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1.
Methods Mol Biol ; 2273: 139-149, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604850

RESUMEN

Ovarian failure is the most common cause of infertility and affects about 1% of young women. One innovative strategy to restore ovarian function may be represented by the development of a bioprosthetic ovary, obtained through the combination of tissue engineering and regenerative medicine.We here describe the two main steps required for bioengineering the ovary and for its ex vivo functional reassembling. The first step aims at producing a 3D bioscaffold, which mimics the natural ovarian milieu in vitro. This is obtained with a whole organ decellularization technique that allows the maintenance of microarchitecture and biological signals of the original tissue. The second step involves the use of magnetic activated cell sorting (MACS) to isolate purified female germline stem cells (FGSCs). These cells are able to differentiate in ovarian adult mature cells, when subjected to specific stimuli, and can be used them to repopulate ovarian decellularized bioscaffolds. The combination of the two techniques represents a powerful tool for in vitro recreation of a bioengineered ovary that may constitute a promising solution for hormone and fertility function restoring. In addition, the procedures here described allow for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.


Asunto(s)
Células Madre Oogoniales/trasplante , Ovario/crecimiento & desarrollo , Ingeniería de Tejidos/métodos , Animales , Bioingeniería/métodos , Ingeniería Biomédica , Técnicas de Cultivo de Célula/métodos , Matriz Extracelular/metabolismo , Femenino , Fertilidad , Humanos , Células Madre Oogoniales/metabolismo , Organoides/crecimiento & desarrollo , Medicina Regenerativa , Porcinos , Andamios del Tejido/química
2.
Nat Commun ; 12(1): 1020, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589611

RESUMEN

The extracellular matrix (ECM) is unique to each tissue and capable of guiding cell differentiation, migration, morphology, and function. The ECM proteome of different developmental stages has not been systematically studied in the human pancreas. In this study, we apply mass spectrometry-based quantitative proteomics strategies using N,N-dimethyl leucine isobaric tags to delineate proteome-wide and ECM-specific alterations in four age groups: fetal (18-20 weeks gestation), juvenile (5-16 years old), young adults (21-29 years old) and older adults (50-61 years old). We identify 3,523 proteins including 185 ECM proteins and quantify 117 of them. We detect previously unknown proteome and matrisome features during pancreas development and maturation. We also visualize specific ECM proteins of interest using immunofluorescent staining and investigate changes in ECM localization within islet or acinar compartments. This comprehensive proteomics analysis contributes to an improved understanding of the critical roles that ECM plays throughout human pancreas development and maturation.


Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Páncreas/metabolismo , Proteoma/genética , Adolescente , Adulto , Niño , Preescolar , Cromatografía Liquida , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/clasificación , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Feto , Técnica del Anticuerpo Fluorescente , Ontología de Genes , Humanos , Masculino , Persona de Mediana Edad , Anotación de Secuencia Molecular , Organogénesis/genética , Páncreas/crecimiento & desarrollo , Proteoma/clasificación , Proteoma/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem
3.
Respir Res ; 22(1): 38, 2021 Feb 05.
Artículo en Inglés | MEDLINE | ID: mdl-33546680

RESUMEN

Pulmonary fibrosis has been identified as a main factor leading to pulmonary dysfunction and poor quality of life in post-recovery Severe Acute Respiratory Syndrome (SARS) survivor's consequent to SARS-Cov-2 infection. Thus there is an urgent medical need for identification of readily available biomarkers that in patients with SARS-Cov-2 infection are able to; (1) identify patients in most need of medical care prior to admittance to an intensive care unit (ICU), and; (2) identify patients post-infection at risk of developing persistent fibrosis of lungs with subsequent impaired quality of life and increased morbidity and mortality. An intense amount of research have focused on wound healing and Extracellular Matrix (ECM) remodelling of the lungs related to lung function decline in pulmonary fibrosis (PF). A range of non-invasive serological biomarkers, reflecting tissue remodelling, and fibrosis have been shown to predict risk of acute exacerbations, lung function decline and mortality in PF and other interstitial lung diseases (Sand et al. in Respir Res 19:82, 2018). We suggest that lessons learned from such PF studies of the pathological processes leading to lung function decline could be used to better identify patients infected with SARS-Co-V2 at most risk of acute deterioration or persistent fibrotic damage of the lung and could consequently be used to guide treatment decisions.


