Senescence-induced endothelial-to-mesenchymal transition accelerates the subretinal fibrosis in neovascualr age-related macular degeneration.

Choriocapillary endothelial cells (CapECs) in nAMD exhibited elevated senescence gene set variation analysis scores and upregulated differentially expressed genes enriched in senescence and mesenchymal transition pathways.

• Single-cell RNA sequencing data analysis was used to investigate the role of senescent CapECs in subretinal fibrosis.
• SenMayo gene set variation analysis (GSVA) scores were elevated in CapECs from nAMD samples.
• Upregulated differentially expressed genes (DEGs) were enriched in both senescence and mesenchymal transition pathways.
• A higher proportion of senescence-like CapECs (17.68%) was observed in nAMD tissue.

Senescence-like CapECs in nAMD showed increased mesenchymal scores and elevated TGFB1 expression.

• Senescence-like CapECs comprised 17.68% of the CapEC population in nAMD.
• These senescence-like CapECs demonstrated increased mesenchymal scores compared to non-senescent CapECs.
• TGFB1 expression was upregulated in the senescence-like CapEC subpopulation in nAMD.
• These findings were derived from single-cell RNA sequencing data analysis.

Senescent endothelial cells in laser-induced fibrotic models and bleomycin/doxorubicin-treated human umbilical vein endothelial cells underwent enhanced endothelial-to-mesenchymal transition.

• In vitro assays using bleomycin and doxorubicin were used to induce senescence in human umbilical vein endothelial cells (HUVECs).
• Laser-induced subretinal fibrosis models in Brown-Norway rats and INK-ATTAC mice were used for in vivo validation.
• Senescent endothelial cells in both in vitro and in vivo models demonstrated enhanced EndMT compared to non-senescent controls.
• These results validated the mechanistic link between cellular senescence and EndMT.

Early intervention with dasatinib plus quercetin (D+Q) senolytic treatment markedly reduced subretinal fibrosis and restored retinal function in laser-induced fibrosis models.

• Senolytic treatment consisted of dasatinib plus quercetin (D+Q).
• Experiments were conducted in Brown-Norway rats and INK-ATTAC mice with laser-induced subretinal fibrosis.
• Early intervention timing was emphasized as important for the therapeutic effect.
• Both reduction of subretinal fibrosis and restoration of retinal function were observed following D+Q treatment.

Selective clearance of p16INK4A-positive senescent cells markedly reduced subretinal fibrosis and restored retinal function.

• INK-ATTAC mice, which allow selective elimination of p16INK4A-positive cells, were used for this experiment.
• Selective removal of p16INK4A-positive cells produced effects comparable to D+Q senolytic treatment.
• Both subretinal fibrosis reduction and retinal function restoration were observed after p16INK4A-positive cell clearance.
• This finding supports a causal role for p16INK4A-expressing senescent cells in subretinal fibrosis development.

Endothelial cells from old mice exhibited heightened EndMT and more severe subretinal fibrosis compared to those from young mice.

• Young mice were defined as 2-3 months of age and old mice as 18 months of age; both were C57BL/6J strain.
• Laser-induced subretinal fibrosis models were used to compare fibrosis severity between age groups.
• Old mice showed heightened EndMT in endothelial cells compared to young mice.
• More severe subretinal fibrosis was observed in old mice than in young mice, implicating aging as a contributing factor.