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1.
J Vis Exp ; (163)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-33044462

RESUMO

Laminin-111 (Ln1) is an essential part of the extracellular matrix in epithelia, muscle and neural systems. We have previously demonstrated that the microstructure of Ln1 alters the way that it signals to cells, possibly because Ln1 assembly into networks exposes different adhesive domains. In this protocol, we describe three methods to generate polymerized Ln1.


Assuntos
Laminina/metabolismo , Transdução de Sinais , Matriz Extracelular/metabolismo , Fractais , Laminina/química , Polimerização
2.
Sci Rep ; 10(1): 10192, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576846

RESUMO

Tumour spheroids have the potential to be used as preclinical chemo-sensitivity assays. However, the production of three-dimensional (3D) tumour spheroids remains challenging as not all tumour cell lines form spheroids with regular morphologies and spheroid transfer often induces disaggregation. In the field of pancreatic cancer, the MiaPaCa-2 cell line is an interesting model for research but it is known for its difficulty to form stable spheroids; also, when formed, spheroids from this cell line are weak and arduous to manage and to harvest for further analyses such as multiple staining and imaging. In this work, we compared different methods (i.e. hanging drop, round-bottom wells and Matrigel embedding, each of them with or without methylcellulose in the media) to evaluate which one allowed to better overpass these limitations. Morphometric analysis indicated that hanging drop in presence of methylcellulose leaded to well-organized spheroids; interestingly, quantitative PCR (qPCR) analysis reflected the morphometric characterization, indicating that same spheroids expressed the highest values of CD44, VIMENTIN, TGF-ß1 and Ki-67. In addition, we investigated the generation of MiaPaCa-2 spheroids when cultured on substrates of different hydrophobicity, in order to minimize the area in contact with the culture media and to further improve spheroid formation.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Pancreáticas/patologia , Esferoides Celulares/patologia , Linhagem Celular Tumoral , Colágeno/metabolismo , Meios de Cultura/metabolismo , Combinação de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Laminina/metabolismo , Metilcelulose/química , Proteoglicanas/metabolismo
3.
Arch Biochem Biophys ; 689: 108443, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32485152

RESUMO

Human peroxidasin 1 (PXDN) is a homotrimeric multidomain heme peroxidase and essential for tissue development and architecture. It has a biosynthetic function and catalyses the hypobromous acid-mediated formation of specific covalent sulfilimine (SN) bonds, which cross-link type IV collagen chains in basement membranes. Currently, it is unknown whether and which domain(s) [i.e. leucine-rich repeat domain (LRR), immunoglobulin domains, peroxidase domain, von Willebrand factor type C domain] of PXDN interact with the polymeric networks of the extracellular matrix (ECM), and how these interactions integrate and regulate the enzyme's cross-linking activity, without imparting oxidative damage to the ECM. In this study, we probed the interactions of four PXDN constructs with different domain compositions with components of a basement membrane extract by immunoprecipitation. Strong binding of the LRR-containing construct was detected with the major ECM protein laminin. Analysis of these interactions by surface plasmon resonance spectroscopy revealed similar kinetics and affinities of binding of the LRR-containing construct to human and murine laminin-111, with calculated dissociation constants of 1.0 and 1.5 µM, respectively. The findings are discussed with respect to the recently published in-solution structures of the PXDN constructs and the proposed biological role of this peroxidase.


Assuntos
Membrana Basal/metabolismo , Laminina/metabolismo , Peroxidases/metabolismo , Animais , Células HEK293 , Humanos , Leucina/química , Leucina/metabolismo , Camundongos , Peroxidases/química , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo
4.
Am J Physiol Renal Physiol ; 318(6): F1520-F1530, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390516

