Prognostic significance of circulating level of GRP78 in pulmonary arterial hypertension

Abstract

Background

Endoplasmic reticulum (ER) stress is associated with pulmonary arterial hypertension (PAH), and glucose-regulated protein 78 (GRP78) is an important marker of ER stress in PAH. Most studies have shown that its expression is increased at both lung and right ventricle as well as pulmonary vascular cells during ER stress. Currently, circulating GRP78 levels are also shown to be significantly increased in several diseases and can be used as a disease biomarker.

Methods

Culture media from pulmonary artery smooth muscle cells (PASMCs) and cardiomyocytes with different trigger interventions were collected. Plasma was harvested from control, monocrotaline (MCT)-treated and Sugen-hypoxia (SuHx) treated rats. Plasma was also collected from 66 PAH patients and 40 healthy controls. GRP78 levels were measured by ELISA.

Results

The extracellular GRP78 levels were significantly increased in hypoxia-treated PASMCs and cardiomyocytes compared with the normoxia group. Circulating levels of GRP78 were significantly increased in the MCT and SuHx groups compared with the control group. Circulating GRP78 levels are higher in PAH patients. In subgroup analyses, GRP78 was higher in the Idiopathic PAH (IPAH), Congenital heart disease-associated PAH (CHD-PAH), world heart World Health Organization Function Class (WHO-FC) III/IV, and intermediate-high-risk groups. Cox regression analysis (hazard ratio, HR) and Kaplan-Meier survival analysis showed that GRP78 was a prognostic risk factor for event-free survival (HR 1.422 per 1 µg/mL, 95% CI 1.188–1.702).

Conclusion

In PAH, circulating GRP78 is significantly elevated and serves as an incremental prognostic biomarker for event-free survival.

Introduction

Pulmonary arterial hypertension (PAH) is a chronic progressive malignant disease characterized by severe pulmonary vascular remodeling [1]. In recent years, with the introduction of new targeted drugs for PAH and the gradual standardization of clinical management, PAH patients’ event-free survival rate and life quality have been significantly improved [2]. Although several targeted agents are available to improve hemodynamics, these agents do not reverse pulmonary vascular remodeling, and long-term patient outcomes are suboptimal. Therefore, it is still very important to explore new markers, diagnostic and therapeutic targets for PAH, which can guide the treatment strategy and the prognosis of patients.

The endoplasmic reticulum (ER) is an important organelle for intracellular protein synthesis and modification, and is very sensitive to stimuli both inside and outside the cell [3].Various PAH disease triggers such as hypoxia, oxidative stress, genetic mutations, inflammation, and cytokines, will disturb ER function, disrupt cellular homeostasis and affect ion transport and proper protein folding, leading to the occurrence of ER stress [4]. ER stress has found in various types of PAH patients as well as in PH animal models [5–7]. Studies have shown that ER stress involved in the occurrence and development of PAH, not only participated in pulmonary vasculature, but also detected in the right heart and circulating monocytes [8, 9]. ER stress inhibitors have proved to ameliorate pulmonary artery remodeling and right heart failure in numerous PH animal models [10]. Although animal studies suggest that inhibiting ER stress have an excellent result on ameliorate PAH, we still need further evidence to indicating how it works in human and seeking some detailed information on how to examine it through a convenient way. While animal studies suggest that inhibition of ER stress has a promising effect on PAH improvement, we still need further evidence of its role in humans and seek some details on how to examine it in a convenient way.

