RESUMEN
Calcineurin inhibitors, such as Cyclosporin (CsA), are the mainstay of anti-rejection therapy in solid organ transplants but can paradoxically induce progressive nephropathy characterised by renal dysfunction and interstitial fibrosis. Lysyl oxidases (LOXs), a group of enzymes that catalyse extracellular matrix (ECM) crosslinking, were shown to implicate in tissue scarring. It is hypothesized that inhibition of these enzymes may render therapeutic effects against CsA-induced nephropathy. In this study, 6-to-8 weeks old C57BL/6 J mice were administered saline or CsA (30 mg/kg/day s.c) for 16 weeks. At 8 weeks, CsA-treated animals were divided into 5 groups respectively treated with: (1) vehicle, (2) PXS-5505 (Pan-LOX inhibitor), (3) PXS-5382 (LOX-like 2 inhibitor), (4) PXS-5505 for 4 weeks then PXS-5382 for 4 weeks (sequential therapy), and (5) Telmisartan (standard therapy). Our results indicate that CsA administration significantly increased the levels of blood urea nitrogen, glomerular and tubular injury, tubulointerstitial fibrosis, inflammation and oxidative stress in mouse kidney. These changes were associated with upregulated mRNA expression of LOX and LOXL2. Administration of Pan-LOX or LOXL2 inhibitors or the sequential therapy suppressed the expression of ECM proteins (α-SMA, FN and COL1A), matrix metalloproteases (MMP)2 and 9, inflammatory markers (TNFα and MCP-1) and TGF-ß1-Smad3 signalling. Among all regimens including telmisartan, only Pan-LOX inhibitor PXS-5505 was able to attenuate uraemia. Collectively, our study suggests that Pan-LOX and LOXL2 inhibition can attenuate progressive nephropathy due to CsA administration.
Asunto(s)
Aminoácido Oxidorreductasas/antagonistas & inhibidores , Ciclosporina/efectos adversos , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Enfermedades Renales/tratamiento farmacológico , Sustancias Protectoras/uso terapéutico , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Aminoácido Oxidorreductasas/metabolismo , Animales , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/patología , Proteínas de la Matriz Extracelular/metabolismo , Fibrosis , Humanos , Enfermedades Renales/inducido químicamente , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Masculino , Ratones , Sustancias Protectoras/farmacología , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
Metastasis is a major cause of cancer-related deaths. Tumor-intrinsic properties can determine whether tumor metastasis occurs or not. Here, by comparing the gene expression patterns in primary colorectal cancer (CRC) patients with or without metastasis, we found that Collagen Triple Helix Repeat Containing 1 (CTHRC1) in primary CRC served as a metastasis-associated gene. Animal experiments verified that CTHRC1 secreted by CRC cells promoted hepatic metastasis, which was closely correlated with macrophage infiltration. Depletion of macrophages by liposomal clodronate largely abolished the promoting effect of CTHRC1 on CRC liver metastasis. Furthermore, we demonstrated that CTHRC1 modulated macrophage polarization to M2 phenotypes through TGF-ß signaling. A mechanistic study revealed that CTHRC1 bound directly to TGF-ß receptor II and TGF-ß receptor III, stabilized the TGF-ß receptor complex, and activated TGF-ß signaling. The combination treatment of CTHRC1 monoclonal antibody and anti-PD-1 blocking antibody effectively suppressed CRC hepatic metastasis. Taken together, our data demonstrated that CTHRC1 is an intrinsic marker of CRC metastasis and further revealed that CTHRC1 promoted CRC liver metastasis by reshaping infiltrated macrophages through TGF-ß signaling, suggesting that CTHRC1 could be a potential biomarker for the early prediction of and a therapeutic target of CRC hepatic metastasis.