RESUMO

Tensin2 (Tns2), an integrin-linked protein, is enriched in podocytes within the glomerulus. Previous studies have revealed that Tns2-deficient mice exhibit defects of the glomerular basement membrane (GBM) soon after birth in a strain-dependent manner. However, the mechanisms for the onset of defects caused by Tns2 deficiency remains unidentified. Here, we aimed to determine the role of Tns2 using newborn Tns2-deficient mice and murine primary podocytes. Ultrastructural analysis revealed that developing glomeruli during postnatal nephrogenesis exhibited abnormal GBM processing due to ectopic laminin-α2 accumulation followed by GBM thickening. In addition, analysis of primary podocytes revealed that Tns2 deficiency led to impaired podocyte-GBM interaction and massive expression of laminin-α2 in podocytes. Our study suggests that weakened podocyte-GBM interaction due to Tns2 deficiency causes increased mechanical stress on podocytes by continuous daily filtration after birth, resulting in stressed podocytes ectopically producing laminin-α2, which interrupts GBM processing. We conclude that Tns2 plays important roles in the podocyte-GBM interaction and maintenance of the glomerular filtration barrier.


Assuntos
Membrana Basal Glomerular/metabolismo , Taxa de Filtração Glomerular , Podócitos/metabolismo , Tensinas/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Adesão Celular , Células Cultivadas , Membrana Basal Glomerular/ultraestrutura , Laminina/genética , Laminina/metabolismo , Camundongos Knockout , Podócitos/ultraestrutura , Estresse Mecânico , Tensinas/deficiência , Tensinas/genética
5.
Proc Natl Acad Sci U S A ; 117(19): 10131-10141, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32350144

RESUMO

Over the course of the aging process, fibroblasts lose contractility, leading to reduced connective-tissue stiffness. A promising therapeutic avenue for functional rejuvenation of connective tissue is reprogrammed fibroblast replacement, although major hurdles still remain. Toward this, we recently demonstrated that the laterally confined growth of fibroblasts on micropatterned substrates induces stem-cell-like spheroids. In this study, we embedded these partially reprogrammed spheroids in collagen-I matrices of varying densities, mimicking different three-dimensional (3D) tissue constraints. In response to such matrix constraints, these spheroids regained their fibroblastic properties and sprouted to form 3D connective-tissue networks. Interestingly, we found that these differentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and actomyosin contractility. In addition, the rejuvenated fibroblasts show increased matrix protein (fibronectin and laminin) deposition and collagen remodeling compared to the parental fibroblast tissue network. Furthermore, we show that the partially reprogrammed cells have comparatively open chromatin compaction states and may be more poised to redifferentiate into contractile fibroblasts in 3D-collagen matrix. Collectively, our results highlight efficient fibroblast rejuvenation through laterally confined reprogramming, which has important implications in regenerative medicine.


Assuntos
Diferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Medicina Regenerativa , Rejuvenescimento/fisiologia , Idoso , Animais , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Laminina/metabolismo , Camundongos , Células NIH 3T3 , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
6.
Life Sci ; 255: 117763, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32389831

RESUMO

AIMS: To explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration and the possibility of cotransplantation hUCMSCs and endothelial cells (ECs) for angiogenesis and pulp regeneration in vivo. MATERIALS AND METHODS: hUCMSCs and human umbilical vein endothelial cells (HUVECs) were cocultured for matrigel angiogenesis assay in vitro and Matrigel plug assay in vivo. Next, we used the transwell coculture system to coculture hUCMSCs and HUVECs in vitro for RNA- sequencing (RNA-seq). Last, encapsulated hUCMSCs and HUVECs in scaffolds were injected into the root segments, and transplanted into immunodeficient mice for dental pulp regeneration. KEY FINDINGS: In vitro Matrigel angiogenesis assay and in vivo Matrigel plug assay indicated that cocultured hUCMSCs and HUVECs promote vascular formation of HUVECs, especially in 1:5 (hUCMSCs:HUVECs) coculture group. The RNA-seq result indicated that cocultured HUVECs exhibited high Hif-1 signaling pathway activity. We performed the cell transfection assay to knock down HIF1A-AS2 in HUVECs and then coculture with hUCMSCs, and the expression of VEGFA, HIF1A and PECAM1 were reduced. In pulp regeneration assay, Cotransplantation of hUCMSCs and HUVECs (1,5) group showed pulp-like tissue regeneration. SIGNIFICANCE: Cocultured hUCMSCs and HUVECs can promote vascular formation of HUVECs, and the optimal coculture ration is 1:5 (hUCMSCs:HUVECs). hUCMSCs promote angiogenesis of HUVECs through the long noncoding RNA HIF1A-AS2-activation of the Hif-1 signaling pathway. Cotransplantation of hUCMSCs and HUVECs can regenerate dental pulp-like tissue in vivo.


