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1.
PLoS One ; 15(1): e0220019, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31945053

RESUMO

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes ß1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility.


Assuntos
Colágeno/farmacologia , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Adesões Focais/efeitos dos fármacos , Laminina/farmacologia , Mecanotransdução Celular , Proteoglicanas/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colágeno/química , Combinação de Medicamentos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Adesões Focais/ultraestrutura , Expressão Gênica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Laminina/química , Modelos Biológicos , Proteoglicanas/química , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Propriedades de Superfície , Microambiente Tumoral/efeitos dos fármacos
2.
Carbohydr Polym ; 226: 115302, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31582049

RESUMO

Hydrogels could be promising wound healing dressings that maintain a moist environment in the wound site and accelerate wound healing. However, the lack of antibacterial effect, suitable mechanical property and adhesiveness limits their applications. Here, we designed a quaternized chitosan-Matrigel-polyacrylamide (QCS-M-PAM) hydrogel with multi-functions. The morphology, swelling ratio, mechanical test, antimicrobial property, hemostatic performance and biocompatibility of the hybrid hydrogel were investigated in vitro and vivo. The hybrid hydrogel showed a three-dimensional (3D) microporous structure, high swelling ratio, excellent stretchable and compressive property, similar modulus to human skin, good adhesiveness, and low cytotoxicity. The results of histology and molecular testing in vivo demonstrated that the hybrid hydrogel could significantly enhance wound healing, collagen deposition, and induce skin adnexal regeneration by upregulating anti-inflammatory factors, and downregulating proinflammatory factors. Together, the present antibacterial hydrogels with hemostatic and adhesive properties are considered to have promising potential used as wound dressings for full-thickness skin defect.


Assuntos
Resinas Acrílicas/farmacologia , Curativos Hidrocoloides , Quitosana/farmacologia , Colágeno/farmacologia , Hidrogéis/farmacologia , Laminina/farmacologia , Proteoglicanas/farmacologia , Cicatrização/efeitos dos fármacos , Resinas Acrílicas/química , Adesividade , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antifúngicos/química , Antifúngicos/farmacologia , Células Cultivadas , Quitosana/química , Colágeno/química , Combinação de Medicamentos , Humanos , Hidrogéis/química , Laminina/química , Camundongos , Proteoglicanas/química , Cicatrização/fisiologia
3.
Nat Protoc ; 14(11): 3082-3100, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31554955

RESUMO

Blood vessels are fundamental to animal life and have critical roles in many diseases, such as stroke, myocardial infarction and diabetes. The vasculature is formed by endothelial cells that line the vessel and are covered with mural cells, specifically pericytes in smaller vessels and vascular smooth muscle cells (vSMCs) in larger-diameter vessels. Both endothelial cells and mural cells are essential for proper blood vessel function and can be derived from human pluripotent stem cells (hPSCs). Here, we describe a protocol to generate self-organizing 3D human blood vessel organoids from hPSCs that exhibit morphological, functional and molecular features of human microvasculature. These organoids are differentiated via mesoderm induction of hPSC aggregates and subsequent differentiation into endothelial networks and pericytes in a 3D collagen I-Matrigel matrix. Blood vessels form within 2-3 weeks and can be further grown in scalable suspension culture. Importantly, in vitro-differentiated human blood vessel organoids transplanted into immunocompromised mice gain access to the mouse circulation and specify into functional arteries, arterioles and veins.


