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1.
Life Sci ; 256: 118026, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615187

RESUMO

AIM: We aimed to determine the biological processes and pathways involved in cervical carcinogenesis associated with high-risk human papillomavirus (HPV) infection. MATERIALS AND METHODS: Total RNA was extracted from three formalin-fixed paraffin-embedded (FFPE) samples each of normal cervix, HPV-infected low-grade squamous intraepithelial lesion (LSIL), high-grade SIL (HSIL) and squamous cell carcinoma (SCC). Transcriptomic profiling by microarrays was conducted followed by downstream Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. RESULTS: We examined the difference in GOs enriched for each transition stage from normal cervix to LSIL, HSIL, and SCC, and found 307 genes to be differentially expressed. In the transition from normal cervix to LSIL, the extracellular matrix (ECM) genes were significantly downregulated. The MHC class II genes were significantly upregulated in the LSIL to HSIL transition. In the final transition from HSIL to SCC, the immunoglobulin heavy locus genes were significantly upregulated and the ECM pathway was implicated. CONCLUSION: Deregulation of the immune-related genes including MHC II and immunoglobulin heavy chain genes were involved in the transitions from LSIL to HSIL and SCC, suggesting immune escape from host anti-tumour response. The extracellular matrix plays an important role during the early and late stages of cervical carcinogenesis.


Assuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Genes MHC da Classe II/genética , Infecções por Papillomavirus/complicações , Lesões Intraepiteliais Escamosas Cervicais/patologia , Neoplasias do Colo do Útero/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/virologia , Regulação para Baixo , Matriz Extracelular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Estadiamento de Neoplasias , Infecções por Papillomavirus/genética , Lesões Intraepiteliais Escamosas Cervicais/genética , Lesões Intraepiteliais Escamosas Cervicais/virologia , Transcriptoma , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
2.
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
3.
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
4.
Proc Natl Acad Sci U S A ; 117(32): 19033-19044, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32709748

RESUMO

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).


Assuntos
Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/citologia , Microgéis/química , Neovascularização Fisiológica , Animais , Proliferação de Células , Células Imobilizadas/química , Células Imobilizadas/citologia , Células Imobilizadas/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Membro Posterior/irrigação sanguínea , Membro Posterior/metabolismo , Membro Posterior/cirurgia , Humanos , Integrinas/genética , Integrinas/metabolismo , Isquemia/fisiopatologia , Isquemia/cirurgia , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus
5.
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
6.
Nat Commun ; 11(1): 2604, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451392

RESUMO

Matrix deposition is essential for wound repair, but when excessive, leads to hypertrophic scars and fibrosis. The factors that control matrix deposition in skin wounds have only partially been identified and the consequences of matrix alterations for the mechanical properties of wounds are largely unknown. Here, we report how a single diffusible factor, activin A, affects the healing process across scales. Bioinformatics analysis of wound fibroblast transcriptome data combined with biochemical and histopathological analyses of wounds and functional in vitro studies identify that activin promotes pro-fibrotic gene expression signatures and processes, including glycoprotein and proteoglycan biosynthesis, collagen deposition, and altered collagen cross-linking. As a consequence, activin strongly reduces the wound and scar deformability, as identified by a non-invasive in vivo method for biomechanical analysis. These results provide mechanistic insight into the roles of activin in wound repair and fibrosis and identify the functional consequences of alterations in the wound matrisome at the biomechanical level.


Assuntos
Subunidades beta de Inibinas/metabolismo , Pele/lesões , Pele/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Cicatriz/patologia , Cicatriz/fisiopatologia , Cicatriz Hipertrófica/patologia , Cicatriz Hipertrófica/fisiopatologia , Colágeno/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose , Humanos , Subunidades beta de Inibinas/genética , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pele/patologia , Transcriptoma , Regulação para Cima , Cicatrização/genética , Cicatrização/fisiologia
7.
Plast Reconstr Surg ; 146(3): 552-562, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32459729