Asunto(s)
/metabolismo , Matriz Extracelular/metabolismo , Fibrosis Pulmonar/metabolismo , Cicatrización de Heridas/fisiología , Animales , Biomarcadores/metabolismo , Humanos , Pulmón/metabolismo , Fibrosis Pulmonar/diagnóstico
4.
Am J Physiol Renal Physiol ; 320(3): F273-F284, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33427062

RESUMEN

Peritoneal dialysis (PD)-related peritoneal fibrosis (PF) is characterized by progressive extracellular matrix (ECM) accumulation in peritoneal mesothelial cells (PMCs) during long-term use of high glucose (HG)-based dialysates. Activation of the renin-angiotensin system (RAS) has been shown to be associated with PF. The aim of this study was to explore the underlying mechanism of the RAS in HG-induced PF. We treated C57BL/6 mice and a human PMC line with HG to induce a PF model and to stimulate ECM accumulation, respectively. RAS activity was blocked using valsartan or angiotensin II (ANGII) type 1 receptor siRNA. The major findings were as follows. First, mice in the HG group exhibited increased collagen deposition and expression of ECM proteins, including α-smooth muscle actin (α-SMA) and collagen type I in the peritoneum. Consistent with the in vivo data, HG upregulated α-SMA expression in human peritoneal mesothelial cells (HPMCs) in a time- and dose-dependent manner. Second, HG stimulation led to RAS activation in HPMCs, and inactivation of RAS decreased the expression of ECM proteins in vivo and in vitro, even during HG stimulation. Finally, RAS-mediated ECM production was associated with lipid accumulation in HPMCs and depended on the dysregulation of the low-density lipoprotein receptor (LDLr) pathway. HG-stimulated HPMCs showed increased coexpression of LDLr and α-SMA, whereas blockade of RAS activity reversed the effect. Furthermore, inhibition of LDLr signaling decreased α-SMA and collagen type I expression in HPMCs when treated with HG and ANG II. In conclusion, increased intracellular RAS activity impaired lipid homeostasis and induced ECM accumulation in HPMCs by disrupting the LDLr pathway, which contributed to PF.


Asunto(s)
Matriz Extracelular/metabolismo , Fibrosis Peritoneal/metabolismo , Peritoneo/metabolismo , Receptores de LDL/metabolismo , Sistema Renina-Angiotensina , Actinas/metabolismo , Animales , Línea Celular , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/patología , Glucosa , Humanos , Masculino , Ratones Endogámicos C57BL , Oxidación-Reducción , Fibrosis Peritoneal/inducido químicamente , Fibrosis Peritoneal/genética , Fibrosis Peritoneal/patología , Peritoneo/patología , Receptores de LDL/genética , Sistema Renina-Angiotensina/genética , Transducción de Señal
5.
Cell Prolif ; 54(2): e12976, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33393124

