Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.079
Filtrar
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.
Carbohydr Polym ; 225: 115199, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31521317

RESUMO

Proteoglycans (PGs) are proteins which are vital components located in the extracellular matrix, cell surface or intracellular granules. They are linked to polysaccharides called glycosaminoglycans. There are several aspects associated with PGs, such as cell signaling and organization of the extracellular matrix (ECM), making them pivotal participants in many tissue compositions. In teeth, PGs also play an essential role, as many of its components have elaborate ECM structures. However, lack of information on how PGs constitute the various tissues of the tooth and on their roles makes it difficult to elicit the major importance associated with this class of proteins. This review seeks to detail how proteoglycans are involved in many aspects of tooth organization and development, and as far as we are concerned, this has not been performed yet. We have also exemplified the participation of small leucine-rich proteoglycans, a special class of PGs seen in dental trauma cases.


Assuntos
Proteoglicanas , Traumatismos Dentários/metabolismo , Dente/crescimento & desenvolvimento , Dente/ultraestrutura , Animais , Humanos , Ortodontia , Proteoglicanas/química , Proteoglicanas/classificação , Proteoglicanas/fisiologia , Traumatismos Dentários/cirurgia
6.
Food Chem ; 301: 125302, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31387034

RESUMO

The autolysis of sea cucumber is caused by depolymerisation of collagen fibres and unfolding of fibrils. In order to highlight the role of collagenase in sea cucumber autolysis, collagen fibres from sea cucumber were hydrolysed with collagenase type I. Electron microscopy (EM) results indicated the collagenase caused partial depolymerisation of collagen fibres into fibrils due to the fracture of proteoglycan interfibrillar bridges, as well as uncoiling of collagen fibrils. Chemical analysis and SDS-PAGE both indicated collagenase induced a time-dependent release of glycosaminoglycans (GAGs) and soluble proteins, which further demonstrated the degradation of proteoglycan interfibrillar bridges. Collagenase also degraded collagens by releasing soluble hydroxyproline (Hpy), with the dissolution rate of Hyp reaching 11.11% after 72 h. Fourier transform infrared analysis showed that collagenase caused the reduction of intermolecular interactions and structural order of collagen. Hence, collagenase participated in the autolysis of sea cucumber by deteriorating both macromolecular and monomeric collagens.


Assuntos
Colágeno/química , Colagenases/química , Stichopus/química , Animais , Autólise , Colágeno/metabolismo , Colagenases/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosaminoglicanos/metabolismo , Hidrólise , Hidroxiprolina/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteoglicanas/química , Proteoglicanas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Stichopus/anatomia & histologia
7.
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
8.
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
9.
Carbohydr Res ; 478: 25-32, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31042589

RESUMO

Polysaccharide peptides (or protein-bound polysaccharides, PSPs) are commonly found in mushrooms and plants and possess important nutritional properties and health benefits. The pathogenic bacterium Streptococcus zooepidemicus does not inherently produce PSPs but secretes the capsular polysaccharide hyaluronan. However, in a previous investigation of the catalytic mechanism of UDP-glucose dehydrogenase (UGDH), a PSP of peptide-bound hyaluronan was found to be produced by S. zooepidemicus through the in vivo expression of a mutant of the gene encoding UGDH. In the present study, this hyaluronan-derived PSP was structurally characterized by FT-IR, NMR, and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the data confirmed that the polysaccharide backbone, hyaluronan, is covalently bound to the side-chain peptides via an amide linkage. More importantly, the bacterial production of a PSP via this genetic modification method should inspire further research on the in vitro enzymatic synthesis of PSPs or even naturally occurring polysaccharide derivatives and may provide a theoretical foundation for investigating the in vivo synthetic mechanism of PSPs.


Assuntos
Ácido Hialurônico/biossíntese , Proteoglicanas/biossíntese , Streptococcus equi/metabolismo , Configuração de Carboidratos , Ácido Hialurônico/química , Ácido Hialurônico/isolamento & purificação , Proteoglicanas/química , Proteoglicanas/isolamento & purificação , Streptococcus equi/genética
10.
Biomed Tech (Berl) ; 64(5): 591-600, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30951496

RESUMO

The articular cartilage tissue is an essential component of joints as it reduces the friction between the two bones. Its load-bearing properties depend mostly on proteoglycan distribution, which can be analyzed through the study of the presence of sulfated glycosaminoglycan (sGAG). Currently, sGAG distribution in articular cartilage is not completely known; it is calculated by means of laboratory tests that imply the inherent inaccuracy of a manual procedure. This paper presents an easy-to-use desktop software application for obtaining the sGAG distribution profile in tissue. This app uses color images of stained cartilage tissues taken under a microscope, so researchers at the Trinity Centre for Bioengineering (Dublin, Ireland) can understand the qualitative distribution of sGAG with depth in the studied tissues.


