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1.
Nat Commun ; 11(1): 4520, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908154

RESUMO

Tumor extracellular matrix has been associated with drug resistance and immune suppression. Here, proteomic and RNA profiling reveal increased collagen levels in lung tumors resistant to PD-1/PD-L1 blockade. Additionally, elevated collagen correlates with decreased total CD8+ T cells and increased exhausted CD8+ T cell subpopulations in murine and human lung tumors. Collagen-induced T cell exhaustion occurs through the receptor LAIR1, which is upregulated following CD18 interaction with collagen, and induces T cell exhaustion through SHP-1. Reduction in tumor collagen deposition through LOXL2 suppression increases T cell infiltration, diminishes exhausted T cells, and abrogates resistance to anti-PD-L1. Abrogating LAIR1 immunosuppression through LAIR2 overexpression or SHP-1 inhibition sensitizes resistant lung tumors to anti-PD-1. Clinically, increased collagen, LAIR1, and TIM-3 expression in melanoma patients treated with PD-1 blockade predict poorer survival and response. Our study identifies collagen and LAIR1 as potential markers for immunotherapy resistance and validates multiple promising therapeutic combinations.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD8-Positivos/imunologia , Colágeno/metabolismo , Resistencia a Medicamentos Antineoplásicos/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Receptores Imunológicos/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Humanos , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA-Seq , Receptores Imunológicos/genética
2.
Nat Commun ; 11(1): 3642, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686688

RESUMO

Carpal tunnel syndrome (CTS) is the most common peripheral nerve entrapment syndrome, affecting a large proportion of the general population. Genetic susceptibility has been implicated in CTS, but the causative genes remain elusive. Here, we report the identification of two mutations in cartilage oligomeric matrix protein (COMP) that segregate with CTS in two large families with or without multiple epiphyseal dysplasia (MED). Both mutations impair the secretion of COMP by tenocytes, but the mutation associated with MED also perturbs its secretion in chondrocytes. Further functional characterization of the CTS-specific mutation reveals similar histological and molecular changes of tendons/ligaments in patients' biopsies and the mouse models. The mutant COMP fails to oligomerize properly and is trapped in the ER, resulting in ER stress-induced unfolded protein response and cell death, leading to inflammation, progressive fibrosis and cell composition change in tendons/ligaments. The extracellular matrix (ECM) organization is also altered. Our studies uncover a previously unrecognized mechanism in CTS pathogenesis.


Assuntos
Síndrome do Túnel Carpal , Proteína de Matriz Oligomérica de Cartilagem , Animais , Síndrome do Túnel Carpal/etiologia , Síndrome do Túnel Carpal/genética , Síndrome do Túnel Carpal/metabolismo , Síndrome do Túnel Carpal/patologia , Proteína de Matriz Oligomérica de Cartilagem/genética , Proteína de Matriz Oligomérica de Cartilagem/metabolismo , Condrócitos/patologia , Estresse do Retículo Endoplasmático/fisiologia , Matriz Extracelular/patologia , Humanos , Inflamação , Ligamentos/citologia , Ligamentos/patologia , Mutação , Osteocondrodisplasias/genética , Osteocondrodisplasias/patologia , Tendões/citologia , Tendões/patologia , Tenócitos/patologia
3.
Int J Nanomedicine ; 15: 4625-4637, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636622

