GLI1+ Cells Contribute to Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: The precise origin of newly formed ACTA2+ (alpha smooth muscle actin-positive) cells appearing in nonmuscularized vessels in the context of pulmonary hypertension is still debatable although it is believed that they predominantly derive from preexisting vascular smooth muscle cells (VSMCs). METHODS: Gli1Cre-ERT2; tdTomatoflox mice were used to lineage trace GLI1+ (glioma-associated oncogene homolog 1-positive) cells in the context of pulmonary hypertension using 2 independent models of vascular remodeling and reverse remodeling: hypoxia and cigarette smoke exposure. Hemodynamic measurements, right ventricular hypertrophy assessment, flow cytometry, and histological analysis of thick lung sections followed by state-of-the-art 3-dimensional reconstruction and quantification using Imaris software were used to investigate the contribution of GLI1+ cells to neomuscularization of the pulmonary vasculature. RESULTS: The data show that GLI1+ cells are abundant around distal, nonmuscularized vessels during steady state, and this lineage contributes to around 50% of newly formed ACTA2+ cells around these normally nonmuscularized vessels. During reverse remodeling, cells derived from the GLI1+ lineage are largely cleared in parallel to the reversal of muscularization. Partial ablation of GLI1+ cells greatly prevented vascular remodeling in response to hypoxia and attenuated the increase in right ventricular systolic pressure and right heart hypertrophy. Single-cell RNA sequencing on sorted lineage-labeled GLI1+ cells revealed an Acta2high fraction of cells with pathways in cancer and MAPK (mitogen-activated protein kinase) signaling as potential players in reprogramming these cells during vascular remodeling. Analysis of human lung-derived material suggests that GLI1 signaling is overactivated in both group 1 and group 3 pulmonary hypertension and can promote proliferation and myogenic differentiation. CONCLUSIONS: Our data highlight GLI1+ cells as an alternative cellular source of VSMCs in pulmonary hypertension and suggest that these cells and the associated signaling pathways represent an important therapeutic target for further studies.

Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice.

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.

Lung Fibrosis Is Linked to Increased Endothelial Cell Activation and Dysfunctional Vascular Barrier Integrity.

Pulmonary fibrosis can be a fatal disease characterized by progressive lung scarring. It is still poorly understood how the pulmonary endothelium is involved in the disease pathogenesis. Differences of the pulmonary vasculature between patients and donors were analysed using transmission electron microscopy, immunohistochemistry and single-cell-RNA-sequencing. Vascular barrier resistance, endothelial-immune cell adhesion, and sensitivity to an inflammatory milieu were studied in-vitro. Integrity and activation markers were measured by ELISA in human plasma. Transmission electron microscopy demonstrated abnormally swollen endothelial cells in fibrotic lungs as compared to donors. A more intense CD31 and vWF and patchy VE-Cadherin staining in fibrotic lungs supported the presence of a dysregulated endothelium. Integrity markers CD31, VE-Cadherin, Thrombomodulin and VEGFR-2 and activation marker von-Willebrand-Factor gene expression was increased in different endothelial subpopulations (e.g. arterial, venous, gCap, aCap) in pulmonary fibrosis. This was associated with a heightened sensitivity of fibrotic endothelial cells to TNF-α or IFN-γ and elevated immune cell adhesion. The barrier strength was overall reduced in endothelial cells from fibrotic lungs. vWF and IL-8 were increased in the plasma of patients, while VE-Cadherin, Thrombomodulin and VEGFR-2 were decreased. VE-Cadherin staining was also patchy in biopsy tissue and was decreased in plasma samples of PF patients six months after the initial diagnosis. Our data demonstrate highly abnormal endothelial cells in PF. The vascular compartment is characterized by hyper-activation and increased immune cell adhesion, as well as dysfunctional endothelial barrier function. Re-establishing endothelial cell homeostasis and function might represent a new therapeutic option for fibrotic lung diseases.

A comprehensive map of proteoglycan expression and deposition in the pulmonary arterial wall in health and pulmonary hypertension.

