The goal of the project is to detect senescence-associated modifications of extracellular matrix that promote deposition of Alzheimer's amyloid-beta in brain blood vessel walIs. Progressive cross-linking of vascular matrix by lysyl osidase-like2/related protein (LOX-L2/RP), over-expressed in aging, over the years may contribute to vascular amyloidosis by impairing clearance of amyloidogenic proteins and peptides. The potential of LOX-L2/RP to cross-link amyloidogenic proteins and to immobilize them in matrix has not been explored. The most common vascular amyloidosis in aged persons is amyIoidosis-beta of Alzheimer's type. It causes life threatening hemorrhages and strokes. Hence, a LOX-L2/RP-associated pathomechanism of cerebrovascular amyloid-beta productipn could be targeted by specific therapeutic approach. We hypothesize that enzymatic crosslinking of matrix proteins by LOX-Lr/RP secreted by senescent vascular cells. reduces clearance of soluble amyloid-beta peptide (ABeta), crosslinks ABeta to matrix and causes aggregation of ABeta. Thus, amyloid would be deposited at sites of over-production of both ABeta and LOX-L2/RP. To test the hypothesis we created a cell culture model of vascular amyloidosis--established Iines of brain vascular smooth muscle cells which secrete and accumulate ABeta. The lines, derived from mice Tg2576, transgenic with human amyloid-beta precursor protein (AbetaPP) with Swedish mutation, display distinct levels of expression of LOX-L2/RP and other lysyl oxidases. Using these cell lines, not transfected, or transfected with LOX-L2 expression vector, as well as cell free assays. we wiII study immobilization and aggregation of ABeta in matrix by ELISA and by light confocal and electron microscopy. The effect of LOX-RP on aggregation of ABeta will be tested in cell-free system. Correlation between LOX-L2 expression and initial vascular amyloidosis will be studied in brain sections by confocal microscopy. The study is expected to generate data and tools for future larger proposal on the mechanisms of amyloid-beta deposition promoted by enzymatic lysine cross-linking, that might be a target of therapy in brain vascular amyloidosis-beta. 1
| Frackowiak, Janusz; Potempska, Anna; LeVine, Harry et al. (2005) Extracellular deposits of A beta produced in cultures of Alzheimer disease brain vascular smooth muscle cells. J Neuropathol Exp Neurol 64:82-90 |