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Anticuerpos Monoclonales/farmacología , Neoplasias Colorrectales/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Neoplasias Hepáticas/secundario , Macrófagos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Estadificación de Neoplasias , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Glaucoma is a progressive optic neuropathy in more than 25 % of cases in patients with permanent blindness. The microRNA is implicated in modulating the cellular function of the trabecular meshwork (TM). The aim of this study is to investigate the role of miR-137 in glaucoma and illustrate the potential molecular mechanisms. We show that miR-137 was down-regulated in H2O2-induced human trabecular meshwork cells (HTMCs), and overexpression of miR-137 attenuated H2O2-induced cell growth inhibition, apoptosis and elevated extracellular matrix (ECM) protein expression. In addition, miR-137 blocked the activation of YAP/TAZ by directly targeting src. Overexpression of src or activation of the YAP/TAZ pathway partly abrogated the effects of miR-137 on H2O2-induced cell viability and apoptosis and dampened the inhibition effect on ECM protein expression. In conclusion, miR-137 promotes cell growth and inhibits extracellular matrix protein expression in H2O2-induced human trabecular meshwork cells via the YAP/TAZ pathway by targeting src. Hence, miR-137 might be used as a novel therapeutic target to treat glaucoma.
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Proliferación Celular/fisiología , Proteínas de la Matriz Extracelular/biosíntesis , Peróxido de Hidrógeno/toxicidad , MicroARNs/biosíntesis , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Malla Trabecular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Humanos , MicroARNs/genética , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Malla Trabecular/efectos de los fármacosRESUMEN
Collagen triple helix repeat containing 1 (CTHRC1), a secreted glycoprotein, is widely expressed in many tissues. It has been recently defined as a novel marker for rheumatoid arthritis (RA), a systemic inflammatory disorder. However, the precise role of CTHRC1 in other chronic inflammatory diseases, like periodontal disease, remains unclear. This research aimed to explore the presence of CTHRC1 in periodontal inflammation, determine the precise role in inflammatory response modulation in periodontal ligament cells (PDLCs), and explore its underlying mechanisms. In vivo gingival crevicular fluid (GCF) and gingivae were obtained from healthy people and chronic periodontitis patients. Maxillary tissues of mice with or without ligature-induced periodontitis were immunostained for CTHRC1. In vitro human PDLCs were treated with tumor necrosis factor alpha (TNF-α) to mimic the inflammatory environment. Small interfering RNA (siRNA) was used to silence CTHRC1. SB203580 was used to inhibit the p38 mitogen-activated protein kinase (MAPK) pathway. CTHRC1 was highly expressed in GCF and gingival tissues of periodontitis patients. Animal models also revealed the same tendency. CTHRC1 knockdown promoted inflammatory cytokine production and activated the p38 MAPK signaling pathway in PDLCs. Inhibiting the p38 MAPK signaling pathway partially attenuated the inflammatory responses. This study revealed that CTHRC1 was highly expressed in periodontitis and suggested that CTHRC1 might play an important role in modulating periodontal inflammation.
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Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/biosíntesis , Técnicas de Silenciamiento del Gen/métodos , Mediadores de Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Ligamento Periodontal/metabolismo , Adolescente , Animales , Células Cultivadas , Niño , Activación Enzimática/fisiología , Proteínas de la Matriz Extracelular/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ligamento Periodontal/patología , Periodontitis/genética , Periodontitis/metabolismo , Periodontitis/patología , Adulto JovenRESUMEN
Neuronal synapse formation is critical for brain development and depends on secreted factors from astrocytes. Here, we report that small extracellular vesicles (EVs) secreted from primary astrocytes, but not from neurons or C6 glioma cells, greatly enhance spine and synapse formation by primary cortical neurons. A comparative proteomics analysis of small EVs from astrocytes, neurons, and C6 glioma cells identified fibulin-2 as a promising EV cargo to regulate synaptogenesis. Treatment of cortical neurons with recombinant fibulin-2 increased the formation of spines and synapses, similar to the effect of small EVs. In addition, treatment of neurons with fibulin-2 or astrocyte-derived small EVs led to increased phosphorylation of Smad2, an indicator of TGF-ß signaling. Finally, the effects of fibulin-2 and astrocyte-derived small EVs on synapse formation were reversed by inhibiting transforming growth factor ß (TGF-ß) signaling. These data suggest a model in which astrocyte EVs promote synapse formation via fibulin-2-mediated activation of TGF-ß signaling.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Transducción de Señal , Sinapsis/fisiología , Animales , Astrocitos/citología , Astrocitos/metabolismo , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Células Cultivadas , Espinas Dendríticas/fisiología , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Femenino , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
[Figure: see text].
