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. 2020 Dec 3;15(12):e0242960. doi: 10.1371/journal.pone.0242960

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

William Salibe-Filho 1,*, Thaís L S Araujo 2, Everton G Melo 2, Luiza B C T Coimbra 2, Monica S Lapa 1, Milena M P Acencio 1, Orival Freitas-Filho 3, Vera Luiza Capelozzi 4, Lisete Ribeiro Teixeira 1, Caio J C S Fernandes 1, Fabio Biscegli Jatene 3, Francisco R M Laurindo 5, Mario Terra-Filho 1
Editor: Michael Bader6
PMCID: PMC7714249  PMID: 33270690

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.

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]. After at least three months of anticoagulation, the gold standard treatment for CTEPH is pulmonary endarterectomy (PEA) for eligible patients [2]. The second line treatment is medical therapy or balloon pulmonary angioplasty [1]. Despite hemodynamic improvement and increased median survival after PEA, it is remarkable that 35% of patients remain with pulmonary hypertension [3]. To date, the underlying microvascular biology of poor PEA responses as well CTEPH physiopathology is unclear, with such late-onset PH attributed to small-vessel arteriopathy [2, 4].

Hemodynamic forces in pulmonary circulation contribute to endothelial cell phenotype mainly through shear stress [5]. Overall physiological shear stress can vary from high (15 dynes/cm2) to low (4 dynes/cm2) in lung arterial tree [6, 7]. However, in CTEPH patients, shear stress may reach above 15 dynes/cm2, especially in the unobstructed areas disrupting normal vascular homeostasis. The PH is tightly associated with CTEPH pathogenesis triggered by vascular remodeling [8]. The vessel tonus is mediated by endothelial cells in line with smooth muscle cell phenotype changes. Nitric oxide (NO) is most important vasodilator synthesized by endothelial nitric oxide synthases (eNOS), cytosolic endothelial protein, whose levels are decreased in PH led to increasing in pulmonary vascular resistance (PVR) [9].

eNOS activation depends on cytoplasmic 90kDa heat shock protein (HSP90). In basal condition, 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]. Although NO depletion has been demonstrated in CTEPH patients [11]. 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]. Intracellular HSP70 plays a protective role in cardiovascular diseases such as atherosclerosis [13], and endothelial cell Akt1 phosphorylation [14, 15], p110 PI3K subunits expression [15] and tube formation stimulated by VEGF have been demonstrated [15, 16]. HSP70 is known to be a mainstay in the endothelial cell response. Besides angiogenesis, HSP70 loss of function decreases VEGF-dependent EC migration and proliferation [15, 16]. Recently, we showed that HSP70 expression was decreased in human endothelial cells submitted to low laminar shear stress and in mice aortic arch which is exposed to pathological shear stress [17]. Therefore, HSP70 expression is modulated by hemodynamic patterns associated with the proatherogenic and atheroprotective flow [17]. Further, HSP70 has anti-inflammatory effects [18] and GRP78, an ER resident HSP70, exerts vascular protection through antithrombotic effects [19].

In order to better understanding CTEPH pathogenesis, we hypothesized that global failure in molecular chaperones could be present in CTEPH patients which in turn contributes to endothelial dysfunction observed in this severe pulmonary disease. Our study aimed to understand better the endothelial dysfunction involved in CTEPH patients, based on cytosolic and ER HSP70 and HSP90 expression. Furthermore, the endothelial cell response was analyzed through tube formation and migration. Finally, the pharmacological inhibition of HSP70 was evaluated. Overall, 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.

Materials and methods

Ethics statement

All participants enrolled in the present study signed informed consent before their inclusion.

This study was approved and followed the guidelines of the Ethics Committee for Analysis of Research Projects of the Clinics Hospital of the University of São Paulo School of Medicine (approval number 1.051.734).

Participants

The thromboembolic material of 7 patients who underwent PEA at the Heart Institute (InCor-HCFMUSP, Brazil) was studied. The surgical specimens were immediately incubated with 0.9% saline and 1% penicillin. Patients using PH-specific medication or who had antiphospholipid syndrome were excluded to avoid external interference in endothelial function.