Assuntos
Vasos Sanguíneos/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Engenharia Tecidual/métodos , Diferenciação Celular , Linhagem Celular , Colágeno/química , Combinação de Medicamentos , Endotélio Vascular/citologia , Humanos , Laminina/química , Microvasos/citologia , Neovascularização Fisiológica , Pericitos/citologia , Proteoglicanas/química , Tecidos Suporte/química
4.
Mater Sci Eng C Mater Biol Appl ; 104: 109964, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499990

RESUMO

Choroidal neovascularization (CNV) is the pathological growth of new blood vessels in the sub-retinal pigment epithelial (RPE) space from the choroid through a break in the Bruch's membrane (BM). Despite its importance in studying biological processes and drug discovery, the development of an in vitro CNV model that achieves the physiological structures of native RPE-BM-choroidal capillaries (CC) is still challenging. Here, we develop a novel 3D RPE-BM-CC complex biomimetic system on an ultra-thin, free-standing nanofiber membrane. The thickness of the pristine nanofiber membrane is 2.17 ±â€¯0.81 µm, and the Matrigel-coated nanofiber membrane attains a permeability coefficient of 2.95 ±â€¯0.25 × 10-6 cm/s by 40 kDa FITC-dextran, which is similar to the physiological value of the native BM. On the in vitro 3D RPE-BM-CC complex system, we demonstrate endothelial cell invasion across the 3D RPE-BM-CC complex and the mechanism of the invasion by imposing a hypoxic condition, which is thought to be the major pathological cause of CNV. Furthermore, alleviation of the invasion is achieved by treating with chrysin and anti-VEGF antibody. Thus, the in vitro 3D RPE-BM-CC complex biomimetic system can recapitulate essential features of the pathophysiological environment and be employed for the screening of drug candidates to reduce the number of costly and time-consuming in vivo tests or clinical trials.


Assuntos
Lâmina Basilar da Corioide/patologia , Neovascularização de Coroide/patologia , Hipóxia/patologia , Nanofibras/química , Biomimética/métodos , Linhagem Celular , Colágeno/química , Combinação de Medicamentos , Células Endoteliais/patologia , Flavonoides/química , Humanos , Laminina/química , Permeabilidade/efeitos dos fármacos , Proteoglicanas/química , Epitélio Pigmentado da Retina/patologia
5.
Biophys Chem ; 254: 106246, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31426023

RESUMO

The inhibitory effect of the flavonoid naringenin on plant and human Two-Pore Channels (TPCs) was assessed by means of electrophysiological measurements. By acting on human TPC2, naringenin, was able to dampen intracellular calcium responses to VEGF in cultured human endothelial cells and to impair angiogenic activity in VEGF-containing matrigel plugs implanted in mice. Molecular docking predicts selective binding sites for naringenin in the TPC structure, thus suggesting a specific interaction between the flavonoid and the channel.


Assuntos
Canais de Cálcio/química , Flavanonas/química , Plantas/metabolismo , Animais , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Colágeno/química , Combinação de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Flavanonas/metabolismo , Humanos , Laminina/química , Camundongos , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/metabolismo , Proteoglicanas/química
6.
Biomater Sci ; 7(9): 3906-3917, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31322163

RESUMO

Cardiovascular diseases represent a major socio-economic burden. In recent years, considerable effort has been invested in optimizing cell delivery strategies to advance cell transplantation therapies to restore heart function for example after an infarct. A particular issue is that the implantation of cells using a non-electroconductive matrix potentially causes arrhythmia. Here, we demonstrate that our hydrazide-functionalized nanotubes-pericardial matrix-derived electroconductive biohybrid hydrogel provides a suitable environment for maturation of human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. hiPSC-derived cardiomyocytes exhibited an improved contraction amplitude (>500%) on conductive hydrogels compared to cells cultured on Matrigel®. This was accompanied by increased cellular alignment, enhanced connexin 43 expression, and improved sarcomere organization suggesting maturation of the hiPSC-derived cardiomyocytes. Sarcomeric length of these cells increased from 1.3 to 1.7 µm. Moreover, 3D cell-laden engineered tissues exhibited enhanced calcium handling as well as positive response to external electrical and pharmaceutical stimulation. Collectively, our data indicate that our biohybrid hydrogels consisting of solubilized nanostructured pericardial matrix and electroconductive positively charged hydrazide-conjugated carbon nanotubes provide a promising material for stem cell-based cardiac tissue engineering.


Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Nanotubos de Carbono/química , Pericárdio/química , Tecidos Suporte/química , Biomarcadores/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Colágeno/química , Conexina 43/metabolismo , Combinação de Medicamentos , Condutividade Elétrica , Humanos , Laminina/química , Células-Tronco Mesenquimais/citologia , Tamanho da Partícula , Proteoglicanas/química
7.
Redox Biol ; 26: 101236, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31181457

RESUMO

Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published. In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 µM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment. A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples.


Assuntos
Misturas Complexas/análise , Fibronectinas/análise , Ácido Hipocloroso/química , Laminina/análise , Animais , Membrana Basal/química , Misturas Complexas/química , Misturas Complexas/isolamento & purificação , Fibronectinas/química , Halogenação , Humanos , Laminina/química , Espectrometria de Massas , Camundongos , Oxirredução , Sensibilidade e Especificidade , Tirosina/química
8.
Comput Biol Chem ; 80: 480-497, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31174160

RESUMO

Laminin-111 is a trimeric glycoprotein of the extracellular matrix (ECM) that holds a significant role in cell adhesion, migration and differentiation. Laminin-111 is the most studied laminin isoform, composed of three chains; α1, ß1 and γ1. Phosphorylation is the most common eukaryotic post - translational modification and has regulatory effect on protein function. Using bioinformatic tools we computationally predicted all the possible phosphorylation sites on human laminin α1-chain sequence (LAMA1) according to kinases binding motifs. Thus, we predicted, for the first time, the possibly responsible kinases for fifteen of the nineteen already published experimentally observed phosphorylated residues in LAMA1. Searching the literature extensively, we recorded all the known functional sites (active sites) in LAMA1. We combined the experimentally observed and predicted phosphorylated residues as well as the active sites in LAMA1, generating an analytic phosphorylation map of human laminin α1-chain, which is useful for further analysis. Our results indicated fourteen kinases that might be important for the phosphorylation of human laminin α1-chain, out of which three kinases with reported ecto-phosphorylation activity (PKA, PKC and CKII) were suggested to have a more significant role. Six cancer associated-active sites were correlated with kinases, three out which were correlated with only the above ecto - kinases.


Assuntos
Laminina/química , Laminina/metabolismo , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Biologia Computacional/métodos , Bases de Dados de Proteínas , Humanos , Fosforilação , Processamento de Proteína Pós-Traducional
9.
J Biosci ; 44(2)2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31180068

RESUMO

Laminins are a major constituent of the extracellular matrix (ECM). Laminin-111, the most extensively studied laminin isoform, consists of the α1, the ß1 and the γ1 chain, and is involved in many cellular processes, like adhesion, migration and differentiation. Given the regulatory role of phosphorylation in protein function, it is important to identify the phosphorylation sites of human laminin ß1-chain sequence (LAMB1). Therefore, we computationally predicted all possible phosphorylation sites in LAMB1. For the first time, we identified the possibly responsible kinases for already in vitro experimentally observed phosphorylated residues in LAMB1. All known functional (active) sites of LAMB1, were recorded after an extensive literature search and combined with the experimentally observed and our predicted phosphorylated residues. This generated a detailed phosphorylation map of LAMB1. Five kinases (PKA, PKC, CKII, CKI and GPCR1) were indicated important, while the role of PKA, PKC and CKII, kinases known for ectophosphorylation activity, was highlighted. The activity of PKA and PKC was associated with the active site RIQNLLKITNLRIKFVKLHTLGDNLLDS. Also, predicted phosphorylations inside two amyloidogenic (DSITKYFQMSLE, VILQHSAADIAR) and two anti-cancerous (YIGSR and PDSGR) sites suggested a possible role in the development of the corresponding diseases.