RESUMO

Purpose: Besides the tumor cells themselves, solid tumors are comprised of numerous cell types including infiltrating immune cells such as tumor-associated macrophages (TAMs). TAMs are vital stromal components of host immune system and play a critical role in the development of cancer. TAMs can be divided into two subtypes: M1 tumor-suppressive macrophage and M2 tumor-supportive macrophage. To better address the observations of TAMs functional performance, we describe an in vitro system that mimics the populations of TAMs infiltrated into the tumor mass by using our disintegrable supramolecular gelatin (DSG) hydrogels, which are physically crosslinked by host-guest complexations. Materials and Methods: The host-guest interaction was adopted between the aromatic groups of gelatin and the photocrosslinkable acrylated ß-cyclodextrins (Ac-ß-CDs) to form the DSG hydrogels. The convenient macrophage/endometrial cancer cells heterospheroid 3D model was set up by DSG hydrogels. RT-PCR and Western blot assays were developed to evaluate the efficiencies of inducers on the macrophages. The ELISA and oxygen saturation assays were performed to measure the secretion of VEGF and consumption of oxygen of tumor and/or macrophages, respectively. To determine the antitumor effects of M2 reprogrammed macrophages in vitro and in vivo, migration assay and tumor xenograft model were used, respectively. Results: The host-guest complexations of DSG hydrogels were controllably broken efficiently by soaking into the solution of competitive guest monomers 1-adamantanamine hydrochloride. The DSG hydrogels help IFN-γ reprogram the M2 to M1 and then decrease the tumor/M2 reprogrammed macrophage cells heterospheroid secretion of VEGF and increase the relative oxygen saturation. Significantly, the co-cultural tumor/M2 reprogrammed group from the disintegrated DSG hydrogels reduced the migration of cancer cells in vitro and the tumor growth in vivo. Conclusion: We obtain a TAMs/tumor microenvironment-responsive 3D model based on the novel DSG hydrogels, and will be of utility in cancer therapy and drug discovery.


Assuntos
Neoplasias do Endométrio/patologia , Gelatina/química , Hidrogéis/química , Macrófagos/citologia , Macrófagos/transplante , Animais , Técnicas de Cultura de Células , Neoplasias do Endométrio/terapia , Matriz Extracelular/patologia , Feminino , Gelatina/farmacocinética , Humanos , Hidrogéis/farmacocinética , Camundongos Endogâmicos BALB C , Esferoides Celulares/citologia , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Arterioscler Thromb Vasc Biol ; 40(9): 2212-2226, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640908

RESUMO

OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade do Canal Arterial/metabolismo , Canal Arterial/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Canal Arterial/anormalidades , Permeabilidade do Canal Arterial/genética , Permeabilidade do Canal Arterial/patologia , Células Endoteliais/patologia , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Proteína Quinase C/metabolismo , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
6.
Arterioscler Thromb Vasc Biol ; 40(9): 2195-2211, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32698686

RESUMO

OBJECTIVE: To delineate temporal and spatial dynamics of vascular smooth muscle cell (SMC) transcriptomic changes during aortic aneurysm development in Marfan syndrome (MFS). Approach and Results: We performed single-cell RNA sequencing to study aortic root/ascending aneurysm tissue from Fbn1C1041G/+ (MFS) mice and healthy controls, identifying all aortic cell types. A distinct cluster of transcriptomically modulated SMCs (modSMCs) was identified in adult Fbn1C1041G/+ mouse aortic aneurysm tissue only. Comparison with atherosclerotic aortic data (ApoE-/- mice) revealed similar patterns of SMC modulation but identified an MFS-specific gene signature, including plasminogen activator inhibitor-1 (Serpine1) and Kruppel-like factor 4 (Klf4). We identified 481 differentially expressed genes between modSMC and SMC subsets; functional annotation highlighted extracellular matrix modulation, collagen synthesis, adhesion, and proliferation. Pseudotime trajectory analysis of Fbn1C1041G/+ SMC/modSMC transcriptomes identified genes activated differentially throughout the course of phenotype modulation. While modSMCs were not present in young Fbn1C1041G/+ mouse aortas despite small aortic aneurysm, multiple early modSMCs marker genes were enriched, suggesting activation of phenotype modulation. modSMCs were not found in nondilated adult Fbn1C1041G/+ descending thoracic aortas. Single-cell RNA sequencing from human MFS aortic root aneurysm tissue confirmed analogous SMC modulation in clinical disease. Enhanced expression of TGF-ß (transforming growth factor beta)-responsive genes correlated with SMC modulation in mouse and human data sets. CONCLUSIONS: Dynamic SMC phenotype modulation promotes extracellular matrix substrate modulation and aortic aneurysm progression in MFS. We characterize the disease-specific signature of modSMCs and provide temporal, transcriptomic context to the current understanding of the role TGF-ß plays in MFS aortopathy. Collectively, single-cell RNA sequencing implicates TGF-ß signaling and Klf4 overexpression as potential upstream drivers of SMC modulation.


