Transcriptome analysis and molecular signature of human retinal pigment epithelium.

Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed 'signature' genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases.

cAMP-dependent absorption of chloride across airway epithelium.

Elevated levels of Na and Cl in airway surface liquid may play a major role in the airway pathology of cystic fibrosis (CF) (J. J. Smith, S. M. Travis, E. P. Greenberg, and M. J. Welsh. Cell 85: 229-236, 1996) and could be caused by block of transcellular Cl absorption due to lack of a functional CF transmembrane conductance regulator (CFTR). To test for transcellular absorption of Cl across non-CF epithelium, we studied how fluid absorption was affected by the opening and closing of Cl channels. Forskolin (an activator of CFTR) tripled fluid absorption across primary cultures of bovine tracheal epithelium but had no effect on human cells. However, in both species, fluid absorption was markedly inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate, a blocker of CFTR. Microelectrode studies suggested that the magnitude of the absorptive response to forskolin in bovine cells depended on the size of an inwardly directed electrochemical driving force for Cl movement across the apical membrane. Patch-clamp measurements of bovine cells revealed CFTR in the apical membrane and a cAMP-activated, inwardly rectifying Cl channel in the basolateral membrane. We conclude that a significant fraction of absorbed Cl passes transcellularly in bovine tracheal epithelial cultures, with CFTR as the path of entry in the apical membrane and a novel cAMP-activated Cl channel as the exit route in the basolateral membrane. Our data further indicate that a similar pathway may exist in non-CF human tracheal epithelium.

Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia.

Fluid transport across cultures of bovine tracheal epithelium was measured with a capacitance probe technique. Baseline fluid absorption (Jv) across bovine cells of 3.2 microliter. cm-2. h-1 was inhibited by approximately 78% after 1 h of exposure to suspensions of Pseudomonas aeruginosa, with a concomitant decrease in transepithelial potential (TEP) and increase in transepithelial resistance (Rt). Effects of P. aeruginosa were blocked by amiloride, which decreased Jv by 112% from baseline of 2.35 +/- 1.25 microliter. cm-2. h-1, increased Rt by 101% from baseline of 610 +/- 257 Omega. cm2, and decreased TEP by 91% from baseline of -55 +/- 18.5 mV. Microelectrode studies suggested that effects of P. aeruginosa on amiloride-sensitive Na absorption were due in part to a block of basolateral membrane K channels. In the presence of Cl transport inhibitors [5-nitro-2-(3-phenylpropylamino)-benzoic acid, H2-DIDS, and bumetanide], P. aeruginosa induced a fluid secretion of approximately 2.5 +/- 0.4 microliter. cm-2. h-1 and decreased Rt without changing TEP. However, these changes were abolished when the transport inhibitors were used in a medium in which Cl was replaced by an impermeant organic anion. Filtrates of P. aeruginosa suspensions had no effect on Jv, TEP, or Rt. Mutants lacking exotoxin A or rhamnolipids or with defective lipopolysaccharide still inhibited fluid absorption and altered bioelectrical properties. By contrast, mutations in the rpoN gene encoding a sigma factor of RNA polymerase abolished actions of P. aeruginosa. In vivo, changes in transepithelial salt and water transport induced by P. aeruginosa may alter viscosity and ionic composition of airway secretions so as to foster further bacterial colonization.

Apical and basolateral membrane mechanisms that regulate pHi in bovine retinal pigment epithelium.

pH regulation was studied in fresh explant bovine retinal pigment epithelium-choroid using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and intracellular microelectrodes. Acid recovery was HCO3 dependent, inhibited by apical amiloride and apical or basal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and required apical and basal Na. Alkali recovery was HCO3 dependent and inhibitable by apical or basal DIDS. Three apical and two basolateral transporters were identified. Four contribute to acid extrusion, i.e., apical Na/H exchange, apical H-lactate cotransport, and apical Na-HCO3 cotransport and basolateral Na-HCO3 cotransport. At least two contribute to alkali extrusion, i.e., apical Na-HCO3 cotransport and a basolateral HCO3-dependent, DIDS-inhibitable mechanism, possibly Na-HCO3 cotransport, Cl/HCO3 exchange, or both. The apical Na-HCO3 cotransporter is electrogenic, carrying net negative charge inward. Basal Cl removal or addition of basal HCO3 caused HCO3- and Cl-dependent alkalinizations, respectively. Apical DIDS increased both responses. These cytosolic pH (pHi) regulatory mechanisms are so tightly coupled that changes in pHi can only occur after two or more of them are inhibited. In addition, these mechanisms help provide pathways for transport of Na and HCO3 across the retinal pigment epithelium between the blood and the distal retina.

Contact Effects of Mononuclear Cells on L929 Fibroblast Morphological Phenotype.

We studied the contact interactions of human peripheral blood mononuclear cells (MNC) and transformed mouse fibroblast cell line L929 (L-cells), namely their effects on morphological phenotype of L-cells. The morphological characteristics of the fibroblast, (cell area, nucleus-cytoplasmic ratio, cell spreading, cell shape) were estimated with the aid of fight scanning microscopy, followed by computer image analysis. Contact interaction between fibroblasts and MNC caused normalization of morphological phenotype of the fibroblasts (increase of cell area, shape factor, spreading and decrease of nucleus-cytoplasmic ratio). This phenomenon was revealed by analysis of both average morphological characteristics and population contents. Only a particular subpopulation of L-cells, but not the whole population, was shown to be normalized by the effects of MNC. For elucidation of responsible MNC population, which was capable of influencing on fibroblast morphological phenotype, we separated MNC in several cell types: adherent cells, non-adherent cells, which were separated in E-rosetting cells, and non-E-rosetting cells. Only non-adherent and E-rosetting cells could normalize the morphological phenotype. E-rosetting population consisted of 85% of CD3(+) (T lymphocytes) and 15percnt; of CD56(+)/CD16(+) (natural killers). Supernatants of MNC, and MNC cocultured L-cells, obtained after 24 h incubation in the cell culture medium, were not able to normalize the morphological phenotype of the fibroblasts. They had small denormalizing effect on the fibroblasts (decrease of cell area, shape factor, spreading and increase of nucleus-cytoplasmic ratio). The results of this study indicate that the contact interaction between MNC and fibroblasts may normalize transformed fibroblast morphological phenotype. The dependence of fibroblast functional state on their morphological phenotype imply the presence of regulatory mechanism of contact interaction between fibroblasts and MNC, which determines many live processes in fibroblast.

[A model for the assessment of fibroblast morphology]. / Model' dlia otsenki morfologii fibroblastov.

A model for comprehensive assessment of morphologic parameters of a cell (area, nucleus and cytoplasm area, degree of spreading), based on the use of a scanning device, has been developed. The model comprises an original method for staining the preparations, which permits assessment of the degree of cell spreading by microdensitometry, and updating the equipment and software. The programs help assess the integral and derivative parameters, such as the area of the interface zone of a cell, the shape factor, spreading, and nucleus to cytoplasm ratio. Using this model, the effect of peripheral blood mononuclears on the morphology of transformed murine fibroblasts L929 upon a contact exposure was assessed. Addition of mononuclears to fibroblast culture led to alteration of their morphology: increase of the mean cellular area, degree of spreading, and of shape factor and reduction of the nucleus/cytoplasm ratio. This was true for both the mean values and the population composition.