F.J. Gonzalez-Velasquez, J.W. Reed, J.W. Fuseler, E.E. Matherly, D.D. Soto-Ortega, J.A. Kotarek,  and  M.A. Moss Departmen...
Alzheimer’s Pathology <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><l...
A   and Endothelium <ul><li>Cellular degeneration </li></ul><ul><li>Compromised BBB integrity </li></ul><ul><li>Inflammat...
Research Questions… <ul><li>What form of A   interacts with  endothelial cells to promote activation </li></ul><ul><li>Wh...
Isolation of A   Species  <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><...
<ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South ...
Endothelial Cell Assays <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul>...
<ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South ...
<ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South ...
<ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South ...
… and responsible for A  -stimulated adhesion <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></u...
<ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South ...
<ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South ...
Summary <ul><li>A   is capable of activating endothelial monolayers. </li></ul><ul><li>Specifically, soluble A   aggrega...
Mimic cell-surface growth Correlate growth with physiological activity Publications:  Kotarek and Moss (2008)  Anal. Bioch...
Define inhibitor structure-function relationships Utilize inhibitors to understand, control protein aggregation Elongation...
Acknowledgements <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Uni...
F.J. Gonzalez-Velasquez, J.W. Reed, J.W. Fuseler, E.E. Matherly, D.D. Soto-Ortega, J.A. Kotarek,  and  M.A. Moss Departmen...
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Activation of Brain Endothelium by Soluble Amyloid Protien Aggregates, Melissa Moss, PhD

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  • 05/28/10
  • 05/28/10
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  • Activation of Brain Endothelium by Soluble Amyloid Protien Aggregates, Melissa Moss, PhD

    1. 1. 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
    2. 2. Alzheimer’s Pathology <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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
    3. 3. A  and Endothelium <ul><li>Cellular degeneration </li></ul><ul><li>Compromised BBB integrity </li></ul><ul><li>Inflammatory responses </li></ul><ul><li>Increased vessel-associated monocyte/macrophage cells </li></ul><ul><li>Toxicity </li></ul><ul><li>Increased permeability </li></ul><ul><li>Cytokine secretion </li></ul><ul><li>Increased monocyte adhesion, transmigration </li></ul><ul><li>Upregulated adhesion molecule expression </li></ul><ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>Washington University ADRC Cerebrovasculature Cell Culture
    4. 4. Research Questions… <ul><li>What form of A  interacts with endothelial cells to promote activation </li></ul><ul><li>What signaling pathways are involved </li></ul><ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>monomer nucleus soluble intermediates fibril
    5. 5. Isolation of A  Species <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>Mixture 150 mM NaCl 2-10 mM NaCl Fibrils
    6. 6. <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>A model endothelium exhibiting blood-brain barrier properties was developed. Gonzalez-Velasquez and Moss (2008) J Neurochem , 104 : 500-13.
    7. 7. Endothelial Cell Assays <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>Adhesion Transmigration Permeability permeability coefficient P e % transmigrated cells % adherent cells ~ 0.25x10 -4 cm/s 10-15% 10-15%
    8. 8. <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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
    9. 9. <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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
    10. 10. <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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.
    11. 11. … and responsible for A  -stimulated adhesion <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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 .
    12. 12. <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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 .
    13. 13. <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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 .
    14. 14. Summary <ul><li>A  is capable of activating endothelial monolayers. </li></ul><ul><li>Specifically, soluble A  aggregation intermediates are responsible for endothelial activation. </li></ul><ul><li>Smaller soluble aggregation intermediates are the most potent activators of the endothelium. </li></ul><ul><li>A  activation of endothelial monolayers involves loss of tight junctions and increased expression of ICAM-1 and VCAM-1. </li></ul><ul><li>NF-  B signaling is involved in A  activation of endothelial monolayers. </li></ul><ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>
    15. 15. 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
    16. 16. 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.
    17. 17. Acknowledgements <ul><ul><ul><ul><li>College of Engineering and Computing </li></ul></ul></ul></ul><ul><ul><ul><ul><li>University of South Carolina </li></ul></ul></ul></ul>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 <ul><li>National Science Foundation </li></ul><ul><ul><li>CAREER Award (BES 0644826) </li></ul></ul><ul><li>American Heart Association, Mid-Atlantic Affiliate </li></ul><ul><ul><li>Beginning Grant-In-Aid (0565387U) </li></ul></ul><ul><li>Alzheimer’s Association </li></ul><ul><li>New Investigator Research Grant (NIRG-07-60412) </li></ul><ul><li>USC Complementary and Alternative Medicine Center </li></ul>
    18. 18. 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
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