Assuntos
Aneurisma Aórtico/genética , Fibrilina-1/genética , Perfilação da Expressão Gênica , Síndrome de Marfan/complicações , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Análise de Célula Única , Transcriptoma , Animais , Aorta/metabolismo , Aorta/patologia , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Progressão da Doença , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Predisposição Genética para Doença , Masculino , Síndrome de Marfan/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/patologia , Mutação , Miócitos de Músculo Liso/patologia , Fenótipo , RNA-Seq , Fatores de Tempo , Remodelação Vascular/genética
7.
J Pharmacol Sci ; 143(4): 281-289, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32513569

RESUMO

FoxM1 is a transcriptional regulator involved in tumor development, pulmonary fibrosis, and cardiac fibrosis. However, its role in renal interstitial fibrosis (RIF) has yet to be elucidated. We established a TGF-ß1-stimulated human proximal tubular epithelial cell (HK-2) model in vitro and a unilateral ureteral obstruction (UUO)-induced rat RIF model in vivo. FoxM1 inhibition was achieved by siRNA interference in vitro and by injecting thiostrepton into UUO-induced RIF rats in vivo. The degree of renal damage and fibrosis were determined by histological assessment via hematoxylin and eosin (H&E) staining. Immunohistochemistry, western blots, and qPCR were used to determine the expression levels of FoxM1, Collagen I, E-cadherin, α-SMA, and Snail1. Our results showed that FoxM1 inhibition could ameliorate RIF and reduce the deposition of Collagen I. H&E staining revealed that renal structural damage, inflammatory cell infiltration, and ECM deposition were significantly attenuated by thiostrepton treatment in the UUO rats. Furthermore, FoxM1 downregulation significantly suppressed epithelial-to-mesenchymal transition, as evidenced by decreased protein and mRNA expression levels of α-SMA and Snail1 and a significant increase in protein and mRNA expression levels of E-cadherin. Collectively, these results suggested that FoxM1 inhibition could be a novel therapeutic strategy for the treatment of RIF.


Assuntos
Transição Epitelial-Mesenquimal , Matriz Extracelular/patologia , Proteína Forkhead Box M1/genética , Inativação Gênica , Nefropatias/genética , Rim/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/genética , Fibrose , Humanos , Nefropatias/patologia , Nefropatias/terapia , Masculino , Ratos
8.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510478

RESUMO

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.


Assuntos
Cartilagem Articular/patologia , Condrócitos/patologia , Matriz Extracelular/patologia , Microscopia de Força Atômica/métodos , Osteoartrite/patologia , Elasticidade , Humanos
9.
Arterioscler Thromb Vasc Biol ; 40(7): 1763-1776, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32493168

RESUMO

OBJECTIVE: Vascular calcification is a pathology characterized by arterial mineralization, which is a common late-term complication of atherosclerosis that independently increases the risk of adverse cardiovascular events by fourfold. A major source of calcifying cells is transdifferentiating vascular smooth muscle cells (VSMCs). Previous studies showed that deletion of the collagen-binding receptor, DDR1 (discoidin domain receptor-1), attenuated VSMC calcification. Increased matrix stiffness drives osteogenesis, and DDR1 has been implicated in stiffness sensing in other cell types; however, the role of DDR1 as a mechanosensor in VSMCs has not been investigated. Here, we test the hypothesis that DDR1 senses increased matrix stiffness and promotes VSMC transdifferentiation and calcification. Approach and Results: Primary VSMCs isolated from Ddr1+/+ (wild-type) and Ddr1-/- (knockout) mice were studied on collagen-I-coated silicon substrates of varying stiffness, culturing in normal or calcifying medium. DDR1 expression and phosphorylation increased with increasing stiffness, as did in vitro calcification, nuclear localization of Runx2 (Runt-related transcription factor 2), and expression of other osteochondrocytic markers. By contrast, DDR1 deficient VSMCs were not responsive to stiffness and did not undergo transdifferentiation. DDR1 regulated stress fiber formation and RhoA (ras homolog family member A) activation through the RhoGEF (rho guanine nucleotide exchange factor), Vav2. Inhibition of actomyosin contractility reduced Runx2 activation and attenuated in vitro calcification in wild-type VSMCs. Finally, a novel positive feedforward loop was uncovered between DDR1 and actomyosin contractility, important in regulating DDR1 expression, clustering, and activation. CONCLUSIONS: This study provides mechanistic insights into DDR1 mechanosignaling and shows that DDR1 activity and actomyosin contractility are interdependent in mediating stiffness-dependent increases in VSMC calcification.


