Exploring the Toxicity, Lung Distribution, and Cellular Uptake of Rifampicin and Ascorbic Acid-Loaded Alginate Nanoparticles as Therapeutic Treatment of Lung Intracellular Infections.

Nanotechnology is a very promising technological tool to combat health problems associated with the loss of effectiveness of currently used antibiotics. Previously, we developed a formulation consisting of a chitosan and tween 80-decorated alginate nanocarrier that encapsulates rifampicin and the antioxidant ascorbic acid (RIF/ASC), intended for the treatment of respiratory intracellular infections. Here, we investigated the effects of RIF/ASC-loaded NPs on the respiratory mucus and the pulmonary surfactant. In addition, we evaluated their cytotoxicity for lung cells in vitro, and their biodistribution on rat lungs in vivo after their intratracheal administration. Findings herein demonstrated that RIF/ASC-loaded NPs display a favorable lung biocompatibility profile and a uniform distribution throughout lung lobules. RIF/ASC-loaded NPs were mainly uptaken by lung macrophages, their primary target. In summary, findings show that our novel designed RIF/ASC NPs could be a suitable system for antibiotic lung administration with promising perspectives for the treatment of pulmonary intracellular infections.

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

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.

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 TGFß1 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 TGFß1-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.

Histone demethylase KDM1A represses inflammatory gene expression in preadipocytes.

OBJECTIVE: Persistent inflammation and impaired adipogenesis are frequent features of obesity and underlie the development of its complications. However, the factors behind adipose tissue dysfunction are not completely understood. Previously it was shown that histone demethylase KDM1A is required for adipogenesis. DESIGN AND METHODS: Kdm1a expression was knocked down in 3T3-L1 preadipocytes by siRNA transfection and whole-genome expression profiling was performed by microarray hybridization. The role of NF-κß and C/EBPß was analyzed by incubation with the inhibitor parthenolide and by cebpb knockdown, respectively. RESULTS: Knockdown of kdm1a or rcor2 in 3T3-L1 preadipocytes results in impaired differentiation and induction of inflammatory gene expression. Enhanced expression of il6 in kdm1a knocked down preadipocytes is associated with increased recruitment of C/EBPß and the NF-κß subunit RelA to the il6 promoter. Cebpb knockdown attenuates the induction of il6 expression in kdm1a knocked down cells, whereas simultaneous cebpb knockdown and NF-κß inhibition abrogates it. Dietary-induced and genetic mouse models of obesity display decreased KDM1A in adipose tissue, and this correlates with increased expression of proinflammatory genes and C/EBPß. CONCLUSION: KDM1A represses the expression of inflammatory genes in preadipocytes. Dysregulated kdm1a expression in preadipocytes may thus participate in the development of obesity-associated inflammation.

Pulmonary inflammatory reaction and structural changes induced by cigarette smoke exposure in the Guinea pig.

Cigarette smoke (CS) induces an inflammatory process in the lung that may underlie the development of chronic obstructive pulmonary disease (COPD). The nature and characteristics of this process have not been fully established in animal models. We aimed to evaluate the pulmonary inflammatory reaction and its involvement in structural changes in guinea pigs chronically exposed to CS. 19 Hartley guinea pigs were exposed to 7 cigarettes/day, during 3 or 6 months. 18 control guinea pigs were sham-exposed. Numbers of neutrophils, macrophages and eosinophils and lymphoid follicles were assessed in different lung structures. Airway and vessel morphometry, alveolar space size and collagen deposition were also quantified. After 6 months of exposure, CS-exposed guinea pigs showed increased numbers of neutrophils, macrophages and eosinophils in the airways, intrapulmonary vessels and alveolar septa, as well as lymphoid follicles. Increased numbers of muscularized intrapulmonary vessels were apparent at 3 months. After 6 months of exposure, the airway wall thickened and the alveolar space size increased. Collagen deposition was also apparent in airway walls and alveolar septa after 6 months' exposure. The magnitude of airway wall-thickening correlated with the number of infiltrating inflammatory cells, and the extension of collagen deposition correlated with alveolar space size. We conclude that in the guinea pig, 6 months of CS exposure induces inflammatory cell infiltrate in lung structures, at an intensity that correlates with airway remodelling. These changes resemble those observed in COPD, thus endorsing the pathogenic role of CS and the usefulness of this animal model for its study.

Endothelial progenitor cells undergo an endothelial-to-mesenchymal transition-like process mediated by TGFbetaRI.

AIMS: Endothelial progenitor cells (EPC) have been shown to repair pulmonary endothelium, although they can also migrate into the arterial intima and differentiate into smooth muscle-like (mesenchymal) cells contributing to intimal hyperplasia. The molecular mechanisms by which this process proceeds have not been fully elucidated. Here, we study whether genes involved in the endothelial-to-mesenchymal transition (EnMT) may contribute to the mesenchymal phenotype acquisition of EPC and we evaluate whether transforming growth factor ß1 (TGFß1) is involved in this process. METHODS AND RESULTS: Our results show that co-culture of EPC with smooth muscle cells (SMC) increases the expression of the mesenchymal cell markers α-smooth muscle actin, sm22-α, and myocardin, and decreases the expression of the endothelial cell marker CD31. In the same conditions, we also observed a concomitant increase in the gene expression of the EnMT-related transcription factors: slug, snail, zeb1, and endothelin-1. This indicates that mesenchymal phenotype acquisition occurred through an EnMT-like process. Inhibition of TGFß receptor I (TGFßRI) downregulated snail gene expression, blocked the EnMT, and facilitated the differentiation of EPC to the endothelial cell lineage. Furthermore, TGFßRI inhibition decreased migration of EPC stimulated by SMC without affecting their functionality and adhesion capacity. CONCLUSION: These results indicate that EPC may differentiate into SMC-like cells through an EnMT-like process and that TGFßI plays an important role in the fate of EPC.