Assuntos
Biologia Computacional/métodos , Laminina/química , Mapeamento de Peptídeos/métodos , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Caseína Quinase I/química , Caseína Quinase I/metabolismo , Caseína Quinase II/química , Caseína Quinase II/metabolismo , Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/química , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Expressão Gênica , Humanos , Laminina/genética , Laminina/metabolismo , Fosforilação , Proteína Quinase C/química , Proteína Quinase C/metabolismo
10.
Int J Mol Sci ; 20(13)2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31252620

RESUMO

Psoriasis is a chronic inflammatory skin disease characterized by excessive growth of keratinocytes and hyperkeratosis in the epidermis. An abnormality of the non-lesional epidermis at an early stage of psoriasis is involved in triggering inflammatory cell infiltration into the dermis. Integrin α5ß1 acts as a receptor for fibronectin and has been found to be overexpressed in non-lesional psoriatic epidermis. To investigate whether α5ß1 integrin has a potential as a drug target for psoriasis treatment, the α5ß1 integrin-binding peptide, C16, was used to obstruct the HaCat keratinocyte cellular responses induced by fibronectin (Fn) in culture and psoriasis-like skin inflammation induced in mice by imiquimod (IMQ). The C16 exhibited antagonistic activity against α5ß1 integrin in HaCat cells, with evidence of suppression of the Fn-mediated proliferative, cytoskeletal, and inflammatory responses. Topical treatment with C16 greatly reduced the IMQ-induced epidermal hyperplasia, infiltration of neutrophils/macrophages, and expression of pro-inflammatory mediators in mouse skin. The C16SP (C16-derived short peptide; DITYVRLKF) also exhibited antagonistic activity, suppressing α5ß1 integrin activity in culture, and reducing IMQ-induced skin inflammation. Taken together, this study provides the first evidence that α5ß1 integrin may be a potential drug target for psoriasis. The synthetic C16 peptide may serve as an agent for psoriasis therapy.


Assuntos
Anti-Inflamatórios/uso terapêutico , Laminina/química , Fragmentos de Peptídeos/uso terapêutico , Psoríase/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Linhagem Celular , Feminino , Fibronectinas/farmacologia , Humanos , Imiquimode/toxicidade , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Psoríase/etiologia
11.
Neuroscience ; 410: 97-107, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31059743

RESUMO

The benefits of Cochlear implant (CI) technology depend among other factors on the proximity of the electrode array to the spiral ganglion neurons. Laminin, a component of the extracellular matrix, regulates Schwann cell proliferation and survival as well as reorganization of actin fibers within their cytoskeleton, which is necessary for myelination of peripheral axons. In this study we explore the effectiveness of laminin-coated electrodes in promoting neuritic outgrowth from auditory neurons towards the electrode array and the ability to reduce acoustic and electric auditory brainstem response (i.e. aABR and eABR) thresholds. In vitro: Schwann cells and neurites are attracted towards laminin-coated surfaces with longer neuritic processes in laminin-coated dishes compared to uncoated dishes. In vivo: Animals implanted with laminin-coated electrodes experience significant decreases in eABR and aABR thresholds at selected frequencies compared to the results from the uncoated electrodes group. At 1 month post implantation there were a greater number of spiral ganglion neurons and neuritic processes projecting into the scala tympani of animals implanted with laminin-coated electrodes compared to animals with uncoated electrodes. These data suggest that Schwann cells are attracted towards laminin-coated electrodes and promote neuritic outgrowth/ guidance and promote the survival of spiral ganglion neurons following electrode insertion trauma.


