Transcript GROWTH FACTORS AND CEREBRAL ISCHEMIA

“In Theory, Theory and Practice
are the same, but in Practice
they’re different.”
-Unknown
“You can do combinations.”
-John Connor
Terminator II: Judgement Day
CHILLING OUT WITH GROWTH
FACTORS: NEW THERAPIES FOR
CEREBRAL ISCHEMIA
Jonathon M. Sullivan MD, PhD
Associate Director
Cerebral Resuscitation Laboratory
Dept of Emergency Medicine
Wayne State University
My Colleagues:
Rita Kumar, PhD
Thomas Sanderson PhD
Anthony Lagina
“Tell ‘Em What You’re Gonna Tell ‘Em”
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There’s this thing called brain ischemia.
We hate brain ischemia.
We usually can’t fix it. Yet.
It’s complicated.
Hypothermia seems to help.
Growth factors (including insulin) are promising.
“You can do combinations.”
Science stuff we’ve already done.
Stuff we’re gonna do.
Fade to black. Roll credits. Crowd goes wild.
Brain Ischemia is a Big Deal
• 700,000 suffer a focal ischemic stroke each
year.
– 3d leading cause of death
– Treatment options extremely limited:
• Supportive care
• Thrombolysis and other reperfusion strategies
– Efficacious, not very effective (yet)
– Risky: NNT is low, but so are NNH and NNK.
– 70,000 survive cardiac arrest each year
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Heart resuscitation: better.
Cerebral resuscitation: not so much.
Vast majority have serious brain damage
Hypothermia the only proven neuroprotective strategy.
– Still not universally employed
– Effective in 20-25% of cases
Primum Non Nocere
Global vs. Focal Ischemia
Focal Ischemic Stroke
8 m Global Ischemia at 14 days
Ischemia Cocks the Hammer,
Reperfusion Pulls the Trigger
The Cerebral Ischemia Train Wreck
Single Drug Therapies Have Been
Uniformly Disappointing
• A partial list:
– Calcium Channel Blockers
– Glutamate antagonists
– Desferoxamine
– Cyclooxygenase/lipoxygenase inhibitors
– Steroids, lazaroids
– NXY-059
Monotherapy Will Never Work
• Brain ischemia is the result of multiple,
progressive, independently-lethal
processes.
• Targeting one process (such as
excitoxicity or free radicals) will do little
more than alter the cell death phenotype.
Only One Therapy Has Been Cool
Enough
• Hypothermia has been clinically
demonstrated effective for transient global
brain ischemia.
•Clinical data for focal
ischemic stroke is still
lacking.
•Laboratory data indicates
likely effectiveness for focal
ischemia
Hypothermia Isn’t Monotherapy
• Induces pro-survival signaling processes
– Activation of Akt
– Suppression of cytochrome c release and
caspase activation
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Decreases free radical production
Decreases lipolysis
Alters glutamate receptor composition
Other?
Single Agent
Monotherapy
• Hypothermia is a case in point
• So are peptide growth factors.
GFs in Brain Ischemia: Historical
Perspective
• Siemkowicz 1982: Increased glucose after global
ischemia impedes CMRO2.
• Strong 1985: mitochondria from insulin-treated ischemic
rats maintain respiratory control.
• Marsh 1986: hyperglycemia  cortical acidosis
• LeMay 1988: Insulin administration protects neurologic
function in cerebral ischemia in rats.
• Voll and Auer 1988: Post-ischemic insulin decreases
hippocampal and striatal neuronal death.
• Voll and Auer 1989: Makes rats smarter, too.
• Voll and Auer 1991: Insulin attenuates ischemic brain
damage independent of its hypoglycemic effect.
Growth Factors are Good.
• High dose insulin (12U/kg) decreases lesion volume and
cell death in diabetic rats (focal model). Rizk, Rafols and
Dunbar, 2006
• Topical IGF-1 reduces infarct volume and caspase-3
staining after focal ischemia in rats. Abe et al 2001.
