Non-fibrillar beta-amyloid protein is associated with smooth muscle cells of vessel walls in Alzheimer disease.

Meningeal blood vessels were studied in Alzheimer disease (AD) and control brain specimens obtained from autopsies within 16 hours after death. Serial sections were stained with thioflavine S and Congo red and immunostained for the presence of beta-amyloid precursor protein (beta PP) and beta-protein and for smooth muscle-specific proteins myosin, alpha-actin, and desmin. Isolated blood vessels were studied by immunoblotting for the presence of beta PP, fragments of beta PP, and beta-protein. The arteries that were strongly immunopositive for beta-protein in all layers of the walls were also positive for amyloid fibrils on thioflavine S and Congo red stainings. The focal immunostaining for beta-protein in less affected vessels was located in the tunica media in the cytoplasm of smooth muscle cells or formed granules between myocytes. The cytoplasmic beta-protein and some of the small deposits present between cells were negative for amyloid fibrils. The vessels isolated from specimens containing beta-protein-immunoreactive material contained 3 kD, 4.2-4.5 kD, 8.5-9 kD, and 17.5 kD beta-protein-immunoreactive bands. These bands were not found in the samples assessed as beta-protein-negative by immunocytochemistry. These data indicate that during formation of amyloid in AD vessel walls, nonfibrillar, monomeric, and oligomeric beta-protein accumulate.

Accumulation of Alzheimer amyloid-beta peptide in cultured myocytes is enhanced by serum and reduced by cerebrospinal fluid.

Smooth muscle cells cultured from leptomeningeal vessels from old dogs with amyloid-angiopathy accumulate intracellular deposits that are immunoreactive for amyloid-beta peptide (A beta). We used this cellular model in the present study to examine the influence of sera and cerebrospinal fluid on intracellular accumulation of A beta-immunoreactive deposits and on secretion of soluble A beta into culture media. We found that sera from old dogs significantly increased the percentage of A beta-positive smooth muscle cells in culture. The enhanced accumulation of A beta was associated with (a) lower secretion of A beta into media, (b) altered maturation of amyloid-beta-precursor protein (A betaPP) into A betaPP751-770 with faster electrophoretic mobility, (c) increased accumulation of C-terminal fragments of A betaPP (12-15 kD, 10kD and less), and (d) increased secretion of A betaPP into culture media. These findings suggest that age- or disease-related serum factors increase accumulation of A beta by affecting production and processing of A betaPP In contrast, cerebrospinal fluids reduced accumulation of A beta. Involvement of A beta-carrier proteins-apolipoprotein E and transthyretin-in accumulation of A beta is demonstrated. Accumulation of A beta in cultured smooth muscle cells-a model of beta-amyloidosis-may be regulated by factors that alter production and processing of A betaPP as well as the fate of soluble A beta in extracellular space.

Apolipoprotein E alters metabolism of AbetaPP in cells engaged in beta-amyloidosis.

Canine smooth muscle cells (SMCs), cultured from amyloid-affected brain blood vessels accumulate Alzheimer amyloid-beta peptide (Abeta) intracellularly, either spontaneously or after treatment with apolipoprotein E (apoE). ApoE is codeposited with Abeta, which suggests that apoE participates in Abeta accumulation. We tested the hypothesis that apoE-induced accumulation of Abeta in SMCs is caused by an increased production of amyloid-beta precursor protein (AbetaPP) and/or its altered metabolism. We found that 24 hours of treatment with apoE3 or apoE4 induced intracellular accumulation of Abeta-immunoreactive deposits in SMCs but did not influence AbetaPP production and processing. The treatment with apoE3 or E4 for 3 days resulted in the following: increased Abeta-accumulation; reduced levels of secreted Abeta; increased production and cellular retention of mature AbetaPP770; and reduced culture growth, cell proliferation, and viability. ApoE4, but not apoE3, increased cellular levels of mRNA AbetaPP 770 (the main form produced in SMCs) about ninefold. ApoE3 stimulated production and cellular retention of endogenous apoE. We hypothesize that Abeta accumulation is triggered by apoE, which may bind and immobilize soluble Abeta produced in SMCs. The newly formed Abeta deposits may further accelerate Abeta accumulation by altering metabolism of AbetaPP.

Role of perivascular cells and myocytes in vascular amyloidosis.

Amyloidogenic processing of amyloid-beta precursor protein (APP) by cells of the brain is the major pathologic component of Alzheimer's disease. Amyloid-beta (A beta) is of heterogeneous origin. Perivascular cells of monocyte-macrophage-microglial cell lineage produce fibrillar A beta in the wall of capillaries, whereas parenchymal microglial cells produce fibrillar A beta in the parenchyma of gray matter. Fibrillar A beta deposition by perivascular cells lead to endothelial cell degeneration and death, obliteration of affected capillaries, and reduction of the length of the vascular network. These changes cause local ischemia with neuronal degeneration and death. Smooth muscle cells are the source of A beta in the tunica media of parenchymal and leptomeningeal arteries and veins. Fibrillar A beta in the tunica media of leptomeningeal and parenchymal vessels causes degeneration and necrosis of smooth muscle cells and leads to multiple cortical hemorrhages. Smooth muscle cells isolated from blood vessels with amyloid deposits secrete A beta and accumulate nonfibrillar A beta intracellularly. The amyloidogenic processing of APP can be enhanced by apolipoprotein E, reduced by transthyretin, and modulated by several cytokines.

