Characterizing Lung Particulates Using Quantitative Microscopy in Coal Miners With Severe Pneumoconiosis.

CONTEXT.­: Current approaches for characterizing retained lung dust using pathologists' qualitative assessment or scanning electron microscopy with energy-dispersive spectroscopy (SEM/EDS) have limitations. OBJECTIVE.­: To explore polarized light microscopy coupled with image-processing software, termed quantitative microscopy-particulate matter (QM-PM), as a tool to characterize in situ dust in lung tissue of US coal miners with progressive massive fibrosis. DESIGN.­: We developed a standardized protocol using microscopy images to characterize the in situ burden of birefringent crystalline silica/silicate particles (mineral density) and carbonaceous particles (pigment fraction). Mineral density and pigment fraction were compared with pathologists' qualitative assessments and SEM/EDS analyses. Particle features were compared between historical (born before 1930) and contemporary coal miners, who likely had different exposures following changes in mining technology. RESULTS.­: Lung tissue samples from 85 coal miners (62 historical and 23 contemporary) and 10 healthy controls were analyzed using QM-PM. Mineral density and pigment fraction measurements with QM-PM were comparable to consensus pathologists' scoring and SEM/EDS analyses. Contemporary miners had greater mineral density than historical miners (186 456 versus 63 727/mm3; P = .02) and controls (4542/mm3), consistent with higher amounts of silica/silicate dust. Contemporary and historical miners had similar particle sizes (median area, 1.00 versus 1.14 µm2; P = .46) and birefringence under polarized light (median grayscale brightness: 80.9 versus 87.6; P = .29). CONCLUSIONS.­: QM-PM reliably characterizes in situ silica/silicate and carbonaceous particles in a reproducible, automated, accessible, and time/cost/labor-efficient manner, and shows promise as a tool for understanding occupational lung pathology and targeting exposure controls.

Spatially distinct molecular patterns of gene expression in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies. METHODS: We performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia). RESULTS: We identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF. CONCLUSIONS: Our findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways.

Pathologic Findings in Severe Coal Workers' Pneumoconiosis in Contemporary US Coal Miners.

CONTEXT.­: The pathology of coal workers' pneumoconiosis (CWP) and its most severe form-progressive massive fibrosis (PMF)-in US coal miners has changed in recent years. Severe disease is occurring in younger miners and has been linked to an increase in silica dust exposure. OBJECTIVE.­: To update the description of the pathologic features of CWP in contemporary miners compared to historical miners. DESIGN.­: This study is a retrospective expert classification of lung tissue from 85 historical and contemporary coal miners with PMF. Significant pathologic features were scored by using a standardized instrument with consensus achieved for major findings, including newly defined categories of PMF as coal-type, mixed-type, and silica-type. RESULTS.­: Pathologic features associated with silica dust exposure, including silica-type PMF, mineral dust alveolar proteinosis (MDAP), and immature (early stage) silicotic nodules were increased in contemporary miners. Detailed descriptions of the pathology of contemporary CWP with illustrative figures are provided. CONCLUSIONS.­: Silica-related pathologies are more common in contemporary miners. Severe forms of CWP can be detected by subtyping PMF lesions (if present) or by identification of mature and immature silicotic nodules, coal mine dust-related alveolar proteinosis, and severe inflammation in coal miners' lungs. Silica-type PMF cases showed significantly higher levels of MDAP than either mixed- or coal-type PMF (P < .001). High profusion of birefringent silica/silicate particles was observed more frequently in cases with immature (early stage) silicotic nodules (P = .04). Severe inflammation was also significantly increased in contemporary miners (P = .03). Our findings underscore the urgent need to revise current exposure limits and monitoring of respirable crystalline silica in US coal mines.

Clonal Somatic Mutations in Chronic Lung Diseases Are Associated with Reduced Lung Function.

