Farid Rahimi

S100A8 and S100A9 in Human Arterial Wall IMPLICATIONS FOR ATHEROGENESIS

Atherogenesis is a complex process involving inflammation. S100A8 and S100A9, the Ca2+-binding neutrophil cytosolic proteins, are associated with innate immunity and regulate processes leading to leukocyte adhesion and transmigration. In neutrophils and monocytes the S100A8-S100A9 complex regulates phosphorylation, NADPH-oxidase activity, and fatty acid transport. The proteins have anti-microbial properties, and S100A8 may play a role in oxidant defense in inflammation. Murine S100A8 is regulated by inflammatory mediators and recruits macrophages with a proatherogenic phenotype. S100A9 but not S100A8 was found in macrophages in ApoE-/- murine atherosclerotic lesions, whereas both proteins are expressed in human giant cell arteritis. Here we demonstrate S100A8 and S100A9 protein and mRNA in macrophages, foam cells, and neovessels in human atheroma. Monomeric and complexed forms were detected in plaque extracts. S100A9 was strongly expressed in calcifying areas and the surrounding extracellular matrix. Vascular matrix vesicles contain high levels of Ca2+-binding proteins and phospholipids that regulate calcification. Matrix vesicles characterized by electron microscopy, x-ray microanalysis, nucleoside triphosphate pyrophosphohydrolase assay and cholesterol/phospholipid analysis contained predominantly S100A9. We propose that S100A9 associated with lipid structures in matrix vesicles may influence phospholipid-Ca2+ binding properties to promote dystrophic calcification. S100A8 and S100A9 were more sensitive to hypochlorite oxidation than albumin or low density lipoprotein and immunoaffinity confirmed S100A8-S100A9 complexes; some were resistant to reduction, suggesting that hypochlorite may contribute to protein cross-linking. S100A8 and S100A9 in atherosclerotic plaque and calcifying matrix vesicles may significantly influence redox- and Ca2+-dependent processes during atherogenesis and its chronic complications, particularly dystrophic calcification.

Monocyte Chemoattractant Protein‐1 Plays a Dominant Role in the Chronic Inflammation Observed in Alzheimer's Disease

Chronic neuroinflammation correlates with cognitive decline and brain atrophy in Alzheimers disease (AD), and cytokines and chemokines mediate the inflammatory response. However, quantitation of cytokines and chemokines in AD brain tissue has only been carried out for a small number of mediators with variable results. We simultaneously quantified 17 cytokines and chemokines in brain tissue extracts from controls (n = 10) and from patients with and without genetic forms of AD (n = 12). Group comparisons accounting for multiple testing revealed that monocyte chemoattractant protein‐1 (MCP‐1), interleukin‐6 (IL‐6) and interleukin‐8 (IL‐8) were consistently upregulated in AD brain tissue. Immunohistochemistry for MCP‐1, IL‐6 and IL‐8 confirmed this increase and determined localization of these factors in neurons (MCP‐1, IL‐6, IL‐8), astrocytes (MCP‐1, IL‐6) and plaque pathology (MCP‐1, IL‐8). Logistic linear regression modeling determined that MCP‐1 was the most reliable predictor of disease. Our data support previous work on significant increases in IL‐6 and IL‐8 in AD but indicate that MCP‐1 may play a more dominant role in chronic inflammation in AD.

Inflammatory S100A9 and S100A12 proteins in Alzheimer's disease

Inflammation, insoluble protein deposition and neuronal cell loss are important features of the Alzheimers disease (AD) brain. S100B is associated with the neuropathological hallmarks of AD where it is thought to play a role in neuritic pathology. S100A8, S100A9 and S100A12 comprise a new group of inflammation-associated proteins that are constitutively expressed by neutrophils and inducible in numerous inflammatory cells. We investigated expression of S100B, S100A8, S100A9 and S100A12 in brain samples from sporadic and familial (PS-1) AD cases and controls using immunohistochemistry and Western blot analysis. S100B, S100A9 and S100A12, but not S100A8, were consistently associated with the neuropathological hallmarks of AD. Western blot analysis confirmed significant increases in soluble S100A9 in PS-1 AD compared to controls. S100A9 complexes that were resistant to reduction were also evident in brain extracts. A reactive component of a size consistent with hexameric S100A12 was seen in all cases. This study indicates a potential role for pro-inflammatory S100A9 and S100A12 in pathogenesis caused by inflammation and protein complex formation in AD.