Factors secreted by activated microglia and monocytes reduce amyloidogenesis in vascular smooth muscle cells.

Smooth muscle cells cultured from amyloid-beta-affected arteries accumulate amyloid-beta peptide A beta. We now show that accumulation of "A beta" deposits in this model can be significantly reduced by culture in conditioned media from microglia and monocytes. Reduced A beta accumulation was associated with (i) lower secretion of A beta, (ii) increased secretion, but not cellular levels of amyloid-beta-precursor protein (A beta PP), and (iii) increased cell proliferation and metabolic activity. We suggest that improper regulation of A beta PP metabolism by monokines may facilitate vascular amyloidogenesis.

Apolipoproteins E3 and E4 induce, and transthyretin prevents accumulation of the Alzheimer's beta-amyloid peptide in cultured vascular smooth muscle cells.

Cultured brain vascular smooth muscle cells (SMCs) accumulate beta-peptide in intracytoplasmic granules [9,10,33]. We show here that apoE3 and E4 induces the intracytoplasmic beta-peptide accumulation in cultured human and canine SMCs. The induction is dose-dependent and the accumulated granules also contain apoE and some were thioflavine S-positive. The deposits induced with apoE3 were more abundant though less stable than with apoE4. Transthyretin at physiological concentrations blocked the effects of apoE3/E4. Thus, accumulation of beta-peptide appears to be regulated by beta-peptide carrier proteins.

Deposition of Alzheimer's vascular amyloid-beta is associated with decreased expression of brain L-3-hydroxyacyl-coenzyme A dehydrogenase (ERAB).

L-3-hydroxyacyl-coenzyme A dehydrogenase type II (HADH) was described as an endoplasmic reticulum amyloid beta-peptide-binding protein (ERAB), which enhances Abeta toxicity, and accumulates in neurons in Alzheimer's disease (AD). Hence, HADH/ERAB was suggested to mediate the amyloid-induced neurodegeneration. We estimated the in vivo interactions of HADH and Abeta in an immunocytochemical study of ten Alzheimer's disease and seven normal brains using five monoclonal HADH-specific antibodies. We found no HADH in amyloid plaques or vascular amyloid. The neuronal expression of HADH was not correlated with the severity of amyloid load in neuropil. HADH was expressed in vascular smooth muscle cells in young and old controls and in amyloid-free blood vessels in AD cases, but little or no HADH was in smooth muscle cells in arteries with amyloid deposits. The putative intracellular interaction between HADH and Abeta in amyloid-producing cells was further studied in vascular smooth muscle cells isolated from brain blood vessels with amyloid-beta angiopathy - the cells that were shown previously to accumulate Abeta intracellularly ['Research advances in Alzheimer's disease and related disorders' (1995) 747; Brain Res. 676 (1995) 225; Neurosci. Lett. 183 (1995) 120]. HADH had a mitochondrial localization and did not co-localize with an endoplasmic reticulum marker. Cells that accumulated Abeta were those with low expression of HADH and the proteins did not co-localize. Explanation of the association between low levels of HADH and deposition of Abeta by brain smooth muscle cells requires further studies.

Factors produced by activated macrophages reduce accumulation of Alzheimer's beta-amyloid protein in vascular smooth muscle cells.

Smooth muscle cells (SMCs) isolated from amyloid-angiopathy affected brain vessels accumulate intracellularly amyloid-beta peptide (A beta). Now we demonstrate that accumulation of A beta in SMCs can be reduced by factors secreted by macrophages - IL-1alpha, IL-6, TNF-alpha, TGF-beta1 or PGE2 - probably by stimulating the non-amyloidogenic processing of A beta precursor protein (PP). It is suggested that brain macrophages may regulate A betaPP/A beta metabolism under physiological conditions and prevent beta-amyloidosis. The disturbance of this regulatory function of brain macrophages may result in excessive production and accumulation of A beta.

Secretion and accumulation of Alzheimer's beta-protein by cultured vascular smooth muscle cells from old and young dogs.

Cultured smooth muscle cells isolated from beta-amyloid-affected blood vessels from old dogs accumulate beta-protein at early passages [5,24]. Now, we show that smooth muscle cells derived from amyloid-free brain blood vessels and peripheral arteries from old and young animals are induced by culture conditions to deposit intracellularly fibrillar and non-fibrillar beta-protein. Accumulation of beta-protein is associated with a higher secretion of beta-protein, but not with a higher secretion of beta-amyloid precursor protein (beta APP) or higher cellular content of beta APP. Gradual cessation of proliferative activity was observed in cultures that accumulate beta-protein.

