Ординатура / Офтальмология / Английские материалы / Retinal Vascular Disease_Joussen, Gardner, Kirchhof_2007

8Blood Retinal Barrier

8.1Blood-Retinal Barrier, Retinal Vascular Leakage and Macular Edema

B.E. Phillips, D.A. Antonetti

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Core Messages

The blood-retinal barrier forms a selective barrier, restricting ion, substrate, and water permeability, allowing for proper neuronal function

The blood-retinal barrier has a number of specialized characteristics that allow for barrier formation

Vascular endothelial cells and retinal pigmented epithelial cells physically form the bloodretinal barrier

The tight junction protein complex is essential in establishing the blood-retinal barrier Diabetic retinopathy is characterized by increases in vascular permeability

Diabetic retinopathy causes changes in growth factors and cytokines which modify tight junction protein content

8.1.1 Introduction

Proper retinal function requires the presence of a well-defined blood-retinal barrier (BRB). In many of the leading causes of medical blindness this BRB is compromised. Indeed, retinopathy of prematurity and age related macular degeneration both include production of aberrant vessels with poor barrier properties. Further, diabetic retinopathy, the leading cause of blindness in working age adults, involves progressive vision loss and is closely associated with macular edema [117]. Increased fluid accumulation, as well as lipid and albumin deposits, is believed to be the result of the breakdown of the BRB that normally controls the neuronal environment. Barrier dysfunction results in increased permeability which diagnostically indicates progressive retinopathy [32]. This chapter will review the normal physiology of the BRB, the changes that occur through the course of diabetic retinopathy, and the known underlying molecular mechanisms that may lead to barrier dysfunction. Elucidating the mechanisms of barrier dysfunction in diabetic retinopathy will further our understanding of its pathogenesis and provide future therapeutic targets.

8.1.2Characteristics of the Blood-Retinal Barrier

Essentials

The BRB comprises vascular endothelial and retinal pigmented epithelial cells

Mechanisms of permeability across endothelial and epithelial barriers

Facilitated water and substrate transport Tight junction formation

8.1.2.1 Blood-Retinal Barrier Physiology

The BRB, similar to the blood-brain barrier (BBB), is a physiologic barrier that regulates ion, protein, and water flux into the retina. This barrier allows the neural retina to establish and maintain specific substrate and ion concentrations allowing for proper neuronal function. Additionally, it regulates infiltration of immune competent cells, blood-born toxins, and various hormones that could negatively affect neuronal function and survival. The barrier is physically established by two cell types: vascular endothelial cells that form capillary beds in the ganglion cell layer and outer plexiform layer of the retina and retinal pigmented epithelial cells (RPE) that form a barrier between the choroid capillary plexus and the retina [102, 86]. Combined, these cells regulate the flux of nutrients into the retina from the blood supply.

8.1.2.3 Endocytosis and Facilitated Diffusion

Cells continuously take up and sample extracellular material in a process called pinocytosis (Fig. 8.1.1). Membrane invaginations across the cell surface pinch off forming vesicles that move to the cell interior. These vesicles can move internally to be degraded or through the cell to be released at the basal-lateral membrane leading to non-specific transport of materials across the cell. Pinocytotic vesicles are selectively decreased in endothelial cells located in the BBB [16, 27, 149]. In order to maintain transport

of necessary substrates for neuronal function, specific receptor-facilitated transport mechanisms are used to move materials across the BBB. Receptor mediated endocytosis may occur through clathrin or caveolin mediated endocytosis, which is ATP dependent [189]. A receptor binds to its specific substrate concentrating the substrate within the invagination to reduce transport of non-specific materials (Fig. 8.1.1). The transferrin receptor and the transport of iron across the BBB is an important example of receptor mediated transport across vascular endothelium [20]. Vascular endothelial cells of the BBB have increased mitochondria content, which generates a cell’s energy supply of ATP [126]. Elevated mitochondria levels may be necessary to supply numerous receptor-mediated transport mechanisms.