Assuntos
Polpa Dentária/metabolismo , Células Endoteliais da Veia Umbilical Humana/transplante , Transplante de Células-Tronco Mesenquimais/métodos , Neovascularização Fisiológica/fisiologia , Animais , Técnicas de Cocultura , Colágeno/metabolismo , Polpa Dentária/citologia , Combinação de Medicamentos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Fator 1 Induzível por Hipóxia/metabolismo , Laminina/metabolismo , Camundongos , Proteoglicanas/metabolismo , RNA Longo não Codificante/genética , Regeneração/fisiologia , Cordão Umbilical/citologia
7.
J Biosci Bioeng ; 130(1): 98-105, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32278672

RESUMO

Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7ß1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.


Assuntos
Matriz Extracelular/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Fusão Celular , Linhagem Celular , Colágeno/metabolismo , Combinação de Medicamentos , Integrinas/genética , Integrinas/metabolismo , Laminina/metabolismo , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteoglicanas/metabolismo
8.
Sci Rep ; 10(1): 4249, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144297

RESUMO

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) hold great promise for disease modeling and drug discovery. However, PSC-CMs exhibit immature phenotypes in culture, and the lack of maturity limits their broad applications. While physical and functional analyses are generally used to determine the status of cardiomyocyte maturation, they could be time-consuming and often present challenges in comparing maturation-enhancing strategies. Therefore, there is a demand for a method to assess cardiomyocyte maturation rapidly and reproducibly. In this study, we found that Myomesin-2 (Myom2), encoding M-protein, is upregulated postnatally, and based on this, we targeted TagRFP to the Myom2 locus in mouse embryonic stem cells. Myom2-RFP+ PSC-CMs exhibited more mature phenotypes than RFP- cells in morphology, function and transcriptionally, conductive to sarcomere shortening assays. Using this system, we screened extracellular matrices (ECMs) and identified laminin-511/521 as potent enhancers of cardiomyocyte maturation. Together, we developed and characterized a novel fluorescent reporter system for the assessment of cardiomyocyte maturation and identified potent maturation-enhancing ECMs through this simple and rapid assay. This system is expected to facilitate use of PSC-CMs in a variety of scientific and medical investigations.


Assuntos
Biomarcadores , Diferenciação Celular , Expressão Gênica , Genes Reporter , Laminina/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cálcio , Diferenciação Celular/genética , Biologia Computacional/métodos , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Laminina/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Sarcômeros/metabolismo , Transcrição Genética , Transcriptoma
9.
Am J Respir Cell Mol Biol ; 63(1): 104-117, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32160015

RESUMO

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.


Assuntos
Membrana Basal/fisiopatologia , Células Endoteliais/fisiologia , Hipertensão Pulmonar Primária Familiar/fisiopatologia , Artéria Pulmonar/fisiopatologia , Adulto , Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Feminino , Humanos , Laminina/metabolismo , Masculino , Proteoglicanas/metabolismo , Artéria Pulmonar/metabolismo
10.
Genes Dev ; 34(7-8): 560-579, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32139421

RESUMO

Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Proteínas Cromossômicas não Histona/genética , Deleção de Genes , Distrofia Muscular de Emery-Dreifuss/genética , Animais , Sítios de Ligação/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/patologia , Cromatina/genética , Modelos Animais de Doenças , Genoma Helmíntico/genética , Laminina/genética , Laminina/metabolismo , Músculos/fisiopatologia , Distrofia Muscular de Emery-Dreifuss/fisiopatologia , Mutação , Estrutura Terciária de Proteína/genética , Sarcômeros/química , Sarcômeros/genética , Transcrição Genética/genética
11.
Mol Med Rep ; 21(3): 1491-1500, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016453

