Circulating chaperone GRP78 is elevated in patients with coronary artery disease and reduces vascular inflammation in human coronary endothelial cells
Authors: Repges E , Al Zaidi M , Sommer-Weisel S , Becher M U , Jansen F , Zimmer S , Tiyerili V , Nickenig G , Aksoy A 
Published: October 2021

Abstract

Introduction
Oxidative stress and vascular inflammation play a critical role in atherogenesis. Glucose-Regulated Protein 78kD (GRP78) is a chaperone and main regulator of the endoplasmic-reticulum (ER) stress response which is triggered by a variety of conditions that disturb folding of proteins in the ER. In case of ER Stress, GRP78 activates the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. Circulating chaperones have emerged as both effectors and prognostic markers for various cardiovascular diseases, including Coronary Artery Disease (CAD) and Acute Coronary Syndrome (ACS). The aim of this study was to investigate the role of circulating chaperone GRP78 in CAD.

Methods and results
To investigate the effect of GRP78 we performed in vitro experiments. HCAEC were incubated with ER stress inductor Tunicamycin to obtain GRP78 containing conditioned medium. To confirm the active secretion of GRP78 into the extracellular space, GRP78 protein levels of the medium were detected via Western Blot and ELISA (Graphical Abstract).
For control medium, HCAEC were incubated with Tunicamycin and Brefeldin A, which inhibits the ER-Golgi protein transport and therefore the active secretion of GRP78.
Treatment with GRP78 containing conditioned medium decreases intracellular level of GRP78 (0.35 fold vs. control, p&lt;0.0001). Furthermore, it enhances viability (93.0% vs. 79.6%, p=0.0168) and decreases mRNA expression of markers of vascular inflammation and ER Stress in HCAEC (NF-κB; 0.51 fold vs. control, p=0.001, and CHOP; 0.48 fold vs. control, p&lt;0.0001, respectively). In addition, we observed a decrease of intracellular formation of oxidative stress (0.78 fold, p=0.07).
To confirm that these effects depend on GRP78, we repeated those assays by directly treating with commercially available recombinant form of GRP78 and observed similar outcomes in a dose depending manner.
Furthermore, PCR profiler assays of markers of vascular inflammation with GRP78-treated HCAEC showed a robust increase of chemokine C-X-C motif chemokine ligand 10 (CXCL10), which has been attributed both proatherogenic and protective properties in vascular inflammation (6.51 fold).
Finally, we measured levels of circulating GRP78 in patients undergoing coronary angiography. Mean serum levels of GRP78 were higher in patients with Chronic Coronary Syndrome and ACS as compared to patients without CAD (2553 ng/ml [95% CI: 2152–2953] vs. 2081 ng/ml [95% CI: 1702–2460] vs. 1872 ng/ml [95% CI: 1594–2194], p=0.017, n=514).

Conclusion
GRP78 enhances viability and reduces vascular and oxidative stress HCEAC, suggesting protective properties of circulating chaperones in endothelial inflammation. GRP78 appears to be a possible therapeutic option in order to decelerate progression of cardiovascular diseases and could serve as a biomarker with diagnostic significance.

Funding Acknowledgement
Type of funding sources: Foundation. Main funding source(s): German Heart Foundation e.V. (Kaltenbach Stipendium) Graphical Abstract