RESUMEN
Gene therapy offers a promising avenue for treating ischemic diseases, yet its clinical efficacy is hindered by the limitations of single gene therapy and the high oxidative stress microenvironment characteristic of such conditions. Lipid-polymer hybrid vectors represent a novel approach to enhance the effectiveness of gene therapy by harnessing the combined advantages of lipids and polymers. In this study, we engineered lipid-polymer hybrid nanocarriers with tailored structural modifications to create a versatile membrane fusion lipid-nuclear targeted polymer nanodelivery system (FLNPs) optimized for gene delivery. Our results demonstrate that FLNPs facilitate efficient cellular uptake and gene transfection via membrane fusion, lysosome avoidance, and nuclear targeting mechanisms. Upon encapsulating Hepatocyte Growth Factor plasmid (pHGF) and Catalase plasmid (pCAT), HGF/CAT-FLNPs were prepared, which significantly enhanced the resistance of C2C12 cells to H2O2-induced injury in vitro. In vivo studies further revealed that HGF/CAT-FLNPs effectively alleviated hindlimb ischemia-induced gangrene, restored motor function, and promoted blood perfusion recovery in mice. Metabolomics analysis indicated that FLNPs didn't induce metabolic disturbances during gene transfection. In conclusion, FLNPs represent a versatile platform for multi-dimensional assisted gene delivery, significantly improving the efficiency of gene delivery and holding promise for effective synergistic treatment of lower limb ischemia using pHGF and pCAT.
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Terapia Genética , Isquemia , Lípidos , Polímeros , Animales , Isquemia/terapia , Terapia Genética/métodos , Lípidos/química , Ratones , Polímeros/química , Nanopartículas/química , Factor de Crecimiento de Hepatocito/genética , Línea Celular , Transfección/métodos , Plásmidos/genética , Técnicas de Transferencia de Gen , Masculino , Miembro Posterior/irrigación sanguínea , Catalasa/metabolismoRESUMEN
In this study, we assessed the impact of hepatocyte growth factor (HGF) on corneal endothelial cells (CECs), finding that HGF concentrations of 100-250 ng/mL significantly increased CEC proliferation by 30%, migration by 32% and improved survival under oxidative stress by 28% compared to untreated controls (p < 0.05). The primary objective was to identify non-fibrotic pharmacological strategies to enhance corneal endothelial regeneration, addressing a critical need in conditions like Fuchs' endothelial dystrophy (FED), where donor tissue is scarce. To confirm the endothelial nature of the cultured CECs, Na+/K+-ATPase immunohistochemistry was performed. Proliferation rates were determined through BrdU incorporation assays, while cell migration was assessed via scratch assays. Cell viability was evaluated under normal and oxidative stress conditions using WST-1 assays. To ensure that HGF treatment did not trigger epithelial-mesenchymal transition, which could lead to undesirable fibrotic changes, α-SMA staining was conducted. These comprehensive methodologies provided robust data on the effects of HGF, confirming its potential as a therapeutic agent for corneal endothelial repair without inducing harmful EMT, as indicated by the absence of α-SMA expression. These findings suggest that HGF holds therapeutic promise for enhancing corneal endothelial repair, warranting further investigation in in vivo models to confirm its clinical applicability.