RESUMO

BACKGROUND: Traumatic muscle loss often results in poor functional restoration. Skeletal muscle injuries cannot be repaired without substantial fibrosis and loss of muscle function. Given its regenerative properties, the authors evaluated outcomes of fetal tissue-derived decellularized matrix for skeletal muscle regeneration. The authors hypothesized that fetal matrix would lead to enhanced myogenesis and suppress inflammation and fibrosis. METHODS: Composite tissue composed of dermis, subcutaneous tissue, and panniculus carnosus was harvested from the trunk of New Zealand White rabbit fetuses on gestational day 24 and from Sprague-Dawley rats on gestational day 18 and neonatal day 3, and decellularized using a sodium dodecyl sulfate-based negative-pressure protocol. Six, 10-mm-diameter, full-thickness rat latissimus dorsi wounds were created for each treatment, matrix was implanted (excluding the defect groups), and the wounds were allowed to heal for 60 days. Analyses were performed to characterize myogenesis, neovascularization, inflammation, and fibrosis at harvest. RESULTS: Significant myocyte ingrowth was visualized in both allogeneic and xenogeneic fetal matrix groups compared to neonatal and defect groups based on myosin heavy chain immunofluorescence staining. Microvascular networks were appreciated within all implanted matrices. At day 60, expression of Ccn2, Col1a1, and Ptgs2 were decreased in fetal matrix groups compared to defect. Neonatal matrix-implanted wounds failed to show decreased expression of Col1a1 or Ptgs2, and demonstrated increased expression of Tnf, but also demonstrated a significant reduction in Ccn2 expression. CONCLUSIONS: Initial studies of fetal matrices demonstrate promise for muscle regeneration in a rat latissimus dorsi model. Further research is necessary to evaluate fetal matrix for future translational use and better understand its effects.


Assuntos
Matriz Extracelular/genética , Regulação da Expressão Gênica , Desenvolvimento Muscular/genética , Músculo Esquelético/lesões , Prenhez , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Animais Recém-Nascidos , Western Blotting , Matriz Extracelular/metabolismo , Feminino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Gravidez , RNA/genética , Coelhos , Ratos , Ratos Sprague-Dawley
8.
Nat Commun ; 11(1): 1723, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32265444

RESUMO

Metaplastic breast carcinoma (MBC) is a highly aggressive form of triple-negative cancer (TNBC), defined by the presence of metaplastic components of spindle, squamous, or sarcomatoid histology. The protein profiles underpinning the pathological subtypes and metastatic behavior of MBC are unknown. Using multiplex quantitative tandem mass tag-based proteomics we quantify 5798 proteins in MBC, TNBC, and normal breast from 27 patients. Comparing MBC and TNBC protein profiles we show MBC-specific increases related to epithelial-to-mesenchymal transition and extracellular matrix, and reduced metabolic pathways. MBC subtypes exhibit distinct upregulated profiles, including translation and ribosomal events in spindle, inflammation- and apical junction-related proteins in squamous, and extracellular matrix proteins in sarcomatoid subtypes. Comparison of the proteomes of human spindle MBC with mouse spindle (CCN6 knockout) MBC tumors reveals a shared spindle-specific signature of 17 upregulated proteins involved in translation and 19 downregulated proteins with roles in cell metabolism. These data identify potential subtype specific MBC biomarkers and therapeutic targets.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma de Células Escamosas/metabolismo , Proteoma/metabolismo , Sarcoma/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/secundário , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Transição Epitelial-Mesenquimal/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Redes e Vias Metabólicas/genética , Metaplasia/genética , Metaplasia/metabolismo , Camundongos , Pessoa de Meia-Idade , Mutação , Biossíntese de Proteínas/genética , Proteoma/genética , Proteômica , Sarcoma/genética , Sarcoma/secundário , Fuso Acromático/genética , Fuso Acromático/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
9.
Proc Natl Acad Sci U S A ; 117(18): 9896-9905, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321834

RESUMO

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix-cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvß1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin ß1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Integrina beta1/genética , Trombospondina 1/genética , Fatores de Transcrição/genética , Remodelação Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aorta/crescimento & desenvolvimento , Aorta/metabolismo , Artérias Carótidas/crescimento & desenvolvimento , Artérias Carótidas/metabolismo , Microambiente Celular/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Adesões Focais/genética , Humanos , Integrina beta1/metabolismo , Mecanotransdução Celular , Camundongos , Neointima/genética , Neointima/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Trombospondina 1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rap de Ligação ao GTP/genética
10.
PLoS One ; 15(4): e0231594, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32315343

