CLEC3B acts as an endogenous brake on pathological angiogenesis in diabetic retinopathy by targeting the PI3K/ Akt/mTOR signaling cascade

ABSTRACT

Objective: To investigate the role of C-type lectin domain family 3 member B (CLEC3B) in high glucose-induced
proliferation, migration, and tube formation of HRECs, and to verify the role of the PI3K/Akt signaling pathway in
this process.
Methods: Vitreous humor was collected from patients with proliferative diabetic retinopathy (PDR) and control individuals with epiretinal membrane/macular hole (ERM/MH). HRECs were treated with normal glucose (5 mM) or high glucose (25 mM). TMT proteomics of human vitreous and proteomics of high glucose-induced HRECs were detected. CLEC3B expression was measured by ELISA. CLEC3B was overexpressed or knocked down using plasmid transfection. The PI3K/Akt inhibitor LY294002 was also applied. Cell proliferation was assessed by CCK-8 and EdU assays; migration was evaluated by scratch and Transwell assays; tube formation was examined using Matrigel. Protein expression of CLEC3B, p-PI3K, p-Akt, p-mTOR, VEGF-A, HIF-1α, and iNOS was detected by western blotting.
Results: Proteomics result revealed significant differential expression of eight proteins including TF and FTL. PFN1, F5, and SELENOP were excluded due to low detection rates and high variability. CLEC3B showing the most significant difference (P<0.01), was selected as the core target and was enriched in the PI3K/Akt signaling pathway. ELISA result shows CLEC3B protein levels were significantly decreased in the vitreous of PDR patients compared with controls (P<0.0001). High glucose downregulated CLEC3B expression in HRECs (P<0.05). High glucose significantly enhanced HREC proliferation, migration, and tube formation, whereas CLEC3B overexpression reversed these effects, and CLEC3B knockdown further aggravated them (P<0.05). Western blotting showed that CLEC3B overexpression suppressed the activation of the PI3K/Akt/mTOR pathway and reduced the expression of VEGF-A, HIF-1α, and iNOS (P<0.05). No significant differences in these functional and protein changes were observed between the CLEC3B overexpression group and the LY294002 treatment group. Importantly, the inhibitory effects of LY294002 were partially reversed by CLEC3B knockdown.
Conclusion: CLEC3B inhibits high glucose-induced proliferation, migration, and angiogenic activity in HRECs, which is associated with the suppression of the PI3K/Akt signaling pathway and downstream angiogenic factors. CLEC3B may represent a novel protective target in diabetic retinopathy.