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MOLECULAR COMPOSITION OF THE BLOOD–RETINAL BARRIER
Specialized Retinal Vessels Control Flux into Neural Tissue
The blood vessels of the CNS, including the retina, are different from blood vessels in other regions of the body. Vessels of the CNS lack fenestrations observed in highly permeable vessels such as the glomerular capillary wall (22) and choroid capillary plexus (23). These fenestrations are a thinning of the capillary wall, bringing the apical and basal membranes in close apposition and promoting transcellular flux. This loss of endothelial cell fenestrations is one of the initial developmental steps of BBB formation (24, 25). Additionally, the vessels of the CNS have reduced pinocytotic vesicles, also reducing transcellular transport (26–28). These structural features allow specific control of transcellular permeability through specific mechanisms such as receptor-mediated endocytosis, and provide tight control of the fluids, nutrients, and metabolic precursors that enter the neural parenchyma.
Overview of Tight Junction Proteins
Paracellular flux is controlled by the presence and composition of the junctional complex, which includes both tight junctions and adherens junctions. In epithelial cells the adherens and tight junctions are easily discernable, but in the endothelium of the BBB and BRB these complexes cannot be differentiated at the ultrastructural level (29, 30). Many studies of the tight junction have been carried out in epithelial cells and a number of reviews have detailed the molecular components of the tight junctions including (31–34), and recently addressed by our group (1). Here the overall structure of the tight junction is briefly reviewed and the changes that occur to the tight junction in diabetes are addressed.
Tight junctions are now known to be complex structures comprised of over 40 structural and regulatory proteins. Figure 2 provides a schematic of the vascular structure in the retina and a highly simplified view of the tight junction complex. (For a more detailed review of the tight junction proteins in the retina see the following review and text (1, 35) ). Current concepts of the tight junctions suggest an organization of this complex array of proteins. Tight junctions are composed of both transmembrane proteins that create the connection to the adjoining cell, and organizing proteins that bind to multiple junctional proteins and link these proteins to the cytoskeleton.
The tight junction transmembrane proteins include junction adhesion molecules or JAM, occludin, tricellulin, and the claudin gene family. The most well-studied mem- brane-associated proteins that organize the junctional complex are the zonula occludens isoforms or ZO-1, -2, and -3 proteins. Recent studies using siRNA for the ZO isoforms reveals that this protein is necessary for proper assembly of the junctional complex (36), confirming a role for these proteins as a scaffold. Other studies are beginning to elucidate the molecular function of the transmembrane proteins of the tight junction.
Claudins Confer Tight Junction Barrier Properties
Cell-culture experiments utilizing siRNA (37) or mutational analysis (38) and in vivo analysis of transgenic mice have demonstrated that claudins confer barrier properties to the tight junctions (39–44). Claudins are a multigene family comprised of at least 24
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Fig. 2. Blood–retinal barrier is induced by glia and pericytes. Blood vessels are comprised of endothelial cells (E) and pericytes (P) that are contacted by glial processes (G). Together, the glia and pericytes induce the tight junction complex (inset). This highly simplified version of the tight junction emphasizes the transmembrane proteins occludin (longer cytoplasmic carboxy-tail) and claudins that create the barrier. The zonula occludens protein (ZO) connects these transmembrane proteins to the cytoskeleton. Tight junctions are now known to have over 40 proteins, and the contribution of these proteins to retinal endothelial barrier properties and the changes in these proteins that occur in diabetes are just beginning to be addressed. However, occludin phosphorylation state, cellular distribution, and content are all altered in diabetes.
isoforms (45). The claudins are tetraspan transmembrane proteins with two extracellular loops and a short carboxy intracellular tail that interacts with the PDZ-1 domains of ZO-1, -2, and -3 (46). The barrier property of a given tissue depends on the expression pattern of claudins. Claudins interact in both, a homologous and heterologous fashion,
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creating the barrier and simultaneously forming ion specific pores within the barrier (47). Thus, charge-selective paracellular pores across the tight junction may be formed.
The expression pattern of claudins in the retina has only been partially characterized. The expression of Claudin 5 is largely restricted to the vasculature (41) and has been identified in retinal (48) and brain microvessels (49). Gene deletion of Claudin 5 results in permeability to molecules less than 800 Da and neonatal lethality (50). In chick retinal pigment epithelium, expression of Claudins 1, 2, and 5 was demonstrated by embryonic Day 14 at both the protein and mRNA level (51). A comprehensive study of the expression pattern of claudins in the retina and changes in claudin expression in diabetic retinopathy is needed.
Occludin Regulates Barrier Properties
While claudins confer barrier properties to the tight junctions, occludin appears to regulate the cells response to the external signals that control barrier properties. Occludin content correlates well with barrier properties, and is higher in cells with a tighter barrier, such as the retinal endothelial cells of arterioles and capillaries, and lower in cells known to be more permeable, such as venous endothelial cells and endothelial cells of nonneuronal tissues (48, 52, 53). Since occludin was the first transmembrane tight junction protein identified, and a number of expression studies suggested occludin contributed to barrier properties (54, 55) it was thought that this protein was a structural protein in tight junctions.
Studies of occludin knock-out mice changed the view of occludin. Occludin knock-out mice are viable and tight junctions appear normal, as assessed by electron microscopy. Studies of visceral endoderm cells originating from embryonic stem cells lacking occludin support the observations in knockout mice, that occludin is not required to maintain TJ structure (56). However, the occluding-deficient mice do possess a diverse number of abnormalities, including postnatal growth retardation, male infertility, and inability of females to suckle their young, suggesting that this protein has an important regulatory function in several tissues that possess tight junctions. Additionally, salivary gland abnormalities, thinning of compact bone, brain calcium deposits, and hyperplasia of the gastric epithelium are other consequences of occludin gene deletion in mice (57,58). The role of occludin in regulation of epithelial cell division was further supported by the ability of exogenous occludin expression to revert the phenotype of raf transformed cells (59). In studies conducted in our laboratory, antisense RNA to occludin induced the RPE cell line ARPE19 to increase cell division by approximately twofold (manuscript submitted). Therefore, occludin contributes to control of cell division in cells that express tight junctions. Understanding this process is just beginning and a role for occludin in controlling angiogenesis of the blood vessels of the CNS has not been investigated.
Recent studies using small inhibitory (siRNA) to reduce occludin expression reveal a regulatory role for this protein in the cell’s response to changes in the environment as well as a direct contribution to barrier properties. A stable epithelial cell line with occludin gene expression almost completely reduced through siRNA, demonstrated increased permeability to small organic cations, such as ethanolamine and arginine (60). But even more intriguing was the lack of normal response to cholesterol depletion. Cholesterol depletion dramatically reduced the electrical resistance of the cell monolayer