Leukocyte Mediators of Vascular Leakage

AZ, heparin-binding protein (HBP/CAP37) is an inactive serine protease consisting of 225 amino acid residues and is a highly glycosylated molecule of 37 kDa. AZ is stored in the azurophilic granules of neutrophils [26]. Upon binding of neutrophils to the activated endothelium, b2-integrin ligation with endothelial ICAM-1 causes AZ release [25]. It is a multifunctional protein with diverse roles in host defense and inflammation [27]. AZ is a chemoattractant for monocytes and T cells and induces monocytes to differentiate into macrophages [28]. Furthermore, AZ stimulates endothelial cells via an unknown receptor to detach and aggregate [25].

AZ induces Ca2+-dependent cytoskeletal rearrangement and intercellular gap formation in endothelial cell monolayers in vitro and increases macromolecular permeability [25]. Moreover, AZ blockade prevents neutrophil-induced endothelial hyperpermeability, emphasizing the crucial role of AZ in vascular responses during inflammation [25].

The serine protease inhibitor, aprotinin, binds AZ and abolishes its ability to disrupt endothelial junctions [29]. Aprotinin is used clinically to protect patients undergoing extensive surgery, that is, cardiopulmonary bypass, from leukocyte sequestration in organs and fluid loss from the vasculature [30]. Recent in vivo results show that aprotinin is an effective inhibitor of the AZ-induced retinal vascular leakage. Aprotinin treatment also significantly decreases VEGF-induced leakage and BRB breakdown in experimentally induced diabetes, suggesting a possible role for AZ in these events. Aprotinin, as a broad inhibitor, also blocks other serine proteases, such as neutrophil-derived elastase, cathepsin G, proteinase 3, and some proteases in coagulation and fibrinolysis pathways, including plasmin and kallikrein [29, 31, 32]. Some of these proteases may be involved in retinal vascular leakage [33], and since aprotinin does not exclusively block AZ, there is potential involvement of other proteases in the VEGF-induced retinal vascular leakage or the BRB breakdown seen in early diabetes. Furthermore, since aprotinin is known to be anti-inflammatory and an inhibitor of leukocyte recruitment, aprotinin’s protective function against BRB breakdown could in part be due to its anti-inflammatory properties. Taken together, aprotinin or similar inhibitors of AZ might be useful in the treatment of retinal vascular leakage in DR.

Aprotinin is in clinical use for patients undergoing extensive cardiothoracic and orthopedic surgery. These patients often develop neutrophil sequestration in organs and massive leakage of fluid from the vasculature, and aprotinin can help to reduce blood loss and blood transfusion requirements postoperatively [30, 32]. Inhibition of AZ was proposed by Gautam et al. [25] as a possible mechanism of action for aprotinin in these clinical settings, considering the crucial role of AZ in neutrophil-evoked permeability. Two recent reports show an increased risk of renal [34] and cardiovascular toxicity, including myocardial infarction and stroke [35], following aprotinin administration in major surgeries. These systemic side effects of aprotinin might in part be due to its limited specificity in vivo. To date, there is no selective inhibitor of AZ. However, upon availability of such inhibitors, their intravitreal delivery in DR might lower the risk for systemic side effects.