The purpose of this study was to map retinal blood vessels with increased paracellular permeability due to diabetes. A histologic technique that uses the plant lectin ConA was used to determine the spatial relationship between tight junction protein expression and localized increases in paracellular vascular permeability.
26 ConA binds to α-
d-mannosyl and α-
d-glucosyl glycoprotein residues, which are located in abundance on the abluminal side of brain and retinal vascular endothelial cells, but infrequently on the luminal side.
28 When AlexaFluor 488–conjugated ConA is perfused after fixation with 2% paraformaldehyde it binds to regions where the basement membrane is exposed to the vascular lumen, labeling regions of paracellular permeability. This perfusion technique was originally used to identify regions of increased paracellular permeability in the tracheal vasculature after induction of acute inflammation with substance P.
26 In that study, ConA binding corresponded to regions of plasma leakage identified by electron microscopy for monastral blue extravasation, which confirmed that ConA binding corresponded to paracellular permeability. Another study also described ConA binding associated with increased vascular permeability in the dystrophic rat model for proliferative retinopathy.
29
ConA binding was more extensive in retinas of STZ diabetic rats than in retinas of age-matched control animals. This study demonstrates that paracellular permeability increases in the retinas of STZ diabetic rats as early as 2 weeks after the induction of diabetes. The vessels most consistently having an increase in labeling were the superficial postcapillary venules and regions of arterioles. Some retinas had extensive labeling in the capillary bed of the outer plexiform layer after 1 month of STZ diabetes. These data show that paracellular permeability to a 52- to 104-kDa solute increased soon after the onset of diabetes in STZ diabetic rats, implying that significant gaps appeared between the vascular endothelial cells. In addition, vascular permeability occurred throughout the retina rather than at focal points. Other studies have described focal increases in vascular permeability to endogenous albumin in rat retinas and postmortem tissue after diabetes.
16 30 However, these studies examined albumin extravasation rather than the accessibility of the vascular basement membrane. Clinical studies tend to identify focal points of increased vascular leakage on the macro scale, but it is likely that generalized increases in permeability to water, giving rise to macular edema, are not so easily detected by fluorescein angiography. Therefore, the difference in findings may be due to the sensitivity of the ConA technique and the ability to view the entire retina using this approach, rather than sampling histologic sections.
There was an inverse relationship between occludin immunoreactivity and paracellular permeability. This relationship was demonstrated by the pattern of occludin immunoreactivity in normal retinas, which was high in the arterioles and capillaries and progressively decreased in venules with propinquity to the optic disc.
25 Furthermore, ConA labeling was only present in the venules of control retinas. On treatment with VEGF or diabetes, occludin immunoreactivity decreased at the plasma membrane with a concomitant increase in ConA labeling. Occludin protein content measured by Western blot was reduced by approximately 35% after 3 months of STZ diabetes.
6 Taken together, these data suggest that reduced occludin content and increased vascular permeability are directly related in diabetes. However, relocation of occludin from the plasma membrane to the cytoplasm may be the mechanism responsible for the onset of permeability, because VEGF did not reduce total occludin content in rat retinas over a similar time course, as observed in Western blot analysis,
31 but dramatically reduced occludin immunoreactivity at the cell borders. The possibility that the changes in occludin immunoreactivity occur in response to increased vascular permeability cannot be ruled out, but this causal relationship seems less attractive because of the absence of a likely mechanism. The subcellular redistribution of occludin protein was noted previously in the vasculature of 1-month STZ diabetic rat retinas.
25 Redistribution of occludin and ZO-1 also occurs in cultured brain microvascular endothelial cells in response to VEGF
32 and hypoxia.
33 In addition, platelet-derived growth factor, also induces tight junction protein redistribution and increased permeability in Madin-Darby canine kidney cells.
34 These data are consistent with the hypothesis that occludin migrates into a different cellular compartment after stimulation with growth factor, allowing paracellular permeability.
34 Therefore, redistribution of tight junction proteins from the plasma membrane into the cytoplasm may be part of the mechanism that regulates paracellular permeability at the blood–retinal barrier.
The distribution of claudin-5 immunofluorescence was not affected by diabetes or VEGF. Claudin-5, or transmembrane protein deleted in velocardiofacial syndrome (TMVCF)
35 36 is ubiquitously expressed by endothelial cells.
37 In the current work, we found that claudin-5 immunofluorescence did not change in response to diabetes or VEGF injection, demonstrating that occludin redistribution is a specific response. However, these data do not exclude a role for claudin-5 in retinal vascular permeability. Similar conclusions have been made in other studies of claudins. Hypoxia induced a 2.6-fold increase in permeability to sucrose in bovine brain microvessel endothelial cells, accompanied by altered occludin, ZO-1, and ZO-2 immunoreactivity, but the protein expression and distribution of claudin-1 remained unchanged.
38 Brain vascular claudin-1 expression was also unchanged in an inflammatory pain model, despite an increase in brain vascular permeability and associated changes in ZO-1.
39 Morcos et al.
40 described the distribution of claudin-1 in the retina; however, our study is the first demonstration of immunoreactivity for the endothelial cell–specific claudin-5 in the retina.
In conclusion, this is the first study to provide direct identification of regions of paracellular vascular permeability in the retina and, to our knowledge, is the first study to map the vessels with increased paracellular permeability due to diabetes. Increased permeability begins with the arterioles and postcapillary venules of the superficial vasculature followed later by the capillary network in the outer plexiform layer. These data suggest that redistribution of the transmembrane tight junction protein occludin from the plasma membrane to the cell interior regulates paracellular permeability. Future studies of the molecular mechanism of occludin redistribution will shed light on the cellular events that regulate permeability of the blood–retinal barrier.
The authors thank Thomas W. Gardner for useful comments during preparation of the manuscript.