Introduction

Glucose-Regulated Protein 78kD (GRP78) is a chaperone and the main regulator of the ER-stress response which is triggered by a variety of conditions that disturb folding of proteins in the ER. Upon ER Stress, GRP78 activates the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. A prolonged activation of the UPR triggers inflammation, thus contributing to the progression of cardiovascular diseases. Recently, extracellular secretion of GRP78 was described. However, the pathophysiological relevance of secreted GRP78 in atherosclerosis and endothelial cell inflammation remains to be elucidated. 

Aim of this study is to investigate the role of GRP78 secretion in endothelial cells.

Methods and Results

First, we sought to investigate if vascular cells secrete GRP78 during ER Stress. Human coronary artery endothelial cells (HCAEC) were treated with the ER stress inductors tunicamycin or thapsigargin for up to 48h. After ER Stress induction, Western Blot and ELISA experiments detected an increased intracellular GRP78 expression. Intriguingly, prolonged ER Stress also promoted extracellular secretion of GRP78. Unbiased proteomic analysis of the HCAEC secretome confirmed that after ER-Stress induction, GRP78 is one of the most highly upregulated extracellular proteins (2.43-fold). Co-incubation with Brefeldin A, an inhibitor of ER-Golgi protein transport, abolished extracellular secretion (Fig.1). Hence, ER-Stress-induced GRP78 secretion is an actively regulated process.

Next, the effect of GRP78 containing conditioned medium (CM) on HCAEC was analyzed. Treatment with GRP78 containing CM decreased GRP78 mRNA expression in target cells (0.35-fold vs. control [+BFA], p<0.0001). Furthermore, it increased viability (93.0 % vs. 79.6%, p=0.017) and decreased the rate of apoptosis (1.14 vs. 3.24, p=0.0025). Moreover, expression of markers of vascular inflammation and ER Stress (e.g., NF-κB and CHOP) were decreased when compared to control CM with additional BFA treatment (Fig.2).

However, the CM used does not allow the protective properties to be clearly attributed to GRP78 alone. Thus, we chose two further approaches to produce CM in which we used Antibodies against GRP78 on the one hand and siRNA-Transfection against GRP78on the other.

After treatment with non-GRP78 containing CM, apoptosis and expression of markers of vascular inflammation and ER stress (e.g., ICAM-1, ATF-4) were increased compared to control CM. Furthermore, higher levels of reactive oxygen species were measured (0.74-fold vs. control [+IgG], p=0.0001) (Fig.2).

Thus, GRP78 is responsible for the beneficial effects that are observed after CM treatment. 

Conclusion

Endothelial ER Stress promotes GRP78 secretion. Presence of GRP78 in conditioned medium ameliorates subsequent ER Stress and endothelial inflammation, which play a critical role in atherogenesis. Further investigations are important to shed light on the mechanism of release and the effect on target cells.