lymphand angiogenesis [76]. VEGF-C expression is higher in blood vessel endothelium than in lymphatic endothelium; conversely, VEGFR-3 expression is higher in lymphatic endothelium [73]. In comparison, VEGFR-2 expression is similar in both endothelial cell types [77]. However, the differential contribution of VEGF-C/VEGFR-2 interaction to lymphand angiogenesis is not well understood.

VEGF is closely tied to the pathogenesis of DR. It plays a key role in the leukocytemediated breakdown of the BRB as well as retinal neovascularization [78]. Recent evidence ties VEGF with inflammation [79]. VEGF increases endothelial ICAM-1 expression, facilitating leukocyte adhesion [80] and BRB breakdown in diabetic retinal vessels [23].

Within the first 2 weeks of experimental diabetes in rats, retinal VEGF levels increased with associated upregulation of ICAM-1 in retinal endothelial cells and its ligands, the b2-integrins, on the surface of peripheral blood neutrophils [81, 82]. These molecular events result in increased adhesion of leukocytes, predominantly neutrophils, with a concomitant increase in retinal vascular permeability. Analogously, intravitreal VEGF injection induces retinal vascular changes that are quite similar to those seen in experimental diabetes, namely retinal leukostasis and the concomitant BRB breakdown [78], while blockade of VEGF abolishes retinal leukostasis and vascular leakage in experimentally induced diabetes [81, 83, 84].

Recent evidence shows that in addition to being the principle cytokine in growth and leakiness of neovascular membranes, VEGF also regulates RPE function [64]. The leading treatment of neovascular diseases is based on VEGF inhibition, using monoclonal antibody fragments. These anti-VEGF therapies are efficacious not only for reducing neovascularization but also for resolving retinal edema. However, recent evidence suggests that VEGF is required for normal retinal physiology, raising concerns about the long-term use of the VEGF inhibition strategy.

This motivated a search for endogenous antagonists of VEGF. A recent study revealed natriuretic peptides (NP), cyclic peptide hormones with diuretic, natriuretic, and vasodilatory properties, which antagonize not only choroidal neovascularization but also the breakdown of the outer BRB [85]. Understanding the role of endogenous antagonists of VEGF in the retinal barrier function will help to develop new strategies in the management of DR.

ANTI-VEGF PROPERTIES OF NATRIURETIC PEPTIDES

Inhibition of VEGF is currently under investigation in clinical trials, where retinal leakage and edema is a complication [86], such as DR [87], macular edema, [88], and retinal vein occlusion [89]. The rationale in these therapies is that removal of VEGF and the edematous fluid from the intraocular environment might be beneficial. However, VEGF has also protective properties for the retina [90], suggesting that VEGF is required for normal retinal physiology. This raises concerns about the long-term use of VEGF inhibition strategy. Furthermore, the simple removal of VEGF also eliminates the potential antiproliferative effects associated with VEGFR-1 activation [91], which might explain the lack of success in some cases.

Two endogenous anti-VEGF agents have been identified in the eye. Tombran-Tink et al. [92] reported the expression of pigment epithelium-derived factor (PEDF), produced and secreted by the RPE. PEDF was initially identified as a neurotrophic factor secreted by fetal human RPE cells, but later, vascular quiescence and permeability were also found to depend on the balance between VEGF and PEDF [93]. Molecules that interfere with the VEGF signaling pathways are attractive candidates for prevention of BRB breakdown. PEDF blocks the VEGF-induced TEER breakdown via the activation of juxtamembrane proteases to digest the VEGFR-2 receptor [64]. Thus, VEGF signaling is inhibited by limiting the available VEGFR-2 receptors. PEDF’s anti-VEGF and antipermeability effects in the RPE could potentially be utilized to treat retinal vascular leakage or edema.

