SiRNA silencing of VEGF, IGFs, and their receptors in human retinal microvascular endothelial cells

Abstract

Vascular endothelial growth factor (VEGF) is a potent mitogen that regulates proliferation, migration, and tube formation of endothelial cells (EC). VEGF has recently become a target for severe retinopathy of prematurity (ROP) therapy. We tested the hypothesis that a specific VEGF isoform and/or receptor acts synergistically with insulin-like growth factor (IGF)-I to alter normal retinal microvascular EC angiogenesis and RNA interference can be used to reverse VEGF effects. We used small interfering RNA (SiRNA) transfection to target VEGF isoforms, IGFs, and their receptors in human retinal microvascular endothelial cells (HRECs). Media was collected at 24 and 48 hours post transfection for measurement of VEGF, sVEGFR-1 and IGF-1 levels; and HRECs were assessed for migration, tube formation, VEGF signaling genes, oxidative stress, and immune-reactivity. At 24 hours post transfection VEGF increased with VEGFR-2; sVEGFR-1 decreased with VEGF₁₆₅, VEGFR-2, and IGF-1R; and IGF-I increased with VEGF₁₈₉, VEGFR-1, IGF-2R, IGF+VEGF₁₆₅, and IGF+VEGF₁₂₁. IGF-I transfection with each VEGF isoform reduced sphere- forming and migration capacities with robust upregulation of caspase-9, COX-2, MAPK, PKC, and VEGF receptors. At 48 hours, the effects were reversed with a majority of genes downregulated, except with IGF-I and NP-1 transfection. Using RNA interference for targeted inhibition of VEGF isoforms in conjunction with IGF-I may be preferable for suppression of HREC overgrowth in vasoproliferative retinopathies such as ROP.

Keywords: Angiogenesis; insulin-like growth factor; retinal endothelial cells; small interfering RNAs; vascular endothelial growth factor.

Figure 1

Uptake of VEGF SiRNA at 24 (A) and 48 (B) hours post transfection. Effect of selective SiRNA inhibition of VEGF isoforms, VEGF receptors, and/or IGF-I on lactate dehydrogenase activity, in the media of proliferating human retinal endothelial cells (HRECs) at 24 (C) and 48 (D) hours post transfection. Data are expressed as mean ± SEM (n=12 samples/group; *P<0.05, **P<0.01 vs. control). Images were captured at 4× magnification.

Figure 2

Expression of total VEGF in cells at 24 (A) and 48 (B) hours post transfection. Effect of selective SiRNA inhibition of VEGF isoforms, VEGF receptors, and/or IGF-I on VEGF levels in the media of proliferating human retinal endothelial cells (HRECs) at 24 (C) and 48 (D) hours transfection. Data are expressed as mean ± SEM (n=12 samples/group; *P<0.05, ***P<0.001 vs. control). Images were captured at 20× magnification.

Figure 3

Expression of total VEGFR-1 in cells at 24 (A) and 48 (B) hours post transfection. Effect of selective SiRNA inhibition of VEGF isoforms, VEGF receptors, and/or IGF-I on sVEGFR-1 levels in the media of proliferating human retinal endothelial cells (HRECs) at 24 (C) and 48 (D) hours post transfection. Data are expressed as mean ± SEM (n=12 samples/group; *P<0.05, ***P<0.01 vs. control). Images were captured at 20× magnification.

Figure 4

Expression of total IGF-I in cells at 24 (A) and 48 (B) hours post transfection. Effect of selective SiRNA inhibition of VEGF isoforms, VEGF receptors, and/or IGF-I on IGF-I levels in the media of proliferating human retinal endothelial cells (HRECs) at 24 (C) and 48 (D) hours post transfection. Data are expressed as mean ± SEM (n=12 samples/group; *P<0.05, **P<0.01, ***P<0.001 vs. control). Images were captured at 20× magnification.

Figure 5

Representative sample of migration capacity of human retinal endothelial cells (HRECs) at 24 (A) and 48 (B) hours post VEGF transfection. Cells were stained with calcein AM fluorescent dye, the plates were imaged, and the number of cells that migrated to the bottom was determined at 24 (C) and 48 (D) hours. Data are expressed as mean ± SEM (n=4 samples/group; *P<0.05, **P<0.01 vs. control). Images were captured at 4× magnification.

Figure 6

Representative sample of tube formation capacity of human retinal endothelial cells (HRECs) at 24 (A) and 48 (B) hours post transfection. Cells were resuspended in serum-free media and seeded onto 96 well plates. The plates were placed incubated under their growth conditions for 7 days and scored for the presence or absence of spheres at 24 (C) and 48 (D) hours. Data are expressed as mean ± SEM (n=96 samples/group; *P<0.05, **P<0.01, ***P<0.001 vs. control). Images were captured at 20× magnification.

Figure 7

Representative sample of oxidative stress in of human retinal endothelial cells (HRECs) at 24 (C) and 48 (D) hours post VEGF transfection. Panels (A and B) represent control non-transfected cells at 24 and 48 hours, respectively. Oxidative stress was determined using the Image-It lipid peroxidation assay. Images show intense green staining in the SiRNA exposed cells suggesting lipid peroxidation. Images were captured at 20× magnification.