Rationale: Constantly exposed to the external environment and mutagens such as tobacco smoke, human lungs have one of the highest somatic mutation rates among all human organs. However, the relationship of these mutations to lung disease and function is not known. Objectives: To identify the prevalence and significance of clonal somatic mutations in chronic lung diseases. Methods: We analyzed the clonal somatic mutations from 1,251 samples of normal and diseased noncancerous lung tissue RNA sequencing with paired whole-genome sequencing from the Lung Tissue Research Consortium. We examined the associations of somatic mutations with lung function, disease status, and computationally deconvoluted cell types in two of the most common diseases represented in our dataset, chronic obstructive pulmonary disease (COPD; 29%) and idiopathic pulmonary fibrosis (IPF; 13%). Measurements and Main Results: Clonal somatic mutational burden was associated with reduced lung function in both COPD and IPF. We identified an increased prevalence of clonal somatic mutations in individuals with IPF compared with normal control subjects and individuals with COPD independent of age and smoking status. IPF clonal somatic mutations were enriched in disease-related and airway epithelial-expressed genes such as MUC5B in IPF. Patients who were MUC5B risk variant carriers had increased odds of developing somatic mutations of MUC5B that were explained by increased expression of MUC5B. Conclusions: Our identification of an increased prevalence of clonal somatic mutation in diseased lung that correlates with airway epithelial gene expression and disease severity highlights for the first time the role of somatic mutational processes in lung disease genetics.

Immunological and metabolic characterization of environmental Mycobacterium chimaera infection in a murine model.

Mycobacterium chimaera causes pulmonary disease, but little is known of gradations in isolate virulence. Previously, 17 M. chimaera isolates were screened for survival in THP1 macrophages. "M. chimaera 1" was categorized as "more virulent" because it showed the greatest survival in macrophages, whereas "M. chimaera 2" was categorized as "less virulent" with reduced survival. Herein, we infected C3HeB/FeJ mice to compare the in vivo immune responses to M. chimaera 1 and 2. Unlike macrophages, significantly lower M. chimaera 1 counts were recovered from mouse lung tissue and BAL cells with less lung histopathologic changes compared to M. chimaera 2. Compared to M. chimaera 2, significantly more IL-1ß, IL-6, and TNFα was produced early after M. chimaera 1 infection. LC-MS metabolomics analyses of BAL fluid revealed divergence in sphingolipid, phospholipid metabolism between M. chimaera 1 versus M. chimaera 2 mice. From pan-GWAS analyses, virulence and organizing DNA/molecular structure genes were associated with more virulent M. chimaera isolates. Vigorous lung-specific immune responses to M. chimaera 1 may influence effective bacterial control, but for a different isolate M. chimaera 2, subvert immune control. Continued studies of the gradations in virulence among the same NTM species will advance our understanding of NTM pathogenesis.

Historical shift in pathological type of progressive massive fibrosis among coal miners in the USA.

BACKGROUND: Pneumoconiosis among coal miners in the USA has been resurgent over the past two decades, despite modern dust controls and regulatory standards. Previously published studies have suggested that respirable crystalline silica (RCS) is a contributor to this disease resurgence. However, evidence has been primarily indirect, in the form of radiographic features. METHODS: We obtained lung tissue specimens and data from the National Coal Workers' Autopsy Study. We evaluated specimens for the presence of progressive massive fibrosis (PMF) and used histopathological classifications to type these specimens into coal-type, mixed-type and silica-type PMF. Rates of each were compared by birth cohort. Logistic regression was used to assess demographic and mining characteristics associated with silica-type PMF. RESULTS: Of 322 cases found to have PMF, study pathologists characterised 138 (43%) as coal-type, 129 (40%) as mixed-type and 55 (17%) as silica-type PMF. Among earlier birth cohorts, coal-type and mixed-type PMF were more common than silica-type PMF, but their rates declined in later birth cohorts. In contrast, the rate of silica-type PMF did not decline in cases from more recent birth cohorts. More recent year of birth was significantly associated with silica-type PMF. CONCLUSIONS: Our findings demonstrate a shift in PMF types among US coal miners, from a predominance of coal- and mixed-type PMF to a more commonly encountered silica-type PMF. These results are further evidence of the prominent role of RCS in the pathogenesis of pneumoconiosis among contemporary US coal miners.

