normal dogs. American Journal of Veterinary
  Research (1999) 60 636-642.
• Gorio A, Gokmen N, Erbayraktar S, Yilmaz O,
  Madaschi L, Cichetti C, Di Guilo AM, Vardar E,
  Cerami A, Brines M. Recombinant human
  erythropoietin counteracts secondary injury and
  markedly enhances neurological recovery from
  experimental spinal cord trauma. PNAS, USA (2002)
  99 9450-9455.

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References

• Goldman SA Nedergarrd M. Erythropoietin
  strikes a new cord. Nature (2002) 8 785-787.
• Bocker-Meffert S, Rosenstiel P, Rohl C, Warneke
  N, Held-Feindt J, Sievers J, Lucius R.
  Erythropoietin and VEGF Promote Neural Outgrowth
  from Retinal Explants in Postnatal Rats.
  Investigative Ophthalmology and Visual Science
  (2002) 43 2021-26.
• Wang CY Baldwin Jr. AS. NF-kappa-B
  antiapoptosis induction of TRAF1 and TRAF2 and
  c-IAP1 and c-IAP2 to suppress caspase-8
  activation. Science (1998) 281 1680-4.
• Barinaga M. Stroke-damaged neurons may commit
  cellular suicide. Science (1998) 281 1302.
• Brenner C and Kroemer G. Mitochondriathe Death
  Signal Integrators. Science (2000) 289 1150

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References

• Li H, Kolluri SK, Gu J, Dawson MI, Cau X, Hobbs
  PD, Lin B, Chen G, Lu J, Lin F, Xie Z, Fontana
  JA, Reed JC, Zhang X. Cytochrome c Release and
  Apoptosis Induced by Mitochondrial targeting of
  Nuclear Orphan Receptor TR3. Science (2000)
  289 1159.
• Pray L. Life or Death in Cells. The Scientist
  (2001) 15 1.
• Adams JM Cory S. The Bcl-2 Protein Family
  Arbiters of Cell Survival. Science (1998) 281
  1322-5.
• Grasso G, Buemi M, Alafaci C, Sfacteria C,
  Passalacqua M, Sturiale A, Calapai G, De Vico G,
  Piedimonte G, Salpietro F, Tomasello F.
  Beneficial effects of systemic administration of
  recombinant human erythropoietin in rabbits
  subjected to subarachnoid hemorrhage. PNAS, USA
  (2002) 99 5627-5631.