Effets des facteurs induits par l’hypoxie dans l’ischémie cérébrale : quand l’EPO dope le cerveau
Effects of hypoxia-inducible factors in cerebral ischemia: when EPO dopes the brain
Myriam BERNAUDIN, CR1 CNRS “Hypoxia and cerebrovascular pathophysiology” CERVOxy group, Neurodegenerescence: models and therapeutic strategies UMR-CNRS 6185, CYCERON, CAEN, France
Cerebral ischemia artery wall
emboli
artery wall
blood
Embolic stroke
blood
thrombus atherosclerosis
Thrombolotic stroke
¾ 1st cause of morbidity ¾ 3rd cause of mortality Therapeutics : - rt-PA : thrombolytic treatment - Prevention hypertension diabete…
Hypoxic inducible factors and cerebral ischemia Neuronal death Glial reaction Inflammation Angiogenesis Neurogenesis
O2 Energy
Excitatory amino acids, free oxygen radicals
Cytokines Organ response
Hypoxia Molecular response: HIF-1 EPO, VEGF…
HIF-1α
HIF-2α
3d Marti et al. Am. J. Pathol. (2000)
EPO was the first target gene for HIF-1 to be identified and is still one of the most best-characterized genes activated by hypoxia.
Erythropoietin (EPO)
- glycoprotein of 34kDa; kidney, liver … - growth factor specific for erythroblast differentiation and proliferation - effect on the platelet maturation (Krantz, 1991) - angiogenic (Anagnoustou et al., 1990 ; Carlini et al., 1995 ; Nagai et al., 1995 ; Ribatti et al., 1999)
- gene cloned in Human by Jacobs et al. (1985) - 79-94% of homology between species - transcriptional factor = HIF-1 (hypoxia inducible factor-1)
HIF-1 activation mechanisms induced by hypoxia Normoxia
O2
CO2
2-oxoglutarate
succinate
HIF-1α
pVHL P OH HIF-1α
P OH HIF-1α
Prolyl-4-hydroxylases (HPH)
E3 Ubiquitin Ligase
PROTEASOME
Degradation
Ub
(Fe2+)
Hypoxia
HIF-1α
Iron chelators (DFX) CoCl2
O2 2-oxoglutarate
P
OH
succinate N
HIF-1α
Asparaginyl hydroxylase (FIH-1) « Factor inhibiting HIF-1 » (Fe2+)
Transcriptional activation
ARNT
CO2
HIF-1α
P
P300 /CBP
P300 /CBP
HIF-1 α P P
BTM
ARNT HRE
Target genes
u ro ns As tro cyt es RB E4
EPO: cellular sources and targets Hypoxia
Neurons
0h 2h 6h 12h 24h
Ne
NMDA 10 µM
EPO
EPO
β-actin 100 pA
Astrocytes
5 sec
0h 2h 6h 12h 24h u ro ns As tro cyt es RB E4
Bernaudin et al., Glia (2000)
Ne
EPOR
Bernaudin et al., JCBFM (1999)
EPO Hypoxia Cortex CoCl2 0h 6h DFX
EPO β-actin
β-actin
DFX, CoCl2 Bernaudin et al., Glia (2000)
EPO may have autocrine and paracrine roles in the brain.
LDH activity release (% )
EPO effect on neurons in vitro
NMDA
Bernaudin et al., JCBFM (1999)
EPO pretreatment protects cortical neurons from an excitotoxic stress.
