Key Words: Blood–retinal barrier (BRB); blood–brain barrier (BBB); claudin; occluding; permeability; tight junctions.

FORMATION OF THE BLOOD–RETINAL BARRIER

The central nervous system (CNS), including the brain and retina, require the development of a blood–neural barrier for proper neuronal function. In the retina this barrier includes the retinal pigment epithelium (RPE) and the vascular network that creates the capillary plexus in the ganglion cell layer and a deeper plexus extending from the inner plexiform layer through the inner nuclear layer to the outer plexiform layer (1). The blood–brain and blood–retinal barrier (BRB) provide tight control of the nutrients, metabolic intermediates and fluid that enter the neural parenchyma and is often compromised in disease states. Formation of the blood–brain and BRBs requires a complex interaction of multiple cell types including the endothelial cells of the blood vessels, astrocytes, Müller cells, and pericytes.

Glia-Endothelial Interaction

Blood vessels in the CNS differentiate to form the BBB and BRB through signaling from the neural environment. One of the first demonstrations of the ability of neural tissue to induce formation of the BBB was achieved by Stewart and Wiley in 1981. The authors found that by transplanting the avascular neural tissue of Stage 13 quail brains into the coelomic cavity of 3-day chick embryos, the invading capillaries took on BBB characteristics (2). Namely, the invading capillaries were able to exclude circulating trypan blue, tight junctions were increased as observed by electron microscopy, pinocytotic vesicles decreased, mitochondrial number increased to a density equal to that in the endothelium of neural capillaries, and two BBB markers, alkaline phosphatase and butyryl cholinesterase were elevated. However, somites grafted to the brain did not induce BBB of invading capillaries. These historic studies demonstrate that a component of neural tissue induces the formation of the BBB, supporting the theory that the capillary environment is critical for induction of capillary differentiation and development of the appropriate barrier properties. A similar experimental paradigm was utilized by Janzer et al. to reveal that astrocytes are capable of induction of the blood–brain/blood–retinal barriers (3). Injection of astrocytes into the anterior chamber of the rat eye induced barrier properties as determined by reduced flux of the albumin-binding dye, Evans blue, in the vessels that invaded the astrocyte aggregates, as well as the iris proper. The astrocytes were also capable of inducing barrier properties in the vessels of chick chorioallantoic membranes. These studies support a role of astrocytes in the regulation of endothelial cell differentiation to the BRB and BRB. In the light of these experiments, it is interesting to note that while a number of epithelial cell lines develop strong ionic, solute, and fluid barriers such as the transformed retinal pigment epithelium cell line, ARPE19, there are no endothelial cell lines that reflect the very tight barrier of the BBB or BRB. However, a number of external signals such as astrocyte-conditioned media, angiopoietin 1, or glucocorticoids, can induce barrier properties in isolated retinal, and brain endothelial cells.

In vitro experiments provide evidence that glia directly contribute to endothelial differentiation of the BBB and BRB. These in vitro models were pioneered by Rubin

et al., who demonstrated that astrocyte-conditioned media, along with cAMP analogues, stimulate barrier properties in endothelial cells (4). Wolberg et al. further established that the combination of astrocytes and cAMP stimulate barrier properties and tight junction complexity in endothelial cells (5). The role of cAMP signaling in the regulation of retinal endothelial barrier properties and alterations to this signaling pathway that may occur in diabetic retinopathy requires further study.

Astrocytes and Müller cells contact and ensheath the vascular plexus of the superficial region of the retina, but the deep capillary bed contacts only Müller cells. Tout et al. demonstrated that Müller cells are capable of inducing BRB properties similar to astrocyte induction. Transplantation of Müller cells into the anterior chamber of the rat eye cause Müller cell aggregates to form. These aggregates are vascularized and the invading blood vessels develop barrier properties preventing the flux of horseradish peroxidase or Evans Blue dye, similar to transplanted astrocytes but superior to those formed by meningeal cells (6). Müller cell induction of BRB properties was further supported by co-culture experiments with retinal endothelial cells. Müller cell co-culture across 0.4 µm pore transwell filters increases barrier properties of endothelial cells demonstrated by decreased insulin flux and increased transendothelial electrical resis-tance (7). Müller cell-conditioned media, however, did not provide the same effect (7). Further, injection of Müller cells into the anterior chamber of the eye failed to restrict horseradish peroxidase permeability across adjacent vessels (8). Thus, while astrocytes and Müller cells may both induce barrier properties, these studies may indicate important differences between these glial cells regarding barrier induction or maintenance.