Changes in the extracellular matrix of pulmonary arteries (PAs) are a key aspect of vascular remodeling in pulmonary hypertension (PH). Yet, our understanding of the alterations affecting the proteoglycan (PG) family remains limited. We sought to investigate the expression and spatial distribution of major vascular PGs in PAs from healthy individuals and various PH groups (chronic obstructive pulmonary disease: PH-COPD, pulmonary fibrosis: PH-PF, idiopathic: IPAH). PG regulation, deposition, and synthesis were notably heightened in IPAH, followed by PH-PF, with minor alterations in PH-COPD. Single-cell analysis unveiled cell-type and disease-specific PG regulation. Agrin expression, a basement membrane PG, was increased in IPAH, with PA endothelial cells (PAECs) identified as a major source. PA smooth muscle cells (PASMCs) mainly produced large-PGs, aggrecan and versican, and small-leucine-like proteoglycan (SLRP) biglycan, whereas the major PGs produced by adventitial fibroblasts were SLRP decorin and lumican. In IPAH and PF-PH, the neointima-forming PASMC population increased the expression of all investigated large-PGs and SLRPs, except fibroblast-predominant decorin (DCN). Expression of lumican, versican, and biglycan also positively correlated with collagen 1α1/1α2 expression in PASMCs in patients with IPAH and PH-PF. We demonstrated that transforming growth factor-beta (TGF-ß) regulates versican and biglycan expression, indicating their contribution to vessel fibrosis in IPAH and PF-PH. We furthermore show that certain circulating PG levels display a disease-dependent pattern, with increased decorin and lumican across all patient groups, while versican was elevated in PH-COPD and IPAH and biglycan reduced in IPAH. These findings suggest unique compartment-specific PG regulation in different forms of PH, indicating distinct pathological processes.NEW & NOTEWORTHY Idiopathic pulmonary arterial hypertension (IPAH) pulmonary arteries (PAs) displayed the greatest proteoglycan (PG) changes, with PH associated with pulmonary fibrosis (PH-PF) and PH associated with chronic obstructive pulmonary disease (PH-COPD) following. Agrin, an endothelial cell-specific PG, was solely upregulated in IPAH. Among all cells, neo-intima-forming smooth muscle cells (SMCs) displayed the most significant PG increase. Increased levels of circulating decorin, lumican, and versican, mainly derived from SMCs, and adventitial fibroblasts, may serve as systemic indicators of pulmonary remodeling, reflecting perivascular fibrosis and neointima formation.

The endothelium in lung fibrosis: a core signaling hub in disease pathogenesis?

Pulmonary fibrosis (PF) is a progressive chronic lung disease characterized by excessive deposition of extracellular matrix (ECM) and structural destruction, associated with a severe 5-year mortality rate. The onset of the disease is thought to be triggered by chronic damage to the alveolar epithelium. Since the pulmonary endothelium is an important component of the alveolar-capillary niche, it is also affected by the initial injury. In addition to ensuring proper gas exchange, the endothelium has critical functional properties, including regulation of vascular tone, inflammatory responses, coagulation, and maintenance of vascular homeostasis and integrity. Recent single-cell analyses have shown that shifts in endothelial cell (EC) subtypes occur in PF. Furthermore, the increased vascular remodeling associated with PF leads to deteriorated outcomes for patients, underscoring the importance of the vascular bed in PF. To date, the causes and consequences of endothelial and vascular involvement in lung fibrosis are poorly understood. Therefore, it is of great importance to investigate the involvement of EC and the vascular system in the pathogenesis of the disease. In this review, we will outline the current knowledge on the role of the pulmonary vasculature in PF, in terms of abnormal cellular interactions, hyperinflammation, vascular barrier disorders, and an altered basement membrane composition. Finally, we will summarize recent advances in extensive therapeutic research and discuss the significant value of novel therapies targeting the endothelium.

Pentastatin, a matrikine of the collagen IVα5, is a novel endogenous mediator of pulmonary endothelial dysfunction.

