Abstract 11: Thioredoxin-Interacting Protein Is Required for VEGF-Induced Angiogenesis Through Regulation of VEGFR2 Internalization

Abstract

Introduction— Thioredoxin-interacting protein (TXNIP) is an arrestin-like scaffold protein. We have shown previously that it is necessary for the transactivation of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) as well as promoting the migration and survival of endothelial cells (ECs). However, its roles in VEGF-induced angiogenesis and in vivo studies of TXNIP function have not been elucidated. Hypothesis— TXNIP regulates VEGF-mediated angiogenesis through modulation of angiogenic signaling pathways in ECs. Methods and Results— To determine the functions of TXNIP in ECs, we generated endothelial-specific TXNIP knockout (EC-TXNIP KO) mice (TXNIPflox/flox: Tie2-Cre/+). These mice displayed impaired capillary growth of the retinal vasculature compared to control mice. Furthermore, aortic rings from EC-TXNIP KO mice exhibited fewer and shorter vascular sprouts than those in control mice. To investigate the role of TXNIP in the regulation of VEGF-induced angiogenesis, we determined the subcellular localization of TXNIP in human umbilical vein EC (HUVEC). Immunofluorescence and cell fractionation studies revealed that upon VEGF stimulation (10ng/ml). TXNIP translocated from cytoplasm to the plasma membrane. There was a 9 fold increase of membrane associated TXNIP with a peak at 15 minutes compared to non-VEGF treatment cells. We hypothesized that membrane associated TXNIP may modulate VEGFR2 internalization and thereby affect VEGF-induced signaling and angiogenesis. To investigate this, we performed in vitro cell surface biotinylation assays in HUVEC. VEGFR2 internalization was decreased by 65% in TXNIP siRNA knockdown cells compared to control siRNA treated cells following VEGF stimulation. Consistent with this result, VEGF-induced phosphorylation of VEGFR2, PLCγ and ERK1/2 was decreased by knockdown of TXNIP. Significantly, TXNIP knockdown inhibited VEGF-induced proliferation and tube formation in vitro. Conclusion— Our results suggest that TXNIP can modulate VEGF-induced angiogenesis and signaling by regulation of VEGFR2 internalization.