ABSTRACT
Compromised lysosome function is implicated in the pathology of many neurodegenerative diseases, including Alzheimer's disease (AD). Familial Alzheimer's disease (fAD) is caused primarily by mutations in the presenilin encoding genes, but the underlying mechanism remains obscure. Loss of the conserved C. elegans presenilin orthologue SEL-12 results in increased mitochondrial calcium, which promotes neurodegeneration. Here, we find that sel-12 mutant lysosomes, independent of SEL-12 proteolytic activity, are significantly enlarged and more alkaline due to increased ER-to-mitochondrial calcium signaling and concomitant mitochondrial oxidative stress. These defects and their dependence on mitochondrial calcium are recapitulated in human fAD fibroblasts, demonstrating a conserved role for mitochondrial calcium in presenilin-mediated lysosome dysfunction. sel-12 mutants also have increased contact surface area between the ER, mitochondria, and lysosomes, suggesting sel-12 has an additional role in modulating organelle contact and communication. Overall, we demonstrate that SEL-12 maintains lysosome acidity and lysosome health by controlling ER-to-mitochondrial calcium signaling.
ABSTRACT
Objective. Photon-counting micro-computed tomography (micro-CT) is a major advance in small animal preclinical imaging. Small molecule- and nanoparticle-based contrast agents have been widely used to enable the differentiation of liver tumors from surrounding tissues using photon-counting micro-CT. However, there is a notable gap in the application of these market-available agents to the imaging of breast and ovarian tumors using photon-counting micro-CT. Herein, we have used photon-counting micro-CT to determine the effectiveness of these contrast agents in differentiating ovarian and breast tumor xenografts in live, intact mice.Approach. Nude mice carrying different types of breast and ovarian tumor xenografts (AU565, MDA-MB-231 and SKOV-3 human cancer cells) were injected with ISOVUE-370 (a small molecule-based agent) or Exitron Nano 12000 (a nanoparticle-based agent) and subjected to photon-counting micro-CT. To improve tumor visualization using photon-counting micro-CT, we developed a novel color visualization method, which changes color tones to highlight contrast media distribution, offering a robust alternative to traditional material decomposition methods with less computational demand.Main results. Ourin vivoexperiments confirm the effectiveness of this color visualization approach, showing distinct enhancement characteristics for each contrast agent. Qualitative and quantitative analyses suggest that Exitron Nano 12000 provides superior vasculature enhancement and better quantitative consistency across scans, while ISOVUE-370 delivers a more comprehensive tumor enhancement but with significant variance between scans due to its short blood half-time. Further, a paired t-test on mean and standard deviation values within tumor volumes showed significant differences between the AU565 and SKOV-3 tumor models with the nanoparticle-based contrast agent (p-values < 0.02), attributable to their distinct vascularity, as confirmed by immunohistochemical analysis.Significance. These findings underscore the utility of photon-counting micro-CT in non-invasively assessing the morphology and anatomy of different tumor xenografts, which is crucial for tumor characterization and longitudinal monitoring of tumor progression and response to treatments.
Subject(s)
Contrast Media , Photons , X-Ray Microtomography , Animals , Mice , Humans , X-Ray Microtomography/instrumentation , Cell Line, Tumor , Female , Breast Neoplasms/diagnostic imaging , Mice, Nude , Ovarian Neoplasms/diagnostic imaging , Ovarian Neoplasms/pathology , NanoparticlesABSTRACT
Photon-counting micro computed tomography (micro-CT) offers new potential in preclinical imaging, particularly in distinguishing materials. It becomes especially helpful when combined with contrast agents, enabling the differentiation of tumors from surrounding tissues. There are mainly two types of contrast agents in the market for micro-CT: small molecule-based and nanoparticle-based. However, despite their widespread use in liver tumor studies, there is a notable gap in research on the application of these commercially available agents for photon-counting micro-CT in breast and ovarian tumors. Herein, we explored the effectiveness of these agents in differentiating tumor xenografts from various origins (AU565, MDA-MB-231, and SKOV-3) in nude mice, using photon-counting micro-CT. Specifically, ISOVUE-370 (a small molecule-based agent) and Exitrone Nano 12000 (a nanoparticle-based agent) were investigated in this context. To improve tumor visualization, we proposed a novel color visualization method for photon-counting micro-CT, which changes color tones to highlight contrast media distribution, offering a robust alternative to traditional material decomposition methods with less computational demand. Our in vivo experiments confirm its effectiveness, showing distinct enhancement characteristics for each contrast agent. Qualitative and quantitative analyses suggested that Exitrone Nano 12000 provides superior vasculature enhancement and better quantitative consistency across scans, while ISOVUE-370 gives more comprehensive tumor enhancement but with a significant variance between scans due to its short blood half-time. This variability leads to high sensitivity to timing and individual differences among mice. Further, a paired t-test on mean and standard deviation values within tumor volumes showed significant differences between the AU565 and SKOV-3 tumor models with the nanoparticle-based (p-values < 0.02), attributable to their distinct vascularity, as confirmed by immunohistochemistry. These findings underscore the utility of photon-counting micro-CT in non-invasively assessing the morphology and anatomy of different tumor xenografts, which is crucial for tumor characterization and longitudinal monitoring of tumor development and response to treatments.