Lymphangioleiomyomatosis and Other Cystic Lung Diseases.

Cysts and cavities in the lung are commonly encountered on chest imaging. It is necessary to distinguish thin-walled lung cysts (≤2 mm) from cavities and characterize their distribution as focal or multifocal versus diffuse. Focal cavitary lesions are often caused by inflammatory, infectious, or neoplastic processes in contrast to diffuse cystic lung diseases. An algorithmic approach to diffuse cystic lung disease can help narrow the differential diagnosis, and additional testing such as skin biopsy, serum biomarkers, and genetic testing can be confirmatory. An accurate diagnosis is essential for the management and disease surveillance of extrapulmonary complications.

Pathology and Mineralogy of the Pneumoconioses.

Pneumoconioses represent the spectrum of lung diseases caused by inhalation of respirable particulate matter small enough (typically <5-µm diameter) to reach the terminal airways and alveoli. Pneumoconioses primarily occur in occupational settings where workers perform demanding and skilled manual labor including mining, construction, stone fabrication, farming, plumbing, electronics manufacturing, shipyards, and more. Most pneumoconioses develop after decades of exposure, though shorter latencies can occur from more intense particulate matter exposures. In this review, we summarize the industrial exposures, pathologic findings, and mineralogic features of various well-characterized pneumoconioses including silicosis, silicatosis, mixed-dust pneumoconiosis, coal workers' pneumoconiosis, asbestosis, chronic beryllium disease, aluminosis, hard metal pneumoconiosis, and some less severe pneumoconioses. We also review a general framework for the diagnostic work-up of pneumoconioses for pulmonologists including obtaining a detailed occupational and environmental exposure history. Many pneumoconioses are irreversible and develop due to excessive cumulative respirable dust inhalation. Accurate diagnosis permits interventions to minimize ongoing fibrogenic dust exposure. A consistent occupational exposure history coupled with typical chest imaging findings is usually sufficient to make a clinical diagnosis without the need for tissue sampling. Lung biopsy may be required when exposure history, imaging, and testing are inconsistent, there are unusual or new exposures, or there is a need to obtain tissue for another indication such as suspected malignancy. Close collaboration and information-sharing with the pathologist prior to biopsy is of great importance for diagnosis, as many occupational lung diseases are missed due to insufficient communication. The pathologist has a broad range of analytic techniques including bright-field microscopy, polarized light microscopy, and special histologic stains that may confirm the diagnosis. Advanced techniques for particle characterization such as scanning electron microscopy/energy dispersive spectroscopy may be available in some centers.

Dichotomous role of integrin-ß5 in lung endothelial cells.

Pulmonary arterial hypertension (PAH) is a progressive, devastating disease, and its main histological manifestation is an occlusive pulmonary arteriopathy. One important functional component of PAH is aberrant endothelial cell (EC) function including apoptosis-resistance, unchecked proliferation, and impaired migration. The mechanisms leading to and maintaining physiologic and aberrant EC function are not fully understood. Here, we tested the hypothesis that in PAH, ECs have increased expression of the transmembrane protein integrin-ß5, which contributes to migration and survival under physiologic and pathological conditions, but also to endothelial-to-mesenchymal transition (EnMT). We found that elevated integrin-ß5 expression in pulmonary artery lesions and lung tissue from PAH patients and rats with PH induced by chronic hypoxia and injection of CD117+ rat lung EC clones. These EC clones exhibited elevated expression of integrin-ß5 and its heterodimerization partner integrin-αν and showed accelerated barrier formation. Inhibition of integrin-ανß5 in vitro partially blocked transforming growth factor (TGF)-ß1-induced EnMT gene expression in rat lung control ECs and less in rat lung EC clones and human lung microvascular ECs. Inhibition of integrin-ανß5 promoted endothelial dysfunction as shown by reduced migration in a scratch assay and increased apoptosis in synergism with TGF-ß1. In vivo, blocking of integrin-ανß5 exaggerated PH induced by chronic hypoxia and CD117+ EC clones in rats. In summary, we found a role for integrin-ανß5 in lung endothelial survival and migration, but also a partial contribution to TGF-ß1-induced EnMT gene expression. Our results suggest that integrin-ανß5 is required for physiologic function of ECs and lung vascular homeostasis.

