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1.
Cell Physiol Biochem ; 53(1): 87-100, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31204440

RESUMO

BACKGROUND/AIMS: Different components of the tumor microenvironment can be either tumor-promoting or tumor-suppressive agents depending on factors which are not fully understood. Fibulins are components of the extracellular matrix from different tissues and constitute a clear example of this dual function. In fact, fibulins may either support tumor growth or abolish progression of malignant cells depending on the crosstalk between tumor cells and their surrounding stroma through mechanisms that remain to be elucidated. Among all fibulins, fibulin-5 contains a particular structural hallmark which consists in the presence of a RGD motif within its architecture. Previous reports have highlighted the importance of the interaction of this motif with integrins, and not only in normal functions but also in a tumor context. METHODS: Site-Directed Mutagenesis technique was employed to introduce the change RGD to RGE (RGD-to-RGE) within Fbln5 cDNA sequence. Cell proliferation was measured using the MTT assay or by counting Ki-67 positive cell nuclei. Cell adhesion was analysed using culture plates coated with different extracellular matrix components. Cell invasion was evaluated using 24-well Matrigel-coated invasion chambers, and mammosphere formation was monitored using ultralow attachment culture plates. BALB/c mice were employed to induce subcutaneous tumors. RESULTS: The RGD-to-RGE change alters the capacity of breast cancer cells to adhere to different extracellular matrix proteins as well as to αvß3 and α5ß1 integrins, and promotes protumor effects using different cell-based assays. Moreover, 4T1 cells, a mouse breast cancer cell line model, shows an increased capacity to generate tumors when exogenously expresses fibulin-5 with a RGD-to-RGE change, and such capacity is similar to that shown for 4T1 cells with an interfered Fbln5 gene. CONCLUSION: These data highlight the importance of the RGD motif of fibulin-5 to induce antitumor effects and provide new insights into the involvement of fibulins in tumor processes.


Assuntos
Adesão Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Oligopeptídeos/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Oligopeptídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Transplante Homólogo , Vimentina/metabolismo
2.
Biomed Pharmacother ; 113: 108594, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30849639

RESUMO

The healing of acute wounds is vital to humans and is a well-orchestrated process that involves systemic and local factors. However, there is a lack of effective and safe clinical therapies. The collagen triple helix repeat containing 1 (CTHRC1) protein is a type of exocrine protein that has been recently reported to contribute to tissue repair. Our aim is to validate the promoting effects of CTHRC1 on the healing of acute wounds and to elucidate the underlying molecular mechanism. Therefore, we first established acute wound healing mouse models and confirmed that CTHRC1 accelerates the healing process of acute wounds. Then, we characterized wound macrophages using a polyvinylalcohol (PVA) sponge model and used Western blotting to investigate the molecular mechanism. We found that CTHRC1 increased the M2 macrophage population and the TGF-ß expression level as a result of the activation of the TGF-ß and Notch pathways, which eventually contributed to the promotion of wound healing. Inhibition of the Notch pathway showed attenuated M2 macrophage recruitment, and it decreased the TGF-ß expression level. These results substantiate our hypothesis that CTHRC1 promotes wound healing by recruiting M2 macrophages and regulating the TGF-ß and Notch pathways.


Assuntos
Proteínas da Matriz Extracelular/farmacologia , Macrófagos/efeitos dos fármacos , Receptores Notch/metabolismo , Pele/lesões , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/efeitos dos fármacos , Ferimentos Perfurantes/tratamento farmacológico , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Pele/imunologia , Pele/metabolismo , Cicatrização/imunologia , Ferimentos Perfurantes/imunologia , Ferimentos Perfurantes/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(6): 2042-2051, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30659152

