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1.
J Imaging ; 8(1)2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-35049846

RESUMO

Vegetation alters soil fabric by providing biological reinforcement and enhancing the overall mechanical behaviour of slopes, thereby controlling shallow mass movement. To predict the behaviour of vegetated slopes, parameters representing the root system structure, such as root distribution, length, orientation and diameter, should be considered in slope stability models. This study quantifies the relationship between soil physical characteristics and root growth, giving special emphasis on (1) how roots influence the physical architecture of the surrounding soil structure and (2) how soil structure influences the root growth. A systematic experimental study is carried out using high-resolution X-ray micro-computed tomography (µCT) to observe the root behaviour in layered soil. In total, 2 samples are scanned over 15 days, enabling the acquisition of 10 sets of images. A machine learning algorithm for image segmentation is trained to act at 3 different training percentages, resulting in the processing of 30 sets of images, with the outcomes prompting a discussion on the size of the training data set. An automated in-house image processing algorithm is employed to quantify the void ratio and root volume ratio. This script enables post processing and image analysis of all 30 cases within few hours. This work investigates the effect of stratigraphy on root growth, along with the effect of image-segmentation parameters on soil constitutive properties.

2.
Front Immunol ; 12: 752916, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34956184

RESUMO

C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway (AP) of complement and treatment options remain inadequate. Factor H (FH) is a potent regulator of the AP. An in-depth analysis of FH-related protein dimerised minimal (mini)-FH constructs has recently been published. This analysis showed that addition of a dimerisation module to mini-FH not only increased serum half-life but also improved complement regulatory function, thus providing a potential treatment option for C3G. Herein, we describe the production of a murine version of homodimeric mini-FH [mHDM-FH (mFH1-5^18-20^R1-2)], developed to reduce the risk of anti-drug antibody formation during long-term experiments in murine models of C3G and other complement-driven pathologies. Our analysis of mHDM-FH indicates that it binds with higher affinity and avidity to WT mC3b when compared to mouse (m)FH (mHDM-FH KD=505 nM; mFH KD=1370 nM) analogous to what we observed with the respective human proteins. The improved binding avidity resulted in enhanced complement regulatory function in haemolytic assays. Extended interval dosing studies in CFH-/- mice (5mg/kg every 72hrs) were partially effective and bio-distribution analysis in CFH-/- mice, through in vivo imaging technologies, demonstrates that mHDM-FH is preferentially deposited and remains fixed in the kidneys (and liver) for up to 4 days. Extended dosing using an AAV- human HDM-FH (hHDM-FH) construct achieved complete normalisation of C3 levels in CFH-/- mice for 3 months and was associated with a significant reduction in glomerular C3 staining. Our data demonstrate the ability of gene therapy delivery of mini-FH constructs to enhance complement regulation in vivo and support the application of this approach as a novel treatment strategy in diseases such as C3G.

3.
Sci Rep ; 11(1): 1415, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446811

RESUMO

Advances in fluorescence imaging coupled with the generation of near infrared probes have significantly improved the capabilities of non-invasive, real-time imaging in whole animals. In this study we were able to overcome a limitation of in vivo fluorescence imaging and have established a dual cell tracking method where two different cell types can be monitored according to the spectral signature of the cell labelling fluorophore. Using a mouse model of acute liver injury, we have characterised the in vivo migration patterns of wild type and transgenic neutrophils with impaired chemotaxis. Here, we were able to demonstrate that IVIS provides a sensitive multiplexing technology to differentiate two different cell populations based on the spectral signature of the cell labelling fluorophores. This spectral unmixing methodology has the potential to uncover multidimensional cellular interactions involved in many diseases such as fibrosis and cancer. In vivo spectral un-mixing provides a useful tool for monitoring multiple biological process in real-time in the same animal.


Assuntos
Movimento Celular , Rastreamento de Células , Corantes Fluorescentes/química , Neutrófilos , Animais , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Neutrófilos/citologia , Neutrófilos/metabolismo
5.
Nat Metab ; 2(11): 1350-1367, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33168981

RESUMO

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-ß1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease.