RESUMO

Lumbar laminectomy is commonly deemed as the most valid surgery for a series of lumbar illnesses, such as lumbar disc herniation, which could lead to spinal canal stenosis. However, epidural fibrosis is one of the most common complications that limits the application of lumbar laminectomy, which is mainly caused by proliferation of local fibroblasts. Laminins are glycoproteins that consist of α, ß and γ chains, which serve a crucial role in biological cell behaviors, such as adhesion, differentiation, migration and proliferation, especially the isoform with the fifth α chain­laminin α5. The PI3K/AKT/mTOR signaling pathway was demonstrated to be associated with various biological functions in cells. The aim of the present study was to explore whether laminin α5 is an important factor in epidural fibrosis by modulating the proliferation of fibroblasts through the activation of PI3K/AKT/mTOR signaling pathway. In the animal model, the results of the hematoxylin­eosin staining, cell counting, Masson's trichrome staining and immunohistochemical staining showed laminin α5 to be positively associated with epidural fibrosis. Furthermore, to verify the assumption that laminin α5 could modulate fibroblast proliferation through the PI3K/AKT/mTOR signal pathway, fibroblasts were transfected with laminin α5­small interfering (si)RNA. The results of western blotting (proliferating cell nuclear antigen and cyclin D1), the Cell Counting Kit­8 and EdU incorporation assays indicated that the proliferative level of fibroblasts decreased, and the expression of phosphorylated (p)­focal adhesion kinase 1, p­AKT and p­mTOR was reduced. Subsequently, laminin α5 was overexpressed and the change in cell proliferation and expression of associated proteins contrasted with that observed in siRNA. The results demonstrated that laminin α5 could interfere the activation of the PI3K/AKT/mTOR signaling pathway. Finally, the inhibition of the PI3K/AKT/mTOR signaling pathway by LY294002 resulted in decreased fibroblast proliferation. In conclusion, laminin α5 could modulate fibroblast proliferation in epidural fibrosis through the PI3K/AKT/mTOR signaling pathway.


Assuntos
Dura-Máter/metabolismo , Dura-Máter/patologia , Laminina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Biomarcadores , Biópsia , Proliferação de Células , Células Cultivadas , Fibroblastos/metabolismo , Fibrose , Humanos , Laminina/genética , Masculino , Ratos
12.
Brain Struct Funct ; 225(2): 805-816, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32072250

RESUMO

Evidence suggests that extracellular matrix molecules of perivascular basal laminae help orchestrate the molecular assemblies at the gliovascular interface. Specifically, laminin and agrin are thought to tether the dystrophin-associated protein (DAP) complex to the astrocytic basal lamina. This complex includes α-syntrophin (α-Syn), which is believed to anchor aquaporin-4 (AQP4) to astrocytic endfoot membrane domains. We have previously shown that the size of the perivascular AQP4 pool differs considerably between brain regions in an α-Syn-dependent manner. Also, both AQP4 and α-Syn occur at higher densities in endfoot membrane domains facing pericytes than in endfoot membrane domains facing endothelial cells. The heterogeneous distribution of AQP4 at the regional and capillary level has been attributed to a direct interaction between AQP4 and α-Syn. This would be challenged (1) if the microdistributions of laminin and agrin fail to align with those of DAP and AQP4 and (2) if targeted deletion of α-Syn leads to a loss of laminin and/or agrin. Here, we provide the first detailed and quantitative analysis of laminin and agrin in brain basal laminae of mice. We show that the microdistributions of these molecules vary in a fashion that is well aligned with the previously reported microdistribution of AQP4. We also demonstrate that the expression patterns of laminin and agrin are insensitive to targeted deletion of α-Syn, suggesting that α-Syn deletion affects AQP4 directly and not indirectly via laminin or agrin. These data fill remaining voids in the current model of how key molecules are assembled and tethered at the gliovascular interface.