RESUMO

We challenge the conventional designation of structural matrix proteins primarily as supporting scaffolds for resident cells. The extracellular matrix protein tropoelastin is classically regarded as a structural component that confers mechanical strength and resilience to tissues subject to repetitive elastic deformation. Here we describe how tropoelastin inherently induces a range of biological responses, even in cells not typically associated with elastic tissues and in a manner unexpected of typical substrate-dependent matrix proteins. We show that tropoelastin alone drives mesenchymal stem cell (MSC) proliferation and phenotypic maintenance, akin to the synergistic effects of potent growth factors such as insulin-like growth factor 1 and basic fibroblast growth factor. In addition, tropoelastin functionally surpasses these growth factors, as well as fibronectin, in allowing substantial media serum reduction without loss of proliferative potential. We further demonstrate that tropoelastin elicits strong mitogenic and cell-attractive responses, both as an immobilized substrate and as a soluble additive, via direct interactions with cell surface integrins αvß3 and αvß5. This duality of action converges the long-held mechanistic dichotomy between adhesive matrix proteins and soluble growth factors and uncovers the powerful, untapped potential of tropoelastin for clinical MSC expansion and therapeutic MSC recruitment. We propose that the potent, growth factor-like mitogenic and motogenic abilities of tropoelastin are biologically rooted in the need for rapid stem cell homing and proliferation during early development and/or wound repair.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Tropoelastina/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos , Fator de Crescimento Insulin-Like I/efeitos dos fármacos , Integrina alfaVbeta3 , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores de Vitronectina , Cicatrização
4.
PLoS Pathog ; 14(5): e1007026, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29775486

RESUMO

Innate immune recognition is classically mediated by the interaction of host pattern-recognition receptors and pathogen-associated molecular patterns; this triggers a series of downstream signaling events that facilitate killing and elimination of invading pathogens. In this report, we provide the first evidence that peroxidasin (PXDN; also known as vascular peroxidase-1) directly binds to gram-negative bacteria and mediates bactericidal activity, thus, contributing to lung host defense. PXDN contains five leucine-rich repeats and four immunoglobulin domains, which allows for its interaction with lipopolysaccharide, a membrane component of gram-negative bacteria. Bactericidal activity of PXDN is mediated via its capacity to generate hypohalous acids. Deficiency of PXDN results in a failure to eradicate Pseudomonas aeruginosa and increased mortality in a murine model of Pseudomonas lung infection. These observations indicate that PXDN mediates previously unrecognized host defense functions against gram-negative bacterial pathogens.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Peroxidase/metabolismo , Peroxidase/farmacologia , Animais , Antibacterianos/imunologia , Antibacterianos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/imunologia , Feminino , Bactérias Gram-Negativas/imunologia , Imunidade Inata/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/imunologia , Infecções Respiratórias/imunologia , Transdução de Sinais
5.
Cell Physiol Biochem ; 46(4): 1305-1316, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29689558

RESUMO

BACKGROUND/AIMS: Fibulin-3, an extracellular matrix glycoprotein, inhibits vascular oxidative stress and remodeling in hypertension. Oxidative stress is prevalent in chronic kidney disease (CKD) patients and is an important mediator of osteo-/chondrogenic transdifferentiation and calcification of vascular smooth muscle cells (VSMCs) during hyperphosphatemia. Therefore, the present study explored the effects of Fibulin-3 on phosphate-induced vascular calcification. METHODS: Experiments were performed in primary human aortic smooth muscle cells (HAoSMCs) treated with control or with phosphate without or with additional treatment with recombinant human Fibulin-3 protein or with hydrogen peroxide as an exogenous source of oxidative stress. RESULTS: Treatment with calcification medium significantly increased calcium deposition in HAoSMCs, an effect significantly blunted by additional treatment with Fibulin-3. Moreover, phosphate-induced alkaline phosphatase activity and mRNA expression of osteogenic and chondrogenic markers MSX2, CBFA1, SOX9 and ALPL were all significantly reduced by addition of Fibulin-3. These effects were paralleled by similar regulation of oxidative stress in HAoSMCs. Phosphate treatment significantly up-regulated mRNA expression of the oxidative stress markers NOX4 and CYBA, down-regulated total antioxidant capacity and increased the expression of downstream effectors of oxidative stress PAI-1, MMP2 and MMP9 as well as BAX/BLC2 ratio in HAoSMCs, all effects blocked by additional treatment with Fibulin-3. Furthermore, the protective effects of Fibulin-3 on phosphate-induced osteogenic and chondrogenic markers expression in HAoSMCs were reversed by additional treatment with hydrogen peroxide. CONCLUSIONS: Fibulin-3 attenuates phosphate-induced osteo-/ chondrogenic transdifferentiation and calcification of VSMCs, effects involving inhibition of oxidative stress. Up-regulation or supplementation of Fibulin-3 may be beneficial in reducing the progression of vascular calcification during hyperphosphatemic conditions such as CKD.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Glicerofosfatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Osteogênese/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo
6.
Mol Med Rep ; 17(4): 6130-6137, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29436660

