RESUMEN
BACKGROUND: The accumulation of glucose degradation products (GDPs) during peritoneal dialysis (PD) can lead to immature angiogenesis in the peritoneum. However, the effect of GDPs on angiogenesis, at concentrations observed in dialysate effluent, has not been widely investigated. We do not know how the inflammation observed in PD-related peritonitis affects angiogenesis of the peritoneum. METHODS: Human umbilical vessel endothelial cells (HUVEC) and human umbilical aortic smooth muscle cells (HUASMC) were used to examine the response to the three main GDPs found in peritoneal dialysate (methylglyoxal (MGO), 3-deoxyglucosone (3-DG), and 5-hydroxymethylfurfural (5-HMF). Supernatant from lipopolysaccharide (LPS)-activated murine macrophage cell lines (RAW 264.7 cells) were used to stimulate angiogenesis in the peritoneum. Changes in the expression of vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor B (PDGFB) in HUVEC, and PDGF-receptor beta (PDGF-Rß) in HUASMC, were examined by real-time PCR, Western blot, and ELISA. RESULTS: In HUVECs, the expression of PDGFB mRNA and protein were decreased by exposure to MGO, 3-DG, and 5-HMF at concentrations observed in dialysate effluent. A subsequent decrease in secreted PDGF-BB was observed. In HUASMCs, MGO and 5-HMF increased the expression of VEGF-A mRNA and protein, while 5-HMF decreased the expression of PDGF-Rß. VEGF-A is upregulated, and PDGF-Rß is downregulated, by conditioned medium of LPS-stimulated macrophages in HUASMCs. CONCLUSIONS: The GDPs of PD effluent cause an imbalance of angiogenic factors in endothelial cells and vascular smooth muscle cells that may lead to immature angiogenesis in the peritoneum.
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Inductores de la Angiogénesis , Soluciones para Diálisis , Glucosa , Neovascularización Patológica , Diálisis Peritoneal , Animales , Humanos , Ratones , Soluciones para Diálisis/farmacología , Células Endoteliales/metabolismo , Glucosa/farmacología , Glucosa/metabolismo , Lipopolisacáridos/farmacología , Óxido de Magnesio/metabolismo , Músculo Liso Vascular/química , Miocitos del Músculo Liso/química , Neovascularización Patológica/metabolismo , Diálisis Peritoneal/efectos adversos , Peritoneo/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Piruvaldehído/farmacología , Piruvaldehído/metabolismo , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Células RAW 264.7RESUMEN
Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.
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Técnicas Biosensibles/instrumentación , GMP Cíclico/análisis , Donantes de Óxido Nítrico/química , Transferencia de Energía por Resonancia de Bioluminiscencia , Endotelio Vascular/química , Endotelio Vascular/citología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lentivirus/genética , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/citología , Cultivo Primario de CélulasRESUMEN
Atherosclerosis is a lipid-driven chronic inflammatory disease that leads to the formation of plaques in the inner lining of arteries. Plaques form over a range of phenotypes, the most severe of which is vulnerable to rupture and causes most of the clinically significant events. In this study, we evaluated the efficacy of nanoparticles (NPs) to differentiate between two plaque phenotypes based on accumulation kinetics in a mouse model of atherosclerosis. This model uses a perivascular cuff to induce two regions of disturbed wall shear stress (WSS) on the inner lining of the instrumented artery, low (upstream) and multidirectional (downstream), which, in turn, cause the development of an unstable and stable plaque phenotype, respectively. To evaluate the influence of each WSS condition, in addition to the final plaque phenotype, in determining NP uptake, mice were injected with NPs at intermediate and fully developed stages of plaque growth. The kinetics of artery wall uptake were assessed in vivo using dynamic contrast-enhanced magnetic resonance imaging. At the intermediate stage, there was no difference in NP uptake between the two WSS conditions, although both were different from the control arteries. At the fully-developed stage, however, NP uptake was reduced in plaques induced by low WSS, but not multidirectional WSS. Histological evaluation of plaques induced by low WSS revealed a significant inverse correlation between the presence of smooth muscle cells and NP accumulation, particularly at the plaque-lumen interface, which did not exist with other constituents (lipid and collagen) and was not present in plaques induced by multidirectional WSS. These findings demonstrate that NP accumulation can be used to differentiate between unstable and stable murine atherosclerosis, but accumulation kinetics are not directly influenced by the WSS condition. This tool could be used as a diagnostic to evaluate the efficacy of experimental therapeutics for atherosclerosis.