Cell isolation and culture

Endothelial cells were isolated from thrombi obtained from patients who underwent PEA. These cells were referred to as the CTEPH-EC group. The tissues were cut into 2x2-cm sections and treated with 0.2% collagenase type II (Worthington) for 15 min at room temperature [20] followed by centrifugation for 5 min at 4°C at 1500 rpm. This supernatant and the pellet suspended in a specific medium for endothelial cells, EGM-2MV (Clonetics, Lonza, cat. number CC-3202), were placed 25-cm2 culture bottles (Techno Plastic Products AG- TPP, cat. number 90028) and incubated in 5% CO2 at 37°C. The culture medium was changed 3 days after the procedure and then every 48 hours. Human Pulmonary Artery Endothelial Cells (Thermo Fisher Scientific, cat. number C0085C) were used as control cell cultures and referred to as the HPAEC group.

Flow cytometry analysis

The cultures with endothelial morphology were marked with anti-CD31 (BD Pharmingen, Alexa Fluor 647, mouse anti-human CD31, cat. number 558094) and anti-CD90 (Abcam, PE, ab957000) [21]. Then, the cells were quantified using the flow cytometry method (BD Excalibur). For the apoptosis experiments, annexin V-FITC (Oncogene, cat # PF032) was used. The supernatant was centrifuged, and the marker was incubated. After, the cells were stained using the protocol described by manufacturing and were analyzed in the flow cytometry.

Immunohistochemistry experiments

Histological sections were produced with 3-mm thickness containing representative samples of endothelium extracted from the thrombus of patients with CTEPH, which was previously stored at -80°C. Transplanted lungs were used as control tissue by using pieces of the pulmonary artery of the donor. All the samples underwent immunohistochemical analysis for the identification of cytoplasmic HSP70 (Abcam, anti-Hsp70 antibody (3A3), #ab5439) and annexin 2 (Abcam, anti-annexin A2 antibody C-terminal, #ab185957), according to the manufacturer’s protocol. Histomorphometric quantification of the expression of the HSP70 and annexin A2 markers and total area quantification randomly of 5 different fields was performed by using a microscope-coupled image analyzer. The system consists of an Olympus-5 camera coupled to an Olympus microscope, from which the images are viewed on a monitor and evaluated on a digital imaging system (Software Image Pro-Plus 6.0).

Shear stress assay

The experiments were performed on a 100-mm plate [22]. The cells were maintained in an atmosphere at 5% CO2 at 37°C. The shear stress system was maintained at 5 and 15 dynes/cm2 for 24 hours to simulate the flows exerted in the pulmonary vessels [23]. One plate was sustained to static conditions and used as a control. After 24 hours, the following variables were analyzed: HSP70, HSP90, GRP94, alpha 5 integrin, annexin A2, and PDI by Western blot in the shear stress (5 and 15 dynes/cm2) and static conditions. All samples were run on the same gel, and the best representative images were chosen to the figures.

The cells were lysed in 20 mM HEPES pH = 7.2, 150 mM NaCl, 1 mM EGTA, 1.5 mM MgCl2, 10% glycerol, and 1% Triton-containing proteases and phosphatase inhibitors for 30 min at 4°C. Sonicated lysates were centrifuged for 10 min at 12,000 rpm at 4°C, and the supernatant was frozen at -80°C. Forty-sixty micrograms of total protein underwent SDS-PAGE, transferred onto a nitrocellulose membrane, milk blocked, and incubated with the indicated antibodies. The antibody dilution varied from 1:1000–1:5000, including anti-PDI (MA3-019, Thermo Scientific), anti-GRP78 (ab21685, Abcam), GRP94 (ab52031, Abcam), HSP70 (ab5439, Abcam), anti-HSP90 (sc-13119, Santa Cruz Biotechnology), anti-integrin alpha 5 (ab150361, Abcam), anti-caveolin-1 (Cell Signaling Technology), anti-annexin A2 (ab185957, Abcam), and anti-β actin (AC-74, Sigma).

Wound healing assay

The wound healing migration assay was performed following previously published methods [24]. HPAEC and CTEPH-EC were seeded at 1.5x105 cells in 12-well-plate. For each cell type, we used one basal and other with 30μM VER-155008 and grow cells for 4h. Then, cells were washed twice with HEPES and serum-starved for 2 hours in EBM-2 medium (Lonza) with 30 μM VER-155008 or DMSO. We made one scratch lengthwise per well with a sterile P200 tip and cultivate cells in presence of EGM-2 medium (Lonza) with 30 uM VER-155008 or DMSO. Cells were incubated for 18 hours at 37°C and 5% CO2. Images were acquired at time 0 and 18h with a Zeiss microscope and 10X objective. The wound area was determined with Adobe Photoshop CS6, and the migration ratio was calculated by the equation %Healed = [(Area of original wound—Area of wound during healing)/Area of original wound]X100 [25].