Glucose-regulated protein 78 (GRP78) is a key sensor and effector of ER stress and with vital roles in this process. In experimental animal models, GRP78 has been shown to be a marker of ER stress in lung tissue and right heart. It’s expression also increase in pulmonary vascular cells and cardiomyocytes under conditions of hypoxia, growth factors or other PAH inducers [11, 12]. GRP78 is a key node of ER stress in the pulmonary vasculature and can either promote or inhibit smooth muscle cell proliferation, endothelial barrier disruption, and vascular remodeling. In the right ventricle, GRP78 is essential for cardiomyocyte survival and contractile function, but both excessively high and abnormally low expression can aggravate ventricular remodeling and heart failure, indicating that its level must be finely regulated. However, in addition to its role in the ER, it can also be detected in the cytoplasm, membrane and mitochondria, and secreted outside the cell. During ER stress, increased GRP78 production and altered subcellular distribution are accompanied by increased secretion into the blood circulation [13]. Recent studies have shown that secreted GRP78 can be used as a biomarker for various diseases, such as cancer, diabetes, metabolic syndrome and atherosclerosis [14, 15]. In addition, secreted GRP78 is still biologically active and can act as a ligand to cell surface receptors [16].

Therefore, although ER stress showed to involve in PAH, the specific role of circulating GRP78 in PAH has not been investigated. Here, we examined circulating GRP78 levels in 2 different PH rat models and extracellular levels of GRP78 in pulmonary artery smooth muscle cells (PASMCs) and cardiomyocytes in response to different disease-inducing agents to provide translational support for a cellular origin of circulating GRP78. More importantly, we systematically analyzed circulating GRP78 levels across PAH subgroups, characterized its association with NT-proBNP and hemodynamic parameters, and for the first time demonstrated its independent prognostic value for event-free survival in PAH patients.

Methods

Animal models

Eighteen male SD rats, weighing about 200 g, were purchased from SJA laboratory animal company (Changsha, China) and raised in the Experimental Animal Center and raised followed the guidance of Animal Experiment Guidelines of Central South University. After adapting the environment for around 1 week, rats were equally divided into 3 groups: MCT rat group, SuHx rat group and control group. MCT rats received monocrotaline (MCT, sigma, 60 mg/Kg) intraperitoneally on day 1. SuHx rats received weekly subcutaneous injections of Su5416 (Med Chem express, 20 mg/kg) and were left in a hypoxic chamber (10% oxygen), while control rats received vehicle injections for 3 weeks. After 3 weeks, right heart catheter (RHC) was done to measure right ventricular systolic blood pressure (RVSP). Hemodynamic and histological validation data are shown in Supplemental Figure S1A–B. At the time of anesthesia, we used 2% isoflurane inhalation, and at the time of execution of the animals we used 200 mg/kg sodium pentobarbital intraperitoneally and made sure that the animals were unconscious throughout the experiment. This approach is consistent with internationally recognized ethical guidelines for the care and use of laboratory animals and is designed to ensure the well-being of the animals and to minimize animal suffering during experimental procedures and euthanasia. Both MCT exposure and the SuHx protocol have been reported to activate ER stress pathways in pulmonary vascular and right ventricular tissues, supporting their relevance for studying GRP78 regulation in vivo. All animal procedures complied with institutional guidelines for the care and use of laboratory animals and were approved by the institutional animal ethics committe (No. 2021141).

Cells and treatments

Rat PASMCs were isolated as previously described [30, 31]. The whole pulmonary artery and its branches were collected and the inner layer was scraped. After cutting, small pieces of pulmonary artery around 1mm² were placed on the bottom of the culture flask. After about 3–7 days, PASMCs will grow out. PASMCs were used after characterization by immunofluorescence. PASMCs were treated with 10ng/ml platelet-derived growth factor-BB (PDGF-BB) or hypoxia (1% oxygen) for 24–48 h. H9C2 cardiomyocytes were purchased from Shanghai Fuheng Biotechnology (FH1004) and hypoxia (1% oxygen) for 24–48 h. Supernatants of both cells were obtained for further ELISA analysis. Western blot validation of intracellular GRP78 induction by hypoxia and PDGF-BB is provided in Supplemental Figure S1C–D. PDGF-BB is a PAH-relevant mitogen and has been reported to perturb ER homeostasis and activate ER stress signaling in vascular cells, which may increase GRP78 expression and extracellular release.

