Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain.

The use of murine cerebrovascular endothelial and smooth muscle cells has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. Instead, cerebrovascular cells from larger mammals are preferred and several methods describing the isolation of endothelial and smooth muscle cells from human, canine, rat, and guinea pig have been published. In recent years, several transgenic mouse lines showing CAA pathology have been established; consequently murine cerebrovascular cells derived from these animals can serve as a key cellular model to study CAA. Here, we describe a procedure for isolating murine microvessels that yields healthy smooth muscle and endothelial cell populations and produce sufficient material for experimental purposes. Murine smooth muscle cells isolated using this protocol exhibit the classic "hill and valley" morphology and are immunoreactive for the smooth muscle cell marker α-actin. Endothelial cells display a "cobblestone" pattern phenotype and show the characteristic immunostaining for the von Willebrand factor and the factor VIII-related antigen. In addition, we describe methods designed to preserve these cells by storage in liquid nitrogen and reestablishing viable cell cultures. Finally, we compare our methods with protocols designed to isolate and maintain human cerebrovascular cell cultures.

Endogenous amyloid-ß is necessary for hippocampal synaptic plasticity and memory.

OBJECTIVE: The goal of this study was to investigate the role of endogenous amyloid-ß peptide (Aß) in healthy brain. METHODS: Long-term potentiation (LTP), a type of synaptic plasticity that is thought to be associated with learning and memory, was examined through extracellular field recordings from the CA1 region of hippocampal slices, whereas behavioral techniques were used to assess contextual fear memory and reference memory. Amyloid precursor protein (APP) expression was reduced through small interfering RNA (siRNA) technique. RESULTS: We found that both antirodent Aß antibody and siRNA against murine APP reduced LTP as well as contextual fear memory and reference memory. These effects were rescued by the addition of human Aß42, suggesting that endogenously produced Aß is needed for normal LTP and memory. Furthermore, the effect of endogenous Aß on plasticity and memory was likely due to regulation of transmitter release, activation of α7-containing nicotinic acetylcholine receptors, and Aß42 production. INTERPRETATION: Endogenous Aß42 is a critical player in synaptic plasticity and memory within the normal central nervous system. This needs to be taken into consideration when designing therapies aiming at reducing Aß levels to treat Alzheimer disease.

His-tag binding by antibody C706 mimics ß-amyloid recognition.

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of ß-amyloid (Aß) plaques. Aggregation of the Aß(42) peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti-Aß monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aß peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2(1) and C2. Although the Fab was crystallized in the presence of Aß(16), no peptide was observed in the crystals. The antigen-binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly-His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4-His5) stack against tryptophan residues in the central pocket of the antigen-binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his-tag binding by C706 resembles the Aß recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B-cell epitope of Aß. By similarity, residues Phe-Arg-His of Aß would be a major portion of the C706 epitope.

Macroautophagy is not directly involved in the metabolism of amyloid precursor protein.

Alterations in the metabolism of amyloid precursor protein (APP) are believed to play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate that APP is proteolytically processed in endosomal-autophagic-lysosomal compartments. In this study, we used both in vivo and in vitro paradigms to determine whether alterations in macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff disease, had mTOR-independent increases in the autophagic vacuole (AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse models; however, discrepancies between LC3-II and APP-CTFs were seen between primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick type C1) lysosomal storage models. APP-CTFs were proportionately higher than LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease, although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous murine Aß40 from RIPA-soluble extracts was increased in brains of all three mice. The in vivo relationship between AV and APP-CTF accumulation was also seen in cultured neurons treated with agents that impair primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A and vinblastine) lysosomal flux. However, Aß secretion was unaffected by agents that induced autophagy (rapamycin) or impaired AV clearance, and LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive lysosomes. These data suggest that neuronal macroautophagy does not directly regulate APP metabolism but highlights the important anti-amyloidogenic role of lysosomal proteolysis in post-secretase APP-CTF catabolism.

TNF-alpha drives remodeling of blood vessels and lymphatics in sustained airway inflammation in mice.

