TGFbeta1 enhances formation of cellular Abeta/apoE deposits in vascular myocytes

Brain injury increases the risk of Alzheimer's disease (AD) through unknown mechanisms. We studied deposition of amyloid-beta protein (Abeta) in cells exposed to transforming growth factor beta1 (TGFbeta1), a cytokine that regulates cell metabolism during brain injury, and apolipoproteinE (apoE), the major lipid transporter in the brain. The studies were conducted by using brain vascular smooth muscle cells that are engaged in beta-amyloidosis in vivo and produce Abeta in cell culture. We found that cell treatment with TGFbeta1 together with apoE4 strongly increased the amount of cellular Abeta. The intracellular Abeta co-localized with apoE but not with TGFbeta, similarly as in vascular beta-amyloid. Some cellular Abeta/apoE deposits increased in size and persisted in culture even after the TGFbeta1 and apoE4 were removed. The appearance of cellular deposits of Abeta was associated with increased production of the amyloid-beta precursor protein and cellular retention of its mature form. The results suggest that the concomitant presence of apoE and TGFbeta1 can trigger vascular beta-amyloidosis by inducing intracellular formation of stable Abeta/apoE deposits.     

The effect of oxidative stress on amyloid precursor protein processing in cells engaged in β-amyloidosis is related to apolipoprotein E genotype

The reduced antioxidative defense in allele 4 carriers is suggested to contribute to -amyloidosis in Alzheimers disease and Downs syndrome. We studied the effect of oxidative stress on accumulation of amyloid- peptide (A) in vascular smooth muscle cells (SMCs) that are engaged in production of amyloid- in vivo. Previously, we found that oxidative stress caused by ferrous ions induced accumulation of A-apolipoprotein E deposits in lysosomes and was associated with a greater oxidative protein damage in 4 carriers. Here, we demonstrate that ferrous ions induce formation of A deposits also in vascular tunica media in organotypic cultures of whole brain vessels, suggesting the role of oxidative stress in development of vascular -amyloidosis. Cellular accumulation of A in SMCs treated with ferrous ions was associated with a greater accumulation of C-terminal amyloid precursor protein (APP) fragments in 4/4 than in 3/3 myocytes and reduced the amount of soluble APP in 3/3, but not 4/4, cultures. Antioxidant vitamin E prevented these effects, and, when applied alone, diminished the amount of APP C-terminal fragments and increased the amount of secreted APP in 3/3, but not 4/4, cells. C-terminal APP-immunoreactive material was accumulated in lysosomes partly with A- and N-terminal APP immunoreactivities. These results suggest that the increased accumulation of APP and its fragments in lysosomes may yield additional amounts of cellular A, particularly in 4 carriers. We hypothesize that the altered processing of APP in SMCs locally exposed to oxidative stress facilitates cellular deposition of A and contribute to the increased risk of development of -amyloidosis in 4/4 carriers.     

Lysosomal deposition of Abeta in cultures of brain vascular smooth muscle cells is enhanced by iron

Recently, we found that brain vascular smooth muscle cells from Tg2576 mice over-expressed the APP transgene in culture, secreted amyloid-beta peptide (Abeta) and accumulated Abeta intracellularly. Now we detected this intracellular Abeta inside lysosomes, which were also rich in C-terminal domain of APP, but not in endoplasmic reticulum, Golgi apparatus, or trans-Golgi network. Treatment of cultures with ferrous ions (50-150 microM) increased the proportion of muscle cells with Abeta immunoreactive granules and the amounts of intracellular Abeta1-40 and Abeta1-42 in a dose-dependent manner. This increase of intracellular Abeta1-40 by iron was inhibited by alpha-tocopherol, but not by a water-soluble antioxidant melatonin. The increase of intracellular Abeta1-42 by iron was not inhibited by alpha-tocopherol or melatonin. Cell treatment with iron did not alter the lysosomal localization of Abeta immunoreactivity. Cell treatment with iron (II and III), copper (II), zinc (II) and aluminum (III) increased cellular levels of carbonyls. However, the effect of zinc on Abeta accumulation in cultures was weak, and there were no effects of copper and aluminum. The data suggest that iron may be the factor that triggers vascular amyloidosis. Lysosomal accumulation of APP and Abeta initiates deposition of amyloid in blood vessels in Tg2576 mice.     

The effect of oxidative stress on accumulation of apolipoprotein E3 and E4 in a cell culture model of beta-amyloid angiopathy (CAA)

Apolipoprotein E (apoE) is a multifunctional molecule that is active during brain development, maintenance, and injury. Allele epsilon 4 of apoE is recognized as a risk factor for beta-amyloidosis, but the responsible mechanisms are not clear. Recently, we showed that vascular smooth muscle cells (SMCs) from epsilon 4/ epsilon 4 carriers are the most susceptible to oxidative protein damage that was associated with the appearance of apoE-Abeta-immunoreactive granules in cells. Here, we demonstrate that apoE4 is more readily accumulated in SMCs treated with ferrous ions than is apoE3. ApoE accumulated in lysosomes in the form of monomers, dimers, apoE-containing complexes, and apoE fragments. ApoE4 and apoE4-containing complexes persisted in SMCs longer than apoE3 and its complexes. Both isoforms of apoE stimulated formation of apoE-Abeta deposits and increased immobilization of iron in cultures treated with ferrous ions. The accumulation of apoE-Abeta deposits in lysosomes was associated with the appearance of lipid peroxidation products such as malondialdehyde and 4-hydroxynonenal-2-nonenal. The higher cellular accumulation of apoE4 than apoE3 in SMCs exposed to oxidative stress may facilitate development of beta-amyloid angiopathy that is more frequent in epsilon 4/ epsilon 4 carriers.     

Secretion and accumulation of Abeta by brain vascular smooth muscle cells from AbetaPP-Swedish transgenic mice

Alzheimer amyloid-beta is deposited in the neuropil and in brain blood vessels in transgenic Tg2576 mice that overexpress human amyloid-beta precursor protein (AbetaPP) containing the Swedish mutation (AbetaPP-Swe). Because the AbetaPP transgene in Tg2576 mice is placed behind the PrP promoter, all amyloid-beta, including vascular amyloid, is considered to be of neuronal origin. We studied the expression of the transgenic AbetaPP in smooth muscle cells cultured from brain blood vessels from Tg2576 mice. We found that brain vascular smooth muscle cells overexpressed human AbetaPP-Swe approximately 4 times the physiological levels of mouse AbetaPP. The cultured cells secreted abundant Abeta1-40 and Abeta1-42 and formed intracellular Abeta-immunoreactive granules. The percentage of cells containing intracellular Abeta and the amount of intracellular Abeta were significantly higher in cultures obtained from 14-month-old than from 4-month-old mice, as tested on first or second passages. During cell senescence in culture, intracellular accumulation of Abeta and C-terminal fragments of AbetaPP increased in cells derived from both 4- and 14-month-old mice. Vascular muscle cells from Tg2576 mice appear to be a valuable model of the intracellular accumulation of Abeta. We suggest that vascular muscle cells may be involved in the production of cerebrovascular amyloid in Tg2576 mice.