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Aterosclerosis/prevención & control , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Adhesión Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Rodamiento de Leucocito/efectos de los fármacos , Leucocitos/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oligonucleótidos Antisentido/farmacología , Animales , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Receptor 1 de Quimiocinas CX3C/genética , Moléculas de Adhesión Celular Neuronal/deficiencia , Moléculas de Adhesión Celular Neuronal/genética , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Relacionadas con Receptor de LDL/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Masculino , Ratones Transgénicos , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Placa Aterosclerótica , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteína Reelina , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Transducción de Señal , Células U937RESUMEN
The family with sequence similarity 20, member C (Fam20C), a Golgi casein kinase, has been recently regarded as a potential therapeutic target for the treatment of triple negative breast cancer (TNBC). Lacking enzyme activity center has been becoming an obstacle to the development of small-molecule inhibitors of Fam20C. Herein, we combined in silico high-throughput screening with chemical synthesis methods to obtain a new small-molecule Fam20C inhibitor 3r, which exhibited desired anti-proliferative activities against MDA-MB-231 cells and also inhibited migration. Subsequently, the enzymatic assay, molecular docking, and molecular dynamics (MD) simulations were carried out for validating that 3r could bind to Fam20C. In addition, 3r was found to induce apoptosis via the mitochondrial pathway in MDA-MB-231 cells as well as to inhibit cell migration. Moreover, we demonstrated that 3r inhibited tumor growth in vivo and thereby having a good therapeutic potential on TNBC. Taken together, these results suggest that 3r may be a novel Fam20C inhibitor with anti-proliferative and apoptosis-inducing activities, which would shed light on discovering more small-molecule drugs for the future TNBC therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Quinasa de la Caseína I/antagonistas & inhibidores , Descubrimiento de Drogas , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Antineoplásicos/síntesis química , Antineoplásicos/química , Quinasa de la Caseína I/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Peel of Citrus reticulata, a Chinese herbal drug with functions of regulating Qi and expelling phlegm, has been used for the treatment of lung related diseases in Chinese medicine for a long time. Its detailed effects on collagen in anti-idiopathic pulmonary fibrosis (IPF) is still unclear. AIM OF THE STUDY: To explore the effects of citrus alkaline extract (CAE) on collagen synthesis, crosslinking and deposition in pulmonary fibrosis and understand the possible signal pathways involved in the activity. MATERIALS AND METHODS: CAE was prepared from C. reticulata. Bleomycin-induced pulmonary fibrosis mouse model was applied. Pulmonary fibrosis of lung was estimated with histopathology analysis, and collagen deposition was evaluated with immunohistochemistry. Collagen crosslinking related biomarkers and enzymes were analyzed with chemical methods, immunohistochemical and western blot analyses. RESULTS: CAE oral administration lowered hydroxyproline content, inhibited the collagen deposition including expressions of collagen I and III, and relieved bleomycin-induced pulmonary fibrosis in mice model. The productions of a collagen crosslink pyridinoline and crosslinking related enzymes including lysyl oxidase (LOX), lysyl oxidase-like protein 1 (LOXL1) in lung were suppressed by CAE treatment. Furthermore, the protein expressions of TGF-ß1 and Smad3 levels in lungs were also downregulated by CAE. CONCLUSIONS: This study demonstrated that CAE inhibited collagen synthesis, crosslinking and deposition, and ameliorated bleomycin-induced pulmonary fibrosis. Preliminary mechanism study revealed that CAE exerted its bioactivity at least via downregulation of TGF-ß1/Smad3 pathway. Our findings provided a great potential in fighting IPF based on CAE.
