Partial characterization of the human retinal endothelial cell tight and adherens junction complexes.
OBJECTIVE
In diabetic retinopathy and macular edema, the blood-retinal barrier fails to function properly, and there is transvascular leakage of proteins and solutes. The tight junction protein occludin and the adherens junction protein cadherin-5 have been shown to be critical to maintaining the endothelial barrier and regulating paracellular transport of large vessel endothelia. However, the expression and distribution of these junction proteins in the retinal endothelium is not well characterized.


METHODS
Human and bovine retinal endothelial cells were isolated as described previously. Western blot analysis and flow cytometry techniques were used to assay for the presence of occludin, zonula occludens-1 (ZO-1), cadherin-5, and beta-catenin. The subcellular localization of the proteins was visualized by immunohistochemistry performed on cultured human retinal endothelial cells and cryosections of bovine retina.


RESULTS
Western blot analysis and flow cytometry techniques found occludin, ZO-1, cadherin-5, and beta-catenin in cultured human retinal endothelial cells. Immunofluorescence staining of cultured retinal endothelial cells and cryosections of bovine retina showed junctional localization of occludin, ZO-1, cadherin-5, and beta-catenin.


CONCLUSIONS
This report demonstrates the expression of occludin and cadherin-5 in retinal endothelial cells and their localization to sites of cell-cell contact. Expression of their respective regulatory proteins, ZO-1 and beta-catenin, at sites of cell-cell contact suggests that occludin and cadherin-5 play a role in maintaining the retinal endothelial barrier.
Russ
P K
PK
Department of Biomedical Engineering, School of Engineering, Vanderbilt University, Nashville, Tennessee 37235, USA.
Davidson
M K
MK
Hoffman
L H
LH
Haselton
F R
FR
D000818:Animals; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001813:Blood-Retinal Barrier; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism*; D002417:Cattle; D002450:Cell Communication; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism*; D012171:Retinal Vessels / Q000166:cytology / Q000378:metabolism*; D019108:Tight Junctions / Q000502:physiology*; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D000099152:Cadherin 5
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015820:Cadherins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D062793:Occludin; D010750:Phosphoproteins; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D000099152:Cadherin 5; C495270:CTNNB1 protein, human; C568985:OCLN protein, human; C569042:TJP1 protein, human