Olga Tura-Ceide

Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.

RATIONALE Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available. OBJECTIVES To investigate the role of the soluble guanylate cyclase (sGC)-cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC-cGMP axis is a treatment target for these conditions. METHODS Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done. MEASUREMENTS AND MAIN RESULTS The functionally essential sGC β1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils. CONCLUSIONS The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.

Circulating Progenitor Cells and Vascular Dysfunction in Chronic Obstructive Pulmonary Disease

Background In chronic obstructive pulmonary disease (COPD), decreased progenitor cells and impairment of systemic vascular function have been suggested to confer higher cardiovascular risk. The origin of these changes and their relationship with alterations in the pulmonary circulation are unknown. Objectives To investigate whether changes in the number of circulating hematopoietic progenitor cells are associated with pulmonary hypertension or changes in endothelial function. Methods 62 COPD patients and 35 controls (18 non-smokers and 17 smokers) without cardiovascular risk factors other than cigarette smoking were studied. The number of circulating progenitors was measured as CD45+CD34+CD133+ labeled cells by flow cytometry. Endothelial function was assessed by flow-mediated dilation. Markers of inflammation and angiogenesis were also measured in all subjects. Results Compared with controls, the number of circulating progenitor cells was reduced in COPD patients. Progenitor cells did not differ between control smokers and non-smokers. COPD patients with pulmonary hypertension showed greater number of progenitor cells than those without pulmonary hypertension. Systemic endothelial function was worse in both control smokers and COPD patients. Interleukin-6, fibrinogen, high sensitivity C-reactive protein, vascular endothelial growth factor and tumor necrosis factor were increased in COPD. In COPD patients, the number of circulating progenitor cells was inversely related to the flow-mediated dilation of systemic arteries. Conclusions Pulmonary and systemic vascular impairment in COPD is associated with cigarette smoking but not with the reduced number of circulating hematopoietic progenitors. The latter appears to be a consequence of the disease itself not related to smoking habit.

Gene expression profile of angiogenic factors in pulmonary arteries in COPD: relationship with vascular remodeling.

Pulmonary vessel remodeling in chronic obstructive pulmonary disease (COPD) involves changes in smooth muscle cell proliferation, which are highly dependent on the coordinated interaction of angiogenic-related growth factors. The purpose of the study was to investigate, in isolated pulmonary arteries (PA) from patients with COPD, the gene expression of 46 genes known to be modulators of the angiogenic process and/or involved in smooth muscle cell proliferation and to relate it to vascular remodeling. PA segments were isolated from 29 patients and classified into tertiles, according to intimal thickness. After RNA extraction, the gene expression was assessed by RT-PCR using TaqMan low-density arrays. The univariate analysis only showed upregulation of angiopoietin-2 (ANGPT-2) in remodeled PA (P < 0.05). The immunohistochemical expression of ANGPT-2 correlated with intimal enlargement (r = 0.42, P < 0.05). However, a combination of 10 factors in a multivariate discriminant analysis model explained up to 96% of the classification of the arteries. A network analysis of 46 genes showed major decentralization. In this network, the metalloproteinase-2 (MMP-2) was shown to be the bridge between intimal enlargement and fibrogenic factors. In COPD patients, plasma levels of ANGPT-2 were higher in current smokers or those with pulmonary hypertension. We conclude that an imbalance in ANGPT-2, combined with related factors such as VEGF, β-catenin, and MMP-2, may partially explain the structural derangements of the arterial wall. MMP-2 may act as a bridge channeling actions from the main fibrogenic factors.

Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient

Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G > A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1–45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems.