Assuntos
Aterosclerose/enzimologia , Transdiferenciação Celular , Receptor com Domínio Discoidina 1/metabolismo , Matriz Extracelular/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Osteogênese , Calcificação Vascular/enzimologia , Proteína rhoA de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/patologia , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Receptor com Domínio Discoidina 1/deficiência , Receptor com Domínio Discoidina 1/genética , Modelos Animais de Doenças , Matriz Extracelular/patologia , Mecanotransdução Celular , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/patologia
10.
Ann Rheum Dis ; 79(7): 975-984, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32371389

RESUMO

OBJECTIVE: Calcification of cartilage with basic calcium phosphate (BCP) crystals is a common phenomenon during osteoarthritis (OA). It is directly linked to the severity of the disease and known to be associated to hypertrophic differentiation of chondrocytes. One morphogen regulating hypertrophic chondrocyte differentiation is Wnt3a. METHODS: Calcification and sulfation of extracellular matrix of the cartilage was analysed over a time course from 6 to 22 weeks in mice and different OA grades of human cartilage. Wnt3a and ß-catenin was stained in human and murine cartilage. Expression of sulfation modulating enzymes (HS2St1, HS6St1) was analysed using quantitative reverse transcription PCR (RT-PCR). The influence of BCP crystals on the chondrocyte phenotype was investigated using quantitative RT-PCR for the marker genes Axin2, Sox9, Col2, MMP13, ColX and Aggrecan. Using western blot for ß-catenin and pLRP6 we investigated the activation of Wnt signalling. The binding capacity of BCP for Wnt3a was analysed using immunohistochemical staining and western blot. RESULTS: Here, we report that pericellular matrix sulfation is increased in human and murine OA. Wnt3a co-localised with heparan sulfate proteoglycans in the pericellular matrix of chondrocytes in OA cartilage, in which canonical Wnt signalling was activated. In vitro, BCP crystals physically bound to Wnt3a. Interestingly, BCP crystals were sufficient to induce canonical Wnt signalling as assessed by phosphorylation of LRP6 and stabilisation of ß-catenin, and to induce a hypertrophic shift of the chondrocyte phenotype. CONCLUSION: Consequently, our data identify BCP crystals as a concentrating factor for Wnt3a in the pericellular matrix and an inducer of chondrocyte hypertrophy.


Assuntos
Fosfatos de Cálcio/metabolismo , Diferenciação Celular/genética , Condrócitos/patologia , Osteoartrite/genética , Proteína Wnt3A/metabolismo , Animais , Cartilagem Articular/citologia , Condrócitos/metabolismo , Matriz Extracelular/patologia , Humanos , Hipertrofia , Camundongos , Osteoartrite/patologia , Via de Sinalização Wnt/genética
11.
Prostate ; 80(9): 715-726, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32364250

RESUMO

BACKGROUND: Tumor microenvironment or stroma has the potency to regulate the behavior of malignant cells. Fibroblast-like cells are abundant in tumor stroma and they are also responsible for the synthesis of many extracellular matrix components. Fibroblast-cancer cell interplay can modify the functions of both cell types. METHODS: We applied mass spectrometry and proteomics to unveil the matrisome in 3D spheroids formed by DU145 prostate cancer cells, PC3 prostate cancer cells, or prostate-derived fibroblasts. Similarly, DU145/fibroblast and PC3/fibroblast coculture spheroids were also analyzed. Western blot analysis and immunofluorescence were used to confirm the presence of specific proteins in spheroids. Cancer dissemination was studied by utilizing "out of spheroids" migration and invasion assays. RESULTS: In the spheroid model cancer cell-fibroblast interplay caused remarkable changes in the extracellular matrix and accelerated the invasion of DU145 cells. Fibroblasts produced structural matrix proteins, growth factors, and matrix metalloproteinases. In cancer cell/fibroblast cocultures basement membrane components, including laminins (α3, α5, ß2, and ß3), heparan sulfate proteoglycan (HSPG2 gene product), and collagen XVIII accumulated in a prominent manner when compared with spheroids that contained fibroblasts or cancer cells only. Furthermore, collagen XVIII was intensively processed to different endostatin-containing isoforms by cancer cell-derived cathepsin L. CONCLUSIONS: Fibroblasts can promote carcinoma cell dissemination by several different mechanisms. Extracellular matrix and basement membrane proteins provide attachment sites for cell locomotion promoting adhesion receptors. Growth factors and metalloproteinases are known to accelerate cell invasion. In addition, cancer cell-fibroblast interplay generates biologically active fragments of basement membrane proteins, such as endostatin.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Proteínas de Membrana/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Autoantígenos/metabolismo , Membrana Basal/metabolismo , Catepsina L/metabolismo , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Masculino , Espectrometria de Massas , Invasividade Neoplásica , Colágenos não Fibrilares/metabolismo , Células PC-3 , Proteômica/métodos , Esferoides Celulares
12.
Proc Natl Acad Sci U S A ; 117(21): 11432-11443, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381732