Channel-facilitated transport is a mechanism for the diffusion of specific substrates across the BBB. This is mediated by transmembrane proteins located in the cell surface that allow for the flux of specific materials across the cell membrane. The GLUT-1 glucose transporter is highly and selectively ex pressed in vascular endothelial cells of the BBB and supplies the neuronal tissue with necessary glucose [128]. Together these mechanisms provide the necessary substrates for neuronal function.

8.1.2.4 Water Transport

Facilitated transport of water out of the neuronal extracellular space is essential to maintain retinal function. RPE cells regulate water content and lactic acid removal generated by high metabolic rates in the retina. RPE cells transport water out of the retina and into the choroid capillary plexus. The force gen-

RBC

Proteins

Vesicle

Tight Junction

YY

Fig. 8.1.1. A side view of two adjacent vascular endothelial cells is depicted. Paracellular flux of blood-born proteins between the cells is inhibited by the tight junction complex. Non-specific transport of materials through the cell is pinocytosis. Pinocytotic vesicles are greatly reduced in barrier forming cells. To compensate for reduced permeability, receptor mediated transcytosis allows for specific substrate transport across the barrier

erated by water flux helps to maintain retinal attachment [153]. Lactic acid and water transport across the RPE are closely linked. The movement of water out of the retina is accomplished through the use of a number of ion channels and a specific water channel, aquaporin-1 [153, 158]. Aquaporin-1 is a transmembrane protein found in water permeable tissues that forms a transcellular channel for water transport [1, 123, 153]. Water transport is dependent on the pumping of Cl– and K+ across the RPE basolateral membrane into the choroid capillary plexus [18, 107 – 109, 140]. The flow of these ions is driven by the apical Na+/K+ATPase pump that elevates internal K+ levels allowing the subsequent flux of Cl–. In addition, lactic acid is taken up by the RPE from the neuronal extracellular space by the monocarboxylate trans- porter-1 and then transported from the RPE cell interior to the choroid capillary plexus by monocarboxylate transporter-3 [15, 130, 194]. Increased acidification of the RPE cell due to lactic acid buildup activates the Cl– and K+ pumps on the apical membrane, with subsequent diffusion across the basolateral membrane that ultimately drives water flux through aquaporin channels. The reuptake of Cl– from the choroid capillary plexus by HCO3–/Cl– exchange pumps is activated with extracellular acidification [158]. In summary, elevated retinal metabolic rates increase lactic acid production. The RPE responds to this elevated metabolism by increasing lactate transport into RPE cells, which increases water transport from the retina to the choroid capillary plexus allowing for compensatory removal of by-products from the retina. This regulation of transcellular transport requires precise control of paracellular flux between the two compartments, which is achieved by well-developed tight junctions.

8.1.2.5 Tight Junctions

The tight junctions are a protein complex that forms a continuous belt around the circumference of the cell in order to regulate paracellular flux of solutes and water (Fig. 8.1.1). The tight junction can be observed with electron microscopy as points of increased protein density and close apposition between adjacent cell membranes (Fig. 8.1.2) [48, 19]. The distance between the cell membranes is spanned by strands comprising transmembrane proteins [143], which will be explored later in this chapter. Tight junctions can be found in epithelial cell layers such as the RPE cells, and in the vascular endothelial cells of the BBB and BRB, both of which have well-developed tight junction complexes uncharacteristic of peripheral vasculature [116, 120, 177]. Rat models demonstrate occludin protein expression, a transmembrane tight junction protein, can be ini-

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Fig. 8.1.2. An electron micrograph of two vascular endothelial cells. The black arrows point out the tight junction complex between the two cell membranes

tially observed in BBB vascular endothelial cells 1 week postnatally, which correlates with the maturation of the BBB [73]. Developmentally the BBB vascular endothelial cells and the RPE show increased expression of TJ proteins over time that correlates with decreases in permeability and the establishment of the BBB and BRB [40, 73, 134, 139, 155, 183, 185].

