The tyrosine phosphatase DEP-1 induces cytoskeletal rearrangements, aberrant cell-substratum interactions and a reduction in cell proliferation

The receptor protein tyrosine phosphatase density-enhanced phosphatase-1 (DEP-1) has been implicated in aberrant cancer cell growth and immune cell function, however, its function within cells has yet to be properly elucidated. To investigate the cellular function of DEP-1, stable cell lines inducibly expressing DEP-1 were generated. Induction of DEP-1 expression was found to decrease PDGF-stimulated tyrosine phosphorylation of a number of cellular proteins including the PDGF receptor, and to inhibit growth factor-stimulated phosphorylation of components of the MAPK pathway, indicating that DEP-1 antagonised PDGF receptor signalling. This was supported by data showing that DEP-1 expression resulted in a reduction in cell proliferation. DEP-1-expressing cells had fewer actin-containing microfilament bundles, reduced vinculin and paxillin-containing adhesion plaques, and were defective in interactions with fibronectin. Defective cell-substratum adhesion correlated with lack of activation of FAK in DEP-1-expressing cells. Time-lapse interference reflection microscopy of live cells revealed that although small focal contacts at the leading edge were generated in DEP-1-expressing cells, they failed to mature into stable focal adhesions, as found in control cells. Further motility analysis revealed that DEP-1-expressing cells retained limited random motility, but showed no chemotaxis towards a gradient of PDGF. In addition, cell-cell contacts were disrupted, with a change in the localisation of cadherin from discrete areas of cell-cell contact to large areas of membrane interaction, and there was a parallel redistribution of beta-catenin. These results demonstrate that DEP-1 is a negative regulator of cell proliferation, cell-substratum contacts, motility and chemotaxis in fibroblasts.

The effects of high glucose and atorvastatin on endothelial cell matrix production

Background Statins are known to enhance atherosclerotic plaque stability through influences on extracellular matrix homeostasis. Net matrix production reflects the relative balance of matrix production and degradation through enzymes such as matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of MMP (TIMPs). The effects of statins on endothelial cell production of these parameters following co-exposure with a proatherogenic stimulus such as high glucose are not known. Methods Human endothelial cells were exposed for 72 h to 5 mM> (control) or 25 mM (high) glucose +/- atorvastatin (1 mumol/l). Extracellular matrix homeostasis was assessed by measuring matrix metalloproteinase (MMP)-2 secretion, tissue inhibitor of MMP (TIMP)-1 and -2 secretion and net collagen IV production. Results were expressed as percentage +/- SEM of control values. Results Exposure to high glucose increased cellular collagen IV expression to 190.1 +/- 11.7% (P < 0.0001) of control levels. No change in MMP-2 secretion (111.6 +/- 5.2%; P > 0.05) was observed but both TIMP-1 and TIMP-2 expression were increased to 136.3 +/- 6.4% and 144.0 +/- 27.5%, respectively (both P < 0.05). The presence of atorvastatin in high glucose conditions reduced collagen IV expression to 136.1 +/- 20.6%. This was paralleled by increased secretion of MMP-2 to 145.8 +/- 7.8% (P < 0.01), increased TIMP-2 expression to 208.0 +/- 21.3% (P < 0.005 compared with high glucose) but no change in TIMP-1 expression (155.1 +/- 14.6%) compared with high glucose alone. The presence of atorvastatin in control conditions did not affect levels of collagen IV expression (114.5 +/- 13.2%). Conclusions Endothelial cell exposure to high glucose was associated with a MMP/TIMP profile that increased extracellular matrix production which was attenuated by concurrent exposure to atorvastatin. Consequently, a mechanism by which the atherosclerotic plaque regression that is observed in patients taking these drugs has been demonstrated.

