Low-molecular-weight fucoidan enhances the proangiogenic phenotype of endothelial progenitor cells

Endothelial progenitor cell (EPC) transplantation is a potential means of inducing neovascularization in vivo. However, the number of circulating EPC is relatively small, it may thus be necessary to enhance their proangiogenic properties ex vivo prior to injection in vivo. Fucoidan has previously been shown to potentiate in vitro tube formation by mature endothelial cells in the presence of basic fibroblast growth factor (FGF-2). We therefore examined whether fucoidan, alone or combined with FGF-2, could increase EPC proangiogenic potency in vitro. EPC exposure to 10 microg/ml fucoidan induced a proangiogenic phenotype, including cell proliferation (p < 0.01) and migration (p < 0.01); moreover, differentiation into vascular cords occurred in the presence of FGF-2 (p < 0.01). This latter effect correlated with upregulation of the cell-surface #alpha6 integrin subunit of the laminin receptor (p < 0.05). Compared to untreated HUVEC, untreated EPC #alpha6 expression and adhesion to laminin were enhanced two-fold. Fucoidan treatment further enhanced HUVEC but not EPC adhesion to laminin. These results show that fucoidan enhances the proangiogenic properties of EPC and suggest that ex vivo fucoidan preconditioning of EPC might lead to increased neovascularization when injected into ischemic tissues.     

Store-operated calcium entry modulates neuronal network activity in a model of chronic epilepsy

Store-operated Ca²⁺ entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca²⁺ stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca²⁺ concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.     

Cyclooxygenase-2 and tissue inhibitor of matrix metalloproteinases-1 confer the antimigratory effect of cannabinoids on human trabecular meshwork cells

Cannabinoids have received considerable attention as potential antiglaucomatous drugs. Recently, prostaglandins (PG) have been suggested to contribute to this effect. Within the factors conferring the development of glaucoma, depletion of the aqueous humor outflow-regulating trabecular meshwork (TM) cells elicited by migration from the outflow system is considered to play a pivotal role. This study therefore investigates the impact of two cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), on the migration of human TM cells and the involvement of the PG-synthesizing enzyme cyclooxygenase-2 (COX-2) and one of its potential downstream targets, the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), to this response. Using Boyden chamber assays cannabinoids were shown to elicit an antimigratory effect that was reversed by antagonists for CB₁ as well as CB₂ receptors and accompanied by upregulation of COX-2 and TIMP-1 expression and PGE₂ synthesis. Knockdown of cannabinoid-induced COX-2 or TIMP-1 expression by siRNA or inhibition of COX-2 activity by NS-398 led to a significant suppression of this antimigratory action. Migration was also diminished by the major COX-2 product PGE₂ and by recombinant TIMP-1. Experiments using selective E prostanoid (EP) receptor agonists and antagonists revealed that decreased migration by PGE₂, THC and MA was mediated via EP₂ and EP₄ receptors. Finally, the cannabinoid-mediated increases of TIMP-1 levels were abolished by NS-398, and PGE₂ was shown to elicit a concentration-dependent increase of TIMP-1. Collectively, this data demonstrate a COX-2-dependent upregulation of TIMP-1 conferring the antimigratory action of cannabinoids. A decreased migration reducing TM cell loss in glaucoma might be involved in the antiglaucomatous action of cannabinoids.     

Properties of HTLV-I transformed CD8 T-cells in response to HIV-1 infection

HIV-1 infection studies of primary CD8⁺ T-cells are hampered by difficulty in obtaining a significant number of targets for infection and low levels of productive infection. Further, there exists a paucity of CD8-expressing T-cell lines to address questions pertaining to the study of CD8⁺ T-cells in the context of HIV-1 infection. In this study, a set of CD8⁺ T-cell clones were originated through HTLV-I transformation in vitro, and the properties of these cells were examined. The clones were susceptible to T-cell tropic strains of the virus and exhibited HIV-1 production 20-fold greater than primary CD4⁺ T-cells. Productive infection resulted in a decrease in expression of CD8 and CXCR4 molecules on the surface of the CD8⁺ T-cell clones and antibodies to these molecules abrogated viral binding and replication. These transformed cells provide an important tool in the study of CD8⁺ T-cells and may provide important insights into the mechanism(s) behind HIV-1 induced CD8⁺ T-cell dysfunction.     