RESUMO

The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.


Assuntos
Glioblastoma/patologia , Receptores de Hialuronatos/metabolismo , Actinas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Técnicas de Inativação de Genes , Glioblastoma/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Técnicas de Cultura de Órgãos , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
13.
PLoS Pathog ; 16(5): e1008516, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32413091

RESUMO

Lyme disease, caused by Borrelia burgdorferi, B. afzelii and B. garinii, is a chronic, multi-systemic infection and the spectrum of tissues affected can vary with the Lyme disease strain. For example, whereas B. garinii infection is associated with neurologic manifestations, B. burgdorferi infection is associated with arthritis. The basis for tissue tropism is poorly understood, but has been long hypothesized to involve strain-specific interactions with host components in the target tissue. OspC (outer surface protein C) is a highly variable outer surface protein required for infectivity, and sequence differences in OspC are associated with variation in tissue invasiveness, but whether OspC directly influences tropism is unknown. We found that OspC binds to the extracellular matrix (ECM) components fibronectin and/or dermatan sulfate in an OspC variant-dependent manner. Murine infection by isogenic B. burgdorferi strains differing only in their ospC coding region revealed that two OspC variants capable of binding dermatan sulfate promoted colonization of all tissues tested, including joints. However, an isogenic strain producing OspC from B. garinii strain PBr, which binds fibronectin but not dermatan sulfate, colonized the skin, heart and bladder, but not joints. Moreover, a strain producing an OspC altered to recognize neither fibronectin nor dermatan sulfate displayed dramatically reduced levels of tissue colonization that were indistinguishable from a strain entirely deficient in OspC. Finally, intravital microscopy revealed that this OspC mutant, in contrast to a strain producing wild type OspC, was defective in promoting joint invasion by B. burgdorferi in living mice. We conclude that OspC functions as an ECM-binding adhesin that is required for joint invasion, and that variation in OspC sequence contributes to strain-specific differences in tissue tropism displayed among Lyme disease spirochetes.


Assuntos
Borrelia burgdorferi/metabolismo , Dermatan Sulfato/metabolismo , Matriz Extracelular/metabolismo , Artropatias/metabolismo , Articulações/metabolismo , Doença de Lyme/metabolismo , Animais , Antígenos de Bactérias , Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Dermatan Sulfato/genética , Matriz Extracelular/genética , Matriz Extracelular/microbiologia , Matriz Extracelular/patologia , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Artropatias/genética , Artropatias/microbiologia , Artropatias/patologia , Articulações/microbiologia , Articulações/patologia , Doença de Lyme/genética , Doença de Lyme/microbiologia , Doença de Lyme/patologia , Camundongos , Camundongos SCID , Mutação , Especificidade de Órgãos
14.
Phytomedicine ; 72: 153232, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32460034

RESUMO

BACKGROUND: In chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure. PURPOSE: We isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis. STUDY DESIGN: Unilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-ß1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms. METHODS: Western blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA. RESULTS: PAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect. CONCLUSION: PAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Matriz Extracelular/efeitos dos fármacos , Nefropatias/tratamento farmacológico , Rim/patologia , Triterpenos/farmacologia , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/genética , Animais , Estudos de Casos e Controles , Linhagem Celular , Relação Dose-Resposta a Droga , Matriz Extracelular/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Ratos Sprague-Dawley , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Triterpenos/química , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia
15.
Ann Rheum Dis ; 79(8): 1111-1120, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32409323

