ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized [33]. Thus tight junctions are considered to be a large complex composed of at least 40 known proteins. Within the tight junction proteins, claudins with 20–27 kDa are the most indispensable proteins because they are solely capable of forming tight junction strands. The claudin family consists of 24 members, and, in general, more than two claudin members are expressed in epithelial and endothelial cells. Claudins are tetraspam proteins with a cytoplasmic N-terminus, two extracellular loops, and a C-terminus [30]. They have a PDZ (PSD-95/Dlg/ZO-1) binding motif at their C-terminus which is tethered to a PDZ domain of scaffold proteins such as ZO-1 and ZO-2. Since claudin family is solely able to form tight junction strands, endothelial permeability, in terms of the barrier function, depends on claudin expression.

In the endothelial cell forming of the BBB and/or the BRB, expression of claudin-1, claudin-3, claudin-5, and claudin-12 was identified by immunostaining and Western blot analyses [31]. Despite four isoforms of claudin being expressed in the BBB, claudin- 5-deficient mice died in the first day after birth [34]. Furthermore, claudin-5 is shown to be indispensable for the BBB because claudin-5 functions as a barrier against small molecules. Expression of claudin-5 is regulated by a transcription factor SOX-18 in endothelial cells [35]. Recently VE-cadherin has been shown to upregulate claudin-5 expression by inhibition of transcriptional factor Fox01 [36]. Claudin-5 is phosphorylated at threonine 207 by PKA [37, 38] and Rho-A [39]. Regarding claudin-3, it has been reported that the canonical Wnt signal upregulates claudin-3 expression in cultured mouse brain microvascular endothelial cells, although the signal is very low after birth [40]. On the other hand, the regulation and functions of the other isoforms of the claudin family expressed in the BBB are yet unknown.

MAJOR CYTOKINES DERIVED FROM GLIAL CELLS AFFECTING TIGHT JUNCTIONS OF THE BRB

As major cytokines involved in the pathogenesis of the diabetic retinopathy, TNF- a, IL-1b, VEGF, GDNF, and IL-6 have been identified and characterized as described below. These cytokines were identified within vitreous specimens and their concentrations were significantly elevated in diabetic retinopathy [16–23].

TNF-a

TNF-a, a multifunctional proinflammatory cytokine belonging to the tumor necrosis factor (TNF) superfamily, is mainly secreted by macrophages and binds and functions through its specific receptors TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. Functionally, TNF-a is involved in the regulation of a wide spectrum of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism, and coagulation. TNF-a has also been implicated in a variety of diseases, including autoimmune diseases, insulin resistance, and cancer [41, 42]. In diabetic retinopathy, TNF-a is identified as playing a role in promoting angiogenesis by altering endothelial cell morphology and stimulates mesenchymal cells to generate extracellular matrix proteins [43–45]. The susceptibility to diabetic retinopathy has been associated with the TNF-a gene polymorphism and expression of HLA-DR3 and HLA-DR-4 phenotypes [45]. As a possible

contribution of TNF-a in the pathogenesis of the diabetic retinopathy, TNF-a induces adhesion of leukocytes to vascular endothelium by mediating increased production of adhesion molecules, such as ICAM-1 and platelet endothelial adhesion molecule-1 (PECAM-1) [12–16]. TNF-a is also known to affect the tight junctions between epithelium cells, thus increasing the flow of solutes across the epithelium [46].

IL-1b

IL-1b is a member of the interleukin 1 cytokine family. IL-1b and eight other interleukin 1 family genes form a cytokine gene cluster on chromosome 2. This is produced by activated macrophages as a proprotein, which becomes active through the proteolytic process by caspase 1 (CASP1/ICE). IL-1b is a pivotal mediator of the inflammatory response and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis [47]. Similar to TNF-a, IL-1b also induces ICAM-1- and PECAM-1-induced leukostasis during the initial stage of the diabetic retinopathy [12– 16]. IL-1b, in addition to acting directly, induces VEGF [48], TNF-a [49], and PEG2, and PEG2, in turn, can induce VEGF [50], emphasizing the complex interaction. Thus, TNF-a and IL-1b can increase vascular endothelial permeability.

VEGF

Vascular endothelial growth factor (VEGF) is a hypoxia-induced angiogenic and vasopermeability factor which is mainly involved in the pathogenesis of diabetic retinopathy by playing a role of leukocyte-mediated breakdown of the BRB and retinal neovascularization [51–55]. Based upon an experimental diabetes rat model, retinal VEGF levels increase with associated upregulation of ICAM-1 in retinal endothelia cells and its ligands, the b2-integrins, on the surfaces of peripheral blood neutrophils [56]. These molecular events result in an increased adhesion of leukocytes, predominantly neutrophils, and a concomitant increase in retinal vascular permeability. In experimental models, the intravitreal injection of VEGF in fact induced the retinal vascular changes including retinal leukostasis and concomitant BRB breakdown [57, 58]. In turn, these changes were abolished by the addition of inhibitors of VEGF, ICAM-1, or b2-integrin [59, 60]. In terms of the effects of VEGF on tight junctions of the BRB, in diabetes, several types of advanced glycation end-derivatives (AGEs), which are formed by a nonenzymatic reaction under hyperglycemic conditions, increase the expression of VEGF, and hypoxia induces VEGF expression. These conditions result in the disruption of the BRB, in diabetic retinopathy, because VEGF affects the expression of claudin-5 [61] and occludin [62].

GDNF

Glial cell line–derived neurotrophic factor (GDNF) was originally identified as a neurotrophic differentiation factor for dopaminergic neurons in the central nervous system and retina, and much has subsequently been learned about the neuroprotective effects of GDNF [63]. In a series of studies, we demonstrated that BRB-forming capillary endothelial cells express GDNF family receptor a1, a receptor for GDNF, and that GDNF enhances the barrier function of tight junctions in cultured endothelial