Hyperglycemia‐Induced Microglial CTSS Disrupts Blood‐Retinal Barrier Integrity via PAR2 ‐Dependent Downregulation of Endothelial MFSD2A

ABSTRACT Background Diabetic retinopathy (DR), a leading cause of blindness in diabetes, involves dysregulated neuroimmune crosstalk. While microglial activation and endothelial dysfunction are established in DR, the molecular mechanisms linking innate immunity to blood‐retinal barrier (BRB) breakdown remain elusive. Methods We combined single‐cell RNA sequencing (scRNA‐seq) of diabetic mouse retinas with in vivo models, retinal endothelial MFSD2A overexpression, and microglial Cathepsin S (CTSS) knockdown via adeno‐associated virus (AAV). Human retinal microvascular endothelial cells (HRMECs) and human microglial cells (HMC3) were subjected to high‐glucose conditions combined with in vitro systems (transwell co‐cultures, holographic 3D tomography). Mechanistic studies employed siRNA knockdown, plasmid overexpression, exogenous CTSS supplementation, and transcytosis assays. Results scRNA‐seq revealed diabetes‐induced CTSS upregulation in retinal microglia ( p = 0.0031) and MFSD2A downregulation in endothelial cells ( p = 0.01). Microglial CTSS promoted M1 polarization ( p < 0.001) and pro‐inflammatory cytokine secretion (IL‐1β and TNF‐α, p < 0.05), while Ctss knockdown attenuated these effects ( p < 0.05). CTSS activated endothelial PAR2, suppressing endothelial MFSD2A and enhancing caveolin‐1‐mediated transcytosis. Endothelial MFSD2A overexpression reduced vascular permeability independently of tight junction modulation (ZO‐1, Occludin, Claudin‐5, p > 0.05). Microglia‐endothelial crosstalk was disrupted in diabetes, with ultrastructural alterations (cytoplasmic shrinkage, microglial amoeboid transformation) promoting cytotoxic interactions and exacerbate vascular endothelial damage. Conclusions Hyperglycemia induces retinal microglial activation and CTSS upregulation, disrupting microglia‐endothelial crosstalk via the PAR2 pathway, suppressing endothelial MFSD2A expression, enhancing transcytosis, compromising iBRB integrity, and accelerating DR progression. Therapeutic strategies targeting microglial CTSS or endothelial MFSD2A represent promising avenues for preserving vision in diabetic patients.