Citrullination or conversion of peptidyl-arginine to peptidyl-citrulline is PTM of proteins catalyzed by peptidyl-arginine deiminases (PAD). It alters the structure and function of proteins e.g. by modification of the active site. Increased levels of PAD and citrullinated proteins are linked to many pathological conditions, especially autoimmune disorders and cancer. The physiological role of this process is still poorly understood, particularly in endothelial cells (ECs), which regulate important processes including hemostasis and angiogenesis.This study aimed to determine how pharmacological inhibition of citrullination affects the functions of human vascular ECs. Two models were used: an immortalized microvascular endothelial line (HMEC-1) and primary umbilical vein ECs (HUVEC). We used three irreversible PADs inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. To verify the research goal, we performed: a cytotoxicity test of the tested inhibitors; analysis of citrullination level of histone H3 (H3cit); assessment of the impact of PADs inhibition on the angiogenic potential (pseudocapillary structures formation, wound healing, MMPs activity and analysis of ECs secretory profile). Gene expression was analyzed by qPCR and protein levels by Western blotting. To explain the differential cytotoxicity of the inhibitors, a comet assay as well as analyzes of the interaction of the inhibitors with DNA were performed using fluorescence spectroscopy, and the results were verified by in silico modeling.
All inhibitors effectively decreased H3cit in ECs and decreased cells migration formation of pseudocapillary structures. Importantly, we report a decrease in the production/secretion of the key angiogenesis regulator – VEGFA. Additionally, activation of the PI3K/Akt pathway was decreased, which may explain the antiangiogenic effects. Our results indicate that PAD inhibitors significantly modulate the functions of ECs, significantly reducing their angiogenic potential.