“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, PhDAssociate Director

Cerebral Resuscitation LaboratoryDept of Emergency Medicine

Wayne State University

My Colleagues:

Rita Kumar, PhD

Thomas Sanderson PhD

Anthony Lagina

“Tell ‘Em What You’re Gonna Tell ‘Em”

• 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• 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

• 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

MetabolicEffects

Genetic Responses Translational Control

SurvivalSignaling

Glucose Bulk of data says no.

Excitotoxicityincr GABA, decreased glutamate

release, altered GlutR

Oxidative Stress? changes in SOD, Glutathione

No strong in vivo data

Calpain Proteolysis No good data

Genetic ModulationLots of in vitro data, surprisingly

little in vivo data.

Mitochondrial and ER stress, Protein Synth, Apoptosis

Coming right up.

Potential Salutary Effects of GFs in Ischemia:

GFs in Ischemia:Translational Control Mechanisms

eIF2-α Kinases: The Usual Suspects

HRI PKRGCN-2 PERK

I want a lawyer.

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 DeathPro-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

PI3K

INSULIN

Akt

Bad pBad Sequestered

PS

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

R30 + Ins 20U/kg + WMSanderson, Murariu, Kumar, Owen, Krause, Sullivan 2008

Bad Data

• Sanderson and Sullivan: our very own White Whale.

• NEVER saw a change in Bad phosphorylation or translocation.

p112 Bad

p136 BadTotal Bad

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

THERAPEUTIC HYPOTHERMIA

Anti-ROSGFsProteaseInhibition

Other

AGGRESSIVE REPERFUSION

The Potential for GF Treatment in Cerebral Ischemia: Combined Therapy

CVRI-Supported Research• “Combination Therapy for Focal Ischemic Stroke.”

– 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 PhDAnthony Lagina MDKarin Przyklenk PhD

Mike DeograciaSuzanne White MD, FACEP

Joseph Dunbar PhDJose Rafols PhD

James Rillema PhDGary 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)