Inflammation is associated with blood vessel and lymphatic vessel proliferation and remodeling. The microvasculature of the mouse trachea provides an ideal opportunity to study this process, as Mycoplasma pulmonis infection of mouse airways induces widespread and sustained vessel remodeling, including enlargement of capillaries into venules and lymphangiogenesis. Although the mediators responsible for these vascular changes in mice have not been identified, VEGF-A is known not to be involved. Here, we sought to determine whether TNF-alpha drives the changes in blood vessels and lymphatics in M. pulmonis-infected mice. The endothelial cells, but not pericytes, of blood vessels, but not lymphatics, were immunoreactive for TNF receptor 1 (TNF-R1) and lymphotoxin B receptors. Most TNF-R2 immunoreactivity was on leukocytes. Infection resulted in a large and sustained increase in TNF-alpha expression, as measured by real-time quantitative RT-PCR, and smaller increases in lymphotoxins and TNF receptors that preceded vessel remodeling. Substantially less vessel remodeling and lymphangiogenesis occurred when TNF-alpha signaling was inhibited by a blocking antibody or was silenced in Tnfr1-/- mice. When administered after infection was established, the TNF-alpha-specific antibody slowed but did not reverse blood vessel remodeling and lymphangiogenesis. The action of TNF-alpha on blood vessels is probably mediated through direct effects on endothelial cells, but its effects on lymphangiogenesis may require inflammatory mediators from recruited leukocytes. We conclude that TNF-alpha is a strong candidate for a mediator that drives blood vessel remodeling and lymphangiogenesis in inflammation.

Complexes of amyloid-beta and cystatin C in the human central nervous system.

A role for cystatin C (CysC) in the pathogenesis of Alzheimer's disease (AD) has been suggested by the genetic linkage of a CysC gene (CST3) polymorphism with late-onset AD, the co-localization of CysC with amyloid-beta (Abeta) in AD brains, and binding of CysC to soluble Abeta in vitro and in mouse models of AD. This study investigates the binding between Abeta and CysC in the human central nervous system. While CysC binding to soluble Abeta was observed in AD patients and controls, a SDS-resistant CysC/Abeta complex was detected exclusively in brains of neuropathologically normal controls, but not in AD cases. The association of CysC with Abeta in brain from control individuals and in cerebrospinal fluid reveals an interaction of these two polypeptides in their soluble form. The association between Abeta and CysC prevented Abeta accumulation and fibrillogenesis in experimental systems, arguing that CysC plays a protective role in the pathogenesis of AD in humans and explains why decreases in CysC concentration caused by the CST3 polymorphism or by specific presenilin 2 mutations can lead to the development of the disease. Thus, enhancing CysC expression or modulating CysC binding to Abeta have important disease-modifying effects, suggesting a novel therapeutic intervention for AD.

Cystatin C inhibits amyloid-beta deposition in Alzheimer's disease mouse models.

Using transgenic mice expressing human cystatin C (encoded by CST3), we show that cystatin C binds soluble amyloid-beta peptide and inhibits cerebral amyloid deposition in amyloid-beta precursor protein (APP) transgenic mice. Cystatin C expression twice that of the endogenous mouse cystatin C was sufficient to substantially diminish amyloid-beta deposition. Thus, cystatin C has a protective role in Alzheimer's disease pathogenesis, and modulation of cystatin C concentrations may have therapeutic implications for the disease.

Murine cerebrovascular cells as a cell culture model for cerebral amyloid angiopathy: isolation of smooth muscle and endothelial cells from mouse brain.

The use of murine cerebrovascular cells, that is, endothelial and smooth muscle cells, has not been widely employed as a cell culture model for the investigation of cellular mechanisms involved in cerebral amyloid angiopathy (CAA). Difficulties in isolation and propagation of murine cerebrovascular cells and insufficient yields for molecular and cell culture studies have deterred investigators from using mice as a source for cerebrovascular cells in culture. To date, most of the literature has described isolation of smooth muscle cells or endothelial cells from human, canine, rat, guinea pig, or other large animals. In recent years, several transgenic mice have been established that show CAA pathology; therefore, it is necessary to re-examine the use of mouse cerebrovascular cells as an important model for cell culture studies. We have optimized the isolation procedure of (1) murine microvessels, (2) smooth muscle cells, and (3) endothelial cells to yield a sufficient population of cells for experimentation purposes. Comparisons with rat and human isolation procedures are also noted. Murine smooth muscle cells isolated using the methodology described herein exhibit the classic "hill and valley" morphology and are immunoreactive for smooth muscle cell-specific alpha-actin, whereas endothelial cells demonstrate a more "cobblestone" appearance and stain for von Willebrand factor or factor VIII-related antigen.