RESUMO

Recurrence and poorly differentiated (grade 3 and above) and atypical cell type endometrial cancer (EC) have poor prognosis outcome. The mechanisms and characteristics of recurrence and distal metastasis of EC remain unclear. The extracellular matrix (ECM) of the reproductive tract in women undergoes extensive structural remodelling changes every month. Altered ECMs surrounding cells were believed to play crucial roles in a cancer progression. To decipher the associations between ECM and EC development, we generated a PAN-ECM Data list of 1516 genes including ECM molecules (ECMs), synthetic and degradation enzymes for ECMs, ECM receptors, and soluble molecules that regulate ECM and used RNA-Seq data from The Cancer Genome Atlas (TCGA) for the studies. The alterations of PAN-ECM genes by comparing the RNA-Seq expressions profiles of EC samples which have been grouped as tumorigenesis and metastasis group based on their pathological grading were identified. Differential analyses including functional enrichment, co-expression network, and molecular network analysis were carried out to identify the specific PAN-ECM genes that may involve in the progression of EC. Eight hundred and thirty-one and 241 PAN-ECM genes were significantly involved in tumorigenesis (p-value <1.571e-15) and metastasis (p-value <2.2e-16), respectively, whereas 140 genes were in the intersection of tumorigenesis and metastasis. Interestingly, 92 of the 140 intersecting PAN-ECM genes showed contrasting fold changes between the tumorigenesis and metastasis datasets. Enrichment analysis for the contrast PAN-ECM genes indicated pathways such as GP6 signaling, ILK signaling, and interleukin (IL)-8 signaling pathways were activated in metastasis but inhibited in tumorigenesis. The significantly activated ECM and ECM associated genes in GP6 signaling, ILK signaling, and interleukin (IL)-8 signaling pathways may play crucial roles in metastasis of EC. Our study provides a better understanding of the etiology and the progression of EC.


Assuntos
Carcinogênese/genética , Neoplasias do Endométrio/genética , Matriz Extracelular/genética , Proteínas de Neoplasias/genética , Biologia Computacional , Progressão da Doença , Neoplasias do Endométrio/patologia , Endométrio/metabolismo , Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Neoplásica , RNA-Seq , Receptores de Superfície Celular/genética , Transdução de Sinais/genética
11.
Invest Ophthalmol Vis Sci ; 61(4): 40, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32340032

RESUMO

Purpose: To determine whether high glucose (HG) compromises internalization of lysyl oxidase (LOX) through excess binding of LOX with extracellular matrix (ECM) proteins. Methods: To determine whether HG promotes binding of LOX with ECM proteins, fibronectin (FN) and collagen IV (Coll IV), total or ECM-only proteins from rat retinal endothelial cells grown in normal (N; 5 mM) or HG (30 mM) medium were analyzed by coimmunoprecipitation and Western blot (WB). In parallel, coimmunostaining was performed to determine changes in LOX binding to FN or Coll IV. To determine the effect of HG on extracellular LOX levels, medium in which cells were grown for 1, 3, 5, and 7 days were assessed for LOX levels. Results: WB analysis using total protein showed LOX overexpression and elevated levels of LOX bound to Coll IV or FN in HG condition. Similarly, a significant increase in LOX bound to FN or Coll IV was observed in ECM-only protein. These data were supported by Z-stack confocal microscopy images from coimmunostaining. Furthermore, immunostaining performed on ECM layer revealed increased presence of LOX bound to Coll IV or FN. Additionally, when media from cells grown in HG was monitored, a maximal increase in LOX level was observed by day 3, which declined by day 7. Conclusions: Findings indicate that HG promotes binding of LOX to FN and Coll IV extracellularly that results in reduced LOX internalization, attenuation of negative feedback, and upregulation of LOX expression associated with diabetic retinopathy.


