EPO, the growth magical factor

NEURO-PROTECTIVE
EFFECTS OF
ERYTHROPOIETIN
Reza Nejat, M.D.,
Anesthesiologist, FCCM
former Assistant Professor, SBMU,
Bazarganan Hospital,Tehran,
IRAN
2

Neuro-protective effects of EPO
 Viault (1890)
 Carnot and Deflandre (1906)
 There must be hormone to increase
erythropoiesis in high altitude, it
regulates erythropoiesis
 Jacobson (1957) and Nathan (1964):
 the kidney is the major site but not the
sole site of Epo production.
 Goldwasser and his team (1977):
 prepared 8 mg of highly purified human
Epo
3

 EPO:
 Is required for:
 survival,
 Proliferation,
 differentiation
 of erythroid progenitor cells in
bone marrow, and
 Prevents apoptosis in progenitor
cells
4

 EPO:
 Produced by renal interstitial
cells having a neuron-like shape
and express marker antigens
found in neuronal cells
 Ito cells in the liver produces
EPO; very similar to the EPO-
producing renal fibroblast-like
interstitial cells,
5

 Epo and Epo-R
 EPO-R
 belongs to the cytokine receptor superfamily
 consists of:
 an extracellular domain,
 a transmembrane domain,
 an intracellular domain.
6

Cold Spring Harb Perspect Med 2013;3:a011619
7

 EPO and EPO-R can be
found in the:
 Nervous system,
 Cardiovascular system,
 Digestive system,
 Endocrine system,
 Female and male
reproductive system,
 Respiratory system
 Spleen
8

 EPO production and secretion:
 regulated by the tissue oxygen
supply (kidney, liver, brain)
 via activation of the hypoxia-
inducible factor 1 (HIF-1)
pathway
9

 HIF:
 a heterodimeric protein
α- and β-subunits
 HIF α-subunits (1, 2, 3):
regulated by oxygen tension,
in normoxia hydroxylated,
 degraded by pVHL
in hypoxia not hydroxylated
 HIF β-subunit:
constitutively expressed.
10

the expression of EPO-R is:
not sensitive to hypoxia???
regulated by:
pro-inflammatory cytokines:
TNFα, IL-1β
erythropoietin
probably other unidentified factors
11

In EPO or EPO-R knock-out
mice:
Apoptosis increases prior to the
onset of anemia
There seems to be a functional
role of EPO/EPO-R signaling
rather than erythropoiesis
12

Cytokine-Jak-STAT signaling
is an evolutionary ancient
mechanism
13

 auto-phosphorylation
of JAK-2 results in
activation of several
signaling pathways:
RAS/MAPK with the
ability to activate
STAT5
up-regulating
antiapoptotic proteins
Bcl-2 and Bcl-XL
EPO+EPO-R
activates
JAK-2
RAS/MAPK
STAT5
upregulation of
Bcl-2, Bcl-XL
14

of JAK-2 results in
signaling pathways:
PI3K/AKT:
inhibits pro-apoptotic
molecules and prevents
release of cytochrome
c from mitochondria
PI3K/AKT
inhibition of
activation of
JAK-2
EPO+EPO-R
GSK-3β
caspase -3/-9
BAD
15

of JAK-2 results in
signaling pathways:
PI3K/AKT:
Phosphorylation of
GATA-1
Inhibition of BAD, GSK
and caspase-3/-9
results in aborting
apoptosis
PI3K/AKT
inhibition of
activation of
JAK-2
EPO+EPO-R
GSK-3β
caspase -3/-9
BAD
16

 auto-phosphorylation of
JAK-2 results in activation
of several signaling
pathways:
 PI3K/AKT:
Down-regulation of NF-ƙB:
suppresses pro-inflammatory
cytokines likeTNF-α and IL-6
and simultaneously
increases anti-inflammatory
cytokine IL-10 level.
EPO+EPO-R
JAK-2
NF-ƙB
downregulation
TNF-α
IL-6
IL-10
anti-
inflammatory
effect
17

of JAK-2 results in
signaling pathways:
PI3K/AKT:
Expression of eNOS
(endothelial Nitric
Oxide Synthase)
eNOS
PI3K/AKT
EPO+EPO-R
JAK-2
NO
&vasodilatation
NADPH oxidase inhibition
&
ROS
18

 a multicenter double blinded
clinical study in Germany:
EPO had no cell-protective
effect or even might be
hazardous in humans.
 Ehrenreich H, Weissenborn K, Prange H.
Recombinant Human Erythropoietin in the
Treatment of Acute Ischemic Stroke. Stroke. 2009;
40: e647-e656
19

the modulatory effect of EPO
on the central respiratory
commands:
 acute (less than 1 hour) and
chronic EPO administration:
attenuated and abolished
hypoxia-induced central
respiratory depression
MEK½ and PI3K pathways
mediates this response
Repir Physiol Neurobiol. 2015; 206: 36-40
20