Asunto(s)
Citrus/química , Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Extractos Vegetales/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Administración Oral , Álcalis/química , Aminoácido Oxidorreductasas/antagonistas & inhibidores , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos/metabolismo , Animales , Bleomicina/toxicidad , Colágeno Tipo III/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/metabolismo , Hidroxiprolina/metabolismo , Ratones Endogámicos C57BL , Extractos Vegetales/administración & dosificación , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Proteína smad3/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
Matrix Gla protein (MGP), an extracellular matrix protein, has been widely reported to participate in the tumorigenic process and is abnormally expressed in several tumors. However, the role of MGP in colorectal cancer (CRC) remains unknown. Chemotherapy resistance represents a significant limitation in the treatment of CRC. Here, a comprehensive bioinformatics analysis revealed that MGP, which is overexpressed in CRC, might act as one of the critical genes conferring resistance to oxaliplatin (OXA). Furthermore, we found that MGP overexpression in tumor tissue might be correlated with cancer stage and patient prognosis, consistent with the bioinformatics analysis. The upregulation of MGP may act as an independent risk factor for CRC. The knockdown of MGP or inhibition of MGP expression significantly increased the sensitivity of the CRC cell lines to OXA. Suppression of MGP may reverse OXA resistance by upregulating copper transporter 1 (CTR1) and downregulating ATP7A and ATP7B. When used in combination with OXA, the inhibition of MGP reduced cancer cell proliferation, invasion, and migration and increased cell apoptosis in vitro. Suppression of MGP or OXA treatment alone significantly inhibited tumor growth in the CRC mouse model. Additionally, we found that OXA might promote the antitumor immune response in vivo. In summary, our study is the first to provide evidence that MGP expression confers OXA chemotherapy resistance in CRC and provides novel strategies to overcome chemotherapy resistance in CRC.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Oxaliplatino/farmacología , Animales , Antineoplásicos/uso terapéutico , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Bases de Datos Genéticas , Resistencia a Antineoplásicos/fisiología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Oxaliplatino/uso terapéutico , Proteína Gla de la MatrizRESUMEN
Systemic sclerosis a chronic, fibrotic disorder associated with high disease-specific mortality and morbidity. Cutaneous manifestations include dermal thickening and obliteration of dermal adipose tissue. Accumulation of low-molecular-weight hyaluronan, which signals through the receptor for hyaluronan-mediated motility, RHAMM, leads to progressive fibrosis and is correlated with increased severity of systemic sclerosis. The purpose of this study is to test the efficacy of two function-blocking RHAMM peptides, NPI-110 and NPI-106, in reducing skin fibrosis in a bleomycin-induced mouse model of systemic sclerosis. NPI-110 reduced visible measures of fibrosis (dermal thickness and collagen production, deposition, and organization) and profibrotic gene expression (Tgfb1, c-Myc, Col1a1, Col3a1). NPI-110 treatment also increased the expression of the antifibrotic adipokines perilipin and adiponectin. Both RHAMM peptides strongly reduced dermal RHAMM expression, predicting that dermal fibroblasts are peptide targets. Transcriptome and cell culture analyses using Rhamm-/- and Rhamm-rescued dermal fibroblasts reveal a TGFß1/RHAMM/MYC signaling axis that promotes fibrogenic gene expression and myofibroblast differentiation. RHAMM functionâblocking peptides suppress this signaling and prevent TGFß1-induced myofibroblast differentiation. These results suggest that inhibiting RHAMM signaling will offer a treatment method for cutaneous fibrosis in systemic sclerosis.