RESUMEN

BACKGROUND: In mammals, early pregnancy is a critical vulnerable period during which complications may arise, including pregnancy failure. Establishment of a maternal endometrial acceptance phenotype is a prerequisite for semiheterogeneous embryo implantation, comprising the rate-limiting step of early pregnancy. METHODS: Confocal fluorescence, immunohistochemistry and western blot for nuclear and cytoplasmic protein were used to examine the activation of yes-associated protein (YAP) in uterine tissue and primary endometrial cells. The target binding between miR16a and YAP was verified by dual-luciferase reporter gene assay. The mouse pregnancy model and pseudopregnancy model were used to investigate the role of YAP in the maternal uterus during early pregnancy in vivo. RESULTS: We showed that YAP translocates into the nucleus in the endometrium of cattle and mice during early pregnancy. Mechanistically, YAP acts as a mediator of ECM rigidity and cell density, which requires the actomyosin cytoskeleton and is partially dependent on the Hippo pathway. Furthermore, we found that the soluble factor IFNτ, which is a ruminant pregnancy recognition factor, also induced activation of YAP by reducing the expression of miR-16a. CONCLUSIONS: This study revealed that activation of YAP is necessary for early pregnancy in bovines because it induced cell proliferation and established an immunosuppressive local environment that allowed conceptus implantation into the uterine epithelium.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endometrio/metabolismo , Matriz Extracelular/metabolismo , Interferón Tipo I/metabolismo , Proteínas Gestacionales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antagomirs/metabolismo , Bovinos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Endometrio/citología , Molécula de Adhesión Celular Epitelial/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Interferón Tipo I/farmacología , Masculino , Ratones , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Musculares/metabolismo , Embarazo , Proteínas Gestacionales/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Útero/metabolismo , Útero/patología
6.
Nat Commun ; 12(1): 462, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469019

RESUMEN

Clostridioides difficile is a bacterial pathogen that causes a range of clinical disease from mild to moderate diarrhea, pseudomembranous colitis, and toxic megacolon. Typically, C. difficile infections (CDIs) occur after antibiotic treatment, which alters the gut microbiota, decreasing colonization resistance against C. difficile. Disease is mediated by two large toxins and the expression of their genes is induced upon nutrient depletion via the alternative sigma factor TcdR. Here, we use tcdR mutants in two strains of C. difficile and omics to investigate how toxin-induced inflammation alters C. difficile metabolism, tissue gene expression and the gut microbiota, and to determine how inflammation by the host may be beneficial to C. difficile. We show that C. difficile metabolism is significantly different in the face of inflammation, with changes in many carbohydrate and amino acid uptake and utilization pathways. Host gene expression signatures suggest that degradation of collagen and other components of the extracellular matrix by matrix metalloproteinases is a major source of peptides and amino acids that supports C. difficile growth in vivo. Lastly, the inflammation induced by C. difficile toxin activity alters the gut microbiota, excluding members from the genus Bacteroides that are able to utilize the same essential nutrients released from collagen degradation.


Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Infecciones por Clostridium/inmunología , Microbioma Gastrointestinal/inmunología , Factor sigma/metabolismo , Animales , Antibacterianos/efectos adversos , Proteínas Bacterianas/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Bacteroides/efectos de los fármacos , Bacteroides/metabolismo , /inmunología , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/patología , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Nutrientes/metabolismo , Proteolisis , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación , RNA-Seq , Factor sigma/genética , Factor sigma/inmunología , Transcriptoma/inmunología
7.
Life Sci ; 269: 119001, 2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33421527

RESUMEN

AIMS: Osteoarthritis (OA) is a common joint disease and the main cause of disability. We sought to determine the effective concentration of emodin on chondrocytes and to identify the dosage of emodin that induces a comparable therapeutic effect with the COX-2 inhibitor drug, celecoxib that is currently used to treat OA. MATERIAL AND METHODS: In vitro experiments induced inflammation of chondrocytes by IL-1ß, and an osteoarthritis model was established in vivo by cutting rat anterior cruciate ligament. Western Blot, Real-time PCR, HE staining, Safranin O-green staining and immunohistochemistry were performed to detect MMP-3, MMP-13, ADAMTS-4, iNOS and COL2A1 on the chondrocytes or the tibial plateau. The cytokine activity and content in serum of six groups of rats were measured by kit. RESULTS: It was found that the surface layer of the cartilage was thicker and smoother after the administration of emodin. Tissue expression of MMP-3, MMP-13, ADAMTS-4 and iNOS were significantly (p < 0.05) decreased in chondrocytes and cartilage treated with different doses of emodin, and the content of COL2A1 was reversed. Emodin also significantly decreased the blood levels of COX-2 and PGE2. The effective emodin in vitro was 5 µmol/L, whereas emodin at 80 mg/kg was equivalent to celecoxib in vivo. CONCLUSION: Emodin reduces the expression of cartilage matrix degradation biomarkers, thereby reducing the degradation of cartilage matrix and protecting the knee joint cartilage. Emodin at 5 µmol/L shows the best concentration to treat chondrocytes, and the protective effect of emodin at 80 mg/kg is comparable to that of celecoxib.