Study population

The cohort recruited 66 PAH participants from the Second Xiangya Hospital between July 2018 and July 2020. Inclusion criteria for the PAH group were: (1) age > 18 years; (2) RHC diagnosis, mPAP ≥ 25mmHg, PVR ≥ 3 wood units, PAWP ≤ 15mmHg; if there were established etiologies, including left heart failure, mitral valve disease, severe lung disease with hypoxemia, portal hypertension, or obstructive sleep apnea, patients with PAH were excluded. Detailed disease history and demographic data such as sex, age, height, weight, body mass index (BMI), PAH subtype, PAH-targeted therapy and WHO functional class, 6-minute walk distance, echocardiography, and RHC records were collected. Another 40 healthy controls were recruited from the physical examination center during the same period, and pulmonary hypertension was excluded by transthoracic echocardiography. The smaller number of controls relative to PAH patients reflects the strict inclusion and exclusion criteria applied during recruitment, which required the absence of any cardiopulmonary disease, malignancy, or significant comorbidities, and ensured comparability in age and sex distribution with the patient group. Unequal group sizes are common in case-control studies and do not compromise statistical validity when appropriate non-parametric methods are used, as applied throughout this analysis. The research protocol was approved by the Ethics Committee of the Second Xiangya Hospital of Central South University (No. 2020-085), and written informed consent was obtained from all participants. All procedures were carried out in accordance with the Declaration of Helsinki.

Follow-up of PAH patients

PAH patients were followed up every 6 months via medical record review and telephone contact until March 31, 2022. The primary end point was the first clinical worsening event, defined as all-cause mortality, lung transplantation, hospitalization due to right heart failure, escalation of PAH-targeted therapy, or deterioration in WHO functional class. The primary outcome is reported as event-free survival. All of this information was obtained by reviewing medical records and phone calls.

Sample and measurements

NT-proBNP was measured in the hospital laboratory using a commercially available ECLIA assay (Roche Diagnostics), according to the manufacturer’s instructions. Peripheral blood samples from PAH patients and healthy controls, rat serum samples, and conditioned media from cultured cells were aliquoted and stored at − 80 °C until analysis (avoiding repeated freeze–thaw cycles). GRP78 concentrations were measured using a commercially available ELISA kit (ENZO Life Sciences, ADI-900-214) according to the manufacturer’s protocol. All samples were assayed in duplicate.

Statistical analysis

Normal distribution data were analyzed by student’s t test between two groups or one-way analysis of variance between several group. Nonparametric Kruskal-Wallis H test was used for variance inhomogeneity. Correlations between clinical, echocardiographic, and hemodynamic parameters were assessed using Spearman’s rank correlation analysis, given the non-normal distribution of several variables. To account for multiple testing, P values derived from correlation analyses were adjusted using the Benjamini–Hochberg false discovery rate (FDR) method. To assess the HR of event-free survival with 95% confidence intervals, univariate and multivariable COX proportional hazards models were used. To specifically assess the incremental prognostic value of circulating GRP78 beyond NT-proBNP, nested Cox proportional hazards models were constructed. A base model including log-transformed NT-proBNP was compared with an extended model additionally including GRP78. Model improvement was assessed using the likelihood ratio test (LRT) for nested models, along with changes in the Akaike information criterion (AIC) and Harrell’s concordance index (C-index). The proportional hazards assumption was evaluated using Schoenfeld residuals and was satisfied for all models. Statistical assessments and plots were done by using GraphPad Prism 6.0 (GraphPad Software Inc., USA) or R (version 4.0.1).