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Movimiento Celular , Proliferación Celular , Endotelio Corneal , Factor de Crecimiento de Hepatocito , Cicatrización de Heridas , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/farmacología , Endotelio Corneal/efectos de los fármacos , Endotelio Corneal/metabolismo , Humanos , Cicatrización de Heridas/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Estrés Oxidativo/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Distrofia Endotelial de Fuchs/tratamiento farmacológico , Distrofia Endotelial de Fuchs/metabolismo , Distrofia Endotelial de Fuchs/patología , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Platelets play a crucial role in tissue regeneration, and their involvement in liver regeneration is well-established. However, the specific contribution of platelet-derived Transforming Growth Factor Beta 1 (TGFß1) to liver regeneration remains unexplored. This study investigated the role of platelet-derived TGFß1 in initiating liver regeneration following 2/3 liver resection. Using platelet-specific TGFß1 knockout (Plt.TGFß1 KO) mice and wild-type littermates (Plt.TGFß1 WT) as controls, the study assessed circulating levels and hepatic gene expression of TGFß1, Platelet Factor 4 (PF4), and Thrombopoietin (TPO) at early time points post-hepatectomy (post-PHx). Hepatocyte proliferation was quantified through Ki67 staining and PCNA expression in total liver lysates at various intervals, and phosphohistone-H3 (PHH3) staining was employed to mark mitotic cells. Circulating levels of hepatic mitogens, Hepatocyte Growth Factor (HGF), and Interleukin-6 (IL6) were also assessed. Results revealed that platelet-TGFß1 deficiency significantly reduced total plasma TGFß1 levels at 5 h post-PHx in Plt.TGFß1 KO mice compared to controls. While circulating PF4 levels, liver platelet recruitment and activation appeared normal at early time points, Plt.TGFß1 KO mice showed more stable circulating platelet numbers with higher numbers at 48 h post-PHx. Notably, hepatocyte proliferation was significantly reduced in Plt.TGFß1 KO mice. The results show that a lack of TGFß1 in platelets leads to an unbalanced expression of IL6 in the liver and to strongly increased HGF levels 48 h after liver resection, and yet liver regeneration remains reduced. The study identifies platelet-TGFß1 as a regulator of hepatocyte proliferation and platelet homeostasis in the early stages of liver regeneration.
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Plaquetas , Hepatectomía , Regeneración Hepática , Ratones Noqueados , Trombopoyetina , Factor de Crecimiento Transformador beta1 , Animales , Regeneración Hepática/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/genética , Ratones , Plaquetas/metabolismo , Trombopoyetina/metabolismo , Interleucina-6/metabolismo , Interleucina-6/genética , Proliferación Celular , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Hígado/metabolismo , Hepatocitos/metabolismo , Masculino , Factor Plaquetario 4/metabolismo , Factor Plaquetario 4/genética , Ratones Endogámicos C57BLRESUMEN
Non-small cell lung cancer (NSCLC) is characterized by several molecular alterations that contribute to its development and progression. These alterations include the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), human epidermal growth factor receptor 2 (HER2), and mesenchymal-epithelial transition factor (c-MET). Among these, the hepatocyte growth factor (HGF)/c-MET signaling pathway plays a crucial role in NSCLC. In spite of this, the involvement of the HGF/c-MET signaling axis in remodeling the tumor microenvironment (TME) remains relatively unexplored. This review explores the biological functions of the HGF/c-MET signaling pathway in both normal and cancerous cells, examining its multifaceted roles in the NSCLC tumor microenvironment, including tumor cell proliferation, migration and invasion, angiogenesis, and immune evasion. Furthermore, we summarize the current progress and clinical applications of MET-targeted therapies in NSCLC and discuss future research directions, such as the development of novel MET inhibitors and the potential of combination immunotherapy.
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Carcinoma de Pulmón de Células no Pequeñas , Factor de Crecimiento de Hepatocito , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas c-met , Transducción de Señal , Microambiente Tumoral , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas Proto-Oncogénicas c-met/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , AnimalesRESUMEN
Alcoholic liver disease (ALD) is a form of hepatic inflammation. ALD is mediated by gut leakiness. This study evaluates the anti-inflammatory effects of ASCs overexpressing interferon-beta (ASC-IFN-ß) on binge alcohol-induced liver injury and intestinal permeability. In vitro, ASCs were transfected with a non-viral vector carrying the human IFN-ß gene, which promoted hepatocyte growth factor (HGF) secretion in the cells. To assess the potential effects of ASC-IFN-ß, C57BL/6 mice were treated with three oral doses of binge alcohol and were administered intraperitoneal injections of ASC-IFN-ß. Mice treated with binge alcohol and administered ASC-IFN-ß showed reduced liver injury and inflammation compared to those administered a control ASC. Analysis of intestinal tissue from ethanol-treated mice administered ASC-IFN-ß also indicated decreased inflammation. Additionally, fecal albumin, blood endotoxin, and bacterial colony levels were reduced, indicating less gut leakiness in the binge alcohol-exposed mice. Treatment with HGF, but not IFN-ß or TRAIL, mitigated the ethanol-induced down-regulation of cell death and permeability in Caco-2 cells. These results demonstrate that ASCs transfected with a non-viral vector to induce IFN-ß overexpression have protective effects against binge alcohol-mediated liver injury and gut leakiness via HGF.