Tube formation assay

Tube formation assays were performed based on experiments described previously [26]. Each well of a pre-cooled 48-well plate was briefly coated with 150 μL Matrigel matrix (Corning, Tewksbury, MA, USA) and allowed to polymerize for 5 minutes at room temperature followed by 30 minutes at 37°C. Primary HPAECs or CTEPH-EC isolated from CTEPH patients (3 x 104 cells per well) were seeded to the coated plate and incubated for 5h30min in EGM-2 alone or EGM-2 containing 50μM VER-155008 in a humidified incubator at 37°C with 5% of CO2. Five images were obtained from each well using TissueFAXS imaging system (TissueGnostics, Vienna, Austria).

Statistical analysis

Data are reported as the mean ± standard error of the mean. One-way analysis of variance (ANOVA) with the Newman-Keuls post-hoc test was performed for comparisons between 3 or more groups; the unpaired Student t test was used for comparisons between 2 groups. The analysis (#) was performed to compare the condition of static and low shear stress using unpaired Student t test. In both cases, the level of significance was 0.05. All statistical tests were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA).

Results

CTEPH-ECs were isolated from the pulmonary arteries of patients who underwent PEA (Fig 1A). CTEPH- EC presented as a monolayer composed of rounded cells whose morphology was altered, with a larger size than HPAEC under static condition (Fig 1B). After exposure to high laminar shear stress, their round phenotype was shifted to an elongated one (Fig 1B, 15 dynes/cm2). Low laminar shear stress did not alter the phenotype of CTEPH-EC cells compared with static condition (Fig 1B, 5 dynes/cm2). Flow cytometry analysis revealed the presence of CD31 and absence of CD90, endothelial and mesenchymal cells markers, respectively (Fig 1C). Of note, cell viability evaluated through apoptosis was not altered in the CTEPH-EC group compared with control HPAEC (Fig 1D).

Fig 1. Characterization of endothelial cells from CTEPH patient.

Fig 1

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(A). Representative image from PEA samples. (B) Images of HPAEC and CTEPH-EC in static condition and after exposition to low (5 dynes/cm2) and high shear stress (15 dynes/cm2). (C) Flow cytometry analyses of CD31 and CD90 expression in HPAEC and CTEPH-EC (n = 5). (D) Annexin V quantification of HPAEC and CTEPH-EC in static condition (n = 5).

CTEPH-EC had diminished expression of constitutively expressed cytoplasmic HSC70 (HSPA8) and stress-inducible HSP70 (HSPA1A/B), both collectively refereed here as HSP70, under static condition (Fig 2A). The high laminar shear stress in HPAEC upregulated HSP70 expression, whereas in CTEPH-EC HSP70 amount was slightly decreased. The low laminar shear stress had a similar effect on both cells. In static conditions, HSP90 was also downregulated in CTEPH-EC compared with HPAEC, while fibronectin receptor alpha5 integrin was unchanged (Fig 2B).

Fig 2. Cytoplasmic protein expression in HPAEC and CTEPH-EC.

Fig 2

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(A) Representative blot of HSP70 expression in static, low, and high magnitude shear stress (0, 5, and 15 dynes/cm2). Graphs are presented as the mean ± standard error. (*) HPAEC vs HPAEC 15, (#) CTEPH-EC vs CTEPH-EC 5, (&) HPAEC 15 vs CTEPH-EC 15, P < 0.05.(n = 6–7). (B) HSP90 (n = 6) and alpha 5 integrin (n = 4) expression in static condition. Data are presented as the mean ± standard error. (*) HPAEC vs CTEPH-EC, P < 0.05. All of the samples were normalized to ß-actin and run in the same gel.

Further characterization of molecular chaperone levels in CTEPH-EC showed that the ER HSP90 member (GRP94) was decreased in static condition and was not affected by applied shear stress (Fig 3A). ER HSP70 member (GRP78/Bip) was upregulated by high and low laminar shear stress in HPAEC, while only low shear stress increases GRP78 expression in CTEPH-EC (Fig 3B). However, not all ER chaperones were affected in CTEPH-EC, since protein disulfide isomerase (PDIA1), an important redox chaperone catalyst, had no change independent of condition (Fig 3B).

Fig 3. Endoplasmic reticulum chaperones are downregulated in CTEPH-EC.

