OTHER MEDIATORS OF LEUKOCYTE RECRUITMENT IN DR

Due to the immune-privileged status of the eye, few immune cells transmigrate into the retina under physiological conditions. However, in DR, large numbers of immune cells cross the BRB and migrate into the neuronal retina. The infiltrating leukocytes are believed to be the cause of considerable harm to the neurons. Recent results indicate a critical role for a new molecule, the vascular adhesion protein 1 (VAP-1), in the retinas of diabetic animals [36].

VAP-1 is an endothelial adhesion molecule involved in leukocyte recruitment [37, 38]. It is a homodimeric sialylated glycoprotein expressed on the endothelium of human tissues such as skin, brain, lung, liver, and heart under both normal and inflamed conditions [39–42]. Increased levels of both soluble and membrane-associated VAP-1 are reported in diabetes [43].

In addition to being an adhesion molecule, VAP-1 is also an enzyme. Indeed, VAP-1 is the only known adhesion molecule that also has catalytic activity. It has characteristics of semicarbazide-sensitive amine oxidases (SSAO), enzymes that catalyze the deamination of primary amines such as methylamine and aminoacetone [44, 45]. SSAO’s active site generates toxic formaldehyde and methylglyoxal, hydrogen peroxide and ammonia [45], reactive chemicals, and major reactive oxygen species [43].

VAP-1 is expressed on the retinal endothelium, and it plays a critical role in the recruitment of leukocytes to the eye during DR [36], acute inflammation [46], and laserinduced neovascularization [47]. The fact that VAP-1 is expressed in the human eye [48] suggests that it could become an attractive molecular target in the prevention and treatment of ocular inflammatory diseases, such as DR.

To detect molecular changes or early endothelial injury at the BRB, we recently introduced a new noninvasive molecular imaging approach [49, 50]. In this technique, fluorescent microspheres (MSs), of slightly less than cellular dimensions, are conjugated with ligands or antibodies to one or more endothelial surface molecules of interest [50, 51]. After systemic injection, the interactions of these MSs with the endothelium of the retinal and choroidal vessels of live animals is studied by scanning laser ophthalmoscopy (SLO) in normal or diabetic animals. These new approaches will likely advance our understanding of the cellular and molecular events that lead to BRB breakdown, for instance in early DR.

STRUCTURAL COMPROMISE OF THE BRB

The inner and outer BRB can also be compromised due to structural changes. A common cause of the structural damage underlying BRB breakdown is neovascularization, or the growth of new vessels. Neovascularization in the eye is a leading cause of vision loss. It occurs in the proliferative stage of diabetic retinopathy, where retinal vessels grow, likely secondary to ischemia. While normal retinal vessels have the BRB function, the neovascular vessels are leaky for proteins and also prone to bleeding, allowing accumulation of fluid within the extracellular spaces of the neurosensory retina. The ensuing damage to the cells of the neuronal retina can result in permanent vision loss. Neovascularization occurs in consequence of the intraocular release of the pro-angiogenic cytokine and vasopermeability agent, VEGF [52].

Fig. 4. Vascular endothelial growth factor (VEGF) isoforms and their endothelial receptors.

Vascular Endothelial Growth Factor

A key mediator of permeability as well as neovascularization is VEGF. VEGF expression is primarily triggered through hypoxia [53], but growth factors [54], inflammatory molecules [55], oxidative stress [56], and advanced glycation end products [57] can also induce VEGF production. A major source of VEGF in the eye is the RPE [58]. Under normal conditions, VEGF secretion from the RPE stimulates choriocapillaris endothelium development [59]. The role of VEGF in endothelial cell biology has been extensively studied. VEGF receptors activate multiple signaling pathways including survival [60], migration [61], mitogenesis [62], and permeability [63]. Recent studies show expression of VEGF receptors on RPE [64] and that VEGF modulates RPE barrier properties through the VEGFR-2 receptor [65].

The VEGF family consists of five members that bind to and activate three distinct receptors [66, 67] (Fig. 4). VEGF-A binds to both VEGFR-1 and VEGFR-2, while placental growth factor (PlGF) and VEGF-B bind only to VEGFR-1. VEGF-C and VEGF- D are the only known ligands for VEGFR-3 and do not bind to VEGFR-1 [68, 69].

VEGF-A is upregulated in various physiological and pathological conditions, causing endothelial permeability [70], lymph- [71], and angiogenesis [72]. VEGF-A induces proliferation and migration of the lymphatic endothelium through the VEGFR-2 [73]. Proteolytically processed VEGF-C binds to and activates VEGFR-2, while the unprocessed precursor form of VEGF-C signals through VEGFR-3 [74]. Both VEGF-C and VEGF- D primarily affect development of lymphatic vasculature through VEGFR-3 activation, but they also participate in angiogenesis through VEGFR-2 [75]. For instance, a soluble VEGFR-2 form that is secreted by corneal epithelial cells selectively suppresses the physiologic growth of lymphatics; however, it does not address the interdependency of