Activation of Brain Endothelium

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 Engineering University of South Carolina Activation of Brain Endothelium by Soluble Amyloid Protein Aggregates

Alzheimer’s Pathology ,[object Object],[object Object],Alzheimer’s Brain Pathology A  Fibrils Nichols et al. (2002) Biochemistry , 41: 6115-27. Courtesy of D. Dickson, Mayo Clinic, Jacksonville. Cerebral Amyloid Angiopathy http://www.adrc.wustl.edu/adrc2.html Amyloid Plaques

A  and Endothelium ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Washington University ADRC Cerebrovasculature Cell Culture

Research Questions… ,[object Object],[object Object],[object Object],[object Object],monomer nucleus soluble intermediates fibril

Isolation of A  Species ,[object Object],[object Object],Mixture 150 mM NaCl 2-10 mM NaCl Fibrils

[object Object],[object Object],A model endothelium exhibiting blood-brain barrier properties was developed. Gonzalez-Velasquez and Moss (2008) J Neurochem , 104 : 500-13.

Endothelial Cell Assays ,[object Object],[object Object],Adhesion Transmigration Permeability permeability coefficient P e % transmigrated cells % adherent cells ~ 0.25x10 -4 cm/s 10-15% 10-15%

[object Object],[object Object],Soluble A  aggregates selective activate endothelial cells. Gonzalez-Velasquez and Moss (2008) J Neurochem , 107 : 466-77; Gonzalez-Velasquez, Kotarek, and Moss (2008) J Neurochem , 104 : 500-14.  MIX  MON  SOL  FIB

[object Object],[object Object],Stimulation of both adhesion and permeability are more pronounced in the presence of smaller soluble A  aggregation intermediates. Gonzalez-Velasquez and Moss (2008) J Neurochem , 107 : 466-77; Gonzalez-Velasquez, Kotarek, and Moss (2008) J Neurochem , 104 : 500-14.  Adhesion  Permeability

[object Object],[object Object],Treatment of HBMVEC monolayers with an A  reaction mixture modifies ZO-1 localization. Background CONT TNF 2.5  M 5  M 10  M Gonzalez-Velasquez and Moss (2008) J Neurochem , 107 : 466-77.

… and responsible for A  -stimulated adhesion ,[object Object],[object Object],VCAM MON FIB SOL CONT ICAM ICAM-1 and VCAM-1 expression is selectively upregulated… Gonzalez-Velasquez et al (2010) J Adh Sci Tech , in press .

[object Object],[object Object],CONT TNF MON SOL MIX FIB NF-  B nuclear translocation is selectively stimulated by soluble A  aggregates. Gonzalez-Velasquez et al (2010) Curr Alzheimers Res , under review .

[object Object],[object Object],Inhibiting NF-  B nuclear translocation abrogates A  -induced adhesion, transmigration, and permeability.  Control  MG 132 Gonzalez-Velasquez et al (2010) Curr Alzheimers Res , under review .

Summary ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Mimic cell-surface growth Correlate growth with physiological activity Publications: Kotarek and Moss (2008) Anal. Biochem. 378: 15-24. Kotarek and Moss (2009) In Preparation. Employ novel techniques to quantify A  aggregation POPC POPC/DPPC monomer aggregate biotin tag Au electrode surface unlabeled soluble aggregate avidin biotinylated Ab 1-40 monomer unlabeled A  1-40 monomer supported phospholipid bilayer (SPB) A  1-40 aggregation intermediate

Define inhibitor structure-function relationships Utilize inhibitors to understand, control protein aggregation Elongation Association Identify, characterize inhibitors that target specific growth mechanisms Publications: Reyes Barcelo et al (2009) J Biol Eng 3:5. Davis et al (2009) Mol Pharmacol, 76: 405-413.

Acknowledgements ,[object Object],[object Object],Graduate Students Adriana Reyes Barcelo Joseph Kotarek Will Reed Deborah Soto-Ortega Chen Suo Jui-Heng Tseng Kelly Wilson Research Associate Francisco Gonzalez-Velasquez Undergraduate Students Sukhi Guram Emily Matherly Brandon Murphy Apoorva Srivastava Andreea Stoichita ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Activation of Brain Endothelium

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