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Etanol , Interferón beta , Hepatopatías Alcohólicas , Células Madre Mesenquimatosas , Ratones Endogámicos C57BL , Permeabilidad , Animales , Humanos , Interferón beta/metabolismo , Hepatopatías Alcohólicas/metabolismo , Hepatopatías Alcohólicas/patología , Hepatopatías Alcohólicas/genética , Ratones , Células Madre Mesenquimatosas/metabolismo , Etanol/efectos adversos , Células CACO-2 , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Masculino , Tejido Adiposo/metabolismo , Hígado/metabolismo , Hígado/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patologíaRESUMEN
Macrophages have been recognized as pivotal players in the progression of MASLD/MASH. However, the molecular mechanisms underlying their multifaceted functions in the disease remain to be further clarified. In the current study, we developed a new mouse model with YAP activation in macrophages to delineate the effect and mechanism of YAP signaling in the pathogenesis of MASLD/MASH. Genetically modified mice, featuring specific depletion of both Mst1 and Mst2 in macrophages/monocytes, were generated and exposed to a high-fat diet for 12 weeks to induce MASLD. Following this period, livers were collected for histopathological examination, and liver non-parenchymal cells were isolated and subjected to various analyses, including single-cell RNA-sequencing, immunofluorescence and immunoblotting and qRT-PCR to investigate the impact of YAP signaling on the progression of MASLD. Our data revealed that Mst1/2 depletion in liver macrophages enhanced liver inflammation and fibrosis in MASLD. Using single-cell RNA-sequencing, we showed that YAP activation via Mst1/2 depletion upregulated the expressions of both pro-inflammatory genes and genes associated with resolution/tissue repair. We observed that YAP activation increases Kupffer cell populations (i.e., Kupffer-2 and Kupffer-3) which are importantly implicated in the pathogenesis of MASLD/MASH. Our data indicate that YAP activation via Mst1/2 deletion enhances both the pro-inflammatory and tissue repairing functions of Kupffer-1 and -2 cells at least in part through C1q. These YAP-regulatory mechanisms control the plasticity of liver macrophages in the context of MASLD/MASH. Our findings provide important evidence supporting the critical regulatory role of YAP signaling in liver macrophage plasticity and the progression of MASLD. Therefore, targeting the Hippo-YAP pathway may present a promising therapeutic strategy for the treatment of MASH.
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Cirrosis Hepática , Hígado , Macrófagos , Proteínas Serina-Treonina Quinasas , Serina-Treonina Quinasa 3 , Proteínas Señalizadoras YAP , Animales , Ratones , Proteínas Señalizadoras YAP/metabolismo , Macrófagos/metabolismo , Hígado/metabolismo , Hígado/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones Endogámicos C57BL , Masculino , Transducción de Señal , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genética , Inflamación/metabolismo , Inflamación/patología , Macrófagos del Hígado/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genéticaRESUMEN
Corneal defects can lead to stromal scarring and vision loss, which is currently only treatable with a cadaveric corneal transplant. Although in situ-forming hydrogels have been shown to foster regeneration of the cornea in the setting of stromal defects, the cross-linking, biomechanical, and compositional parameters that optimize healing have not yet been established. This, Corneal defects are also almost universally inflamed, and their rapid closure without fibrosis are critical to preserving vision. Here, an in situ forming, bioorthogonally cross-linked, nanocluster (NC)-reinforced collagen and hyaluronic acid hydrogel (NCColHA hydrogel) with enhanced structural integrity and both pro-regenerative and anti-inflammatory effects was developed and tested within a corneal defect model in vivo. The NCs serve as bioorthogonal nanocross-linkers, providing higher cross-linking density than polymer-based alternatives. The NCs also serve as delivery vehicles for prednisolone (PRD) and the hepatocyte growth factor (HGF). NCColHA hydrogels rapidly gel within a few minutes upon administration and exhibit robust rheological properties, excellent transparency, and negligible swelling/deswelling behavior. The hydrogel's biocompatibility and capacity to support cell growth were assessed using primary human corneal epithelial cells. Re-epithelialization on the NCColHA hydrogel was clearly observed in rabbit eyes, both ex vivo and in vivo, with expression of normal epithelial biomarkers, including CD44, CK12, CK14, α-SMA, Tuj-1, and ZO-1, and stratified, multilayered morphology. The applied hydrogel maintained its structural integrity for at least 14 days and remodeled into a transparent stroma by 56 days.