Assuntos
Retinopatia Diabética/metabolismo , Matriz Extracelular/genética , Hiperglicemia/metabolismo , Proteína-Lisina 6-Oxidase/genética , Regulação para Cima/genética , Animais , Western Blotting/métodos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Endoteliais , Fibronectinas/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Hiperglicemia/fisiopatologia , Ligação Proteica/genética , Ratos
12.
Aquat Toxicol ; 222: 105468, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32199137

RESUMO

The extracellular matrix (ECM) is a non-cellular and three-dimensional structure, constituted by a macromolecular dynamic network that involves the cells in all animal tissues, including embryonic ones. Several studies with vertebrates and cell cultures have reported deleterious effects of ultraviolet-B (UVB) radiation on the components associated with the ECM. However, studies focusing on the UVB radiation effects on ECM components of crustaceans during embryonic development are very scarce. Thus, the aim of this study was to identify the coding sequences of components associated with the ECM and to evaluate the effect of UVB radiation on embryos of the ecologically-important decapod Macrobrachium olfersii. To evaluate the modulation of these ECM components during embryonic development, the transcript levels of Col4α1, Itgß, Lamα, Mmp1 and Timp in M. olfersii embryos were analyzed at early developmental stages (E1, E3 and E4), intermediate developmental stage (E7) and late developmental stages (E10 and E14). In addition, embryos at E7, which correspond to a landmark of crustacean development, were analyzed after 12 h of UVB exposure to verify UVB effects on the ECM components. The ECM component sequences were similar to other decapods, suggesting conservation of these genes among crustaceans. The results showed modulations of the ECM components of M. olfersii embryos that reflect the need for each component in the cellular mechanisms, necessary for normal embryonic development. After UVB exposure, embryos showed opacity of embryonic tissues and it was found the overexpression of Col4α1, Itgß, Mmp1 and Timp transcript levels (1.82-, 1.52-, 2.34- and 6.27-fold, respectively). These impairments can compromise important events for normal embryonic development, such as growth of optic lobes, caudal papilla, ramification of appendages and differentiation of organic systems. The results presented here, together with the effects on morphology, cell proliferation, differentiation, and apoptosis demonstrated previously, strengthen the knowledge of the complex impacts of UVB radiation on freshwater embryos. Nevertheless, our results encourage further investigations focusing on the assessment of UVB effects on different organisms in order to better understand the myriad of UVB effects on ECM components.


Assuntos
Embrião não Mamífero/efeitos da radiação , Desenvolvimento Embrionário/efeitos da radiação , Matriz Extracelular/efeitos da radiação , Palaemonidae/efeitos da radiação , Transcrição Genética/efeitos da radiação , Raios Ultravioleta , Animais , Apoptose/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/genética , Matriz Extracelular/genética , Água Doce/química , Palaemonidae/genética , Palaemonidae/crescimento & desenvolvimento
13.
Artigo em Inglês | MEDLINE | ID: mdl-32130070

RESUMO

Cellular communication network (CCN) proteins are matricellular proteins that coordinate signaling among extracellular matrix, secreted proteins, and cell surface receptors. Their specific in vivo function is context-dependent, but they play profound roles in pathological conditions, such as fibrosis and cancers. Anti-CCN therapies are in clinical consideration. Only recently, however, has the function of these complex molecules begun to emerge. This review summarizes and interprets our current knowledge regarding these fascinating molecules and provides experimental evidence for their utility as therapeutic targets.


Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Comunicação Celular , Microambiente Celular , Matriz Extracelular/metabolismo , Junções Intercelulares/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Proteínas de Sinalização Intercelular CCN/genética , Matriz Extracelular/genética , Matriz Extracelular/patologia , Fibrose , Regulação Neoplásica da Expressão Gênica , Humanos , Junções Intercelulares/genética , Junções Intercelulares/patologia , Neoplasias/genética , Neoplasias/patologia , Microambiente Tumoral
14.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118695, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32169420

