Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
1.
ACS Appl Bio Mater ; 3(12): 8757-8767, 2020 Dec 21.
Article in English | MEDLINE | ID: mdl-35019647

ABSTRACT

High-aspect-ratio and hierarchically nanostructured surfaces are common in nature. Synthetic variants are of interest for their specific chemical, mechanic, electric, photonic, or biologic properties but are cumbersome in fabrication or suffer from structural collapse. Here, we replicated and directly biofunctionalized robust, large-area, and high-aspect-ratio nanostructures by nanoimprint lithography of an off-stoichiometric thiol-ene-epoxy polymer. We structured-in a single-step process-dense arrays of pillars with a diameter as low as 100 nm and an aspect ratio of 7.2; holes with a diameter of 70 nm and an aspect ratio of >20; and complex hierarchically layered structures, all with minimal collapse and defectivity. We show that the nanopillar arrays alter mechanosensing of human hepatic cells and provide precise spatial control of cell attachment. We speculate that our results can enable the widespread use of high-aspect-ratio nanotopograhy applications in mechanics, optics, and biomedicine.

2.
Pharmacol Rev ; 74(1): 141-206, 2022 01.
Article in English | MEDLINE | ID: mdl-35017176

ABSTRACT

The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.


Subject(s)
Drug Discovery , Drug-Related Side Effects and Adverse Reactions , Drug Development , Drug Evaluation, Preclinical , Humans , Multicenter Studies as Topic
3.
Gastroenterology ; 161(6): 1982-1997.e11, 2021 12.
Article in English | MEDLINE | ID: mdl-34425095

ABSTRACT

BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.


Subject(s)
Fatty Liver/enzymology , Fumarate Hydratase/metabolism , Insulin Resistance , Liver/enzymology , Macrophages/enzymology , MicroRNAs/metabolism , NF-E2-Related Factor 2/metabolism , Obesity/enzymology , Animals , Carboxy-Lyases/genetics , Carboxy-Lyases/metabolism , Citric Acid Cycle , Disease Models, Animal , Fatty Liver/genetics , Fumarate Hydratase/genetics , Fumarates/metabolism , Humans , Hydro-Lyases/genetics , Hydro-Lyases/metabolism , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , NF-E2-Related Factor 2/genetics , Obesity/genetics , Oxidative Stress , RAW 264.7 Cells , Reactive Oxygen Species/metabolism , Signal Transduction , Succinates/metabolism
4.
Adv Sci (Weinh) ; 8(16): e2100106, 2021 08.
Article in English | MEDLINE | ID: mdl-34165908

ABSTRACT

Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold-free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi-omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R2 = 0.97). Integration of multi-omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFß signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.


Subject(s)
Adipogenesis/physiology , Adipose Tissue/pathology , Cell Culture Techniques/methods , Cells, Cultured , Humans , Signal Transduction/physiology
5.
Am J Physiol Cell Physiol ; 320(5): C822-C841, 2021 05 01.
Article in English | MEDLINE | ID: mdl-33439778

ABSTRACT

Adipocytes are specialized cells with pleiotropic roles in physiology and pathology. Several types of fat cells with distinct metabolic properties coexist in various anatomically defined fat depots in mammals. White, beige, and brown adipocytes differ in their handling of lipids and thermogenic capacity, promoting differences in size and morphology. Moreover, adipocytes release lipids and proteins with paracrine and endocrine functions. The intrinsic properties of adipocytes pose specific challenges in culture. Mature adipocytes float in suspension culture due to high triacylglycerol content and are fragile. Moreover, a fully differentiated state, notably acquirement of the unilocular lipid droplet of white adipocyte, has so far not been reached in two-dimensional culture. Cultures of mouse and human-differentiated preadipocyte cell lines and primary cells have been established to mimic white, beige, and brown adipocytes. Here, we survey various models of differentiated preadipocyte cells and primary mature adipocyte survival describing main characteristics, culture conditions, advantages, and limitations. An important development is the advent of three-dimensional culture, notably of adipose spheroids that recapitulate in vivo adipocyte function and morphology in fat depots. Challenges for the future include isolation and culture of adipose-derived stem cells from different anatomic location in animal models and humans differing in sex, age, fat mass, and pathophysiological conditions. Further understanding of fat cell physiology and dysfunction will be achieved through genetic manipulation, notably CRISPR-mediated gene editing. Capturing adipocyte heterogeneity at the single-cell level within a single fat depot will be key to understanding diversities in cardiometabolic parameters among lean and obese individuals.