8.1.2.6 P-Glycoprotein

The BBB and BRB selectively transport soluble substrates and water across the barrier, but there are a number of lipophilic molecules that can freely diffuse across the cell membrane. The entry and accumulation of these potentially harmful compounds is restricted by multidrug-resistance proteins, including P-glycoproteins, by actively pumping specific lipophilic molecules from the cell back into the blood stream in an ATP dependent manner [96, 138]. A P- glycoprotein homozygous knock-out mouse possesses greatly elevated levels of lipophilic tracers in the brain [145, 146]. P-glycoproteins are located in BBB endothelial cells as well as a large number of different epithelial tissues including the liver, kidney, intestine, pancreas, and brain choroid plexus [14, 133]. Recently, P-glycoprotein has been shown to be expressed in retinal vascular endothelial cells, suggesting a role in the BRB [100]. Together, the tight junctions and multi-drug resistance proteins form an effective balance that reduces permeability but allows passage of specific substrates required for proper neuronal function.

Fig. 8.1.3. A fundus photograph from a patient with non-prolif- erative diabetic retinopathy displaying vascular leakage. The red spots are vascular hemorrhages. The black arrow points out a lipoprotein plaque located in the macula of the eye. The buildup of lipoprotein exudates leads to macula edema, negatively affecting vision

reous fluorometry measurements, occurs in diabetic patients and diagnostically indicates progressive retinopathy [32].

While solute flux is most often measured as indicator of permeability, an increase in water permeability also occurs in diabetic retinopathy causing macular edema [166, 172, 176]. Macular edema is an increase in the thickness of the central macular region due to water accumulation and is associated with vision loss [31]. The net movement of water and solutes out of capillaries is governed by Strling’s law and subsequent adaptations reviewed by Salathe [35, 142]. Starling’s law states the net movement of fluid out of capillaries is determined by the sum of hydrostatic and oncotic pressures, predominantly intralumenal hydrostatic and oncotic pressure. The flux of solutes is controlled by both diffusive flux and convective forces generated as the fluid travels across the endothelial barrier. Permeability of the endothelial wall to paracellular flux of water and solutes is governed by the junctional complex. Therefore, changes in accumulation of water and solutes in the retina may be due to changes in wall permeability or may be caused by changes in hydrostatic or oncotic pressure and these parameters should be considered in the interpretation of experimental results.

8.1.3.2 Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) contributes to a number of phenotypic changes seen with diabetic retinopathy. Diabetic animal models and humans with diabetes have elevated levels of VEGF in the vitreous of the retina [3, 4, 99, 119]. VEGF was originally identified as a potent permeabilizing agent on endothelial cells [150] and increases both solute and water flux in endothelial cell culture [38] and solute flux in the retina [9]. VEGF also plays a role in angiogenesis and is associated with neovascularization in diabetic retinopathy [83, 99, 147].

The cause of elevated VEGF in diabetic retinopathy has not been rigorously demonstrated but may be due to retinal ischemia after capillary non-perfu- sion and occlusion [2, 51, 147] or may be due to activation of an inflammatory response possibly through activation of the receptor for advanced glycation end products (AGEs) [156]. The exact mechanism that leads to capillary occlusion is not clear, but a number of observations have been made. Intercellular adhesion molecule-1 (ICAM-1) expression is elevated in diabetes [98], and allows for increased leukostasis [113]. Leukostasis is the first step of diapedesis but while no evidence exists for leukocyte infiltration in diabetic retinopathy, adhesion of leukocytes to endothelial cells may prevent normal blood flow. Leukostasis increases with diabetic reti-

8.1 Blood-Retinal Barrier, Retinal Vascular Leakage and Macular Edema

nopathy [82, 112] and inhibition of ICAM-1 has been shown to block leukostasis and improve retinopathy prognosis [79, 113]. Vasoconstriction of the vessels may further facilitate the ability of leukostasis to reduce capillary blood flow. A VEGF-induced model of retinopathy showed that VEGF alone leads to vessel narrowing [75]. Additionally, the vasoconstrictor endothelin-1 is upregulated in diabetic retinopathy and is associated with decreased blood flow [23, 163, 193]. Recently, it has been demonstrated that the inhibition of the endothelin-1 receptor blocks increases in VEGF, ICAM, and leukostasis [103]. The involvement of VEGF is further complicated by the number of VEGF and VEGF receptor isoforms and their different interactions [83]. Understanding VEGF’s multiple roles will allow for better pharmacological techniques of treatment.