Assuntos
Implantes Cocleares/normas , Laminina/administração & dosagem , Neurônios/fisiologia , Órgão Espiral/fisiologia , Animais , Animais Recém-Nascidos , Sobrevivência Celular/fisiologia , Células Cultivadas , Eletrodos Implantados/normas , Laminina/química , Masculino , Órgão Espiral/citologia , Distribuição Aleatória , Ratos , Ratos Endogâmicos BN , Ratos Sprague-Dawley
12.
J Biotechnol ; 298: 35-44, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-30980843

RESUMO

Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesion sequence, or a scrambled VKAIV sequence, were incorporated by genetic engineering in the structure of an ELP, expressed in Escherichia coli and purified. The transition temperatures of the ELP-IKVAV and ELP-VKAIV were determined to be 23 °C. Although the phase transition was fully reversible for ELP-VKAIV, we observed an irreversible aggregation for ELP-IKVAV. The corresponding aggregates shared some characteristics with amyloid-like polypeptides. The two ELPs were then reacted with functionalized polyethylene glycol (PEG) to form hydrogels. These hydrogels were characterized for rheological properties, tested with cultures of rat primary sensory neurons, and implanted subcutaneously in mice for 4 weeks. Sensory neurons cultured on high IKVAV concentration hydrogels (20%) formed longer neurite than those of neurons grown on hydrogels containing the scrambled IKVAV sequence. Finally, in vivo evaluation showed the absence of detectable inflammation. In conclusion, this functionalized ELP-IKVAV biomaterial shows interesting properties for tissue engineering requiring neurotization.


Assuntos
Elastina/química , Hidrogéis/química , Peptídeos/química , Engenharia Tecidual , Sequência de Aminoácidos/genética , Animais , Elastina/genética , Elastina/isolamento & purificação , Elastina/farmacologia , Hidrogéis/farmacologia , Laminina/química , Laminina/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Peptídeos/genética , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Ratos , Reologia , Células Receptoras Sensoriais/química , Células Receptoras Sensoriais/efeitos dos fármacos
13.
Methods Mol Biol ; 1952: 219-232, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30825178

RESUMO

Matrigel is extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma in C57BL/6 mice, a tumor rich in extracellular matrix (ECM) proteins. It consists mainly of laminin (approximately 60%), collagen IV (approximately 30%), and nidogen-1/entactin (approximately 8%), while it also contains heparan sulfate proteoglycans, such as perlecan, other ECM proteins, as well as growth factors bound to the ECM. Matrigel mimics the physiological cell matrix and is the most commonly used matrix substrate to study in vitro and in vivo angiogenesis. Here, we describe the in vivo Matrigel plug assay and how it can be used for both qualitative and quantitative assessment of angiogenesis.


Assuntos
Colágeno/química , Células Endoteliais/citologia , Laminina/química , Glicoproteínas de Membrana/química , Neovascularização Fisiológica , Proteoglicanas/química , Animais , Separação Celular/métodos , Combinação de Medicamentos , Citometria de Fluxo/métodos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Microtomia/métodos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos
14.
Biomed Mater ; 14(3): 035014, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30769335

RESUMO

INTRODUCTION: Calcific aortic valve disease (CAVD) is the most common acquired heart valve disease with complex underlying pathomechanisms that are yet not fully understood. Three-dimensional (3D) cell culture models as opposed to conventional two-dimensional (2D) techniques may reveal new aspects of CAVD and serve as a transitional platform between conventional 2D cell culture and in vivo experiments. METHODS: Here we report on fabrication and characterization of a novel 3D hydrogel derived from cell-free native aortic valves. A detailed analysis containing protein composition, rheological behavior, cytotoxic and proliferative effects as well as results of 3D cell culture experiments are presented. Moreover, this aortic valve derived hydrogel (AVdH) is compared to commercially available biological extracellular matrix (ECM) components to evaluate and classify AVdH with respect to other currently used ECM solutions, i.e. Collagen type I and Matrigel®. RESULTS: On the biochemical level, a complex composition of native proteins was detected. Using different techniques, including mass spectrometry with Gene Ontology network and enrichment analysis, different fundamental biological functions of AVdH were identified, including peptidase-, peptidase inhibitor-, growth- and binding activity. No cytotoxic effects were detected and AVdH showed positive effects on cell growth and proliferation in vitro when compared to Collagen type I and Matrigel®. CONCLUSION: These results suggest AVdH as an organotypic ECM supporting sophisticated 3D cell culture model studies, while mimicking the native environment of the aortic valve to a greater level for enhanced in vitro analyses.