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Adipoquinas/metabolismo , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Receptores de Hialuranos/antagonistas & inhibidores , Péptidos/farmacología , Esclerodermia Sistémica/tratamiento farmacológico , Piel/patología , Animales , Bleomicina/administración & dosificación , Bleomicina/toxicidad , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibrosis , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ratones , Ratones Noqueados , Péptidos/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/metabolismo , Esclerodermia Sistémica/inducido químicamente , Esclerodermia Sistémica/patología , Transducción de Señal/efectos de los fármacos , Piel/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismoAsunto(s)
Aneurisma de la Aorta Abdominal , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Caracteres Sexuales , Animales , Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Aneurisma de la Aorta Abdominal/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Ratones , Proteína-Lisina 6-Oxidasa/metabolismoRESUMEN
BACKGROUND: Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation. METHODS: In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays. RESULTS: We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 . CONCLUSIONS: The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.
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Sistemas CRISPR-Cas , Diferenciación Celular , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Técnicas de Sustitución del Gen/métodos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis , Fosfoproteínas/antagonistas & inhibidores , Proliferación Celular , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiones Promotoras GenéticasRESUMEN
The outbreak of the highly contagious and deadly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease 2019 (COVID-19), has posed a serious threat to public health across the globe, calling for the development of effective diagnostic markers and therapeutics. Here, we report a highly reliable severity diagnostic biomarker, acetylated 676th lysine transforming growth factor-beta-induced protein (TGFBIp K676Ac). TGFBIp K676Ac was consistently elevated in the blood of patients with SARS-CoV-2 pneumonia (n = 113), especially in patients in the intensive care unit (ICU) compared to non-ICU patients. Patients' blood samples showed increased cytokines and lymphopenia, which are exemplary indicators of SARS-CoV-2 pneumonia. Treatment with TGFBIp neutralizing antibodies suppressed the cytokine storm. The increased level of TGFBIp K676Ac in ICU patients suggests the promise of this protein as a reliable severity diagnostic biomarker for severe SARS-CoV-2 disease.
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Betacoronavirus/patogenicidad , Infecciones por Coronavirus/diagnóstico , Síndrome de Liberación de Citoquinas/diagnóstico , Proteínas de la Matriz Extracelular/inmunología , Leucocitos Mononucleares/inmunología , Neumonía Viral/diagnóstico , Procesamiento Proteico-Postraduccional , Insuficiencia Respiratoria/diagnóstico , Factor de Crecimiento Transformador beta/inmunología , Acetilación , Anticuerpos Neutralizantes/farmacología , Betacoronavirus/inmunología , Biomarcadores/sangre , COVID-19 , Estudios de Casos y Controles , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/patología , Síndrome de Liberación de Citoquinas/sangre , Síndrome de Liberación de Citoquinas/inmunología , Síndrome de Liberación de Citoquinas/patología , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Expresión Génica , Humanos , Unidades de Cuidados Intensivos , Recuento de Leucocitos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Leucocitos Mononucleares/virología , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/virología , Lisina/metabolismo , FN-kappa B/genética , FN-kappa B/inmunología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/inmunología , Neumonía Viral/patología , Cultivo Primario de Células , Pronóstico , Insuficiencia Respiratoria/sangre , Insuficiencia Respiratoria/inmunología , Insuficiencia Respiratoria/patología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Vascular calcification (VC) is a major risk factor for increasing cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS), a representative uremic toxin, is closely associated with VC in CKD patients. Matrix Gla protein (MGP) plays pivotal role in VC as a calcification inhibitor. The aim of this work was to explore whether MGP was involved in IS-induced VC. Here, we demonstrated the role of MGP in the IS-induced osteogenic differentiation of human aortic smooth muscle cells (HASMCs). The methods included Von Kossa staining, immunohistochemistry, Alizarin Red staining, quantitative real-time PCR and western blotting. MGP was decreased in calcified arteries both in CKD patients and rats. In vitro, IS suppressed MGP expression in HASMCs by activating ROS/NF-κB signaling in parallel with osteogenic differentiation, which was mitigated by inhibiting ROS and NF-κB with diphenyleneiodonium and Bay11-7082. Further investigation showed that IS induced NF-κB-responsive microRNA (miR)-155-5p mediating MGP downregulation. Overexpression of miR-155-5p with mimics aggravated IS-induced MGP reduction and osteogenic differentiation. In contrast, these conditions were diminished by silencing miR-155-5p. We demonstrate that IS promotes the HASMCs phenotype switch by suppressing MGP expression via ROS/NF-κB/miR-155-5p signaling and provide a new insight for the pathogenesis of IS-induced VC.
