Abstract
The accumulation of fibrillar amyloid-beta protein (A beta) in cerebral blood vessels, a condition known as cerebral amyloid angiopathy (CAA), is a key pathological feature of Alzheimer's disease and certain related disorders and is intimately associated with cerebrovascular cell death both in vivo and in vitro. Moreover, severe CAA leads to loss of vessel wall integrity and cerebral hemorrhage. Although the basis for these latter pathological consequences in CAA remains unresolved alterations in local proteolytic mechanisms may be involved. Here we show that pathogenic forms of A beta stimulate the expression of plasminogen activator activity in cultured human cerebrovascular smooth muscle (HCSM) cells, an in vitro model of CAA. RNase protection assays and plasminogen zymography showed that urokinase-type plasminogen activator (uPA) was responsible for this activity. There was preferential accumulation of uPA on the HCSM cell surface that was mediated through a concomitant increase in expression of the uPA receptor. In the presence of plasminogen there was robust degradation of A beta that was added to the HCSM cells resulting in restoration of cell viability. This suggests that increased expression of uPA may initially serve as a protective mechanism leading to localized degradation and clearance of the pathogenic stimulus A beta. On the other hand, chronic expression of uPA and plasminogen activation led to a profound loss of HCSM cell attachment. This suggests that a similar prolonged effect in vivo in the cerebral vessel wall may contribute to loss of integrity and cerebral hemorrhage in CAA.
Highlights
Orrhage with amyloidosis Dutch-type [1]
Similar to these in vivo observations, we have reported that amyloid- protein (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 amyloid -protein precursor (APP) in cultured human cerebrovascular smooth muscle (HCSM) cells, an in vitro model of cerebral amyloid angiopathy (CAA) (20 –22)
The present results suggest that enhanced urokinase-type plasminogen activator (uPA) expression and subsequent plasminogen activation may initially be a protective response by HCSM cells to promote localized degradation of A, the pathogenic stimulus, and inhibit the progression of CAA
Summary
Materials—Wild-type and Dutch mutant A peptides were synthesized by solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acid chemistry, purified by reverse-phase high pressure liquid chromatography, and structurally characterized as described previously [44]. Solubilized monomeric wild-type or Dutch mutant A40, at a final concentration of 25 M, was added to the cultures in serum-free medium and incubated at 37 °C for up to 6 days. Cell Culture Plasminogen Activation Assay—HCSM cells were grown in 48-well tissue culture plates and incubated for 2 to 6 days in phenol red-free, serum-free culture medium in absence or presence of A peptides. Purified Glu-plasminogen was added at a final concentration of 0.5 M to the collected conditioned medium samples and the fresh phenol red-free, serum-free culture medium on the HCSM cells. The medium samples and cells were incubated for 30 min at 37 °C, and the generated plasmin activity in the samples was measured using the chromogenic substrate tosyl-Gly-Pro-Lys-p-nitroanilide in a Vmax microtiter plate reader as described previously [29]. Using avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals), cDNA was made using ϳ500 ng of HCSM cell mRNA, 60 pg/l oligo(dT), 500 nM dNTP, incubated for 1 h at 41 °C. cDNA templates for riboprobes were generated using the following primers for uPA (GenBankTM accession number X02419; nucleotides 3350 – 4459): forward, 5Ј-GCTCTGTCACCTACGTGTGTG-3Ј; and reverse, 5Ј-GGAACCTTGCCTAGA-
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