AUTHOR=Birch Cierra A. , Wedegaertner Helen , Orduña-Castillo Lennis B. , Gonzalez Ramirez Monica L. , Qin Huaping , Trejo JoAnn 

TITLE=Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

JOURNAL=Frontiers in Molecular Biosciences

VOLUME=Volume 10 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2023.1211597

DOI=10.3389/fmolb.2023.1211597

ISSN=2296-889X

ABSTRACT=Introduction:  Dysfunction of the endothelium impairs its’ protective role and promotes inflammation and progression of vascular diseases. Activated protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic.  Despite recent advancements in developing new APC variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance APC and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses.

Methods: Quantitative multiplexed mass spectrometry analysis of APC treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. 

Results: Here we report that APC modulates phosphorylation of tumor necrosis factor (TNF)- signaling pathway components and attenuates of TNF- induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the GPCR co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. 

Discussion:  This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete -arrestin-2-driven signaling pathways modulated by specific GPCR co-receptors and GRKs.