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Apolipoproteínas E/genética , Aterosclerosis/diagnóstico por imagen , Ácido Fólico/administración & dosificación , Gadolinio/química , Miocitos del Músculo Liso/química , Placa Aterosclerótica/diagnóstico por imagen , Animales , Aterosclerosis/genética , Velocidad del Flujo Sanguíneo , Medios de Contraste/administración & dosificación , Medios de Contraste/química , Medios de Contraste/farmacocinética , Diagnóstico Diferencial , Modelos Animales de Enfermedad , Femenino , Ácido Fólico/química , Ácido Fólico/farmacocinética , Gadolinio/farmacocinética , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Nanopartículas , Placa Aterosclerótica/genética , Resistencia al Corte , Estrés MecánicoRESUMEN
AIMS: Vascular calcification is a common complication in patients with chronic kidney disease and associated with increased morbidity and mortality. The role of TRPM7 in vascular smooth muscle cell (VSMC) transformation during vascular calcification is not clear. We aim to investigate the effects of phosphate and indoxyl sulphate on the expression of TRPM7 and calcification-related molecules in VSMC. MAIN METHODS: Human aortic smooth muscle cells (HASMC) were treated with phosphate (3.3 mM) or indoxyl sulphate (500 µM and 1000 µM). 2-APB, a channel blocker of TRPM7 was added simultaneously in blocking experiment. Cells were then examined grossly and alizarin red solution was employed for calcification assessment. Lastly, cells were harvested for gene expression and protein abundance analysis. KEY FINDINGS: Phosphate treatment induced significant increase in BMP2, RUNX2, BMP7, vitamin D receptor (VDR), calcium sensing receptor (CaSR) and TRPM7, but 1-alpha hydroxylase, klotho, DKK1 and sclerostin were not changed. The addition of 2-APB prevented increase of BMP2, RUNX2, BMP7, VDR, CaSR and TRPM7. Indoxyl sulphate treatment was associated with decrease in TRPM7 and DKK1, but increase in RUNX2, BMP2 and VDR were noted. There were no significant alterations in BMP7, CaSR, klotho,1-alpha hydroxylase and sclerostin. Co-treatment with 2-APB reversed the increase in VDR. SIGNIFICANCE: Both phosphate and indoxyl sulphate induced calcification in VSMC but it was more prominent in phosphate. TRPM7 was upregulated by phosphate but downregulated in indoxyl sulphate treatment. Vascular calcification was reduced by blocking TRPM7 with 2-APB and there was partial anti-calcification effect in indoxyl sulphate.
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Indicán/farmacología , Músculo Liso Vascular/fisiología , Miocitos del Músculo Liso/fisiología , Fosfatos/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Canales Catiónicos TRPM/fisiología , Calcificación Vascular/fisiopatología , Proteína Morfogenética Ósea 2/análisis , Proteína Morfogenética Ósea 7/análisis , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/análisis , Humanos , Músculo Liso Vascular/química , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Calcitriol/análisis , Insuficiencia Renal Crónica/complicaciones , Canales Catiónicos TRPM/análisis , Canales Catiónicos TRPM/antagonistas & inhibidores , Calcificación Vascular/inducido químicamente , Calcificación Vascular/etiologíaRESUMEN
Regulation of vascular smooth muscle cell (VSMC) proliferation is essential to maintain vascular homeostasis. Hypoxia induces abnormal proliferation of VSMCs and causes vascular proliferative disorders, such as pulmonary hypertension and atherosclerosis. As several cyclin/cyclin-dependent kinase (CDK) complexes and CDK inhibitors (CKIs) control cell proliferation, in this study, we investigated CKIs involved in the hypoxia-induced proliferation process of human primary pulmonary artery smooth muscle cells to understand the underlying molecular mechanism. We demonstrated that p15, p16, and p21 are downregulated in pulmonary artery smooth muscle cells when exposed to hypoxia. In addition, we identified novel hypoxia-induced microRNAs (hypoxamiRs) including miR-497, miR-1268a, and miR-665 that are upregulated under hypoxia and post-transcriptionally regulate p15, p16, and p21 genes, respectively, by directly targeting their 3'UTRs. These miRNAs promoted the proliferation of VSMCs, and their inhibition decreased VSMC proliferation even in hypoxic conditions. Overall, this study revealed that miRNA-mediated regulatory mechanism of CKIs is essential for hypoxia-induced proliferation of VSMCs. These findings provide insights for a better understanding of the pathogenesis of vascular proliferative disorders.