Asunto(s)
Cartílago Articular/patología , Emodina/farmacología , Matriz Extracelular/metabolismo , Articulación de la Rodilla/patología , Sustancias Protectoras/farmacología , Proteína ADAMTS4/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Ciclooxigenasa 2/sangre , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Emodina/administración & dosificación , Matriz Extracelular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Metaloproteinasas de la Matriz/metabolismo , Óxido Nítrico/sangre , Óxido Nítrico Sintasa de Tipo II/sangre , Óxido Nítrico Sintasa de Tipo II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/patología
8.
Life Sci ; 269: 119085, 2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33482190

RESUMEN

Pulmonary fibrosis (PF), which is characterized by excessive matrix formation, may ultimately lead to irreversible lung damage and thus death. Fibroblast activation has been regarded as a central event during PF pathogenesis. In our previous study, we confirmed that the miR-627/high-mobility group box protein 1 (HMGB1)/Nuclear factor kappa beta (NF-κB) axis modulates transforming growth factor beta 1 (TGFß1)-induced pulmonary fibrosis. In the present study, we investigated the upstream factors leading to miR-627 dysregulation in the process of pulmonary fibroblast activation and PF. The lncRNA MIR155 host gene (MIR155HG) was found to be abnormally upregulated in pulmonary fibrosis tissues and TGFß1-stimulated normal human primary lung fibroblasts (NHLFs). By directly binding to miR-627, MIR155HG inhibited miR-627 expression. MIR155HG overexpression enhanced TGFß1-induced increases in HMGB1 protein expression and p65 phosphorylation, NHLF proliferation, and extracellular matrix (ECM) deposition. In contrast, miR-627 overexpression attenuated the TGFß1-induced changes in NHLFs and significantly reversed the effects of MIR155HG overexpression. Under TGFß1 stimulation, miR-627 inhibition promoted, whereas JSH-23 treatment inhibited NF-κB activation; in NHLFs, NF-κB overexpression upregulated, whereas JSH-23 treatment downregulated MIR155HG expression. In tissue samples, HMGB1 protein levels and p65 phosphorylation were increased; MIR155HG was negatively correlated with miR-627 and positively correlated with HMGB1. In conclusion, we validated that the MIR155HG/miR-627/HMGB1/NF-κB axis formed a regulatory loop that modulates TGFß1-induced NHLF activation. Considering the critical role of NHLF activation in PF pathogenesis, the NF-κB/MIR155HG/miR-627/HMGB1 regulatory loop could exert a vital effect on PF pathogenesis. Further in vivo and clinical investigations are required to confirm this model.


Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/citología , Proteína HMGB1/metabolismo , MicroARNs/genética , FN-kappa B/metabolismo , Fibrosis Pulmonar/patología , ARN Largo no Codificante/genética , Estudios de Casos y Controles , Proliferación Celular , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Proteína HMGB1/genética , Humanos , Pulmón/citología , Pulmón/metabolismo , FN-kappa B/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo
9.
Int J Mol Sci ; 22(1)2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-33466312

