The ANGPTL3-integrin α5 axis drives retinal vascular leakage in diabetic retinopathy

Fig. 1. ANGPTL3 is elevated in the diabetic ocular microenvironment. (A) Bioinformatics analysis showing an upregulation of ANGPTL3 and ANGPTL4 in aqueous humor. (B, C) Western blot analysis of ANGPTL3 and ANGPTL4 in retinal tissue from the DR group and controls. (D, E) Immunofluorescence staining for ANGPTL3 in retinal sections from control and STZ-diabetic mice (nuclei counterstained with DAPI)

Fig. 2. Genetic targeting of ANGPTL3 rescues diabetic retinal vascular leakage. (A) Body weight of the mice. (B and C) IPGTT and corresponding AUC. (D and E) Representative FFA and quantification of extravascular/intravascular fluorescence intensity ratio. (F and G) Representative OCT images and quantification of retinal layer thickness. (H) PAS staining of retinal vasculature (scale bar = 50 μm). (I) Quantification of AC-Cap, EC, and PC based on PAS-stained images in (H)

Fig. 3. ANGPTL3 overexpression exacerbates STZ-induced retinal vascular leakage in diabetic mice. (A) Plasma ANGPTL3 levels following AAV-mediated ANGPTL3 overexpression. (B and C) IPGTT and AUC following ANGPTL3 overexpression. (D and E) Representative FFA images and quantification of vascular leakage in ANGPTL3-overexpressing mice. *P < 0.05, **P < 0.01

Fig. 4. ANGPTL3 knockout preserves retinal endothelial barrier function by upregulating tight junction proteins. (A) Representative TEM images showing tight junction structures in retinal capillary endothelial cells of control, diabetic, and Angptl3 knockout mice. (B) Immunofluorescence staining of retinal sections for tight junction proteins ZO-1 and VE-cadherin. Scale bars = 10 μm. (C and D) Quantification of ZO-1 and VE-cadherin fluorescence intensity from (B). (E) Proliferation assay of HUVECs treated with recombinant human ANGPTL3. (F) Western blot analysis of ZO-1 and VE-cadherin protein levels in HUVECs following ANGPTL3 treatment. (G-H) Quantification of protein expression from (F). *P < 0.05, **P < 0.01

Fig. 5. ANGPTL3 compromises the endothelial barrier via integrin α5-dependent signaling. (A) Protein interaction network analysis. (B) Protein docking simulation (C) Co-IP assay between integrin α5 and ANGPTL3 in HUVECs. (D) Quantification of fluorescence intensity in endothelial permeability. (E) Representative immunofluorescence images of HUVECs stained for ZO-1 and VE-cadherin. Scale bars = 20 μm. (F and G) Quantification of ZO-1 and VE-cadherin fluorescence intensity is shown in (E). *P < 0.05, **P < 0.01

Fig. 6. Therapeutic targeting of ANGPTL3 with evinacumab rescues retinal vascular leakage without altering systemic metabolism. (A) Flowchart of the study. (B) Fasting blood glucose level. (C) Body weight of the mice. (D-E) Representative FFA images and quantification of vascular leakage. *P < 0.05, **P < 0.01

Fig. 7. Schematic of the proposed mechanism for ANGPTL3-mediated BRB disruption in DR. Under diabetic conditions, increased ANGPTL3 interacts with endothelial integrin α5, leading to the downregulation of tight junction proteins (ZO-1, VE-cadherin). This compromises BRB integrity and causes vascular leakage. Both genetic knockout of Angptl3 and local inhibition with evinacumab rescue this phenotype by preserving tight junction structure and function