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Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , MicroARNs/genética , Músculo Liso Vascular/citología , Arteria Pulmonar/citología , Regiones no Traducidas 3' , Animales , Células COS , Hipoxia de la Célula , Línea Celular , Proliferación Celular , Chlorocebus aethiops , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Homeostasis , Humanos , Músculo Liso Vascular/química , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/citología , Arteria Pulmonar/químicaRESUMEN
Restenosis is still one of the main challenges in small-diameter vascular regeneration, and effective modulation of vascular smooth muscle cells (SMCs) is essential to cope with the related issues. As one of microRNAs (miRNAs) in vascular systems, miRNA-145 can regulate SMCs in the normal contractile phenotype, and inhibit the excessive proliferation and intimal hyperplasia. Herein, VAPG peptide-modified trimethyl chitosan-g-poly(ethylene glycol) (TMC-g-PEG-VAPG) was developed specially for target-delivery of miRNA-145 to SMCs to fulfill the proper function. The TMC-g-PEG-VAPG/miRNA-145 complexes exhibited low cytotoxicity, and TMC-g-PEG-VAPG with relatively higher molecular weight of chitosan (50â¯kDa) could significantly enhance cellular uptake in SMCs. Moreover, loading with TMC-g-PEG-VAPG/miRNA-145 complexes, the electrospun membranes of poly(ethylene glycol)-b-poly(L-lactide-co-ε-caprolactone) were capable of controlling SMCs at gene and protein levels on day 3 by targeting Krüppel-like factor 4 to increase the expression of myocardin and α-smooth muscle actin. Furthermore, miRNA-145 released from the electrospun membranes also showed in vitro bioactivity of modulating the contractile phenotype of SMCs in the prolonged duration, at least 56 days. The functional electrospun membranes containing TMC-g-PEG-VAPG/miRNA-145 complexes may have a great potential in the application of small-diameter blood vessel regeneration.
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Quitosano/análogos & derivados , Membranas Artificiales , MicroARNs/química , Miocitos del Músculo Liso/citología , Oligopéptidos/química , Poliésteres/química , Polietilenglicoles/química , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quitosano/química , Electroquímica , Factor 4 Similar a Kruppel , MicroARNs/genética , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/efectos de los fármacos , RatasRESUMEN
BACKGROUND: Patients with uremia have an excessive mortality from cardiovascular disease (CVD). Arterial remodeling is mainly responsible for uremia-induced CVD and has been well studied, yet venous remodeling is poorly understood. Here we investigate the histopathology and proteomic profiles of venous remodeling in uremic patients. METHODS: Forearm cephalic veins were isolated from nine uremic patients during surgeries for arteriovenous fistula, and from nine healthy controls when applying surgical debridement. Hematoxylin-eosin, Masson's trichrome, von Kossa, and immunohistochemistry (IHC) against proliferating cell nuclear antigen were stained for histopathology. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was executed to explore the proteome of the veins. The core regulatory protein was validated by western blot, IHC, and immunofluorescence. RESULTS: Phlebosclerosis, characterized by intimal rarefaction and medial thickening with disordered proliferation of vascular smooth muscle cells (VSMCs), was the prominent pathological manifestation of peripheral veins in uremic patients, while inflammatory cell infiltration, atherosclerosis or calcification were not obviously detected. iTRAQ analysis showed that 350 proteins were significantly changed in phlebosclerosis of uremic patients compared with healthy controls, of which integrin-ß1 (ITGß1) exhibited the strongest regulatory ability by intermolecular interaction network analysis. The enhanced ITGß1 expression was mainly co-expressed with the disordered proliferation of VSMCs while a little with vascular endothelial cells in the forearm cephalic veins of uremic patients. CONCLUSIONS: Phlebosclerosis is the prominent pathological manifestation in peripheral veins of uremic patients. This pathological alteration mainly attributes to the disordered proliferation of VSMCs, which is potentially mediated by ITGß1.