In vitro production of beta-amyloid in smooth muscle cells isolated from amyloid angiopathy-affected vessels.

Our recent results indicate that in Alzheimer's disease (AD) amyloid angiopathy, smooth muscle cells are responsible for beta-amyloid deposition in the vascular wall. Aged dogs have been shown to develop beta-amyloid angiopathy similar to that in AD. Thus, we used brain and peripheral vessels from aged and young dogs to isolate cells of the vascular wall: smooth muscle cells, fibroblasts, and endothelial cells, and to study their ability to produce beta-protein. We demonstrate that only myocytes from aged animals cultured for up to 4 weeks accumulate beta-protein-immunoreactive material intracellularly, in the form of fibrillar and amorphous deposits.

The influence of thymus factor X (TFX) and of anti-TFX globulin on the growth of Lewis lung carcinoma in mice.

The influence of thymus factor X--TFX (Polfa) and an anti-TFX rabbit gammaglobulin (RATFX) on the growth of Lewis lung carcinoma in mice was studied. The preparations were administered subcutaneously into the peritumoral region. Tumor growth was significantly retarded in the RATFX-treated groups, while a low dose TFX therapy was ineffective. No significant differences in peritumoral inflammatory reaction in treated and untreated mice were found.

Amyloid-beta impairs development of neuronal progenitor cells by oxidative mechanisms.

Neuronal progenitor cells (NPCs) are being considered for treatment of neurodegenerative diseases associated with beta-amyloidosis: Alzheimer's disease (AD) and Down syndrome (DS). However, the neurotoxic properties of amyloid-beta peptide (Abeta) may impair survival and differentiation of transplanted NPCs. Hence, we studied the influence of Abeta on development of human NPCs--proliferation, migration, formation of colonies of neurons, formation processes--in culture. Pre-fibrillized human Abeta1-40 blocked development of neuronal colonies. NPC development was impaired in the presence of soluble Abeta1-40 (1.75-7 microM), and NPC differentiation into large and small neurons was altered, as demonstrated by morphometry. Antioxidant vitamin E partially abolished these effects, but not the reduced formation of neuronal processes. NPCs cultured with 7 microM Abeta1-40 accumulated Abeta monomers and oligomers and contained higher levels of protein carbonyls and lipid peroxidation products HNE and MDA. We suggest that Abeta1-40 impairs development of NPCs by oxidative damage. Hence, a prerequisite of successful neuroreplacement therapy using NPCs in AD and DS/AD may be removal of amyloid-beta and antioxidative treatment.

Acid alpha-naphthyl acetate esterase assay in murine lymphocytes.

Acid alpha-naphthyl acetate esterase (ANAE) activity was assayed in cell homogenates and in intact cells by an endpoint colorimetric method, in which sodium dodecyl sulfate was used to stop the reaction. Each method of cell disruption and enzyme solubilization tested here caused a partial loss of the ANAE activity in lymphocyte preparations. The majority of the ANAE activity in lymphocytes was found to be membrane bound. The ANAE activity in thymocytes was over two times lower than that obtained for lymph node and spleen lymphocytes. Macrophages were found to contain about 18 times higher ANAE activity than mature lymphocytes.

Changes of dry mass of blood lymphocytes in the course of mouse estrous cycle and their dependence on the sexual steroid hormones.

The dry mass of blood lymphocytes in female mice has been found to underline the variations according to the phases of estrous cycle. Ovariectomy caused disappearance of cyclic changes and reduced the mean dry mass of lymphocytes. Whereas estradiol was effective in restoration of the lymphocyte dry mass to the values characteristic for non-operated animals, progesterone failed to produce any noticeable effect. It is concluded that estradiol is responsible for the changes of lymphocyte dry mass observed during the successive phases of estrous cycle.

Ultrastructure of the microglia that phagocytose amyloid and the microglia that produce beta-amyloid fibrils.

The function of microglia associated with beta-amyloid deposits still remains a controversial issue. On the basis of recent ultrastructural data, microglia were postulated to be cells that form amyloid fibrils, not phagocytes that remove amyloid deposits. In this electron microscopic study, we examined the ability of microglia to ingest and digest exogenous amyloid fibrils in vitro. We demonstrate that amyloid fibrils are ingested by cultured microglial cells and collected and stored in phagosomes. The ingested, nondegraded amyloid remains within phagosomes for up to 20 days, suggesting a very limited effectiveness of microglia in degrading beta-amyloid fibrils. On the other hand, we showed that in microglial cells of classical plaques in brain cortex of patients with Alzheimer's disease, amyloid fibrils appear first in altered endoplasmic reticulum and deep infoldings of cell membranes. These differences in intracellular distribution of amyloid fibrils in microglial cells support our observations that microglial cells associated with amyloid plaques are engaged in production of amyloid, but not in phagocytosis.