and activates Rho, which is likely involved in cytoskeletal rearrangements conferring the cell’s response. Both of these cholesterol responses are blocked in cells with occludin siRNA. These data suggest that occludin contributes to barrier properties and the ability of the cells to respond to environmental changes in the regulation of barrier properties.

Alterations in Occludin in Diabetic Retinopathy

Occludin in the retinal vasculature undergoes changes in phosphorylation, localization, and content with diabetes, which can be recapitulated in primary endothelial cell culture and VEGF treatment. Streptozotocin-induced diabetes reduces occludin content (61) and immunostaining at the endothelial cell borders (48, 61, 62), while permeability to FITC-labeled albumin increases. This change in occludin content can be observed in primary retinal endothelial cells in response to VEGF treatment (61) and depends on urokinase plasminogen activator, suggesting extracellular proteolytic activity contributes to occludin degradation and permeability (63). Similarly, diabetes reduces occludin content in the brain vasculature (64).

Changes in occludin also occur in the RPE in response to permeabilizing factors. Treatment of RPE cells with hepatocyte growth factor (HGF) reduced tight junctions, decreased TER, and increased diffusion of fluorescently labeled marker from the apical to basolateral membrane. After 6 h of HGF treatment, occludin, Claudin-1, and α-catenin were redistributed from the membrane to the cytoplasm and ZO-1 immunostaining was reduced (65). Interestingly, overexpression of HGF in RPE for 28 days resulted in chronic retinal detachment and retinal inflammation (66).

Phosphorylation of occludin may provide a mechanism by which junctional properties are regulated. Treatment of endothelial cells with VEGF (67, 68), histamine (69), oxidized phospholipids (70), monocyte chemoattractant protein-1 (MCP-1 or CCL2) (71, 72), or shear stress (73) increased both serine/threonine phosphorylation of occludin and permeability. Furthermore, diabetes increases occludin phosphorylation in the rat retina similar to the VEGF-induced increase in BREC (68). Conversely, in epithelial cells occludin phosphorylation may be associated with barrier tightening (74). In these studies, dephosphorylation of occludin through protein phosphatase 2A and 1 are associated with promotion of barrier properties. These discrepancies may represent differences in the systems used or differences in the sites phosphorylated. Clearly, site identification and mutational analysis is necessary to pursue the functional meaning of occludin phosphorylation.

VE-CADHERIN AND DIABETIC RETINOPATHY

This chapter has focused on the tight junction complex, which is essential for the well-developed barrier properties of the BRB. However, adherens junction proteins also contribute to the barrier properties of the BRB. As mentioned earlier, the tight junction and adherens junction complex are not readily discernable at the ultrastructural level, as viewed by electron microscopy. However, vascular endothelial cadherin (VE-cadherin or cadherin 5) is a vascular constrained transmembrane protein of the adherens junction and is expressed in the retinal vasculature (75). Injection of antibodies to VE-cadherin increases retinal permeability in vivo and causes primary retinal endothelial cells to

separate in culture. Further, diabetes causes a reduction in VE-cadherin protein through matrix metalloprotease activity contributing to changes in permeability (76). Also, in vascular endothelial cells without tight junctions, VEGF stimulation leads to VE-cadherin phosphorylation and endocytosis that controls endothelial permeability (77). The tight junction complex and adherens junction may be viewed as resistors that act in series, each with distinct barrier properties. Diabetes alters both these complexes, contributing to increased permeability. The system is made more complex since there is a clear interaction of the two junctional complexes as demonstrated by the interaction of ZO-1 first with the adherens junction, from which point ZO-1 then organizes polymerization of the claudins of the tight junctions (36).

PERMEABILITY IN DIABETIC RETINOPATHY

Diabetic retinopathy is characterized by increased vascular permeability as can be observed by fluorescein angiography. This change in permeability occurs during the early stages of the onset of diabetic retinopathy. Table 1 is a compilation of studies demonstrating that this change in vascular permeability can be recapitulated in rodent models of diabetes. There are limited studies that have failed to observe a difference in accumulation of marker in rodent models in diabetes but the majority of studies have observed an increase of 1.5- to 4.5-fold. This accumulation can be due to either increased flux into the retina, or decreased flux out of the retina. Further, passive flux is controlled by hydrostatic and oncotic pressure, as well as changes in vascular permeability (for a detailed discussion of fluid and solute movement across a vascular bed see (78) ). Thus, changes in marker accumulation may be the result of a variety of vascular changes other than permeability alone. Indeed, increased solute accumulation in the retina elevates the tissue oncotic pressure, retaining fluid and likely contributing to macular edema. However, direct changes in endothelial cell culture permeability demonstrate a similar response to vascular endothelial growth factor addition (79, 80) suggesting the in vivo response is, at least in part, due to increased vessel permeability. A recent longitudinal study demonstrates the rate of diabetic retinopathy at 14 years is still over 70% (81), and while the rate of proliferative retinopathy is on the decline, the rate of macular edema is on the rise (82).