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Hidrogeles , Hidrogeles/química , Hidrogeles/farmacología , Animales , Conejos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Córnea/efectos de los fármacos , Regeneración/efectos de los fármacos , Humanos , Reactivos de Enlaces Cruzados/química , Colágeno/química , Factor de Crecimiento de Hepatocito/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/químicaRESUMEN
BACKGROUND: Numerous studies have shown that various cytokines are important factors affecting bone mineral density (BMD), but the causality between the two remains uncertain. METHODS: Genetic variants associated with 41 circulating cytokines from a genome-wide association study (GWAS) in 8,293 Finns were used as instrumental variables (IVs) for a two-sample Mendelian randomization (MR) analysis. Inverse variance weighting (IVW) was employed as the primary method to investigate whether the 41 cytokines were causally associated with BMD at five different sites [total body bone mineral density (TB-BMD), heel bone mineral density (HE-BMD), forearm bone mineral density (FA-BMD), femoral neck bone mineral density (FN-BMD), and lumbar spine bone mineral density (LS-BMD)]. Weighted median and MR-Egger were chosen to further confirm the robustness of the results. We performed MR pleiotropy residual sum and outlier test (MR-PRESSO), MR-Egger regression, and Cochran's Q test to detect pleiotropy and sensitivity testing. RESULTS: After Bonferroni correction, two circulating cytokines had a strong causality with BMD at corresponding sites. Genetically predicted circulating hepatocyte growth factor (HGF) levels and HE-BMD were negatively correlated [ß (95 % CI) -0.035(-0.055, -0.016), P=0.00038]. Circulating macrophage inflammatory protein-1α (MIP-1α) levels and TB-BMD were negatively correlated [ß(95 %CI): -0.058(-0.092, -0.024), P=0.00074]. Weighted median and MR-Egger results were in line with the IVW results. We also found suggestive causal relationship (IVW P<0.05) between seven circulating cytokines and BMD at corresponding sites. No significant pleiotropy or heterogeneity was observed in our study. CONCLUSION: Our MR analyses indicated a causal effect between two circulating cytokines and BMD at corresponding sites (HGF and HE-BMD, MIP-1α and TB-BMD), along with suggestive evidence of a potential causality between seven cytokines and BMD at the corresponding sites. These findings would provide insights into the prevention and treatment of osteoporosis, especially immunoporosis.
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Densidad Ósea , Citocinas , Estudio de Asociación del Genoma Completo , Factor de Crecimiento de Hepatocito , Análisis de la Aleatorización Mendeliana , Humanos , Densidad Ósea/genética , Citocinas/sangre , Masculino , Femenino , Factor de Crecimiento de Hepatocito/sangre , Factor de Crecimiento de Hepatocito/genética , Polimorfismo de Nucleótido Simple/genética , Persona de Mediana Edad , Cuello Femoral/metabolismo , Quimiocina CCL3/sangre , Quimiocina CCL3/genéticaRESUMEN
Background and Objectives: Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), often necessitates long-term treatment and hospitalizations and also may require surgery. The macrophage-stimulating 1 (MST1) rs3197999 polymorphism is strongly associated with the risk of IBD but its exact clinical correlates remain under investigation. We aimed to characterize the relationships between the MST1 rs3197999 genotype and the clinical characteristics in children and adolescents with IBD within a multi-center cross-sectional study. Materials and Methods: Clinical data included serum C-reactive protein (CRP), albumin, activity indices (PUCAI, PCDAI), anthropometric data, pharmacotherapy details, surgery, and disease severity. Genotyping for rs3197999 was carried out using TaqMan hydrolysis probes. Results: The study included 367 pediatric patients, 197 with Crohn's disease (CD) (40.6% female; a median age of 15.2 years [interquartile range 13.2-17.0]) and 170 with ulcerative colitis (UC) (45.8% female; a median age of 15.1 years [11.6-16.8]). No significant relationships were found between MST1 genotypes and age upon first biologic use, time from diagnosis to biological therapy introduction, PUCAI, PCDAI, or hospitalizations for IBD flares. However, in IBD, the height Z-score at the worst flare was negatively associated with the CC genotype (p = 0.016; CC: -0.4 [-1.2-0.4], CT: -0.1 [-0.7-0.8], TT: 0.0 [-1.2-0.7)]). The TT genotype was associated with higher C-reactive protein upon diagnosis (p = 0.023; CC: 4.3 mg/dL [0.7-21.8], CT 5.3 mg/dL [1.3-17.9], TT 12.2 mg/dL [3.0-32.9]). Conclusions: This study identified links between MST1 rs3197999 and the clinical characteristics of pediatric IBD: height Z-score and CRP. Further studies of the associations between genetics and the course of IBD are still warranted, with a focus on more extensive phenotyping.
