Lysosomal cholesterol accumulation in aged astrocytes impairs cholesterol delivery to neurons and can be rescued by cannabinoids.

Cholesterol is crucial for the proper functioning of eukaryotic cells, especially neurons, which rely on cholesterol to maintain their complex structure and facilitate synaptic transmission. However, brain cells are isolated from peripheral cholesterol by the blood-brain barrier and mature neurons primarily uptake the cholesterol synthesized by astrocytes for proper function. This study aimed to investigate the effect of aging on cholesterol trafficking in astrocytes and its delivery to neurons. We found that aged astrocytes accumulated high levels of cholesterol in the lysosomal compartment, and this cholesterol buildup can be attributed to the simultaneous occurrence of two events: decreased levels of the ABCA1 transporter, which impairs ApoE-cholesterol export from astrocytes, and reduced expression of NPC1, which hinders cholesterol release from lysosomes. We show that these two events are accompanied by increased microR-33 in aged astrocytes, which targets ABCA1 and NPC1. In addition, we demonstrate that the microR-33 increase is triggered by oxidative stress, one of the hallmarks of aging. By coculture experiments, we show that cholesterol accumulation in astrocytes impairs the cholesterol delivery from astrocytes to neurons. Remarkably, we found that this altered transport of cholesterol could be alleviated through treatment with endocannabinoids as well as cannabidiol or CBD. Finally, according to data demonstrating that aged astrocytes develop an A1 phenotype, we found that cholesterol buildup is also observed in reactive C3+ astrocytes. Given that reduced neuronal cholesterol affects synaptic plasticity, the ability of cannabinoids to restore cholesterol transport from aged astrocytes to neurons holds significant implications in aging and inflammation.

The Exosome-like Vesicles of Giardia Assemblages A, B, and E Are Involved in the Delivering of Distinct Small RNA from Parasite to Parasite.

The genetically related assemblages of the intestinal protozoa parasite Giardia lamblia are morphologically indistinguishable and are often derived from specific hosts. The Giardia assemblages are separated by large genetic distances, which might account for their relevant biological and pathogenic differences. In this work, we analyzed the RNAs cargo released into exosomal-like vesicles (ElVs) by the assemblages A and B, which differentially infect humans, and the assemblage E, which infects hoofed animals. The RNA sequencing analysis revealed that the ElVs of each assemblage contained distinct small RNA (sRNA) biotypes, suggesting a preference for specific packaging in each assemblage. These sRNAs were classified into three categories, ribosomal-small RNAs (rsRNAs), messenger-small RNAs (msRNAs), and transfer-small RNAs (tsRNAs), which may play a regulatory role in parasite communication and contribute to host-specificity and pathogenesis. Uptake experiments showed, for the first time, that ElVs were successfully internalized by the parasite trophozoites. Furthermore, we observed that the sRNAs contained inside these ElVs were first located below the plasma membrane but then distributed along the cytoplasm. Overall, the study provides new insights into the molecular mechanisms underlying the host-specificity and pathogenesis of G. lamblia and highlights the potential role of sRNAs in parasite communication and regulation.

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.

New Player in Endosomal Trafficking: Differential Roles of Smad Anchor for Receptor Activation (SARA) Protein.

The development and maintenance of multicellular organisms require specialized coordination between external cellular signals and the proteins receiving stimuli and regulating responses. A critical role in the proper functioning of these processes is played by endosomal trafficking, which enables the transport of proteins to targeted sites as well as their return to the plasma membrane through its essential components, the endosomes. During this trafficking, signaling pathways controlling functions related to the endosomal system are activated both directly and indirectly. Although there are a considerable number of molecules participating in these processes, some are more known than others for their specific functions. Toward the end of the 1990s, Smad anchor for receptor activation (SARA) protein was described to be controlling and to facilitate the localization of Smads to transforming growth factor ß (TGF-ß) receptors during TGF-ß signaling activation, and, strikingly, SARA was also identified to be one of the proteins that bind to early endosomes (EEs) participating in membrane trafficking in several cell models. The purpose of this review is to analyze the state of the art of the contribution of SARA in different cell types and cellular contexts, focusing on the biological role of SARA in two main processes, trafficking and cellular signaling, both of which are necessary for intercellular coordination, communication, and development.

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.

Transcriptomic profiling of platelet senescence and platelet extracellular vesicles.