Fig 3

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(A) Representative blot of the GRP94 expression in static, low, and high magnitude shear stress (0, 5, and 15 dynes/cm2). Data are presented as the mean ± standard error: (*) HPAEC vs CTEPH-EC, (&) HPAEC 15 vs CTEPH-EC 15. (n = 5–7). (B) GRP78 and PDI expression before and after shear stress exposure as described in methods. Data are presented as the mean ± standard error: (*) HPAEC vs HPAEC 15, (#) CTEPH-EC vs CTEPH-EC 5 (&) HPAEC 15 vs CTEPH-EC 15, P < 0.05. (n = 6–7). All of the samples were normalized to ß-actin and run in the same gel.

To analyze pathophysiological implications of the above results, we performed immunohistochemistry for HSP70 and annexin A2 in CTEPH pulmonary arteries in CTEPH patients tissue using lung donors as a control. The results were close to those found in the CTEPH-EC, showing HSP70 downregulation [22% in controls vs 13% in CTEPH (P < 0.05)] (Fig 4A and 4B). In contrast, annexin A2 expression was upregulated in CTEPH [1.5% control vs 9.8% CTEPH (P < 0.05)] (Fig 4C and 4D).

Fig 4. Expression of HSP70 and annexin A2 in the internal part of the pulmonary artery from CTEPH patients and the pulmonary artery from lung transplant donors (control).

Fig 4

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(A) Immunohistochemistry analyses of HSP70 and (C) Annexin A2 expression. (B, D) Quantification of images from (A) and (C), respectively. Data are presented as the mean ± standard error. Control: n = 4 // Patients: n = 5. (*) Control vs CTEPH, P < 0.05.

Endothelial dysfunction was proposed to be an essential component in CTEPH [4], however it has not been directly investigated in CTEPH patients. We took advantage of our successful isolation of endothelial cells to evaluate its physiological responses. Interestingly, CTEPH-EC presented strong impairment in migration (Fig 5) and tube formation (Fig 6), with less total branches and more cells in each node consistent with frustrated angiogenic response. Moreover, both responses were significantly decreased by the specific [27] HSP70 inhibitor VER155008 (Figs 5 and 6), clearly showing that HSP70 plays an essential function in migration and angiogenesis both in HPAEC and CTEPH-EC.

Fig 5. Migration capacity of HPAEC and CTEPH-EC in basal and with HSP70 inhibitor VER-155008.

Fig 5

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(A) Representative images of HPAECs and CTEPH-ECs basal and after VER-155008. (B) Quantification of the migration. Data are presented as the mean ± standard error: (*) HPAEC Basal vs HPAEC VER, (#) HPAEC Basal vs CTEPH-EC Basal, (&) CTEPH-EC Basal vs CTEPH-EC VER, P < 0.05. (n = 3).

Fig 6. Tube formation: Representative light microscopic images of the structures formed by HPAEC and CTEPH-EC seeded and incubated for 5h30 min in the absence or presence of 50 uM VER-155008 (n = 6).

Fig 6

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Discussion

Our results shed a new light of molecular chaperones functions in CTEPH, showing novel insights into the pathophysiology of this disease condition. Overall, the collective downregulation of HSP70, HSP90, GRP94, and GRP78 expression in CTEPH-EC is a novel hallmark of endothelial cell dysfunction. Furthermore, in vivo HSP70 expression was decreased in CTEPH tissues, and endothelial function was shown to be supported by this chaperone. To the best of our knowledge, this report is the first to implicate HSPs, particularly HSP70, in CTEPH pathophysiology.

It has been suggested that CTEPH-related endothelial cells exhibit autophagy failure [21]. Together with our data, this further points to a possible disruption of proteostasis in this condition. We investigated HSP70, a molecular chaperone that works as an insular component in protein folding and also cooperates with HSP90, mediates protein folding or client stability [12]. Of note, both chaperones are the hub of a network sustaining cancer cell survival, in which HSP90 inhibition has been explored as a basis for personalized treatment [28]. The possibility of a protective role for the intracellular HSP70 family in cardiovascular diseases [29] opens the possibility that therapeutic increases in the expression of this chaperone could mitigate endothelial dysfunction. In CTEPH patient endothelial cells, the expression of HSP70 was substantially downregulated in cell response to high shear stress (Fig 2A) and in vivo tissue expression (Fig 4), corroborating the findings of endothelial dysfunction and loss of protective effects.