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Factor de Crecimiento de Hepatocito , Enfermedades Inflamatorias del Intestino , Humanos , Femenino , Masculino , Adolescente , Niño , Estudios Transversales , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/sangre , Enfermedades Inflamatorias del Intestino/genética , Proteína C-Reactiva/análisis , Genotipo , Enfermedad de Crohn/genética , Colitis Ulcerosa/genética , Colitis Ulcerosa/sangre , Polimorfismo Genético , Polimorfismo de Nucleótido Simple , Predisposición Genética a la Enfermedad , Proteínas Proto-OncogénicasRESUMEN
Adipocyte-cancer cell interactions promote tumor development and progression. Previously, we identified adipsin (CFD) and its downstream effector, hepatocyte growth factor (HGF), as adipokines that enhance adipocyte-breast cancer stem cell interactions. Here, we show that adipsin-dependent adipocyte maturation and the subsequent upregulation of HGF promote tumor invasion in breast cancers. Mature adipocytes, but not their precursors, significantly induced breast tumor cell migration and invasion in an adipsin expression-dependent manner. Promoters of tumor invasion, galectin 7 and matrix metalloproteinases, were significantly upregulated in cancer cells cocultured with mature adipocytes; meanwhile, their expression levels in cancer cells cocultured with adipocytes were reduced by adipsin knockout (Cfd KO) or a competitive inhibitor of CFD. Tumor growth and distant metastasis of mammary cancer cells were significantly suppressed when syngeneic mammary cancer cells were transplanted into Cfd KO mice. Histological analyses revealed reductions in capsular formation and tumor invasion at the cancer-adipocyte interface in the mammary tumors formed in Cfd KO mice. These findings indicate that adipsin-dependent adipocyte maturation may play an important role in adipocyte-cancer cell interaction and breast cancer progression.
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Adipocitos , Neoplasias de la Mama , Movimiento Celular , Factor D del Complemento , Invasividad Neoplásica , Animales , Femenino , Humanos , Ratones , Adipocitos/metabolismo , Adipocitos/patología , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Técnicas de Cocultivo , Factor D del Complemento/metabolismo , Factor D del Complemento/genética , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Ratones NoqueadosRESUMEN
The pathway for axon regeneration in Caenorhabditis elegans is activated by SVH-1, a growth factor belonging to the HGF/plasminogen family. SVH-1 is a dual-function factor that acts as an HGF-like growth factor to promote axon regeneration and as a protease to regulate early development. It is important to understand how SVH-1 is converted from a protease to a growth factor for axon regeneration. In this study, we demonstrate that cytidine deaminase (CDD) SVH-17/CDD-2 plays a role in the functional conversion of SVH-1. We find that the codon exchange of His-755 to Tyr in the Asp-His-Ser catalytic triad of SVH-1 can suppress the cdd-2 defect in axon regeneration. Furthermore, the stem hairpin structure around the His-755 site in svh-1 mRNA is required for the activation of axon regeneration by SVH-1. These results suggest that CDD-2 promotes axon regeneration by transforming the function of SVH-1 from a protease to a growth factor through modification of svh-1 mRNA.
