Galectin-3 requires VEGF to activate VEGF receptor 2 in retinal endothelial cells

Abstract

Purpose : Galectin-3 is a carbohydrate-binding protein that modulates vascular endothelial growth factor receptor 2 (VEGFR2) signal transduction, but the molecular mechanism of Gal3 in VEGF/VEGFR2 signal transduction has not been fully elucidated. To better define the role of VEGF in Gal3-induced VEGFR2 signaling, we determined whether exogenous Gal3 requires VEGF to activate VEGFR2 signaling and if exogenous VEGF requires any Gal3 to activate VEGFR2.

Methods : To examine the interaction between Gal3 and VEGFR2, Gal3 was conjugated to cyanogen beads and used for immunoprecipitation (IP). VEGFR2 activation was inhibited using the small molecule VEGFR2- selective tyrosine kinase inhibitor, sunitinib malate (10 μM). Semi-quantitative PCR was used to measure the expression levels of E-selectin and VCAM-1 mRNA, genes that are downstream of VEGFR2. The knockdown of Gal3 was achieved in human retinal microvascular endothelial cells (HRECs) using siGal3, with nontargeting siRNA as a control. HREC surface proteins were labeled with NHS-SS-biotin and isolated using avidin beads. VEGF neutralization was attained with ranibizumab (10 μg/ml). The ability of exogenous Gal3 (50 μg/ml) to activate VEGFR2 kinase was measured at 0, 5, 10, 30, and 60 min after addition. Levels of total and phosphorylated VEGFR2 (Y1175), CD31, and tubulin were examined using western blot. HREC migration was determined using a wound-healing assay, in which confluent HRECs were mechanically scratched and cell migration was measured 15 hours after the addition of VEGF (10 ng/ml) or Gal3 (50 μg/ml).

Results : VEGFR2 was IP-ed with Gal3. Exogenous Gal3 induced an increase in E- selectin (0.99 ± 0.07 vs 15.3 ± 1.2, p<0.004. N=3) and VCAM-1 (1 ± 0.03 vs 3.4 ± 0.3, p<0.02. N=3) expression compared to the control. Inhibition of VEGFR2 activation by sunitinib significantly reduced exogenous Gal3-induction of E-selectin (91%, p<0.001. N=3) and VCAM-1 (67%, p<0.02. N=3). Gal3 was knockdown by over 90% (0.064 ± 0.001 vs 1 ± 0.14, p<0.05. N=3). Loss of endogenous Gal3 did not alter VEGF-induced VEGFR2 internalization or migration in HRECs. Neutralization of VEGF significantly inhibited the migration of exogenous Gal3-induced HREC migration compared to the control (1.17 ± 0.059 vs 0.97 ± 0.066, p<0.05. N=6).

Conclusions : Any VEGF is necessary for Gal3 induced kinase activation and HREC migration but endogenous Gal3 is not sufficient for VEGF/VEGFR2 signal transduction.

This is a 2021 ARVO Annual Meeting abstract.