• Patients with lower IGF-1 and IGFBP-3 levels are at
higher risk of stroke. Johnson et al 2005; Bondanelli et al 2006, Denti
et al 2004.
• Expression of GFs and GFRs is enhanced in resistant
neurons after ischemia, but reduced in SVNs. Hwang et al,
2004.
How Do GFs Work in Cerebral
Ischemia?
• Short answer: nobody really knows.
• Single mechanism unlikely.
• Two places to look for answers:
– Mechanisms of GF effect
– Mechanisms of ischemic damage
Growth Factor Signaling Systems:
The Obligatory Oversimplified
Cartoon
NGF
BDNF
GF Signaling Systems: Summary
GF
Metabolic
Effects
Genetic Responses
Survival
Signaling
Translational Control
Potential Salutary Effects of GFs in Ischemia:
Glucose
Bulk of data says no.
Excitotoxicity
incr GABA, decreased glutamate
release, altered GlutR
Oxidative Stress
? changes in SOD, Glutathione
No strong in vivo data
Calpain Proteolysis
No good data
Genetic Modulation
Lots of in vitro data, surprisingly
little in vivo data.
Mitochondrial and ER stress,
Protein Synth, Apoptosis
Coming right up.
GFs in Ischemia:Translational Control Mechanisms
eIF2-α Kinases: The Usual
Suspects
I want a
lawyer.
HRI
GCN-2
PKR
PERK
ER Stress and Translational
Control
eIF2α(P) Localizes to SVNs During
Early Reperfusion
DeGracia, Sullivan, Neumar, Alousi, Hikade, Pittman, White, Rafols, Krause.
Salutary Effects of GFs in Ischemia:
Translational Control
Sullivan, Alousi, Hickade, Bahu, Rafols, Krause, White
GFs in ER Stress
• Effect of insulin on eIF2α(P) is highly
compelling, however….
• Virtually no other published data in vivo.
• Some in vitro data that GFs are salutary in
setting of ER stress
• Watch Dr. Kumar.
Salutary Effects of GFs in Ischemia:
Programmed Cell Death
Apoptosis: For Real?
Focal Ischemic Neuronal Death
Pro-Apoptosis
Anti-Apoptosis
DNA laddering observed in
penumbra within a few hours
Necrotic phenotypes predominate
in the core.
Transcription of caspase 3
observed in vulnerable neurons
Caspase inhibitors convert
“apoptotic death phenotypes to
“necrotic” phenotypes.
Activated caspase 3 seen by 1-3 h
of reperfusion
Caspase 3 inhibitors decrease
caspase cleavage products,
reduce tissue damage, improve
outcome in rodent stroke models.
Apoptosis: For Real?
Global Ischemic Neuronal Death
Pro-Apoptosis
Anti-Apoptosis
TUNEL staining observed in both
animals and humans.
Apoptotic bodies not observed by
most investigators.
Caspase inhibitors protect against
neuronal death.
“Necrosis” morph typical in primate
neurons, even w. caspase activation.
Activated caspase 3 seen as early as
1 hr of reperfusion.
Little evidence of chromatin condens’n
at light level (mixed results from EM).
Caspase 3 mRNA appears early in
SVNs.
PARP breaks down between 24 and
72 hours.
BAX and BclXs expressed in
vulnerable CA1 neurons.
Manichean Biology
EITHER
OR
Intrinsic Apoptotic Bad-ness
Intrinsic Apoptosis and Bcl-2 Family
Proteins: the Movie
GF-Mediated Survival Aktion
TRANSLATION
So Perfect, It Had to Be Wrong
INSULIN
PI3K
PS
Akt
Bad
pBad
Sequestered
Survival
Insulin Maintains Hippocampal
Architecture and Function
Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008
Insulin-Akt Dose-Response
Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008
Insulin Induces Akt Phosphorylation
Through PI3K
Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008
p-Akt in CA1 Hippocampus
Sham
R30
R30 +
Ins 20U/kg
Sanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008
R30 + Ins 20U/kg + WM
Bad Data
p112 Bad
p136 Bad
Total Bad
• Sanderson and Sullivan: our very own White
Whale.
