Histamine, ZO-1 and increased blood-retinal barrier permeability in diabetic retinopathy.
OBJECTIVE
First, to develop an improved retinal capillary endothelial cell culture system which exhibits some of the physiologic features of the bloodretinal barrier; second, to use this model to determine how histamine and chemical conditions of diabetes effect expression of the tight junction protein, ZO-1; and third, to discuss application of the Henle-Koch postulates to the problem of diabetic retinopathy.


METHODS
Bovine retinal capillary endothelial cells were exposed to varying serum and growth factor concentrations, as well as astrocyte-conditioned medium, in order to establish a model of the blood-retinal barrier. Cells were also exposed to varying concentrations of histamine, and of insulin and glucose. The expression of ZO-1 tight junction protein was determined by immunocytochemistry and immunoblotting.


RESULTS
Modified concentrations of growth factors reduced endothelial cell proliferation, without reducing viability. Astrocyte conditioned medium increased ZO-1 protein content. Histamine reduced ZO-1 protein content. Both high glucose (20mM) and low insulin (10(-12)M) reduced ZO-1 protein content compared to control conditions (5mM glucose and 10(-9) M insulin).


CONCLUSIONS
Control of culture conditions results in a more physiologic in vitro model of the blood-retinal barrier. Soluble factors from astrocytes promote tight junction formation. Both histamine and chemical conditions of diabetes diminish tight junction formation. These factors may mediate blood-retinal barrier breakdown in diabetic retinopathy. Henle-Koch postulates for diabetic retinopathy are presented.
Gardner
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D000818:Animals; D001253:Astrocytes / Q000166:cytology; D001813:Blood-Retinal Barrier; D002196:Capillaries; D002199:Capillary Permeability / Q000502:physiology*; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003930:Diabetic Retinopathy / Q000209:etiology / Q000503:physiopathology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005947:Glucose / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D015151:Immunoblotting; D007328:Insulin / Q000494:pharmacology; D008565:Membrane Proteins / Q000096:biosynthesis*; D010750:Phosphoproteins / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012171:Retinal Vessels / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
D017077:Culture Media, Conditioned; D007004:Hypoglycemic Agents; D007328:Insulin; D008565:Membrane Proteins; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D006632:Histamine; D005947:Glucose