RESUMO

Lumbar disc disease (LDD) is common in aged populations, and it is primarily caused by intervertebral disc degeneration (IDD). Cartilage intermediate layer protein (CILP), which is specifically expressed in intervertebral discs (IVDs), is suspected to be associated with IDD. However, it remains unclear whether CILP contributes to IDD in humans. Furthermore, the regulation of CILP in human IVDs is poorly understood, especially by mechanical stimuli, which are regarded as primary factors promoting IDD. To address these issues, the present study collected nucleus pulposus (NP) cells from patients undergoing lumbar spinal surgery for degenerative disc disease (DDD). Subsequently, CILP expression was measured in human NP cells in response to mechanical stimuli, including cyclic compressive stress and cyclic tensile strain (CTS), by reverse transcription­quantitative polymerase chain reaction and western blotting. Aggrecan and collagen II, which are the main components of the extracellular matrix (ECM) and traditional degenerative markers for IDD, were detected following the treatment with CILP small interfering (si)RNA or recombinant human CILP (rhCILP) at various concentrations to determine whether CILP contributes to IDD by negatively regulating expression of the ECM. The results revealed that CILP expression in loaded NP cells was significantly increased compared with that in non­loaded cells under compressive loading, and that it was markedly decreased in cells under tensile loading, in contrast with the expression of aggrecan and collagen II in response to the same stimuli. Furthermore, CILP siRNA effectively inhibited CILP expression and significantly increased the expression of aggrecan and collagen II. In addition, treatment of NP cells with a high concentration of rhCILP resulted in significantly decreased expression of aggrecan and collagen II. In conclusion, these results demonstrated for the first time, to the best of our knowledge, that in human NP cells, CILP is regulated by mechanical stress and that its expression affects ECM synthesis. Therefore, CILP represents a promising therapeutic target for preventing loss of the matrix during IDD as a novel treatment strategy.


Assuntos
Proteínas da Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Pirofosfatases/genética , Estresse Mecânico , Agrecanas/genética , Agrecanas/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Humanos , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/etiologia , Degeneração do Disco Intervertebral/metabolismo , Deslocamento do Disco Intervertebral/etiologia , Deslocamento do Disco Intervertebral/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/metabolismo , Pirofosfatases/metabolismo , Pirofosfatases/farmacologia , Interferência de RNA
7.
Chem Biol Interact ; 278: 92-100, 2017 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-29042256

RESUMO

Transforming growth factor-ß-induced protein (TGFBIp), an extracellular protein, is expressed on several cell types in response to TGF-ß stimulation. Human umbilical vein endothelial cell (HUVEC)-derived TGFBIp functions as a mediator of sepsis. Screening of bioactive compound libraries is an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases (drug repositioning). Dabrafenib (DAB), a B-Raf inhibitor, was initially used for treating metastatic melanoma. The present study determined whether DAB modulated TGFBIp-mediated septic responses in HUVECs and in mice. Antiseptic functions of DAB were examined by measuring permeability, leukocyte adhesion and migration, and proinflammatory protein activation in TGFBIp-stimulated HUVECs and mice. In addition, beneficial effects of DAB on survival rate were examined using a mouse model of sepsis. We found that DAB inhibited TGFBIp-induced vascular barrier disruption, cell adhesion molecule (CAM) expression, and neutrophil adhesion/transendothelial migration toward human endothelial cells. DAB also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that DAB exerts anti-inflammatory effects by inhibiting hyperpermeability, CAM expression, and leukocyte adhesion and migration, indicating its utility for treating vascular inflammatory diseases.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Leucócitos/efeitos dos fármacos , Oximas/farmacologia , Oximas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Sepse/tratamento farmacológico , Fator de Crescimento Transformador beta/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos/citologia , Leucócitos/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Permeabilidade/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Sepse/mortalidade , Sepse/patologia , Taxa de Sobrevida , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
PLoS One ; 12(7): e0179729, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28704392