RESUMO

OBJECTIVES: FBXO6, a component of the ubiquitin E3 ligases, has been shown to bind high mannose N-linked glycoproteins and act as ubiquitin ligase subunits. Most proteins in the secretory pathway, such as matrix metalloproteinases, are modified with N-glycans and play important roles in the development of osteoarthritis (OA). However, whether FBXO6 exerts regulatory effects on the pathogenesis of OA remains undefined. METHODS: The expression of FBXO6 was examined in the cartilage of human and multiple mouse OA models. The role of FBXO6 in cartilage degeneration was analysed with global FBXO6 -/- mice, transgenic Col2a1-CreERT2;FBXO6f/f mice. The FBXO6 interacting partner MMP14 and its regulatory transcriptional factor SMAD2/3 were identified and validated in different pathological models as well as SMAD2 -/- mice. RESULTS: The expression of FBXO6 decreased in the cartilage from human OA samples, anterior cruciate ligament transaction (ACLT) -induced OA samples, spontaneous OA STR/ort samples and aged mice samples. Global knockout or conditional knockout of FBXO6 in cartilage promoted experimental OA process. The molecular mechanism study revealed that FBXO6 decreased MMP14 by ubiquitination and degradation, leading to inhibited proteolytic activation of MMP13. Interestingly, FBXO6 expression is regulated by transforming growth factor ß (TGFß)-SMAD2/3 signalling pathway. Therefore, the overexpression of FBXO6 protected mice from post-injury OA development. CONCLUSIONS: TGFß-SMAD2/3 signalling pathway suppressed MMP13 activation by upregulating of FBXO6 transcription and consequently promoting MMP14 proteasomal degradation. Inducement of FBXO6 expression in OA cartilage might provide a promising OA therapeutic strategy.


Assuntos
Matriz Extracelular/patologia , Metaloproteinase 14 da Matriz/metabolismo , Osteoartrite/patologia , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Matriz Extracelular/metabolismo , Humanos , Camundongos , Osteoartrite/metabolismo , Ubiquitinação/fisiologia
16.
Breast Cancer Res ; 22(1): 41, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32370801

RESUMO

BACKGROUND: In utero endocrine disruption is linked to increased risk of breast cancer later in life. Despite numerous studies establishing this linkage, the long-term molecular changes that predispose mammary cells to carcinogenic transformation are unknown. Herein, we investigated how endocrine disrupting compounds (EDCs) drive changes within the stroma that can contribute to breast cancer susceptibility. METHODS: We utilized bisphenol A (BPA) as a model of estrogenic endocrine disruption to analyze the long-term consequences in the stroma. Deregulated genes were identified by RNA-seq transcriptional profiling of adult primary fibroblasts, isolated from female mice exposed to in utero BPA. Collagen staining, collagen imaging techniques, and permeability assays were used to characterize changes to the extracellular matrix. Finally, gland stiffness tests were performed on exposed and control mammary glands. RESULTS: We identified significant transcriptional deregulation of adult fibroblasts exposed to in utero BPA. Deregulated genes were associated with cancer pathways and specifically extracellular matrix composition. Multiple collagen genes were more highly expressed in the BPA-exposed fibroblasts resulting in increased collagen deposition in the adult mammary gland. This transcriptional reprogramming of BPA-exposed fibroblasts generates a less permeable extracellular matrix and a stiffer mammary gland. These phenotypes were only observed in adult 12-week-old, but not 4-week-old, mice. Additionally, diethylstilbestrol, known to increase breast cancer risk in humans, also increases gland stiffness similar to BPA, while bisphenol S does not. CONCLUSIONS: As breast stiffness, extracellular matrix density, and collagen deposition have been directly linked to breast cancer risk, these data mechanistically connect EDC exposures to molecular alterations associated with increased disease susceptibility. These alterations develop over time and thus contribute to cancer risk in adulthood.