Results

Analysis of Circulating GRP78 in Animal Models of PH and PAH Patients

Higher circulating level of GRP78 in PH animal models and increased extracellular GRP78 in hypoxia treated PASMCs and cardiomyocytes

Both monocrotaline (MCT) induced or Sugen-Hypoxia (SuHx) PH animal models showed significantly increased circulating GRP78 level compared with control group (MCT vs. control: P = 0.033; SuHx vs. control: P = 0.003; Fig. 1A). Successful establishment of the PH phenotype was confirmed by elevated RVSP and pulmonary vascular remodeling on HE staining (Supplemental Figure S1A–B). Further, to test whether pulmonary vasculature or the heart is related to the increase of circulating GRP78, PASMCs and H9C2 cardiomyocytes were treated with different PAH specific triggers and then extracellular GRP78 in the culture medium were measured. Meanwhile, a cytokine PDGF-BB, which is elevated in PAH and causes PASMCs proliferation, was used to treat PASMC. Although there was a small increase in extracellular GRP78, the results were not statistically different after PDGF-BB treatment. (Fig. 1B). When PASMCs and H9C2 cardiomyocytes were exposed to hypoxia, increased extracellular GRP78 were showed in both cells (PASMCs: P = 0.0286; H9C2: P = 0.0079; Fig. 1C, D).Furthermore, intracellular GRP78 protein expression was confirmed to be upregulated by both hypoxia and PDGF-BB stimulation in PASMCs and H9C2 cells by western blot (Supplemental Figure S1C–D).

Fig. 1.

Comparison of GRP78 levels in different in vivo and in vitro PAH models. (A) Comparisons between circulating GRP78 between control, MCT treated and Sugen-Hypoxia (SuHx) treated rats; (B) Comparisons of GRP78 in the cell culture medium between control or PDGF-BB treated PASMCs; (C) Comparisons of GRP78 in the cell culture medium between normoxia or hypoxia treated PASMCs; (D) Comparisons of GRP78 in the cell culture medium between normoxia or hypoxia treated H9C2 cells. In in vivo experiments, n = 6 for each group. For PDGF-BB or Hypoxia treated PASMCs, n = 4 for each group and for hypoxia treated H9C2 cells, n = 5 for each group. Statistical analysis was performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparisons test for three-group comparisons and the Mann–Whitney U test for two-group comparisons. Exact p values are shown in the figure

Comparison of baseline characteristics and circulating GRP78 between PAH and healthy groups

Circulating GRP78 levels were tested by ELISA. 66 PAH patients and 40 healthy controls were enrolled. Baseline characteristics are shown in Table 1. PAH patients exhibited higher circulating GRP78 levels while compared with the healthy control group (3.20 ± 2.03 vs. 2.54 ± 0.66, P < 0.001) (Fig. 2A). Considering the inclusion of several PAH subtypes, comparisons between subgroups were measured. It indicated that GRP78 levels were elevated in IPAH (P < 0.001) and CHD-PAH patients (P = 0.019), but not in CTD-PAH, compared with healthy controls (Fig. 2B). GRP78 was also evaluated in different PAH risk stratification and World Health Organization Functional Class (WHO-FC). Compared with patients with low-risk PAH, GRP78 levels were higher in intermediate-risk (P = 0.028) and high-risk patients (P = 0.028) (Fig. 2C). Meanwhile, circulating GRP78 was much higher in WHO-FCs III and IV than in WHO-FCs I and II (P = 0.009) (Fig. 2D).

Table 1.