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Antebrazo/irrigación sanguínea , Integrina beta1/análisis , Enfermedades Vasculares Periféricas/etiología , Proteómica/métodos , Uremia/complicaciones , Remodelación Vascular , Venas/química , Venas/patología , Estudios de Casos y Controles , Proliferación Celular , Células Endoteliales/química , Células Endoteliales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/química , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/patología , Enfermedades Vasculares Periféricas/metabolismo , Enfermedades Vasculares Periféricas/patología , Esclerosis , Uremia/diagnósticoRESUMEN
We investigated the suitability of immuno-SERS (iSERS) microscopy for imaging of smooth muscle cells (SMCs) in atherosclerotic plaques. Localization of SMCs is achieved by using SERS-labelled antibodies direct against alpha-smooth muscle actin (SMA). The staining quality of the false-colour iSERS images obtained by confocal Raman microscopy with point mapping is compared with wide-field immunofluorescence images. Both direct (labelled primary antibody) and indirect iSERS staining (unlabelled primary and labelled secondary antibody) techniques were employed. Direct iSERS staining yields results comparable to indirect IF staining, demonstrating the suitability of iSERS in research on atherosclerosis and paving the way for future multiplexed imaging experiments.
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Actinas/aislamiento & purificación , Aterosclerosis/diagnóstico por imagen , Técnicas Biosensibles , Placa Aterosclerótica/diagnóstico por imagen , Actinas/química , Aterosclerosis/patología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulinas/química , Microscopía Confocal , Microscopía Fluorescente , Miocitos del Músculo Liso/química , Placa Aterosclerótica/patologíaRESUMEN
Chronic inflammation and proliferation play important roles in atherosclerosis progression. This study aimed to identify the mechanisms responsible for the anti-inflammatory and antiproliferative effects of melatonin on tumor necrosis factor-α (TNF-α)- and platelet-derived growth factor-BB (PDGF-BB)-treated rat aortic smooth muscle cells (RASMCs). Melatonin reduced TNF-α-induced RASMC inflammation by decreasing vascular cell adhesion molecule-1 (VCAM-1) expression and nuclear factor-kappa B (NF-κB) P65 activity by inhibiting P38 mitogen-activated protein kinase phosphorylation ( P < 0.05). Additionally, melatonin inhibited PDGF-BB-induced RASMC proliferation by reducing mammalian target of rapamycin (mTOR) phosphorylation ( P < 0.05) but not migration in vitro. Melatonin also reduced TNF-α- and PDGF-BB-induced reactive oxygen species (ROS) production ( P < 0.05). Furthermore, melatonin treatment (prevention and treatment groups) significantly repressed high cholesterol diet-stimulated atherosclerotic lesions in vivo (19.59 ± 4.11%, 20.28 ± 5.63%, 32.26 ± 12.06%, respectively, P < 0.05). Taken together, the present study demonstrated that melatonin attenuated TNF-α-induced RASMC inflammation and PDGF-BB-induced RASMC proliferation in cells and reduced atherosclerotic lesions in mice. These results showed that melatonin has anti-inflammatory and antiproliferative properties and may be a novel therapeutic target in atherosclerosis.
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Antiinflamatorios/farmacología , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Melatonina/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Animales , Aorta , Becaplermina/antagonistas & inhibidores , Becaplermina/farmacología , Proliferación Celular/efectos de los fármacos , Masculino , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/patología , FN-kappa B/análisis , Fosforilación/efectos de los fármacos , Células RAW 264.7 , Ratas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
OBJECTIVES: The study aimed to investigate the expression of the integrin isoforms α7A and ß1A, expressed by myogenic precursor cells, and α7B and ß1D, expressed by mature muscle cells in the cremaster of patients affected by an undescended testis. METHODS: Fifteen samples of cremaster were obtained from patients undergoing surgery for an undescended testis. Thirty control specimens of cremaster were harvested from patients with congenital hydrocele or inguinal hernia. Immunofluorescent analysis was carried out using anti-α7A, ß1A, α7B, and ß1D integrin antibodies. Sections were observed using confocal laser scanning microscopy. RESULTS: As compared with controls, a significant loss of a α7B (p = 0.0355) and ß1D (p = 0.0069) integrins and a higher expression of α7A (p = 0.0003) and ß1A (p = 0.0150) was detected in the cremaster of patients affected by an undescended testis. CONCLUSIONS: Our data document a critical alteration of the cytoskeleton of cremasteric smooth muscle cells in patients with an undescended testis. This might explain the altered function in smooth muscle cells in cremaster implied during testicular descent. We therefore speculate that the postnatal splicing of α7A to α7B and of ß1A to ß1D integrins is delayed. This could account for the common clinical scenario of spontaneous descent of the testes in the first months of life.