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Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Regulación de la Expresión Génica/efectos de los fármacos , Indicán/farmacología , Músculo Liso Vascular/patología , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Insuficiencia Renal Crónica/complicaciones , Calcificación Vascular/patología , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , MicroARNs/genética , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , FN-kappa B/genética , Osteogénesis , Ratas , Ratas Sprague-Dawley , Calcificación Vascular/etiología , Calcificación Vascular/metabolismo , Proteína Gla de la MatrizRESUMEN
BACKGROUND: As a substrate of apoER2, Reelin has been verified to exert neuroprotection by preventing memory impairment. Pinocembrin is the most abundant natural flavonoid found in propolis, and it has been used to exert neuroprotection, blood-brain barrier protection, anti-oxidation, and inflammation diminishing, both in vitro and in vivo. However, the roles and molecular mechanisms of pinocembrin in neurobehavioral outcomes and neuronal repair after vascular dementia are still under investigation. PURPOSE: To explore the role of pinocembrin in the involvement of the Reelin-dab1 signaling pathway in improving memory impairment, both in cell culture and animals experiments. MATERIAL AND METHODS: Behavioral tests were conducted on day 48 to confirm the protection of pinocembrin against cognitive impairment. Cell and molecular biology experiments demonstrated that the Reelin-dab1 pathway mediates the underlying mechanism of cognitive improvement by pinocembrin. RESULTS: It was showed that pinocembrin alleviated learning and memory deficits induced by vascular dementia, by inducing the expression of Reelin, apoER2, and p-dab1 in the hippocampus. The expression of Reelin and p-dab1 was both inhibited following Reelin RNA interference in SH-SY5Y prior to oxygen glucose deprivation (OGD) injury, suggesting that Reelin played a core role in pinocembrin's effect on OGD in vitro. CONCLUSION: Pinocembrin improves the cognition via the Reelin-dab1 signaling pathway.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Disfunción Cognitiva/tratamiento farmacológico , Demencia Vascular/tratamiento farmacológico , Proteínas de la Matriz Extracelular/metabolismo , Flavanonas/farmacología , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Conducta Animal/efectos de los fármacos , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Moléculas de Adhesión Celular Neuronal/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Demencia Vascular/metabolismo , Demencia Vascular/patología , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Humanos , Masculino , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Proteína Reelina , Serina Endopeptidasas/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Tumor growth and invasion occurs through a dynamic interaction between cancer and stromal cells, which support an aggressive niche. MicroRNAs are thought to act as tumor messengers to "corrupt" stromal cells. We previously demonstrated that miR-9, a known metastamiR, is released by triple negative breast cancer (TNBC) cells to enhance the transition of normal fibroblasts (NFs) into cancer-associated fibroblast (CAF)-like cells. EGF containing fibulin extracellular matrix protein 1 (EFEMP1), which encodes for the ECM glycoprotein fibulin-3, emerged as a miR-9 putative target upon miRNA's exogenous upmodulation in NFs. Here we explored the impact of EFEMP1 downmodulation on fibroblast's acquisition of CAF-like features, and how this phenotype influences neoplastic cells to gain chemoresistance. Indeed, upon miR-9 overexpression in NFs, EFEMP1 resulted downmodulated, both at RNA and protein levels. The luciferase reporter assay showed that miR-9 directly targets EFEMP1 and its silencing recapitulates miR-9-induced pro-tumoral phenotype in fibroblasts. In particular, EFEMP1 siRNA-transfected (si-EFEMP1) fibroblasts have an increased ability to migrate and invade. Moreover, TNBC cells conditioned with the supernatant of NFs transfected with miR-9 or si-EFEMP1 became more resistant to cisplatin. Overall, our results demonstrate that miR-9/EFEMP1 axis is crucial for the conversion of NFs to CAF-like cells under TNBC signaling.