Assuntos
Disruptores Endócrinos/toxicidade , Matriz Extracelular/patologia , Glândulas Mamárias Animais/patologia , Efeitos Tardios da Exposição Pré-Natal/patologia , Células Estromais/patologia , Animais , Compostos Benzidrílicos/toxicidade , Estrogênios não Esteroides/farmacologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Feminino , Fibroblastos/imunologia , Fibroblastos/patologia , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Fenóis/toxicidade , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/imunologia , Transcriptoma
17.
Arterioscler Thromb Vasc Biol ; 40(7): e193-e202, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32404005

RESUMO

Vascular calcification is a ubiquitous pathology of aging. Oxidative stress, persistent DNA damage, and senescence are major pathways driving both cellular and tissue aging, and emerging evidence suggests that these pathways are activated, and even accelerated, in patients with vascular calcification. The DNA damage response-a complex signaling platform that maintains genomic integrity-is induced by oxidative stress and is intimately involved in regulating cell death and osteogenic differentiation in both bone and the vasculature. Unexpectedly, a posttranslational modification, PAR (poly[ADP-ribose]), which is a byproduct of the DNA damage response, initiates biomineralization by acting to concentrate calcium into spheroidal structures that can nucleate apatitic mineral on the ECM (extracellular matrix). As we start to dissect the molecular mechanisms driving aging-associated vascular calcification, novel treatment strategies to promote healthy aging and delay pathological change are being unmasked. Drugs targeting the DNA damage response and senolytics may provide new avenues to tackle this detrimental and intractable pathology.


Assuntos
Envelhecimento/patologia , Artérias/patologia , Aterosclerose/patologia , Dano ao DNA , Estresse Oxidativo , Placa Aterosclerótica , Calcificação Vascular/patologia , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Apatitas/metabolismo , Artérias/efeitos dos fármacos , Artérias/metabolismo , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismo , Senescência Celular , Dano ao DNA/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Mediadores da Inflamação/metabolismo , Osteogênese , Estresse Oxidativo/efeitos dos fármacos , Poli Adenosina Difosfato Ribose/metabolismo , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/genética , Calcificação Vascular/metabolismo
18.
Arterioscler Thromb Vasc Biol ; 40(7): 1651-1663, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32404006

RESUMO

OBJECTIVE: SMAD3 pathogenic variants are associated with the development of thoracic aortic aneurysms. We sought to determine the role of SMAD3 in lineage-specific vascular smooth muscle cells (VSMCs) differentiation and function. Approach and Results: SMAD3 c.652delA, a frameshift mutation and nonsense-mediated decay, was introduced in human-induced pluripotent stem cells using CRISPR-Cas9. The wild-type and SMAD3-/- (c.652delA) human-induced pluripotent stem cells were differentiated into cardiovascular progenitor cells or neural crest stem cells and then to lineage-specific VSMCs. Differentiation, contractility, extracellular matrix synthesis, and TGF-ß (transforming growth factor-ß) signaling of the differentiated VSMCs were analyzed. The homozygous frameshift mutation resulted in SMAD3 deficiency and was confirmed in human-induced pluripotent stem cells by Sanger sequencing and immunoblot analysis. In cardiovascular progenitor cell-VSMCs, SMAD3 deletion significantly disrupted canonical TGF-ß signaling and decreased gene expression of VSMC markers, including SM α-actin, myosin heavy chain 11, calponin-1, SM22α, and key controlling factors, SRF and myocardin, but increased collagen expression. The loss of SMAD3 significantly decreased VSMC contractility. In neural crest stem cells-VSMCs, SMAD3 deficiency did not significantly affect the VSMC differentiation but decreased ELN (elastin) expression and increased phosphorylated SMAD2. Expression of mir-29 was increased in SMAD3-/- VSMCs, and inhibition of mir-29 partially rescued ELN expression. CONCLUSIONS: SMAD3-dependent TGF-ß signaling was essential for the differentiation of cardiovascular progenitor cell-VSMCs but not for the differentiation of neural crest stem cell-VSMCs. The lineage-specific TGF-ß responses in human VSMCs may potentially contribute to the development of aortic root aneurysms in patients with SMAD3 mutations.


