Pathologic Amyloid β‐Protein Cell Surface Fibril Assembly on Cultured Human Cerebrovascular Smooth Muscle Cells

Abstract

Cerebrovascular amyloid β‐protein (Aβ) deposition is a key pathological feature of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis‐Dutch type (HCHWA‐D). Aβ1–40 containing the E22Q HCHWA‐D mutation, but not wild‐type Aβ1–40, potently induces several pathologic responses in cultured human cerebrovascular smooth muscle cells, including cellular degeneration and a robust increase in the levels of cellular Aβ precursor. In the present study, we show by several quantitative criteria, including thioflavin T fluorescence binding, circular dichroism spectroscopy, and transmission electron microscopic analysis, that at a concentration of 25 µM neither HCHWA‐D Aβ1–40 nor wild‐type Aβ1–40 appreciably assembles into β‐pleated sheet‐containing fibrils in solution over a 6‐day incubation period. In contrast, at the same concentrations, HCHWA‐D Aβ1–40, but not wild‐type Aβ1–40, selectively binds and assembles into abundant fibrils on the surfaces of cultured human cerebrovascular smooth muscle cells. The simultaneous addition of an equimolar concentration of the dye Congo red prevents the cell surface fibril assembly of HCHWA‐D Aβ1–40. Moreover, Congo red effectively blocks the key pathologic responses induced by HCHWA‐D Aβ1–40 in these cells. The present findings suggest that the surface of human cerebrovascular smooth muscle cells may selectively orchestrate the assembly of pathogenic Aβ fibrils and that cell surface Aβ fibril formation plays an important role in causing the pathologic responses in these cells.

Localization of a Fibrillar Amyloid β-Protein Binding Domain on Its Precursor

“…Similar to these in vivo observations, we have reported that A␤- , the more pathogenic form of the wild-type peptide, causes severe cellular degeneration accompanied by a marked increase in the level of cell-associated A␤PP in cultured human cerebrovascular smooth muscle (HCSM) cells (20 -22). In more recent studies we demonstrated that mutations associated with familial forms of cerebral amyloid angiopathy (E22Q Dutch, E22K Italian, and D23N Iowa) markedly enhance both the fibrillogenic and cerebrovascular pathogenic properties of A␤ toward cultured HCSM cells (23)(24)(25)(26). These experiments showed that these pathogenic forms of A␤ assemble into an elaborate network of fibrils on the surfaces of HCSM cells.…”

“…For preparation of amyloid fibrils, A␤ peptides or amylin were resuspended to a final concentration of 1.25 mM in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 and incubated at 37°C for 3 days. The ␤-sheet, fibrillar structure of each peptide was confirmed by circular dichroism spectroscopy and electron microscopy as previously described (24). For cell culture experiments, lyophilized A␤ peptide was first resuspended to a concentration of 250 M in sterile distilled water.…”

Amyloid β-Protein Stimulates the Expression of Urokinase-type Plasminogen Activator (uPA) and Its Receptor (uPAR) in Human Cerebrovascular Smooth Muscle Cells

“…Similar to these in vivo observations, we have reported that A␤42, the more pathogenic form of the wild-type peptide, causes cellular degeneration accompanied by a marked increase in the level of cell-associated A␤PP in cultured human cerebrovascular smooth muscle (HCSM) cells, an in vitro model of CAA (20 -22). In more recent studies we demonstrated that mutations within A␤ that are associated with familial forms of CAA (E22Q Dutch, E22K Italian, and D23N Iowa) markedly enhance both the fibrillogenic and cerebrovascular pathogenic properties of A␤ toward cultured HCSM cells (23)(24)(25)(26). These investigations showed that CAA mutant forms of A␤ assemble into an extensive network of fibril-like structures on the surfaces of HCSM cells.…”

“…For preparation of amyloid fibrils, A␤ was resuspended to a final concentration of 1.25 mM in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, and incubated at 37°C for 3 days. Circular dichroism spectroscopy and electron microscopy confirmed the ␤-sheet, fibrillar structure of A␤ as described previously (24). For cell culture experiments lyophilized A␤ peptide was first resuspended to a concentration of 250 M in sterile distilled water.…”

“…Our previous studies (20,23,24,27) showed that wild-type A␤40 is not toxic to HCSM cells whereas Dutch mutant A␤40 is highly pathogenic for these cultured cerebrovascular cells. At the designated time points secreted or cell surface-associated plasminogen activator activity was measured using a chromogenic substrate assay as described under "Experimental Procedures."…”