RESUMEN

Despite modern surgical trauma care, bleeding contributes to one-third of trauma-related death. A significant improvement was obtained through the introduction of tranexamic acid (TXA), which today is widely used in emergency and elective orthopedic surgery to control bleeding. However, concerns remain regarding potential adverse effects on bone turnover and regeneration. Therefore, we employed standardized cell culture systems including primary osteoblasts, osteoclasts, and macrophages to evaluate potential effects of TXA on murine bone cells. While osteoblasts derived from calvarial digestion were not affected, TXA increased cell proliferation and matrix mineralization in bone marrow-derived osteoblasts. Short-term TXA treatment (6 h) failed to alter the expression of osteoblast markers; however, long-term TXA stimulation (10 days) was associated with the increased expression of genes involved in osteoblast differentiation and extracellular matrix synthesis. Similarly, whereas short-term TXA treatment did not affect gene expression in terminally differentiated osteoclasts, long-term TXA stimulation resulted in the potent inhibition of osteoclastogenesis. Finally, in bone marrow-derived macrophages activated with LPS, simultaneous TXA treatment led to a reduced expression of inflammatory cytokines and chemokines. Collectively, our study demonstrates a differential action of TXA on bone cells including osteoanabolic, anti-resorptive, and anti-inflammatory effects in vitro which suggests novel treatment applications.


Asunto(s)
Médula Ósea/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Ácido Tranexámico/farmacología , Animales , Médula Ósea/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogénesis/efectos de los fármacos
10.
Cell Prolif ; 54(2): e12987, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33415745

RESUMEN

OBJECTIVES: Inappropriate or excessive compression applied to intervertebral disc (IVD) contributes substantially to IVD degeneration. The actomyosin system plays a leading role in responding to mechanical stimuli. In the present study, we investigated the roles of myosin II isoforms in the compression stress-induced senescence of nucleus pulposus (NP) cells. MATERIAL AND METHODS: Nucleus pulposus cells were exposed to 1.0 MPa compression for 0, 12, 24 or 36 hours. Immunofluorescence and co-immunoprecipitation analysis were used to measure the interaction of myosin IIA and IIB with actin. Western blot analysis and immunofluorescence staining were used to detect nuclear expression and nuclear localization of MRTF-A. In addition, the expression levels of p-RhoA/RhoA, ROCK1/2 and p-MLC/MLC were measured in human NP cells under compression stress and in degenerative IVD tissues. RESULTS: Compression stress increased the interaction of myosin IIA and actin, while the interaction of myosin IIB and actin was reduced. The actomyosin cytoskeleton remodelling was involved in the compression stress-induced fibrotic phenotype mediated by MRTF-A nuclear translocation and inhibition of proliferation in NP cells. Furthermore, RhoA/ROCK1 pathway activation mediated compression stress-induced human NP cells senescence by regulating the interaction of myosin IIA and IIB with actin. CONCLUSIONS: We for the first time investigated the regulation of actomyosin cytoskeleton in human NP cells under compression stress. It provided new insights into the development of therapy for effectively inhibiting IVD degeneration.


Asunto(s)
Miosina Tipo IIA no Muscular/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Estrés Mecánico , Actinas/metabolismo , Actomiosina/metabolismo , Células Cultivadas , Senescencia Celular , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Metaloproteinasa 3 de la Matriz/metabolismo , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/genética , Núcleo Pulposo/citología , Núcleo Pulposo/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transactivadores/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/metabolismo
11.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33498156

RESUMEN

Excessive cross-linking is a major factor in the resistance to the remodelling of the extracellular matrix (ECM) during fibrotic progression. The role of TGFß signalling in impairing ECM remodelling has been demonstrated in various fibrotic models. We hypothesised that increased ECM cross-linking by TGFß contributes to skin fibrosis in Systemic Sclerosis (SSc). Proteomics was used to identify cross-linking enzymes in the ECM of primary human dermal fibroblasts, and to compare their levels following treatment with TGFß-1. A significant upregulation and enrichment of lysyl-oxidase-like 1, 2 and 4 and transglutaminase 2 were found. Western blotting confirmed the upregulation of lysyl hydroxylase 2 in the ECM. Increased transglutaminase activity in TGFß-1 treated ECM was revealed from a cell-based assay. We employed a mass spectrometry-based method to identify alterations in the ECM cross-linking pattern caused by TGFß-1. Cross-linking sites were identified in collagens I and V, fibrinogen and fibronectin. One cross-linking site in fibrinogen alpha was found only in TGFß-treated samples. In conclusion, we have mapped novel cross-links between ECM proteins and demonstrated that activation of TGFß signalling in cultured dermal fibroblasts upregulates multiple cross-linking enzymes in the ECM.


Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Aminoácido Oxidorreductasas/metabolismo , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/química , Dermis/citología , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Femenino , Fibrinógeno/química , Fibrinógeno/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Transglutaminasas/metabolismo
12.
Methods Mol Biol ; 2221: 53-70, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32979198

RESUMEN

Co-culture of chondrocytes and mesenchymal stromal cells (MSCs) has been shown to be beneficial in engineering cartilage tissue in vitro. In these co-cultures, MSCs increase the proliferation and matrix deposition of chondrocytes. The MSCs accomplish this beneficial effect by so-called trophic actions. Thus, large cartilage constructs can be made with a relatively small number of chondrocytes. In this chapter, we describe different methods for making co-cultures of MSCs and chondrocytes. We also provide detailed protocols for analyzing MSC-chondrocyte co-cultures with cell tracking, proliferation assays, species-specific polymerase chain reactions (PCR), rheological analysis, compression analysis, RNA-sequencing analysis, short tandem repeats analysis, and biochemical examination.


Asunto(s)
Cartílago/citología , Condrocitos/citología , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Animales , Bovinos , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrogénesis , Técnicas de Cocultivo , Matriz Extracelular/metabolismo , Humanos , Andamios del Tejido
13.
Methods Mol Biol ; 2217: 27-37, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215374

RESUMEN

Focal adhesions in planar substrates constitute an excellent cellular resource to evaluate different parameters related to cell morphology, cytoskeletal organization, and adhesive strength. However, their intrinsic heterogeneity in terms of size, molecular composition, orientation, and so on complicates their analysis. Here, we describe a simple and straightforward ImageJ/Fiji-based method to quantify several parameters that describe the morphology and relative composition of focal adhesions. This type of analysis can be implemented in various ways and become useful for drug and shRNA screenings.


Asunto(s)
Citoesqueleto de Actina/ultraestructura , Matriz Extracelular/ultraestructura , Adhesiones Focales/ultraestructura , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagen Molecular/métodos , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Animales , Células CHO , Adhesión Celular , Línea Celular Tumoral , Cricetulus , Matriz Extracelular/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Humanos , Ratones , Células 3T3 NIH , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Faloidina/química
14.
Methods Mol Biol ; 2217: 39-44, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215375

RESUMEN

Focal adhesions are force sensitive structures that dynamically alter their composition, protein-protein interactions, and signaling in response to external mechanical stimuli. These dynamic changes are critical for focal adhesion function and are required for cellular mechanosensing. Here, we describe a simple protocol that allows for isolation of the focal adhesion complex from adherent cells in culture in response to different mechanical stimuli applied at adhesion sites. By combining this assay with approaches such as proteomics or western blot analysis, one can study the force-dependent changes in focal adhesion composition, protein-protein interactions and signaling.


Asunto(s)
Bioensayo , Materiales Biocompatibles Revestidos/química , Matriz Extracelular/química , Fibroblastos/química , Fibronectinas/química , Adhesiones Focales/química , Animales , Fenómenos Biomecánicos , Adhesión Celular , Línea Celular , Materiales Biocompatibles Revestidos/metabolismo , Reactivos de Enlaces Cruzados/química , Embrión de Mamíferos , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibronectinas/metabolismo , Adhesiones Focales/metabolismo , Humanos , Imidas/química , Imanes , Mecanotransducción Celular/fisiología , Ratones , Propionatos/química , Unión Proteica
15.
Methods Mol Biol ; 2217: 57-69, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215377