Basic characteristics of healthy control group and patients with PAH

	Control (n = 40)	PAH (n = 66)	p-value
Age years	42.23 ± 12.70	38.42 ± 11.50	0.116
Sex female, n(%)	26 (65)	56 (70)	0.313
BMI kg/m2	21.69 ± 2.46	21.19 ± 2.99	0.378
SBP mmHg	114.50 ± 13.41	115.08 ± 14.29	0.837
HR beats/min	84 ± 12	86 ± 16	0.460
Comorbidities(n)
Diabetes	7	5	0.118
NT-proBNP pg/ml		1114.00 (276.35-4344.42)
WHO-FC n(%)
I		1 (1.5)
II		27 (40.9)
III		34 (51.5)
IV		4 (6.1)
Echocardiography
RAS	28.5(26.0–30.0)	45.0(39.0–50.0)	< 0.001
RVD	29.5(28.0–30.0)	47.0(42.0-51.3)	< 0.001
LAS	30.0(28.3–32.8)	32.0 (28.0-37.3)	0.025
LVD	46.0(45.0–48.0)	39.0 (36.8–44.0)	< 0.001
PA	21.0(20.0–22.0)	33.0 (27.8–37.3)	< 0.001
TAPSE	19.0(17.3–19.8)	12.0(10.0–15.0)	< 0.001
6MWD m		455.5 ± 108.8
Haemodynamic parameters
mPAP mmHg		51.77 ± 10.56
mRAP mmHg		8.0 (6.0-10.3)
CI L/min/m2		2.2 (1.7–2.9)
SvO2%		66.0 (60.0–70.0)
SVI mL/beat/m2		27.4 (20.6–33.8)
PAC mL/mmHg		1.1 (0.7–1.8)
PVR wood units		11.1 (7.4–18.2)
PAH therapy n(%)
ERAs		55 (83.3)
PDE-5i		49 (74.2)
Prostacyclin analogue		2 (3.0)
Selexipeg		3 (4.5)
Combination therapy		51 (77.3)
PAH subtype n(%)
IPAH		17 (25.76)
CHD-PAH		43 (65.15)
CTD-PAH		6 (9.09)

Data are presented as mean ± standard deviation, median (P25–P75), or number (percentage). Comparisons between the two groups were performed using Student’s t test for normally distributed continuous variables, the Mann–Whitney U test for non-normally distributed continuous variables, and the chi-square test or Fisher’s exact test for categorical variables, as appropriate

BMI Body mass index, SBP systolic blood pressure, HR heart rate, NT-proBNP N-terminal pro-brain natriuretic peptide, WHO-FC World Health Organization functional class, RAS systolic right atrium diameter, RVD diastolic right ventricle dimension, LAS systolic left atrium diameter, LVD left ventricular end-diastolic dimension, PA pulmonary artery diameter, TAPSE tricuspid annular plane systolic excursion, 6MWD six-minute walking distance, mPAP Mean pulmonary arterial pressure, mRAP Mean right atrial pressure, CI cardiac index, PVR Pulmonary vascular resistance, SvO₂ mixed venous oxygen saturation, SVI Stroke volume index, PAC pulmonary arterial compliance, PAH, ERA endothelin receptor antagonist, PDE-5i phosphodiesterase type 5 (PDE5) inhibitor, PAH pulmonary arterial hypertension, IPAH idiopathic PAH, CHD-PAH congenital heart disease-related PAH, CTD-PAH connective tissue disease-related PAH

Fig. 2.

Comparisons of circulating GRP78 levels. A between healthy control and PAH patients; B between healthy control and different PAH subgroups; C between different risk stratification; D. between different WHO-FC. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by Tukey’s test or Welch’s ANOVA followed by Games–Howell post hoc test, whereas comparisons between two groups were performed using Welch’s t-test. Exact p values are shown in the figures.

Association of circulating GRP78 with NT-proBNP in PAH

The correlations of GRP78 with NT-proBNP, echocardiographic or hemodynamic parameters are shown in Fig. 3. In PAH patients, using Spearman correlation analysis with Benjamini–Hochberg false discovery rate correction across pairwise comparisons, circulating GRP78 was significantly associated only with NT-proBNP (r = 0.52, FDR-adjusted P < 0.001). No other clinical, echocardiographic, or hemodynamic variables remained significantly correlated with GRP78 after multiple-testing correction.

Fig. 3.

