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Viral Vasculopathy
Maria Acena Nagel
Overview
I. Background
II. VZV vasculopathy
Clinical, morphological,
and immunological features
III. Cerebrovascular adventitial fibroblast
studies
Varicella
zoster
Latency
cranial nerve
ganglia
dorsal root
ganglia
autonomic
ganglia
Varicella
Latency
VZV reactivation
from neurons
Peripheral spread
to skin
Central spread
to cerebral arteries
Productive virus infection in cerebral arteries
multinucleated
giant cells
herpesvirions
Cowdry A
inclusions
VZV DNA
VZV antigen
Gilden et al., Neurology, 1996
How common is VZV vasculopathy?
VZV persists in >95% of the world population
VZV
Virgin et al., Cell, 2009
VZV reactivation
 1 in 3 individuals will develop zoster; 2/3 will
continue to shed VZV in saliva up to
12 years after zoster
 30% increased risk of stroke after zoster
 4.5x higher if ophthalmic-distribution zoster
 an unknown number will develop stroke
from VZV infection of arteries without rash
Kang et al., Stroke, 2009
Lin et al., Neurology, 2010
Nagel et al., J Inect Dis, 2011
VZV is present in cerebral arteries of
an assymptomatic diabetic subject without zoster
Nagel et al., Arch Neurol, 2012
Clinical features
 Stroke is most often ischemic, sometimes hemorrhagic
 Large and small vessels are involved
 Time from rash to neurological s/s ~ 4 months
 Time from neurological s/s to diagnosis ~ 4 months
 Only 2/3 of patients have rash
 Only 2/3 of patients have increased cells in CSF
 Clinical diagnosis is confirmed by detection of
VZV DNA or anti-VZV IgG antibody in CSF
Nagel et al., Neurology, 2007; 2008
VZV travels transaxonally to infect cerebral arteries
Trigeminal
Ganglia
VZV
Morphological/immunological
features of VZV vasculopathy
Cerebral artery morphology
lumen
intima
IEL
media
adventitia
nerve fibers
normal
early
VZV vasculopathy
chronic 1
chronic 2
Nagel et al., Neurology, 2011
normal
VZV vasculopathy
early
chronic 1
chronic 2
Nagel et al., Neurology, 2011
Leukocytes in early case of VZV vasculopathy
correlates with the thickened intima
CD45
Immune response in arteries
from subjects with VZV vasculopathy
VZV VASCULOPATHY
ADVENTITIA
EARLY
VZV
CD45
CD3, 4, 8
CD68
CD15
MEDIA
INTIMA
ADVENTITIA ASSOCIATED
WITH THICKENED INTIMA
CD45
CD3, 4, 8
CD68
CD3, 4, 8
CD68
CD20
CHRONIC
CD45
CD3, 4, 8
CD68
VZV
CD45
CD3, 4, 8
CD68
VZV-infected arteries from subjects
with VZV vasculopathy
 Thickened neointima, composed of SMA+ and
SMA+/myosin+ cells not endothelial cells
 Internal elastic lamina is duplicated/disrupted
 VZV is present in adventitia in early case and
media and intima in later cases supporting
transmural spread of virus
 The media is more severely disrupted in later
cases
 Inflammatory cells are associated with thickened
intima
Pathogenesis of VZV vasculopathy
Trigeminal
Ganglia
VZV
myofibroblast
accumulation
disruption of
media
Primary human cerebrovascular
adventitial fibroblast studies
Human cerebrovascular
adventitial fibroblasts
Establish quiescence
Infect with VZV
qPCR and ICC for
migratory,
proliferative,
& invasive
trasncripts/proteins
Migration/invasion
assay on transwell chambers
RNA/protein analysis
Functional analysis
VZV infection of human cerebrovascular
adventitial fibroblasts (6 dpi)
fold difference (VZV/mock-infected)
Fold difference (VZV/mock-infected)
1000
chemokine/cytokines
adhesion
proliferation/migration
invasion
100
10
1
0.1
n=3
IL8 protein is elevated in VZV-infected human
cerebrovascular adventitial fibroblasts
IL8 concentration (pg/mL)
250
mock-infected
VZV-infected
ELISA
200
150
100
50
0
VZV
IL8
DAPI
VZV-infected cells
bystander cells
mock-infected cells
Conditioned media from VZV-infected cerebrovascular
adventitial fibroblasts attract neutrophils
Neutrophil migration (%)
35
IL8 (20 ng/mL)
VZV
mock
VZV+Acyclovir
VZV+PAA
30
25
20
15
10
5
0
0
20
40
60
Time (minutes)
80
100
VZV-infected artery from a patient with early VZV vasculopathy
has cells expressing IL8 in adventitia
100x
600x
100x
Tonsil (+)
Atherosclerosis (-)
Normal (-)
Abundant neutrophils are in adventitia of a VZV-infected artery
from a patient with early VZV vasculopathy
VZV-infected cerebrovascular
adventitial fibroblasts
 Feasible model for studying early VZV
vasculopathy
 IL8 is characteristic of early VZV vasculopathy
 Neutrophils are abundant in early
VZV vasculopathy
Ongoing/future studies
How common is VZV vasculopathy?
VZV infection in stroke patients (CDC)
Screening of cerebral arteries for VZV
What are other methods for diagnosis?
Saliva testing for VZV
PET imaging with labeled antiviral
How does VZV persist and evade immune clearance?
Immunosenescence
Infection of immunoprivileged media
Modulation of innate immune response
How does thickened intima form?
Source of thickened intima, mitogenic factors
Extracellular ATP/purinergic signalling
Consider VZV vasculopathy in your
stroke differential since it is
treatable with antivirals!
Alexander
Choe
Igor
Traktinskiy
ACKNOWLEDGMENTS
Nagel Lab
Igor Traktinskiy
Alexander Choe
Eugene Azarkh, PhD
April Rempel
UCH-CVPR Laboratories
Kurt Stenmark, MD
Maria Frid, PhD
VZV Labs
Don Gilden, MD
Randy Cohrs, PhD
Ravi Mahalingam, PhD
NIH
NINDS K08 067070
NIA RO1 AG006127
NIA PO1 AG032958
Neuropathologists
Bette Kleinschmidt-DeMasters, MD - UCH
Tessa Hedley-Whyte, MD - Harvard
Eve VanEgmond, MD - Henry Ford
Hospital