Physical disruption of cell-cell contact induces VEGF expression in RPE cells.
Mol Vis
; 23: 431-446, 2017.
Article
em En
| MEDLINE
| ID: mdl-28761317
ABSTRACT
PURPOSE:
To investigate the role of RPE cell-cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19).METHODS:
Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell-cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, ß-catenin, and claudin-1 in RPE cultures.RESULTS:
Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, ß-catenin, and claudin-1 successfully localized to cell-cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, ß-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell-cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell-cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells.CONCLUSIONS:
VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in the cytoplasm of RPE cells.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Adesão Celular
/
Comunicação Celular
/
Fator A de Crescimento do Endotélio Vascular
/
Epitélio Pigmentado da Retina
/
Junções Intercelulares
Limite:
Adult
/
Humans
/
Male
Idioma:
En
Revista:
Mol Vis
Assunto da revista:
BIOLOGIA MOLECULAR
/
OFTALMOLOGIA
Ano de publicação:
2017
Tipo de documento:
Article