RESUMEN

Integrins are heterodimeric adhesion receptors that maintain cell-extracellular matrix (ECM) interactions in diverse tissue microenvironments. They mediate cell adhesion and signaling through the assembly of large cytoplasmic multiprotein complexes that focally connect with the cytoskeleton. Integrin adhesion complexes (IAC) are specialized by the type of integrin-ECM contact and are sensitive to mechanical forces. Thus, they encrypt context-dependent information about the microenvironment in their composition. Signals mediated through IACs modulate many aspects of cell behavior, which allows cells to adapt to their surroundings. To gain insights into their function, IACs have been isolated from cultured cells and explored by proteomics. IACs are insoluble by nature and held together by transient/weak interactions, which makes it challenging to isolate intact IACs. Usually all IACs coupled to a specified ECM, which may employ different integrins, are isolated. Here we describe an alternative method based on proximity-dependent biotin identification (BioID), where specific integrin interaction partners are labeled in live cells and isolated without the need to isolate intact IACs.


Asunto(s)
Bioensayo , Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Escherichia coli/metabolismo , Cadenas alfa de Integrinas/metabolismo , Cadenas beta de Integrinas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Biotina/química , Biotina/metabolismo , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Adhesión Celular , Membrana Celular/química , Membrana Celular/metabolismo , Perros , Proteínas de Escherichia coli/genética , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Cadenas alfa de Integrinas/clasificación , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/clasificación , Cadenas beta de Integrinas/genética , Células de Riñón Canino Madin Darby , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Represoras/genética , Coloración y Etiquetado/métodos , Transfección
16.
Methods Mol Biol ; 2217: 71-81, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215378

RESUMEN

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.


Asunto(s)
Adhesiones Focales/metabolismo , Inmunohistoquímica/métodos , Cadenas alfa de Integrinas/metabolismo , Integrina beta1/metabolismo , Complejos Multiproteicos/metabolismo , Oligonucleótidos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Anticuerpos/química , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Adhesiones Focales/ultraestructura , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Cadenas alfa de Integrinas/química , Integrina beta1/química , Microscopía Fluorescente , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Complejos Multiproteicos/química , Músculo Liso/metabolismo , Músculo Liso/ultraestructura , Oligonucleótidos/síntesis química , Unión Proteica
17.
Methods Mol Biol ; 2217: 149-179, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215381

RESUMEN

Cell-surface adhesion receptors mediate interactions with the extracellular matrix (ECM) to control many fundamental aspects of cell behavior, including cell migration, survival, and proliferation. Integrin adhesion receptors recruit structural and signaling proteins to form multimolecular adhesion complexes that link the plasma membrane to the actomyosin cytoskeleton. The assembly and turnover of adhesion complexes are tightly regulated, governed in part by the networks of physical protein interactions and functional signaling associations between components of the adhesome. Proteomic profiling of adhesion complexes has begun to reveal their molecular complexity and diversity. To interrogate the composition of cell-ECM adhesions, we detail herein an approach for the network analysis of adhesion complex proteomes. Integration of these proteomic data with adhesome databases in the context of predicted protein interactions enables the mapping of experimentally defined adhesion complex networks. Computational analysis of resultant network models can identify subnetworks of putative functionally linked adhesion protein communities. This approach provides a framework to predict functional adhesion protein relationships and generate new mechanistic hypotheses for further experimental testing.


Asunto(s)
Biología Computacional/métodos , Integrinas/metabolismo , Complejos Multiproteicos/metabolismo , Redes Neurales de la Computación , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animales , Adhesión Celular , Movimiento Celular , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Integrinas/genética , Complejos Multiproteicos/genética , Unión Proteica , Proteoma/genética , Programas Informáticos
18.
Methods Mol Biol ; 2217: 183-195, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215382

RESUMEN

Surface nanopatterning allows for the creation of spatially controlled binding sites for extracellular matrix ligands and the modulation of receptor binding sites. Here we describe the preparation of gold nanopatterned substrates using diblock micellar nanolithography to immobilize integrin ligands at defined spacing and combined with molecular tension sensors to measure molecular forces as function of integrin lateral clustering.