Assuntos
Valva Aórtica/fisiologia , Materiais Biomiméticos , Técnicas de Cultura de Células , Hidrogéis/química , Engenharia Tecidual/métodos , Animais , Valva Aórtica/patologia , Estenose da Valva Aórtica/terapia , Calcinose/terapia , Proliferação de Células , Sistema Livre de Células , Colágeno/química , Combinação de Medicamentos , Matriz Extracelular/química , Doenças das Valvas Cardíacas/terapia , Cinética , Laminina/química , Proteoglicanas/química , Reologia , Ovinos , Software
15.
Int J Mol Sci ; 20(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626121

RESUMO

Laminin (Ln)-332 consists of α3, ß3, and γ2 chains, which mediate epithelial cell adhesion to the basement membrane. Ln-γ2, a component of Ln-332, is frequently expressed as a monomer in the invasion front of several types of malignant tissues without simultaneous expression of Ln-α3 and/or Ln-ß3 chains. Moreover, monomeric Ln-γ2 induces tumor cell proliferation and migration in vitro. These unique biological activities indicate that monomeric Ln-γ2 could be a candidate biomarker for early cancer surveillance. However, the present immune method for monomeric Ln-γ2 detection can only predict its expression, since no antibody that specifically reacts with monomeric γ2, but not with heterotrimeric γ2 chain, is commercially available. We have, therefore, developed monoclonal antibodies to specifically detect monomeric Ln-γ2, and devised a highly sensitive method to measure serum monomeric Ln-γ2 levels using a fully automated chemiluminescent immunoassay (CLIA). We evaluated its diagnostic value in sera from patients with several digestive cancers, including hepatocellular carcinoma (HCC), and found serum monomeric Ln-γ2 to be a clinically available biomarker for HCC surveillance. The combination of monomeric Ln-γ2 and prothrombin induced by Vitamin K Absence II (PIVKA-II) may be more sensitive for clinical diagnosis of HCC than any currently used combination.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Laminina/metabolismo , Neoplasias Hepáticas/metabolismo , Animais , Especificidade de Anticorpos , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/patologia , Humanos , Laminina/sangue , Laminina/química , Neoplasias Hepáticas/patologia , Medições Luminescentes
16.
Sci Rep ; 9(1): 12, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626885

RESUMO

Vasculogenesis is the de novo formation of a vascular network from individual endothelial progenitor cells occurring during embryonic development, organogenesis, and adult neovascularization. Vasculogenesis can be mimicked and studied in vitro using network formation assays, in which endothelial cells (ECs) spontaneously form capillary-like structures when seeded in the appropriate microenvironment. While the biochemical regulators of network formation have been well studied using these assays, the role of mechanical and topographical properties of the extracellular matrix (ECM) is less understood. Here, we utilized both natural and synthetic fibrous materials to better understand how physical attributes of the ECM influence the assembly of EC networks. Our results reveal that active cell-mediated matrix recruitment through actomyosin force generation occurs concurrently with network formation on Matrigel, a reconstituted basement membrane matrix regularly used to promote EC networks, and on synthetic matrices composed of electrospun dextran methacrylate (DexMA) fibers. Furthermore, modulating physical attributes of DexMA matrices that impair matrix recruitment consequently inhibited the formation of cellular networks. These results suggest an iterative process in which dynamic cell-induced changes to the physical microenvironment reciprocally modulate cell behavior to guide the formation and stabilization of multicellular networks.


