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S. Jhamb, A. Simon, S. Roy; Cx43+/- Knockout Mice Exhibit Reduced Heterogeneous Gap Junctions in Vascular Cells of Retinal Capillaries. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2645.
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Diabetes or high glucose has been shown to downregulate Cx43 expression and gap junction intercellular communication. However, its influence on heterogeneous gap junctions in retinal vascular cells is unknown. In this study, we have determined whether reduced Cx43 expression in Cx43+/- KO mice affects Cx43/Cx40 colocalization and the number of heterogeneous gap junctions in vascular cells of retinal capillaries.
Retinas from WT and Cx43+/- KO mice eyes were isolated, fixed in paraformaldehyde, and subjected to trypsin digestion to isolate the retinal vascular network. Trypsin digestion was performed in 3% trypsin buffer at 37°C for approximately 2 hours to remove the vitreous and glia. The retinal trypsin digest preparations were then subjected to double immunostaining for Cx43 and Cx40 using secondary antibodies conjugated to rhodamine or FITC, respectively. At least ten random images from each retina were digitally photographed under a fluorescence microscope and analyzed for Cx43 and Cx40 plaques. In addition, Cx43/Cx40 heterogeneous plaques were analyzed from superimposed images. In parallel, protein isolated from retinas of contralateral eyes of Cx43+/- KO and WT mice were subjected to Western Blot analysis to determine Cx43 and Cx40 protein expression.
Western Blot analysis showed significant reduction in Cx43 protein level in the Cx43+/- mice compared to that of WT mice (52%±7% of control, p<0.05). Also, Cx43+/- mice compared to that of WT mice exhibited reduced Cx43 immunostaining in the retinal vessels (53%±7% of control, p<0.05) while Cx40 immunostaining showed no change (93%±9% of control). Cx40 localization appeared more pericellular than that of Cx43, while Cx43 localization was more on cell boundaries. The number of Cx43/Cx40 heterogeneous gap junction plaques was significantly reduced in the Cx43+/- mice compared to those of WT mice (42%±22% of control, P=0.001).
Our finding indicates that decreased Cx43 expression reduces the number of Cx43/Cx40 heterogeneous gap junctions in endothelial cells of retinal capillaries. Thus, high glucose-induced downregulation of Cx43 expression may affect cell-cell communication via heterogeneous gap junctions in diabetic retinopathy.
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