Assuntos
Agrina/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Matriz Extracelular/metabolismo , Laminina/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Capilares/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , RNA Mensageiro/metabolismo
13.
BMC Res Notes ; 13(1): 90, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32085798

RESUMO

OBJECTIVES: The laminins (LM) are a family of basement membranes glycoproteins with essential roles in supporting epithelia, endothelia, nerves and muscle adhesion, and in regulating a range of processes including cell migration, stem cell maintenance and differentiation. However, surprisingly little is known about the mechanisms of turnover and remodelling of LM networks due to lack of appropriate tools to study these processes at the necessary resolution. Recently, the nematode C. elegans ortholog of human the LMß1 chain was labelled at the C-terminus with the photoconvertible fluorophore Dendra2. Here we used genome editing to establish a similar system in a mammalian cell line as proof of concept for future mammalian models. RESULTS: CRISPR-Cas9 was used to introduce the Dendra2 sequence at the C-terminus of LMß1 in the human lung adenocarcinoma cell line A549. Despite expression of the tagged protein within cells, no detectable LMß1-Dendra2 protein was deposited to the extracellular matrices or conditioned media of edited cells. Moreover, the edited cells displayed reduced proliferation rates. Together, these data suggest that, in humans, addition of C-terminal Dendra2 tag to LMß1 inhibits LM secretion, and is not a viable approach for use in animal models.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Laminina/química , Laminina/metabolismo , Células A549 , Edição de Genes , Humanos
14.
Cell Mol Biol Lett ; 25: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32042281

RESUMO

Background: Precise coordination of cytoskeletal components and dynamic control of cell adhesion and migration are required for crucial cell processes such as differentiation and morphogenesis. We investigated the potential involvement of αII-spectrin, a ubiquitous scaffolding element of the membrane skeleton, in the adhesion and angiogenesis mechanism. Methods: The cell models were primary human umbilical vein endothelial cells (HUVECs) and a human dermal microvascular endothelial cell line (HMEC-1). After siRNA- and shRNA-mediated knockdown of αII-spectrin, we assessed its expression and that of its partners and adhesion proteins using western blotting. The phenotypes of the control and spectrin-depleted cells were examined using immunofluorescence and video microscopy. Capillary tube formation was assessed using the thick gel Matrigel matrix-based method and a microscope equipped with a thermostatic chamber and a Nikon Biostation System camera. Results: Knockdown of αII-spectrin leads to: modified cell shape; actin cytoskeleton organization with the presence of peripheral actin patches; and decreased formation of stress fibers. Spectrin deficiency affects cell adhesion on laminin and fibronectin and cell motility. This included modification of the localization of adhesion molecules, such as αVß3- and α5-integrins, and organization of adhesion structures, such as focal points. Deficiency of αII-spectrin can also affect the complex mechanism of in vitro capillary tube formation, as demonstrated in a model of angiogenesis. Live imaging revealed that impairment of capillary tube assembly was mainly associated with a significant decrease in cell projection length and stability. αII-spectrin depletion is also associated with significantly decreased expression of three proteins involved in capillary tube formation and assembly: VE-cadherin, MCAM and ß3-integrin. Conclusion: Our data confirm the role of αII-spectrin in the control of cell adhesion and spreading. Moreover, our findings further support the participation of αII-spectrin in capillary tube formation in vitro through control of adhesion molecules, such as integrins. This indicates a new function of αII-spectrin in angiogenesis.


Assuntos
Citoesqueleto de Actina/metabolismo , Capilares/metabolismo , Adesão Celular/fisiologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Espectrina/metabolismo , Antígenos CD/metabolismo , Antígeno CD146/metabolismo , Caderinas/metabolismo , Capilares/crescimento & desenvolvimento , Adesão Celular/genética , Diferenciação Celular/genética , Movimento Celular/genética , Forma Celular , Células Endoteliais/citologia , Fibronectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Cadeias alfa de Integrinas/metabolismo , Integrina beta3/metabolismo , Laminina/metabolismo , Morfogênese/genética , Morfogênese/fisiologia , Neovascularização Fisiológica/genética , RNA Interferente Pequeno , Espectrina/deficiência , Espectrina/genética , Fibras de Estresse/metabolismo
15.
Invest Ophthalmol Vis Sci ; 61(2): 9, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32049341