Assuntos
Epitélio/patologia , Cirrose Hepática/genética , Cirrose Hepática/patologia , Macrófagos/patologia , Proteínas Proto-Oncogênicas c-rel/genética , Animais , Polaridade Celular/genética , Marcação de Genes , Hepatócitos/patologia , Hidroxiprolina/metabolismo , Cirrose Hepática/prevenção & controle , Regeneração Hepática/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose/genética , Comunicação Parácrina/genética , Fosfofrutoquinase-2/genética , Proteínas Proto-Oncogênicas c-rel/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-rel/metabolismo
6.
JCI Insight ; 5(4)2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32102985

RESUMO

Neutrophils are the most abundant inflammatory cells at the earliest stages of wound healing and play important roles in wound repair and fibrosis. Formyl peptide receptor 1 (FPR-1) is abundantly expressed on neutrophils and has been shown to regulate their function, yet the importance of FPR-1 in fibrosis remains ill defined. FPR-1-deficient (fpr1-/-) mice were protected from bleomycin-induced pulmonary fibrosis but developed renal and hepatic fibrosis normally. Mechanistically, we observed a failure to effectively recruit neutrophils to the lungs of fpr1-/- mice, whereas neutrophil recruitment was unaffected in the liver and kidney. Using an adoptive transfer model we demonstrated that the defect in neutrophil recruitment to the lung was intrinsic to the fpr1-/- neutrophils, as C57BL/6 neutrophils were recruited normally to the damaged lung in fpr1-/- mice. Finally, C57BL/6 mice in which neutrophils had been depleted were protected from pulmonary fibrosis. In conclusion, FPR-1 and FPR-1 ligands are required for effective neutrophil recruitment to the damaged lung. Failure to recruit neutrophils or depletion of neutrophils protects from pulmonary fibrosis.


Assuntos
Infiltração de Neutrófilos/fisiologia , Fibrose Pulmonar/fisiopatologia , Receptores de Formil Peptídeo/fisiologia , Animais , Bleomicina/toxicidade , Humanos , Ligantes , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Receptores de Formil Peptídeo/genética , Receptores de Formil Peptídeo/metabolismo
7.
Circ Res ; 126(2): 243-257, 2020 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-31805812

RESUMO

RATIONALE: ENG (endoglin) is a coreceptor for BMP (bone morphogenetic protein) 9/10 and is strongly expressed in endothelial cells. Mutations in ENG lead to the inherited vascular disorder hereditary hemorrhagic telangiectasia characterized by local telangiectases and larger arteriovenous malformations (AVMs); but how ENG functions to regulate the adult vasculature is not understood. OBJECTIVE: The goal of the work was to determine how ENG maintains vessel caliber in adult life to prevent AVM formation and thereby protect heart function. METHODS AND RESULTS: Genetic depletion of endothelial Eng in adult mice led to a significant reduction in mean aortic blood pressure. There was no evidence of hemorrhage, anemia, or AVMs in major organs to explain the reduced aortic pressure. However, large AVMs developed in the peripheral vasculature intimately associated with the pelvic cartilaginous symphysis-a noncapsulated cartilage with a naturally high endogenous expression of VEGF (vascular endothelial growth factor). The increased blood flow through these peripheral AVMs explained the drop in aortic blood pressure and led to increased cardiac preload, and high stroke volumes, ultimately resulting in high-output heart failure. Development of pelvic AVMs in this region of high VEGF expression occurred because loss of ENG in endothelial cells leads to increased sensitivity to VEGF and a hyperproliferative response. Development of AVMs and associated progression to high-output heart failure in the absence of endothelial ENG was attenuated by targeting VEGF signaling with an anti-VEGFR2 (VEGF receptor 2) antibody. CONCLUSIONS: ENG promotes the normal balance of VEGF signaling in quiescent endothelial cells to maintain vessel caliber-an essential function in conditions of increased VEGF expression such as local hypoxia or inflammation. In the absence of endothelial ENG, increased sensitivity to VEGF drives abnormal endothelial proliferation in local regions of high VEGF expression, leading to AVM formation and a rapid injurious impact on heart function.