Assuntos
Aneurisma da Aorta Torácica/metabolismo , Diferenciação Celular , Linhagem da Célula , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Smad3/deficiência , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/fisiopatologia , Células Cultivadas , Elastina/genética , Elastina/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Mutação da Fase de Leitura , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fosforilação , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Fator de Crescimento Transformador beta/metabolismo , Remodelação Vascular , Vasoconstrição
19.
Parasitol Res ; 119(6): 1989-1995, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32291469

RESUMO

Recent advances in chronic toxoplasmosis understanding became the focus of discussion about behavioral abnormalities, which could be explained by cyst location and neuronal impairment in specific brain areas. Perineuronal nets (PNNs) are specialized extracellular matrices that surround the neuronal body and proximal dendrites and play key roles in neuronal circuitry maintenance and stabilization. Its impairment can lead to abnormal synaptic functioning with behavioral repercussions. In this context, we analyzed the impact of Toxoplasma gondii infection on neuronal integrity in the Corpus striatum of chronically infected mice. C57BL/6 and Balb/c female mice were infected with T. gondii ME49 cysts. Brain sections were submitted to immunohistochemistry with Wisteria floribunda agglutinin (WFA) for PNN labeling followed by quantification of tissue cyst and labeled neuronal cells 30 days after infection. Our results revealed that C57BL/6 exhibited a significant decrease in PNN-positive (WFA+) labeled neurons and an expressively higher number of tissue cysts than Balb/c mice. It was also possible to observe that the number of T. gondii tissue cysts and the number of WFA+ neurons were inversely correlated for C57BL/6-infected mice. However, no correlation was observed for Balb/c mice. These data suggest how the impact of parasite dissemination in the brain and host characteristics can influence neuronal integrity impairment during infection by decreasing WFA+ neurons. This might be a plausible pathway in which the presence of T. gondii contributes to behavioral changes in the infected host.


Assuntos
Corpo Estriado/patologia , Neurônios/patologia , Toxoplasmose/patologia , Animais , Doença Crônica , Matriz Extracelular/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Lectinas de Plantas , Receptores de N-Acetilglucosamina , Toxoplasma , Toxoplasmose/metabolismo
20.
Life Sci ; 253: 117685, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32315726

RESUMO

AIMS: Cumulative evidence suggests that long-chain non-coding RNA (lncRNA) is involved in the pathogenesis of osteoarthritis (OA). The present study aimed to explore the regulatory role and related mechanisms of HOX transcript antisense intergenic RNA (HOTAIR) in OA. MATERIAL AND METHODS: The OA mouse model was constructed by the medial meniscus (DMM) method, and Interleukin (IL)-1ß-induced chondrocytes were used to simulate OA in vitro. KEY FINDINGS: Results found that HOTAIR was significantly up-regulated in articular cartilage tissues of OA mice and IL-1ß-induced chondrocytes, accompanied by down-regulation of miR-20b and increased expression of the phosphatase and tensin homolog (PTEN). HOTAIR silencing improved cartilage tissue damage in OA mice, and promoted the expression of collagen II and aggrecan in cartilage tissue, while inhibited the expression of matrix metalloproteinases (MMP)-13 and ADAMTS-5. Overexpression of HOTAIR inhibited the proliferation of IL-1ß-induced chondrocytes and promoted apoptosis and extracellular matrix (ECM) degradation, whereas the effect of HOTAIR knockdown was reversed. Bioinformatics software and luciferase reporter experiments confirmed that HOTAIR could negatively regulate miR-20b, and PTEN was a target gene of miR-20b. An increase in PTEN expression induced by HOTAIR overexpression could be reversed by the introduction of miR-20b mimic. HOTAIR overexpression significantly reversed miR-20 mimic-mediated inhibition of apoptosis and ECM degradation in IL-1ß-induced chondrocytes, whereas the introduction of si-HOTAIR eliminated anti-miR-20b-mediated apoptosis and ECM degradation. SIGNIFICANCE: HOTAIR can participate in OA by promoting chondrocyte apoptosis and ECM degradation, which may be related to its targeted regulation of miR-20b/PTEN axis.


Assuntos
Condrócitos/patologia , MicroRNAs/genética , Osteoartrite/fisiopatologia , PTEN Fosfo-Hidrolase/genética , RNA Longo não Codificante/genética , Animais , Apoptose/genética , Artrite Experimental/genética , Artrite Experimental/fisiopatologia , Cartilagem Articular/patologia , Progressão da Doença , Matriz Extracelular/patologia , Interleucina-1beta/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/genética
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