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