PURPOSE: To investigate whether high glucose (HG) alters expression of connexin 30.2 (Cx30.2) and influences gap junction intercellular communication (GJIC) in retinal endothelial cells and promotes vascular lesions characteristic of diabetic retinopathy (DR). METHODS: Western blot analysis and immunostaining were performed to determine Cx30.2 protein expression and localization in rat retinal endothelial cells (RRECs) grown in normal (N; 5 mM) or HG (30 mM) medium for 7 days. Concurrently, GJIC was assessed in cells grown in N or HG medium and in cells transfected with Cx30.2 siRNA. Similarly, retinal Cx30.2 expression was assessed in nondiabetic and diabetic rats. Additionally, the effect of reduced Cx30.2 on development of acellular capillaries (ACs) and pericyte loss (PL) was studied in retinas of Cx30.2 knockout mice. RESULTS: Cx30.2 was identified in RRECs in vitro and in vascular cells of retinal capillaries. RRECs grown in HG exhibited significantly reduced Cx30.2 protein levels consistent with decreased Cx30.2 immunostaining compared with those grown in N medium. Cells grown in HG and cells transfected with Cx30.2 siRNA exhibited significantly diminished dye transfer compared with N or nontransfected cells. Importantly, Cx30.2 protein level and immunostaining were decreased in diabetic retinas compared with nondiabetic retinas. Retinal capillaries of Cx30.2 knockout mice exhibited increased numbers of ACs and PL compared with those of wild-type mice. CONCLUSIONS: These results indicate that HG- or diabetes-induced downregulation of Cx30.2 expression and decrease in GJIC activity play a critical role in the development of retinal vascular lesions in early DR.
PURPOSE: To investigate whether high glucose (HG) alters expression of connexin 30.2 (Cx30.2) and influences gap junction intercellular communication (GJIC) in retinal endothelial cells and promotes vascular lesions characteristic of diabetic retinopathy (DR). METHODS: Western blot analysis and immunostaining were performed to determine Cx30.2 protein expression and localization in rat retinal endothelial cells (RRECs) grown in normal (N; 5 mM) or HG (30 mM) medium for 7 days. Concurrently, GJIC was assessed in cells grown in N or HG medium and in cells transfected with Cx30.2 siRNA. Similarly, retinal Cx30.2 expression was assessed in nondiabetic and diabeticrats. Additionally, the effect of reduced Cx30.2 on development of acellular capillaries (ACs) and pericyte loss (PL) was studied in retinas of Cx30.2 knockout mice. RESULTS:Cx30.2 was identified in RRECs in vitro and in vascular cells of retinal capillaries. RRECs grown in HG exhibited significantly reduced Cx30.2 protein levels consistent with decreased Cx30.2 immunostaining compared with those grown in N medium. Cells grown in HG and cells transfected with Cx30.2 siRNA exhibited significantly diminished dye transfer compared with N or nontransfected cells. Importantly, Cx30.2 protein level and immunostaining were decreased in diabetic retinas compared with nondiabetic retinas. Retinal capillaries of Cx30.2 knockout mice exhibited increased numbers of ACs and PL compared with those of wild-type mice. CONCLUSIONS: These results indicate that HG- or diabetes-induced downregulation of Cx30.2 expression and decrease in GJIC activity play a critical role in the development of retinal vascular lesions in early DR.
Authors: Joanna Gemel; Xianming Lin; Raymond Collins; Richard D Veenstra; Eric C Beyer Journal: Biochem Biophys Res Commun Date: 2008-02-20 Impact factor: 3.575