ANGPTL2 overexpression promotes angiogenic responses in human retinal microvascular endothelial cells with accompanying integrin α5β1 molecular changes

Figures

• 

  Figure 1. Integrin α5β1 expression is elevated in diabetic retinal tissues and shows a directionally lower pattern in the ANGPTL2 knockdown comparison group. (A) RT-qPCR analysis of integrin α5 and integrin β1 mRNA levels in retinal tissues from four groups (Control, STZ, STZ + sh-NC, STZ + sh-ANGPTL2), normalized to β-actin (n = 3 biological replicates per group, each representing an individual retina). (B) Representative western blot images and quantification of integrin α5 and integrin β1 protein expression normalized to β-actin (n = 3 biological replicates per group). Integrin α5: Control vs. STZ, p = 1.18 × 10−6; STZ + sh-NC vs. STZ + sh-ANGPTL2, p = 0.00002; STZ vs. STZ + sh-NC, ns. Integrin β1: Control vs. STZ, p = 7.72 × 10−8; STZ + sh-NC vs. STZ + sh-ANGPTL2, p = 4.00 × 10−7; STZ vs. STZ + sh-NC, ns. (C) Immunofluorescence staining for integrin α5 (red) with Hoechst 33342 nuclear counterstain (blue); merged images and quantification of integrin α5-positive area (%) from three separate animals (one representative section per animal; animal-level values used for statistical analysis) are shown. Scale bar, 50 μm. Control vs. STZ, p = 0.0002; STZ + sh-NC vs. STZ + sh-ANGPTL2, p =…

• 

  Figure 2. PI3K/AKT phosphorylation changes in diabetic retinal tissues are associated with ANGPTL2 expression levels across comparison groups. Representative western blot images showing p-PI3K, total PI3K, p-AKT, total AKT, and β-actin in retinal tissues from four groups (Control, STZ, STZ + sh-NC, STZ + sh-ANGPTL2). Quantification of p-PI3K/PI3K and p-AKT/AKT ratios (n = 3 biological replicates per group, each representing an individual retina) is shown. p-PI3K/PI3K: Control vs. STZ, p = 1.49 × 10−6; STZ + sh-NC vs. STZ + sh-ANGPTL2, p = 0.00001; STZ vs. STZ + sh-NC, ns. p-AKT/AKT: Control vs. STZ, p = 0.0006; STZ + sh-NC vs. STZ + sh-ANGPTL2, p = 0.0010; STZ vs. STZ + sh-NC, ns. Data are presented as mean ± SD.

• 

  Figure 3. ANGPTL2 overexpression enhances angiogenic responses in HRMECs. (A) RT-qPCR of ANGPTL2 mRNA in Control, OE-NC, and OE-ANGPTL2 HRMECs, normalized to β-actin (n = 3 independent experiments). OE-NC vs. OE-ANGPTL2, p < 1 × 10−15. (B) Western blot and quantification of ANGPTL2 protein (n = 3 independent experiments). OE-NC vs. OE-ANGPTL2, p = 9.37 × 10−8. (C) CCK-8 cell viability assay (n = 3 independent experiments, 9 replicates per group). OE-NC vs. OE-ANGPTL2, p = 1.00 × 10−13. (D) Representative wound healing images at 0 h and 48 h with quantification of wound closure ratio (%) (n = 3 independent experiments, 9 replicates per group). Scale bar, 100 μm. OE-NC vs. OE-ANGPTL2, p < 1 × 10−15. (E) Representative Transwell invasion images with quantification of invading cell number per field (n = 3 independent experiments, 9 replicates per group). Scale bar, 50 μm. OE-NC vs. OE-ANGPTL2, p = 2.20 × 10−14. (F) Representative tube formation images with quantification of tube number (n = 3 independent experiments, 9 replicates per group). Scale bar, 100 μm. OE-NC vs. OE-ANGPTL2, p = 1.31 × 10−13. Data are presented as mean ± SD.

• 

  Figure 4. ANGPTL2 overexpression is accompanied by upregulation of angiogenic markers and integrin α5β1 in HRMECs. (A) Western blot of VEGF, p-VEGFR2 (Tyr1059), and total VEGFR2 with quantification (n = 3 independent experiments). VEGF/β-actin: OE-NC vs. OE-ANGPTL2, p = 0.00001. p-VEGFR2/VEGFR2: OE-NC vs. OE-ANGPTL2, p = 0.0098. (B) Western blot of integrin α5 and integrin β1 protein levels in Control, OE-NC, and OE-ANGPTL2 HRMECs with quantification (n = 3 independent experiments). Integrin α5: OE-NC vs. OE-ANGPTL2, p = 4.66 × 10−5 (one-way ANOVA with Tukey post hoc). Integrin β1: OE-NC vs. OE-ANGPTL2, p = 1.13 × 10−5 (one-way ANOVA with Tukey post hoc). Data are presented as mean ± SD.

• 

  Figure 5. ANGPTL2 overexpression is accompanied by PI3K/AKT phosphorylation changes in HRMECs. Representative western blot images of p-PI3K, total PI3K, p-AKT, total AKT, and β-actin in Control, OE-NC, and OE-ANGPTL2 HRMECs. Quantification of p-PI3K/PI3K and p-AKT/AKT ratios (n = 3 independent experiments) is shown. No significant difference was detected between Control and OE-NC groups. p-PI3K/PI3K: OE-NC vs. OE-ANGPTL2, p = 0.00061. p-AKT/AKT: OE-NC vs. OE-ANGPTL2, p = 0.00006. ns, not significant (Control vs. OE-NC). Data are presented as mean ± SD.