RESUMO

Purpose: Variant B precursor cysteine protease inhibitor cystatin C, a known recessive risk factor for developing exudative age-related macular degeneration (AMD), presents altered intracellular trafficking and reduced secretion from retinal pigment epithelial (RPE) cells. Because cystatin C inhibits multiple extracellular matrix (ECM)-degrading cathepsins, this study evaluated the role of this mutation in inducing ECM-related functional changes in RPE cellular behavior. Methods: Induced pluripotent stem cells gene-edited bi-allelically by CRISPR/Cas9 to express the AMD-linked cystatin C variant were differentiated to RPE cells and assayed for their ability to degrade fluorescently labeled ECM proteins. Cellular migration and adhesion on multiple ECM proteins, differences in transepithelial resistance and polarized protein secretion were tested. Vessel formation induced by gene edited cells-conditioned media was quantified using primary human dermal microvascular epithelial cells. Results: Variant B cystatin C-expressing induced pluripotent stem cells-derived RPE cells displayed a significantly higher rate of laminin and fibronectin degradation 3 days after seeding on fluorescently labeled ECM (P < 0.05). Migration on matrigel, collagen IV and fibronectin was significantly faster for edited cells compared with wild-type (WT) cells. Both edited and WT cells displayed polarized secretion of cystatin C, but transepithelial resistance was lower in gene-edited cells after 6 weeks culture, with significantly lower expression of tight junction protein claudin-3. Media conditioned by gene-edited cells stimulated formation of significantly longer microvascular tubes (P < 0.05) compared with WT-conditioned media. Conclusions: Reduced levels of cystatin C lead to changes in the RPE ability to degrade, adhere, and migrate supporting increased invasiveness and angiogenesis relevant for AMD pathology.


Assuntos
Cistatina C/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/citologia , Movimento Celular/fisiologia , Células Cultivadas , Cistatina C/genética , Cistatina C/metabolismo , Fibronectinas/metabolismo , Edição de Genes , Humanos , Laminina/metabolismo , Mutação Puntual/genética
16.
J Neurochem ; 153(3): 377-389, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31950499

RESUMO

PrPC is a glycoprotein capable to interact with several molecules and mediates diverse signaling pathways. Among numerous ligands, laminin (LN) is known to promote neurite outgrowth and memory consolidation, while amyloid-beta oligomers (Aßo) trigger synaptic dysfunction. In both pathways, mGluR1 is recruited as co-receptor. The involvement of PrPC /mGluR1 in these opposite functions suggests that this complex is a key element in the regulation of synaptic activity. Considering that sleep-wake cycle is important for synaptic homeostasis, we aimed to investigate how sleep deprivation affects the expression of PrPC and its ligands, laminin, Aßo, and mGluR1, a multicomplex that can interfere with neuronal plasticity. To address this question, hippocampi of control (CT) and sleep deprived (SD) C57BL/6 mice were collected at two time points of circadian period (13 hr and 21 hr). We observed that sleep deprivation reduced PrPC and mGluR1 levels with higher effect in active state (21 hr). Sleep deprivation also caused accumulation of Aß peptides in rest period (13 hr), while laminin levels were not affected. In vitro binding assay showed that Aßo can compete with LN for PrPC binding. The influence of Aßo was also observed in neuritogenesis. LN alone promoted longer neurite outgrowth than non-treated cells in both Prnp+/+ and Prnp0/0 genotypes. Aßo alone did not show any effects, but when added together with LN, it attenuated the effects of LN only in Prnp+/+ cells. Altogether, our findings indicate that sleep deprivation regulates the availability of PrPC and Aß peptides, and based on our in vitro assays, these alterations induced by sleep deprivation can negatively affect LN-PrPC interaction, which is known to play roles in neuronal plasticity.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Laminina/metabolismo , Plasticidade Neuronal/fisiologia , Proteínas PrPC/metabolismo , Privação do Sono/metabolismo , Animais , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout
17.
Biochem Biophys Res Commun ; 524(2): 288-294, 2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-31987497