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Fibroblastos Asociados al Cáncer/metabolismo , Transformación Celular Neoplásica/genética , Proteínas de la Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias de la Mama Triple Negativas/genética , Animales , Antineoplásicos/farmacología , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cisplatino/farmacología , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones SCID , MicroARNs/metabolismo , Cultivo Primario de Células , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
High-grade serous ovarian carcinoma (HGSOC) is one of the most lethal gynecological malignancies; however, the precise molecular mechanisms have not been fully characterized. Fibulin5 (FBLN5) is an extracellular matrix (ECM) glycoprotein, and plays a crucial role in maintaining the stability of ECM structures, regulating cell proliferation and tumorigenesis. In the present study, the expression of FBLN5, as determined by western blot analysis and immunohistochemistry, was significantly increased in normal fallopian tube (FT) samples compared with that in HGSOC samples, and decreased FBLN5 expression was associated with unfavorable prognosis of HGSOC. Functional characterization revealed that FBLN5 overexpression significantly inhibited migration, invasion and proliferation abilities of ovarian cancer cells in vitro. Furthermore, micro (mi)RNA27a3p (miR27a3p) was revealed to be increased in HGSOC, and dualluciferase reporter assay indicated that miR27a3p was functioned as a negative regulator of FBLN5 by directly binding with its 3'untranslated region. Collectively, FBLN5 expression was associated with prognosis, proliferation, and metastasis in HGSOC. We hypothesized that FBLN5 was targeted by miR27a3p and may serve as a biomarker and provide a new therapeutic approach for the treatment of HGSOC.
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Cistadenocarcinoma Seroso/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , MicroARNs/metabolismo , Neoplasias Ováricas/metabolismo , Adulto , Anciano , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Progresión de la Enfermedad , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Femenino , Humanos , MicroARNs/genética , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
Lysyl oxidase (LOX) is involved in fibrosis by catalyzing collagen cross-linking. Previous work observed that Triptolide (TPL) alleviated radiation-induced pulmonary fibrosis (RIPF), but it is unknown whether the anti-RIPF effect of TPL is related to LOX. In a mouse model of RIPF, we found that LOX persistently increased in RIPF which was significantly lowered by TPL. Excessive LOX aggravated fibrotic lesions in RIPF, while LOX inhibition mitigated RIPF. Irradiation enhanced the transcription and synthesis of LOX by lung fibroblasts through IKKß/NFκB activation, and siRNA knockdown IKKß largely abolished LOX production. By interfering radiation induced IKKß activation, TPL prevented NFκB nuclear translocation and DNA binding, and potently decreased LOX synthesis. Our results demonstrate that the anti-RIPF effect of TPL is associated with reduction of LOX production which mediated by inhibition of IKKß/NFκB pathway.