EPO receptor expression after cerebral ischemia
Control
1d
ZI
3d
7d
EPO effect in cerebral ischemia
12
Infarcted area (mm2)
Vehicle
Vehicle rMoEpo
rMoEpo
10 8 * * *
6
*
4
* *
2
*
0 -2
-4
-3
-2
-1
0
1
*
2
3
4
Distance from Bregma (mm)
Epo pre-treatment (i.c.v.) reduced of 47% the infarcted volume
EPO and cerebral ischemia: pleiotropic roles EPO / EPO-R
Neuroprotection
Neurorepair
Control
rMoEpo
Endothelial cells Proliferation (% of control)
• Glial reaction • Inflammation • Angiogenesis …
*
60
40
*
20
0 0
Bernaudin et al., JCBFM (1999)
*
150
300 bFGF rHuEpo (pM) (2ng/ml)
When EPO dopes the brain
EPO
Inflammatory cells
Capillary
Neuronal death Hemorrhage Trauma Epilepsy Parkinson disease ALS EAE Retina ischemia Schizophrenia?
Neurogenesis
Target
Bernaudin and Petit, M&S (2000)
Neuroprotective effect of EPO: potential mechanisms Reduced-glutamate exocytosis Ca2+ influx
Anti-apoptotic pathways JAK2, MAPK, Stat5 NF-kB, ERK, PI3K/Akt Bcl-2 Bcl-XL XIAP C-IAP2
Reduced-NO / anti-oxydant BBB integrity Reduced eNOS
Nat Rev Neurosci, 2005
SSS, NIH SS, Barthel index, Ranking scale S100β assay
Stroke 0d
1d
2d
3d
7d
18 d
30 d
MRI
rhEPO (Roche, EPO-β) (100 000 UI) i.v. 3.3X104 IU/50ml/30min for 3 days or saline (0.9% NaCl) Safety part : 13 patients Efficacy part : 40 patients
Inclusion criteria Age < 80 years-old Stroke at the MCA EPO administration < 8 h
EPO in clinic
Science et Avenir, mars 2004
EPO and stroke: detrimental chronic effect
Infarct volume (mm3) at 24 h
* T-test P< 0,001
*
60 50 40 30 20 10 0
Control
Tg-EPO -/+
Tg-EPO +/+
Chronic EPO expression increases infarct volume Increased cerebral infarct volumes in polyglobulic mice overexpressing erythropoietin. Wiessner C et al., 2001. J Cereb Blood Flow Metab.
Modified EPO molecules: no hematopoietic effect
¾ CEPO = carbamylated EPO
Hypoxic inducible factors and cerebral ischemia Hypoxia and Neuroprotection
Neuroprotection Tolerance and neuroprotection
Hypoxie Neurogenesis
Angiogenesis
Functional recovery
¾ Hypoxic preconditioning
Adaptation to hypoxia : tolerance to cerebral ischemia HYPOXIA 8%O2
ISCHEMIA Reoxygenation 24 h
Penumbra
ISCHEMIA Core
Endogenous mechanisms
Neuroprotection
¾ Neonatal rat, hypoxia 3h Gidday et al., Neurosci Lett (1994) ¾ Adult mice, hypoxia 1h, 3h, 6h Bernaudin et al., JCBFM (2002) To study the endogenous molecular mechanisms that may explain how hypoxic preconditioning protects against subsequent ischemia To provide novel therapeutic targets for treatment of cerebral ischemia and other brain disorders
Adaptation to hypoxia : tolerance to cerebral ischemia ¾ In the neonatal model Reoxygenation
Hypoxia
HIF-1α
¾ In the adult model Hypoxia C
1h
3h
VEGF
5
2.5
4 Fold change
24 h C 3h
Fold change
0h C 3h
EPO
3.0
2.0 1.5 1.0 0.5
3 2 1 0
0.0 Control
0hr R
6hr R 18hr R Hypoxia 3hr
24hr R
Control
0hr R
6hr R 18hr R Hypoxia 3hr
Hypoxia C
1h
3h
6h
EPO
6h
HIF-1α
VEGF
Hypoxic preconditioning: HIF-1, EPO & VEGF implication Prass et al., Stroke (2003)
24hr R
Genes increased by hypoxic preconditioning ¾ In the neonatal model
¾ In the adult model
EPO VEGF GLUT-1 Adrenomedullin Prolyl-4-hydroxylase α MAP kinase phosphatase-1
EPO VEGF GLUT-1 Adrenomedullin RTP801 P21 Tyrosine hydroxylase
HIF-1 dependant
¾ Common in both models Hypoxia HIF-1 EPO
VEGF
ADM
MTF-1
Egr-1
?
