activation of brain endothelium by soluble amyloid protien aggregates, melissa moss, phd
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Activation of Brain Endothelium by Soluble Amyloid Protein
Aggregates
F.J. Gonzalez-Velasquez, J.W. Reed, J.W. Fuseler,E.E. Matherly, D.D. Soto-Ortega, J.A. Kotarek,
and M.A. Moss
Department of Chemical EngineeringUniversity of South Carolina
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College of Engineering and ComputingUniversity of South Carolina
Alzheimer’s Pathology
Alzheimer’s Brain
Pathology
A Fibrils
Nichols et al. (2002)Biochemistry, 41: 6115-27.
Courtesy of D. Dickson,Mayo Clinic, Jacksonville.
Cerebral AmyloidAngiopathy
http://www.adrc.wustl.edu/adrc2.html
Amyloid Plaques
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College of Engineering and ComputingUniversity of South Carolina
A and Endothelium
Cellular degeneration
Compromised BBB integrity
Inflammatory responses
Increased vessel-associated monocyte/macrophage cells
ToxicityIncreased permeabilityCytokine secretionIncreased monocyte
adhesion, transmigration
Upregulated adhesion molecule expression
Washington University ADRC
Cerebrovasculature Cell Culture
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College of Engineering and ComputingUniversity of South Carolina
Research Questions…
What form of A interacts with endothelial cells to promote activation
What signaling pathways are involved
monomer
nucleus
soluble intermediate
s
fibril
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College of Engineering and ComputingUniversity of South Carolina
Isolation of A Species
Mixture
150 mMNaCl
2-10 mMNaCl
Fibrils
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College of Engineering and ComputingUniversity of South Carolina
A model endothelium exhibitingblood-brain barrier properties was developed.
Gonzalez-Velasquez and Moss (2008) J Neurochem, 104: 500-13.
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College of Engineering and ComputingUniversity of South Carolina
Endothelial Cell Assays
Adhesion Transmigration Permeability
permeabilitycoefficient
Pe
%transmigrated
cells
%adherent
cells
~ 0.25x10-4 cm/s10-15%10-15%
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College of Engineering and ComputingUniversity of South Carolina
Soluble Aaggregates selective activate endothelial cells.
MIX MON SOL FIB
Gonzalez-Velasquez and Moss (2008) J Neurochem, 107: 466-77;Gonzalez-Velasquez, Kotarek, and Moss (2008) J Neurochem, 104: 500-14.
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College of Engineering and ComputingUniversity of South Carolina
Stimulation of bothadhesion andpermeability aremore pronouncedin the presence of smaller soluble Aaggregationintermediates.
Gonzalez-Velasquez and Moss (2008) J Neurochem, 107: 466-77;Gonzalez-Velasquez, Kotarek, and Moss (2008) J Neurochem, 104: 500-14.
Adhesion Permeability
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College of Engineering and ComputingUniversity of South Carolina
Treatment of HBMVEC monolayers with anAreaction mixture modifies ZO-1
localization.
Background CONT TNF
2.5 M 5 M 10 MGonzalez-Velasquez and Moss (2008) J Neurochem, 107: 466-77.
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…and responsible for A-stimulated adhesion
College of Engineering and ComputingUniversity of South Carolina
VCAM
MON FIB SOLCONT
ICAM
ICAM-1 and VCAM-1 expression is selectively upregulated…
Gonzalez-Velasquez et al (2010) J Adh Sci Tech, in press.
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College of Engineering and ComputingUniversity of South Carolina
CONT TNF
MON SOL
MIX
FIB
NF-B nuclear translocation is
selectively stimulated by soluble
Aaggregates.Gonzalez-Velasquez et al (2010) Curr Alzheimers Res, under review.
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College of Engineering and ComputingUniversity of South Carolina
CONT MON MIX FIB0
10
20
30
40
***
% A
dhes
ion
CONT MON MIX FIB0
10
20
30
40
***
% T
rans
mig
rati
on
Inhibiting NF-B nucleartranslocation abrogatesA-induced adhesion,transmigration, andpermeability.
Control MG 132
Gonzalez-Velasquez et al (2010) Curr Alzheimers Res, under review.
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College of Engineering and ComputingUniversity of South Carolina
Summary
• A is capable of activating endothelial monolayers.
• Specifically, soluble A aggregation intermediates are responsible for endothelial activation.
• Smaller soluble aggregation intermediates are the most potent activators of the endothelium.
• A activation of endothelial monolayers involves loss of tight junctions and increased expression of ICAM-1 and VCAM-1.
• NF-B signaling is involved in A activation of endothelial monolayers.
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Mimic cell-surface growth
Correlate growth withphysiological activity
Publications: Kotarek and Moss (2008) Anal. Biochem. 378: 15-24.Kotarek and Moss (2009) In Preparation.
biotin tag
Au
electrode surface
unlabeled soluble aggregateavidin
biotinylated Ab1-40
monomer unlabeled A1-40 monomer
Employ novel techniques to quantifyAaggregation
POPC
POPC/DPPC
monomer
aggregate
supported phospholipid bilayer (SPB)
A1-40 aggregation intermediate
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Define inhibitorstructure-
functionrelationships
Utilize inhibitors tounderstand, controlprotein aggregation
Elongation
Association
Identify, characterizeinhibitors that targetspecific growthmechanisms
Publications: Reyes Barcelo et al (2009) J Biol Eng 3:5.Davis et al (2009) Mol Pharmacol, 76: 405-413.
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College of Engineering and ComputingUniversity of South Carolina
Graduate StudentsAdriana Reyes BarceloJoseph KotarekWill ReedDeborah Soto-OrtegaChen SuoJui-Heng TsengKelly Wilson
Research AssociateFrancisco Gonzalez-Velasquez
Undergraduate StudentsSukhi GuramEmily MatherlyBrandon MurphyApoorva SrivastavaAndreea Stoichita
AcknowledgementsNational Science Foundation
CAREER Award (BES 0644826)
American Heart Association, Mid-Atlantic AffiliateBeginning Grant-In-Aid (0565387U)
Alzheimer’s AssociationNew Investigator Research Grant (NIRG-07-60412)
USC Complementary and Alternative Medicine Center
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Activation of Brain Endothelium by Soluble Amyloid Protein
Aggregates
F.J. Gonzalez-Velasquez, J.W. Reed, J.W. Fuseler,E.E. Matherly, D.D. Soto-Ortega, J.A. Kotarek,
and M.A. Moss
Department of Chemical EngineeringUniversity of South Carolina