ER stress is known to associate with monocrotaline-induced PH pathology [30]. Feaver et al. have studied atherosclerotic lesions and ER stress, demonstrating that the shear stress to which cells are exposed causes changes in the expression of GRP94 and GRP78 [19]. Here we found that the expression of GRP94 and GRP78 was decreased. These cells are unable to positively regulate these molecular chaperones in CTEPH-EC (Fig 3). These results suggest a possible specific CTEPH response and the decreased levels of HSP70 and HSP90 could be a possible trigger for disease development. These findings also provide a potential therapeutic option considering the availability of drugs capable of modulating the release of HSP70 and GRP78 [31, 32]. In Alzheimer’s disease, a reduction of HSP70 has been demonstrated, and it has been proposed that increases in HSP70 levels induced by the YC-1 have protective effects [33]. Based on our results, upregulating HSP70 as a way to interfere with the vascular remodeling in CTEPH patients deserves further investigation.

Endothelial cell migration is a vital component of normal vascular development. Changes in this property are evaluated as an inadequate physiological response in pathophysiological situations [24]. Importantly, CTEPH-EC fails to upregulate HSP70 by high shear stress (15dynes/cm2) which is deleterious in pulmonary circulation (Fig 2A). Furthermore, endothelial cell response analyzed though tube formation and migration (Figs 5 and 6) were strongly impaired in CTEPH-EC. Finally, CTEPH-EC dysfunction was enhanced by pharmacological HSP70 inhibition.

Others have shown that low shear stress is beneficial in pulmonary arteries [8]. Our data corroborate this view. Moser et al. [34] described the involvement of pulmonary circulation that goes beyond the mechanical obstruction. This phenomenon was later identified as a secondary pulmonary arteriopathy or small-vessel disease that was more evident in the unobstructed pulmonary arteries [4]. A study using an animal shunt model with hyperdynamic flow revealed that endothelial cell involvement led to disordered proliferation of pulmonary arterioles [35]. Our data open the possibility that changes caused by high flow in unobstructed areas also compromise the expression of HSPs, and failures of this positive regulation suggests that this arteriopathy promotes protein imbalance.

The changes in annexin A2 observed in our results deserve further discussion. Plasminogen activation to release plasmin on the cell surface is mediated by annexin A2 through direct binding of tissue plasminogen activator (t-PA) and plasminogen, which is synthesized by endothelium [36, 37]. Thus, annexin A2 translocation to the endothelial membrane is a key regulatory step in vascular fibrinolysis [38]. Antibodies against annexin A2 in antiphospholipid syndrome, together with inhibition of plasmin release, promote thrombus formation [39]; of note, 20% of CTEPH patients carry antiphospholipid antibodies [4]. Fibrinolysis defects are described in CTEPH pathophysiology, however, such changes remain controversial [40]. Upregulated annexin A2 expression (Fig 4) in our cells may represent an attempt to enhance fibrinolysis.

Together, our data showed for the first time that high-flow shear stress decreases the expression of heat shock proteins in endothelial cells of CTEPH patients. These results show that HSP70 failure supports endothelial dysfunction in these cells, suggesting that this chaperone may orchestrate a new pathophysiological pathway in CTEPH disease and highlight HSP70 as a new potential biomarker. These findings compose a new model to understand endothelial dysfunction in this disease. Microvascular involvement with a decrease expression in essential chaperones, such as HSP70 and HSP90, has been poorly explores and opens a new perspective in the understanding of this severe pulmonary vascular disease.

Supporting information

S1 File

(PDF)

Click here for additional data file. (1.6MB, pdf)

Acknowledgments

We thank Marcos Naoyuki Samano, MD, PhD, who provided the pulmonary arteries of lung donors.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) in the form of a research grant awarded to TLSA (18/13739-8) and fellowships awarded to TLSA (19/20435-8, 15/06210-2), LBCTC (20/11249-3) and EGM (19/25503-9), Centro de Pesquisa, Inovação e Difusão FAPESP (CEPID “Processos Redox em Biomedicina”) in the form of a research grant awarded to FRML (13/07937-8), and Fundação Zerbini and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) (Finance Code 001) in the form of funds used for the purchase of materials and reagents. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Michael Bader

13 Nov 2019

PONE-D-19-24096

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

PLOS ONE

Dear Dr Salibe-Filho,

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PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors showed a paradoxical reduction in the level of Hsp70 protein in shear-stress exposed pulmonary artery endothelial cells from chronic thromboembolic pulmonary hypertension. While their findings are interesting and intriguing, however, I have some concerns:

1) The authors claimed that the decreased hsp70 level was due to shear-stress induced proteostatic dysregulation. Though hsp70 is a major player in the protein quality control system, other proteins including CHIP, an E3 ubiquitin ligase play crucial roles. The authors cannot justify decreased hsp70 to increased protein degradation. If the authors want use the word proteostasis, they will have to back it up with data that decreased hsp70 expression is due to transcriptional or post transcriptional mechanism, both of which can be due to enhanced hsp70 degradation by ubiquitin-proteasomal signaling pathways.