8.1.3.3 Apoptosis and Cell Loss

One of the early changes of diabetic retinopathy is the loss of pericytes and endothelial cells. Pericytes are smooth muscle cells that form a sheath around vascular endothelial cells. Pericytes direct vascular development by prohibiting endothelial growth, allowing for vessel stabilization, and providing necessary factors for angiogenesis [29, 53, 74, 121]. Genetic and pharmacological studies that prevent proper pericyte interaction have demonstrated increased endothelial cell growth and abnormal vascular development under these conditions [95, 168]. These pathological changes may provide a strong

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um size. Finally, large pores formed by cell division 8 I or cell death may also contribute to paracellular flux [65]. Application of this model to diabetic retinopathy would implicate VEGF increasing both the number of medium and large pores. The medium pores are increased due to tight junction disassembly, and the large pores are increased due to the number of dividing endothelial cells in proliferative diabetic retinopathy. The large pores are further increased with endothelial cell drop out due to apoptosis. The existence of multiple changing pores that lead to increased permeability may require alternate approaches for

therapy.

8.1.4The Structure and Role of the Tight Junctions

Essentials

Cell polarity is necessary for assembly

Occludin and claudins create the barrier Changes in junctional proteins alter barrier properties

8.1.4.1 Tight Junction

The tight junction protein complex allows for the establishment of the BRB, restricting paracellular diffusion of blood-born compounds into the neuronal tissues. Understanding the normal function of the tight junction and the pathological changes that lead to increased permeability is necessary to understand disease progression. This section will cover in detail the establishment and composition of the tight junction complex and the alterations that occur in diabetes.

8.1.4.2 Polarity and Tight Junction Assembly

Establishment of cell polarity is the first step in development of the tissue barrier. The endothelial cells of the vascular retina and RPE develop distinct apical versus basal lateral surfaces. This development of cell polarity orients the cell with its surroundings and is an essential component of development, asymmetrical cell division, cell migration, and the formation of asymmetrical cell morphology [45, 61, 84]. Polarity initiation is an essential process for the formation of the cell barrier. It organizes the cytoskeleton, junctional complexes, and apical/basal cell membrane proteins in a cell tissue layer [22, 76, 122, 151]. Epithelial cell polarity has been more widely studied than endothelial polarity. The initiating step in cell polarization is contact with a basement membrane or an adjacent cell [122]. The cell

then begins to establish junctional complexes. The first junction to form is a primitive spot like junction that consists of adherene junction proteins [54, 55, 114, 160]. The protein zonula occludens-1 (ZO-1) in the spot like junctions then recruits junction adhesion molecule (JAM) and additional tight junction proteins [54, 55]. These proteins are recruited apically above the adherene junction, and go on to form the tight junction, which is the most apical junction formed between mammalian cells [114]. The transition from a spot like junction to a continuous adherens and tight junction requires the Par6/ Par3/atypical PKC (aPKC) polarity complex [160]. Par6 (Partioning defect protein 6) was first identified through mutational analysis in C. elegans as being required for cell polarity [66]. Genetic studies of Drosophila and mammalian epithelial cells show a loss of cell polarization and tight junction formation with Par6 mutations [129, 192]. Similarly, a non-function- al kinase dead aPKC caused Par-6, TJ, and apical/basal protein mislocalization preventing barrier formation [161]. The role of the Par6/Par3/aPKC complex in tight junction formation and maintenance has only begun to be studied and still requires additional investigation, particularly as it relates to endothelial junction formation.