It should be emphasized that rodents lack a macula, and thus cannot completely model macular edema, which in humans is closely correlated with loss of vision (83). However, the changes in vessel permeability observed in rodents and in the early stages of retinopathy in humans may be a first step toward macular edema. This altered barrier may set the stage for an ensuing event that leads to focal edema or hemorrhaging of retinal vessels. Much of the current research has focused on this change in vessel permeability. Future studies will need to address the issue as to the causes for progression from general leaky blood vessels, as observed by fluorescein angiography, to focal edema associated with vision loss. A recent study using mass spectrometry has identified carbonic anhydrase I from red blood cells in the vitreous of patients with hemorrhagic retinopathy, and further demonstrated that carbonic anhydrase stimulates the kallikrein pathway leading to bradykinin-induced endothelial permeability (84). Thus, once established, hemorrhaging vessels further drive vascular permeability.

NEUROINFLAMMATION IS A CENTRAL PROCESS IN THE

PATHOLOGY OF DIABETIC RETINOPATHY

The well-established elevation of vascular endothelial growth factor (85) may be part of a larger neuroinflammation in diabetic retinopathy. Neuroinflammation has been assigned various definitions; however, for the purpose of this chapter, neuroinflammation is defined as elevated expression of cytokines and chemokines, microglial activation, and

leukostasis without necessarily including infiltration. Neuroinflammation has been identified as a component of a variety of neurodegenerative diseases. For example, elevated IL-1 has been found in ischemia, stroke, and in chronic CNS diseases such as Alzheimer’s, Down’s syndrome, multiple sclerosis, Parkinson’s, HIV-associated dementia, amyotrophic lateral sclerosis, and epilepsy (reviewed in (86) ). Further, IL-18 has been identified in ischemic stroke, multiple sclerosis and axonal injury (reviewed in (87) ). Both of these interleukins are capable of generating a cascade of downstream responses similar to that observed in diabetic retinopathy. This inflammatory cascade includes elevated cytokines and chemokines such as MCP, a permeabilizing chemokine, and VEGF, induction of adhesion molecules such as PECAM and ICAM in endothelium and consequent leukostasis, adhesion of monocytes and polymorphonuclear cells leading to respiratory burst and generation of reactive oxygen species. Furthermore, the inflammatory response may include elevated FasL contributing to endothelial apoptosis (88).

A number of studies support the hypothesis that diabetic retinopathy includes neuroinflammation. In a study of 543 Type I diabetic patients, elevated IL-6, TNF, and C-reactive peptide individually or combined was associated with retinopathy and cardiovascular disease, as was an association with elevated HbA1c, LDL, triglycerides, and blood pressure (89). Indeed, these inflammatory markers were specifically associated with nonproliferative diabetic retinopathy (NPDR). Another study of 93 patients revealed that levels of RANTES (chemokine CCL5) and SDF-1 (CXCL12) were elevated in serum associated with severe NPDR compared to nonsevere NPDR (90). Immunocytochemistry of one retina revealed RANTES, MCP-1 (CCL2), and ICAM were elevated. IL-1β and TNFα were increased in the serum and vitreous of patients with proliferative diabetic retinopathy (91). Antibodies associated with heat shock protein 65 are elevated in Type 1 diabetes with retinopathy recapitulating an increase in serum levels of this inflammatory marker in artherosclerotic disease (92). Together, these studies provide compelling evidence that inflammatory markers, and in particular IL-1β, is elevated in the serum and vitreous of patients with proliferative and nonproliferative diabetic retinopathy.

Abundant data in animals further support a role for neuroinflammation in diabetic retinopathy. IL-1β protein was elevated in the retina of diabetic animals at 2 months (93, 94). Gerhardinger et al. demonstrated that IL-1β mRNA was elevated in 6-month diabetic animals and by performing microchip array analysis of Muller cells isolated from control and diabetic retinas identified a series of acute phase and inflammatory markers that are responsive to IL-1 (95). A series of elegant experiments demonstrated that leukostasis increases during streptozotocin-induced diabetes in mouse and rat models, and that deletion of the gene for the endothelial protein ICAM or its leukocyte binding partner CD18 ameliorated leukostasis after 11 months of diabetes and rectified vascular lesions in a galactosemic model (96). Furthermore, in a 1-week model of diabetes, vascular permeability, leukostasis, CD18 and ICAM expression as well as NF-κB activation were all normalized by high-dose aspirin, or the COX-2 inhibitor, meloxicam, and by a soluble TNF receptor/Fc hybrid (entanercept), suggesting TNF and COX contribute to diabetic retinopathy (97). Together, these animal models demonstrate a role for neuroinflammation in diabetic retinopathy and that interfering with this inflammatory response can alter the disease.