Background—: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin–expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin–expressing cells. Methods and Results—: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2 ^Δ140Ex1/+ rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable. Conclusions—: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications.

We report on an artificially intelligent nanoarray based on molecularly modified gold nanoparticles and a random network of single-walled carbon nanotubes for noninvasive diagnosis and classification of a number of diseases from exhaled breath. The performance of this artificially intelligent nanoarray was clinically assessed on breath samples collected from 1404 subjects having one of 17 different disease conditions included in the study or having no evidence of any disease (healthy controls). Blind experiments showed that 86% accuracy could be achieved with the artificially intelligent nanoarray, allowing both detection and discrimination between the different disease conditions examined. Analysis of the artificially intelligent nanoarray also showed that each disease has its own unique breathprint, and that the presence of one disease would not screen out others. Cluster analysis showed a reasonable classification power of diseases from the same categories. The effect of confounding clinical and environmental factors on the performance of the nanoarray did not significantly alter the obtained results. The diagnosis and classification power of the nanoarray was also validated by an independent analytical technique, i.e., gas chromatography linked with mass spectrometry. This analysis found that 13 exhaled chemical species, called volatile organic compounds, are associated with certain diseases, and the composition of this assembly of volatile organic compounds differs from one disease to another. Overall, these findings could contribute to one of the most important criteria for successful health intervention in the modern era, viz. easy-to-use, inexpensive (affordable), and miniaturized tools that could also be used for personalized screening, diagnosis, and follow-up of a number of diseases, which can clearly be extended by further development.

Rationale: Patients with idiopathic pulmonary arterial hypertension (IPAH) present circulating autoantibodies against vascular wall components. Pathogenic antibodies may be generated in tertiary (ectopic) lymphoid tissues (tLTs). Objectives: To assess the frequency of tLTs in IPAH lungs, as compared with control subjects and flow-induced PAH in patients with Eisenmenger syndrome, and to identify local mechanisms responsible for their formation, perpetuation, and function. Methods: tLT composition and structure were studied by multiple immunostainings. Cytokine/chemokine and growth factor expression was quantified by real-time polymerase chain reaction and localized by immunofluorescence. The systemic mark of pulmonary lymphoid neogenesis was investigated by flow cytometry analyses of circulating lymphocytes. Measurements and Main Results: As opposed to lungs from control subjects and patients with Eisenmenger syndrome, IPAH lungs contained perivascular tLTs, comprising B- and T-cell areas with high endothelial venules and dendritic cells. Lymphocyte survival factors, such as IL-7 and platelet-derived growth factor-A, were expressed in tLTs as well as the lymphorganogenic cytokines/chemokines, lymphotoxin-α/-β, CCL19, CCL20, CCL21, and CXCL13, which might explain the depletion of circulating CCR6⁺ and CXCR5⁺ lymphocytes. tLTs were connected with remodeled vessels via an ER-TR7⁺ stromal network and supplied by lymphatic channels. The presence of germinal center centroblasts, follicular dendritic cells, activation-induced cytidine deaminase, and IL-21⁺PD1⁺ follicular helper T cells in tLTs together with CD138⁺ plasma cell accumulation around remodeled vessels in areas of immunoglobulin deposition argued for local immunoglobulin class switching and ongoing production. Conclusions: We highlight the main features of lymphoid neogenesis specifically in the lungs of patients with IPAH, providing new evidence of immunological mechanisms in this severe condition.

Rationale: C-kit⁺ cells, including bone marrow (BM)–derived progenitors and mast cells, may participate in vascular remodelling. Because recent studies suggest that c-kit may be a target for innovative therapies in experimental pulmonary hypertension, we investigated the contribution of c-kit⁺ cells in human idiopathic pulmonary arterial hypertension (IPAH). Objectives: To investigate the contribution of c-kit⁺ cells in human IPAH. Methods: Single c-kit, CXCL12/SDF-1α, CXCR4, CD34, and multiple c-kit, α-smooth muscle actin (α-SMA) and tryptase immunostainings were performed in IPAH lungs. C-kit mRNA expression was quantified by real-time polymerase chain reaction in microdissected pulmonary arteries from patients with IPAH and control subjects. Phenotype and function of circulating progenitors were analyzed by flow cytometry. Plasma levels of soluble c-kit and CXCL12/SDF-1α were measured by ELISA. Measurements and Main Results: Infiltration of c-kit⁺ cells in pulmonary arterial lesions was associated with an increase in c-kit mRNA expression (P < 0.01 compared with control subjects). Both c-kit⁺/tryptase⁺ mast cells and c-kit⁺/tryptase⁻ BM-derived cells were increased in pulmonary arteries of patients with IPAH compared with control subjects (106.6 ± 54.5 vs. 28 ± 16.8/mm² and 143.8 ± 101.1 vs. 23.3 ± 11.9/mm²; all P < 0.01). Plasma-soluble c-kit was increased in IPAH compared with control subjects (27.4 ± 12.4 vs. 19.5 ± 5.8 ng/ml; P < 0.05). Two populations of circulating BM-derived cells (lin-CD34^highCD133^high [c-kit^highCXCR4^low] and lin-CD34^lowCD133⁻ [c-kit^lowCXCR4^high]) were increased in IPAH compared with control subjects (P = 0.01). Pulmonary arterial lesions were associated with vasa vasorum expansion expressing CXL12/SDF-1α that may recruit c-kit⁺ cells. Conclusions: In IPAH, c-kit⁺ cells infiltrate pulmonary arterial lesions and may participate to vascular remodeling. Therefore, c-kit may represent a potential target for innovative PAH therapy.