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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
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
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
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
College of Engineering and ComputingUniversity of South Carolina
Isolation of A Species
Mixture
150 mMNaCl
2-10 mMNaCl
Fibrils
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.
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%
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.
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
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.
…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.
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.
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.
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.
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
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.
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
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