Another endogenous anti-VEGF factor in the eye is the atrial natriuretic peptide (ANP) [85]. Natriuretic peptides are cyclic peptide hormones with diuretic, natriuretic, and vasodilatory properties. The NP family consists of three members: atrial NP (ANP), brain NP (BNP), and C-type NP (CNP). The action of NPs is mediated through two types of receptors: guanylate cyclase type A, which reacts with ANP and BNP, and guanylate cyclase type B, which is CNP specific [94, 95]. Binding of NPs to these receptors results in cGMP production, which activates protein kinase G and subsequent target genes [96]. Although primarily produced by the cardiac atria, ANPs are used in the treatment of various disorders, including hypertension, renal insufficiency, and congestive heart failure. Interestingly, ANP is also expressed in the inner plexiform layer and RPE of the human retina [97].

Recent results indicate that ANP plays an important role in neovascular diseases of the eye, as it antagonizes not only neovascularization but also the breakdown of the outer BRB [85]. VEGF-A produces a significant TEER drop in the outer BRB within 2 h posttreatment. This response reaches its peak by 5 h and lasts approximately 48 h [98]. In the presence of ANP, however, TEER levels remain at baseline values by 2 h despite VEGF administration, showing the protective function of ANP in the outer BRB. Furthermore, the ANP response is polar, as only apical but not basolateral administration of ANP reverses apical VEGF response [85]. Isatin, a universal NP receptor antagonist, completely reverses the inhibitory effects of ANP with respect to the VEGF-induced TEER reduction, indicating that ANP receptor-mediated signaling is critical in this event. These data indicate that ANP acts by inhibiting VEGF signaling pathways in RPE cells. The recent linking of the expression of natriuretic peptides and the barrier function of the RPE and the retinal vessels might lead to new therapeutic strategies in reducing retinal edema. This is because natriuretic peptides are already in use in vascular disorders, and thus, detailed knowledge of their dosage and toxicity exists. However, future work will need to address the impact of these peptides on immune regulation and other aspects of DR development.

Proposed Model of BRB Breakdown in DR

A working model for how BRB breakdown might occur in early DR involves interaction of leukocytes via their b2-integrins to the endothelial ICAM-1. The resulting release of the serine protease, AZ, from leukocytes causes an increase in BRB permeability (Fig. 5). This is backed by the fact that recombinant AZ injected intravitreally significantly increases

Fig. 5. Leukocyte-induced BRB permeability in early DR. b2-integrin ligation with endothelial ICAM-1 (1) initiates signaling (2) that leads to release of AZ containing granulae (3). AZ binds to unidentified endothelial ligands and causes rapid opening of the BRB leading to leakage of plasma proteins (4).

BRB permeability as quantified by EB technique. AZ appears to be also an attractive target for controlling BRB permeability, as for instance systemic injection of aprotinin, a broad protease inhibitor, 1 h before the AZ injection completely blocks the increase in leakage. More striking is that the AZ-induced leakage is rather rapid, with a peak BRB leakage approximately 1 h after intravitreal AZ injection, suggesting a key role for AZ in diabetic BRB breakdown.

Key Role of AZ in VEGF-Induced Leakage

VEGF causes leukocyte accumulation in retinal vessels as well as protein leakage into the retinal parenchyma. Since VEGF is a key permeability factor in DR, the question arises, what portion of the VEGF-induced leakage is a direct effect of VEGF on the endothelium rather than through downstream mediators. Of course, the editors have the discretion to correct potential grammatical errors of the newly suggested sentence, however, the suggested sentence by the editors did not meet the intended scientific meaning. Whether AZ is a downstream mediator of VEGF’s action is addressed by an experiment showing suppression of VEGF-induced retinal vascular leakage by AZ blockade. Intravitreal injection of VEGF together with systemic application of aprotinin completely prevents VEGF’s permeability increase.

Interestingly, VEGF-induced leakage peaks around 6 h after its intravitreal injection [99]. In comparison, AZ-induced effect is more immediate, and its highest level is