RESUMO

Cardiac fibroblasts (CFs) are necessary to maintain extracellular matrix (ECM) homeostasis in the heart. Normally, CFs are quiescent and secrete small amounts of ECM components, whereas, in pathological conditions, they differentiate into more active cells called cardiac myofibroblasts (CMF). CMF conversion is characteristic of cardiac fibrotic diseases, such as heart failure and diabetic cardiomyopathy. TGF-ß1 is a key protein involved in CMF conversion. SMADs are nuclear factor proteins activated by TGF-ß1 that need other proteins, such as forkhead box type O (FoxO) family members, to promote CMF conversion. FoxO1, a member of this family protein, is necessary for TGF-ß1-induced CMF conversion, whereas the role of FoxO3a, another FoxO family member, is unknown. FoxO3a plays an important role in many fibrotic processes in the kidney and lung. However, the participation of FoxO3a in the conversion of CFs into CMF is not clear. In this paper, we demonstrate that TGF-ß1 decreases the activation and expression of FoxO3a in CFs. FoxO3a regulation by TGF-ß1 requires activated SMAD3, ERK1/2 and Akt. Furthermore, we show that FoxO1 is crucial in the FoxO3a regulation induced by TGF-ß1, as shown by overexpressed FoxO1 enhancing and silenced FoxO1 suppressing the effects of TGF-ß1 on FoxO3a. Finally, the regulation of TGF-ß1-induced CMF conversion was enhanced by FoxO3a silencing and suppressed by inhibited FoxO3a degradation. Considering these collective findings, we suggest that FoxO3a acts as a negative regulator of the CMF conversion that is induced by TGF-ß1.


Assuntos
Proteína Forkhead Box O3/genética , Miocárdio/metabolismo , Proteína Smad3/genética , Fator de Crescimento Transformador beta1/genética , Animais , Diferenciação Celular/genética , Matriz Extracelular/genética , Proteína Forkhead Box O3/antagonistas & inibidores , Inativação Gênica , Homeostase/genética , Humanos , Miocárdio/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Cultura Primária de Células , Ratos
15.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118703, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32179057

RESUMO

The heart contains an abundant fibroblast population that may play a role in homeostasis, by maintaining the extracellular matrix (ECM) network, by regulating electrical impulse conduction, and by supporting survival and function of cardiomyocytes and vascular cells. Despite an explosion in our understanding of the role of fibroblasts in cardiac injury, the homeostatic functions of resident fibroblasts in adult hearts remain understudied. TGF-ß-mediated signaling through the receptor-activated Smads, Smad2 and Smad3 critically regulates fibroblast function. We hypothesized that baseline expression of Smad2/3 in fibroblasts may play an important role in cardiac homeostasis. Smad2 and Smad3 were constitutively expressed in normal mouse hearts and in cardiac fibroblasts. In cultured cardiac fibroblasts, Smad2 and Smad3 played distinct roles in regulation of baseline ECM gene synthesis. Smad3 knockdown attenuated collagen I, collagen IV and fibronectin mRNA synthesis and reduced expression of the matricellular protein thrombospondin-1. Smad2 knockdown on the other hand attenuated expression of collagen V mRNA and reduced synthesis of fibronectin, periostin and versican. In vivo, inducible fibroblast-specific Smad2 knockout mice and fibroblast-specific Smad3 knockout mice had normal heart rate, preserved cardiac geometry, ventricular systolic and diastolic function, and normal myocardial structure. Fibroblast-specific Smad3, but not Smad2 loss modestly but significantly reduced collagen content. Our findings suggest that fibroblast-specific Smad3, but not Smad2, may play a role in regulation of baseline collagen synthesis in adult hearts. However, at least short term, these changes do not have any impact on homeostatic cardiac function.


Assuntos
Matriz Extracelular/genética , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Animais , Colágeno/biossíntese , Colágeno/genética , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Frequência Cardíaca/genética , Homeostase/genética , Humanos , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Transdução de Sinais/genética
16.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118693, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198023

RESUMO

Talin2 plays an important role in transduction of mechanical signals between extracellular matrix and actin cytoskeleton. Recent studies showed that talin2 is localized to invadopodia and regulates their maturation, subsequently cancer cell invasion and metastasis. However, the molecular mechanism whereby talin2 mediates invadopodium maturation is unknown. Here we show that ablation of talin2 in MDA-MB-231 cells inhibited the secretion of matrix metallopeptidase 9 (MMP9), a proteinase involved in extracellular matrix degradation in invadopodium maturation and metastasis. Furthermore, re-expression of talin2WT in talin2-KO cells rescued MMP9 secretion, but talin2S339C, a mutant with reduced ß-integrin binding, did not, indicating that the talin2-ß-integrin interaction is involved in the MMP9 secretion. Moreover, ablation of talin2 caused an accumulation of enlarged MMP9 vesicles. These vesicles co-localized with enlarged early, late endosomes and autophagosomes, suggesting talin2 controls MMP9 trafficking process. Therefore, these data suggest that talin2 regulates extracellular matrix degradation and invadopodium maturation by mediating MMP9 secretion.