Characterization of Immune Cells From the Lungs of Patients With Chronic Non-Tuberculous Mycobacteria or Pseudomonas aeruginosa Infection.

Little is known of the lung cellular immunophenotypes in patients with non-tuberculous mycobacterial lung disease (NTM-LD). Flow-cytometric analyses for the major myeloid and lymphoid cell subsets were performed in less- and more-diseased areas of surgically resected lungs from six patients with NTM-LD and two with Pseudomonas aeruginosa lung disease (PsA-LD). Lymphocytes, comprised mainly of NK cells, CD4+ and CD8+ T cells, and B cells, accounted for ~60% of all leukocytes, with greater prevalence of T and B cells in more-diseased areas. In contrast, fewer neutrophils were found with decreased number in more-diseased areas. Compared to NTM-LD, lung tissues from patients with PsA-LD demonstrated relatively lower numbers of T and B lymphocytes but similar numbers of NK cells. While this study demonstrated a large influx of lymphocytes into the lungs of patients with chronic NTM-LD, further analyses of their phenotypes are necessary to determine the significance of these findings.

Pathology and Mineralogy Demonstrate Respirable Crystalline Silica Is a Major Cause of Severe Pneumoconiosis in U.S. Coal Miners.

Rationale: The reasons for resurgent coal workers' pneumoconiosis and its most severe forms, rapidly progressive pneumoconiosis and progressive massive fibrosis (PMF), in the United States are not yet fully understood. Objectives: To compare the pathologic and mineralogic features of contemporary coal miners with severe pneumoconiosis with those of their historical counterparts. Methods: Lung pathology specimens from 85 coal miners with PMF were included for evaluation and analysis. We compared the proportion of cases with pathologic and mineralogic findings in miners born between 1910 and 1930 (historical) with those in miners born in or after 1930 (contemporary). Results: We found a significantly higher proportion of silica-type PMF (57% vs. 18%; P < 0.001) among contemporary miners compared with their historical counterparts. Mineral dust alveolar proteinosis was also more common in contemporary miners compared with their historical counterparts (70% vs. 37%; P < 0.01). In situ mineralogic analysis showed that the percentage (26.1% vs. 17.8%; P < 0.01) and concentration (47.3 × 108 vs. 25.8 × 108 particles/cm3; P = 0.036) of silica particles were significantly greater in specimens from contemporary miners compared with their historical counterparts. The concentration of silica particles was significantly greater when silica-type PMF, mineral dust alveolar proteinosis, silicotic nodules, or immature silicotic nodules were present (P < 0.05). Conclusions: Exposure to respirable crystalline silica appears causal in the unexpected surge of severe disease in contemporary miners. Our findings underscore the importance of controlling workplace silica exposure to prevent the disabling and untreatable adverse health effects afflicting U.S. coal miners.

Histopathologic Analysis of Surgically Resected Lungs of Patients with Non-tuberculous Mycobacterial Lung Disease: a Retrospective and Hypothesis-generating Study.