Regulation of S100A8 by Glucocorticoids

S100A8 (A8) has roles in inflammation, differentiation and development and is associated with oxidative defense. Murine A8 (mA8) is up-regulated in macrophages, fibroblasts, and microvascular endothelial cells by LPS. Glucocorticoids (GCs) amplified LPS-induced mA8 in these cells. Relative to stimulation by LPS, GCs increased mA8 gene transcription and mRNA half-life. Enhancement required new protein synthesis, IL-10 and products of the cyclooxygenase-2 pathway, and both ERK1/2 and p38 MAPK. Protein kinase A positively and protein kinase C negatively regulated this process. Promoter analysis indicated element(s) essential for LPS and dexamethasone enhancement colocated within the region −178 to 0 bp. In the absence of glucocorticoid response elements, NF1 motif at −58 is a candidate for mediation of enhancement. Gel shift analysis detected no differences between LPS- and LPS/dexamethasone-treated complexes within this region. GCs increased constitutive levels of A8 and S100A9 (A9) mRNA in human monocytes. The synovial membrane of rheumatoid patients treated with high dose i.v. methylprednisolone contained higher numbers of A8/A9-positive macrophages than pre- or posttreatment samples. Results support the proposal that A8 has anti-inflammatory properties that may be independent of hetero-complex formation with A9 and may also enable localized defense in the absence of overriding deleterious host responses.

FGF‐2, IL‐1β and TGF‐β regulate fibroblast expression of S100A8

Growth factors, including fibroblast growth factor‐2 (FGF‐2) and transforming growth factor‐β (TGF‐β) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca2+‐binding proteins and are now accepted as markers of inflammation. They are expressed by keratinocytes and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon γ (IFNγ), tumour‐necrosis factor (TNF) and TGF‐β did not induce the S100A8 gene in murine fibroblasts whereas FGF‐2 induced mRNA maximally after 12 h. The FGF‐2 response was strongly enhanced and prolonged by heparin. Interleukin‐1β (IL‐1β) alone, or in synergy with FGF‐2/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF‐2 and IL‐1β was partially dependent on the mitogen‐activated‐protein‐kinase pathway and dependent on new protein synthesis. FGF‐2‐responsive elements were distinct from the IL‐1β‐responsive elements in the S100A8 gene promoter. FGF‐2‐/heparin‐induced, but not IL‐1β‐induced responses were significantly suppressed by TGF‐β, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8‐positive fibroblast‐like cells 2 and 4 days post injury; numbers declined by 7 days. Up‐regulation of S100A8 by FGF‐2/IL‐1β, down‐regulation by TGF‐β, and its time‐dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.

Pleiotropic Roles of S100A12 in Coronary Atherosclerotic Plaque Formation and Rupture

Macrophages, cytokines, and matrix metalloproteinases (MMP) play important roles in atherogenesis. The Ca2+-binding protein S100A12 regulates monocyte migration and may contribute to atherosclerosis by inducing proinflammatory cytokines in macrophages. We found significantly higher S100A12 levels in sera from patients with coronary artery disease than controls and levels correlated positively with C-reactive protein. S100A12 was released into the coronary circulation from ruptured plaque in acute coronary syndrome, and after mechanical disruption by percutaneous coronary intervention in stable coronary artery disease. In contrast to earlier studies, S100A12 did not stimulate proinflammatory cytokine production by human monocytes or macrophages. Similarly, no induction of MMP genes was found in macrophages stimulated with S100A12. Because S100A12 binds Zn2+, we studied some functional aspects that could modulate atherogenesis. S100A12 formed a hexamer in the presence of Zn2+; a novel Ab was generated that specifically recognized this complex. By chelating Zn2+, S100A12 significantly inhibited MMP-2, MMP-9, and MMP-3, and the Zn2+-induced S100A12 complex colocalized with these in foam cells in human atheroma. S100A12 may represent a new marker of this disease and may protect advanced atherosclerotic lesions from rupture by inhibiting excessive MMP-2 and MMP-9 activities by sequestering Zn2+.

Deletion of Amino Acid Transporter ASCT2 (SLC1A5) Reveals an Essential Role for Transporters SNAT1 (SLC38A1) and SNAT2 (SLC38A2) to Sustain Glutaminolysis in Cancer Cells

Many cancer cells depend on glutamine as they use the glutaminolysis pathway to generate building blocks and energy for anabolic purposes. As a result, glutamine transporters are essential for cancer growth and are potential targets for cancer chemotherapy with ASCT2 (SLC1A5) being investigated most intensively. Here we show that HeLa epithelial cervical cancer cells and 143B osteosarcoma cells express a set of glutamine transporters including SNAT1 (SLC38A1), SNAT2 (SLC38A2), SNAT4 (SLC38A4), LAT1 (SLC7A5), and ASCT2 (SLC1A5). Net glutamine uptake did not depend on ASCT2 but required expression of SNAT1 and SNAT2. Deletion of ASCT2 did not reduce cell growth but caused an amino acid starvation response and up-regulation of SNAT1 to replace ASCT2 functionally. Silencing of GCN2 in the ASCT2(−/−) background reduced cell growth, showing that a combined targeted approach would inhibit growth of glutamine-dependent cancer cells.