 The report of a phase II double blinded
placebo controlled study in infants with
moderate to severe hypoxic/ischemic
encephalopathy:
 EPO could:
1) diminish MRI brain injury, and
2) improve the motor function of
the infants after 1 year, yet
3) the mortality did not differ
significantly
Pediatrics. 2016; 137(6): e20160191
21

 EPO-R knock-out mice:
Low level of neural progenitor
cells
Low neurogenesis
22

High baseline concentration
of EPO-R is expressed as an
autocrine/paracrine system
in:
 astrocytes that surround
capillaries in white matter,
 purkinje neurons,
 the choroid plexus
 the ependymal cells
 limbic system and hippocampus
23

 EPO and EPO-R:
Expressed in low levels in
healthy brain
Increase markedly following
injuries
24

EPO: Does it pass through BBB?
cannot penetrate BBB:
in hypoxia/ischemia permeability of
BBB increases, and
peripherally administered EPO:
can be found in CSF in rats, primates
and humans.
through the extracellular
pathways.
25

 the absolute source of EPO in the
CNS is not the blood.
 it is produced de novo in the CNS
 tissue hypoxia in the CNS increases:
EPO concentration
EPO-R expression
26

 EPO expression in the nervous system
is regulated by:
 the tissue oxygen supply and via
activation of the (HIF-1) pathway
 In non-hypoxic circumstances:
mechanical damage
Infection
metabolic stress (glucose , insulin?)
oxidative stress
elevated temperature
Intense neural activity
enriched environment
 pro-inflammatory cytokines
27

 CNS lesions:
 primary injury:
Necrotic core, Penumbra
Infiltration of inflammatory
cells
 secondary injury:
Propagation of necrotic core
and lesion in the penumbra
 Final pathway:
resorption of cellular debris
gliosis vs regenerating a
functional tissue
28

 Ischemic/hypoxic and other
types of brain injuries:
lack of oxygen and nutrients
pro-inflammatory mediators in
neurovascular unit:
TNF-α, IL-6, ICAM-1
brain edema and hemorrhagic
transformation
neural cell apoptosis and death
29

Reperfusion
restoration of perfusion in
the ischemic lesion
 oxygen stress
 free radical formation
 excitotoxicity,
 nitric oxide production
30

 In brain injuries the
leakiness of BBB 
due to:
inflammatory
mediators,
reactive oxygen
species (ROS),
VEGF,
MMP,
microRNA
31

 HIF-1α in hypoxic brain
insult:
  expression ofVEGF,
VEGFR and MMP-9, AQP-4:
  BBB hyer-permeability
  Brain edema
 inhibition of HIF-1α:
  cerebral edema through:
  MMP-9 and AQP-4
32

 Neural stem cells in
mammals:
 SGZ, SVZ, OB
 In cortical injuries:
 neuroblasts originating
from SVZ stem/progenitor
cells migrates to the
penumbra:
VEGF, IGF-1, SDF-1/CXCR-4
and Ang-1/Tie-2 signals
33

 MMPs:
 With several significant
physiological
potentials involved in:
 growth,
 development,
 tissue repair and wound
healing
 synaptic plasticity
 neurite growth
 myelinogenesis.
34

 MMPs:
 up-regulated and activated
in ischemic and other brain
injuries,
 Its latent form is activated
by:
 Endogenous and exogenous
plasminogen activator
 Furin
 free radicals
35

 Angiogenesis:
 proliferation of mature endothelial and endothelial
progenitor cells (EPC) located in the penumbra in the
first 12-24 hour after the insult
 VEGF leads the process
36

 VEGF:
 a family of cytokines,
 induce angiogenesis
through proliferation,
sprouting, migration of
the endothelial cells and
 New vascular tube
formation by these cells.
 Found in pericytes in the
border of brain lesions
37

 VEGF:
 After binding withVEGFR-2 increases
vascular permeability through activating
cGMP and a NO-dependent pathway
38

 AQP-4
 integral membrane proteins
plays important roles in:
mediating water
homeostasis
bidirectional passive trans-
cellular water transfer in
response to osmotic
gradient
 the expression of this channel
is modulated by HIF-1 and
VEGF
39

AQP-4 expression has an
impact on BBB integrity
40

 Strategy against CNS
insults:
Decrease apoptosis and
supporting the cells
restore delivery of
oxygen and nutrients
(angiogenesis)
suppressing edema and
saving the integrity of
BBB, {ASAP}
supporting neurogenesis
and synaptogenesis
(ECM)
41

 hypoxia dose-dependently
makes astrocytes secret
EPO
EPO has a trophic paracrine
effect on:
Neurons,
Astrocytes,
Microglia.
42