MicroRNA Dysregulation in Pulmonary Arteries from Chronic Obstructive Pulmonary Disease. Relationships with Vascular Remodeling

Abstract Pulmonary vascular remodeling is an angiogenic‐related process involving changes in smooth muscle cell (SMC) homeostasis, which is frequently observed in chronic obstructive pulmonary disease (COPD). MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA expression levels of many genes, leading to the manifestation of cell identity and specific cellular phenotypes. Here, we evaluate the miRNA expression profiles of pulmonary arteries (PAs) of patients with COPD and its relationship with the regulation of SMC phenotypic change. miRNA expression profiles from PAs of 12 patients with COPD, 9 smokers with normal lung function (SK), and 7 nonsmokers (NS) were analyzed using TaqMan Low‐Density Arrays. In patients with COPD, expression levels of miR‐98, miR‐139‐5p, miR‐146b‐5p, and miR‐451 were upregulated, as compared with NS. In contrast, miR‐197, miR‐204, miR‐485‐3p, and miR‐627 were downregulated. miRNA‐197 expression correlated with both airflow obstruction and PA intimal enlargement. In an in vitro model of SMC differentiation, miR‐197 expression was associated with an SMC contractile phenotype. miR‐197 inhibition blocked the acquisition of contractile markers in SMCs and promoted a proliferative/migratory phenotype measured by both cell cycle analysis and wound‐healing assay. Using luciferase assays, Western blot, and quantitative PCR, we confirmed that miR‐197 targets the transcription factor E2F1. In PAs from patients with COPD, levels of E2F1 were increased as compared with NS. In PAs of patients with COPD, remodeling of the vessel wall is associated with downregulation of miR‐197, which regulates SMC phenotype. The effect of miR‐197 on PAs might be mediated, at least in part, by the key proproliferative factor, E2F1.

Effect of targeted therapy on circulating progenitor cells in precapillary pulmonary hypertension

BACKGROUND Endothelial dysfunction is key in the development of pulmonary hypertension (PH) and is associated with reduced number of circulating progenitor cells. Studies to date evaluating levels of circulating progenitor cells in PH have provided conflicting results. Current treatment of pulmonary arterial hypertension (PAH) and medical treatment of chronic thromboembolic pulmonary hypertension (CTEPH) targets endothelium dependent signalling pathways. The effect of PAH-targeted therapy on circulating progenitor cells has not been clearly established. OBJECTIVES To investigate whether levels of circulating progenitor cells in treatment-naïve patients with PAH or CTEPH differ from healthy subjects and to assess the effect of PAH-targeted therapy on the circulating levels of these progenitors. METHODS Thirty controls, 33 PAH and 11 CTEPH treatment-naïve patients were studied. Eighteen patients with PAH and 9 with CTEPH were re-evaluated 6-12months after starting PAH-targeted therapy. Levels of progenitors were measured by flow cytometry as CD45+CD34+ and CD45+CD34+CD133+ cells. RESULTS Compared with controls, the number of circulating progenitor cells was reduced in PAH but not in CTEPH. After 6-12months of treatment, levels of circulating progenitors increased in PAH and remained unchanged in CTEPH. Patients with lower exercise tolerance presented lower levels of circulating progenitors. No other relation was found between levels of progenitors and clinical or hemodynamic parameters. CONCLUSIONS Patients with PAH, but not those with CTEPH, present reduced levels of circulating progenitor cells. PAH-targeted therapy increases levels of progenitors in PAH but not in CTEPH, suggesting different involvement of progenitor cells in the pathobiology of these pulmonary hypertensive disorders.

Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype.