Non-tuberculous mycobacterial lung disease (NTM-LD) is most commonly due to species within the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAbC). Surgical lung resection, typically a lobectomy or segmentectomy, is occasionally undertaken for individuals with recalcitrant but localized NTM-LD. Since the growth characteristics of MAC (slow growers) and MAbC (rapid growers) as well as their drug susceptibility patterns are significantly different, the objective of this study is to characterize and compare the histopathologic features of the resected lungs due to these two major NTM groups. From 1996 to 2017, 356 patients with NTM-LD due to MAC (n=270), MAbC (n=54), or both (n=32) underwent a total of 404 lobar resections (with the lingula counted as a separate lobe) at the University of Colorado Hospital. We analyzed by microscopy the existing surgical lung tissue sections for bronchiolitis, bronchiolectasis, bronchiectasis, non-necrotizing granuloma (airway, parenchymal, and total), necrotizing granuloma (airway, parenchymal, and total), peri-airway fibrosis, fibrous pleuritis, and lymphoid follicles. There were no significant differences in the presence or absence of most of the histopathologic features of surgically removed lungs due to MAC, MAbC, or both MAC + MAbC. However, there were significantly more necrotizing granulomas (airway, parenchymal, and total) and fibrous pleuritis in MAC compared to MAbC lung diseases. Since necrotizing granulomas may be a sign of inadequate control of the infection, we posit that their presence may be an indication of increased chronicity, increased virulence of MAC compared to MAbC, and/or impaired host immunity against the NTM. Futures studies to determine the root cause of such differences in histopathologic findings in MAC versus MAbC lung disease may spawn new leads on differential pathogenic mechanisms with different NTM, with the goal of aiming for more targeted therapy against both the NTM and the lung damage induced by them.

Histopathological Correlation of Acute on Chronic Eosinophilic Pneumonitis Caused by Vaporized Cannabis Oil Inhalation.

Whether eosinophilic pneumonitis represents a true manifestation of e-cigarette, or vaping, product use-associated lung injury remains uncertain, and this ambiguity stems from a lack of histopathological data. We present a previously healthy young woman whose asthma-like symptoms and histopathologic finding of eosinophilic pneumonitis were caused by inhalation of vaporized cannabis hash oil concentrates. This report provides compelling evidence that eosinophilic pneumonitis can result from cannabis hash oil inhalation.

Rituximab and antimetabolite treatment of granulomatous and lymphocytic interstitial lung disease in common variable immunodeficiency.

BACKGROUND: Granulomatous and lymphocytic interstitial lung disease (GLILD) is a life-threatening complication in patients with common variable immunodeficiency (CVID), but the optimal treatment is unknown. OBJECTIVE: Our aim was to determine whether rituximab with azathioprine or mycophenolate mofetil improves the high-resolution computed tomography (HRCT) chest scans and/or pulmonary function test results in patients with CVID and GLILD. METHODS: A retrospective chart review of clinical and laboratory data on 39 patients with CVID and GLILD who completed immunosuppressive therapy was performed. Chest HRCT scans, performed before therapy and after the conclusion of therapy, were blinded, randomized, and scored independently by 2 radiologists. Differences between pretreatment and posttreatment HRCT scan scores, pulmonary function test results, and lymphocyte subsets were analyzed. Whole exome sequencing was performed on all patients. RESULTS: Immunosuppressive therapy improved patients' HRCT scan scores (P < .0001), forced vital capacity (P = .0017), FEV1 (P = .037), and total lung capacity (P = .013) but not their lung carbon monoxide diffusion capacity (P = .12). Nine patients relapsed and 6 completed retreatment, with 5 of 6 of these patients (83%) having improved HRCT scan scores (P = .063). Relapse was associated with an increased number of B cells (P = .016) and activated CD4 T cells (P = .016). Four patients (10%) had pneumonia while undergoing active treatment, and 2 patients (5%) died after completion of therapy. Eight patients (21%) had a damaging mutation in a gene known to predispose (TNFRSF13B [n = 3]) or cause a CVID-like primary immunodeficiency (CTLA4 [n = 2], KMT2D [n = 2], or BIRC4 [n = 1]). Immunosuppression improved the HRCT scan scores in patients with (P = .0078) and without (P < .0001) a damaging mutation. CONCLUSIONS: Immunosuppressive therapy improved the radiographic abnormalities and pulmonary function of patients with GLILD. A majority of patients had sustained remissions.