ABSTRACT
Rationale: Trastuzumab (TZM) is a monoclonal antibody that targets the human epidermal growth factor receptor (HER2) and is clinically used for the treatment of HER2-positive breast tumors. However, the tumor microenvironment can limit the access of TZM to the HER2 targets across the whole tumor and thereby compromise TZM's therapeutic efficacy. An imaging methodology that can non-invasively quantify the binding of TZM-HER2, which is required for therapeutic action, and distribution within tumors with varying tumor microenvironments is much needed. Methods: We performed near-infrared (NIR) fluorescence lifetime (FLI) Forster Resonance Energy Transfer (FRET) to measure TZM-HER2 binding, using in vitro microscopy and in vivo widefield macroscopy, in HER2 overexpressing breast and ovarian cancer cells and tumor xenografts, respectively. Immunohistochemistry was used to validate in vivo imaging results. Results: NIR FLI FRET in vitro microscopy data show variations in intracellular distribution of bound TZM in HER2-positive breast AU565 and AU565 tumor-passaged XTM cell lines in comparison to SKOV-3 ovarian cancer cells. Macroscopy FLI (MFLI) FRET in vivo imaging data show that SKOV-3 tumors display reduced TZM binding compared to AU565 and XTM tumors, as validated by ex vivo immunohistochemistry. Moreover, AU565/XTM and SKOV-3 tumor xenografts display different amounts and distributions of TME components, such as collagen and vascularity. Therefore, these results suggest that SKOV-3 tumors are refractory to TZM delivery due to their disrupted vasculature and increased collagen content. Conclusion: Our study demonstrates that FLI is a powerful analytical tool to monitor the delivery of antibody drug tumor both in cell cultures and in vivo live systems. Especially, MFLI FRET is a unique imaging modality that can directly quantify target engagement with potential to elucidate the role of the TME in drug delivery efficacy in intact live tumor xenografts.
ABSTRACT
Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.
Subject(s)
Breast Neoplasms , Iron , Animals , Female , Humans , Mice , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Endosomes/metabolism , Iron/metabolism , Mitochondria/metabolism , MitophagyABSTRACT
Organoid models have been used to address important questions in developmental and cancer biology, tissue repair, advanced modelling of disease and therapies, among other bioengineering applications. Such 3D microenvironmental models can investigate the regulation of cell metabolism, and provide key insights into the mechanisms at the basis of cell growth, differentiation, communication, interactions with the environment and cell death. Their accessibility and complexity, based on 3D spatial and temporal heterogeneity, make organoids suitable for the application of novel, dynamic imaging microscopy methods, such as fluorescence lifetime imaging microscopy (FLIM) and related decay time-assessing readouts. Several biomarkers and assays have been proposed to study cell metabolism by FLIM in various organoid models. Herein, we present an expert-opinion discussion on the principles of FLIM and PLIM, instrumentation and data collection and analysis protocols, and general and emerging biosensor-based approaches, to highlight the pioneering work being performed in this field.
Subject(s)
Microscopy , Organoids , Humans , Drug DiscoveryABSTRACT
Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.
Subject(s)
Globins , HMGB2 Protein , Animals , Mice , Cytoglobin/genetics , DNA Damage , Globins/genetics , Globins/metabolism , HMGB2 Protein/genetics , HMGB2 Protein/metabolism , Transcription Factors/geneticsABSTRACT
Genomics and proteomics have been central to identify tumor cell populations, but more accurate approaches to classify cell subtypes are still lacking. We propose a new methodology to accurately classify cancer cells based on their organelle spatial topology. Herein, we developed an organelle topology-based cell classification pipeline (OTCCP), which integrates artificial intelligence (AI) and imaging quantification to analyze organelle spatial distribution and inter-organelle topology. OTCCP was used to classify a panel of human breast cancer cells, grown as 2D monolayer or 3D tumor spheroids using early endosomes, mitochondria, and their inter-organelle contacts. Organelle topology allows for a highly precise differentiation between cell lines of different subtypes and aggressiveness. These findings lay the groundwork for using organelle topological profiling as a fast and efficient method for phenotyping breast cancer function as well as a discovery tool to advance our understanding of cancer cell biology at the subcellular level.