RESUMO

Successful induction of milk protein synthesis relies on prolactin/STAT5. In mice, both laminin and ß1 integrin were necessary for STAT5 activity induced by prolactin treatment, resulting in transcriptional activation of ß-casein. However, the mechanism by which ß1 integrin increases the bovine milk protein synthesis is not well known. In order to display the crosstalk between integrin signaling and lactogenic signaling, we investigated the mechanism by which laminin mediated lactogenic effects via interaction with ß1 integrin on bovine mammary epithelial cells (BMECs). Therefore, localization of ß1 integrin was examined by immunofluorescence. The mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting. The results showed that ß1 integrin were detected in basal mammary cells and basal membrane surface of adherent BMECs. However, basal distribution of ß1 integrin was not sufficient to increase ß-casein synthesis in the absence of integrin activation by laminin. A lactogenic hormone cocktail of insulin, hydrocortisone, and prolactin stimulated overall lactogenic effects, including upregulated expression of ß1 integrin, activation of prolactin/STAT5 signaling, and consequent increase of ß-casein synthesis. In response to a 24 h prolactin treatment, the abundance of STAT5, ß1 integrin, and ß-casein in BMECs with laminin was higher compared to that with a control substratum. Meanwhile, laminin-dependent lactogenic effects were inhibited by blocking ß1 integrin function, resulting in attenuated STAT5 activity and decreased ß-casein synthesis. These results indicated that ß1 integrin was a key mediator of the laminin-dependent prolactin/STAT5 signaling, which regulated the sustained STAT5 activity necessary for ß-casein expression in BMECs.


Assuntos
Bovinos/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Proteínas do Leite/metabolismo , Prolactina/metabolismo , Fator de Transcrição STAT5/metabolismo , Animais , Células Epiteliais/metabolismo , Feminino , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Biossíntese de Proteínas , Transdução de Sinais
18.
Cells ; 9(2)2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31991580

RESUMO

During embryonic development, the heart undergoes complex morphogenesis from a liner tube into the four chambers consisting of ventricles, atria and valves. At the same time, the cardiomyocytes compact into a dense, aligned, and highly vascularized myocardium. The extracellular matrix (ECM) is known to play an important role in this process but understanding of the expression and organization remains incomplete. Here, we performed 3D confocal imaging of ECM in the left ventricle and whole heart of embryonic chick from stages Hamburger-Hamilton 28-35 (days 5-9) as an accessible model of heart formation. First, we observed the formation of a fibronectin-rich, capillary-like networks in the myocardium between day 5 and day 9 of development. Then, we focused on day 5 prior to vascularization to determine the relative expression of fibronectin, laminin, and collagen type IV. Cardiomyocytes were found to uniaxially align prior to vascularization and, while the epicardium contained all ECM components, laminin was reduced, and collagen type IV was largely absent. Quantification of fibronectin revealed highly aligned fibers with a mean diameter of ~500 nm and interfiber spacing of ~3 µm. These structural parameters (volume, spacing, fiber diameter, length, and orientation) provide a quantitative framework to describe the organization of the embryonic ECM.


Assuntos
Matriz Extracelular/metabolismo , Ventrículos do Coração/embriologia , Coração/embriologia , Animais , Capilares/embriologia , Embrião de Galinha , Colágeno Tipo IV/metabolismo , Fibronectinas/análise , Fibronectinas/metabolismo , Ventrículos do Coração/metabolismo , Laminina/análise , Laminina/metabolismo , Microscopia Confocal , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo
19.
Molecules ; 25(3)2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31979355