BACKGROUND: Platelets (PLTs) are derived from megakaryocytes during PLT shedding. Senescent or activated PLTs are expanded in vascular and neurological diseases and release PLT extracellular vesicles (PL-EVs). A systematic analysis of regular messenger RNA (mRNA) and small RNA composition in PLTs and PL-EVs during in vitro PLT senescence has not yet been published. STUDY DESIGN AND METHODS: We isolated PLTs, total PL-EVs, and PL-EV subsets on Days 0 and 5 from human stored donor platelet concentrates. Isolated mRNA species and microRNA (miRNA) species were analyzed by microarrays and deep sequencing. Correlation of mRNA and miRNA species (miR) and miRNA target analyses were performed using bioinformatics. RESULTS: During in vitro PLT senescence, residual PLT mRNA species were decreased and partially converted to miRNA species. Residual mRNAs included encoded genes relevant for atherosclerosis, inflammation (matrix metallopeptidase 14 [MMP-14], granulin [GRN], angiopoietin like 2 [ANGPTL2]), and neurotransmission (dopamine receptor 2 [DRD2], γ-aminobutyric acid type A receptor ρ3 [GABRR3]). Compared with senescent PLTs, PL-EVs have up-regulated their miRNA species involved in "diabesity" and in vascular and metabolic disease (miR-144-3p, miR-486-5p, miR-142-5p, miR-451a, miR-25-3p, miR-145-5p, and let-7f-5p). The 100 highest expressed PL-EV miRNA species determined by microarrays were compared with the 100 highest expressed PL-EV miRNA species detected by deep sequencing. This approach resulted in 66 overlaps. The regulated miRNAs (assessed by both methods) were related to neurological disorders, including targets for Alzheimer's disease (e.g., ß-site amyloid precursor protein APP-cleaving enzyme 1 [BACE1], translocase of outer mitochondrial membrane 40 homolog [TOMM40], neuron navigator 3 [NAV3]). CONCLUSION: During in vitro senescence, PLTs degrade large RNA species. Concomitantly, they up-regulate a distinct set of known small RNA species involved in atherosclerosis, inflammation, and neurodegeneration. PL-EVs enrich miRNA species, likely supporting the role of PLTs and PL-EVs in vascular homeostasis and as carriers of neurodegenerative disease-related miRNA cargo.

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.].

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.

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, ß-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.

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.

Epigenetic regulation of adipogenesis.

PURPOSE OF REVIEW: Epigenetic regulation plays an essential role in cell differentiation, by allowing the establishment and maintenance of the gene-expression pattern of the mature cell type. Because of its importance in chronic diseases, adipogenesis is one of the best-studied differentiation processes. The hormonal and transcriptional cascades governing the differentiation of the adipocytes are well known, but the role of epigenetic mechanisms is only starting to emerge. In this review, we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade. RECENT FINDINGS: The advent of high-throughput technologies has made possible the exhaustive analysis of the epigenetic phenomenons taking place during adipogenesis. The cooperative recruitment of CCAAT/enhancer-binding protein (C/EBPß) and other early proadipogenic transcription factors to transcription factor hotspots shortly after induction of adipogenesis is required to establish a transient epigenomic state that then informs the recruitment of the later adipogenic transcription factors peroxisome proliferator-activated receptor (PPARγ) and C/EBPα to their target genes. SUMMARY: Epigenetic marks and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism.

Histone demethylase LSD1 regulates adipogenesis.

Epigenetic mechanisms, in particular the enzymatic modification of histones, are a crucial element of cell differentiation, a regulated process that allows a precursor cell basically to turn into a different cell type while maintaining the same genetic equipment. We have previously described that the promoters of adipogenic genes display significant levels of dimethylation at the Lys(4) of histone H3 (H3K4) in preadipocytes, where these genes are still silenced, thus maintaining the chromatin of the precursor cell in a receptive state. Here, we show that the expression of several histone demethylases and methyltransferases increases during adipogenesis, suggesting an important role for these proteins in this process. Knockdown of the H3K4/K9 demethylase LSD1 results in markedly decreased differentiation of 3T3-L1 preadipocytes. This outcome is associated with decreased H3K4 dimethylation and increased H3K9 dimethylation at the promoter of transcription factor cebpa, whose expression must be induced >200-fold upon stimulation of differentiation. Thus, our data suggest that LSD1 acts to maintain a permissive state of the chromatin in this promoter by opposing the action of a H3K9 methyltransferase. Knockdown of H3K9 methyltransferase SETDB1 produced the opposite results, by decreasing H3K9 dimethylation and increasing H3K4 dimethylation levels at the cebpa promoter and favoring differentiation. These findings indicate that the histone methylation status of adipogenic genes as well as the expression and function of the proteins involved in its maintenance play a crucial role in adipogenesis.