Correlation matrix of circulating GRP78 and clinical parameters in patients with pulmonary arterial hypertension. Spearman correlation coefficients (r) are shown. Color intensity represents the strength and direction of correlations (blue, positive; red, negative). P values were adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) method across all pairwise correlations. Echocardiographic parameters: RAS, systolic right atrium diameter; RVD, diastolic right ventricle dimension; LAS, systolic left atrium diameter; LVD, left ventricular end-diastolic dimension, PA, pulmonary artery diameter; TAPSE, tricuspid annular plane systolic excursion; RVFAC, right ventricular fractional area change; S’: tricuspid annular plane systolic velocity; Hemodynamic parameters: PVR, pulmonary vascular resistance; mPAP, mean pulmonary artery pressure; mRAP, mean right atrium pressure; mRVP, mean right ventricular pressure.

Predictive value of circulating GRP78 for PAH survival

At a median follow-up of 20 (15–37) months, 30 patients met the primary endpoint. Stratified according to the median circulating GRP78 level. patients with higher GRP78 levels exhibited significantly poorer event-free survival compared with those with lower GRP78 levels (Fig. 4, log-rank P = 0.047). When GRP78 levels were included as a continuous variable in Cox proportional hazards analysis, univariate Cox regression analysis showed HR (95% CI) of 1.422 (1.188–1.703) for every 1 µg/ml increase in GRP78 (P < 0.001; Table 2). Log-transformed NT-proBNP was also a significant predictor of event-free survival (HR 1.558, 95% CI 1.124–2.159; P = 0.008). To assess the incremental prognostic value of circulating GRP78 beyond NT-proBNP, three Cox proportional hazards models were constructed (Table 3): Model 1 included ln(NT-proBNP) alone (AIC 219.7; C-index 0.628), Model 2 included GRP78 alone (AIC 215.1; C-index 0.644), and Model 3 included both variables (AIC 216.2; C-index 0.668). The addition of GRP78 to ln(NT-proBNP) significantly improved model fit compared with Model 1 (LRT χ²=5.50, p = 0.019), with an increase in Harrell’s C-index of 0.040. In contrast, adding ln(NT-proBNP) to GRP78 did not improve model fit (LRT χ²=0.85, p = 0.357), suggesting that GRP78 captures prognostic information beyond that provided by NT-proBNP, whereas the converse is not true. These findings indicate that circulating GRP78 confers incremental prognostic value beyond NT-proBNP in patients with PAH.

Fig. 4.

Event-free survival of PAH patients stratified by high and low circulating GRP78 levels. Patients were grouped according to median value of plasma GRP78 levels (3.0835 µg/ml) for predicting event-free survival (P = 0.047, log-rank test). Numbers at risk are shown below the plot

Table 2.

Univariate Cox analysis for event-free survival in PAH patients

	Univariable model
	HR (95% CI)	p-value
Age	0.999 (0.969–1.030)	0.952
Gender	1.409 (0.645–3.077)	0.390
BMI	0.948 (0.831–1.082)	0.430
GRP78	1.422 (1.188–1.702)	< 0.001
ln (NT-proBNP)	1.558 (1.124–2.159)	0.008
mPAP	1.023 (0.988–1.059)	0.193
mRAP	1.087 (0.991–1.191)	0.076
PVR	1.026 (0.988–1.064)	0.183
CI	0.878 (0.581–1.327)	0.537
SvO2	0.978 (0.938–1.020)	0.303
SVI	0.991 (0.962–1.020)	0.541
PAC	0.656 (0.429–1.004)	0.052

GRP78, glucose regulated protein 78; NT-proBNP, N-terminal pro-brain natriuretic peptide; mPAP, Mean pulmonary arterial pressure; mRAP, Mean right atrial pressure; CI, cardiac index; PVR, Pulmonary vascular resistance; SvO₂, mixed venous oxygen saturation; SVI, Stroke volume index; PAC, pulmonary arterial compliance

Table 3.