Subject(s)
Adipocytes/physiology , Adipose Tissue/physiology , Adipogenesis , Adipose Tissue/cytology , Animals , Cell Communication , Cell Culture Techniques , Cell Line , Cell Survival , Humans , Phenotype , Species Specificity , Spheroids, Cellular , Tissue Culture Techniques
6.
Sci Adv ; 7(1)2021 01.
Article in English | MEDLINE | ID: mdl-33187978

ABSTRACT

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.


Subject(s)
Antiviral Agents/pharmacology , Azetidines/pharmacology , COVID-19/mortality , Enzyme Inhibitors/pharmacology , Janus Kinases/antagonists & inhibitors , Liver/virology , Purines/pharmacology , Pyrazoles/pharmacology , SARS-CoV-2/pathogenicity , Sulfonamides/pharmacology , Adult , Aged , Aged, 80 and over , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/virology , Cytokine Release Syndrome , Cytokines/metabolism , Drug Evaluation, Preclinical , Female , Gene Expression Profiling , Humans , Interferon alpha-2/metabolism , Italy , Janus Kinases/metabolism , Liver/drug effects , Male , Middle Aged , Patient Safety , Platelet Activation , Proportional Hazards Models , RNA-Seq , Spain , Virus Internalization/drug effects
7.
Sci Transl Med ; 12(532)2020 02 26.
Article in English | MEDLINE | ID: mdl-32102936

ABSTRACT

Obesity and insulin resistance are risk factors for nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease worldwide. Because no approved medication nor an accurate and noninvasive diagnosis is currently available for NAFLD, there is a clear need to better understand the link between obesity and NAFLD. Lipid accumulation during obesity is known to be associated with oxidative stress and inflammatory activation of liver macrophages (LMs). However, we show that although LMs do not become proinflammatory during obesity, they display signs of oxidative stress. In livers of both humans and mice, antioxidant nuclear factor erythroid 2-related factor 2 (NRF2) was down-regulated with obesity and insulin resistance, yielding an impaired response to lipid accumulation. At the molecular level, a microRNA-targeting NRF2 protein, miR-144, was elevated in the livers of obese insulin-resistant humans and mice, and specific silencing of miR-144 in murine and human LMs was sufficient to restore NRF2 protein expression and the antioxidant response. These results highlight the pathological role of LMs and their therapeutic potential to restore the impaired endogenous antioxidant response in obesity-associated NAFLD.


Subject(s)
Antioxidants , Insulin Resistance , Kupffer Cells , Non-alcoholic Fatty Liver Disease , Animals , Humans , Liver , Mice , MicroRNAs , NF-E2-Related Factor 2 , Obesity
8.
Front Pharmacol ; 10: 1093, 2019.
Article in English | MEDLINE | ID: mdl-31616302

ABSTRACT

Drug-induced liver injury (DILI) is a major concern for the pharmaceutical industry and constitutes one of the most important reasons for the termination of promising drug development projects. Reliable prediction of DILI liability in preclinical stages is difficult, as current experimental model systems do not accurately reflect the molecular phenotype and functionality of the human liver. As a result, multiple drugs that passed preclinical safety evaluations failed due to liver toxicity in clinical trials or postmarketing stages in recent years. To improve the selection of molecules that are taken forward into the clinics, the development of more predictive in vitro systems that enable high-throughput screening of hepatotoxic liabilities and allow for investigative studies into DILI mechanisms has gained growing interest. Specifically, it became increasingly clear that the choice of cell types and culture method both constitute important parameters that affect the predictive power of test systems. In this review, we present current 3D culture paradigms for hepatotoxicity tests and critically evaluate their utility and performance for DILI prediction. In addition, we highlight possibilities of these emerging platforms for mechanistic evaluations of selected drug candidates and present current research directions towards the further improvement of preclinical liver safety tests. We conclude that organotypic and microphysiological liver systems have provided an important step towards more reliable DILI prediction. Furthermore, we expect that the increasing availability of comprehensive benchmarking studies will facilitate model dissemination that might eventually result in their regulatory acceptance.

9.
Chem Res Toxicol ; 33(1): 38-60, 2020 01 21.
Article in English | MEDLINE | ID: mdl-31576743

ABSTRACT

Despite extensive breakthroughs in chemistry, molecular biology, and genetics in the last decades, the success rates of drug development projects remain low. To improve predictions of clinical efficacy and safety of new compounds, a plethora of 3D culture methods of human cells have been developed in which the cultured cells retain physiologically and functionally relevant phenotypes for multiple weeks. Here, we critically review current paradigms for organotypic cultures of human liver, gut, and kidney such as perfused microchips, spheroids, and hollow fiber bioreactors and discuss their utility for understanding drug pharmacokinetics, metabolism, and toxicity. Furthermore, bioprinting and the microfluidic integration of different tissue models to mimic systemic drug effects are highlighted as promising technological trends. In the last part of the review, we discuss important considerations regarding the choice of culture substratum material to limit adverse effects such as drug absorption while facilitating the phenotypic maintenance of cultured cells. We conclude that recent advances in organotypic and microphysiological culture models of human tissues can improve drug development and contribute to an amelioration of clinical attrition rates. However, further validation, benchmarking, and consolidation efforts are needed to achieve more widespread dissemination and eventually regulatory acceptance of these novel tools.


Subject(s)
Gastrointestinal Tract/metabolism , Kidney/metabolism , Liver/metabolism , Models, Biological , Pharmaceutical Preparations/metabolism , Animals , Drug Evaluation, Preclinical , Drug-Related Side Effects and Adverse Reactions , Humans , Pharmacokinetics
10.
Genesis ; 56(9): e23246, 2018 09.
Article in English | MEDLINE | ID: mdl-30114334

ABSTRACT

Development of the embryonic head is driven by the activity of gene regulatory networks of transcription factors. LHX1 is a homeobox transcription factor that plays an essential role in the formation of the embryonic head. The loss of LHX1 function results in anterior truncation of the embryo caused by the disruption of morphogenetic movement of tissue precursors and the dysregulation of WNT signaling activity. Profiling the gene expression pattern in the Lhx1 mutant embryo revealed that tissues in anterior germ layers acquire posterior tissue characteristics, suggesting LHX1 activity is required for the allocation and patterning of head precursor tissues. Here, we used LHX1 as an entry point to delineate its transcriptional targets and interactors and construct a LHX1-anchored gene regulatory network. Using a gain-of-function approach, we identified genes that immediately respond to Lhx1 activation. Meta-analysis of the datasets of LHX1-responsive genes and genes expressed in the anterior tissues of mouse embryos at head-fold stage, in conjunction with published Xenopus embryonic LHX1 (Xlim1) ChIP-seq data, has pinpointed the putative transcriptional targets of LHX1 and an array of genetic determinants functioning together in the formation of the mouse embryonic head.


Subject(s)
Gene Regulatory Networks , Genes, Homeobox , Head/embryology , LIM-Homeodomain Proteins/metabolism , Transcription Factors/metabolism , Animals , Embryonic Stem Cells/metabolism , Gene Expression Profiling , Germ Cells/physiology , Transcription, Genetic , Xenopus laevis/embryology
SELECTION OF CITATIONS
SEARCH DETAIL