Assuntos
Metaloproteinase 9 da Matriz/genética , Podossomos/genética , Talina/genética , Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Podossomos/fisiologia , Ligação Proteica/genética , Transporte Proteico/genética
17.
Cancer Sci ; 111(4): 1047-1057, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32060987

RESUMO

The roles of cancer-associated fibroblasts (CAF) in the progression of various types of cancers are well established. CAF promote cancer progression through pleiotropic mechanisms, including the secretion of soluble factors and extracellular matrix, physical interactions with cancer cells, and the regulation of angiogenesis, immunity and metabolism. Their contribution to therapeutic resistance is also well appreciated. Therefore, CAF have been considered as a therapeutic target in cancer. However, recent studies in autochthonous pancreatic cancer models suggest that specific subset(s) of CAF exhibit cancer-restraining roles, indicating that CAF are functionally and molecularly heterogeneous, which is supported by recent single-cell transcriptome analyses. While cancer-promoting CAF (pCAF) have been extensively studied, the nature and specific marker(s) of cancer-restraining CAF (rCAF) have remained uncharacterized. Interestingly, a recent study provided insight into the nature of rCAF and suggested that they may share molecular properties with pancreatic stellate cells (PSC) and mesenchymal stem/stromal cells (MSC). Complicating this finding is that PSC and MSC have been shown to promote the formation of a tumor-permissive and tumor-promoting environment in xenograft tumor models. However, these cells undergo significant transcriptional and epigenetic changes during ex vivo culture, which confounds the interpretation of experimental results based on the use of cultured cells. In this short review, we describe recent studies and hypotheses on the identity of rCAF and discuss their analogy to fibroblasts that suppress fibrosis in fibrotic diseases. Finally, we discuss how these findings can be exploited to develop novel anticancer therapies in the future.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Transformação Celular Neoplásica/genética , Neoplasias/genética , Neovascularização Patológica/genética , Animais , Fibroblastos Associados a Câncer/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Matriz Extracelular/genética , Xenoenxertos , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Neoplasias/patologia , Neovascularização Patológica/patologia , Células Estreladas do Pâncreas/metabolismo , Microambiente Tumoral/genética
18.
Am J Med Sci ; 359(2): 79-83, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32039769

RESUMO

BACKGROUND: The hexosamine biosynthesis pathway (HBP) is hypothesized to mediate many of the adverse effects of hyperglycemia. We have shown previously that increased flux through this pathway leads to induction of the growth factor transforming growth factor-α (TGF-α) and to insulin resistance in cultured cells and transgenic mice. TGF-ß is regulated by glucose and is involved in the development of diabetic nephropathy. We therefore hypothesized that the HBP was involved in the regulation of TGF-ß by glucose in rat vascular and kidney cells. METHODS: A plasmid containing the promoter region of TGF-ß1 cloned upstream of the firefly luciferase gene was electroporated into rat aortic smooth muscle, mesangial, and proximal tubule cells. Luciferase activity was measured in cellular extracts from cells cultured in varying concentrations of glucose and glucosamine. RESULTS: Glucose treatment of all cultured cells led to a time- and dose-dependent stimulation in TGF-ß1 transcriptional activity, with high (20 mM) glucose causing a 1.4- to 2.0-fold increase. Glucose stimulation did not occur until after 12 hours and disappeared after 72 hours of treatment. Glucosamine was more potent than glucose, with 3 mM stimulating up to a 4-fold increase in TGFß1-transcriptional activity. The stimulatory effect of glucosamine was also dose-dependent but was slower to develop and longer lasting than that of glucose. CONCLUSIONS: The metabolism of glucose through the HBP mediates extracellular matrix production, possibly via the stimulation of TGF-ß in kidney cells. Hexosamine metabolism therefore, may play a role in the development of diabetic nephropathy.