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Músculos Abdominales/química , Antígenos CD/análisis , Criptorquidismo/metabolismo , Cadenas alfa de Integrinas/análisis , Integrina beta1/análisis , Miocitos del Músculo Liso/química , Músculos Abdominales/patología , Músculos Abdominales/cirugía , Estudios de Casos y Controles , Preescolar , Criptorquidismo/patología , Criptorquidismo/cirugía , Citoesqueleto/química , Citoesqueleto/patología , Técnica del Anticuerpo Fluorescente , Humanos , Lactante , Masculino , Microscopía Confocal , Miocitos del Músculo Liso/patologíaRESUMEN
Inherited mitochondrial DNA (mtDNA) mutations cause mitochondrial disease, but mtDNA mutations also occur somatically and accumulate during ageing. Studies have shown that the mutation load of some inherited mtDNA mutations decreases over time in blood, suggesting selection against the mutation. However, it is unknown whether such selection occurs in other mitotic tissues, and where it occurs within the tissue. Gastrointestinal epithelium is a canonical mitotic tissue rapidly renewed by stem cells. Intestinal crypts (epithelium) undergo monoclonal conversion with a single stem cell taking over the niche and producing progeny. We show: (1) that there is a significantly lower mtDNA mutation load in the mitotic epithelium of the gastrointestinal tract when compared to the smooth muscle in the same tissue in patients with the pathogenic m.3243A>G and m.8344A>G mutations; (2) that there is considerable variation seen in individual crypts, suggesting changes in the stem cell population; (3) that this lower mutation load is reflected in the absence of a defect in oxidative phosphorylation in the epithelium. This suggests that there is selection against inherited mtDNA mutations in the gastrointestinal stem cells that is in marked contrast to the somatic mtDNA mutations that accumulate with age in epithelial stem cells leading to a biochemical defect. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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ADN Mitocondrial/genética , Células Epiteliales/química , Mucosa Gástrica/química , Mucosa Intestinal/química , Mitocondrias/genética , Miopatías Mitocondriales/genética , Mutación , Células Madre/química , Adulto , Estudios de Casos y Controles , Senescencia Celular/genética , Células Epiteliales/patología , Femenino , Mucosa Gástrica/patología , Predisposición Genética a la Enfermedad , Herencia , Humanos , Mucosa Intestinal/patología , Persona de Mediana Edad , Mitocondrias/patología , Miopatías Mitocondriales/patología , Mitosis , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/patología , Fosforilación Oxidativa , Linaje , Fenotipo , ARN de Transferencia de Leucina/genética , ARN de Transferencia de Lisina/genética , Selección Genética , Células Madre/patologíaRESUMEN
Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.
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Sistemas CRISPR-Cas , Mapeo Epitopo/métodos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Transactivadores/análisis , Transactivadores/metabolismo , Animales , Embrión de Mamíferos , Epítopos/análisis , Ratones , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/químicaRESUMEN
Follistatin like-1 (Fstl1) is a secreted glycoprotein and can be up-regulated by TGF-ß1. To better study the function of Fstl1 in lung development, we examined Fstl1 expression in the developing lung, in a cell type specific manner, using a tamoxifen inducible Fstl1-reporter mouse strain. Our results show that Fstl1 is ubiquitously expressed at saccular stage in the developing lung. At E18.5, Fstl1 expression is robust in most type of mesenchymal cells, including airway smooth muscle cells surrounding airways, vascular smooth muscle cells, endothelial cells, and vascular pericytes from blood vessel, but not PDGFRα+ fibroblasts in the distal alveolar sacs. Meanwhile, relative weak and sporadic signals of Fstl1 expression are observed in epithelium, including a subgroup of club cells in proximal airways and a few type II alveolar epithelial cells in distal airways. Our data help to understand the critical role of Fstl1 in lung development and lung disease pathogenesis.