RESUMO

Dapagliflozin, empagliflozin, tofogliflozin, selective inhibitors of sodium-glucose cotransporter 2 (SGLT2), is used clinically to reduce circulation glucose levels in patients with type 2 diabetes mellitus by blocking the reabsorption of glucose by the kidneys. Dapagliflozin is metabolized and inactivated by UGT1A9. Empagliflozin is metabolized and inactivated by UGT1A9 and by other related isoforms UGT2B7, UGT1A3, and UGT1A8. Tofogliflozin is metabolized and inactivated by five different enzymes CYP2C18, CYP3A4, CYP3A5, CYP4A11, and CYP4F3. Dapagliflozin treatment of HCT116 cells, which express SGLT2 but not UGT1A9, results in the loss of cell adhesion, whereas HepG2 cells, which express both SGLT2 and UGT1A9, are resistant to the adhesion-related effects of dapagliflozin. PANC-1 and H1792 cells, which do not express either SGLT2 or UGT1A9, are also resistant to adhesion related effects of dapagliflozin. On the other hand, either empagliflozin or tofogliflozin treatment of HCT116, HepG2, PANC-1, and H1792 cells are resistant to the adhesion-related effects as observed in dapagliflozin treated HCT116 cells. Knockdown of UGT1A9 by shRNA in HepG2 cells increased dapagliflozin sensitivity, whereas the overexpression of UGT1A9 in HCT116 cells protected against dapagliflozin-dependent loos of cell adhesion. Dapagliflozin treatment had no effect on cellular interactions with fibronectin, vitronectin, or laminin, but it induced a loss of interaction with collagen I and IV. In parallel, dapagliflozin treatment reduced protein levels of the full-length discoidin domain receptor I (DDR1), concomitant with appearance of DDR1 cleavage products and ectodomain shedding of DDR1. In line with these observations, unmetabolized dapagliflozin increased ADAM10 activity. Dapagliflozin treatment also significantly reduced Y792 tyrosine phosphorylation of DDR1 leading to decrement of DDR1 function and detachment of cancer cells. Concomitant with these lines of results, we experienced that CEA in patients with colon cancer, which express SGLT2 but not UGT1A9, and type 2 diabetes mellitus treated by dapagliflozin in addition to chemotherapy was decreased (case 1). CEA in patients with colon cancer, which express SGLT2 but not UGT1A9, and type 2 diabetes mellitus was treated by dapagliflozin alone after radiation therapy was decreased but started to rise after cessation of dapagliflozin (case 2). CA19-9 in two of patients with pancreatic cancer and type 2 diabetes mellitus was resistant to the combination therapy of dapagliflozin and chemotherapy (case 3 and 4 respectively). PIVKAII in patients with liver cancer and type 2 diabetes mellitus, and CYFRA in patients with squamous lung cancer and type 2 diabetes mellitus was also resistant the combination therapy of dapagliflozin and chemotherapy (case 5 and 6 respectively). Taken together, these data suggest a potential role for dapagliflozin anticancer therapy against colon cancer cells that express SGLT2, but not UGT1A9.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Antineoplásicos/farmacologia , Compostos Benzidrílicos/farmacologia , Adesão Celular/efeitos dos fármacos , Receptor com Domínio Discoidina 1/metabolismo , Glucosídeos/farmacologia , Proteínas de Membrana/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Antígeno Carcinoembrionário/metabolismo , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Fibronectinas/metabolismo , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/metabolismo , Humanos , Laminina/metabolismo , Fosforilação , RNA Interferente Pequeno , Vitronectina/metabolismo
20.
Biochem Biophys Res Commun ; 523(2): 522-526, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-31902587

RESUMO

Isolation of trabecular meshwork stem cells in vitro provides the foundation of a novel treatment for glaucoma. Trabecular meshwork stem cells (TMSCs) of the fetal calve were extracted and cultured for this experiment. TMSCs were isolated through side population cell sorting. TMSCs were then identified using immunofluorescent staining. Extracellular matrix (ECM) expression in TM cells derived from TMSCs was evaluated with Western blot. Our results showed a positive expression of stem cell markers Notch1 and OCT-3/4 in TMSCs, but no TM cells markers TIMP3 or AQP1. In contrast, primary TM cells expressed these TM cell markers but no stem cell markers. Our result confirmed that there are expression of ECM components, such as fibronectin, laminin, collagen I and collagen IV in TM cells differentiated from TMSCs. CONCLUSION: TM cells derived from TMSCs can secrete ECM components which is important for sustain the physiological function.


Assuntos
Matriz Extracelular/metabolismo , Células-Tronco/citologia , Malha Trabecular/citologia , Animais , Biomarcadores/metabolismo , Bovinos , Separação Celular/métodos , Células Cultivadas , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Células-Tronco/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo
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