Deposition of basement membrane components, such as collagen IVα5, is associated with altered endothelial cell function in pulmonary hypertension. Collagen IVα5 harbors a functionally active fragment within its C-terminal noncollageneous (NC1) domain, called pentastatin, whose role in pulmonary endothelial cell behavior remains unknown. Here, we demonstrate that pentastatin serves as a mediator of pulmonary endothelial cell dysfunction, contributing to pulmonary hypertension. In vitro, treatment with pentastatin induced transcription of immediate early genes and proinflammatory cytokines and led to a functional loss of endothelial barrier integrity in pulmonary arterial endothelial cells. Mechanistically, pentastatin leads to ß1-integrin subunit clustering and Rho/ROCK activation. Blockage of the ß1-integrin subunit or the Rho/ROCK pathway partially attenuated the pentastatin-induced endothelial barrier disruption. Although pentastatin reduced the viability of endothelial cells, smooth muscle cell proliferation was induced. These effects on the pulmonary vascular cells were recapitulated ex vivo in the isolated-perfused lung model, where treatment with pentastatin-induced swelling of the endothelium accompanied by occasional endothelial cell apoptosis. This was reflected by increased vascular permeability and elevated pulmonary arterial pressure induced by pentastatin. This study identifies pentastatin as a mediator of endothelial cell dysfunction, which thus might contribute to the pathogenesis of pulmonary vascular disorders such as pulmonary hypertension.NEW & NOTEWORTHY This study is the first to show that pentastatin, the matrikine of the basement membrane (BM) collagen IVα5 polypeptide, triggers rapid pulmonary arterial endothelial cell barrier disruption, activation, and apoptosis in vitro and ex vivo. Mechanistically, pentastatin partially acts through binding to the ß1-integrin subunit and the Rho/ROCK pathway. These findings are the first to link pentastatin to pulmonary endothelial dysfunction and, thus, suggest a major role for BM-matrikines in pulmonary vascular diseases such as pulmonary hypertension.

SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. METHODS: After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach. RESULTS: C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-ß1 (transforming growth factor ß1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. CONCLUSIONS: This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

Impairment of the NKT-STAT1-CXCL9 Axis Contributes to Vessel Fibrosis in Pulmonary Hypertension Caused by Lung Fibrosis.

Rationale: Pulmonary hypertension (PH) is a common, severe comorbidity in interstitial lung diseases such as pulmonary fibrosis (PF), and it has limited treatment options. Excessive vascular fibrosis and inflammation are often present in PH, but the underlying mechanisms are still not well understood. Objectives: To identify a novel functional link between natural killer T (NKT) cell activation and vascular fibrosis in PF-PH. Methods: Multicolor flow cytometry, secretome, and immunohistological analyses were complemented by pharmacological NKT cell activation in vivo, in vitro, and ex vivo. Measurements and Main Results: In pulmonary vessels of patients with PF-PH, increased collagen deposition was linked to a local NKT cell deficiency and decreased IL-15 concentrations. In a mouse model of PH caused by lung fibrosis, pharmacological NKT cell activation using a synthetic α-galactosylceramide analog (KRN7000) restored local NKT cell numbers and ameliorated vascular remodeling and right ventricular systolic pressure. Supplementation with activated NKT cells reduced collagen deposition in isolated human pulmonary arterial smooth muscle cells (hPASMCs) and in ex vivo precision-cut lung slices of patients with end-stage PF-PH. Coculture with activated NKT cells induced STAT1 signaling in hPASMCs. Secretome analysis of peripheral blood mononuclear cells identified CXCL9 and CXCL10 as indicators of NKT cell activation. Pharmacologically, CXCL9, but not CXCL10, potently inhibited collagen deposition in hPASMCs via the chemokine receptor CXCR3. Conclusions: Our results indicate that the absence of NKT cells impairs the STAT1-CXCL9-CXCR3 axis in PF-PH and that restoration of this axis by NKT cell activation may unravel a novel therapeutic strategy to target vascular fibrosis in interstitial lung disease.

[Genetic diagnostics and molecular approaches in pulmonary arterial hypertension]. / Genetische Diagnostik und molekulare Ansätze bei pulmonalarterieller Hypertonie.

