Introduction:
Pulmonary arterial hypertension (PAH) is driven by vascular remodelling due to inflammation and cellular stress, including endoplasmic reticulum stress (ER stress). The main ER-stress chaperone, glucose-regulated protein 78 kDa (GRP78), is known to have protective effects in inflammatory diseases through extracellular signalling. The aim of this study is to investigate its significance in PAH.  

Methods:

Pulmonary arterial smooth muscle cells (PASMC) were stimulated with compounds that induce ER stress (tunicamycin and thapsigargin), after which the secretion of GRP78 into the cell medium was analyzed by western blot. New PASMC were treated with ER-Stress conditioned medium of donor PASMC. Effects on viability (alamarblue), reactive oxygen species formation (ROS, DCFDA assay) were analyzed by fluorescency-based microplate assay. Expression of markers of vascular inflammation and ER Stress was assessed by quantitative PCR.
Blood samples of patients with Nice Group I PAH presenting to our outpatient clinic were collected. Plasma levels of GRP78 were measured by ELISA. Patients were risk stratified according to current ESC guidelines (NT-proBNP, 6-minutes-walking-distance, SvO₂, cardiac index, right atrial pressure, WHO functional class). 

Results:
We found that when ER stress was induced in PASMC, there was also a time-dependent extracellular secretion of GRP78. After 48 hours, secretion of GRP78 was found to be most pronounced. We next treated naïve PASMC with conditioned medium (CM) from the ER-stressed donor PASMC. Incubation with CM from ER-stressed PASMC reduced the viability, oxidative stress and expression of inflammatory and ER-stress markers in target cells. These effects were abrogated when the donor cells were co-treated with Brefeldin A to inhibit active secretion of GRP78. Importantly, viability was only reduced when PASMC were pre-treated with PAH-promoting factors like PDGF, suggesting that GRP78 secretion exhibits no toxic effects under basal conditions.
Direct treatment of PASMC with recombinant GRP78 modulated the expression of key inflammatory markers. Additionally, we measured GRP78 plasma levels in 19 PAH patients (Nice Group I) and correlated the levels to risk stratification according to ESC guidelines. Mean age of patients was 62.5 ± 17 years and 52 % were female. The majority of patients were classified as WHO FC II (42.1%) or III to IV (47.3%) and were predominantly receiving PAH combination therapy (68.4%). Elevated plasma levels of GRP78 were associated with a favourable risk stratification (low-risk patients: 1346 ± 299 ng/ml vs. high-risk patients: 796 ± 203 ng/ml, p = 0.0008).

Conclusion:

In conclusion, GRP78 is actively secreted by PASMC under ER stress and exhibits protective effects from the hallmarks of PAH in vitro. Circulating GRP78 may serve as biomarker for risk adjudication of patients with PAH.