Gary A' Rosenberg, M'D'

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Stroke Research

• Gary A. Rosenberg, M.D.
• Department of Neurology
• University of New Mexico
• Health Sciences Center

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Stroke

• Third leading cause of death
• 550,000 new strokes a year in US
• One third disabled
• 4 million stroke survivors with disabilities in
  US
• Economic cost in US 20 to 40 billion/yr

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Questions

• What is the role of the blood vessels in stroke?
• What is the blood-brain barrier?
• How do enzymes disrupt the blood-brain barrier
  and lead to hemorrhage?
• What can be done to prevent the disruption of the
  blood-brain barrier?

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Brain Attack Rx with rtPA (27-year old
women with CVA)
Pre-Rx
3 Hrs Post-Rx
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Thrombolysis and Hemorrhage

• rtPA is first FDA approved therapy for stroke
• Morbidity lower at 3 months
• Risk of hemorrhage increased from 0.6 to 6.4

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80 year old man with CVA, who was treated with
tissue plasminogen activator
Intracerebral hemorrhage 15 hrs after rtPA
CT normal before rtPA
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Cerebral vessels are end arteries
Few anastomoses between arteries and veins
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Cerebral Circulation

• Anterior
• Internal carotid
• Anterior cerebral
• Middle Cerebral
• Posterior
• Vertebral
• Basilar
• Posterior cerebral

Netter drawing
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Cerebral Thrombosis with Infarction
Subacute infarct
Acute infarct
From Greenfields Neuropathology
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Reperfusion Injury

• 74 year old hypertensive women with multiple TIAs
  due to carotid stenosis
• gt80 stenosis of the internal carotid seen on MRA
• Endarterectomy was performed

Stenosed Carotid
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Hemorrhagic Transformation Following
Endarterectomy
5 days after surgery
7 days after surgery
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What is the mechanism of bleeding?
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Cascade of events in ischemiaShort therapeutic
window
Therapeutic window
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(No Transcript)
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Neutral Proteases Involved in Neuroinflammation

Cysteine
Serine Metalloproteinases
Caspases (uPA/tPA)
MMPs
Plasmin ADAMs

Apoptosis Extracellular Matrix
Remodeling
Angiogenesis and Apoptosis
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Matrix MetalloproteinaseProteins
Minimal Domain Matrilysin (MMP-7)
Zn
Pre-Pro
Catalytic Domain
HP
Hemopexin Domain Stromelysins (MMP-3,-10,-11
-13)
Zn
Pre-Pro
H
Hinge Hemopexin
HP
Transmembrane Domain MT1-MMP (MMP-14)
Zn
Pre-Pro
H
TMD
Fur
Furin-binding
Fibronectin Domain Gelatinases (MMP-2 -9)
Zn
Pre-Pro
H
FN
Fibronectin-binding
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Gene Control of MMP Genes Involved in
Inflammation
FOS/JUN TNF-?
LPS/Free Radicals
MMPs, Plasmin Free Radicals
AP-1NF-?B
mRNA ? proMMP ? MMP
(AP-1 sites in all NF-?B in MMP-9)
Transcription Translation Activation
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Evolution of Tight Junctions (TJs) at the
Blood-Brain Interfaces

No TJs Glia TJs Glia
TJs Endothelia TJs
Blood Glia End Feet Neuropil
(Adapted from Abbott, NJ The Blood-Brain Barrier
in Health and Disease, 1986)
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Anatomy of the BBB
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Capillary Basal Lamina
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Astrocytes, Laminin, Pericytes
(Simard et al J Neurosci 239254, 2003)
Laminin (white) Endothelial cell (RECA red)
Astrocytes around blood vessel
Laminin (white) GFAP (green) Desmin in Pericytes
(red)
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Biphasic Opening of the BBB in Stroke

• Repefusion of blood into an ischemic region
  causes a biphasic disruption of the blood-brain
  barrier
• Early opening is reversible
• Delayed opening related to hemorrhages due to
  neuroinflammation

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Early Reversible BBB Opening in Occurs 4 Hr after
Reperfusion (90-min MCAO in Normotensive Rat WKY)
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Activation of Gelatinases (90 min MCAO in SHR
with 3 Hr Reperfusion)
In Situ Zymogram
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MMPI (BB-1101) Reduces Early Opening of the BBB
Zymograms shows elevated MMP-2
Sucrose space increase reduced by BB-1101
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mRNA of MMP-2, MT1-MMP, and Furin Increased in
Early BBB Opening