Assuntos
Cartilagem Articular/fisiologia , Glicosaminoglicanos/química , Proteoglicanas/química , Cartilagem Articular/química , Cor , Suporte de Carga
11.
Int J Biol Macromol ; 132: 641-650, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30940591

RESUMO

Two proteoglycans (HPP and LPP) with different ratios of protein/polysaccharide were extracted from S. glabra. The chemical compositions, relative average molecular weights, monosaccharide compositions, FT-IR spectra, and rheological properties of the two proteoglycans were determined. The results exhibited that the two proteoglycans had pseudoplastic fluids properties and displayed shear-thinning behavior. The apparent viscosity of the two proteoglycans both increased with increasing concentrations. The temperature had different effects on the viscosity of the two proteoglycans. As temperature increased from 25 to 85 °C, the viscosity of LPP descended while the HPP's viscosity rose first and then dropped slightly. The effects of CaCl2 addition on the two samples were like that of the temperature. The viscosities of HPP and LPP had different tolerances to acidity and alkalinity. HPP solution was more sensitive to pH changes due to its high protein content. The addition of sucrose increased the viscosities of samples. The modulus G' and G″ of HPP and LPP were increased with the increase of oscillation frequency, while the crossover points of G' and G″ values decreased with the increasing concentrations of HPP and LPP. The above data presented that the two proteoglycans could be promising candidates for food industries and pharmacological applications.


Assuntos
Magnoliopsida/química , Proteoglicanas/química , Reologia , Concentração de Íons de Hidrogênio , Peso Molecular , Monossacarídeos/análise , Sacarose/química , Temperatura , Viscosidade
12.
Cell Mol Life Sci ; 76(19): 3899-3914, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30993352

RESUMO

The P3H1/CRTAP/PPIB complex is essential for prolyl 3-hydroxylation and folding of procollagens in the endoplasmic reticulum (ER). Deficiency in any component of this ternary complex is associated with the misfolding of collagen and the onset of osteogenesis imperfecta. However, little structure information is available about how this ternary complex is assembled and retained in the ER. Here, we assessed the role of the KDEL sequence of P3H1 and probed the spatial interactions of PPIB in the complex. We show that the KDEL sequence is essential for retaining the P3H1 complex in the ER. Its removal resulted in co-secretion of P3H1 and CRTAP out of the cell, which was mediated by the binding of P3H1 N-terminal domain with CRTAP. The secreted P3H1/CRTAP can readily bind PPIB with their C-termini close to PPIB in the ternary complex. Cysteine modification, crosslinking, and mass spectrometry experiments identified PPIB surface residues involved in the complex formation, and showed that the surface of PPIB is extensively covered by the binding of P3H1 and CRTAP. Most importantly, we demonstrated that one disease-associated pathological PPIB mutation on the binding interface did not affect the PPIB prolyl-isomerase activity, but disrupted the formation of P3H1/CRTAP/PPIB ternary complex. This suggests that defects in the integrity of the P3H1 ternary complex are associated with pathological collagen misfolding. Taken together, these results provide novel structural information on how PPIB interacts with other components of the P3H1 complex and indicate that the integrity of P3H1 complex is required for proper collagen formation.


Assuntos
Ciclofilinas/química , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas da Matriz Extracelular/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Mutação , Prolil Hidroxilases , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoglicanas/química , Proteoglicanas/genética , Deleção de Sequência
13.
Prog Mol Biol Transl Sci ; 163: 361-381, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31030754