EPO:
Protects the neural
cells against reduced
oxygen tension,
ecitotoxicity and ROS
or other free radicals
43

 EPO
facilitates energy production
in mitochondria:
stabilizing mitochondrial
membrane potential
Inhibiting free radical
production in mitochondria
44

EPO in the nervous system:
 apoptosis,
 inflammatory responses
 re-establishment of compromised
functions by support of :
1) proliferation,
2) Migration, and
3) differentiation
of progenitor cells to compensate
for the lost or injured cells
45

 rhEPO could protect BBB:
 By up-regulating theTJ
proteins
  MMP
  glial cell inflammatory
reactions,
 TNF-α levels and
  NF–кβ activation
46

 EPO protects BBB:
 againstVEGF-induced injury
and the resulting hyper-
permeability in early phases
of brain insults
 have cytoprotective effect
on endothelial cells in
ischemic insults
 inhibit AQP-4-induced
astrocyte swelling through
activating JNK and MAPK
47

 Endogenous EPO was
attributed to:
 Induce neural
stem/progenitor cells to
proliferate, migrate and
differentiate in addition to
survive
 in rats, EPO-R is more
concentrated on neural
progenitor cells (NPCs)
than on the mature ones
48

 astrocyte-derived EPO
 anti-apoptotic factor for
microglia:
without having any influence on
pro-inflammatory potentials
 dose-dependent proliferative
effect on microglia
  Bcl/Bax ratio in the microglia
 prevents activation of caspase-
3 and -9
49

 EPO completes the loop of
a physiological feedback
pathway by:
 inducing MMP-2 and -9
which promotes angiogenesis
and migration of neuronal
progenitor cells
 limiting MMPs activity to a
restricted area throughTIMPs
expression by astrocytes
50

 EPO through activating
PI3K/Akt pathway
regulates:
TIMP-1 gene transcription,
TIMP-1 mRNA induction and
TIMP-1 expression
51

 EPO supports
regenerating neurons
and astroglial cells by:
regulating MMP-2, MMP-9
andVEGF
increasingVEGF receptors
1, 2 and 3 in hypoxia
52

 EPO increases survival of
cultured endothelial cells
through:
 activating Akt1 pathway,
 stabilizing mitochondrial
membrane potentials and
 inhibiting caspase 3 and-9
53

 EPO has pro-angiogenic
property:
 Induces endothelial cell to:
 proliferate,
 migrate,
 produce nitric oxide (NO),
 degrade ECM delicately,
 differentiate
 Mobilizes the endothelial
progenitor cells,
54

 Effective synaptogenesis
needs high degree of
plasticity through
regulating the
consistency of perineural
net and ECM remodeling:
 Role of MMPs in cognition,
memory, learning
55

 activation of EPOR in
cultured young rat
cerebellar and hippocampal
neurons:
reduces glutamate release
 by inhibiting calcium-
dependent exocytosis of this
excitatory amino acid
56

 rhEPO in reperfusion injury:
  up-regulation of IL-1β and
IL-18, MMP-2 and MMP-9
 protects against oxygen
toxicity and free radicals
(ROS, RNS) through:
  pro-inflammatory
mediators
57

 rhEPO attenuates ischemia-
induced inflammation by:
 reducing neuronal death
rather than by direct effects
upon EPO-R–expressing
inflammatory cells.
 rhEPO rescues neurons
within the penumbra from
apoptosis
58

 EPO :
TNF-α, IL-6, and
monocyte chemo-attractant
protein-1 (MCP-1)
 TNF-α, IL-1:
Inhibits EPO production
59

 EPO, in neonatal rats:
 after hypoxic/ischemic injury
stimulated:
 oligodendrogenesis
 attenuated white matter damage
 EPO, in adult rats:
 after stroke
 amplified myelinating oligodendrocytes
 increased myelinated axons in peri-infarct
white matter and
 improved functional outcome
60

 EPO, in MCAO in adult
mice brains:
 reduced demyelination
 astrocyte activation, and
 decreased the protein level
of β-APP.
 inhibited Nogo-A and MAG
protein levels after cerebral
ischemia:
 attenuating axonal injury
61

62

EPO:
 increased water permeability
of astrocyte AQP-4 induced by
group I mGluR agonists
63

64

65

66

May 29, 2018 June 3, 2018
67

June 14, 2018 June 21, 2018
68

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76

 Now, we have a new vision
of EPO’s effect in the
nervous system:
anti-apoptotic,
anti-oxidant,
anti-inflammatory
neuro-protective by
stimulation of :
Angiogenesis
Neurogenesis
77

Fundamentals of EKG
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EPO, the growth magical factor

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