ABSTRACT
Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.
ABSTRACT
Förster resonance energy transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2-10 nm range. FRET is currently being extended to in vivo optical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the three-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated-intensified charge-coupled device, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fDE of the FRET efficiency E and the fraction of donor molecules involved in FRET, fD, are described in detail for both methodologies. Dynamic in vivo FRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of a near-infrared-labeled transferrin FRET pair and benchmarked against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI-FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required nine measurements (six of which are used for calibration) acquired from three mice, MFLI-FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice.
ABSTRACT
BACKGROUND: Activation of vascular smooth muscle cell (VSMC) inflammation is vital to initiate vascular disease. The role of human-specific long noncoding RNAs in VSMC inflammation is poorly understood. METHODS: Bulk RNA sequencing in differentiated human VSMCs revealed a novel human-specific long noncoding RNA called inflammatory MKL1 (megakaryoblastic leukemia 1) interacting long noncoding RNA (INKILN). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation as well as human atherosclerosis and abdominal aortic aneurysm. The transcriptional regulation of INKILN was verified through luciferase reporter and chromatin immunoprecipitation assays. Loss-of-function and gain-of-function studies and multiple RNA-protein and protein-protein interaction assays were used to uncover a mechanistic role of INKILN in the VSMC proinflammatory gene program. Bacterial artificial chromosome transgenic mice were used to study INKILN expression and function in ligation injury-induced neointimal formation. RESULTS: INKILN expression is downregulated in contractile VSMCs and induced in human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB (nuclear factor kappa B) site within its proximal promoter. INKILN activates proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks interleukin-1ß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1 and the luciferase activity of an NF-κB reporter. Furthermore, INKILN knockdown enhances MKL1 ubiquitination through reduced physical interaction with the deubiquitinating enzyme USP10 (ubiquitin-specific peptidase 10). INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in bacterial artificial chromosome transgenic mice. CONCLUSIONS: These findings elucidate an important pathway of VSMC inflammation involving an INKILN/MKL1/USP10 regulatory axis. Human bacterial artificial chromosome transgenic mice offer a novel and physiologically relevant approach for investigating human-specific long noncoding RNAs under vascular disease conditions.
Subject(s)
Aortic Aneurysm, Abdominal , RNA, Long Noncoding , Animals , Humans , Mice , Aortic Aneurysm, Abdominal/metabolism , Cell Proliferation , Cells, Cultured , Inflammation/genetics , Inflammation/metabolism , Luciferases/metabolism , Mice, Transgenic , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , NF-kappa B/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Ubiquitin Thiolesterase/metabolismABSTRACT
Through digital imaging, microscopy has evolved from primarily being a means for visual observation of life at the micro- and nano-scale, to a quantitative tool with ever-increasing resolution and throughput. Artificial intelligence, deep neural networks, and machine learning are all niche terms describing computational methods that have gained a pivotal role in microscopy-based research over the past decade. This Roadmap is written collectively by prominent researchers and encompasses selected aspects of how machine learning is applied to microscopy image data, with the aim of gaining scientific knowledge by improved image quality, automated detection, segmentation, classification and tracking of objects, and efficient merging of information from multiple imaging modalities. We aim to give the reader an overview of the key developments and an understanding of possibilities and limitations of machine learning for microscopy. It will be of interest to a wide cross-disciplinary audience in the physical sciences and life sciences.
ABSTRACT
Förster Resonance Energy Transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2-10 nm range. FRET is currently being extended to in vivo optical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the 3-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated ICCD, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product f D E of the FRET efficiency E and the fraction of donor molecules involved in FRET, f D , are described in detail for both methodologies. Dynamic in vivo FRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of near infrared-labeled transferrin FRET pair and benchmarked against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required 9 measurements (6 of which are used for calibration) acquired from three mice, MFLI FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice.
