Vacuolar protein sorting 35 regulates retinal neurovascular function via astrocyte-mediated inflammatory response in a diabetic mouse model

To explore how vacuolar protein sorting 35 (VPS35) regulates astrocytic inflammation, impairs retinal endothelial function and drives early diabetic retinopathy (DR) neurovascular lesions in diabetic mouse model.

A streptozotocin (STZ)-induced C57BL/6J diabetic mouse model was established. Retinal tissues were collected from 0 to 12wk after successful induction of diabetes. Protein expression levels of VPS35, glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 2 (EAAT2), leucine-rich repeat kinase 2 (LRRK2), and vascular endothelium-associated proteins were assessed by Western blotting (WB). The interaction between VPS35 and LRRK2 was verified by co-immunoprecipitation. Vascular leakage and astrocyte activation were evaluated by fundus fluorescein angiography and retinal flat-mount immunofluorescence staining. Primary astrocytes were cultured in vitro and subjected to high-glucose stimulation or VPS35 knockdown. Cell activation, expression of glutamate transport-associated proteins, and inflammatory cytokine expression were examined. Supernatant fluid from the primary astrocytes was applied to human umbilical vein endothelial cells (HUVECs) to assess cell migration, proliferation, and tube formation capacity. The expression of proteins related to the nuclear factor kappa-B (NF-κB) pathway was also evaluated.

In diabetic mice, retinal VPS35 protein expression exhibited a progressive decline beginning at 4wk post-diabetes onset, whereas GFAP protein expression increased significantly. By 8wk, marked astrocyte activation was observed, accompanied by retinal microvascular leakage and a reduction in vascular area. In vivo and in vitro experiments further confirmed that under high-glucose conditions, retinal VPS35 and EAAT2 protein levels were markedly decreased, while GFAP and LRRK2 protein levels were significantly elevated. Co-immunoprecipitation verified the physical interaction between VPS35 and LRRK2 in astrocytes. Finally, in vitro experiments demonstrated that both high-glucose stimulation and VPS35 knockdown led to astrocyte activation, upregulation of inflammatory cytokine expression, downregulation of EAAT2 and AMPA receptor subunit GLUA2, and upregulation of LRRK2. Treatment of HUVECs with supernatant fluid from these astrocytes enhanced cell migration but significantly inhibited cell proliferation and tube formation. WB analysis revealed markedly increased levels of NF-κB and phosphorylated NF-κB in the treated HUVECs.

During early DR in mice model, decreased retinal VPS35 protein expression induces astrocyte-mediated inflammatory responses and glutamate transport dysfunction. Through the interaction between VPS35 and LRRK2, paracrine inflammatory cytokines subsequently activate the NF-κB signaling pathway in vascular endothelial cells, leading to endothelial dysfunction and further driving DR-associated neurovascular injury. This study provides novel insights into the pathogenesis of DR and highlights the potential of VPS35 as a target for early intervention in DR.