RESUMO

Coriolus versicoloris is one of the well-known traditional medicinal mushrooms used in China for over 2000 years. Polysaccharopeptide (PSP) is identified as the major bioactive component, which can be obtained from the mycelium or fermentation broth of Coriolus versicolor. The polysaccharide content in PSP is ~60% and the peptide content in PSP is ~10-30%. The main monosaccharides found in PSP include glucose, mannose, and a small amount of galactose, xylose, and fucose. ß-Glucan is one of the identified components in PSP with the established immunomodulatory function. PSP was approved by the authority and has been used clinically in Japan and China since 1970s. PSP is helpful in improving the survival and quality of life in patients suffering cancers, hepatopathy, hyperlipidemia, chronic bronchitis, and other complex diseases. In this article, the preclinical and clinical studies of PSP are summarized over the past 41 years based on a literature search covering the CNKI, VIP, and Wanfang databases. Current studies support PSP as an immunotherapeutic. PSP activates and enhances the function and recognition ability of immune cells, strengthens the phagocytosis of macrophages, increases the expressions of cytokines and chemokines such as tumor necrosis factor-α (TNF-α), interleukins (IL-1ß and IL-6), histamine, and prostaglandin E, stimulates the filtration of both dendritic cells and T-cells into tumors, and ameliorates the adverse events associated with chemotherapy. In recent years, immunotherapy has been widely used in cancer treatment. However, to use PSP as an immunotherapeutic at world stage, further chemical, biochemical and pharmacological studies of PSP are needed.


Assuntos
Agaricales/química , Imunoterapia , Proteoglicanas/farmacologia , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , China , Humanos , Fatores Imunológicos/farmacologia , Proteoglicanas/química
14.
Int J Mol Sci ; 20(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901841

RESUMO

Synergizing integrin and cell-membrane heparan sulfate proteoglycan signaling on biomaterials through peptidic sequences is known to have beneficial effects in the attachment and behavior of osteoblasts; however, controlling the exact amount and ratio of peptides tethered on a surface is challenging. Here, we present a dual molecular-based biointerface combining integrin (RGD) and heparin (KRSR)-binding peptides in a chemically controlled fashion. To this end, a tailor-made synthetic platform (PLATF) was designed and synthesized by solid-phase methodologies. The PLATF and the control linear peptides (RGD or KRSR) were covalently bound to titanium via silanization. Physicochemical characterization by means of contact angle, Raman spectroscopy and XPS proved the successful and stable grafting of the molecules. The biological potential of the biointerfaces was measured with osteoblastic (Saos-2) cells both at short and long incubation periods. Biomolecule grafting (either the PLATF, RGD or KRSR) statistically improved (p < 0.05) cell attachment, spreading, proliferation and mineralization, compared to control titanium. Moreover, the molecular PLATF biointerface synergistically enhanced mineralization (p < 0.05) of Saos-2 cells compared to RGD or KRSR alone. These results indicate that dual-function coatings may serve to improve the bioactivity of medical implants by mimicking synergistic receptor binding.


Assuntos
Membrana Celular/metabolismo , Integrinas/metabolismo , Oligopeptídeos/metabolismo , Osteoblastos/metabolismo , Proteoglicanas/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Fenômenos Químicos , Materiais Revestidos Biocompatíveis/química , Matriz Extracelular/metabolismo , Integrinas/química , Oligopeptídeos/química , Proteoglicanas/química , Análise Espectral
15.
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
16.
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
17.
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
18.
Inflammopharmacology ; 27(1): 175-187, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30600472

RESUMO

Osteoarthritis (OA) causes articular cartilage destruction, initiating pain and inflammation in the joints, resulting in joint disability. Medications are available to manage these symptoms; however, their effects on the disease progression are limited. Loss of proteoglycans (PGs) was reported to contribute articular cartilage destruction in OA. Therapeutics approaches were previously studied in the animal models of OA. In the present study, we investigated the oral efficacy of four dosages of PGs (25 mg/kg, 50 mg/kg, 100 mg/kg and 200 mg/kg), isolated from the bramble shark cartilage, in an animal model of OA. Indomethacin was used as a bioequivalent formulation. Primarily, the mass spectrum analysis of the purified PGs obtained from bramble shark cartilage revealed the presence of two unique peptides including AGWLSDGSVR and LDGNPINLSK, that showed sequence similarity with aggrecan core-protein and epiphycan, respectively. The levels of C-reactive protein and uric acid in the OA rats were reduced when treated with PGs. Histopathology analysis displayed less cartilage erosion and neovascularization in OA rats treated with PGs. The X-ray imaging presented higher bone density with 200 mg/kg dosage of PG treatment in OA rats. The expressions of the inflammatory modulators including TNF-α, IL-1ß, MMP13, NOS2, IL-10 and COX-2 were found to be moderated with PG treatment. In addition, PG treatment maintained the activities of antioxidant enzymes, including SOD and catalase in the joint tissues with a higher GSH content, in a dose-dependent manner. Taken together, our preliminary findings report the anti-osteoarthritic properties of PGs and recommend to evaluate its efficacy and safety in randomized trials.


