Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells

<p>Abstract</p> <p>Background</p> <p>Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer <it>in vitro</it>. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC) which are widely used as model for endothelial cell-related studies.</p> <p>Results</p> <p>We here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP) and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na) and for longer incubation periods 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin.</p> <p>Conclusions</p> <p>pCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible <it>in vitro </it>model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins</p

Fluorescence detection of cervical intraepithelial neoplasia for photodynamic therapy with the topical agents 5-aminolevulinic acid and benzoporphyrin-derivative monoacid ring

ObjectiveThe aim of this study was to determine whether 2 photosensitizers, benzoporphyrin-derivative monoacid ring and 5-aminolevulinic acid, are selectively absorbed by dysplastic cervical cells after topical administration.Study designThis phase I clinical trial involved 18 women with biopsy-proven cervical intraepithelial neoplasia at the Beckman Laser Institute, Irvine, Calif. Colposcopically directed cervical biopsy specimens obtained after 1.5, 3, or 6 hours of exposure to a randomly assigned photosensitizer were evaluated for selective drug absorption with hematoxylin and eosin staining and fluorescence microscopy.ResultsAfter exposure to 5-aminolevulinic acid, cervical tissue showed maximal fluorescence in dysplastic cells relative to normal cells, with negligible stromal fluorescence. According to our detection methods benzoporphyrin-derivative monoacid ring demonstrated nonselective, diffusion-driven uptake, with fluorescence appearing in the superficial cells, followed by nonselective drug absorption in the remaining cells and stroma of the epithelium.ConclusionOur data demonstrated selective absorption of 5-aminolevulinic acid by dysplastic cervical cells. This agent therefore represents a promising photosensitizing prodrug for the treatment of cervical intraepithelial neoplasia with photodynamic therapy

Multiethnic Meta-Analysis Identifies Ancestry-Specific and Cross-Ancestry Loci for Pulmonary Function

Nearly 100 loci have been identified for pulmonary function, almost exclusively in studies of European ancestry populations. We extend previous research by meta-analyzing genome-wide association studies of 1000 Genomes imputed variants in relation to pulmonary function in a multiethnic population of 90,715 individuals of European (N = 60,552), African (N = 8429), Asian (N = 9959), and Hispanic/Latino (N = 11,775) ethnicities. We identify over 50 additional loci at genome-wide significance in ancestry-specific or multiethnic meta-analyses. Using recent fine-mapping methods incorporating functional annotation, gene expression, and differences in linkage disequilibrium between ethnicities, we further shed light on potential causal variants and genes at known and newly identified loci. Several of the novel genes encode proteins with predicted or established drug targets, including KCNK2 and CDK12. Our study highlights the utility of multiethnic and integrative genomics approaches to extend existing knowledge of the genetics of lung function and clinical relevance of implicated loci

Multiethnic meta-analysis identifies ancestry-specific and cross-ancestry loci for pulmonary function

Nearly 100 loci have been identified for pulmonary function, almost exclusively in studies of European ancestry populations. We extend previous research by meta-analyzing genome-wide association studies of 1000 Genomes imputed variants in relation to pulmonary function in a multiethnic population of 90,715 individuals of European (N = 60,552), African (N = 8429), Asian (N = 9959), and Hispanic/Latino (N = 11,775) ethnicities. We identify over 50 additional loci at genome-wide significance in ancestry-specific or multiethnic meta-analyses. Using recent fine-mapping methods incorporating functional annotation, gene expression, and differences in linkage disequilibrium between ethnicities, we further shed light on potential causal variants and genes at known and newly identified loci. Several of the novel genes encode proteins with predicted or established drug targets, including KCNK2 and CDK12. Our study highlights the utility of multiethnic and integrative genomics approaches to extend existing knowledge of the genetics of l

Multiethnic meta-analysis identifies ancestry-specific and cross-ancestry loci for pulmonary function

Nearly 100 loci have been identified for pulmonary function, almost exclusively in studies of European ancestry populations. We extend previous research by meta-analyzing genome-wide association studies of 1000 Genomes imputed variants in relation to pulmonary function in a multiethnic population of 90,715 individuals of European (N = 60,552), African (N = 8429), Asian (N = 9959), and Hispanic/Latino (N = 11,775) ethnicities. We identify over 50 additional loci at genome-wide significance in ancestry-specific or multiethnic meta-analyses. Using recent fine-mapping methods incorporating functional annotation, gene expression, and differences in linkage disequilibrium between ethnicities, we further shed light on potential causal variants and genes at known and newly identified loci. Several of the novel genes encode proteins with predicted or established drug targets, including KCNK2 and CDK12. Our study highlights the utility of multiethnic and integrative genomics approaches to extend existing knowledge of the genetics of lung function and clinical relevance of implicated loci

Aldosterone Modulates Endothelial Permeability and Endothelial Nitric Oxide Synthase Activity by Rearrangement of the Actin Cytoskeleton

Aldosterone (Aldo) is involved in vascular remodeling and inflammation; however, the mechanisms are imperfectly defined. We hypothesized that Aldo alters endothelial integrity and modifies paracellular permeability. Human umbilical vein endothelial cells were exposed to Aldo (10(-9) mol/L) and alterations in paracellular permeability, assembly of tight and adherens junctions and activation of intracellular signaling pathways were determined. Aldo increased endothelial permeability for molecules ≤ 70 kDa within 60 minutes. A transient loss of cortical actin with formation of actin stress fibers and disruption of continuous adherens and tight junction strands accompanied these changes. Mineralocorticoid receptor blockade, inhibition of RhoA, or disruption of extracellular-regulated protein kinase1/2 signaling pathways attenuated the Aldo-related effects. Moreover, Aldo-induced cytoskeletal rearrangement led to rapid dephosphorylation of protein kinase B and subsequent deactivation of endothelial nitric oxide synthase. Ex vivo tracer flux experiments with Evans blue-conjugated albumin demonstrated a concordant response to Aldo in freshly isolated umbilical arteries. Furthermore, low-dose cortisol (3 × 10(-10) to 3 × 10(-9) mol/L) mimics the effect of Aldo on endothelial integrity, and Aldo, by upregulating11β-hydroxysteroid dehydrogenase type 2, might even aggravate this deleterious effect of low-dose cortisol. We suggest that these mechanisms may contribute to the vasculopathy induced by inappropriate mineralocorticoid receptor activation