High Glucose Alters Cx43 Expression and Gap Junction Intercellular Communication in Retinal M??ller Cells: Promotes M??ller Cell and Pericyte Apoptosis

摘要

Purpose.: To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal M??ller cells, and promotes M??ller cell and pericyte loss. Methods.: Retinal M??ller cells (rMC-1) and cocultures of rMC-1 and retinal pericytes were grown in normal (N) or HG (30 mM glucose) medium. Additionally, rMC-1 transfected with Cx43 small interfering RNA (siRNA) were grown as cocultures with pericytes, and rMC-1 transfected with Cx43 plasmid were grown in HG. Expression of Cx43 was determined by Western blotting and immunostaining and GJIC was assessed by scrape-loading dye transfer (SLDT) technique. Apoptosis was analyzed by TUNEL or differential staining assay, and Akt activation by assessing Akt phosphorylation. Results.: In monocultures of rMC-1 and cocultures of rMC-1 and pericytes, Cx43 protein level, number of Cx43 plaques, GJIC, and Akt phosphorylation were significantly reduced in HG medium. Number of TUNEL-positive cells was also significantly increased in rMC-1 monocultures and in rMC-1 and pericyte cocultures grown in HG medium. Importantly, when rMC-1 transfected with Cx43 siRNA were grown as cocultures with pericytes, a significant decrease in GJIC, and increase in TUNEL-positive cells was observed, concomitant with decreased Akt phosphorylation. Upregulation of Cx43 rescued rMC-1 from HG-induced apoptosis. Conclusions.: Gap junction communication between M??ller cells and pericytes is essential for their survival. Downregulation of Cx43 that is HG induced and impairment of GJIC activity in M??ller cells contributes to loss of glial and vascular cells associated with the pathogenesis of diabetic retinopathy.