(386f) Optical Image Analysis Facilitates An Understanding of Amyloid-β Protein Aggregate Activation of Brain Microvascular Endothelial Cells

Reed, J. W., University of South Carolina
Fuseler, J. W., University of South Carolina School of Medicine
Matherly, E. E., University of South Carolina
Kotarek, J. A., University of South Carolina
Soto-Ortega, D., University of South Carolina
Gonzalez-Velasquez, F. J., University of South Carolina
Moss, M. A., University of South Carolina

Cerebral amyloid angiopathy (CAA) presents in the majority of Alzheimer's disease (AD) patients. CAA is strongly associated with disruption of the blood-brain barrier (BBB) and increased adhesion of circulating immune cells. These events weaken blood vessels and promote AD pathogenesis. In AD, the fibrillar cerebrovascular deposits characteristic of CAA are composed of the amyloid-β protein (Aβ). Monomeric Aβ self-assembles to create soluble aggregates, which grow further to yield the fibrillar aggregates that deposit in the cerebrovasculature. To better understand the role of Aβ in degradation of the BBB, we used quantitative immunocytochemical assays to evaluate monolayers of human brain microvascular endothelial cells (HBMVEC) challenged with different Aβ aggregate preparations. Fluorophore integrated optical density (IOD), which was calculated using image analysis tools from the Metamorph software package, quantifies the level of protein expression from immunocytochemical images. Quiescent cells express low levels of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and display basal levels of nuclear factor-κB (NF-κB) uniformly throughout the cytoplasm and in lower concentrations within the nucleus. Cellular activation leads to increased levels of ICAM-1 and VCAM-1 and to translocation of NF-κB to the nucleus. Thus, modified cellular activity was assessed by comparing cell surface adhesion molecule IOD and NF-κB IOD to the total cellular area and nuclear area, respectively. Increased expression of both ICAM-1 and VCAM-1 was detected after exposure to micromolar concentrations of Aβ, and this response was paralleled by NF-κB activation. Quantitative comparisons of activation with different Aβ aggregate preparations revealed that fibrillar and monomeric forms of Aβ induced noticeable changes in HBMVEC monolayers, while smaller soluble Aβ aggregates induced a more significant stimulation. A short peptide, MG-132, blocks the proteasomal degradation required for nuclear translocation of NF-κB. When HBMVEC monolayers were incubated with MG-132 prior to Aβ exposure, nuclear NF-κB IOD diminished significantly. Furthermore, inhibition of NF-κB activation also prevented the increased expression of both ICAM-1 and VCAM-1, confirming the role of NF-κB signaling in this signaling pathway. The quantitative immunocytochemical approach confirms the selective activation of human brain endothelium by soluble Aβ aggregates and offers conclusive evidence for NF-κB signaling in this process. This technique will enable the recognition other cellular signaling pathways involved in the pathogenesis of AD in order to identify targets for AD therapeutic intervention.