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Axones , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Citidina Desaminasa , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Axones/metabolismo , Axones/fisiología , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Regeneración Nerviosa/genética , Regeneración Nerviosa/fisiología , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Regeneración/genéticaRESUMEN
E3112 is a recombinant human hepatocyte growth factor which is under development for the treatment of acute liver failure. Pharmacokinetics (PK) evaluation in experimental animals is important and thus a simple assay for the determination of E3112 in rat and monkey serum has been validated using a commercially available enzyme-linked immunosorbent assay (ELISA) kit. E3112 in rat and monkey serum was quantifiable from 0.313 ng/mL to 15.0 ng/mL without prozone effects. Dilution integrity enabled accurate assay up to 500,000-fold dilution. Accuracy and precision were within the acceptance criteria. PK of E3112 was investigated after intravenous administration to rats and monkeys. PK of E3112 was similar between male and female animals in both species. Nonlinear PK of E3112 was observed in rats after intravenous bolus dose at 1-100 mg/kg while nonlinear PK was not significant in monkeys after intravenous infusion at 0.5-25 mg/kg. These findings suggest that the assay of E3112 in serum using a commercially available ELISA kit was validated and successfully applied to PK studies in rats and monkeys.
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Ensayo de Inmunoadsorción Enzimática , Factor de Crecimiento de Hepatocito , Proteínas Recombinantes , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Masculino , Ratas , Femenino , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/administración & dosificación , Humanos , Factor de Crecimiento de Hepatocito/farmacocinética , Factor de Crecimiento de Hepatocito/sangre , Ratas Sprague-Dawley , Macaca fascicularis , Inyecciones Intravenosas , Relación Dosis-Respuesta a Droga , Reproducibilidad de los Resultados , Administración IntravenosaRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematological disorder characterized by an increased proliferation of immature T lymphocytes precursors. T-ALL treatment includes chemotherapy with strong side effects, and patients that undergo relapse display poor prognosis. Although cell-intrinsic oncogenic pathways are well-studied, the tumor microenvironment, like inflammatory cellular and molecular components is less explored in T-ALL. We sought to determine the composition of the inflammatory microenvironment induced by T-ALL, and its role in T-ALL progression. We show in two mouse T-ALL cell models that T-ALLs enhance blood neutrophils and resident monocytes, accompanied with a plasmatic acute secretion of inflammatory molecules. Depleting neutrophils using anti-Ly6G treatment or resident monocytes by clodronate liposomes treatment does not modulate plasmatic inflammatory molecule secretion and mice survival. However, inhibiting the secretion of inflammatory molecules by microenvironment with NECA, an agonist of adenosine receptors, diminishes T-ALL progression enhancing mouse survival. We uncovered Hepatocyte Growth Factor (HGF), T-ALL-driven and the most decreased molecule with NECA, as a potential therapeutic target in T-ALL. Altogether, we identified a signature of inflammatory molecules that can potentially be involved in T-ALL evolution and uncovered HGF/cMET pathway as important to target for limiting T-ALL progression.
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Progresión de la Enfermedad , Factor de Crecimiento de Hepatocito , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Microambiente Tumoral , Animales , Factor de Crecimiento de Hepatocito/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Ratones , Ratones Endogámicos C57BL , Línea Celular Tumoral , Inflamación/patología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Neutrófilos/metabolismo , Neutrófilos/efectos de los fármacos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/patologíaRESUMEN
Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, ß-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.
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Calpaína , Adhesión Celular , Células Epiteliales , Animales , Perros , beta Catenina/metabolismo , Cadherinas/metabolismo , Calcio/metabolismo , Calpaína/metabolismo , Movimiento Celular , Sistemas CRISPR-Cas , Células Epiteliales/metabolismo , Células Epiteliales/citología , Factor de Crecimiento de Hepatocito/metabolismo , Células de Riñón Canino Madin Darby , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , ProteolisisRESUMEN
Postoperative peritoneal adhesion (PPA) is a prevalent complication of abdominal surgery, posing a significant hindrance to postsurgical recovery. Although several strategies have been developed to alleviate and prevent adhesions, their efficacy remains unsatisfactory. For the first time, we studied the therapeutic effect and mechanism of our recently developed thermally stable oligonucleotide-based mimetics of hepatocyte growth factor (HGF DNA aptamer) to prevent PPA. The HGF DNA aptamer effectively inhibited canonical TGF-ß1 signaling transduction, partially suppressing mesothelial mesenchymal transition. Additionally, the aptamer, respectively, upregulated and downregulated the expression of tissue plasminogen activator and plasminogen activator inhibitor 1, thereby enhancing fibrinolytic activity. As a pleiotropic factor, the HGF DNA aptamer also enhanced the migratory and proliferative capacities of mesothelial cells. Finally, the aptamer demonstrated a higher level of effectiveness in preventing PPAs than the commercially available antiperitoneal adhesion barrier, Seprafilm. Due to its therapeutic benefits, excellent stability, biosafety, cost-effectiveness, and versatility, the HGF DNA aptamer demonstrates promise for preventing PPA in future clinical settings.