2) The authors assessed the levels of Hsp70 in HPAECs and HPAEC from patients with chronic thromboembolic PH. However they failed to mention the specific isoform of hsp70. So far, there are about 12 -15 hsp70 isoforms, of which most of them are localized to the cytosol.

3) Importantly, what is the relevance of decreased hsp70 in the context of the pathogenesis of chronic thromboembolic PH?

4) The manuscript was written poorly with no flow to the story. It needs to be re-written and thoroughly edited for clarity.

Reviewer #2: this study from Saline-Filho et al evaluates the effects of shear stress on EC isolated from CTEPH patients compared to normal control PAEC. The role of proteostasis in pulmonary disease is in its infancy so studies which will increase our understanding of this are important for the field. However, this work has several major limitations that limit is potential impact on the field.

1. It is very descriptive and there is no mechanistic insight into why CTEPH PAEC cells down regulation molecular chaperones in response to flow. This needs to be addressed.

2. There are actually no studies presented that actually study proteostasis in the manuscript. It would seem that the authors need to evaluate at least a few targets of hsp70/hsp90 or other chaperones and see how these differ in EC isolated from CTEPH patients compared to normal control PAEC.

3. Can over or under expression of hsp70/hsp90 or other chaperones be used to restore proteostasis in EC from CTEPH patients or perturb it in normal control PAEC respectively? This is needed to demonstrate cause and effect relationships.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Dec 3;15(12):e0242960. doi: 10.1371/journal.pone.0242960.r002

Author response to Decision Letter 0

14 Oct 2020

Dear Prof. Michael Bader,

Please find attached to this electronic submission the revised version of our article entitled “Shear stress-exposed pulmonary artery endothelial cells fail to upregulate HSP70 in chronic thromboembolic pulmonary hypertension”, which I and my collaborators Thaís L. S. Araujo, Everton G. Melo, Luiza B. C. T. Coimbra, Monica S. Lapa, Milena M. P. Acencio, Orival Freitas Filho, Vera Luiza Capelozzi, Lisete Ribeiro Teixeira, Caio J. C. S. Fernandes, Fabio Biscegli Jatene, Francisco R. M. Laurindo, and Mario Terra-Filho are submitting for editorial consideration in PLOS ONE.

First of all, we would like to truly thank the reviewers and Editor for their time and effort to provide a thorough and constructive review of our work. We took great care to seriously consider and address all the concerns previously raised. The revised version now submitted has been quite extensively revised. The changes performed in our study have been described point-by-point in the "Answer to Reviewers" file and can be summarized by addition of two new experiments and extensive alterations in written manuscript based on suggestions of reviewers. Furthermore, the two new experiments were realized by Dr. Araujo and her students Everton and Luiza, the formerly dedicated efforts in performed angiogenesis assays while the latter dedicated to wound-healing assays. Now, we add their names in this revised version of our article.

The original blot images of the figures of the manuscript have been uploaded to the supporting information.

Overall, we believe these extensive modifications contributed to improve the paper and we hope it can now be suitable for publication in PLOS ONE.

We hereby expressly restate that this is fully original material not previously disclosed.

We are grateful for your attention to this article.

With best regards,

William Salibe-Filho

Attachment

Submitted filename: Salibe et al response to reviewers.pdf

Click here for additional data file. (5.5MB, pdf)

Decision Letter 1

Michael Bader

13 Nov 2020

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

PONE-D-19-24096R1

Dear Dr. Salibe-Filho,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

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Kind regards,

Michael Bader

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Nice work! The authors should endeavor to determine the underlying mechanisms leading to decreased Hsp70 and Hsp90 expression in chronic thromboembolic induced-PAH. Identifying these mechanisms can provide therapeutic strategies in the treatment of PAH.

Also, the manuscript needs more work. The authors need today particular attention to tenses

Reviewer #2: Thea authors ahem adequately addressed the concerns raised in the prior review. No remaining concerns need to be addressed.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Acceptance letter

Michael Bader

24 Nov 2020

PONE-D-19-24096R1

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

Dear Dr. Salibe-Filho:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. Michael Bader

Academic Editor

PLOS ONE

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