PGF-VEGFR1 signaling mediates the crosstalk of retinal endothelial cells and pericytes in diabetic retinopathy

Abstract

Purpose : To investigate the crosstalk of retinal endothelial cells (REC) and pericytes mediated by placental growth factor-vascular endothelial growth factor receptor 1 (PGF-VEGFR1) signaling in diabetic retinopathy (DR).

Methods : REC and pericytes were treated with 25 mM high glucose (HG); 5 mM normal glucose (NG) acted as a control. HG-induced REC and pericytes apoptosis were examined by TUNEL and active Caspase-3 staining. PGF and VEGFR1 activities were blocked by their respective neutralizing antibodies. The proteins affected by PGF/VEGFR1 blockade were identified using proteomics followed by Western blots or ELISA. The pericytes-conditioned media (per-CM) were harvested for protein identification and REC treatment. The barrier functions of REC monolayers, which were treated with HG, antibody, or per-CM, were examined with Electric Cell-substance Impedance Sensing (ECIS).

Results : Proteomics identified that protective proteins were upregulated by PGF antibody in REC. Upregulations of several antioxidant enzymes (i.e., Prx3, Prx6, and GSTP1) were validated with Western blots. Concomitantly, PGF antibody prevented HG-induced barrier dysfunction of REC monolayers, as determined by a real-time trans-endothelial electrical resistance (TEER) with the ECIS system. Similar to in vitro, the protein levels of three anti-oxidant enzymes were upregulated in the PGF knockout mouse retinas, which were associated with DR attenuation. HG-induced apoptotic pericytes had an activated VEGFR1 signaling, as shown by the co-localization of phosphorylated (p) VEGFR1 and TUNEL (+) or active Caspase-3 (+) cells. VEGFR1 antibody significantly inhibited HG-induced pericytes apoptosis. Additionally, per-CM of VEGFR1 antibody prevented HG-induced retinal EC barrier dysfunction, but per-CM of control IgG or EC-CM of anti-VEGFR1 did not, suggesting that VEGFR1 regulates EC barrier functions indirectly through pericytes.

Conclusions : Our results suggest that PGF-VEGFR1 signaling mediates the crosstalk of REC and pericytes in DR. In REC, PlGF inhibition upregulates protective proteins, which prevent retina from HG-induced cell apoptosis and barrier dysfunction. In pericytes, VEGFR1 inhibition prevents HG-induced apoptosis and cytokine production. Therefore, PGF and VEGFR1 can act as therapeutic targets for the treatment of DR.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.