Assuntos
Malformações Arteriovenosas/metabolismo , Endoglina/genética , Endotélio Vascular/metabolismo , Insuficiência Cardíaca/etiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Malformações Arteriovenosas/complicações , Malformações Arteriovenosas/genética , Malformações Arteriovenosas/patologia , Pressão Sanguínea , Proliferação de Células , Endoglina/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/fisiologia , Endotélio Vascular/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
8.
Gastroenterology ; 157(5): 1398-1412.e9, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31352003

RESUMO

BACKGROUND & AIMS: Methyl-CpG binding protein 2, MECP2, which binds to methylated regions of DNA to regulate transcription, is expressed by hepatic stellate cells (HSCs) and is required for development of liver fibrosis in mice. We investigated the effects of MECP2 deletion from HSCs on their transcriptome and of phosphorylation of MECP2 on HSC phenotype and liver fibrosis. METHODS: We isolated HSCs from Mecp2-/y mice and wild-type (control) mice. HSCs were activated in culture and used in array analyses of messenger RNAs and long noncoding RNAs. Kyoto Encyclopedia of Genes and Genomes pathway analyses identified pathways regulated by MECP2. We studied mice that expressed a mutated form of Mecp2 that encodes the S80A substitution, MECP2S80, causing loss of MECP2 phosphorylation at serine 80. Liver fibrosis was induced in these mice by administration of carbon tetrachloride, and liver tissues and HSCs were collected and analyzed. RESULTS: MECP2 deletion altered expression of 284 messenger RNAs and 244 long noncoding RNAs, including those that regulate DNA replication; are members of the minichromosome maintenance protein complex family; or encode CDC7, HAS2, DNA2 (a DNA helicase), or RPA2 (a protein that binds single-stranded DNA). We found that MECP2 regulates the DNA repair Fanconi anemia pathway in HSCs. Phosphorylation of MECP2S80 and its putative kinase, HAS2, were induced during transdifferentiation of HSCs. HSCs from MECP2S80 mice had reduced proliferation, and livers from these mice had reduced fibrosis after carbon tetrachloride administration. CONCLUSIONS: In studies of mice with disruption of Mecp2 or that expressed a form of MECP2 that is not phosphorylated at S80, we found phosphorylation of MECP2 to be required for HSC proliferation and induction of fibrosis. In HSCs, MECP2 regulates expression of genes required for DNA replication and repair. Strategies to inhibit MECP2 phosphorylation at S80 might be developed for treatment of liver fibrosis.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , Acetaminofen , Animais , Tetracloreto de Carbono , Proliferação de Células , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Colágeno/metabolismo , Reparo do DNA , Replicação do DNA , Células Estreladas do Fígado/patologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Masculino , Proteína 2 de Ligação a Metil-CpG/deficiência , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Serina , Transdução de Sinais
9.
ChemMedChem ; 14(10): 995-999, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30920173

RESUMO

Delving deeper is possible in whole-body in vivo imaging using a super-bright membrane-targeting BODIPY dye (BD). The dye was used to monitor homing of ex vivo fluorescently labelled neutrophils to an injured liver of dark-pigmented C57BL/6 mice. In vivo imaging system (IVIS) data conclusively showed an enhanced signal intensity and a higher signal-to-noise ratio in mice receiving neutrophils labelled with the BD dye relative to those labelled with a gold standard dye at 2 h post in vivo administration of fluorescently labelled cells. Fluorescence-activated cell sorting (FACS) confirmed that BD is nontoxic, and an exceptional cell labelling dye that opens up precision deep-organ in vivo imaging of inflammation in mice routinely used for biomedical research. The origin of enhanced performance is identified with the molecular structure and the distinct localisation of the dye within cells that enable remarkable changes in its optical parameters.


Assuntos
Lesão Pulmonar Aguda/diagnóstico por imagem , Compostos de Boro/química , Corantes Fluorescentes/química , Animais , Compostos de Boro/administração & dosagem , Linhagem Celular , Modelos Animais de Doenças , Citometria de Fluxo , Corantes Fluorescentes/administração & dosagem , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neutrófilos/metabolismo , Imagem Óptica/métodos
10.
Mol Ther ; 25(1): 218-231, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-28129116

RESUMO

The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética/efeitos dos fármacos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Adenosina/administração & dosagem , Adenosina/farmacologia , Animais , Biomarcadores , Tetracloreto de Carbono/efeitos adversos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Masculino , Camundongos , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo
11.
J Hepatol ; 65(1): 75-83, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27067455