Assuntos
Cartilagem/química , Cartilagem/metabolismo , Osteoartrite/tratamento farmacológico , Proteoglicanas/química , Proteoglicanas/farmacologia , Tubarões/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Osteoartrite/metabolismo , Dor/tratamento farmacológico , Dor/metabolismo , Ratos , Ratos Wistar
19.
J Biol Chem ; 294(8): 2628-2641, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30602571

RESUMO

Trametes robiniophila Murr. (Huaier) is a mushroom with a long history of use as a medicinal ingredient in China and exhibits good clinical efficacy in cancer management. However, the antitumor components of Huaier and the underlying molecular mechanisms remain poorly understood. Here, we isolated a proteoglycan with a molecular mass of ∼5.59 × 104 Da from Huaier aqueous extract. We named this proteoglycan TPG-1, and using FTIR and additional biochemical analyses, we determined that its total carbohydrate and protein compositions are 43.9 and 41.2%, respectively. Using biochemical assays and immunoblotting, we found that exposing murine RAW264.7 macrophages to TPG-1 promotes the production of nitric oxide (NO), tumor necrosis factor α (TNFα), and interleukin-6 (IL-6) through Toll-like receptor 4 (TLR4)-dependent activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling. Of note, the TPG-1 treatment significantly inhibited the tumorigenesis of human hepatoma HepG2 cells likely at least in part by increasing serum levels of TNFα and promoting leukocyte infiltration into tumors in nude mice. TPG-1 also exhibited good antitumor activity in hepatoma H22-bearing mice and had no obvious adverse effects in these mice. We conclude that TPG-1 exerts antitumor activity partially through an immune-potentiating effect due to activation of the TLR4-NF-κB/MAPK signaling cassette. Therefore, TPG-1 may be a promising candidate drug for cancer immunotherapy. This study has identified the TPG-1 proteoglycan as an antitumor agent and provided insights into TPG-1's molecular mechanism, suggesting a potential utility for applying this agent in cancer therapy.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor 4 Toll-Like/metabolismo , Trametes/química , Regulação para Cima/efeitos dos fármacos , Animais , Antineoplásicos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Citocinas/biossíntese , Citocinas/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos Nus , NF-kappa B/genética , Proteínas de Neoplasias/genética , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Proteoglicanas/química , Proteoglicanas/farmacologia , Receptor 4 Toll-Like/genética , Ensaios Antitumorais Modelo de Xenoenxerto
20.
J Biol Chem ; 294(9): 3065-3080, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30598510

RESUMO

Betaglycan (BG) is a membrane-bound co-receptor of the TGF-ß family that selectively binds transforming growth factor-ß (TGF-ß) isoforms and inhibin A (InhA) to enable temporal-spatial patterns of signaling essential for their functions in vivo Here, using NMR titrations of methyl-labeled TGF-ß2 with BG's C-terminal binding domain, BGZP-C, and surface plasmon resonance binding measurements with TGF-ß2 variants, we found that the BGZP-C-binding site on TGF-ß2 is located on the inner surface of its extended finger region. Included in this binding site are Ile-92, Lys-97, and Glu-99, which are entirely or mostly specific to the TGF-ß isoforms and the InhA α-subunit, but they are unconserved in other TGF-ß family growth factors (GFs). In accord with the proposed specificity-determining role of these residues, BG bound bone morphogenetic protein 2 (BMP-2) weakly or not at all, and TGF-ß2 variants with the corresponding residues from BMP-2 bound BGZP-C more weakly than corresponding alanine variants. The BGZP-C-binding site on InhA previously was reported to be located on the outside of the extended finger region, yet at the same time to include Ser-112 and Lys-119, homologous to TGF-ß2 Ile-92 and Lys-97, on the inside of the fingers. Therefore, it is likely that both TGF-ß2 and InhA bind BGZP-C through a site on the inside of their extended finger regions. Overall, these results identify the BGZP-C-binding site on TGF-ß2 and shed light on the specificity of BG for select TGF-ß-type GFs and the mechanisms by which BG influences their signaling.


Assuntos
Inibinas/metabolismo , Proteoglicanas/química , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fator de Crescimento Transformador beta2/química , Fator de Crescimento Transformador beta2/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Ratos , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA