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Small animals are widely used as disease models in medical research. Noninvasive imaging modalities with functional capability play an important role in studying the disease state and treatment progress. Photoacoustics, being a noninvasive and functional modality, has the potential for small-animal imaging. However, the conventional photoacoustic tomographic systems use pulsed lasers, making it expensive, bulky, and require long acquisition time. In this work, we propose the use of photoacoustic and ultrasound tomographic imaging with LEDs as the light source and acoustic detection using a linear transducer array. We have demonstrated full-view tomographic imaging of a euthanized mouse and a potential application in liver fibrosis research.
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Técnicas Fotoacústicas , Ultrasonografía , Animales , Teorema de Bayes , Cirrosis Hepática/diagnóstico por imagen , Masculino , Ratones , Técnicas Fotoacústicas/instrumentación , Ultrasonografía/instrumentaciónRESUMEN
Hepatic cirrhosis is a growing health problem with increasing mortality worldwide. So far, there is a lack of early diagnosis and no clinical therapy is approved for the treatment. In this study, we developed a novel theranostic nanomedicine by targeting relaxin (RLX) that is known to possess potent anti-fibrotic properties but simultaneously has poor pharmacokinetics and detrimental off-target effects. We conjugated RLX to PEGylated superparamagnetic iron-oxide nanoparticles (RLX-SPIONs) and examined hepatic stellate cells (HSCs) specific binding/uptake. Thereafter, we assessed the therapeutic efficacy of RLX-SPIONs on human HSCs in vitro and in vivo in CCl4-induced liver cirrhosis mouse model. RLX-SPIONs showed specific binding and uptake in TGFß-activated HSCs, and inhibited TGFß-induced HSCs differentiation, migration and contraction. In vivo, RLX-SPIONs strongly attenuated cirrhosis and showed enhanced contrast in MR imaging. Altogether, this study presents RLX-SPIONs as a novel theranostic nanomedicine that provides new opportunities for the diagnosis and treatment of liver cirrhosis.
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Cirrosis Hepática/diagnóstico por imagen , Cirrosis Hepática/tratamiento farmacológico , Nanopartículas de Magnetita/uso terapéutico , Relaxina/uso terapéutico , Animales , Línea Celular , Modelos Animales de Enfermedad , Humanos , Imagen por Resonancia Magnética , Nanopartículas de Magnetita/química , Masculino , Ratones Endogámicos BALB C , Relaxina/análogos & derivados , Relaxina/farmacocinética , Nanomedicina TeranósticaRESUMEN
Growing evidence suggests that metabolic dysfunction-associated steatotic liver disease (MASLD) is significantly higher in men versus women. Increased prevalence is observed in postmenopausal women, suggesting that age and sex (hormones) influence MASLD development and progression. Molecular data further reveal that sex regulates the innate immune responses with an essential role in MASLD progression. To date, there has been limited focus on the role of innate immune sexual dimorphism in MASLD, and differences between men and women are not considered in the current drug discovery landscape. In this review, we summarize the sex disparities and innate immune sexual dimorphism in MASLD pathogenesis. We further highlight the importance of harnessing sexual dimorphism in identifying therapeutic targets, developing pharmacological therapies, and designing (pre-) clinical studies for the personalized treatment for MASLD.
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Inmunidad Innata , Caracteres Sexuales , Humanos , Animales , Femenino , Masculino , Hígado Graso/inmunología , Enfermedades Metabólicas/inmunología , Enfermedades Metabólicas/tratamiento farmacológicoRESUMEN
In prostate cancer (PCa), cancer-associated fibroblasts (CAFs) promote tumor progression, drug resistance, and metastasis. Although circulating tumor cells are studied as prognostic and diagnostic markers, little is known about other circulating cells and their association with PCa metastasis. Here, we explored the presence of circulating CAFs (cCAFs) in metastatic castration-naïve prostate cancer (mCNPC) patients. cCAFs were stained with fibroblast activation protein (FAP), epithelial cell adhesion molecule (EpCAM), and receptor-type tyrosine-protein phosphatase C (CD45), then FAP+EpCAM- cCAFs were enumerated and sorted using fluorescence-activated cell sorting. FAP+EpCAM- cCAFs ranged from 60 to 776 (389 mean ± 229 SD) per 2 × 108 mononuclear cells, whereas, in healthy donors, FAP+ EpCAM- cCAFs ranged from 0 to 71 (28 mean ± 22 SD). The mCNPC-derived cCAFs showed positivity for vimentin and intracellular collagen-I. They were viable and functional after sorting, as confirmed by single-cell collagen-I secretion after 48 h of culturing. Two cCAF subpopulations, FAP+CD45- and FAP+CD45+, were identified, both expressing collagen-I and vimentin, but with distinctly different morphologies. Collectively, this study demonstrates the presence of functional and viable circulating CAFs in mCNPC patients, suggesting the role of these cells in prostate cancer.
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Endothelin-1/endothelin A receptor (ET-1/ETAR) pathway plays an important role in the progression of liver fibrosis by activating hepatic stellate cells (HSCs) - a key cell type involved in the pathogenesis of liver fibrosis. Inactivating HSCs by blocking the ET-1/ETAR pathway using a selective ETAR antagonist (ERA) represents a promising therapeutic approach for liver fibrosis. Unfortunately, small-molecule ERAs possess limited clinical potential due to poor bioavailability, short half-life, and rapid renal clearance. To improve the clinical applicability, we conjugated ERA to superparamagnetic iron-oxide nanoparticles (SPIONs) and investigated the therapeutic efficacy of ERA and ERA-SPIONs in vitro and in vivo and analyzed liver uptake by in vivo and ex vivo magnetic resonance imaging (MRI), HSCs-specific localization, and ET-1/ETAR-pathway antagonism in vivo. In murine and human liver fibrosis/cirrhosis, we observed overexpression of ET-1 and ETAR that correlated with HSC activation, and HSC-specific localization of ETAR. ERA and successfully synthesized ERA-SPIONs demonstrated significant attenuation in TGFß-induced HSC activation, ECM production, migration, and contractility. In an acute CCl4-induced liver fibrosis mouse model, ERA-SPIONs exhibited higher liver uptake, HSC-specific localization, and ET-1/ETAR pathway antagonism. This resulted in significantly reduced liver-to-body weight ratio, plasma ALT levels, and α-SMA and collagen-I expression, indicating attenuation of liver fibrosis. In conclusion, our study demonstrates that the delivery of ERA using SPIONs enhances the therapeutic efficacy of ERA in vivo. This approach holds promise as a theranostic strategy for the MRI-based diagnosis and treatment of liver fibrosis.
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Activated hepatic stellate cells (HSCs) that secrete large amounts of extracellular matrix (ECM) proteins, primarily collagens, are recognized as the key pathogenic cells in liver diseases. Excessive ECM accumulation results in tissue scarring, referred to as liver fibrosis, that progresses to liver cirrhosis (liver dysfunction) and hepatocellular carcinoma. Recent studies using single cell RNA sequencing have discovered various subpopulations of HSCs with high degree of heterogeneity in quiescent, activated, as well as inactive (identified during disease regression) HSCs. However, little is known about the role of these subpopulations in ECM secretion and cell-cell communication or if they respond differently to different exogenous and endogenous factors. Moreover, how the heterogenous single cell transcriptome translates into the single cell secretome and "communicatome" (cell-cell communication) remains largely underexplored. In this chapter, we describe the method (modified enzyme-linked immunosorbent spot, ELISpot) for analyzing collagen type 1 secretion of HSCs at the single cell level, enabling a deeper understanding into the HSC secretome. In the near future, we aim to develop an integrated platform with which we can study secretome of individual cells identified by immunostaining-based fluorescence-activated cell sorting derived from healthy and diseased liver. Through the use of the VyCAP 6400-microwell chip in combination with their puncher device, we aim to perform single cell phenomics by analyzing and correlating phenotype, secretome, transcriptome, and genome of the single cells.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Células Estrelladas Hepáticas/metabolismo , Secretoma , Fenómica , Cirrosis Hepática/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de la Matriz Extracelular/metabolismo , Neoplasias Hepáticas/patologíaRESUMEN
Chronic liver diseases (CLDs) caused by viral infections, alcohol/drug abuse, or metabolic disorders affect millions of people globally and have increased mortality owing to the lack of approved therapies. Lipoxygenases (LOXs) are a family of multifaceted enzymes that are responsible for the oxidation of polyunsaturated fatty acids (PUFAs) and are implicated in the pathogenesis of multiple disorders including liver diseases. This review describes the three main LOX signaling pathways - 5-, 12-, and 15-LOX - and their involvement in CLDs. We also provide recent insights and future perspectives on LOX-related hepatic pathophysiology, and discuss the potential of LOXs and LOX-derived metabolites as diagnostic biomarkers and therapeutic targets in CLDs.