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Células/metabolismo , Proteínas Relacionadas con la Folistatina/metabolismo , Pulmón/embriología , Animales , Embrión de Mamíferos , Células Endoteliales/química , Células Endoteliales/metabolismo , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/metabolismo , Ratones , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/metabolismo , Pericitos/química , Pericitos/metabolismo , Sistema Respiratorio/citología , Distribución TisularRESUMEN
Smooth muscle cell (SMC) heterogeneity plays an important role in vascular remodeling, a life-threatening hallmark of many vascular diseases. However, the characterization of SMCs at the single-cell level is stymied by drawbacks of contemporary single-cell protein measurements, including antibody probe cross-reactivity, chemical fixation artifacts, limited isoform-specific probes, low multiplexing and difficulty sampling cells with irregular morphologies. To scrutinize healthy vessels for subpopulations of SMCs with proliferative-like phenotypes, we developed a high-specificity, multiplexed single-cell immunoblotting cytometry tool for unfixed, uncultured primary cells. We applied our assay to demonstrate maturation stage profiling of aortic SMCs freshly isolated from individual mice. After ensuring unbiased sampling of SMCs (80-120 µm in length), we performed single-SMC electrophoretic protein separations, which resolve protein signal from off-target antibody binding, and immunoblotted for differentiation markers α-SMA, CNN-1 and SMMHC (targets ranging from 34 kDa to 227 kDa). We identified a subpopulation of immature-like SMCs, supporting the recently-established mechanism that only a subset of SMCs is responsible for vascular remodeling. Furthermore, the low sample requirements of our assay enable single-mouse resolution studies, which minimizes animal sacrifice and experimental costs while reporting animal-to-animal phenotypic variation, essential for achieving reproducibility and surmounting the drawbacks of pooling primary cells from different animals.
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Dispositivos Laboratorio en un Chip , Proteínas Musculares/análisis , Miocitos del Músculo Liso/clasificación , Análisis de la Célula Individual/instrumentación , Animales , Aorta/citología , Células Cultivadas , Electroforesis/instrumentación , Immunoblotting/instrumentación , Ratones , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/fisiología , FenotipoRESUMEN
The mechanical properties of the urinary bladder wall are important to understand its filling-voiding cycle in health and disease. However, much remains unknown about its mechanical properties, especially regarding regional heterogeneities and wall microstructure. The present study aimed to assess the regional differences in the mechanical properties and microstructure of the urinary bladder wall. Ninety (n=90) samples of porcine urinary bladder wall (ten samples from nine different locations) were mechanically and histologically analysed. Half of the samples (n=45) were equibiaxially tested within physiological conditions, and the other half, matching the sample location of the mechanical tests, was frozen, cryosectioned, and stained with Picro-Sirius red to differentiate smooth muscle cells, extracellular matrix, and fat. The bladder wall shows a non-linear stress-stretch relationship with hysteresis and softening effects. Regional differences were found in the mechanical response and in the microstructure. The trigone region presents higher peak stresses and thinner muscularis layer compared to the rest of the bladder. Furthermore, the ventral side of the bladder presents anisotropic characteristics, whereas the dorsal side features perfect isotropic behaviour. This response matches the smooth muscle fibre bundle orientation within the tunica muscularis. This layer, comprising approximately 78% of the wall thickness, is composed of two fibre bundle arrangements that are cross-oriented, one with respect to the other, varying the angle between them across the organ. That is, the ventral side presents a 60°/120° cross-orientation structure, while the muscle bundles were oriented perpendicular in the dorsal side. STATEMENT OF SIGNIFICANCE: In the present study, we demonstrate that the mechanical properties and the microstructure of the urinary bladder wall are heterogeneous across the organ. The mechanical properties and the microstructure of the urinary bladder wall within nine specific locations matching explicitly the mechanical and structural variations have been examined. On the one hand, the results of this study contribute to the understanding of bladder mechanics and thus to their functional understanding of bladder filling and voiding. On the other hand, they are relevant to the fields of constitutive formulation of bladder tissue, whole bladder mechanics, and bladder-derived scaffolds i.e., tissue-engineering grafts.
