Introduction

Oxidative stress and vascular inflammation play a critical role in atherogenesis. Glucose-Regulated Protein, 78kD (GRP78), is a chaperone and the 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 Syndrom (ACS). The aim of this study was to investigate the role of circulating chaperone GRP78 in human coronary endothelial cells.

Methods and Results

We measured serum levels of circulating GRP78 in patients undergoing coronary angiography with CAD, ACS, and without CAD. Mean serum levels of GRP78 were higher in patients with CAD and ACS as compared to patients without CAD (4086 + 2724 ng/ml vs. 3577 + 2499 ng/ml vs. 2508 + 1908 ng/ml, p=0.024; n=120). To investigate the effect of GRP78 we performed in vtiro experiments. HCAEC were incubated with ER stress inductor Tunicamycin to produce 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 (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<0.0001). Furthermore, it enhances viability (93.0 % vs. 79.6%, p=0.0168) and decreases mRNA expression of NF-κB (0.51 fold vs. control, p=0.001) as well as proapoptotic markers of CCAAT-enhancer-binding protein homologous protein (CHOP) (0.48 fold vs. control, p<0.0001) in HCAEC, respectively. In addition, we observed a decrease (0.78 fold, p=0.07) of intracellular formation of oxidative stress. To confirm that these effects depend on GRP78 we repeated those assays by directly treating with commercially available recombinant form of GRP78 as well as stressing with Tunicamycin after treatment with GRP78 and observed similar outcomes in a dose dependent manner.

Conclusion

GRP78 enhances viability and impairs vascular and oxidative stress in human coronary artery endothelial cells, 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.