The effect of connexin43 on the level of vascular endothelial growth factor in human retinal pigment epithelial cells.

BACKGROUND: Connexins (Cx) are the basic units of gap junctions and contribute to cellular integrity by promoting intercellular communication. Disruption of the retinal pigment epithelial monolayer may be an early event in the pathogenesis of age-related macular degeneration, a condition in which vascular endothelial growth factor (VEGF) is known to be of importance. This study was designed to assess the effect of connexin43 (Cx43) expression and gap junctional intercellular communication (GJIC) on the expression and secretion of VEGF from the retinal pigment epithelium under normal cell culture and oxidative stress conditions. METHODS: Stable cell lines of ARPE-19 were produced in which wild-type Cx43 was either over-expressed, down-regulated by targeted shRNA, or functionally inhibited by co-expression of a disease-linked dominant-negative mutant (G21R). Pharmacologic blockade of GJIC was accomplished with flufenamic acid. Oxidant challenge was performed with tert-butyl hydroperoxide (tBH). VEGF gene expression and secretion were assessed by real-time PCR and ELISA respectively. RESULTS: Over-expression of Cx43 in ARPE-19 cells reduced both gene expression and secretion of VEGF. Down-regulation of Cx43 increased gene expression and secretion of VEGF. Increased secretion of VEGF was also observed in ARPE-19 cells expressing a dominant-negative mutant of Cx43, and when GJIC was blocked. Over-expression of Cx43 reduced tBH-induced secretion of VEGF from ARPE-19 cells. CONCLUSIONS: These studies show that Cx43 protects against oxidative stress-induced VEGF secretion in ARPE-19 cells, and thus has important implications in understanding the pathogenesis of age-related macular degeneration.

Cytoprotective effect of lacritin on human corneal epithelial cells exposed to benzalkonium chloride in vitro.

PURPOSE: Benzalkonium chloride (BAK) is the most commonly found preservative in eye drops, and has been shown to cause ocular surface inflammation and toxicity. Lacritin is a human tear glycoprotein secreted from the lacrimal glands that has been found to be cytoprotective. This study was designed to determine if the presence of lacritin confers protection to a cultured human corneal epithelial (HCE) cell line, CRL-11515, and primary HCE cells after exposure to the ocular preservative agent BAK. MATERIALS AND METHODS: Recombinant human lacritin was cloned into intein fusion vectors, expressed in E. coli, and purified on chitin beads and DEAE Sepharose. Metabolic curves were established using the MTT assay after exposure of sub-confluent CRL-11515 cells to BAK or lacritin. Western blot analysis of lipidated LC3 (LC3-II) provided a measure of autophagy in CRL-11515 cells exposed to lacritin and/or BAK. RESULTS: BAK reduced CRL-11515 cellular metabolic activity in a time- and dose-dependent manner. BAK-induced cellular stress was evident by elevated autophagy that increased with rising concentrations of BAK compared to control (p < 0.05). Lacritin increased HCE cell proliferation at an optimal dose of 1 nM. Preconditioning HCE cells with 1 nM lacritin for 24 h prior to BAK exposure significantly dampened levels of LC3-II (p < 0.05) and promoted a significant increase in cellular metabolic activity (p < 0.01) compared to BAK alone. CONCLUSIONS: These results suggest lacritin protects cultured HCE cells stressed with BAK. Lacritin may have the potential to be used as a topical adjunctive therapy in eyes chronically exposed to BAK.

An autocrine γ-aminobutyric acid signaling system exists in pancreatic ß-cell progenitors of fetal and postnatal mice.

Gamma-aminobutyric acid (GABA) is produced and secreted by adult pancreatic ß-cells, which also express GABA receptors mediating autocrine signaling and regulating ß-cell proliferation. However, whether the autocrine GABA signaling involves in ß-cell progenitor development or maturation remains uncertain. By means of immunohistochemistry we analyzed the expression profiles of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD) and the α1-subunit of type-A GABA receptor (GABAARα1) in the pancreas of mice at embryonic day 15.5 (E15.5), E18.5, postnatal day 1 (P1) and P7. Our data showed that at E15.5 the pancreatic and duodenum homeobox-1 (Pdx1) was expressed in the majority of cells in the developing pancreata. Notably, insulin immunoreactivity was identified in a subpopulation of pancreatic cells with a high level of Pdx1 expression. About 80% of the high-level Pdx-1 expressing cells in the pancreas expressed GAD and GABAARα1 at all pancreatic developmental stages. In contrast, only about 30% of the high-level Pdx-1 expressing cells in the E15.5 pancreas expressed insulin; i.e., a large number of GAD/GABAARα1-expressing cells did not express insulin at this early developmental stage. The expression level of GAD and GABAARα1 increased steadily, and progressively more GAD/GABAARα1-expressing cells expressed insulin in the course of pancreatic development. These results suggest that 1) GABA signaling proteins appear in ß-cell progenitors prior to insulin expression; and 2) the increased expression of GABA signaling proteins may be involved in ß-cell progenitor maturation.