Pick Bodies in a Family with Presenilin-1 Alzheimer's Disease

Presenilin‐1 (PS‐1) mutations can cause Picks disease without evidence of Alzheimers disease (AD). We describe a family with a PS‐1 M146L mutation and both Pick bodies and AD. Sarkosyl‐insoluble hyperphosphorylated tau showed three bands consistent with AD, although dephosphorylation showed primarily three‐repeat isoforms. M146L mutant PS‐1 may predispose to both Picks disease and AD by affecting multiple intracellular pathways involving tau phosphorylation and amyloid metabolism. Ann Neurol 2005;57:139–143

Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimers, Parkinsons, and Huntingtons diseases, hereditary amyotrophic lateral sclerosis, and type 2 diabetes. Owing to the metastable nature of these protein assemblies, it is difficult to assess their oligomer size distribution quantitatively using classical methods, such as electrophoresis, chromatography, fluorescence, or dynamic light scattering. Oligomers of amyloidogenic proteins exist as metastable mixtures, in which the oligomers dissociate into monomers and associate into larger assemblies simultaneously. PICUP stabilizes oligomer populations by covalent cross-linking and when combined with fractionation methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or size-exclusion chromatography (SEC), PICUP provides snapshots of the oligomer size distributions that existed before cross-linking. Hence, PICUP enables visualization and quantitative analysis of metastable protein populations and can be used to monitor assembly and decipher relationships between sequence modifications and oligomerization(1). Mechanistically, PICUP involves photo-oxidation of Ru(2+) in a tris(bipyridyl)Ru(II) complex (RuBpy) to Ru(3+) by irradiation with visible light in the presence of an electron acceptor. Ru(3+) is a strong one-electron oxidizer capable of abstracting an electron from a neighboring protein molecule, generating a protein radical(1,2). Radicals are unstable, highly-reactive species and therefore disappear rapidly through a variety of intra- and intermolecular reactions. A radical may utilize the high energy of an unpaired electron to react with another protein monomer forming a dimeric radical, which subsequently loses a hydrogen atom and forms a stable, covalently-linked dimer. The dimer may then react further through a similar mechanism with monomers or other dimers to form higher-order oligomers. Advantages of PICUP relative to other photo- or chemical cross-linking methods(3,4) include short (<or=1 s) exposure to non-destructive visible light, no need for pre facto modification of the native sequence, and zero-length covalent cross-linking. In addition, PICUP enables cross-linking of proteins within wide pH and temperature ranges, including physiologic parameters. Here, we demonstrate application of PICUP to cross-linking of three amyloidogenic proteins the 40- and 42-residue amyloid beta-protein variants (Abeta40 and Abeta42), and calcitonin, and a control protein, growth-hormone releasing factor (GRF).

Zn2+-Aβ40 Complexes Form Metastable Quasi-spherical Oligomers That Are Cytotoxic to Cultured Hippocampal Neurons

Background: The mechanism by which interaction between Aβ and Zn2+ induces Aβ aggregation and cell toxicity is elusive. Results: Zn2+ and Aβ40 form metastable neurotoxic oligomers. Conclusion: Aβ40 binding to Zn2+ leads to formation of small neurotoxic oligomers that become benign upon further self-assembly. Significance: We provide a structure-function analysis of Zn2+-stabilized Aβ40, a neurotoxic species that may contribute to the pathology in AD. The roles of metal ions in promoting amyloid β-protein (Aβ) oligomerization associated with Alzheimer disease are increasingly recognized. However, the detailed structures dictating toxicity remain elusive for Aβ oligomers stabilized by metal ions. Here, we show that small Zn2+-bound Aβ1–40 (Zn2+-Aβ40) oligomers formed in cell culture medium exhibit quasi-spherical structures similar to native amylospheroids isolated recently from Alzheimer disease patients. These quasi-spherical Zn2+-Aβ40 oligomers irreversibly inhibit spontaneous neuronal activity and cause massive cell death in primary hippocampal neurons. Spectroscopic and x-ray diffraction structural analyses indicate that despite their non-fibrillar morphology, the metastable Zn2+-Aβ40 oligomers are rich in β-sheet and cross-β structures. Thus, Zn2+ promotes Aβ40 neurotoxicity by structural organization mechanisms mediated by coordination chemistry.