Abstract 326: Granzyme B Releases Vascular Endothelial Growth Factor from Extracellular Matrix Stores and Induces Vascular Permeability in vivo
Introduction The formation of unstable and leaky neovessels underlies the pathogenesis of a large number of chronic inflammatory diseases. Granzyme B (GZMB) is a serine protease that is expressed and released by a variety of immune cells and accumulates in the extracellular matrix (ECM) during chronic inflammation where it cleaves a number of ECM proteins, including fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN in both normal and diseased tissue. We hypothesize that GZMB cleavage of FN will release VEGF from its extracellular stores and promote vascular permeability as a mechanism that contributes to neovessel leakage during chronic inflammation.
Methods GZMB-mediated VEGF release from either FN coated wells or endogenously produce endothelial cell (EC) matrix was measured by VEGF ELISA. VEGF-release supernatants were used to treat EC and VEGF receptor 2 (VEGFR2) activation was evaluated by immunoblotting for phosphorylated VEGFR2. Evan’s blue was injected intravenously to CD1 mice followed by ear injection of either mouse GZMB, saline control, GZMB + neutralizing mouse VEGF antibody or GZMB+ IgG control (n=5 for each experimental group). Vascular leakage was evaluated by Evan’s blue dye extraction.
Results GZMB effectively releases VEGF from both FN and from EC matrix, while inhibition of GZMB prevented VEGF release. GZMB-mediated VEGF release resulted in significant activation of VEGFR2 in EC monolayer signified by increased VEGFR2 phosphorylation. GZMB ear injection resulted in a significant increase in vascular permeability in vivo. Importantly, co-injection of GZMB and neutralizing mouse VEGF antibody significantly reduced vascular leakage compared to co-injection of GZMB and matching IgG control.
Conclusions and Impact GZMB increases VEGF bioavailability by releasing it from the ECM leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.
- © 2013 by American Heart Association, Inc.