Endogenous and therapeutic transport of activated protein c across the blood-retina barrier protects the retina

Abstract

Abstract Introduction: Protein C (PC) deficiency is a devastating thrombophilia. Its most striking non-thrombotic consequences involve the eye, including inflammation, neovascularization, and early-onset blindness. This suggests a potential yet underexplored physiological role for PC and its active form, activated PC (APC), in maintaining retinal homeostasis. Besides its well-known anticoagulant properties in plasma, APC exerts multiple cytoprotective activities mediated via binding to the endothelial PC receptor (EPCR) and downstream signaling through proteolytic activation of protease-activated receptors. Intraocular administration of pharmacological doses of APC demonstrates anti-inflammatory, anti-angiogenic, blood-barrier-stabilizing, and neuroprotective effects on the retina. A major challenge in studying the physiological roles of PC is the lack of viable animal models with complete PC deficiency, as homozygous deletion of PC is embryonically lethal. The goals of this study were to determine whether PC/APC is locally produced in the retina, whether systemic APC (a large glycoprotein ~62 kDa) can cross the blood-retinal barrier (BRB) from the systemic circulation, and whether this physiological transport mechanism can be leveraged for therapeutic purposes for retinal diseases. Methods: We used a novel severe PC deficiency (SPCD) murine model, generated by breeding PC-deficient and factor VIII-deficient mice to enable viable offspring that lack any endogenous PC ( Levy-Mendelovich et al. IJMS 2024) PC and APC mRNA and protein levels were assessed by RT-qPCR and immunofluorescence in both wild-type (WT) and SPCD mice. Plasma-derived (pd) APC and 3K3A-APC (a recombinant APC variant with full cytoprotective but reduced anticoagulant activity) were administered systemically, and their translocation across the BRB, retinal accumulation, and co-localization with EPCR were evaluated by immunofluorescence. The therapeutic potential of intravenously administered, systemic 3K3A-APC treatment was assessed in an endotoxin (LPS)-induced uveitis model (Palevski et al. IJMS 2022) and compared to intraocular 3K3A-APC administration. Results: PC mRNA was absent from the retina in both WT and SPCD mice but it was robustly expressed in the WT livers, confirming hepatic synthesis as a major source of plasma PC. Immunostaining revealed APC in the retinal tissue of WT mice but not in SPCD mice, validating its systemic origin and transport into the retina. Following intravenous (tail vein) administration, both pdAPC and 3K3A-APC accumulated within multiple retinal layers in SPCD mice, replicating the distribution seen in WT animals. In contrast, inactive PC did not cross the BRB or localize to the retina. Immunofluorescence imaging showed EPCR expression along the retinal vasculature, with partial co-localization with APC after systemic administration, supporting a potential EPCR-mediated transport mechanism. Functionally, systemic administration of 3K3A-APC significantly reduced CD11b⁺ inflammatory cell infiltration into the retina and limited microglial activation in the LPS-induced retinal inflammation model. The anti-inflammatory efficacy of systemic 3K3A-APC matched that of intraocular treatment and reduced inflammatory parameters to levels comparable to uninjured controls. Conclusions: Our study provides the first in vivo demonstration that APC, but not PC, naturally crosses the BRB into the retina via a selective, likely receptor-mediated process involving EPCR. These observations establish a previously unrecognized link between circulating APC and retinal homeostasis and suggest the existence of an active, receptor-mediated mechanism that allows large protective proteins, like APC, to access immune-privileged ocular tissues. It appears that, by leveraging this BRB endogenous transport pathway, the systemically administered APC analog, 3K3A-APC, can cross the BRB and exert potent anti-inflammatory effects in the retina. These results highlight the feasibility of developing non-invasive, systemic APC-based therapies for retinal diseases and lay the groundwork for further exploration of APC and APC variants as naturally occurring, cytoprotective proteins in the eye.