Asunto(s)
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Adhesiones Focales/metabolismo , Integrinas/química , Oligodesoxirribonucleótidos/química , Estereolitografía , Animales , Sitios de Unión , Adhesión Celular , Movimiento Celular , Cloruros/química , Matriz Extracelular/ultraestructura , Fibroblastos/ultraestructura , Fibronectinas/química , Fibronectinas/metabolismo , Adhesiones Focales/ultraestructura , Compuestos de Oro/química , Integrinas/metabolismo , Ligandos , Ratones , Micelas , Microscopía Fluorescente/métodos , Nanoestructuras/química , Polietilenglicoles/química , Polimerizacion , Unión Proteica , Piridinas/química , Propiedades de Superficie
19.
Methods Mol Biol ; 2217: 197-233, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215383

RESUMEN

Integrins are stress-sensing proteins expressed on the surface of cells. They regulate bidirectional signal transduction during cell-cell or cell-extracellular matrix (ECM) contacts. Integrins link the ECM with the cytoplasm through interaction with their ligands. Biophysically, such interactions can be understood as changes in stress fields at specific integrin stress-sensing domains, such as the MIDAS and ADMIDAS domains. Stress changes between ligands and cytoskeletal structures are involved in cancer cell growth by altering signal transduction pathways dependent on integrin activation. In this chapter, previous results regarding integrin activation and tumor cell growth using nanoparticles (NPs) of different materials, sizes and shapes are placed within a framework of polarized NPs in the ECM by external electromagnetic fields, in which the synergic action between polarized NPs and electromagnetic fields activates the integrins. Small size NPs activate integrins via the polar component of the dipole force between NPs and integrin sensing stress sites, while large size NPs exercise a similar action via the radial component. A quantum electrodynamic model also accounts for ECM overstressing by electromagnetic mode trapping between coherent symmetric and antisymmetric quantum states.


Asunto(s)
Citoesqueleto/química , Campos Electromagnéticos , Matriz Extracelular/química , Integrinas/química , Nanopartículas/química , Células A549 , Animales , Sitios de Unión , Adhesión Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Humanos , Integrinas/agonistas , Integrinas/metabolismo , Integrinas/ultraestructura , Ligandos , Células MCF-7 , Mecanotransducción Celular , Microscopía de Fuerza Atómica/métodos , Nanopartículas/metabolismo , Nanopartículas/ultraestructura , Tamaño de la Partícula , Unión Proteica , Teoría Cuántica , Termodinámica
20.
Methods Mol Biol ; 2217: 285-300, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215387

RESUMEN

The extracellular matrix (ECM) is the noncellular compartment of living organisms and is formed of a complex network of cross-linked proteins, which is collectively known as the matrisome. Apart from providing the structure for an organism, cells interact and thereby communicate with the ECM. Cells interact with their surrounding ECM using cell-surface receptors, such as integrins. Upon integrin engagement with the ECM, cytoskeletal proteins are recruited to integrins and form a molecular protein complex known as the integrin adhesome. Global descriptions of the matrisome and integrin adhesome have been proposed using in silico bioinformatics approaches, as well as through biochemical enrichment of matrisome and adhesome fractions coupled with mass spectrometry-based proteomic analyses, providing inventories of their compositions in different contexts. Here, methods are described for the computational downstream analyses of matrisome and adhesome mass spectrometry datasets that are accessible to wet lab biologists, which include comparing datasets to in silico descriptions, generating interaction networks and performing functional ontological analyses.


Asunto(s)
Biología Computacional/métodos , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Redes Reguladoras de Genes , Integrinas/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Adhesión Celular , Células Cultivadas , Bases de Datos Genéticas , Matriz Extracelular/química , Proteínas de la Matriz Extracelular/clasificación , Proteínas de la Matriz Extracelular/genética , Ontología de Genes , Humanos , Integrinas/clasificación , Integrinas/genética , Espectrometría de Masas , Ratones , Anotación de Secuencia Molecular , Familia de Multigenes , Complejos Multiproteicos/clasificación , Complejos Multiproteicos/genética , Unión Proteica
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