MAP4K4 aggravates microvascular anomalies in diabetic retinopathy in a YTHDF2-dependent manner

Signalling pathways that regulate endothelial cell (EC) dysfunction, ischaemia and inflammation play a crucial role in retinal microangiopathy such as diabetic retinopathy. MAP4K4 is highly expressed in ECs. However, the involvement of MAP4K4 in retinal vasculopathy of diabetic retinopathy remains unclear. We analysed publicly available single-cell RNA sequencing (scRNA-seq) data from fibrovascular membranes (FVMs) from eight individuals with proliferative diabetic retinopathy (PDR) and normal retinas from 11 individuals without diabetes. Using db/db mice and human primary retinal endothelial cells (HRMECs), we further investigated the effects of MAP4K4 on retinal microangiopathy and endothelial dysfunction to explore the underlying regulatory mechanisms. The scRNA-seq analysis revealed that MAP4K4 was predominantly expressed in retinal ECs, with elevated expression in FVMs from individuals with PDR compared with normal retinas from individuals without diabetes. This finding was confirmed at the protein level, with MAP4K4 expression and activity being upregulated in both the FVMs of individuals with PDR and the retinas of db/db mice. Inhibition of MAP4K4 using DMX-5804 alleviated retinal microvascular leakage by enhancing the expression and integrity of junctional proteins in both ECs from db/db mice and HRMECs. Additionally, DMX-5804 reduced retinal angiogenesis by inhibiting EC migration and vascular sprouting. Mechanistically, MAP4K4 regulated EC characteristics through NF-κB signalling pathway activity. The exacerbating effect of recombinant MAP4K4 on diabetic retinopathy in db/db mice was mitigated by a p65 inhibitor, confirming the involvement of NF-κB. Moreover, MAP4K4 expression was regulated by YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2), which modulates the stability of MAP4K4 mRNA. Our study highlights the critical role of MAP4K4 in EC dysfunction and diabetic retinal microangiopathy, providing new insights into its molecular pathogenesis. Targeting MAP4K4, particularly through modulation of the YTHDF2/MAP4K4/NF-κB axis, may provide a novel therapeutic strategy for diabetic retinopathy.