RESUMO

BACKGROUND & AIMS: Currently, staging of fibrosis in preclinical rodent liver fibrosis models is achieved histologically. Many animals are used at multiple time-points to assess disease progression or therapeutic responses. Hepatic myofibroblasts promote liver fibrosis therefore quantifying these cells in vivo could assess disease or predict therapeutic responses in mice. We fluorescently labelled a single chain antibody (C1-3) that binds hepatic myofibroblasts to monitor fibrogenesis in vivo. METHODS: CCl4 was used to induce acute liver injury in WT and cRel(-/-) mice. Bile duct ligation was used to model chronic fibrosis. Hepatic myofibroblasts were depleted using a liposome-drug delivery system or chemically with sulfasalazine. An IVIS® spectrum visualised fluorophore-conjugated C1-3 in vivo. RESULTS: IVIS detection of fluorescently labelled-C1-3 but not a control antibody discriminates between fibrotic and non-fibrotic liver in acute and chronic liver fibrosis models. cRel(-/-) mice have a fibro-protective phenotype and IVIS signal is reduced in CCl4 injured cRel(-/-) mice compared to wild-type. In vivo imaging of fluorescently labelled-C1-3 successfully predicts reductions in hepatic myofibroblast numbers in fibrotic liver disease in response to therapy. CONCLUSIONS: We report a novel fluorescence imaging method to assess murine hepatic myofibroblast numbers in vivo during liver fibrosis and after therapy. We also describe a novel liposomal antibody targeting system to selectively deliver drugs to hepatic myofibroblasts in vivo. C1-3 binds human hepatic myofibroblast therefore imaging labelled-C1-3 could be used for clinical studies in man to help stage fibrosis, demonstrate efficacy of drugs that promote hepatic myofibroblast clearance or predict early therapeutic responses. LAY SUMMARY: In response to damage and injury scars develop in the liver and the main cell that makes the scar tissue is the hepatic myofibroblast (HM). C1-3 is an antibody fragment that binds to the scar forming HM. We have fluorescently labelled C1-3 and given it to mice that have either normal or scarred livers (which contain HM) and then used a machine called an in vivo imaging system (IVIS) that takes pictures of different wavelengths of light, to visualise the antibody binding to HM inside the living mouse. Using fluorescently labelled C1-3 we can assess HM numbers in the injured liver and monitor response to therapy. We have also used C1-3 to target drugs encapsulated in lipid carriers (liposomes) to the HM to kill the HM and reduce the liver disease.


Assuntos
Miofibroblastos , Animais , Ductos Biliares , Fluorescência , Humanos , Fígado , Cirrose Hepática , Camundongos
12.
Sci Rep ; 6: 20101, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26831567

RESUMO

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.


Assuntos
Catepsina D/antagonistas & inibidores , Cloroquina/farmacologia , Lisossomos/enzimologia , Pepstatinas/farmacologia , Insuficiência Renal Crônica/enzimologia , Animais , Catepsina D/metabolismo , Colágeno/biossíntese , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Fibrose , Humanos , Lisossomos/patologia , Camundongos , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia
13.
Hepatology ; 57(2): 817-28, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22996371

RESUMO

UNLABELLED: Phosphorylation of the RelA subunit at serine 536 (RelA-P-Ser536) is important for hepatic myofibroblast survival and is mechanistically implicated in liver fibrosis. Here, we show that a cell-permeable competing peptide (P6) functions as a specific targeted inhibitor of RelA-P-Ser536 in vivo and exerts an antifibrogenic effect in two progressive liver disease models, but does not impair hepatic inflammation or innate immune responses after lipopolysaccharide challenge. Using kinase assays and western blotting, we confirm that P6 is a substrate for the inhibitory kappa B kinases (IKKs), IKKα and IKKß, and, in human hepatic myofibroblasts, P6 prevents RelA-P-Ser536, but does not affect IKK activation of IκBα. We demonstrate that RelA-P-Ser536 is a feature of human lung and skin fibroblasts, but not lung epithelial cells, in vitro and is present in sclerotic skin and diseased lungs of patients suffering from idiopathic pulmonary fibrosis. CONCLUSION: RelA-P-Ser536 may be a core fibrogenic regulator of fibroblast phenotype.


Assuntos
Imunidade Inata/efeitos dos fármacos , Cirrose Hepática/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Adulto , Animais , Intoxicação por Tetracloreto de Carbono/tratamento farmacológico , Fibroblastos/metabolismo , Humanos , Quinase I-kappa B/fisiologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Fragmentos de Peptídeos/metabolismo , Fosforilação , Serina , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/farmacologia
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