MT-1
VEGF
?
Bernaudin et al., JBC (2002) and Neurobiol Dis (2005)
Neuroprotection
Hypoxic inducible factors and cerebral ischemia Hypoxia and Repair
Neuroprotection
Angiogenesis
Neurogenesis
Functional recovery
Hypoxia
VEGF and cerebral ischemia VEGF
PECAM
Control
EF5 (hypoxia)
Ischemia
Periphery
Core
20 h
12 h
3d Marti et al. Am. J. Pathol. (2000)
Pharmacological strategies: HIF-1 stabilisation 1d
60d
Time
Functional recovery ?
DFX Ischemia MCAo, rat
HIF-1 activation
Infarct volume (mm3)
* 350 300 250 200 150 100 50 0
Control DFX
DFX reduces brain damage
(Fréret et al., 2006 Eur. J. Neurosci.)
Pharmacological strategies: HIF-1 stabilisation
Staircase
Sensori-motor performances *
Pre-
Post-
Pre-
Post-
(1 week)
(2 months)
(1 week)
(2 months)
Ischemia Solvant
Ischemia DFX
Neurological score
12 10 8 6 4 2 0
30 25
**
20
*
15
Ischemia + vehicle (n=6) Ischemia + DFX (n=7)
*
10
*
5
Days
0 0
1
2
3
7
8
9 10 11 29 30 31 32 56 57 58 59 60
DFX improves functional recovery (Fréret et al., 2006 Eur. J. Neurosci.)
Neurogenesis and hypoxia: facilating endogenous neurogenesis Hypoxia (HIF-1) VEGF, EPO, ? Cerebral stem cells
Ê neuronal differentiation
(Studer et al., J Neurosci 2000; Shingo et al., J Neurosci 2001; Jin et al., PNAS 2002; Sun et al., J Clin Invest 2003; Wang et al., Stroke 2004)
Endogenous neurogenesis ¾ EPO, VEGF ...
Stem cell therapy ¾ NSC, MSC, ES ...
MSC differentiation into neuron-like cells: a role of HIF-1 ? Progenitor
Neuron-like cells
CoCl2 Nestin
¾ Morphological changes ¾ Neuronal marker expression ¾ Response to neurotransmitters ?
% of responding cells to dopamine
Tuj1
MAP2
*
100 80 60 40 20 0 Control
Fura-2
(Pacary et al., 2006 J. Cell. Sci.)
CoCl2
MSC differentiation into neuron-like cells : a role of HIF-1 ? Control a
Ab HIF-1
Cytoplasm Control
CoCl2 3h
CoCl2 3h HIF-1α
HIF-1α
b
mRNA levels/control
Nucleus Control
CoCl2 3h
5 4 3 2 1 0
EPO
HIF-1α/PI
3d Control CoCl2
6h
HIF-1α
c
24h
Adaptation to hypoxia: HIF-1 activation Brain ischemia
HIF-1β
HIF-1α
“O2 Sensor”
Blood flow
Hypoxia Hypoglycemia
HIF-1 binding sites VEGF EPO ADM NOSi Glycolytic enzymes GLUT-1
Neurogenesis
ATP
Target genes BNIP3, Others ...
Angiogenesis Vasodilatation
Apoptosis
Blood flow
Protection, regeneration and functional recovery
Cell survival
Thanks to
Dr. Edwige Petit Dr. Simon Roussel Dr. Pascale Schumann-Bard Dr. Omar Touzani Emilie Pacary, PhD student Thomas Fréret, PhD student Didier Divoux Jérôme Toutain
Pr. Hugo Marti Heidelberg, Germany Pr. Frank R. Sharp Dr. Yang Tang Cincinnati, USA