Asunto(s)
Aptámeros de Nucleótidos , Transición Epitelial-Mesenquimal , Fibrinólisis , Factor de Crecimiento de Hepatocito , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Adherencias Tisulares/prevención & control , Humanos , Fibrinólisis/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Ensayo de Materiales , Tamaño de la Partícula , Complicaciones Posoperatorias/prevención & controlRESUMEN
Mesenchymal stem cells (MSCs) have shown great potential for the treatment of liver injuries, and the therapeutic efficacy greatly depends on their homing to the site of injury. In the present study, we detected significant upregulation of hepatocyte growth factor (HGF) in the serum and liver in mice with acute or chronic liver injury. In vitro study revealed that upregulation of miR-9-5p or miR-221-3p promoted the migration of human MSCs (hMSCs) toward HGF. Moreover, overexpression of miR-9-5p or miR-221-3p promoted hMSC homing to the injured liver and resulted in significantly higher engraftment upon peripheral infusion. hMSCs reduced hepatic necrosis and inflammatory infiltration but showed little effect on extracellular matrix (ECM) deposition. By contrast, hMSCs overexpressing miR-9-5p or miR-221-3p resulted in not only less centrilobular necrosis and venous congestion but also a significant reduction of ECM deposition, leading to obvious improvement of hepatocyte morphology and alleviation of fibrosis around central vein and portal triads. Further studies showed that hMSCs inhibited the activation of hepatic stellate cells (HSCs) but could not decrease the expression of TIMP-1 upon acute injury and the expression of MCP-1 and TIMP-1 upon chronic injury, while hMSCs overexpressing miR-9-5p or miR-221-3p led to further inactivation of HSCs and downregulation of all three fibrogenic and proinflammatory factors TGF-ß, MCP-1, and TIMP-1 upon both acute and chronic injuries. Overexpression of miR-9-5p or miR-221-3p significantly downregulated the expression of α-SMA and Col-1α1 in activated human hepatic stellate cell line LX-2, suggesting that miR-9-5p and miR-221-3p may partially contribute to the alleviation of liver injury by preventing HSC activation and collagen expression, shedding light on improving the therapeutic efficacy of hMSCs via microRNA modification.
Asunto(s)
Células Estrelladas Hepáticas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Células Estrelladas Hepáticas/metabolismo , Animales , Ratones , Trasplante de Células Madre Mesenquimatosas/métodos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/terapia , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Masculino , Tetracloruro de Carbono/efectos adversos , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Ratones Endogámicos C57BL , Movimiento CelularRESUMEN
Although inhibitors targeting the PD1/PD-L1 immune checkpoint are showing comparably good outcomes, a significant percentage of head and neck squamous cell carcinoma (HNSCC) patients do not respond to treatment. Apart from using different treatment strategies, another possibility would be to target other immune checkpoints operating in these non-responding tumors. To obtain an overview of which checkpoint ligands are expressed on HNSCC tumor cells and if these ligands are affected by HGF/MET signaling, we used mRNA sequencing and antibody-based techniques for identifying checkpoint ligands in six HNSCC tumor cell lines. Furthermore, we compared our results to mRNA sequencing data. From the checkpoint ligands we investigated, VISTA was expressed the highest at the RNA level and was also the most ubiquitously expressed. PD-L2 and B7-H3 were expressed comparably lower and were not present in all cell lines to the same extent. B7-H4, however, was only detectable in the Detroit 562 cell line. Concerning the effect of HGF on the ligand levels, PD-L2 expression was enhanced with HGF stimulation, whereas other checkpoint ligand levels decreased with stimulation. B7-H4 levels in the Detroit 562 cell line drastically decreased with HGF stimulation. This is of interest because both the checkpoint ligand and the growth factor are reported to be connected to epithelial-mesenchymal transition in the literature.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello , Factor de Crecimiento de Hepatocito , Proteínas de Punto de Control Inmunitario , Proteínas Proto-Oncogénicas c-met , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello , Humanos , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/genética , Línea Celular Tumoral , Proteínas de Punto de Control Inmunitario/metabolismo , Proteínas de Punto de Control Inmunitario/genética , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Antígenos B7/metabolismo , Antígenos B7/genéticaRESUMEN
Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design.
