The overall goal of this project is to test the hypothesis that amyloid beta-peptide (A?) in different self-assembled forms, monomers, oligomers and fibrils, directly impacts cerebral endothelial functions because it alters 1) the expression of adhesion molecules (e.g. P- and E-selectins) and actin polymerization;2) cellular mechanical properties;and the forces involved in adhesion;and 3) these alterations are resulted from the signaling pathway involving the binding of A? to receptor for advanced glycation end products (RAGE) to induce activations of NADPH oxidase and MAP kinases. Alteration of the cerebral microvascular endothelium is intimately associated with Alzheimer's Disease (AD). Increased deposition of A? in cerebral vasculature and the increased presence of monocytes in the vessel wall and of activated microglia are frequently observed in AD brains. Recent studies show that peripheral monocytes can migrate across the blood-brain barrier (BBB) and differentiate into microglia within the brain parenchyma. In vitro studies demonstrated that A? deposition at the endothelial cell layer enhances the transmigration of monocytes. Thus, increased transmigration of monocytes into brains is thought to drive the disease development towards exacerbation of the oxidative and inflammatory conditions characteristic of the AD brain. Consistent with our hypothesis, our preliminary data show that A? oligomer increases the immunoreactivity of P-selectin at the endothelial surface and actin polymerization within endothelial cells. Since the transmigration of monocytes across the BBB is both a mechanical and a biochemical process, the expression of adhesion molecules and mechanical properties of endothelial cells are the critical factors that require investigation. In this regard, the direct effects of A? on the expression of adhesion molecules and the mechanical properties of endothelial cell have yet to be fully elucidated. Therefore, our study on how A? alters the factors governing the adhesion between monocytes and endothelial surfaces will provide highly relevant and novel biophysical information about the mechanism of progression of AD and development of intervention strategy. To test our central hypothesis, we propose three specific aims:
Aim 1 : Determine the effects of A? monomers, oligomers and fibrils on the expression of P- and E- selectins and actin polymerization in immortalized cerebral endothelial cells (CECs).
Aim 2 : Measure the effects of A? monomers, oligomers and fibrils on the cell mechanical and adhesion properties of CECs.
Aim 3 : Determine the roles of RAGE, NADPH oxidase and MAP kinases (i.e. p38, ERK and JNK) in A2-induced alterations in endothelial adhesion.
These aims will be accomplished by various biophysical and biochemical techniques including quantitative immunofluorescent microscopy, atomic force microscopy and various biochemical approaches.
Alteration of the cerebral microvascular endothelium is intimately associated with Alzheimer's Disease. We use atomic force microscopy to investigate the mechanism underlying the altered adhesion properties of endothelium induced by amyloid beta peptide. The mechanism is important in part of Alzheimer's disease development.
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