8.1.4.3 Tight Junction Structure

The tight junction complex contains at least 40 proteins comprising trans-membrane and internal adapter proteins that regulate paracellular flux [62]. Transmembrane proteins that make up the tight junction are occludin, claudins, and JAMs. Occludin was the first discovered transmembrane protein of the tight junction [57]. Occludin is a 65-kDa transmembrane protein that spans the membrane four times providing two extracellular loops that are able to bind to the occludin molecule on the adjacent cell. Changes in occludin content correlate with vascular permeability making it a likely candidate in regulating the opening and closing of the tight junction [6, 73, 111, 165]. Claudins consist of at least 24 genes that are differentially expressed in various tissues. Similar to occludin, claudins span the membrane 4 times and are able to bind to other claudins and occludin on the adjacent cell. Claudins regulate small charged molecule and ion permeability [124, 167, 171, 181]. The expression of specific claudins confers the charge permeability properties displayed by that tissue. This principle has been elegantly demonstrated by point mutants of claudin 15 that reverse its charge selectivity [26].

JAM is part of a family of proteins, with JAM-A specifically localized to the tight junction [101]. JAM has a single extracellular domain and associates with

a number of adhesion molecules. JAM is specifically involved in tight junction assembly and leukostasis, and may play a number of other roles involving the tight junction [12, 33, 42, 127].

Numerous adapter proteins localize just below the membrane and act as tight junction organizers and cytoskeleton anchors. The first tight junction protein identified was zonula occludens-1 (ZO-1), later joined by ZO-2 and ZO-3 [64, 71, 154]. These proteins are part of a protein family called membraneassociated guanylate kinases (MAGUK), which consist of an SH3 domain, PDZ domains, and a guanylate kinase GuK binding domain [46]. ZO-1 acts as a molecular scaffold that organizes, assembles, and links the tight junction to the cytoskeleton through a number of protein-protein interactions (Fig. 8.1.5) [47, 54, 55, 184]. ZO-1 binds to ZO-3, which then interacts with the polarity complex protein termed proteins associated with Lin7 (Pals-1), Pals1-associ- ated tight junction protein (PATJ), and crumbs-3 (CRB-3) [136]. Pals-1, also containing PDZ domains, interacts with the Par6/Par3/aPKC complex [76], thus connecting these polarity complexes. Changes in the Pals-1 and PATJ expression interfere with establishment of polarity similarly to reduced Par6 content and aPKC activity [151, 135]. ZO-1 also interacts with AF-6, which is negatively regulated by Ras activation [91, 191]. Constitutive Ras activation has been shown to downregulate tight junction protein

expression [24]. In conclusion, formation of the bar-

rier first requires establishment of cell polarity. Cell I 8 culture models and genetic studies have so far uncovered two complexes apparently necessary for recruitment of the tight junction complex. The first,

Par3, Par6, aPKC, and the second, Pals-1, PATJ, and CRB-3, are connected to the ZO-1 tight junction scaffolding proteins. Ultimately, recruitment of the transmembrane tight junction proteins occludin, claudins and JAM establishes the interaction with adjacent cells and confers barrier properties. Elucidating this complex interaction in the retina and brain will likely unveil unique mechanisms specific to the establishment of the blood-neural barrier.

8.1.4.4 Occludin

The majority of known diabetic changes in the tight junction complex involve the protein occludin. Occludin is of particular interest due to correlation between changes in content and permeability. Diabetic animal models have a 35 % decrease in occludin content of the retina with a 62 % increase in vascular permeability [6]. Changes in occludin content may be due to elevated levels of VEGF and hepatic growth factor (HGF) in the vitreous of diabetic patients [3, 80, 110, 152]. VEGF and HGF administration in vitro increased permeability, reduced occludin protein content, and led to tight junction complex internali-

Fig. 8.1.5. A top down view of two adjacent cells displaying tight junction protein interactions. Transmembrane proteins occludin, claudin, and JAM on adjacent cells bind to either partner on the adjacent cell forming the tight junctional strands. Internal scaffolding proteins containing multiple PDZ domains organize transmembrane and polarity complex proteins allowing for junction formation. The tight junction complex is connected to the cytoskeleton through ZO-1 bridging actin and occludin. Occludin binds the guanylate kinase (GuK) binding domain on ZO-1