ABSTRACT
Background: Activation of vascular smooth muscle cells (VSMCs) inflammation is vital to initiate vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is poorly understood. Methods: Bulk RNA-seq in differentiated human VSMCs revealed a novel human-specific lncRNA called IN flammatory M K L1 I nteracting L ong N oncoding RNA ( INKILN ). INKILN expression was assessed in multiple in vitro and ex vivo models of VSMC phenotypic modulation and human atherosclerosis and abdominal aortic aneurysm (AAA) samples. The transcriptional regulation of INKILN was determined through luciferase reporter system and chromatin immunoprecipitation assay. Both loss- and gain-of-function approaches and multiple RNA-protein and protein-protein interaction assays were utilized to uncover the role of INKILN in VSMC proinflammatory gene program and underlying mechanisms. Bacterial Artificial Chromosome (BAC) transgenic (Tg) mice were utilized to study INKLIN expression and function in ligation injury-induced neointimal formation. Results: INKILN expression is downregulated in contractile VSMCs and induced by human atherosclerosis and abdominal aortic aneurysm. INKILN is transcriptionally activated by the p65 pathway, partially through a predicted NF-κB site within its proximal promoter. INKILN activates the proinflammatory gene expression in cultured human VSMCs and ex vivo cultured vessels. Mechanistically, INKILN physically interacts with and stabilizes MKL1, a key activator of VSMC inflammation through the p65/NF-κB pathway. INKILN depletion blocks ILIß-induced nuclear localization of both p65 and MKL1. Knockdown of INKILN abolishes the physical interaction between p65 and MKL1, and the luciferase activity of an NF-κB reporter. Further, INKILN knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme, USP10. INKILN is induced in injured carotid arteries and exacerbates ligation injury-induced neointimal formation in BAC Tg mice. Conclusions: These findings elucidate an important pathway of VSMC inflammation involving an INKILN /MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.
ABSTRACT
3D patient tumor avatars (3D-PTAs) hold promise for next-generation precision medicine. Here, we describe the benefits and challenges of 3D-PTA technologies and necessary future steps to realize their potential for clinical decision making. 3D-PTAs require standardization criteria and prospective trials to establish clinical benefits. Innovative trial designs that combine omics and 3D-PTA readouts may lead to more accurate clinical predictors, and an integrated platform that combines diagnostic and therapeutic development will accelerate new treatments for patients with refractory disease.
Subject(s)
Neoplasms , Humans , Neoplasms/genetics , Neoplasms/therapy , Neoplasms/diagnosis , Precision Medicine , Prospective Studies , Medical OncologyABSTRACT
Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.
ABSTRACT
Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 µm zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 µm of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.
Subject(s)
Breast Neoplasms , Spheroids, Cellular , Breast Neoplasms/drug therapy , Cell Count , Cell Line, Tumor , Drug Discovery , Female , HumansABSTRACT
Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.
Subject(s)
Idiopathic Pulmonary Fibrosis , Animals , Bleomycin/pharmacology , Female , Idiopathic Pulmonary Fibrosis/chemically induced , Idiopathic Pulmonary Fibrosis/genetics , Idiopathic Pulmonary Fibrosis/metabolism , Insulin-Like Growth Factor Binding Proteins , Lung/metabolism , Mice , Organogenesis , Pregnancy , Signal TransductionABSTRACT
The enveloped positive-sense RNA viruses including Zika virus (ZIKV) need host lipids to successfully replicate. The nature of the lipids and the replication step(s) where lipids are utilized often vary amongst viruses. In this study, we demonstrate that ZIKV particle envelope is significantly enriched in distinct sphingolipid species. To determine the role of sphingolipids in ZIKV replication, we leveraged a panel of sphingolipid-deficient cell lines. Notably, knockout of glucosylceramide and lactosylceramide synthase encoding genes (GCSKO; B4G5KO) resulted in a marked decrease in ZIKV titers. GCSKO or pharmacological inhibition of GCS also led to a significant decrease in ZIKV genome replication. Further analysis indicated that GCSKO reduced intracellular virus titers but had minimal impact on ZIKV binding. Restoration of B4G5 expression in B4G5KO cells or supplementing PDMP-treated cells with glucosylceramide led to a significant rescue of ZIKV replication. Altogether, our findings suggest that ZIKV needs glycosphingolipids to facilitate virus replication.
Subject(s)
Zika Virus Infection , Zika Virus , Glucosylceramides/metabolism , Glycosphingolipids/metabolism , Humans , Virus Replication/physiology , Zika Virus/physiologyABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a progressive and often lethal interstitial lung disease of unknown aetiology. IPF is characterised by myofibroblast activation, tissue stiffening, and alveolar epithelium injury. As current IPF treatments fail to halt disease progression or induce regeneration, there is a pressing need for the development of novel therapeutic targets. In this regard, tri-dimensional (3D) models have rapidly emerged as powerful platforms for disease modelling, drug screening and discovery. In this review, we will touch on how 3D in vitro models such as hydrogels, precision-cut lung slices, and, more recently, lung organoids and lung-on-chip devices have been generated and/or modified to reveal distinct cellular and molecular signalling pathways activated during fibrotic processes. Markedly, we will address how these platforms could provide a better understanding of fibrosis pathophysiology and uncover effective treatment strategies for IPF patients.