Smooth Muscle Proliferation in Moyamoya Vasculopathy

Smooth Muscle Proliferation in
Moyamoya Vasculopathy
Dianna M. Milewicz, MD PhD
University of Texas Health Science
Center at Houston

Moyamoya Disease
• Ischemic strokes due to stenosis of the intracranial internal
carotid arteries and their proximal branches associated with
the formation of collaterals
• Increased in prevalence in Japan
– RNF213 mutations
• Familial in 8% of patients
genetic predisposition
• MMD and genetic syndromes:
– Neurofibromatosis
– Noonan syndrome
– Sickle cell anemia
genetic heterogeneity

Cerebrovascular Pathology Associated
with Moyamoya Disease
Occlusion of the artery
Neointimal lesions with
smooth muscle cells
No lipid deposition or
inflammation
Do genetic mutations that increase SMC
proliferation play a role in the pathogenesis of
Moyamoya disease?

ACTA2 Missense Mutations Cause Familial
Thoracic Aortic Aneurysms and Dissections
ACTA2 R149C missense mutation
Penetrance of TAAD: 50%

Aortic Smooth Muscle Cells and
Aortic Media
the Contractile Unit
Actin
Contractile Unit
Myosin

ACTA2 Missense Mutations in FTAAD
G48V
P72Q
N117T R149C
Forty ACTA2 missense mutations
No polymorphic variants in controls
All mutations mutant protein
R179H
1 2 3 4 5 6 7 8 9
R39C R39H R118Q Y135H G160D R212Q
L69Q
W88R
V154A
R258H R258C
G270R
R292G
T353N

ACTA2 Mutations Cause Livedo Reticularis:
Skin Rash Due to Arterial Occlusion
Penetrance of livedo reticularis is 100%

Aortic Arterial Pathology in Patients
with ACTA2 Mutations
Control ACTA2 R149C ACTA2 R149C ACTA2 T353N
ACTA2 Staining
100X

ACTA2 Mutations Lead to Early Onset
Coronary Artery Disease
TAAD
Premature CAD
Premature stroke

Moyamoya Disease and
Early Onset Strokes in
ACTA2 Mutation Carriers
TAAD
Premature CAD
Premature stroke

De Novo ACTA2 R179H Mutations
Smooth Muscle Dysfunction Syndrome
• 15 patients with de novo ACTA2 R179H
• Fully penetrant vascular diseases at young ages:
– Ascending aortic aneurysms
– Moyamoya disease
– PDA
• Loss of SMC function in other organs:
– Fixed dilated pupils
– Hypotonic bladder
– Hypoperistalsis with malrotation
– Patent ductus arteriosus
• Proliferative SMC phenotypes:
– Aortic coarctation
– Primary pulmonary hypertension

Loss of function:
Decreased SMC
contraction
Large arteries:
-high elastin content
- Exposed to high
pressures
Aortic
Aneurysms
ACTA2 Vasculopathy
Gain of function:
Increased SMC
proliferation
Small arteries:
-low elastin content
-exposed to low
pressures
Vascular occlusive diseases
(CAD, stroke)

Characterization of Mouse Model:
Acta2 -/- Mice
Fold change (a.u.)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Acta2 expression
Wt
Acta2 mRNA
α-actin
GAPDH
Acta2 -/-
Previously characterized by Schildmeyer et al
(2000):
Contractility of arteries
Blood pressure
Schildmeyer LA et al (2000). FASEB J.

Acta2 -/- SMCs Proliferate and Migrate
Faster than Wild-type SMCs
Proliferation – ascending SMCs Proliferation – descending SMCs
BrdU incorporation (OD)
1
0.8
0.6
0.4
0.2
0
p

Acta2 -/- Mice: Increased
Neointimal Proliferation with Vascular Injury
ligation site
Neointima Area (µm 2 )
40000
30000
20000
10000
0
Injury
wildtype Acta2-/-
p < 0.01
p < 0.01
- + - +
WT Acta2-/-
% Stenosis
100
80
60
40
20
0
Injury
p < 0.01
+ +
WT Acta2-/-

BrdU incorporation (OD)
Anti-cancer Drug Decreases Proliferation
in vitro and Neointimal Formation in vivo
1.4
1.2
1
0.8
0.6
0.4
0.2
0
p

Loss of function:
Decreased SMC
contraction
Large arteries:
-high elastin content
- Exposed to high
pressures
Aortic
Aneurysms
ACTA2 Vasculopathy
Gain of function:
Increased SMC
proliferation
Small arteries:
-low elastin content
-exposed to low
pressures
Vascular occlusive diseases
(CAD, stroke)

Vasculopathy Associated with Neurofibromatosis
• Affects aorta to small arterioles
• Can cause aneurysms and occlusive diseases, including
Moyamoya disease
• Mouse studies show increased SMC proliferation and
migration and increase neointimal formation
• NF1 mutations

Moyamoya disease and RNF213 Mutations
• Identified through linkage analysis and
association studies as a gene that causes
MMD in Japan.
• “Founder” mutation in Japan (Asia?).
• Function of the gene is unknown.
• 10 – 15% of MMD patients in our U.S. cohort
have RNF213 mutations (unpublished data).

Conclusions
• A subset of ACTA2 mutations predispose to MMD.
• Acta2 deficient mouse models suggest that
occlusive disease is driven by SMC hyperplasia.
– Why do some mutations cause more SMC hyperplasia?
• Anti-cancer drug may prevent or limit MMD lesions
in patients with ACTA2 mutations.
– Can anti-proliferative drugs used to treat MMD?
• NF1 mouse model also suggests that occlusive
disease is due to SMC hyperplasia.
• RNF213 is a major gene for MMD.
– Do RNF213 mutations cause SMC hyperplasia?

Milewicz Lab members
Ellen Regalado
Dongchuan Guo, Ph.D.
Christina Papke, Ph.D.
Jiumei Cao, Ph.D.
Stanford School of Medicine
Gary Steinberg, MD PhD
MD Anderson Cancer Center
Aarif Khakoo, M.D.
Meredith Rees
Texas A&M
Warren Zimmer, Ph.D.
Soon-Mi Lim, Ph.D.
Andreea Trache, Ph.D.
University of Geneva
Giulio Gabbiani, Ph.D.
Christine Chaponnier, Ph.D.
Acknowledgements
Funding
NIH P50HL083794-01
NIH R01HL034786
Vivian L. Smith Foundation