Occludin

JAM-A

Claudin

Actin

GUK

zation in RPE, BBB, and BRB vascular endothelial 8 I cells [25, 77, 78, 179]. Similar increases in retinal vascular permeability and occludin internalization were observed in rat models injected with VEGF [5, 6, 9, 10], and blocked by VEGF receptor inhibitors [132]. Changes in tight junction organization may be regulated by occludin phosphorylation, which is stimulated by VEGF, shear stress, and histamine [5, 39, 72] and closely correlates with regulation of barrier properties. This phosphorylation of occludin is regulated by PKC, which may explain the mechanism of action of PKC inhibitors in regulating vascular permeability [70]. Diabetes has also been characterized as a chronic low grade inflammatory state [30, 41, 50, 52, 58, 60, 67, 131], and cytokines increase permeability and disrupt tight junctions in both endotheli-

al and epithelial cells [178].

Techniques to modify occludin expression and binding properties have been used to better understand its role in the tight junction. Glucocorticoid treatment increases the tight junction gene expression of occludin in primary retinal vascular endothelial cells leading to decreased solute and water permeability [7, 137]. Glucocorticoids also increase transelectrical epithelial resistance (TER) by de novo protein synthesis [196]. Similarly, direct overexpression of full length and COOH truncated occludin led to an increase in TER [8, 105]. Paradoxically, small molecule paracellular flux increased over time [105]. It was hypothesized that occludin acts as a seal when interacting with the tight junction protein ZO-1, but as occludin’s expression outpaced ZO-1, the lack of protein-protein interaction produced more pores in the tight junction [105]. Conversely, overexpression of occludin in fibroblasts, in low calcium media to prevent adherens junction formation, conferred adhesion properties [170]. The role of occludin in regulating barrier properties was further demonstrated in raf1 transformed epithelial cells, which express reduced occludin content and possess poor cell adhesion properties. Loss of tight junction, cell polarity, and control of cell growth are common in epithelial cell cancers [180]. Reintroduction of wildtype occludin or the second extracellular loop of occludin alone in raf-1 transformed cells reestablished wild-type cell adhesion and prevented tumor formation [180]. These data suggest occludin not only confers adhesive properties but also contributes to organization of the tight junction complex.

Studies have also used reduction or ablation of occludin expression to study the role of occludin. A number of studies used small peptides to bind regions of the first or second extracellular loop of occludin in an attempt to impair its function. The studies did not reach a consensus on the function of individual loops, but binding peptides to either loop

decreases TER, increases permeability, and disrupts localization of other tight junction proteins [92, 125, 164, 173, 188]. The discrepancies between peptide experiments are most likely due to the difference in the occludin protein sequence used from varying species. Transgenic mice that lack occludin were generated and the offspring were viable. Electron microscopy revealed tight junction formation occurring in the absence of occludin, and TER of intestinal epithelium were normal. However, the mice displayed a number of aberrant phenotypes including sterility in males, testicular atrophy, females unable to suckle, chronic inflammation of gastric epithelium, calcification of the brain, and reduced bone densities [141]. Acid secretion in the gastric corpus mucosa was later shown to be reduced due to the loss of parietal cells and hyperplasia of mucus cells [148]. These are all regions possessing endothelial or epithelial tight junction barriers suggesting a dysfunction in tight junctions despite their normal appearance by electron microscopy.

While these data point to a role for occludin in establishing barrier properties, recent data highlights the complexity of the tight junction. Tissue culture models reveal no change in epithelial TER or junctional assembly after occludin ablation [106, 195]. Stable MDCK cell lines lacking occludin expression had normal diffusive permeability to 3- and 10-kDa dextran, but demonstrated an increase in small organic cation and mannitol permeability [195]. Interpretation of these permeability changes is complicated by increased expression of claudins 3 and 4 and a decrease in claudin 1 and 7 [195]. Interestingly, occludin ablated cells failed to stimulate exchange of GTP on Rho in response to cholesterol depletion [195]. Cholesterol depletion has been shown to cause a rearrangement of the cytoskeleton, and Rho-GTP is involved in cytoskeleton remodeling [17]. Inhibition of cytoskeleton rearrangement may explain why occludin depleted cells were unable to release apoptotic cells from the cell monolayer [195]. This body of research on occludin demonstrates a role for occludin in barrier function but highlights the broad nature of occludin function and the need for continuing research to fully understand its complex role in cell biology.