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Matriz Extracelular/química , Miocitos del Músculo Liso/química , Estrés Mecánico , Vejiga Urinaria , Animales , Sus scrofa , Vejiga Urinaria/química , Vejiga Urinaria/fisiopatologíaRESUMEN
The uterine tube (UT) is an important and complex organ of the women's reproductive system. In general, the anatomy and basic histology of this organ are well-known. However, the composition and function of the extracellular matrix (ECM) of the UT is still poorly understood. The ECM is a complex supramolecular material produced by cells which is commonly restricted to the basement membrane and interstitial spaces. ECM molecules play not only a structural role, they are also important for cell growth, survival and differentiation in all tissues. In this context, the aim of this study was to evaluate the deposition and distribution of type I and III collagens and proteoglycans (decorin, biglycan, fibromodulin and versican) in human UT during the follicular and luteal phases by using histochemical and immunohistochemical techniques. Our results showed a broad synthesis of collagens (I and III) in the stroma of the UT. The analysis by regions showed, in the mucosa, a specific distribution of versican and fibromodulin in the epithelial surface, whereas decorin and fibromodulin were observed in the lamina propria. Versican and decorin were found in the stroma of the muscular layer, whereas all studied proteoglycans were identified in the serosa. Curiously, biglycan was restricted to the wall of the blood vessels of the serosa and muscular layers. Furthermore, there was an immunoreaction for collagens, decorin, versican and fibromodulin in the UT peripheral nerves. The differential distribution of these ECM molecules in the different layers of the UT could be related to specific structural and/or biomechanical functions needed for the oviductal transport, successful fertilization and early embryogenesis. However, further molecular studies under physiological and pathological conditions are still needed to elucidate the specific role of each molecule in the human UT.
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Proteínas de la Matriz Extracelular/análisis , Matriz Extracelular/química , Trompas Uterinas/química , Ciclo Menstrual , Adulto , Colágeno/análisis , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Trompas Uterinas/metabolismo , Femenino , Humanos , Ciclo Menstrual/metabolismo , Persona de Mediana Edad , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/metabolismo , Proteoglicanos/análisis , Proteoglicanos/metabolismoRESUMEN
BACKGROUND AND AIMS: We aim to identify significant transcriptome alterations of vascular smooth muscle cells (VSMCs) in the aortic wall of myocardial infarction (MI) patients. Providing a robust transcriptomic signature, we aim to highlight the most likely aberrant pathway(s) in MI VSMCs. METHODS AND RESULTS: Laser-captured microdissection (LCM) was used to obtain VSMCs from aortic wall tissues harvested during coronary artery bypass surgery. Microarray gene analysis was applied to analyse VSMCs from 17 MI and 19 non-MI patients. Prediction Analysis of Microarray (PAM) identified 370 genes that significantly discriminated MI and non-MI samples and were enriched in genes responsible for muscle development, differentiation and phenotype regulation. Incorporation of gene ontology (GO) led to the identification of a 21-gene VSMCs-associated classifier that discriminated between MI and non-MI patients with 92% accuracy. The mass spectrometry-based iTRAQ analysis of the MI and non-MI samples revealed 94 proteins significantly differentiating these tissues. Ingenuity Pathway Analysis (IPA) of 370 genes revealed top pathways associated with hypoxia signaling in the cardiovascular system. Enrichment analysis of these proteins suggested an activation of the superoxide radical degradation pathway. An integrated transcriptome-proteome pathway analysis revealed that superoxide radical degradation pathway remained the most implicated pathway. The intersection of the top candidate molecules from the transcriptome and proteome highlighted superoxide dismutase (SOD1) overexpression. CONCLUSIONS: We provided a novel 21-gene VSMCs-associated MI classifier in reference to significant VSMCs transcriptome alterations that, in combination with proteomics data, suggests the activation of superoxide radical degradation pathway in VSMCs of MI patients.