Incremental prognostic value of circulating GRP78 beyond NT-proBNP

Model	Variable	HR (95% CI)	P value	AIC	C -index
Model 1	lnNT-proBNP	1.56(1.12–2.16)	0.008	219.72	0.628
Model 2	GRP78	1.42 (1.19–1.70)	< 0.001	215.08	0.644
Model 3	lnNT-proBNP	1.19 (0.82–1.73)	0.367	216.23	0.668
Model 3	GRP78	1.33 (1.06–1.68)	0.016

GRP78, glucose-regulated protein 78; NT-proBNP, N-terminal pro-brain natriuretic peptide

Model comparison: LRT Model 1 vs. Model 3 (adding GRP78): χ²=5.50, p = 0.019; LRT Model 2 vs. Model 3 (adding ln[NT-proBNP]): χ²=0.85, p = 0.357; ΔC-index (Model 3 − Model 1) = + 0.040

Discussion

This study is the first to report an increase in circulating GRP78 levels in two different animal models of PH. Furthermore, extracellular GRP78 was shown to be increased in PASMCs and H9C2 cardiomyocytes after hypoxia treatment. The most important finding of current study is circulating GRP78 is increased in PAH patients and predicts poor prognosis. These animal and cellular findings provide preliminary, hypothesis-generating evidence for a potential cellular origin of elevated circulating GRP78 in PAH, and should be interpreted as translational context rather than mechanistic proof.

GRP78 is widely expressed throughout the cell, distributed in the ER, mitochondria, cytoplasm and cell membrane, and is also secreted outside the cell [17]. And studies have shown that many cells, including cancer cells, neuronal cells, and cardiomyocytes, are able to secrete it extracellularly and may be a broad source of circulating GRP78 [18]. Moreover, studies have shown that secreted GRP78 can inhibit bortezomib-induced endothelial cell apoptosis by increasing the expression of phosphorylated ERK and AKT [19]. In addition, secreted GRP78 was found to ameliorate ischemia-hypoxia-induced cardiomyocyte apoptosis [20]. Moreover, a recently published study found an increased secretion of GRP78 after ER stress and PDGF-BB triggered PASMCs and have a protective role on oxidative stress, inflammation and ER-stress [21]. Our study showed that, PDGF-BB-intervened PASMCs did not produce increased GRP78 secretion. The difference between our study and the experiments that demonstrated by Muntadher et al. could be the intervene time and the concentration of PDGF-BB. They use a higher PDGF-BB concentration and a longer treatment time. This finding also proved by our hypoxia intervention, elevated levels of extracellular GRP78 were detected in both PASMCs and cardiomyocytes in the culture medium. Compared with PASMCs, hypoxia treated H9C2 cardiomyocytes elicited with an approximately 40% increase in extracellular GRP78. These results suggest that both pulmonary vascular cells and cardiomyocytes may contribute importantly to circulating GRP78 in patients with pulmonary hypertension under various pathological triggers. However, in addition to increasing cell secretion, circulating GRP78 may also be released after cell death, so whether it has function and activity in vivo, whether it is involved in the pathological process of PAH and right heart failure, or whether it only serves as a serum biomarker remains to be further explored.

In PAH patients, circulating GRP78 was increased. In subgroup analyses, GRP78 elevated in both IPAH and CHD-PAH patients. No significant difference observed in CTD-PAH compared to healthy controls, this may due to the small sample size. In addition, GRP78 levels were higher in worse WHO-FC patients. The study also showed that plasma GRP78 was higher in patients stratified at intermediate and high risk than in patients at low risk. There are also some discrepancies between our study and the study demonstrated by Muntadher et al., which showed a lower circulating GRP78 in high-risk PAH patients [21]. However, our study showed that, circulating GRP78 are closely related to the heart function, which might lead to the differences between studies.

Although independent of echocardiographic and hemodynamic parameters, plasma GRP78 correlated with NT-proBNP levels, with higher GRP78 levels predicting lower event-free survival.

