No abstract

This study determined whether retinal degeneration during diabetes includes retinal neural cell apoptosis. Image analysis of retinal sections from streptozotocin (STZ) diabetic rats after 7.5 months of STZ diabetes identified 22% and 14% reductions in the thickness of the inner plexiform and inner nuclear layers, respectively ( P Ͻ 0.001). The number of surviving ganglion cells was also reduced by 10% compared to controls ( P Ͻ 0.001). In situ end labeling of DNA terminal dUTP nick end labeling (TUNEL) identified a 10-fold increase in the frequency of retinal apoptosis in wholemounted rat retinas after 1, 3, 6, and 12 months of diabetes ( P Ͻ 0.001, P Ͻ 0.001, P Ͻ 0.01, and P Ͻ 0.01, respectively).

The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.

Vascular endothelial growth factor (VEGF) may have a physiologic role in regulating vessel permeability and contributes to the pathophysiology of diabetic retinopathy as well as tumor development. We set out to ascertain the mechanism by which VEGF regulates paracellular permeability in rats. Intra-ocular injection of VEGF caused a post-translational modification of occludin as determined by a gel shift from 60 to 62 kDa. This event began by 15 min post-injection and was maximal by 45 min. Alkaline phosphatase treatment revealed this modification was caused by a change in occludin phosphorylation. In addition, the quantity of extracted occludin increased 2-fold in the same time frame. The phosphorylation and increased extraction of occludin was recapitulated in retinal endothelial cells in culture after VEGF stimulation. The data presented herein are the first demonstration of a change in the phosphorylation of this transmembrane protein under conditions of increased endothelial permeability. In addition, intraocular injection of VEGF also caused tyrosine phosphorylation of ZO-1 as early as 15 min and increased phosphorylation 4-fold after 90 min. In conclusion, VEGF rapidly increases occludin phosphorylation as well as the tyrosine phosphorylation of ZO-1. Phosphorylation of occludin and ZO-1 likely contribute to regulated endothelial paracellular permeability.Tissues of the central nervous system, including the brain and retina, depend on intact blood-brain and blood-retinal barriers, respectively, to partition them from the systemic circulation. These barriers contribute to the maintenance of specific neural tissue environments by regulating ion concentrations, water permeability, and delivery of amino acids and sugars and by preventing exposure to circulating antibodies and immune cells. These requirements imply the need for regulation of the blood-brain/retinal barrier, which permits selective delivery of needed substrates in response to varying local tissue demands and systemic metabolic influences. Endothelial cells regulate the blood-brain/retinal barrier through a number of mechanisms including transporter activity, e.g. glucose transport via GLUT-1 and transcytosis. In this report we provide evidence for regulation of paracellular permeability by vascular endothelial growth factor (VEGF) 1 through the rapid phosphorylation of tight junction proteins.The endothelial cells of the blood-brain and blood-retinal barriers contain tight junctions that confer highly selective barrier properties to these vessels. Tight junctions contain at least seven proteins including occludin, zonula occludens 1, 2, and 3 (Z0 -1, -2, or -3), cingulin, the 7H6 antigen, and symplekin (reviewed in Refs. 1-5). Recent studies of an occludin knockout mouse line revealed that claudins may also influence permeability (6), whereas other laboratories have identified novel isoforms of occludin by reverse transcription-polymerase chain reaction (7). Signaling molecules including Rab proteins, large G-proteins, and soluble tyrosine kinas...