Characterization of heat shock protein-specific T cells in atherosclerosis

A role for infection and inflammation in atherogenesis is widely accepted. Arterial endothelium has been shown to express heat shock protein 60 (HSP60) and, since human (hHSP60) and bacterial (GroEL) HSP60s are highly conserved, the immune response to bacteria may result in cross-reactivity, leading to endothelial damage and thus contribute to the pathogenesis of atherosclerosis. In this study, GroEL-specific T-cell lines from peripheral blood and GroEL-, hHSP60-, and Porphyromonas gingivalis-specific T-cell lines from atherosclerotic plaques were established and characterized in terms of their cross-reactive proliferative responses, cytokine and chemokine profiles, and T-cell receptor (TCR) V beta expression by flow cytometry. The cross-reactivity of several lines was demonstrated. The cytokine profiles of the artery T-cell lines specific for GroEL, hHSP60, and P. gingivalis demonstrated Th2 phenotype predominance in the CD4 subset and Tc0 phenotype predominance in the CD8 subset. A higher proportion of CD4 cells were positive for interferon-inducible protein 10 and RANTES, with low percentages of cells positive for monocyte chemoattractant protein 1 and macrophage inflammatory protein la, whereas a high percentage of CD8 cells expressed all four chemokines. Finally, there was overexpression of the TCR V beta 5.2 family in all lines. These cytokine, chemokine, and V beta profiles are similar to those demonstrated previously for P. gingivalis-specific lines established from periodontal disease patients. These results support the hypothesis that in some patients cross-reactivity of the immune response to bacterial HSPs, including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may explain the apparent association between atherosclerosis and periodontal infection.

A small-molecule lead compound for the treatment of Alzheimer's disease

At autopsy, Alzheimer's disease is characterised by the presence of amyloid plaques and neurofibrillary tangles, made up of two peptide sequences, amyloid-beta(1-40) (A beta 40) and amyloid-beta(1-42) (A beta 42). In Tyrode's solution (2 mM Ca2+), 10 mu M A beta 42 peptide almost immediately aggregates and eventually forms p-sheets. This aggregation can be inhibited with 4,5-dianilinophthalimide (DAPH). Ca2+-permeant AMPA receptors are involved in the neuronal Ca2+ influx (neurotoxicity) induced by the A beta 42 peptide in cultured neuronal cells. The Ca2+ influx observed with pre-incubated A beta 42 peptide was inhibited by DAPH. DAPH also inhibits epidermal growth factor receptor kinase, and this will prevent its development for use in Alzheimer's disease. The potential of DAPH as a small-molecule lead compound for the treatment of Alzheimer's disease next requires the separation of the structural requirements that reverse fibril formation and inhibit epidermal growth factor receptor kinase.

Plasma delipidation process induces rapid regression of atherosclerosis and mobilisation of adipose tissue

Cholesterol is a major component of atherosclerotic plaques. Cholesterol accumulation within the arterial intima and atherosclerotic plaques is determined by the difference of cellular cholesterol synthesis and/or influx from apo B-containing lipoproteins and cholesterol efflux. In humans, apo A-I Milano infusion has led to rapid regression of atherosclerosis in coronary arteries. We hypothesised that a multifunctional plasma delipidation process (PDP) would lead to rapid regression of experimental atherosclerosis and probably impact on adipose tissue lipids. In hyperlipidemic animals, the plasma concentrations of cholesterol, triglyceride and phospholipid were, respectively, 6-, 157-, and 18-fold higher than control animals, which consequently resulted in atherosclerosis. PDP consisted of delipidation of plasma with a mixture of butanol-diisopropyl ether (DIPE). PDP removed considerably more lipid from the hyperlipidemic animals than in normolipidemic animals. PDP treatment of hyperlipidemic animals markedly reduced intensity of lipid staining materials in the arterial wall and led to dramatic reduction of lipid in the adipose tissue. Five PDP treatments increased apolipoprotein A1 concentrations in all animals. Biochemical and hematological parameters were unaffected during PDP treatment. These results show that five PDP treatments led to marked reduction in avian atherosclerosis and removal of lipid from adipose tissue. PDP is a highly effective method for rapid regression of atherosclerosis.