A method for measuring mouse respiratory allergic reaction to low-dose chemical exposure to allergens: An environmental chemical of uncertain allergenicity,a typical contact allergen and a non-sensitizing irritant

Our aim was to improve a method for detecting respiratory hypersensitivity by testing three confirmed respiratory allergens (trimellitic anhydride [TMA], phthalic anhydride [PA] and toluene diisocyanate [TDI]), an environmental chemical of uncertain allergenicity (2,4-d-butyl [DB]), a confirmed contact allergen (2,4-dinitrochlorobenzene [DNCB]) and a standard irritant (sodium dodecyl sulfate [SDS]). BALB/c mice were topically sensitized (nine times in 3 weeks) with these chemicals, then challenged via the trachea. One day post-challenge, samples were taken from the mice to assay for total immunoglobulin (IgE and IgG₁) levels in serum and bronchoalveolar lavage fluid (BALF); differential cell counts and cytokine/chemokine levels in BALF; lymphocyte counts and surface antigen expression on B-cells within lung-associated lymph nodes (LNs); ex situ cytokine production by cells from these LNs; and gene expression in BALF (CCR3) and LNs (CCR4, STAT6 and GATA-3). The three confirmed respiratory allergens and DB induced immune response characteristic of immediate-type respiratory reactions, as evidenced by increased total IgE and IgG₁ levels; influx of eosinophils, neutrophils, chemokines and cytokines in BALF; increased surface antigen expression on B-cells in LNs; increased Th2 cytokine production in LNs; and increased respiratory allergy-related gene expression in both BALF and LNs. In contrast, DNCB and SDS treatments yielded, at most, insignificant increases in all respiratory allergic parameters. Thus, the protocol was equally suitable for use with an environmental chemical of unknown allergenicity and for typical respiratory allergens. Since the protocol differentiated respiratory allergens from contact allergens and irritants, it may be useful for detecting respiratory allergy related to environmental chemicals.     

T7 peptide-functionalized nanoparticles utilizing RNA interference for glioma dual targeting

Among all the malignant brain tumors, glioma is the deadliest and most common form with poor prognosis. Gene therapy is regarded as a promising way to halt the progress of the disease or even cure the tumor and RNA interference (RNAi) stands out. However, the existence of the blood–brain barrier (BBB) and blood tumor barrier (BTB) limits the delivery of these therapeutic genes. In this work, the delivery system targeting to the transferrin (Tf) receptor highly expressed on both BBB and glioma was successfully synthesized and would not compete with endogenous Tf. U87 cells stably express luciferase were employed here to simulate tumor and the RNAi experiments in vitro and in vivo validated that the gene silencing activity was 2.17-fold higher with the targeting ligand modification. The dual-targeting gene delivery system exhibits a series of advantages, such as high efficiency, low toxicity, stability and high transaction efficiency, which may provide new opportunities in RNAi therapeutics and nanomedicine of brain tumors.     

Identification of p53-dependent genes potentially involved in UVB-mediated premature senescence of human skin fibroblasts using siRNA technology

Premature senescence of skin human diploid fibroblasts is induced after a series of 10 sublethal exposures to UVB at 2.5 kJ/m(2) with appearance of several biomarkers of cellular senescence like senescence-associated beta-galactosidase activity (SA beta-gal) and cell cycle arrest. Herein it is shown that the induction of UVB-induced premature senescence is associated with a transient increase of protein abundance and DNA-binding activity of p53. Silencing p53 expression with small interfering RNA (siRNA) affected the basal level of SA beta-gal and proliferative potential, but did not prevent UVB-induced increase of SA beta-gal and decrease of DNA synthesis. We used a senescence-specific low-density DNA array and p53 siRNA to study the mRNA abundance of 240 senescence-related genes and identified several potential p53-dependent genes differentially expressed after the repeated exposures to UVB.     

Serial culture of murine primary airway epithelial cells and ex vivo replication of human rhinoviruses

Human rhinoviruses (HRV) are the primary etiological agents in cold infections, and represent a serious risk to individuals with chronic respiratory disease such as asthma. In order to develop treatment options for HRV infections, murine models are a crucial component in the study of infection mechanisms due to the wide array of reagents and techniques available to study murine immunology. We present here a cell culture system for studying isolated murine epithelial cell responses to HRV. Monolayers of primary mouse airway epithelial cells were maintained in a serial culture system, and the identity and purity of the cell population was confirmed via immunostaining (positive for cytokeratin, negative for vimentin). Infection of these cells with a minor group rhinovirus (HRV-1A) was evidenced by increases in viral RNA, de novo synthesis of viral proteins, and production of infectious virus. This model will be useful in experiments to define mechanisms of viral replication and host/virus interactions within airway epithelial cells.