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Hereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN 2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2 -/- rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2 -/- rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2 -/- rat lungs revealed increases in two T-cell populations, a LAG3 + T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2 -/- rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 (general control nonderepressible 2) gene ( EIF2AK4 [eukaryotic translation initiation factor 2 alpha kinase 4]) were recently associated with pulmonary veno-occlusive disease. The aim of this study is to explore the involvement of the GCN2/eIF2α (eukaryotic initiation factor 2α) pathway in the development of PH during PF, in both human disease and in a laboratory animal model. Lung tissue from patients with PF with or without PH was collected at the time of lung transplantation, and control tissue was obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4 -mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies and organ collection were performed 3 weeks after instillation. Only significant results ( P  < 0.05) are presented. In PF lung tissue, GCN2 protein expression was decreased compared with control tissue. GCN2 expression was reduced in CD31 + endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats compared with saline. EIF2AK4 -mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline concentrations) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure compared with wild-type animals. Our data show that GCN2 is dysregulated in both humans and in an animal model of combined PF and PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied.

Background Platelet-derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1 + ) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet-derived growth factor receptor type α (PDGFRα) pathway in progenitor cell-dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1 + cells expressing PDGFRα. PW1 nLacZ reporter mice were used to follow the fate of pulmonary PW1 + progenitor cells in a model of chronic hypoxia-induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1 + progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1 + progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1 + progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell-dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add-on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.

Objective: Excessive accumulation of resident cells within the pulmonary vascular wall represents the hallmark feature of the remodeling occurring in pulmonary arterial hypertension (PAH). Furthermore, we have previously demonstrated that pulmonary arterioles are excessively covered by pericytes in PAH, but this process is not fully understood. The aim of our study was to investigate the dynamic contribution of pericytes in PAH vascular remodeling. Approach and Results: In this study, we performed in situ, in vivo, and in vitro experiments. We isolated primary cultures of human pericytes from controls and PAH lung specimens then performed functional studies (cell migration, proliferation, and differentiation). In addition, to follow up pericyte number and fate, a genetic fate-mapping approach was used with an NG2CreER;mT/mG transgenic mice in a model of pulmonary arteriole muscularization occurring during chronic hypoxia. We identified phenotypic and functional abnormalities of PAH pericytes in vitro, as they overexpress CXCR (C-X-C motif chemokine receptor)-7 and TGF (transforming growth factor)-βRII and, thereby, display a higher capacity to migrate, proliferate, and differentiate into smooth muscle-like cells than controls. In an in vivo model of chronic hypoxia, we found an early increase in pericyte number in a CXCL (C-X-C motif chemokine ligand)-12-dependent manner whereas later, from day 7, activation of the canonical TGF-β signaling pathway induces pericytes to differentiate into smooth muscle-like cells., Conclusions: Our findings reveal a pivotal role of pulmonary pericytes in PAH and identify CXCR-7 and TGF-βRII as 2 intrinsic abnormalities in these resident progenitor vascular cells that foster the onset and maintenance of PAH structural changes in blood lung vessels.

Background: Continuous intravenous epoprostenol was the first treatment approved for pulmonary arterial hypertension (PAH) but administration through a central venous line carries risks of thrombosis and sepsis, particularly in children. We sought to evaluate the safety, efficacy and management of subcutaneous (SC) treprostinil in children with PAH., Methods: Fifty-six children (median age 65, range 1-200 months) were treated with SC treprostinil. Clinical status, echocardiography, NT-proBNP, and site pain and infection were evaluated. Right heart catheterization was performed in 54 patients before starting SC treprostinil infusion and was repeated at 6 months in 31 patients., Results: Treatment was well tolerated in 79% of patients. Site pain resistant to simple analgesics occurred in 12 patients (21%), but could be managed in 9/12 children. At 6 months, 3 patients had died, 4 had received a Potts shunt and 1 underwent lung transplantation. Among the 48 treated patients, 40 (83%) showed significant improvement in WHO functional class, 6 minute walk distance, NT-proBNP and pulmonary vascular resistance (p < 0.01 for all parameters). At last follow-up (median 37 months), ten patients had died, 2 underwent a lung transplantation and 8 underwent a Potts shunt. In 30 of the 36 remaining treated patients, improvement of clinical status was sustained. No children developed sepsis and 12 had minor site infections., Conclusion: Subcutaneous treprostinil infusion is an effective therapy without serious side effects in children with PAH. Site pain can be managed with simple analgesics in most children., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rationale: Pulmonary arterial hypertension is characterized by vascular remodeling and neomuscularization. PW1(+) progenitor cells can differentiate into smooth muscle cells (SMCs) in vitro., Objective: To determine the role of pulmonary PW1(+) progenitor cells in vascular remodeling characteristic of pulmonary arterial hypertension., Methods and Results: We investigated their contribution during chronic hypoxia-induced vascular remodeling in Pw1(nLacZ+/-) mouse expressing β-galactosidase in PW1(+) cells and in differentiated cells derived from PW1(+) cells. PW1(+) progenitor cells are present in the perivascular zone in rodent and human control lungs. Using progenitor markers, 3 distinct myogenic PW1(+) cell populations were isolated from the mouse lung of which 2 were significantly increased after 4 days of chronic hypoxia. The number of proliferating pulmonary PW1(+) cells and the proportion of β-gal(+) vascular SMC were increased, indicating a recruitment of PW1(+) cells and their differentiation into vascular SMC during early chronic hypoxia-induced neomuscularization. CXCR4 inhibition using AMD3100 prevented PW1(+) cells differentiation into SMC but did not inhibit their proliferation. Bone marrow transplantation experiments showed that the newly formed β-gal(+) SMC were not derived from circulating bone marrow-derived PW1(+) progenitor cells, confirming a resident origin of the recruited PW1(+) cells. The number of pulmonary PW1(+) cells was also increased in rats after monocrotaline injection. In lung from pulmonary arterial hypertension patients, PW1-expressing cells were observed in large numbers in remodeled vascular structures., Conclusions: These results demonstrate the existence of a novel population of resident SMC progenitor cells expressing PW1 and participating in pulmonary hypertension-associated vascular remodeling., (© 2016 American Heart Association, Inc.)