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Hepatopatías , Enfermedades Metabólicas , Ácidos Grasos Insaturados , Humanos , Lipooxigenasas/metabolismo , Oxidación-ReducciónRESUMEN
The lysophosphatidic acid (LPA) signaling axis is an important but rather underexplored pathway in liver disease. LPA is predominantly produced by Autotaxin (ATX) that has gained significant attention with an impressive number of ATX inhibitors (type I-IV) reported. Here, we evaluated the therapeutic potential of a (yet unexplored) type IV inhibitor, Cpd17, in liver injury. We first confirmed the involvement of the ATX-LPA signaling axis in human and murine diseased livers. Then, we evaluated the effects of Cpd17, in comparison with the classic type I inhibitor PF8380, in vitro, where Cpd17 showed higher efficacy. Thereafter, we characterized the mechanism-of-action of both inhibitors and found that Cpd17 was more potent in inhibiting RhoA-mediated cytoskeletal remodeling, and phosphorylation of MAPK/ERK and AKT/PKB. Finally, the therapeutic potential of Cpd17 was investigated in CCl4 -induced acute liver injury and diet-induced nonalcoholic steatohepatitis, demonstrating an excellent potential of Cpd17 in reducing liver injury in both disease models in vivo. We conclude that ATX inhibition, by type IV inhibitor in particular, has an excellent potential for clinical application in liver diseases.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Lisofosfolípidos , Ratones , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
Liver fibrosis affects millions of people worldwide and is rising vastly over the past decades. With no viable therapies available, liver transplantation is the only curative treatment for advanced diseased patients. Excessive accumulation of aberrant extracellular matrix (ECM) proteins, mostly collagens, produced by activated hepatic stellate cells (HSCs), is a hallmark of liver fibrosis. Several studies have suggested an inverse correlation between collagen-I degrading matrix metalloproteinase-1 (MMP-1) serum levels and liver fibrosis progression highlighting reduced MMP-1 levels are associated with poor disease prognosis in patients with liver fibrosis. We hypothesized that delivery of MMP-1 might potentiate collagen degradation and attenuate fibrosis development. In this study, we report a novel approach for the delivery of MMP-1 using MMP-1 decorated polymersomes (MMPsomes), as a surface-active vesicle-based ECM therapeutic, for the treatment of liver fibrosis. The storage-stable and enzymatically active MMPsomes were fabricated by a post-loading of Psomes with MMP-1. MMPsomes were extensively characterized for the physicochemical properties, MMP-1 surface localization, stability, enzymatic activity, and biological effects. Dose-dependent effects of MMP-1, and effects of MMPsomes versus MMP-1, empty polymersomes (Psomes) and MMP-1 + Psomes on gene and protein expression of collagen-I, MMP-1/TIMP-1 ratio, migration and cell viability were examined in TGFß-activated human HSCs. Finally, the therapeutic effects of MMPsomes, compared to MMP-1, were evaluated in vivo in carbon-tetrachloride (CCl4)-induced early liver fibrosis mouse model. MMPsomes exhibited favorable physicochemical properties, MMP-1 surface localization and improved therapeutic efficacy in TGFß-activated human HSCs in vitro. In CCl4-induced early liver fibrosis mouse model, MMPsomes inhibited intra-hepatic collagen-I (ECM marker, indicating early liver fibrosis) and F4/80 (marker for macrophages, indicating liver inflammation) expression. In conclusion, our results demonstrate an innovative approach of MMP-1 delivery, using surface-decorated MMPsomes, for alleviating liver fibrosis.
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Metaloproteinasa 1 de la Matriz , Metaloproteinasa 2 de la Matriz , Colágeno , Matriz Extracelular , Células Estrelladas Hepáticas , Humanos , Hígado/patología , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/patología , Metaloproteinasa 13 de la MatrizRESUMEN
Liver diseases are the growing health problem with no clinically approved therapy available. Activated hepatic stellate cells (HSCs) are the key driver cells responsible for extracellular matrix deposition, the hallmark of liver fibrosis. Fibroblast growth factor 2 (FGF2) has shown to possess anti-fibrotic effects in fibrotic diseases including liver fibosis, and promote tissue regeneration. Among the fibroblast growth factor receptors (FGFRs), FGF2 interact primarily with FGFR1, highly overexpressed on activated HSCs, and inhibit HSCs activation. However, FGF2 poses several limitations including poor systemic half-life and stability owing to enzymatic degradation. The aim of this study is to improve the stability and half-life of FGF2 thereby improving the therapuetic efficacy of FGF2 for the treatment of liver fibrosis. We found that FGFR1-3 mRNA levels were overexpressed in cirrhotic human livers, while FGFR1c, 2c, 3c, 4 and FGF2 mRNA levels were overexpressed in TGFß-activated HSCs (LX2 cells) and FGFR1 protein expression was highly increased in TGFß-activated HSCs. Treatment with FGF2 inhibited TGFß-induced HSCs activation, migration and contraction in vitro. FGF2 was conjugated to superparamagnetic iron-oxide nanoparticles (SPIONs) using carbodiimide chemistry, and the resulting FGF2-SPIONs were confirmed by dynamic light scattering (DLS), zeta potential, dot-blot analysis and Prussian Blue iron-staining. In vitro, treatment with FGF2-SPIONs evidenced increased therapeutic effects (attenuated TGFß-induced HSCs activation, migration and contraction) of FGF2 in TGFß-activated HSCs and ameliorated early liver fibrogenesis in vivo in acute carbon tetrachloride (CCl4)-induced liver injury mouse model. In contrast, free FGF2 showed no significant effects in vivo. Altogether, this study presents a promising therapeutic approach using FGF2-SPIONs for the treatment of liver fibrosis.