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Erythropoietin

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... Cichetti, Anna Maria Di Guilo, Enver Vardar, Anthony Cerami, and Michael Brines. ... Randolph JF, Stokol T, Scarlett JM, MacLeod JN. ... – PowerPoint PPT presentation

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Title: Erythropoietin


1
ErythropoietinNeuronal Recovery
  • Matthew Barber Michael Mienaltowski
  • VIPS V Presentation
  • October 11, 2002

2
What is EPO?- hematopoiesis -
3
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.

4
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?
5
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

6
What is EPO?- An alternative therapy? -
  • Erythropoietin has also been shown to
  • Reduce inflammation
  • Inhibit apoptosis
  • Promote growth and development in neural stem
    cells.

7
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
8
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
9
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

10
Nerve Damage Apoptosis
TR3
11
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

12
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

13
Cellular EPO Recovery
  • Possible treatments in the future include IL-1ra
    and especially EPO, the topic of todays
    discussion

14
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

15
EPOs Role InNeuronal Recovery
Goldman Nedergaard. Nature (2002) 8 785-787.
16
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

17
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
18
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
19
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
20
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.

21
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

22
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.

23
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.
24
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

25
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
26
PNASSAH fig 2
A control B control placebo C control
r-hEPO
D SAH E SAH placebo F SAH r-hEPO
EPO eases vasoconstriction
27
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
28
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.

29
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
30
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.

31
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?

32
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.

33
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

34
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.
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