RESUMO

Melanoma inhibitory activity (MIA) affects the differentiation to hyaline cartilage and can inhibit the osteogenic potential of bone morphogenetic protein (BMP)-2 in mesenchymal stem cells (MSC). The aim of this study was to investigate if MIA also inhibits the osteogenic potential of BMP-2 in human articular chondrocytes during redifferentiation, which may lead to a higher grade of differentiation without calcification. HAC of four female patients (mean age: 73.75 ±6.98) were seeded into 3D culture for 28 days; after adding the recombinant proteins, four groups were formed (Control, BMP-2, MIA, BMP-2+MIA). Samples were analysed for gene expression, glycosaminoglycan (GAG) content and histology on day 0, 14 and 28. Collagen type 2 (COL2A1) was significantly increased in the BMP-2 containing groups on day 28; BMP-2 (100-fold, p = 0.001), BMP-2+MIA (65-fold, p = 0.009) and similar to the level of native cartilage. Higher aggrecan (Agg) levels were present in the BMP-2 (3-fold, p = 0.007) and BMP-2+MIA (4-fold, p = 0.002) group after 14 days and in the BMP-2 (9-fold, p = 0.001) group after 28 days. Collagen type 10 (COL10A1) was increased in the BMP-2 containing groups (6-fold, p = 0.006) but these levels were significantly below native cartilage. Alkaline phosphatase (ALP), collagen type 1 (COL1A1) and the glycosaminoglycan (GAG) content did not reveal any relevant differences between groups. BMP-2 is a potent inducer for differentiation of HAC. A significant enhancement of this effect in combination with MIA could not be observed. Furthermore no significant reduction of osteogenic markers during re-differentiation of chondrocytes was present combining BMP-2 and MIA.


Assuntos
Proteína Morfogenética Óssea 2/farmacologia , Técnicas de Cultura de Células/métodos , Condrócitos/citologia , Colágeno Tipo II/genética , Proteínas da Matriz Extracelular/farmacologia , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/metabolismo , Humanos
9.
Mater Sci Eng C Mater Biol Appl ; 75: 349-358, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28415472

RESUMO

Adipose tissue is a rich source of extracellular matrix (ECM) material that can be isolated by delipidating and decellularizing the tissue. However, the current delipidation and decellularization methods either involve tedious and lengthy processes or require toxic chemicals, which may result in the elimination of vital proteins and growth factors found in the ECM. Hence, an alternative delipidation and decellularization method for adipose tissue was developed using supercritical carbon dioxide (SC-CO2) that eliminates the need of any harsh chemicals and also reduces the amount of processing time required. The resultant SC-CO2-treated ECM material showed an absence of nuclear content but the preservation of key proteins such as collagen Type I, collagen Type III, collagen Type IV, elastin, fibronectin and laminin. In addition, other biological factors such as glycosaminoglycans (GAGs) and growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were also retained. Subsequently, the resulting SC-CO2-treated ECM material was used as a bioactive coating on tissue culture plastic (TCP). Four different cell types including adipose tissue-derived mesenchymal stem cells (ASCs), human umbilical vein endothelial cells (HUVECs), immortalized human keratinocyte (HaCaT) cells and human monocytic leukemia cells (THP-1) were used in this study to show that the SC-CO2-treated ECM coating can be potentially used for various biomedical applications. The SC-CO2-treated ECM material showed improved cell-material interactions for all cell types tested. In addition, in vitro scratch wound assay using HaCaT cells showed that the presence of SC-CO2-treated ECM material enhanced keratinocyte migration whilst the in vitro cellular studies using THP-1-derived macrophages showed that the SC-CO2-treated ECM material did not evoke pro-inflammatory responses from the THP-1-derived macrophages. Overall, this study shows the efficacy of SC-CO2 method for delipidation and decellularization of adipose tissue whilst retaining its ECM and its subsequent utilization as a bioactive surface coating material for soft tissue engineering, angiogenesis and wound healing applications.