Assuntos
Endotélio Vascular/citologia , Matriz Extracelular/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Capilares/crescimento & desenvolvimento , Diferenciação Celular , Células Cultivadas , Colágeno/química , Técnicas de Cultura , Dextranos/química , Combinação de Medicamentos , Humanos , Laminina/química , Metacrilatos/química , Morfogênese , Neovascularização Fisiológica , Proteoglicanas/química
17.
Int J Mol Sci ; 20(2)2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30634448

RESUMO

Age-related macular degeneration (AMD) is the eye disease with the highest epidemic incidence, and has great impact on the aged population. Wet-type AMD commonly has the feature of neovascularization, which destroys the normal retinal structure and visual function. So far, effective therapy options for rescuing visual function in advanced AMD patients are highly limited, especially in wet-type AMD, in which the retinal pigmented epithelium and Bruch's membrane structure (RPE-BM) are destroyed by abnormal angiogenesis. Anti-VEGF treatment is an effective remedy for the latter type of AMD; however, it is not a curative therapy. Therefore, reconstruction of the complex structure of RPE-BM and controlled release of angiogenesis inhibitors are strongly required for sustained therapy. The major purpose of this study was to develop a dual function biomimetic material, which could mimic the RPE-BM structure and ensure slow release of angiogenesis inhibitor as a novel therapeutic strategy for wet AMD. We herein utilized plasma-modified polydimethylsiloxane (PDMS) sheet to create a biomimetic scaffold mimicking subretinal BM. This dual-surface biomimetic scaffold was coated with laminin and dexamethasone-loaded liposomes. The top surface of PDMS was covalently grafted with laminin and used for cultivation of the retinal pigment epithelial cells differentiated from human induced pluripotent stem cells (hiPSC-RPE). To reach the objective of inhibiting angiogenesis required for treatment of wet AMD, the bottom surface of modified PDMS membrane was further loaded with dexamethasone-containing liposomes via biotin-streptavidin linkage. We demonstrated that hiPSC-RPE cells could proliferate, express normal RPE-specific genes and maintain their phenotype on laminin-coated PDMS membrane, including phagocytosis ability, and secretion of anti-angiogenesis factor PEDF. By using in vitro HUVEC angiogenesis assay, we showed that application of our membrane could suppress oxidative stress-induced angiogenesis, which was manifested in decreased secretion of VEGF by RPE cells and suppression of vascularization. In conclusion, we propose modified biomimetic material for dual delivery of RPE cells and liposome-enveloped dexamethasone, which can be potentially applied for AMD therapy.


Assuntos
Dexametasona/administração & dosagem , Dimetilpolisiloxanos , Células Epiteliais/metabolismo , Lipossomos , Neovascularização Fisiológica/efeitos dos fármacos , Nylons , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Biotina/química , Biotina/metabolismo , Proliferação de Células , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Dimetilpolisiloxanos/química , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/química , Laminina/metabolismo , Lipossomos/química , Degeneração Macular/terapia , Nylons/química , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
MAGMA ; 32(1): 133-145, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30498884

RESUMO

OBJECTIVE: Perfluorocarbon nanoemulsions (PFCs) tagged with fluorescence dyes have been intensively used to confirm the in vivo 19F magnetic resonance imaging (MRI) localization of PFCs by post mortem histology or flow cytometry. However, only limited data are available on tagged PFCs and the potential dissociation of fluorescence and 19F label after cellular uptake over time. MATERIALS AND METHODS: PFCs were coupled to rhodamine (Rho) or carboxyfluorescein (Cfl) and their fate was analyzed after in vitro uptake by J774, RAW and CHO cells by flow cytometry and 19F MRI. In separate in vivo experiments, the dual-labelled emulsions were intravenously applied into mice and their distribution was monitored in spleen and liver over 24 h. In a final step, time course of fluorescence and 19F signals from injected emulsions were tracked in a local inflammation model making use of a subcutaneous matrigel depot doped with LPS (lipopolysaccharide). RESULTS: Internalization of fluorescence-labelled PFCs was associated with a substantial whitening over 24 h in all macrophage cell lines while the 19F signal remained stable over time. In all experiments, CflPFCs were more susceptible to bleaching than RhoPFCs. After intravenous injection of RhoPFCs, the fluorescence signal in spleen and liver peaked after 30 min and 2 h, respectively, followed by a successive decrease over 24 h, whereas the 19F signal continuously increased during this observation period. Similar results were found in the matrigel/LPS model, where we observed increasing 19F signals in the inflammatory hot spot over time while the fluorescence signal of immune cells isolated from the matrigel depot 24 h after its implantation was only marginally elevated over background levels. This resulted in a massive underestimation of the true PFC deposition in the reticuloendothelial system and at inflammatory hot spots. CONCLUSION: Cellular uptake of fluorescently tagged PFCs leads to a dissociation of the fluorescence and the 19F label signal over time, which critically impacts on interpretation of long-term experiments validated by histology or flow cytometry.