8.1.4.5 Claudins

The claudin tight junction protein family regulate small charged molecule and ion permeability [124, 167, 171, 181]. Different complements of claudins are expressed in various tissues, establishing their cation or anion selectivity. It has been shown that by changing the expression profile of claudins within a tissue, its ion charge permeability preferences can be

changed [171]. Claudin expression changes have been linked to a number of pathologies that display a loss of barrier function [104, 186]. Mutations in clau- din-16 are the cause of familial hypomagnesemia with hypocalciuria and nephrocalcinosis. Loss of claudin-16 prevents reabsorption of Mg2+ and Ca2+ in the kidney’s thick ascending loop of Henle resulting in eventual kidney failure [81]. Claudin-3 is lost with autoimmune encephalomyelitis in the brain and spinal cord [187]. Claudin-4 expression is reduced in intestinal epithelium with collagenous colitis, preventing intestinal water absorption resulting in diarrhea [21]. Loss of claudin-14 has been linked to deafness in human and mouse genetic analysis [13, 182]. Transgenic mice lacking claudin-1 expression die after birth due to transepidermal water loss [56]. Claudin-5 is an endothelial specific form of claudin and is found in the BBB [181] and BRB [9]. Transgenic mice that lack claudin-5 were only able to survive for up to 10 h after birth [124]. Like the occludin transgenic mice, electron microscopy of these mice showed normal appearing tight junctions. However, unlike the occludin ablated mice, the claudin-5 mice had increased permeability of molecular markers smaller than 800 Da. These markers freely diffused across the endothelial blood vessels in the brain [124]. Thus, claudins have been established as providing a clear role for barrier properties through transgenic animals. However, their role in BRB and the changes that may occur in leading to macular edema are not well described.

8.1.4.6 Zonula Occludens

The ZO-1, ZO-2, and ZO-3 proteins are part of a protein family called membrane-associated guanylate kinases (MAGUK) which consist of an SH3 domain, PDZ domains, and a guanylate kinase (GuK) binding domain [46]. ZO-1 acts as a molecular scaffold that organizes and assembles the tight junction [47, 54, 55, 184]. Tissue culture models with depleted ZO-1 protein have tight junctions indistinguishable from wild-type cells expressing ZO-1, similarly to occludin depletion models [106, 141, 169]. A Ca2+ switch assay reveals a delay in tight junction formation in cells lacking ZO-1 with no effect on the adherens junction assembly [106, 169]. Tight junction assembly delay was rescued when the SH3 domain or full length of ZO-1 was reintroduced into the cell [106]. Ablation of ZO-1 caused an increase in ZO-2 and a decrease in cingulin expression, both of which are binding partners of ZO-1 [169]. Cingulin is a scaffolding protein that is believed to mediate tight junction protein recruitment through interactions with ZO-1, ZO-2, JAM, and the cytoskeleton [12, 28, 34]. Direct ablation of either of these proteins had no

effect on tight junction assembly [63, 106]. The TER

and inulin transport of the ZO-1 ablated cells were I 8 identical to wild-type levels after tight junction formation [106].

8.1.5 Summary

This chapter has reviewed the physiologic components of the BRB that establish its ability to selectively restrict substrate permeability into the neuronal tissue. Tight junction formation and maintenance in the vascular endothelial and RPE cells is a required element in barrier formation. Specific tight junction protein components are changed in a large number of pathologies with disrupted barriers, including diabetic retinopathy. The elevation of VEGF in diabetes leads to a reduction in tight junction protein content, increased permeability, and angiogenesis. The ability of glucorticoids to impede VEGF induced changes makes this class of drugs an attractive possible therapeutic, along with other methods of VEGF inhibition. Continued research and understanding of the mechanisms of BRB formation and breakdown in disease pathology will enable development of treatments for diabetic retinopathy and other eye diseases that involve macular edema.

Acknowledgements. I would like to acknowledge Timothy Bennett for the contribution of the picture seen in Fig. 8.1.3 (unpublished data).

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