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 (APC) is a recombinant variant of the physiological anticoagulant APC with pleiotropic cytoprotective properties albeit without the bleeding risks. The anti-inflammatory activities of 3K3A-APC were demonstrated in multiple preclinical injury models, including various neurological disorders. We determined the ability of 3K3A-APC to inhibit ocular inflammation in a murine model of lipopolysaccharide (LPS)-induced uveitis. Leukocyte recruitment, microglia activation, NLRP3 inflammasome and IL-1β levels were assessed using flow cytometry, retinal cryosection histology, retinal flatmount immunohistochemistry and vascular imaging, with and without 3K3A-APC treatment. LPS triggered robust inflammatory cell recruitment in the posterior chamber. The 3K3A-APC treatment significantly decreased leukocyte numbers and inhibited leukocyte extravasation from blood vessels into the retinal parenchyma to a level similar to controls. Resident microglia, which underwent an inflammatory transition following LPS injection, remained quiescent in eyes treated with 3K3A-APC. An inflammation-associated increase in retinal thickness, observed in LPS-injected eyes, was diminished by 3K3A-APC treatment, suggesting its clinical relevancy. Finally, 3K3A-APC treatment inhibited inflammasome activation, determined by lower levels of NLRP3 and its downstream effector IL-1β. Our results highlight the anti-inflammatory properties of 3K3A-APC in ocular inflammation and suggest its potential use as a novel treatment for retinal diseases associated with inflammation.
Purpose: Accumulating evidence suggests that neuroinflammation and immune response are part of the sequence of pathological events leading to optic nerve damage in glaucoma. Changes in tissue temperature due to inflammation can be measured by thermographic imaging. We investigated the ocular surface temperature (OST) profile of glaucomatous eyes to better understand the pathophysiology of these conditions. Methods: Subjects diagnosed with glaucoma (primary open angle glaucoma [POAG] or pseudo exfoliation glaucoma [PXFG]) treated at the Sam Rothberg Glaucoma Center (11/2019–11/2020.) were recruited. Healthy subjects with no ocular disease served as controls. The Therm-App thermal imaging camera was used for OST acquisition. Room and body temperatures were recorded, and the mean temperatures of the medial cantus, lateral cantus, and cornea were calculated with image processing software. Results: Thermographic images were obtained from 52 subjects (52 eyes: 25 POAG and 27 PXFG) and 66 controls (66 eyes). Eyes with glaucoma had a significantly higher OST compared to controls (mean 0.9 ± 0.3°C, p < 0.005). The difference between the two groups remained significant after adjustment for age, sex, intraocular pressure (IOP) and room and body temperatures. Lens status and topical IOP-lowering medication did not significantly affect OST. A subgroup analysis revealed that the OST was higher among eyes with POAG compared to eyes with PXFG, but not significantly. Conclusions: Differences in the OST between glaucomatous and normal eyes strengthens current thinking that inflammation affects the pathophysiology of glaucoma. Longitudinal studies are warranted to establish the prognostic value of thermographic evaluations in these patients.
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