Volumetric and areal bone mineral density measures are associated with cardiovascular disease in older men and women: The health, aging, and body composition study

The associations of volumetric (vBMD) and areal (aBMD) bone mineral density measures with prevalent cardiovascular disease (CVD) and subclinical peripheral arterial disease (PAD) were investigated in a cohort of older men and women enrolled in the Health, Aging, and Body Composition Study. Participants were 3,075 well-functioning white and black men and women (42% black, 51% women), aged 68-80 years. Total hip, femoral neck, and trochanter aBMD were measured using dual-energy X-ray absorptiometry. Quantitative computed tomography was used to evaluate spine trabecular, integral, and cortical vBMD measures in a subgroup (n = 1,489). Logistic regression was performed to examine associations of BMD measures with CVD and PAD. The prevalence of CVD (defined by coronary heart disease, PAD, cerebrovascular disease, or congestive heart failure) was 29.8%. Among participants without CVD, 10% had subclinical PAD (defined as ankle-arm index < 0.9). Spine vBMD measures were inversely associated with CVD in men (odds ratio of integral [ORintegral] = 1.34, 95% confidence interval [CI] 1.10-1.63; ORtrabecular = 1.25, 95% CI 1.02-1.53; ORcortical = 1.36, 95% CI 1.11-1.65). In women, for each standard deviation decrease in integral vBMD, cortical vBMD, or trochanter aBMD, the odds of CVD were significantly increased by 28%, 27%, and 22%, respectively. Total hip aBMD was associated with subclinical PAD in men (OR = 1.39, 95% CI 1.03-1.84) but not in women. All associations were independent of age and shared risk factors between BMD and CVD and were not influenced by inflammatory cytokines (interleukin-6 and tumor necrosis factors-alpha). In conclusion, our results provide further evidence for an inverse association between BMD and CVD in men and women. Future research should investigate common pathophysiological links for osteoporosis and CVD.

Ceramides reduce CD36 cell surface expression and oxidised LDL uptake by monocytes and macrophages

Oxidised LDL accumulates in macrophages following scavenger receptor (SR) uptake. The expression of the SR, CD36, is increased by oxidised LDL. The signalling molecule, ceramide, can modulate intracellular peroxides and increase lipid peroxidation. Ceramide also accumulates in atherosclerotic plaques. Thus, we have examined whether ceramide can modulate CD36 expression and function in human monocyte/macrophages. Addition of synthetic short chain ceramides or the action of sphingomyelinase to generate physiological long chain ceramides in situ caused significant reductions in CD36 expression by monocytes/macrophages which was not due to inhibition of mRNA expression. Inhibition of proteasomal degradation using lactacystin had no effect on CD36 expression, however, flow cytometric analysis of permeabilised cells suggested an intracellular trafficking blockade. Ceramide treated monocytes/macrophages showed dose dependent reduction in oxidised LDL uptake. Taken together, it is suggested that ceramide blocks the transport of CD36 to the membrane of monocytes/macrophages, thereby preventing uptake of oxidised LDL. © 2006 Elsevier Inc. All rights reserved.

Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

Modelling the growth of prion protein aggregates in the brain in variant Creutzfeldt-Jakob disease

Deposition of insoluble prion protein (PrP) in the brain in the form of protein aggregates or deposits is characteristic of the ‘transmissible spongiform encephalopathies’ (TSEs). Understanding the growth and development of these PrP aggregates is important both in attempting to the elucidate of the pathogenesis of prion disease and in the development of treatments designed to prevent or inhibit the spread of prion pathology within the brain. Aggregation and disaggregation of proteins and the diffusion of substances into the developing aggregates (surface diffusion) are important factors in the development of protein aggregates. Mathematical models suggest that if aggregation/disaggregation or surface diffusion is the predominant factor, the size frequency distribution of the resulting protein aggregates in the brain should be described by either a power-law or a log-normal model respectively. This study tested this hypothesis for two different types of PrP deposit, viz., the diffuse and florid-type PrP deposits in patients with variant Creutzfeldt-Jakob disease (vCJD). The size distributions of the florid and diffuse plaques were fitted by a power-law function in 100% and 42% of brain areas studied respectively. By contrast, the size distributions of both types of plaque deviated significantly from a log-normal model in all brain areas. Hence, protein aggregation and disaggregation may be the predominant factor in the development of the florid plaques. A more complex combination of factors appears to be involved in the pathogenesis of the diffuse plaques. These results may be useful in the design of treatments to inhibit the development of protein aggregates in vCJD.