Chronic Hypersensitivity Pneumonitis, an Interstitial Lung Disease with Distinct Molecular Signatures.

Rationale: Chronic hypersensitivity pneumonitis (CHP) is caused by an immune response to antigen inhalation and is characterized by variable histopathological and clinical features. A subset of subjects with CHP have usual interstitial pneumonia and appear to be clinically similar to subjects with idiopathic pulmonary fibrosis (IPF).Objectives: To determine the common and unique molecular features of CHP and IPF.Methods: Transcriptome analysis of lung samples from CHP (n = 82), IPF (n = 103), and unaffected controls (n = 103) was conducted. Differential gene expression was determined adjusting for sex, race, age, and smoking history and using false discovery rate to control for multiple comparisons.Measurements and Main Results: When compared with controls, we identified 413 upregulated and 317 downregulated genes in CHP and 861 upregulated and 322 downregulated genes in IPF. Concordantly upregulated or downregulated genes in CHP and IPF were related to collagen catabolic processes and epithelial development, whereas genes specific to CHP (differentially expressed in CHP when compared with control and not differentially expressed in IPF) were related to chemokine-mediated signaling and immune responsiveness. Using weighted gene coexpression network analysis, we found that among subjects with CHP, genes involved in adaptive immunity or epithelial cell development were associated with improved or reduced lung function, respectively, and that MUC5B expression was associated with epithelial cell development. MUC5B expression was also associated with lung fibrosis and honeycombing.Conclusions: Gene expression analysis of CHP and IPF identified signatures common to CHP and IPF, as well as genes uniquely expressed in CHP. Select modules of gene expression are characterized by distinct clinical and pathological features of CHP.

The hallmarks of severe pulmonary arterial hypertension: the cancer hypothesis-ten years later.

Severe forms of pulmonary arterial hypertension (PAH) are most frequently the consequence of a lumen-obliterating angiopathy. One pathobiological model is that the initial pulmonary vascular endothelial cell injury and apoptosis is followed by the evolution of phenotypically altered, apoptosis-resistant, proliferating cells and an inflammatory vascular immune response. Although there may be a vasoconstrictive disease component, the increased pulmonary vascular shear stress in established PAH is caused largely by the vascular wall pathology. In this review, we revisit the "quasi-malignancy concept" of severe PAH and examine to what extent the hallmarks of PAH can be compared with the hallmarks of cancer. The cancer model of severe PAH, based on the growth of abnormal vascular and bone marrow-derived cells, may enable the emergence of novel cell-based PAH treatment strategies.

Clonally selected primitive endothelial cells promote occlusive pulmonary arteriopathy and severe pulmonary hypertension in rats exposed to chronic hypoxia.

One current concept suggests that unchecked proliferation of clonally selected precursors of endothelial cells (ECs) contribute to severe pulmonary arterial hypertension (PAH). We hypothesized that clonally selected ECs expressing the progenitor marker CD117 promote severe occlusive pulmonary hypertension (PH). The remodelled pulmonary arteries of PAH patients harboured CD117+ ECs. Rat lung CD117+ ECs underwent four generations of clonal expansion to enrich hyperproliferative ECs. The resulting clonally enriched ECs behaved like ECs, as measured by in vitro and in vivo angiogenesis assays. The same primitive ECs showed a limited ability for mesenchymal lineage differentiation. Endothelial differentiation and function were enhanced by blocking TGF-ß signalling, promoting bone morphogenic protein (BMP) signalling. The transplantation of the EC clones caused arterio-occlusive PH in rats exposed to chronic hypoxia. These EC clones engrafted in the pulmonary arteries. Yet cessation of chronic hypoxia promoted lung cell apoptosis and resolution of vascular lesions. In conclusion, this is to the best of our knowledge, the first report that clonally enriched primitive ECs promote occlusive pulmonary arteriopathy and severe PH. These primitive EC clones further give rise to cells of endothelial and mesenchymal lineage as directed by BMP and TGF-ß signaling.