Objective Previous studies have confirmed Slug as a key player in regulating phenotypic changes in several cell models, however, its role in smooth muscle cells (SMC) has never been assessed. The purpose of this study was to evaluate the expression of Slug during the phenotypic switch of SMC in vitro and throughout the development of vascular remodeling. Methods and Results Slug expression was decreased during both cell-to-cell contact and TGFβ1 induced SMC differentiation. Tumor necrosis factor-α (TNFα), a known inductor of a proliferative/dedifferentiated SMC phenotype, induces the expression of Slug in SMC. Slug knockdown blocked TNFα-induced SMC phenotypic change and significantly reduced both SMC proliferation and migration, while its overexpression blocked the TGFβ1-induced SMC differentiation and induced proliferation and migration. Genome-wide transcriptomic analysis showed that in SMC, Slug knockdown induced changes mainly in genes related to proliferation and migration, indicating that Slug controls these processes in SMC. Notably, Slug expression was significantly up-regulated in lungs of mice using a model of pulmonary hypertension-related vascular remodeling. Highly remodeled human pulmonary arteries also showed an increase of Slug expression compared to less remodeled arteries. Conclusions Slug emerges as a key transcription factor driving SMC towards a proliferative phenotype. The increased Slug expression observed in vivo in highly remodeled arteries of mice and human suggests a role of Slug in the pathogenesis of pulmonary vascular diseases.

Poly(ethylmethacrylate-co-diethylaminoethyl acrylate) coating improves endothelial re-population, bio-mechanical and anti-thrombogenic properties of decellularized carotid arteries for blood vessel replacement

Decellularized vascular scaffolds are promising materials for vessel replacements. However, despite the natural origin of decellularized vessels, issues such as biomechanical incompatibility, immunogenicity risks and the hazards of thrombus formation, still need to be addressed. In this study, we coated decellularized vessels obtained from porcine carotid arteries with poly (ethylmethacrylate-co-diethylaminoethylacrylate) (8g7) with the purpose of improving endothelial coverage and minimizing platelet attachment while enhancing the mechanical properties of the decellularized vascular scaffolds. The polymer facilitated binding of endothelial cells (ECs) with high affinity and also induced endothelial cell capillary tube formation. In addition, platelets showed reduced adhesion on the polymer under flow conditions. Moreover, the coating of the decellularized arteries improved biomechanical properties by increasing its tensile strength and load. In addition, after 5 days in culture, ECs seeded on the luminal surface of 8g7-coated decellularized arteries showed good regeneration of the endothelium. Overall, this study shows that polymer coating of decellularized vessels provides a new strategy to improve re-endothelialization of vascular grafts, maintaining or enhancing mechanical properties while reducing the risk of thrombogenesis. These results could have potential applications in improving tissue-engineered vascular grafts for cardiovascular therapies with small caliber vessels.

Correction: Slug Is Increased in Vascular Remodeling and Induces a Smooth Muscle Cell Proliferative Phenotype.

[This corrects the article DOI: 10.1371/journal.pone.0159460.].

Soluble guanylate cyclase stimulation reduces oxidative stress in experimental Chronic Obstructive Pulmonary Disease

Objective Soluble guanylate cyclase (sGC) is a key enzyme of the nitric oxide–cyclic guanosine 3′,5′-monophosphate (NO–cGMP) signaling pathway, and its pharmacological stimulation has been shown to prevent the development of emphysema and pulmonary vascular remodeling in animal models of chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate the effects of sGC stimulation on oxidative stress in the plasma of guinea pigs chronically exposed to cigarette smoke (CS). Methods and results Guinea pigs were exposed to CS or sham for three months, and received either the sGC stimulator BAY 41–2272 or vehicle. Body weight was measured weekly; and markers of oxidative stress in plasma, and airspace size and inflammatory cell infiltrate in lung tissue were analyzed at the end of the study. Compared to sham-exposed guinea pigs, CS-exposed animals gained less body weight and showed higher plasma levels of nitrated tyrosine residues (3-NT), 4-hydroxynonenal (4-HNE), and 8-hydroxydeoxyguanosine (8-OHdG). Treatment with the sGC stimulator led to a body weight gain in the CS-exposed guinea pigs similar to non-exposed and attenuated the increase in 3-NT and 4-HNE. Plasma levels of 3-NT correlated with the severity of inflammatory cell infiltrate in the lung. Conclusion Stimulation of sGC prevents oxidative stress induced by CS exposure and is associated with an attenuated inflammatory response in the lung.