• NEVER saw a change in Bad phosphorylation
or translocation.
Looking Downstream: Cytochrome c
SHAM (Non-Isch)
8 min Isch
+ 240 min Rep
240 min Rep
+ Insulin 2U/kg
From Insulin to Cytochrome c
INSULIN
PI3K
Akt
Bcl-Xl-Bax Binding
Mitochondrial Bax Pores
Cytochrome c Release
Insulin Restores Bcl-XL-Bax
Binding
Sanderson, Kumar, Sullivan, Krause 2008
Insulin Suppresses Mitochondrial
Bax Localization
Sanderson, Kumar, Sullivan, Krause 2008
Insulin Suppresses Release of
Cytochrome c
Sanderson, Kumar, Sullivan, Krause 2008
Insulin Suppresses Release of
Cytochrome c
Sanderson, Kumar, Sullivan, Krause 2008
The Potential for GF Treatment in
Cerebral Ischemia
• Insulin and IGF-1 are already in clinical
use.
• Insulin is inexpensive, familiar to clinicians,
and easily administered.
• Potential limitations:
– 20U/kg? Are you out of your mind?
– Hypoglycemia, hypokalemia
– Fluid shifts, cerebral edema
You Can Do Combinations
• Combination therapy for stroke/brain
ischemia has been identified as a priority
by the NIH.
• Early targeting of mulitple pathways far
more likely to be successful than singly
targeted therapy.
– By “early,” we mean “upon reperfusion.”
• Preclinical evaluation using STAIR criteria.
Building on the Foundation
AGGRESSIVE REPERFUSION
GFs
Anti-ROS
Protease
Inhibition
Other
THERAPEUTIC HYPOTHERMIA
The Potential for GF Treatment in
Cerebral Ischemia: Combined Therapy
CVRI-Supported Research
• “Combination Therapy for Focal Ischemic Stroke.”
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J. Sullivan, A. Lagina, R. Kumar, J. Li
hypothermia + insulin for focal ischemic stroke
MCAO model of focal ischemia
Guided by STAIR recommendations
Dose-response for hypothermia and insulin
Affect on biochemical targets
• Insulin: Akt, cytochrome c release
• Hypothermia: free radicals (4-HNE, MDA), Akt, cyto c release
– Short- and Long-term outcomes:
• Histological
• Neurobehavioral
• Similar study for Global Brain Ischemia (Lagina,
Sullivan, Sanderson) funded by Emergency
Medicine Foundation (IGF-1 + Hypothermia)
“Combination Therapy for Global
Brain Ischemia”
• Lagina, Sullivan; Emergency Medicine
Foundation
• Preclinical investigation of hypothermia + insulin
for global ischemia
• 2v-OH model of global ischemia
• Dose-response for hypothermia and insulin
• Affect on biochemical targets
– Insulin: Akt, cytochrome c release
– Hypothermia: free radicals, Akt
• Short- and Long-term outcomes:
– Histological
– Neurobehavioral
“Tell ‘Em What You Told ‘Em”
• Brain ischemia is a Big Deal, and current
treatment strategies are limited.
• Single drug therapy will never work.
• Hypothermia and growth factors are both
neuroprotective and act on multiple
pathophysiological processes
• Combination therapy
• CVRI-supported preclinical work underway
in our laboratory
Special Thanks:
Tom Sanderson, PhD
Rita Kumar PhD
Anthony Lagina MD
Karin Przyklenk PhD
Mike Deogracia
Suzanne White MD, FACEP
Joseph Dunbar PhD
Jose Rafols PhD
James Rillema PhD
Gary S. Krause MD, MS
Work shown here supported by:
AHA PDFG 0415380Z (Sanderson)
Emerg Med Found CDG (Kumar)
NIH KO8 NS-02008 (Sullivan)
NIH R01 NS4919 (Sullivan)
Emerg Med Found CDG (Sullivan)
NIH R01 NS33196 (Krause)