Objective: To investigate the role of bone morphogenetic proteins (BMPs) on α-B-crystallin (CRYAB) expression and its physiological consequences on endothelial cells (ECs)., Approach and Results: We report that the gene encoding for the small heat shock protein, CRYAB, is a transcriptional target of the BMP signaling pathway. We demonstrate that CRYAB expression is upregulated strongly by BMPs in an EC line and in human lung microvascular ECs and human umbilical vein ECs. We show that BMP signals through the BMPR2-ALK1 pathway to upregulate CRYAB expression through a transcriptional indirect mechanism involving Id1. We observed that the known antiapoptotic effect of the BMPs is, in part, because of the upregulation of CRYAB expression in EC. We also show that cryab is downregulated in vivo, in a mouse model of pulmonary arterial hypertension induced by chronic hypoxia where the BMP pathway is downregulated., Conclusions: We demonstrate a cross-talk between BMPs and CRYAB and a major effect of this regulatory interaction on resistance to apoptosis.

Pulmonary arterial hypertension (PAH) is a rare, severe disease resulting from progressive obliteration of small-caliber pulmonary arteries by proliferating vascular cells. PAH can occur without recognized etiology (idiopathic PAH), be associated with a systemic disease or occur as a heritable form, with BMPR2 mutated in approximately 80% of familial and 15% of idiopathic PAH cases. We conducted a genome-wide association study (GWAS) based on 2 independent case-control studies for idiopathic and familial PAH (without BMPR2 mutations), including a total of 625 cases and 1,525 healthy individuals. We detected a significant association at the CBLN2 locus mapping to 18q22.3, with the risk allele conferring an odds ratio for PAH of 1.97 (1.59-2.45; P = 7.47 × 10(-10)). CBLN2 is expressed in the lung, and its expression is higher in explanted lungs from individuals with PAH and in endothelial cells cultured from explanted PAH lungs.

Background: The various aetiologies and risk factors for pulmonary arterial hypertension (PAH) lead to close phenotypes with small differences. Plasma microparticles have been shown to be increased in vascular pathologies including PAH. The aim of this study was to determine whether the levels of endothelial and platelet-derived microparticles could vary between different forms of PAH: idiopathic PAH (iPAH), heritable PAH associated with BMPR2 (Bone morphogenetic protein receptor, type II) mutation (hPAH) and PAH associated with connective tissue diseases (aPAH)., Materials and Methods: Microparticles were analysed using flow cytometry in plasma from controls and iPAH, hPAH and aPAH patients. Platelet-derived MP (PMP) were defined as CD31(+)/CD41(+) and endothelial-derived MP (EMP) as CD31(+)/CD41(-). Two populations of PMP were isolated according to their size, defining small PMP (0·3-0·5 μm) and large PMP (0·5-0·9 μm). BMPR2 genotype, clinical and biologic parameters were recorded., Results: EMP and small PMP levels in iPAH, hPAH and aPAH were similar and were significantly increased as compared with controls. No differences in large PMP levels were observed. After adjusting for age, sex, proBNP and CRP, EMP and small PMP levels did not correlate with clinical parameters., Conclusions: iPAH, hPAH and aPAH were characterized by increased levels of EMP and of small PMP, a new class of PMP which seems to be differentially produced than large PMP., (© 2012 The Authors. European Journal of Clinical Investigation © 2012 Stichting European Society for Clinical Investigation Journal Foundation.)