Assuntos
Imagem por Ressonância Magnética de Flúor-19/métodos , Flúor/química , Fluorcarbonetos/química , Nanopartículas/química , Animais , Células CHO , Colágeno/química , Meios de Contraste , Cricetulus , Combinação de Medicamentos , Emulsões , Fluoresceínas/química , Corantes Fluorescentes/química , Injeções Intravenosas , Laminina/química , Lipopolissacarídeos/química , Fígado/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteoglicanas/química , Rodaminas/química , Baço/diagnóstico por imagem , Absorção Subcutânea
20.
Acta Biomater ; 84: 169-179, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30508655

RESUMO

Volumetric muscle loss (VML) occurs when skeletal muscle injury is too large for the body to fully self-repair. Typically, fibrotic tissue fills the void, which reduces muscle functionality and limb movement. Although a wide variety of natural and synthetic scaffolds have been studied with the purpose of providing the appropriate structural support, to date no scaffold has significantly restored muscle functionality after VML. Satellite cells, adult stem cells within the muscle capable of restoring smaller injuries, are sensitive to the stiffness and composition of the surrounding environment. Scaffolds that only address structural support are not sufficient to restore functionality and instead need to be designed to both promote satellite cell activation and prevent excessive fibroblast recruitment. The objective of this study was to design a scaffold that mimicked the regenerative environment and determine how the biomechanical properties differentially influence myogenic precursor and connective tissue cells. One of the main extracellular matrix (ECM) molecules upregulated during regeneration is hyaluronic acid (HA). Therefore, thiol-modified HA and poly(ethylene glycol) diacrylate hydrogels were generated and functionalized with peptides based on ECM known to influence regeneration, including fibronectin, laminin and tenascin-C. Scaffolds with different stiffness were created by varying HA content. The influence of HA stiffness and peptide functionalization on myogenic precursor and connective tissue cell proliferation, migration and gene expression was quantified. Our results indicated that HA hydrogels functionalized with the laminin peptide, IKVAV, show potential due to the enhanced promotion of myogenic cell behaviors including migration, proliferation and an increase in relevant transcription factors. STATEMENT OF SIGNIFICANCE: The goal of this study was to identify hyaluronic acid (HA) hydrogels with peptide and stiffness combinations that will direct muscle-derived cells towards regenerating phenotypes. While the interaction of skeletal muscle with RGD-functionalized HA hydrogels has been investigated, none of the other peptides described in this study had been used in the context of HA-based scaffolds and skeletal muscle-derived cells. Notably, the response of cells to variations in mechanics was dependent on ECM coating and lineage. The 3% HA functionalized with the laminin peptide, IKVAV, showed the most promise for future in vivo studies, as these hydrogels best promoted myoblast cell proliferation, attachment and spreading, enhanced migration over connective tissue cells and upregulated transcription factors associated with activated satellite cells.


Assuntos
Matriz Extracelular/química , Ácido Hialurônico/química , Hidrogéis/química , Laminina/química , Desenvolvimento Muscular , Fragmentos de Peptídeos/química , Células Satélites de Músculo Esquelético/metabolismo , Animais , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/citologia
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