ER importantly participates in intracellular protein synthesis and modification. In response to external stimuli such as hypoxia, nutrient deprivation, and Ca2 + metabolic imbalance, ER function is impaired, and a large number of misfolded and unfolded proteins accumulate [22]. The pathogenesis of PAH is complex, involving hypoxia, gene mutation, inflammation, vascular remodeling and other factors, all of which can lead to ER stress, which in turn affects pulmonary vascular cells and cardiomyocytes, aggravating the disease and right heart failure [23, 24]. Although the role of ER stress has been repeatedly demonstrated in animal models, they have focused on the tissue level of ER stress markers, and evidence in PAH patients is still limited.

GRP78 is a key sensor protein and expressed increased sharply during ER stress, and a very important regulator. GRP78 has also been reported to secretly circulate under conditions of ER stress, as well as several disease states as a biomarker of ER stress. Serum GRP78 is elevated in metabolic diseases such as obesity, diabetes, and metabolic syndrome [14, 25, 26]. It is also increased in preclinical atherosclerosis, especially in those with carotid plaque [14]. Another study showed increased circulating GRP78 in coronary artery disease, and Sabirli et al. showed that patients with heart failure with low ejection fraction had significantly lower serum GRP78 concentrations than healthy controls, suggesting an association between circulating GRP78 and heart failure [27–29]. Our study is the first to demonstrate that plasma GRP78 is higher in IPAH and CHD-PAH patients and is moderately correlated with NT-proBNP, suggesting an important role for circulating GRP78 in PAH and right heart failure. Crucially, circulating GRP78 is a predictor of event-free survival, making it a potentially important biomarker for PAH. Furthermore, since it also responds to ER stress levels, it may also be an important factor in future responses to ER stress and the extent of disease treatment. Further studies are needed to assess its ability to monitor treatment response, efficiency of combination therapy, and predict all-cause mortality.

This study has several limitations. First, although we explored potential cellular sources of GRP78 and provided supportive evidence that disease-relevant stress conditions increase GRP78 expression in cardiomyocytes and PASMCs, the definitive tissue or organ origin of circulating GRP78 in PAH remains unresolved. Second, the experimental components of this study were exploratory and should be interpreted as supportive translational evidence rather than mechanistic proof. The in vitro models relied on wild-type cells and simplified stimuli and therefore do not fully capture the chronic, multifactorial pathobiology of Group 1 PAH or the phenotypic complexity of primary cells derived from established disease models. Moreover, we did not comprehensively assess PASMC phenotypic transition or the broader ER-stress response, and other potentially relevant cell types, including endothelial cells and fibroblasts, were not examined. Third, the clinical analysis was limited by the single-center design, the relatively small sample size, and the limited number of outcome events, which constrained the complexity of multivariable prognostic modeling and may affect generalizability. The imbalance between control and PAH sample sizes may also have reduced statistical precision. Accordingly, these findings should be regarded as hypothesis-generating and require validation in larger multicenter cohorts, preferably with longitudinal GRP78 measurements and dedicated mechanistic investigation.

Conclusion

Our study expands on previous reports suggesting that ER stress participates in the pathophysiology of PAH, and provides the possible role of GRP78 as an important PAH biomarker and as a prognostic clue. It is hoped that more rigorous prospective and standardized clinical research will be conducted in the future to observe the dynamic changes of GRP78 during follow-up.

Authors’ contributions

Jiang Li, Jingyuan Chen: Conceptualization. Jingyuan Chen, Wenjie Chen: writing—original draft preparation, methodology. Zilu Li, Haihua Qiu: validation. Jun Luo, Yusi Chen: formal analysis. Jiang Li: data curation; All authors have read and agreed to the published version of the manuscript.

Funding

The work was supported by National Natural Science Foundation of China. (82470055), General Funding Project of Hunan Provincial Health Commission (W20243239, 202103010961), and Changsha Natural Science Foundation (kq2202392).

Data availability

The raw data of this article can be directly available by the corresponding author.

Declarations

Consent for publication

The written informed consent was obtained by all participants.

Competing interests

The authors declare no competing interests.