Asunto(s)
Serina Endopeptidasas , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Humanos , Diseño de Fármacos , Aminoácidos/química , Aminoácidos/metabolismo , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Animales , Factor de Crecimiento de Hepatocito/metabolismo , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/antagonistas & inhibidoresRESUMEN
Chronic inflammation plays a crucial role in coronary artery disease (CAD), but differences in specific cytokine profiles between acute coronary syndrome (ACS) and stable CAD remain unknown. We investigated cytokine differences between these two manifestations of CAD. The study included 308 patients with angiographically detected, hemodynamically significant CAD: 150 patients undergone angiography for ACS, 158 patients undergone angiography for stable CAD. To assess dynamic changes, 116 patients had index angiogram at least 3 months earlier. We measured the serum concentrations of 48 circulating cytokines. The ACS group had decreased interleukin (IL) 4 (p = 0.005), and increased IL-8 (p = 0.008), hepatocyte growth factor (HGF) (p < 0.001) and macrophage colony-stimulating factor (M-CSF) (p = 0.002) levels compared with the stable CAD group. Multivariable logistic regression revealed increased levels of HGF (OR 18.050 [95% CI 4.372-74.517], p < 0.001), M-CSF (OR 2.257 [1.375-3.705], p = 0.001) and IL-6 (OR 1.586 [1.131-2.224], p = 0.007), independently associated with ACS. In the post-angiography group, only diminished platelet-derived growth factor-BB levels in ACS-manifested patients were observed (OR 0.478, [0.279-0.818], p = 0.007). Cytokine profiles differ between ACS and stable CAD. Such differences seem to be mainly reversible within 3 months after ACS. Thus, targeting one or two cytokines only might not offer one-size fits all-therapeutic approach for CAD-associated inflammation.Trial registration: NCT03444259.
Asunto(s)
Síndrome Coronario Agudo , Enfermedad de la Arteria Coronaria , Citocinas , Humanos , Masculino , Femenino , Síndrome Coronario Agudo/sangre , Enfermedad de la Arteria Coronaria/sangre , Citocinas/sangre , Persona de Mediana Edad , Anciano , Angiografía Coronaria , Biomarcadores/sangre , Factor de Crecimiento de Hepatocito/sangreRESUMEN
PURPOSE: This study aimed to investigate the impact of hepatocyte growth factor (HGF) on colonic morphology and gut microbiota in a rat model of short bowel syndrome (SBS). METHODS: SD rats underwent jugular vein catheterization for total parenteral nutrition (TPN) and 90% small bowel resection [TPN + SBS (control group) or TPN + SBS + intravenous HGF (0.3 mg/kg/day, HGF group)]. Rats were harvested on day 7. Colonic morphology, gut microflora, tight junction, and Toll-like receptor-4 (TLR4) were evaluated. RESULTS: No significant differences were observed in the colonic morphological assessment. No significant differences were observed in the expression of tight junction-related genes in the proximal colon. However, the claudin-1 expression tended to increase and the claudin-3 expression tended to decrease in the distal colon of the HGF group. The Verrucomicrobiota in the gut microflora of the colon tended to increase in the HGF group. The abundance of most LPS-producing microbiota was lower in the HGF group than in the control group. The gene expression of TLR4 was significantly downregulated in the distal colon of the HGF group. CONCLUSION: HGF may enhance the mucus barrier through the tight junctions or gut microbiome in the distal colon.