Working memory impairment in a transgenic amyloid precursor protein TgCRND8 mouse model of Alzheimer's disease

The most profound deficits observed in Alzheimer's disease (AD) are in domains of episodic and working memory systems. Transgenic (Tg) mice expressing mutated human amyloid precursor protein (APP) genes offer a model to study the effect of AD pathology on cognition. We reported previously that APP TgCRND8 mice showed deficits in a reference and working memory evaluated in a Morris water-maze test. In this study, we evaluated the working memory of TgCRND8 mice comparing two training paradigms in a six-arm radial water maze. In the first paradigm, the exploration of the maze was constrained, forcing the mice to use a spatial mapping strategy. In the second paradigm, mice were unconstrained in their exploration of the maze. TgCRND8 mice proved to be significantly impaired in spatial working memory in both paradigms as compared with their non-transgenic littermates. The analysis of data revealed that forcing mice to use a spatial strategy during training caused only a moderate improvement in the performance of all mice. However, unconstrained exploration of the maze not only resulted in a fast learning in control mice, but also facilitated the development of a chaining strategy in spatially impaired TgCRND8 mice. In conclusion, TgCRND8 mice showed impairment in spatial working memory but retained a plasticity to choose alternative search strategies.     

Clathrin-mediated endocytic proteins are upregulated in the cortex of the Tg2576 mouse model of Alzheimer's disease-like amyloid pathology

Amyloid-β (Aβ) is cleaved from amyloid precursor protein (APP) predominantly after APP has trafficked through the secretory pathway and then become re-internalised by endocytosis. Clathrin-mediated and, more recently, clathrin-independent endocytosis have both been implicated in this process. Furthermore, endocytic abnormalities have been identified in cases of Alzheimer’s disease (AD), however, the relevance of these changes to the aetiology of the disease remains unclear. We therefore examined the expression of proteins related to these endocytic processes in the cortex of Tg2576 mice that overexpress the Swedish mutation in APP, and consequently overexpress Aβ, to determine if there were any changes in their associated pathways. We identified significant increases in the levels of clathrin, dynamin and PICALM, all proteins intimately involved with the clathrin-mediated endocytic pathway, in the transgenic animals. However, levels of proteins associated with flotillin or caveolin-mediated endocytic pathways remained unchanged. These results emphasise the importance of clathrin-mediated endocytosis in the aetiology of AD and reinforce the results of the recent GWAS studies that identified genes for clathrin-mediated endocytosis as susceptibility genes for AD. Such studies in transgenic mice will allow us to learn more about the role of clathrin-mediated endocytosis in AD.     

Therapeutic angiogenesis for revascularization in peripheral artery disease

Therapeutic angiogenesis for peripheral artery disease (PAD), achieved by gene and cell therapy, has recently raised a great deal of hope for patients who cannot undergo standard revascularizing treatment. Although pre-clinical studies gave very promising data, still clinical trials of gene therapy have not provided satisfactory results. On the other hand, cell therapy approach, despite several limitations, demonstrated more beneficial effects but initial clinical studies must be constantly validated by larger randomized, multi-center, double-blinded, placebo-controlled trials. This review focuses on previous and recent gene and cell therapy studies for limb ischemia, including both experimental and clinical research, and summarizes some important papers published in this field. Moreover, it provides a short comment on combined gene and cell therapy approach on the example of heme oxygenase-1 overexpressing cells with therapeutic properties.     

Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo

Background/aims

TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involved in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation.

Methods

We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC^KM) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by ³H-TdR incorporation.

Results

TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk.

Conclusions

TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.     

Cardiac oxidative stress in a mouse model of neutral lipid storage disease

Cardiac oxidative stress has been implicated in the pathogenesis of hypertrophy, cardiomyopathy and heart failure. Systemic deletion of the gene encoding adipose triglyceride lipase (ATGL), the enzyme that catalyzes the rate-limiting step of triglyceride lipolysis, results in a phenotype characterized by severe steatotic cardiac dysfunction. The objective of the present study was to investigate a potential role of oxidative stress in cardiac ATGL deficiency. Hearts of mice with global ATGL knockout were compared to those of mice with cardiomyocyte-restricted overexpression of ATGL and to those of wildtype littermates. Our results demonstrate that oxidative stress, measured as lucigenin chemiluminescence, was increased ~ 6-fold in ATGL-deficient hearts. In parallel, cytosolic NADPH oxidase subunits p67phox and p47phox were upregulated 4–5-fold at the protein level. Moreover, a prominent upregulation of different inflammatory markers (tumor necrosis factor α, monocyte chemotactant protein-1, interleukin 6, and galectin-3) was observed in those hearts. Both the oxidative and inflammatory responses were abolished upon cardiomyocyte-restricted overexpression of ATGL. Investigating the effect of oxidative and inflammatory stress on nitric oxide/cGMP signal transduction we observed a ~ 2.5-fold upregulation of soluble guanylate cyclase activity and a ~ 2-fold increase in cardiac tetrahydrobiopterin levels. Systemic treatment of ATGL-deficient mice with the superoxide dismutase mimetic Mn(III)tetrakis (4-benzoic acid) porphyrin did not ameliorate but rather aggravated cardiac oxidative stress. Our data suggest that oxidative and inflammatory stress seems involved in lipotoxic heart disease. Upregulation of soluble guanylate cyclase and cardiac tetrahydrobiopterin might be regarded as counterregulatory mechanisms in cardiac ATGL deficiency.     

Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO^•). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO^• to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration.Effects of NO^•, either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO^• down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO^• was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression.In conclusion, during angiogenesis and leukocyte recruitment, NO^• regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO^•, reflecting the functions of the tissue they originate from.     

Abnormal elevated PTEN expression in the mouse antrum of a model of GIST Kit(K641E/K641E)

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Approximately 85% of GISTs harbor activating mutations of the KIT or PDGFRA receptor tyrosine kinases. PTEN and SHIP2 are major phosphatases that dephosphorylate PI(3,4,5)P₃, one of the intracellular signal pathways downstream of KIT. PTEN is an important tumor suppressor, whereas the involvement of SHIP2 in cancer has been proposed based essentially on cell line studies. We have used a mouse model of GIST, i.e. Kit^K641E knock-in mice, resulting in the substitution of a Lys by Glu at position 641 of Kit. In homozygous Kit^K641E mice, PTEN-immunoreactivity (ir) in antrum was found in the hyperplastic Kit-ir layer. The same localization was found for SHIP2. Western blot analysis in antrum showed a large increase in PTEN expression in Kit^K641E homozygous mice as compared to wild type. In contrast, SHIP2 expression was not affected between the two genotypes. Erk1, but not PKB, phosphorylation appears to be upregulated in Kit^K641E homozygous mice. In the human GIST882 imatinib sensitive cell line, both PTEN and SHIP2 were expressed and showed, in part, a nuclear localization. The upregulation of PTEN in antrum in Kit^K641E mice might serve as a feedback mechanism to limit PI 3-kinase activation downstream of Kit in a context of oncogenic mutation.     

Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells

The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2–5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.     

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rβ or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G₀/G₁ phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G₀/G₁ phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G₀/G₁ phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.     

Insights into the role of components of the tumor microenvironment in oral carcinoma call for new therapeutic approaches

The research on oral cancer has focused mainly on the cancer cells, their genetic changes and consequent phenotypic modifications. However, it is increasingly clear that the tumor microenvironment (TME) has been shown to be in a dynamic state of inter-relations with the cancer cells. The TME contains a variety of components including the non-cancerous cells (i.e., immune cells, resident fibroblasts and angiogenic vascular cells) and the ECM milieu [including fibers (mainly collagen and fibronectin) and soluble factors (i.e., enzymes, growth factors, cytokines and chemokines)]. Thus, it is currently assumed that TME is considered a part of the cancerous tissue and the functionality of its key components constitutes the setting on which the hallmarks of the cancer cells can evolve. Therefore, in terms of controlling a malignancy, one should control the growth, invasion and spread of the cancer cells through modifications in the TME components. This mini review focuses on the TME as a diagnostic approach and reports the recent insights into the role of different TME key components [such as carcinoma-associated fibroblasts (CAFs) and inflammation (CAI) cells, angiogenesis, stromal matrix molecules and proteases] in the molecular biology of oral carcinoma. Furthermore, the impact of TME components on clinical outcomes and the concomitant need for development of new therapeutic approaches will be discussed.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

The role of amino acid transporters in GSH synthesis in the blood-brain barrier and central nervous system

Glutathione (GSH) plays a critical role in protecting cells from oxidative stress and xenobiotics, as well as maintaining the thiol redox state, most notably in the central nervous system (CNS). GSH concentration and synthesis are highly regulated within the CNS and are limited by availability of the sulfhydryl amino acid (AA) l-cys, which is mainly transported from the blood, through the blood-brain barrier (BBB), and into neurons. Several antiporter transport systems (e.g., x(c)(-), x(-)(AG), and L) with clearly different luminal and abluminal distribution, Na(+), and pH dependency have been described in brain endothelial cells (BEC) of the BBB, as well as in neurons, astrocytes, microglia and oligodendrocytes from different brain structures. The purpose of this review is to summarize information regarding the different AA transport systems for l-cys and its oxidized form l-cys(2) in the CNS, such as expression and activity in blood-brain barrier endothelial cells, astrocytes and neurons and environmental factors that modulate transport kinetics.     

Evolution of covered stents in the contemporary era: clinical application,materials and manufacturing strategies using nanotechnology

Endovascular stents have revolutionised the field of interventional cardiology. Despite their excellent clinical outcome complications associated with percutaneous stent implantation following the procedure have remained a major drawback in their widespread use. To overcome such limitations, a number of novel endovascular stents have emerged including a covered stent wrapped in a thin membrane sleeve. As well as prevention of complications associated with stenting, covered stents owing to their physical barrier are used as the treatment option of choice for trauma devices during emergency situations and to treat a number of pathological disease states. The aim of this review is to provide the reader with an overall objective outlook in the use of covered stents as a treatment option in a number of vascular complications and addresses their design and materials used in the manufacturing process. In addition, new strategies are highlighted and future prospects with the emergence of novel smart alloys for 3D scaffolds and the use of nanotechnology in the development of nanocomposite materials.