MMP-2 MT1-MMP
FURIN
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MMP-2 in Astrocytic End-feet
(x100)
(Rosenberg et al. Brain Research 2001)
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90 min MCAO with 3 hr Reperfusion in SHR
MMP-2 in Neurons MMP-2 in
Astrocytes MMP-2 co-localized with MT1-MMP
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Tight Junction Proteins (TJPs)

• Zona occluden
• Attached to actin in the endothelial cell
• Occluden
• Anchored to zona occluden and protruding into the
  extracelluar space
• Claudin-5
• Forms tight junctions in the endothelial cell
  cleft

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Claudin and Occludin in Tight Junctions of
Cerebral Vessels
(Van Itallie et al Physiology 19331 ,2004)
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Claudin-5 in Noninfarcted Hemisphere in 3 hr
Reperfusion Injury
Claudin-5
GFAP Merged
(Yang et. al. Submitted)
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Claudin-5 Fragmented in Early Reperfusion Injury
Claudin-5
GFAP
Merged
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Occludin 3 hrs reperfusion
Noninfarcted

Infarcted Occludin

Occludin GFAP


GFAP Merged

Merged
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BB-1101 Blocks Claudin-5 and Occluden Cleavage
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Early Activation of MMP-2 In Reperfusion Injury
Opens the BBB
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Delayed BBB Opening Causes Hemorrhagic
Transformation
10X
40X
4.0T MRI
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Secondary Opening of the BBB(90 min MCAO in SHR)
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Gelatinase B (MMP-9) Induced (90-MCAO)
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Reperfusion Injury at 24 Hrs
Sucrose Space MMP-9 mRNA MMP-2 mRNA
In Situ Zymogram Gel Zymogram
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Claudin-5 Moves From TJ to Astrocytes by 24 hrs
after Ischemia
Claudin-5 GFAP
Claudin-5/GFAP
Nonischemic Ischemic
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MMP-3 and -9 Cause Delayed Proteolytic Disruption
of the BBB
Hypoxia/Ischemia
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Therapeutic Window
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Complicated Migraine

• HPI 32 y.o. man had right-sided migraine with
  visual impairment followed by left-sided weakness
• Exam Mild weakness of the left arm and severe
  weakness of left leg.
• Hospital Course Leg weakness improved over 4
  days

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Migraine-Induced BBB Opening
T1-Pre Gad
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Neurovascular Inflammation in Migraine and
Cortical Spreading Depression (CSD)

• Migraine headaches associated with CSD
• CSD caused by potassium depolarization wave
• Triggers release of inflammatory substrates
• Complicated migraines have transient symptoms
• Hemiparesis
• Hemisensory loss
• Visual aura

Time-Activity Profiles for the Six Regions of
Interest in the Areas with Maximal Serial
Decreases in Regional Blood Flow during
Measurements 7 through 12.
Woods et al. NEJM 331 (25) 1689, 1994
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Neuroinflammation in migraine
(Bolay H et al Nature Medicine  8, 136
1422002)
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Increased in situ gelatinolytic activity in
cortex ipsilateral to CSD
(Gursoy-Ozdemir, Y. et al. J. Clin. Invest.
20041131447-1455)
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CSD Induces MMP-9 Expression
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CSD-Induced BBB Opening to Evans Blue
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CSD-induced plasma protein leakage
suppressed by MMP inhibition or after CSD in
mice lacking MMP-9 gene expression
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Conclusions

• Blood-brain barrier is transiently opened in the
  early stages of stroke and in migraine, but more
  extensively damaged in the later stages of stroke
• Matrix metalloproteinases are proteolytic enzymes
  that attack the proteins that hold the blood
  vessels together causing intracerebral hemorrhage
• Drugs that block the matrix metalloproteinases
  reduce bleeding induced by the clot-busters

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Collaborators and Funding

• Yi Yang
• Thomas Pfefferkorn
• Eduardo Estrada
• Lee Anna Cunningham
• Jim Wallace
• Hideaki Nagase (University of London)
• Andrew Gearing (British Biotech)
• Michael Moskowitz (Harvard)

Supported by grants from the American Heart
Association and the NIH Neurological Institute