Activated Protein C (APC) and 3K3A-APC-Induced Regression of Choroidal Neovascularization (CNV) Is Accompanied by Vascular Endothelial Growth Factor (VEGF) Reduction

Abstract

The activated protein C (APC) ability to inhibit choroidal neovascularization (CNV) growth and leakage was recently shown in a murine model. A modified APC, 3K3A-APC, was designed to reduce anticoagulant activity while maintaining full cytoprotective properties, thus diminishing bleeding risk. We aimed to study the ability of 3K3A-APC to induce regression of CNV and evaluate vascular endothelial growth factor (VEGF) role in APC’s activities in the retina. CNV was induced by laser photocoagulation on C57BL/6J mice. APC and 3K3A-APC were injected intravitreally after verification of CNV presence. CNV volume and vascular penetration were evaluated on retinal pigmented epithelium (RPE)-choroid flatmount by fluorescein isothiocyanate (FITC)-dextran imaging. VEGF levels were measured using immunofluorescence anti-VEGF staining. We found that 3K3A-APC induced regression of pre-existing CNV. VEGF levels, measured in the CNV lesion sites, significantly decreased upon APC and 3K3A-APC treatment. Reduction in VEGF was sustained 14 days post a single APC injection. As 3K3A-APC retained APCs’ activities, we conclude that the anticoagulant properties of APC are not mandatory for APC activities in the retina and that VEGF reduction may contribute to the protective effects of APC and 3K3A-APC. Our results highlight the potential use of 3K3A-APC as a novel treatment for CNV and other ocular pathologies.

3K3A-Activated Protein C Inhibits Choroidal Neovascularization Growth and Leakage and Reduces NLRP3 Inflammasome, IL-1β, and Inflammatory Cell Accumulation in the Retina

“…As we observed robust inflammation on days 4–7 after CNV induction ( Figure 1 , Figure 2 and Figure 3 ), we used a treatment regimen of 2 consecutive intravitreal injections of 1 µg/µL/eye 3K3A-APC, administered on days 4 and 7 post-CNV induction. The concentration of 3K3A-APC was chosen based on previously performed studies [ 18 , 19 ]. Then, 7 days after the last treatment (on day 13), an in-vivo injection of fluorescein isothiocyanate (FITC)-dextran was performed to mark retinal blood vessels fluorescently.…”

“…To our knowledge, our group is the only one studying the effects of 3K3A-APC in the retina [ 18 , 19 ]. We previously showed that intravitreal (IVT) treatment with 3K3A-APC inhibits CNV growth and significantly reduces VEGF levels at CNV sites [ 18 ].…”

“…To our knowledge, our group is the only one studying the effects of 3K3A-APC in the retina [ 18 , 19 ]. We previously showed that intravitreal (IVT) treatment with 3K3A-APC inhibits CNV growth and significantly reduces VEGF levels at CNV sites [ 18 ]. Recently we demonstrated the anti-inflammatory activities of 3K3A-APC in the inflamed retina using an endotoxin-induced uveitis (EIU) murine model [ 19 ].…”

Neutrophil, neutrophil extracellular traps and endothelial cell dysfunction in sepsis

“…Aside from its anticoagulant effects, APC also displays cytoprotective properties, such as blocking inflammatory cytokine release, 128 maintaining endothelial integrity and protecting cells from apoptosis. 129 , 130 So decreased plasma levels of APC are strongly linked with prognosis. Although controversial, APC has been used in severe sepsis.…”

“…Moreover, blocking endothelial NF‐κB signalling inhibits TNF‐α converting enzyme activity which is responsible for EPCR shedding, reducing plasma plasminogen activator inhibitor type‐1 level and restoring plasma level of APC 22,127 (Figure 4). Aside from its anticoagulant effects, APC also displays cytoprotective properties, such as blocking inflammatory cytokine release, 128 maintaining endothelial integrity and protecting cells from apoptosis 129,130 . So decreased plasma levels of APC are strongly linked with prognosis.…”