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Músculo Liso Vascular/química , Infarto del Miocardio/genética , Miocitos del Músculo Liso/química , Transducción de Señal/genética , Transcriptoma , Aorta/química , Estudios de Casos y Controles , Cromatografía Liquida , Perfilación de la Expresión Génica/métodos , Humanos , Infarto del Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteómica/métodos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Superóxidos/metabolismo , Espectrometría de Masas en TándemRESUMEN
Smooth muscle cells (SMCs) and endothelial cells (ECs) are vital cell types composing the vascular medial wall and the atheroprotective inner lining, respectively. Current treatments for cardiovascular disease inhibit SMC hyperplasia but compromise EC integrity, predisposing patients to thrombosis. Therapeutics targeting SMCs without collateral damage to ECs are highly desirable. However, differential (SMC versus EC) disease-associated regulations remain poorly defined. We conducted RNA-seq experiments to investigate SMC-versus-EC differential transcriptomic dynamics, following treatment of human primary SMCs and ECs with TNFα or IL-1ß, both established inducers of SMC hyperplasia and EC dysfunction. As revealed by combined SMC/EC transcriptomes, after TNFα or IL-1ß induction, 174 and 213 genes respectively showed greater up-regulation in SMCs than in ECs (SMC-enriched), while 117 and 138 genes showed greater up-regulation in ECs over SMCs (EC-enriched). Analysis of gene interaction networks identified central genes shared in the two SMC-enriched gene sets, and a distinct group of central genes common in the two EC-enriched gene sets. Significantly, four gene modules (subnetworks) were identified from these central genes, including SMC-enriched JUN and FYN modules and EC-enriched SMAD3 and XPO1 modules. These modules may inform potential intervention targets for selective blockage of SMC hyperplasia without endothelial damage.
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Citocinas/farmacología , Endotelio Vascular/citología , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Músculo Liso Vascular/citología , Análisis de Secuencia de ARN/métodos , Línea Celular , Células Endoteliales/química , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Endotelio Vascular/química , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-1beta/farmacología , Músculo Liso Vascular/química , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/química , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Especificidad de Órganos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Primary Sjögren's syndrome (pSS) is a solitary poorly understood autoimmune inflammatory disease by involvement of the salivary and lacrimal glands resulting in dry mouth and dry eyes. Myoepithelial cells (MECs) are cells knowing for its hybrid epithelial and mesenchymal phenotype that are important components of the salivary gland (SGs) structure aiding the expulsion of saliva from acinar lobules. In this study we investigate possible alteration in the myofilament component of MECs in SGs specimens obtained from pSS patients in comparison with healthy subjects, to evaluate MECs hypothetical involvement in the pathogenesis of pSS. The expression of alpha-smooth muscle actin (α-SMA) and p63, as MECs markers, was evaluated in bioptic specimens from pSS and healthy labial SGs through immunohistochemistry and immunofluorescence analyses; the distribution of MECs markers was quantified using Aperio ScanScope and ImageScope software to provide quantitative assessments of staining levels. Our observations demonstrated that p63 nuclear labeling in pSS MECs is preserved whereas α-SMA cytoplasmic staining is strongly and significantly reduced when compared with healthy SGs; the digital images analysis quantification of the expression of labeled α-SMA and p63 protein in the healthy and pSS MECs salivary tissues, led to results suggesting a loss of mechanical support for acini and ducts in pSS, correlated, probably, with the reduction of salivary flow that features one important aspect of pSS disease.
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Células Epiteliales/ultraestructura , Miofibrillas , Glándulas Salivales/patología , Síndrome de Sjögren/patología , Actinas/análisis , Humanos , Inmunohistoquímica , Proteínas de la Membrana/análisis , Miocitos del Músculo Liso/químicaRESUMEN
Membrane potential is a principal regulator of arterial contractility. Arterial smooth muscle cells express several different types of ion channel that control membrane potential, including KV channels. KV channel activation leads to membrane hyperpolarization, resulting in inhibition of voltage-dependent Ca2+ channels, a reduction in [Ca2+ ]i , and vasodilation. In contrast, KV channel inhibition leads to membrane depolarization and vasoconstriction. The ability of KV channels to regulate arterial contractility is dependent upon the number of plasma membrane-resident channels and their open probability. Here, we will discuss mechanisms that alter the surface abundance of KV channel proteins in arterial smooth muscle cells and the functional consequences of such regulation. Cellular processes that will be described include those that modulate KV channel transcription, retrograde and anterograde trafficking, and protein degradation.