ADAM-17 regulates endothelial cell morphology, proliferation, and in vitro angiogenesis

Abstract

Modulation of angiogenesis is a promising approach for treating a wide variety of human diseases including ischemic heart disease and cancer. In this study, we show that ADAM-17 is an important regulator of several key steps during angiogenesis. Knocking down ADAM-17 expression using lentivirus-delivered siRNA in HUVECs inhibited cell proliferation and the ability of cells to form close contact in two-dimensional cultures. Similarly, ADAM-17 depletion inhibited the ability of HUVECs to form capillary-like networks on top of three-dimensional Matrigel as well as in co-culture with fibroblasts within a three-dimensional scaffold. In mechanistic studies, both baseline and VEGF-induced MMP-2 activation and Matrigel invasion were inhibited by ADAM-17 depletion. Based on our findings we propose that ADAM-17 is part of a novel pro-angiogenic pathway leading to MMP-2 activation and vessel formation.

Fig. 1. Characterization of ADAM-17 silencing in HUVECs

(A) Western blot analyses confirmed efficient inhibition of ADAM-17 expression (p < 0.001) and function (HB-EGF shedding, p<0.01. (B) Fluorescence microscopic image illustrating the morphological differences between EGFP labeled siAD-17/MoAD-17 and siCo/MoCo. ADAM-17 silencing inhibits HUVEC proliferation (C) (* p<0.05), but not apoptosis (D).

Fig. 2. The effect of the inhibition of ADAM-17 activity and expression on HUVEC network formation

Inhibition of ADAM-17 protein expression by RNAi (siAD-17) and ADAM-17 activity with TAPI-1 (siCo + TAPI) causes early destabilization of HUVEC networks on three-dimensional Matrigel, when compared to their respective controls (siCo and siCo + GMneg).

Fig. 3. ADAM-17 silencing blocks capillary formation in HUVEC-fibroblast co-cultures in chitosan-GAG-collagen gels

Capillary like structure are observed in siCo cultures (A, B) but not in siAD-17 cultures (C, D). Because only the HUVECs express EGFP, the fibroblast and the matrix are not visualized.

Fig. 4. ADAM-17 in VEGF signaling

(A) ADAM-17 silencing inhibits basal and VEGF-induced HUVEC invasion through three-dimensional Matrigel. Invading cells were detected by fluorescent labeling using Calcein. (B) ADAM-17 silencing inhibits basal and VEGF-induced MMP-2 activation in HUVEC, but does not affect MT1-MMP and MT3-MMP expression. Representative zymogram (ZY) and Western blots (WB) from three independent experiments. Densitometric analyses of the zymogram in (B): (C) latent MMP-2, (D), intermediate size MMP-2, and (E) fully activated MMP-2 (* p<0.05, ** p<0.01, *** p<0.001).