Assuntos
Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Hexosaminas/biossíntese , Túbulos Renais Proximais/metabolismo , Células Mesangiais/metabolismo , Transcrição Genética/efeitos dos fármacos , Fator de Crescimento Transformador beta1/biossíntese , Animais , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Glucose/metabolismo , Hexosaminas/genética , Humanos , Túbulos Renais Proximais/patologia , Células Mesangiais/patologia , Camundongos , Camundongos Transgênicos , Ratos , Fatores de Tempo , Fator de Crescimento Transformador beta1/genética
19.
Proc Natl Acad Sci U S A ; 117(7): 3748-3758, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015106

RESUMO

Increased expression of extracellular matrix (ECM) proteins in circulating tumor cells (CTCs) suggests potential function of cancer cell-produced ECM in initiation of cancer cell colonization. Here, we showed that collagen and heat shock protein 47 (Hsp47), a chaperone facilitating collagen secretion and deposition, were highly expressed during the epithelial-mesenchymal transition (EMT) and in CTCs. Hsp47 expression induced mesenchymal phenotypes in mammary epithelial cells (MECs), enhanced platelet recruitment, and promoted lung retention and colonization of cancer cells. Platelet depletion in vivo abolished Hsp47-induced cancer cell retention in the lung, suggesting that Hsp47 promotes cancer cell colonization by enhancing cancer cell-platelet interaction. Using rescue experiments and functional blocking antibodies, we identified type I collagen as the key mediator of Hsp47-induced cancer cell-platelet interaction. We also found that Hsp47-dependent collagen deposition and platelet recruitment facilitated cancer cell clustering and extravasation in vitro. By analyzing DNA/RNA sequencing data generated from human breast cancer tissues, we showed that gene amplification and increased expression of Hsp47 were associated with cancer metastasis. These results suggest that targeting the Hsp47/collagen axis is a promising strategy to block cancer cell-platelet interaction and cancer colonization in secondary organs.


Assuntos
Plaquetas/metabolismo , Neoplasias da Mama/metabolismo , Colágeno/metabolismo , Proteínas de Choque Térmico HSP47/metabolismo , Células Neoplásicas Circulantes/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Transição Epitelial-Mesenquimal , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Amplificação de Genes , Proteínas de Choque Térmico HSP47/genética , Humanos , Camundongos SCID , Metástase Neoplásica
20.
Mol Biol Rep ; 47(3): 2149-2159, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32072402

RESUMO

Medial degeneration of aorta wall is the principal feature of aortic dissection (AD). Sirtuin 1 (SIRT1) plays essential protective effect on many aortic-associated disease. However, it is still unclear whether SIRT1participates in the process of medial degeneration-mediated AD. The purpose of this study is to explore the association between SIRT1 and AD process. qRT-PCR was used to evaluate the transcriptional level of genes involved in study. Protein levels and acetylation detection were measured by Western blotting. The regulatory relations between AP-1 and decorin was assessed by luciferase reporter gene assay. Acute aortic dissection (AAD) mice model was constructed by feeding with ß-aminopropionitrile monofumarate (BAPN). Haematoxylin and eosin (HE) and Mallory staining were performed for pathological analysis. In clinical aorta tissue of thoracic aortic dissection (TAD), the expression of SIRT1, activator protein 1 (AP-1) and decorin were in accordant trend. AP-1 expression which acts on Decorin promoter region is possibly regulated in a SIRT1-mediated deacetylation dependent manner. Resveratrol or SRT1720-initiated SIRT1 activation ameliorated BAPN-induced AAD symptoms accompanied by the activation of AP-1/decorin signaling and decorin-mediated programmed cell death 4 (PDCD4) expression by inhibiting miR-21 and miR-181b. These data suggest that SIRT1/AP-1/decorin signal cascades possibly play a part role in the process of AD. Our research demonstrate that activation of SIRT1 protects against AAD symptoms by enhancing AP-1-mediated decorin expression and downstream PDCD4 signaling pathway. Possibly, SIRT1 is served as a protective factor of AD and targeting SIRT1 therapy might be an attractive therapeutic approaches for AD treatment.


Assuntos
Aneurisma Dissecante/genética , Aneurisma Dissecante/metabolismo , Proteínas Reguladoras de Apoptose/genética , Decorina/metabolismo , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Fator de Transcrição AP-1/metabolismo , Acetilação , Aneurisma Dissecante/patologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Decorina/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Camundongos , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição AP-1/genética
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