The recently published new European guidelines for diagnosis and treatment of pulmonary hypertension now offer the so far most extensive description of genetic testing and counselling for pulmonary arterial hypertension patients. In addition, the importance of a clinical screening of healthy mutation carriers is highlighted as well as the genetic testing of patients with a suspicion of pulmonary veno-occlusive disease. We frame the respective parts of the guidelines on genetic testing and counselling in the context of recent data and provide comments. Finally, we give an outlook on novel molecular approaches starting from Sotatercept, addressing ion channels and novel therapeutic developments.

Pirfenidone exacerbates Th2-driven vasculopathy in a mouse model of systemic sclerosis-associated interstitial lung disease.

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterised by severe vasculopathy and fibrosis of various organs including the lung. Targeted treatment options for SSc-associated interstitial lung disease (SSc-ILD) are scarce. We assessed the effects of pirfenidone in a mouse model of SSc-ILD. METHODS: Pulmonary function, inflammation and collagen deposition in response to pirfenidone were assessed in Fra-2-overexpressing transgenic (Fra-2 TG) and bleomycin-treated mice. In Fra-2 TG mice, lung transcriptome was analysed after pirfenidone treatment. In vitro, pirfenidone effects on human eosinophil and endothelial cell function were analysed using flow cytometry-based assays and electric cell-substrate impedance measurements, respectively. RESULTS: Pirfenidone treatment attenuated pulmonary remodelling in the bleomycin model, but aggravated pulmonary inflammation, fibrosis and vascular remodelling in Fra-2 TG mice. Pirfenidone increased interleukin (IL)-4 levels and eosinophil numbers in lung tissue of Fra-2 TG mice without directly affecting eosinophil activation and migration in vitro. A pronounced immune response with high levels of cytokines/chemokines and disturbed endothelial integrity with low vascular endothelial (VE)-cadherin levels was observed in pirfenidone-treated Fra-2 TG mice. In contrast, eosinophil and VE-cadherin levels were unchanged in bleomycin-treated mice and not influenced by pirfenidone. In vitro, pirfenidone exacerbated the IL-4 induced reduction of endothelial barrier resistance, leading to higher leukocyte transmigration. CONCLUSION: This study shows that antifibrotic properties of pirfenidone may be overruled by unwanted interactions with pre-injured endothelium in a setting of high T-helper type 2 inflammation in a model of SSc-ILD. Careful ILD patient phenotyping may be required to exploit benefits of pirfenidone while avoiding therapy failure and additional lung damage in some patients.

Low oxygen levels decrease adaptive immune responses and ameliorate experimental asthma in mice.

BACKGROUND: High-altitude therapy has been used as add-on treatment for allergic asthma with considerable success. However, the underlying mechanisms remain unclear. In order to investigate the possible therapeutic effects of high-altitude therapy on allergic asthma, we utilized a new in vivo mouse model. METHODS: Mice were treated with house dust mite (HDM) extract over 4 weeks and co-exposed to 10% oxygen (Hyp) or room air for the final 2 weeks. Experimental asthma was assessed by airway hyper-responsiveness, mucus hypersecretion and inflammatory cell recruitment. Isolated immune cells from mouse and allergic patients were stimulated in vitro with HDM under Hyp and normoxia in different co-culture systems to analyse the adaptive immune response. RESULTS: Compared to HDM-treated mice in room air, HDM-treated Hyp-mice displayed ameliorated mucosal hypersecretion and airway hyper-responsiveness. The attenuated asthma phenotype was associated with strongly reduced activation of antigen-presenting cells (APCs), effector cell infiltration and cytokine secretion. In vitro, hypoxia almost completely suppressed the HDM-induced adaptive immune response in both mouse and human immune cells. While hypoxia did not affect effector T-cell responses per-se, it interfered with antigen-presenting cell (APC) differentiation and APC/effector cell crosstalk. CONCLUSIONS: Hypoxia-induced reduction in the Th2-response to HDM ameliorates allergic asthma in vivo. Hypoxia interferes with APC/T-cell crosstalk and confers an unresponsive phenotype to APCs.

