Cystatin C Expression is Promoted by VEGFA Blocking, With Inhibitory Effects on Endothelial Cell Angiogenic Functions Including Proliferation, Migration, and Chorioallantoic Membrane Angiogenesis

Abstract

Background Vascular development, including vasculogenesis and angiogenesis, is involved in many diseases. Cystatin C (CST3) is a commonly used marker of renal dysfunction, and we have previously reported that its expression level is associated with variations in the gerbil circle of Willis. Thus, we hypothesized that CST3 may affect endothelial function and angiogenic capacity. In the current study, we sought to determine the influence of CST3 on endothelial function and explore its potential regulatory pathway. Methods and Results We analyzed CST3 and vascular endothelial growth factor A (VEGFA) levels in different developmental stages of gerbils using ELISAs and immunofluorescence (to examine the relationship between CST3 and VEGFA. We used a real‐time cell analyzer, cytotoxicity assays, and the chorioallantoic membrane assay to investigate the function of CST3 in endothelial cells and the chorioallantoic membrane. Additionally, we used Western blotting to explore the downstream targets of CST3. The expression levels of both CST3 and VEGFA were at their highest on day 10 of the embryonic stage. CST3 inhibited endothelial cell proliferation, migration, tube formation, and permeability, as well as vascular development in the chorioallantoic membrane. Blocking of VEGFA dose‐dependently increased CST3 expression in arterial and venous endothelial cells. Furthermore, overexpression and knockdown of CST3 significantly affected the protein levels of p53 and CAPN10 (calpain 10), suggesting that CST3 might play a role in vascular development through these proteins. Conclusions CST3 may be associated with vascular development and angiogenesis, and this effect could be promoted by blocking VEGFA.

Keywords

• cystatin C
• stroke
• vascular endothelial function
• vascular endothelial growth factor