Pathogenic A beta induces the expression and activation of matrix metalloproteinase-2 in human cerebrovascular smooth muscle cells.

Cerebral amyloid angiopathy (CAA) is a major pathological feature of Alzheimer's disease and related disorders. Human cerebrovascular smooth muscle (HCSM) cells, which are intimately associated with CAA, have been used as an in vitro model system to investigate pathologic interactions with amyloid beta protein (A beta). Previously we have shown that pathogenic forms of A beta induce several pathologic responses in HCSM cells including fibril assembly at the cell surface, increase in the levels of A beta precursor, and apoptotic cell death. Here we show that pathogenic A beta stimulates the expression and activation of matrix metalloproteinase-2 (MMP-2). Furthermore, we demonstrate that the increase in MMP-2 activation is largely caused by increased expression of membrane type-1 (MT1)-MMP expression, the primary MMP-2 activator. Finally, treatment with MMP-2 inhibitors resulted in increased HCSM cell viability in the presence of pathogenic A beta. Our findings suggest that increased expression and activation of MMP-2 may contribute to HCSM cell death in response to pathogenic A beta. In addition, these activities may also contribute to loss of vessel wall integrity in CAA resulting in hemorrhagic stroke. Therefore, further understanding into the role of MMPs in HCSM cell degeneration may facilitate designing therapeutic strategies to treat CAA found in AD and related disorders.

Humanin rescues human cerebrovascular smooth muscle cells from Abeta-induced toxicity.

Cerebral amyloid beta-protein (Abeta) angiopathy (CAA) is a key pathological feature of Alzheimer's disease (AD) and related disorders. We have used human cerebrovascular smooth muscle (HCSM) cells as an in vitro model system to investigate the pathogenic mechanisms of the pathology of CAA. It was previously demonstrated that certain pathogenic forms of Abeta induce several pathologic responses in these cells, including fibril assembly at the cell surface, increased levels of Abeta precursor, degradation of HCSM cell alpha-actin and cell death. The recently discovered novel rescue factor humanin (HN) was shown to protect neuronal cells in culture from various AD-relevant insults including treatment with Abeta. In this report we investigated whether the HN peptide could rescue HCSM cells from Abeta-induced toxicity. We found that treatment of HCSM cells with 10 microm HN prevented pathogenic Abeta-induced HCSM cell death using a fluorescent cell viability assay, and degradation of HCSM alpha-actin was diminished shown by quantitative immunoblotting. However, Abeta deposition and fibril formation at the cell surface and increased levels of cell-associated AbetaPP were not affected by treatment with HN as demonstrated by a thioflavin T fluorescence assay and immunochemical methods, respectively. These results suggest that the protective effects of HN occur downstream of these cell surface molecular events. This is the first demonstration of a rescue factor for HCSM cells from Abeta-mediated cell death as well as being the first report to show that neuronal cells and HCSM cells may share a common downstream mechanism in the Abeta-induced cell death pathway.

Abeta does not induce oxidative stress in human cerebrovascular smooth muscle cells.

We investigated whether oxidative stress participates in the pathogenic Abeta-induced degenerative mechanism of cultured human cerebrovascular smooth muscle (HCSM) cells, which are intimately involved in cerebral amyloid angiopathy (CAA). Studies using the cell-permeable dye dichlorofluorescein diacetate suggested that free radicals were not robustly detected in HCSM cells exposed to pathogenic Abeta. Furthermore, examination for oxidatively modified proteins, indicated by the presence of dinitrophenylhydrazone and dityrosine moieties, demonstrated no appreciable difference between pathogenic Abeta-treated and untreated HCSM cells. These findings support the notion that pathogenic Abeta-induced toxicity in HCSM cells and neuronal cells occurs by different mechanisms.