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.

A chromatin perspective of adipogenesis.

The transcriptional cascade governing adipogenesis has been thoroughly examined throughout the years. Transcription factors PPARγ and C/EBPα are universally recognized as the master regulators of adipocyte differentiation and together they direct the establishment of the gene expression pattern of mature adipose cells. However, this familiar landscape has been considerably broadened in recent years by the identification of novel factors that participate in the regulation of adipogenesis, either favoring or inhibiting it, through their effects on chromatin. Epigenetic signals and chromatin-modifying proteins contribute to adipogenesis and, through regulation of the phenotypic maintenance of the mature adipocytes, to the control of metabolism. In this review we intend to summarize the recently described epigenetic events that participate in adipogenesis and their connections with the main factors that constitute the classical transcriptional cascade.

Application of electrophoretic mobility shift assay and chromatin immunoprecipitation in the study of transcription in adipose cells.

Chromatin, long thought to be no more than a scaffold supporting DNA compaction inside the cell nucleus, has emerged in the last few years as a major regulatory element involved in the control of gene expression both acutely during interphase and programmatically throughout complex processes of development and differentiation. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities and offers an excellent model for the study of transcriptional regulation. In this regard, electrophoretic mobility shift assay and immunoprecipitation of chromatin are complementary methods that can be used to study the binding of nuclear proteins to DNA and to characterize how these proteins interact with and modify chromatin to regulate gene expression and, more globally, cell differentiation. This chapter provides some strategies to perform these two assays using 3T3-L1 cells and rodent primary preadipocytes and adipocytes.

Chromatin and chromatin-modifying proteins in adipogenesis.

Long considered scarcely more than an uninteresting energy depot, adipose tissue has recently achieved star status. Far from being mere fat droplets, the adipocytes secrete a number of hormones and bioactive peptides, collectively known as adipokines, which participate in the regulation of a variety of functions, from haemostasis to angiogenesis to energy balance. Adipose tissue constitutes a bona-fide endocrine organ whose main dysfunctions, obesity and lipodystrophy, are related to the development of diabetes, hypertension, or dyslipidemia. The renewed interest in this tissue has prompted an escalation in the number of studies focusing on every aspect of the biology of the adipose cell, in the belief that a detailed knowledge of the mechanisms involved in the differentiation and function of adipocytes may contribute new therapeutical approaches to the treatment of such alarming medical problems. Adipogenesis is the result of an intertwined network of transcription factors and coregulators with chromatin-modifying activities that together, are responsible for the establishment of the gene expression pattern of mature adipocytes. Although the exquisitely regulated transcription factor cascade controlling adipogenesis has been extensively studied, the role of chromatin and chromatin-modifying proteins has become apparent only in recent times.

Histone H3 lysine 4 dimethylation signals the transcriptional competence of the adiponectin promoter in preadipocytes.

Adipogenesis is regulated by a coordinated cascade of sequence-specific transcription factors and coregulators with chromatin-modifying activities that are between them responsible for the establishment of the gene expression pattern of mature adipocytes. Here we examine the histone H3 post-translational modifications occurring at the promoters of key adipogenic genes during adipocyte differentiation. We show that the promoters of apM1, glut4, gpd1, and leptin are enriched in dimethylated histone H3 Lys4 (H3-K4) in 3T3-L1 fibroblasts, where none of these genes are yet expressed. A detailed study of the apM1 locus shows that H3-K4 dimethylation is restricted to the promoter region in undifferentiated cells and associates with RNA polymerase II (pol II) loading. The beginning of apM1 transcription at the early stages of adipogenesis coincides with promoter H3 hyperacetylation and H3-K4 trimethylation. At the coding region, H3 acetylation and dimethylation, as well as pol II binding, are found in cells at later stages of differentiation, when apM1 transcription reaches its maximal peak. This same pattern of histone modifications is detected in mouse primary preadipocytes and adipocytes but not in a related fibroblast cell line that is not committed to an adipocyte fate. Inhibition of H3-K4 methylation by treatment of 3T3-L1 cells with methylthioadenosine results in decreased apM1 gene expression as well as decreased adipogenesis. Taken together, our data indicate that H3-K4 dimethylation and pol II binding to the promoter of key adipogenic genes are distinguishing marks of cells that have undergone determination to a preadipocyte stage.