Recent studies have identified a signal transduction adaptor molecule that promotes production of pro-barrier factors from astrocytes. Src-suppressed C kinase substrate or SSECKS in rodents, also termed gravin in humans, or AKAP12, coordinates signal transduction pathways by binding and organizing signaling molecules such as protein kinase C, protein kinase A, calmodulin, cyclins, and β-adrenergic receptors. In brain, SSECKS colocalizes with GFAP, indicating a glial expression pattern, and a recent report demonstrated that expression of SSECKS contributes to astrocytic induction of the BBB (9). Overexpression of SSECKS reduces expression of vascular endothelial growth factor, apparently through reduced c-Jun and AP1 signaling and promoted angiopoietin-1 production. Angiopoietin-1 is a ligand for the Tie2 receptor, and is known to both stabilize blood vessels and protect vessels from VEGF-induced permeability (10–12). The conditioned media from astrocytes overexpressing SSECKS blocks angiogenesis and promotes barrier properties of endothelial cells to a greater extent than astrocyte-conditioned media from mocktransfected cells. Further, antibodies to angio-poietin-1 blocked this barrier induction and conditioned media from astrocytes with siRNA to SSECKS-created endothelial cell barriers with greater permeability than control astrocyte-conditioned media. Additional studies of SSECKS/AKAP12 used expression studies and siRNA to demonstrate that SSECKS/AKAP12 downregulates HIF1α through the ubiquitin ligase von Hippel-Lindau and proteosomal degradation (13). Together these studies demonstrate that glia play an important role in the induction of the BBB and BRB, but an understanding of the molecular mechanisms by which this differentiation proceeds is only beginning to be elucidated.

Time Course of Blood–Brain Barrier Development

Immunohistochemistry experiments of the developing brain and retina support a role for astrocyte expression of SSECKS in the formation of the BBB and BRB. Expression of the well-established tight junction proteins zonula occludens 1 (ZO-1), vascular endothelial growth factor and SSECKS were compared in developing mouse-brain cerebral cortex or the embryonic precursor, the neopallial cortex (9). Expression of VEGF is present at embryonic Day 11.5 (E11.5) and increases through postnatal Day 3 (P3) but precipitously drops by P19 and is absent in adult. In contrast, ZO-1 is first detected at E15.5, while SSECKS could be detected at E16.5, and both proteins continue to increase expression through P19 and into adulthood. These results correlate well with the induction of the BBB during rat development, which dramatically increases barrier properties at E21 (14). Using measures of electrical resistance across the brain vascular endothelium, a measure of ionic permeability, the brain vessels were found to increase resistance from 310 to 1,215 ohm × cm2, which remained constant through adults. The results support a model in rodents in which SSECKS reduces VEGF expression in the brain as a phase of vessel growth ends and promotes formation of the tight junction complex that is stabilized by postnatal Day 21. However, it should be noted that other measures of permeability were found to demonstrate increased barrier properties after 21 days. Measures of potassium and urea flux revealed a dramatic drop in the influx rate constant from E21 to P2 that continued to slowly decrease to P50, suggesting further barrier changes after birth in rats (15).

Quantitative immunohistochemical analysis of human retina in developing fetus demonstrates a similar pattern of BRB formation (13). VEGF expression is observed at the 18th week of development in the retina but expression of the tight junction proteins occludin, ZO-2, and Claudin-1 as well as SSECKS/AKAP12, and angiopoietin 1 are not detectable at this time point. However, at the 24th week all these proteins were expressed and increased by the 27th week, while VEGF expression began to decrease at this time point and continued to decrease through the 39th week. These data support a role of SSECKS/AKAP12 in BRB formation through promotion of angiopoietin 1 expression, which subsequently promotes tight junction formation. However, angiopoietin 1 expression is not restricted to either the BBB or BRB, suggesting that additional signaling mechanisms contribute to barrier formation.

Pericyte Induction of the Blood–Retinal Barrier

In addition to glia, pericytes also induce barrier properties in retinal vascular endothelial cells. Pericytes are in close contact with the endothelial cells of the retinal capillaries and are encased by a common basal lamina (16). In vitro studies using pericyte and endothelial cell lines demonstrated that pericytes secrete an angiopoietin 1 complex that induces expression of tight junction protein occludin (17). Additionally, coculture of a rat brain endothelial cell line with a primary culture of rat brain pericytes decreases permeability to sodium fluorescein that is, in part, dependent on transforming growth factor β (TGF β), as demonstrated by using a blocking antibody. Finally, barrier induction is partially recapitulated by direct treatment with TGF β (18).

Further evidence for a role of pericytes in the induction or maintenance of the BRB is found in platelet-derived growth factor B (PDGF) B or PDGF receptor β (PDGFR)