Pulmonary fibrosis in Fra-2 transgenic mice is associated with decreased numbers of alveolar macrophages and increased susceptibility to pneumococcal pneumonia.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae, bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae. Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

Role of the Aryl Hydrocarbon Receptor/ARNT/Cytochrome P450 System in Pulmonary Vascular Diseases.

RATIONALE: CYPs (cytochrome p450) are critically involved in the metabolism of xenobiotics and toxins. Given that pulmonary hypertension is strongly associated with environmental exposure, we hypothesize that CYPs play a role in the development and maintenance of pathological vascular remodeling. OBJECTIVE: We sought to identify key CYPs that could link drug or hormone metabolism to the development of pulmonary hypertension. METHODS AND RESULTS: We searched in Medline (PubMed) database, as well as http://www.clinicaltrials.gov, for CYPs associated with many key aspects of pulmonary arterial hypertension including, but not limited to, severe pulmonary hypertension, estrogen metabolism, inflammation mechanisms, quasi-malignant cell growth, drug susceptibility, and metabolism of the pulmonary arterial hypertension-specific drugs. CONCLUSIONS: We postulate a hypothesis where the AhR (aryl hydrocarbon receptor) mediates aberrant cell growth via expression of different CYPs associated with estrogen metabolism and inflammation.

TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2.

Rationale: The bHLH (basic helix-loop-helix) transcription factor TWIST1 (Twist-related protein 1) controls cell proliferation and differentiation in tissue development and disease processes. Recently, endothelial TWIST1 has been linked to pulmonary hypertension (PH) and endothelial-to-mesenchymal transition, yet the role of TWIST1 in smooth muscle cells (SMCs) remains so far unclear.Objectives: To define the role of TWIST1 in SMCs in the pathogenesis of PH.Methods: SMC-specific TWIST1-deficient mice, SMC-specific TWIST1 silencing in rats, mass spectrometry, immunoprecipitation, and chromatin immunoprecipitation were used to delineate the role of SMC TWIST1 in PH.Measurements and Main Results: In pulmonary vessels from patients with PH and rodent PH models, TWIST1 expression was markedly increased and predominantly localized to SMCs. SMC-specific TWIST1 deficiency or silencing attenuated the development of PH and distal vessel muscularization in chronically hypoxic mice and in monocrotaline-treated rats. In vitro, TWIST1 inhibition or silencing prevented pulmonary artery SMC proliferation and migration. Mechanistically, the observed effects were mediated, at least in part, by TWIST1-dependent degradation of GATA-6 (GATA-binding protein 6). BMPR2 (bone morphogenetic protein receptor-2) was identified as a novel downstream target of GATA-6, which directly binds to its promoter. Inhibition of TWIST1 promoted the recruitment of GATA-6 to the BMPR2 promoter and restored BMPR2 functional expression.Conclusions: Our findings identify a key role for SMC TWIST1 in the pathogenesis of lung vascular remodeling and in PH that is partially mediated via reduced GATA-6-dependent BMPR2 expression. Inhibition of SMC TWIST1 may constitute a new therapeutic strategy for the treatment of PH.

Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E2 exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE2 metabolism under the control of TGF-ß and microRNA 218. OBJECTIVE: We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue. METHODS: In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis. RESULTS: We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE2 levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE2 that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro. CONCLUSIONS: These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.

RGS5 Determines Neutrophil Migration in the Acute Inflammatory Phase of Bleomycin-Induced Lung Injury.

The regulator of G protein signaling (RGS) represents a widespread system of controllers of cellular responses. The activities of the R4 subfamily of RGSs have been elucidated in allergic pulmonary diseases. However, the R4 signaling in other inflammatory lung diseases, with a strong cellular immune response, remained unexplored. Thus, our study aimed to discern the functional relevance of the R4 family member, RGS5, as a potential modulating element in this context. Gene profiling of the R4 subfamily showed increased RGS5 expression in human fibrosing lung disease samples. In line with this, RGS5 was markedly increased in murine lungs following bleomycin injury. RGS knock-out mice (RGS-/-) had preserved lung function while control mice showed significant combined ventilatory disorders three days after bleomycin application as compared to untreated control mice. Loss of RGS5 was associated with a significantly reduced neutrophil influx and tissue myeloperoxidase expression. In the LPS lung injury model, RGS5-/- mice also failed to recruit neutrophils into the lung, which was accompanied by reduced tissue myeloperoxidase levels after 24 h. Our in-vitro assays showed impaired migration of RGS5-/- neutrophils towards chemokines despite preserved Ca2+ signaling. ERK dephosphorylation might play a role in reduced neutrophil migration in our model. As a conclusion, loss of RGS5 preserves lung function and attenuates hyperinflammation in the acute phase of bleomycin-induced pulmonary fibrosis and LPS-induced lung injury. Targeting RGS5 might alleviate the severity of exacerbations in interstitial lung diseases.

Basement Membrane Remodeling Controls Endothelial Function in Idiopathic Pulmonary Arterial Hypertension.

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.

PDGFRα and αSMA mark two distinct mesenchymal cell populations involved in parenchymal and vascular remodeling in pulmonary fibrosis.

Pulmonary fibrosis is characterized by pronounced collagen deposition and myofibroblast expansion, whose origin and plasticity remain elusive. We utilized a fate-mapping approach to investigate α-smooth muscle actin (αSMA)+ and platelet-derived growth factor receptor α (PDGFRα)+ cells in two lung fibrosis models, complemented by cell type-specific next-generation sequencing and investigations on human lungs. Our data revealed that αSMA+ and PDGFRα+ cells mark two distinct mesenchymal lineages with minimal transdifferentiation potential during lung fibrotic remodeling. Parenchymal and perivascular fibrotic regions were populated predominantly with PDGFRα+ cells expressing collagen, while αSMA+ cells in the parenchyma and vessel wall showed variable expression of collagen and the contractile protein desmin. The distinct gene expression profile found in normal conditions was retained during pathologic remodeling. Cumulatively, our findings identify αSMA+ and PDGFRα+ cells as two separate lineages with distinct gene expression profiles in adult lungs. This cellular heterogeneity suggests that anti-fibrotic therapy should target diverse cell populations.

FHL-1 is not involved in pressure overload-induced maladaptive right ventricular remodeling and dysfunction.

AIMS: The cytoskeletal signaling protein four and-a-half LIM domains 1 (FHL-1) has recently been identified as a novel key player in pulmonary hypertension as well as in left heart diseases. In this regard, FHL-1 has been implicated in dysregulated hypertrophic signaling in pulmonary arterial smooth muscle cells leading to pulmonary hypertension. In mice, FHL-1-deficiency (FHL-1-/-) led to an attenuated hypertrophic signaling associated with a blunted hypertrophic response of the pressure-overloaded left ventricle (LV). However, the role of FHL-1 in right heart hypertrophy has not yet been addressed. METHODS AND RESULTS: We investigated FHL-1 expression in C57Bl/6 mice subjected to chronic biomechanical stress and found it to be enhanced in the right ventricle (RV). Next, we subjected FHL-1-/- and corresponding wild-type mice to pressure overload of the RV by pulmonary arterial banding for various time points. However, in contrast to the previously published study in LV-pressure overload, which was confirmed here, RV hypertrophy and hypertrophic signaling was not diminished in FHL-1-/- mice. In detail, right ventricular pressure overload led to hypertrophy, dilatation and fibrosis of the RV from both FHL-1-/- and wild-type mice. RV remodeling was associated with impaired RV function as evidenced by reduced tricuspid annular plane systolic excursion. Additionally, PAB induced upregulation of natriuretic peptides and slight downregulation of phospholamban and ryanodine receptor 2 in the RV. However, there was no difference between genotypes in the degree of expression change. CONCLUSION: FHL-1 pathway is not involved in the control of adverse remodeling in the pressure overloaded RV.