The identification of pathological subtypes of Alzheimer's disease using cluster analysis

A culster analysis was performed on 78 cases of Alzheimer's disease (AD) to identify possible pathological subtypes of the disease. Data on 47 neuropathological variables, inculding features of the gross brain and the density and distribution of senile plaques (SP) and neurofibrillary tangles (NFT) were used to describe each case. Cluster analysis is a multivariate statistical method which combines together in groups, AD cases with the most similar neuropathological characteristics. The majority of cases (83%) were clustered into five such groups. The analysis suggested that an initial division of the 78 cases could be made into two major groups: (1) a large group (68%) in which the distribution of SP and NFT was restricted to a relatively small number of brain regions, and (2) a smaller group (15%) in which the lesions were more widely disseminated throughout the neocortex. Each of these groups could be subdivided on the degree of capillary amyloid angiopathy (CAA) present. In addition, those cases with a restricted development of SP/NFT and CAA could be divided further into an early and a late onset form. Familial AD cases did not cluster as a separate group but were either distributed between four of the five groups or were cases with unique combinations of pathological features not closely related to any of the groups. It was concluded that multivariate statistical methods may be of value in the classification of AD into subtypes. © 1994 Springer-Verlag.

Reduced transferrin binding in Down syndrome:a route to senile plaque formation and dementia

Plasma transferrin binding in Down syndrome and Alzheimer's disease is significantly reduced compared with age matched controls and it was thought this may help elucidate a pathological time sequence for the onset of dementia in Down syndrome. In Down syndrome, there was a reduction in gallium and aluminium transferrin binding both with age and the onset of dementia. Non-transferrin bound gallium species were identified as non-transportable phosphate or silicate. Thus, the route of entry of metals into the brain must be via a transferrin mediated complex only. A clear sequence of pathological events has been demonstrated in Down syndrome which shows the pathway to development of plaques and dementia and this is believed to have an immunological origin.

Laminar distribution of the pathological changes in frontal and temporal cortex in 8 patients with progressive supranuclear palsy

OBJECTIVE: To determine the laminar distribution of the pathological changes in the cerebral cortex in progressive supranuclear palsy (PSP). METHOD: The distribution of the abnormally enlarged neurons (EN), surviving neurons, neurofibrillary tangles (NFT), glial inclusions (GI), tufted astrocytes (TA), and neuritic plaques (NP) were studied across the cortex in tau immunolabeled sections of frontal and temporal cortex in 8 cases of PSP. RESULTS: The distribution of the NFT was highly variable with no consistent pattern of laminar distribution. The GI were distributed either in the lower laminae or uniformly across the cortex. Surviving neurons exhibited either a density peak in the upper laminae or a bimodal distribution was present with density peaks in the upper and lower laminae. The EN and glial cell nuclei were distributed primarily in the lower cortical laminae. There were positive correlations between the densities of the EN and glial cell nuclei and negative correlations between the surviving neurons and glial cells. No correlations were present between the densities of the NFT and GI. CONCLUSION: Cortical pathology in PSP predominantly affects the lower laminae but may spread to affect the upper laminae in some cases. The NFT and GI may have different laminar distributions and gliosis occurs concurrently with neuronal enlargement.

The interface between Alzheimer's disease, normal aging, and related disorders

Since the earliest descriptions of Alzheimer's disease (AD), the presence of senile plaques (SP) and neurofibrillary tangles (NFT) have been regarded as the typical pathological hallmarks of the disease. Studies over the last twenty years, however, have reported a considerable degree of heterogeneity within the AD phenotype and as a consequence, an overlap between the pathological features of AD not only with normal aging, but also with disorders related to AD. This review discusses: 1) the degree of heterogeneity within AD, 2) the concept of an 'interface' between disorders, 3) the nature and degree of the interface between AD and normal aging, vascular dementia (VD), the tauopathies, synucleinopathies, and prion disease, and 4) whether the original status of AD should be retained or whether AD, normal aging, and the related disorders should be regarded as representing a 'continuum' of neuropathological change.