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Diterpenos/farmacología , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Quinasa I-kappa B/antagonistas & inhibidores , Fenantrenos/farmacología , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Fibrosis Pulmonar/tratamiento farmacológico , Traumatismos por Radiación/tratamiento farmacológico , Animales , Diterpenos/administración & dosificación , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/administración & dosificación , Compuestos Epoxi/farmacología , Proteínas de la Matriz Extracelular/biosíntesis , Femenino , Quinasa I-kappa B/metabolismo , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Fenantrenos/administración & dosificación , Proteína-Lisina 6-Oxidasa/biosíntesis , Fibrosis Pulmonar/metabolismo , Traumatismos por Radiación/metabolismo , Relación Estructura-ActividadRESUMEN
Cyclovirobuxine D (CVBD) is an alkaloid, which is mainly derived from Buxus microphylla. It has been reported that CVBD has positive effects on breast cancer, gastric cancer and other malignant tumors. However, to the best of our knowledge, there are no reports regarding the effects of CVBD on colorectal cancer (CRC). The purpose of the present study was to determine the anticancer effects of CVBD and further elucidate its molecular mechanism(s). DLD1 and LoVo cell lines were selected to evaluate the antitumor effect of CVBD. Cytotoxicity, viability and proliferation were evaluated by the MTT and colony formation assays. Flow cytometry was used to detect the effects on apoptosis and the cell cycle in CVBDtreated CRC cells. The migration and invasion abilities of CRC cells were examined by wound healing and Transwell assays. In addition, RNA sequencing, bioinformatics analysis and western blotting were performed to investigate the target of drug action and clarify the molecular mechanisms. A xenograft model was established using nude mice, and ultrasound was employed to assess the preclinical therapeutic effects of CVBD in vivo. It was identified that CVBD inhibited the proliferation, migration, stemness, angiogenesis and epithelialmesenchymal transition of CRC cells, and induced apoptosis and Sphase arrest. In addition, CVBD significantly inhibited the growth of xenografts. It is notable that CVBD exerted anticancer effects in CRC cells partly by targeting collagen triple helix repeat containing 1 (CTHRC1), which may be upstream of the AKT and ERK pathways. CVBD exerted anticancer effects through the CTHRC1AKT/ERKSnail signaling pathway. Targeted therapy combining CTHRC1 with CVBD may offer a promising novel therapeutic approach for CRC treatment.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias del Colon/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/patología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Medicamentos Herbarios Chinos/uso terapéutico , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/metabolismo , RNA-Seq , Factores de Transcripción de la Familia Snail/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A central and unique aspect of high-grade serous ovarian carcinoma (HGSC) is the extensive transcoelomic spreading of tumor cell via the peritoneal fluid or malignant ascites. We and others identified tumor-associated macrophages (TAM) in the ascites as promoters of metastasis-associated processes like extracellular matrix (ECM) remodeling, tumor cell migration, adhesion, and invasion. The precise mechanisms and mediators involved in these functions of TAM are, however, largely unknown. We observed that HGSC migration is promoted by soluble mediators from ascites-derived TAM, which can be emulated by conditioned medium from monocyte-derived macrophages (MDM) differentiated in ascites to TAM-like asc-MDM. A similar effect was observed with IL-10-induced alternatively activated m2c-MDM but not with LPS/IFNγ-induced inflammatory m1-MDM. These observations provided the basis for deconvolution of the complex TAM secretome by performing comparative secretome analysis of matched triplets of different MDM phenotypes with different pro-migratory properties (asc-MDM, m2c-MDM, m1-MDM). Mass spectrometric analysis identified an overlapping set of nine proteins secreted by both asc-MDM and m2c-MDM, but not by m1-MDM. Of these, three proteins, i.e., transforming growth factor beta-induced (TGFBI) protein, tenascin C (TNC), and fibronectin (FN1), have been associated with migration-related functions. Intriguingly, increased ascites concentrations of TGFBI, TNC, and fibronectin were associated with short progression-free survival. Furthermore, transcriptome and secretome analyses point to TAM as major producers of these proteins, further supporting an essential role for TAM in promoting HGSC progression. Consistent with this hypothesis, we were able to demonstrate that the migration-inducing potential of asc-MDM and m2c-MDM secretomes is inhibited, at least partially, by neutralizing antibodies against TGFBI and TNC or siRNA-mediated silencing of TGFBI expression. In conclusion, the present study provides the first experimental evidence that TAM-derived TGFBI and TNC in ascites promote HGSC progression.