Shear stress-exposed pulmonary artery endothelial cells fail to upregulate HSP70 in chronic thromboembolic pulmonary hypertension.

William Salibe-Filho,Fabio Biscegli Jatene,Thaís L S Araujo,Caio J C S Fernandes,Everton G Melo,Francisco R M Laurindo,Vera Luiza Capelozzi,Orival Freitas-Filho,Luiza B C T Coimbra,Monica S Lapa,Milena M P Acencio,Mario Terra-Filho,Lisete Ribeiro Teixeira,Michael Bader

doi:10.1371/journal.pone.0242960

Abstract

The pathophysiological mechanisms underlying chronic thromboembolic pulmonary hypertension (CTEPH) are still unclear. Endothelial cell (EC) remodeling is believed to contribute to this pulmonary disease triggered by thrombus and hemodynamic forces disbalance. Recently, we showed that HSP70 levels decrease by proatherogenic shear stress. Molecular chaperones play a major role in proteostasis in neurological, cancer and inflammatory/ infectious diseases. To shed light on microvascular responses in CTEPH, we characterized the expression of molecular chaperones and annexin A2, a component of the fibrinolytic system. There is no animal model that reproduces microvascular changes in CTEPH, and this fact led us to isolated endothelial cells from patients with CTEPH undergoing pulmonary endarterectomy (PEA). We exposed CTEPH-EC and control human pulmonary endothelial cells (HPAEC) to high- (15 dynes/cm2) or low- (5 dynes/cm2) shear stress. After high-magnitude shear stress HPAEC upregulated heat shock protein 70kDa (HSP70) and the HSP ER paralogs 78 and 94kDa glucose-regulated protein (GRP78 and 94), whereas CTEPH-ECs failed to exhibit this response. At static conditions, both HSP70 and HSP90 families in CTEPH-EC are decreased. Importantly, immunohistochemistry analysis showed that HSP70 expression was downregulated in vivo, and annexin A2 was upregulated. Interestingly, wound healing and angiogenesis assays revealed that HSP70 inhibition with VER-155008 further impaired CTEPH-EC migratory responses. These results implicate HSP70 as a novel master regulator of endothelial dysfunction in type 4 PH. Overall, we first show that global failure of HSP upregulation is a hallmark of CTEPH pathogenesis and propose HSP70 as a potential biomarker of this condition.

Highlights

Chronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease, including group 4 of pulmonary hypertension (PH) characterized by an intraluminal thrombus that causes pulmonary artery obliteration resulting in right ventricular overload [1]
Low laminar shear stress did not alter the phenotype of CTEPH-Endothelial cell (EC) cells compared with static condition (Fig 1B, 5 dynes/cm2)
Cell viability evaluated through apoptosis was not altered in the CTEPH-EC group compared with control human pulmonary endothelial cells (HPAEC) (Fig 1D)

Summary

Introduction

Chronic thromboembolic pulmonary hypertension (CTEPH) is a vascular disease, including group 4 of pulmonary hypertension (PH) characterized by an intraluminal thrombus that causes pulmonary artery obliteration resulting in right ventricular overload [1]. Hemodynamic forces in pulmonary circulation contribute to endothelial cell phenotype mainly through shear stress [5]. Caveolin-1 maintains eNOS in the inactive form while HSP90 is associated with eNOS after endothelial cells stimulation with vascular epidermal growth factor (VEGF), estrogen, histamine, statins, and shear stress led to enhanced NO production [10]. ENOS, caveolin and HSP90 expression is unknown in any cell type lung from CTEPH patients. Another important cytosolic HSP is 70kDa heat shock protein (HSP70) which cooperates with HSP90 in protein folding of some protein clients [12]. Our data shed light HSP70 is a novel player in CTEPH disease and bring molecular chaperones as potential specific biomarker to this type 4 PH

Ethics statement

Participants

Results

Discussion