Assuntos
Tecido Adiposo/química , Dióxido de Carbono , Proteínas da Matriz Extracelular , Matriz Extracelular/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Queratinócitos/metabolismo , Dióxido de Carbono/química , Dióxido de Carbono/farmacologia , Linhagem Celular Tumoral , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/farmacologia , Feminino , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Queratinócitos/citologia , Masculino
10.
Eur J Cell Biol ; 96(3): 266-275, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28336087

RESUMO

Dermatopontin (DPT) is a matricellular protein with cardinal roles in cutaneous wound healing. The protein is also reported to be altered in various anomalies including cancer. The present study is aimed to unravel the role of DPT in angiogenesis which is imperative in many physiological and pathological processes. DPT's capabilities on promoting angiogenesis were assessed using various in vitro and ex vivo systems. The results indicated that DPT enhances cell motility and induces lamellipodia formation in endothelial cells. Additionally, we noticed that DPT stimulates tube formation in endothelial cells when plated on a matrigel substrate. However, it was observed that DPT had no effect on the proliferation of endothelial cells even at higher concentrations and prolonged treatment periods. Additional experiments on CAM and aortic arch assays apparently depicted that DPT promotes neovascularisation and tube sprouting, thus unraveling its prominent role in angiogenesis. Further, PCR analysis revealed that endothelial cells are devoid of DPT expression, but when exogenously supplied, modulates the expression of transforming growth factor ß1 and integrin α3ß1 which are reported to have crucial roles in endothelial cell behaviour during angiogenesis. In conclusion, DPT possess vital pro-angiogenic properties and thus retains promising therapeutic values in managing chronic wounds and cancer.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/farmacologia , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Integrina alfa3beta1/metabolismo , Neovascularização Fisiológica , Fator de Crescimento Transformador beta/metabolismo , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Embrião de Galinha , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Integrina alfa3beta1/genética , Proteínas Recombinantes , Fator de Crescimento Transformador beta/genética
11.
Mol Cell Neurosci ; 80: 111-122, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28286248

RESUMO

Schizophrenia is a highly heritable psychiatric disorder linked to a large number of risk genes. The function of these genes in disease etiology is not fully understood but pathway analyses of genomic data suggest developmental dysregulation of cellular processes such as neuronal migration and axon guidance. Previous studies of patient-derived olfactory cells show them to be more motile than control-derived cells when grown on a fibronectin substrate, motility that is dependent on focal adhesion kinase signaling. The aim of this study was to investigate whether schizophrenia patient-derived cells are responsive to other extracellular matrix (ECM) proteins that bind integrin receptors. Olfactory neurosphere-derived cells from nine patients and nine matched controls were grown on ECM protein substrates at increasing concentrations and their movement was tracked for 24h using automated high-throughput imaging. Control-derived cells increased their motility as the ECM substrate concentration increased, whereas patient-derived cell motility was little affected by ECM proteins. Patient and control cells had appropriate integrin receptors for these ECM substrates and detected them as shown by increases in focal adhesion number and size in response to ECM proteins, which also induced changes in cell morphology and cytoskeleton. These observations indicate that patient cells failed to translate the detection of ECM proteins into appropriate changes in cell motility. In a sense, patient cells act like a moving car whose accelerator is jammed, moving at the same speed without regard to the external environment. This focuses attention on cell motility regulation rather than speed as key to impairment of neuronal migration in the developing brain in schizophrenia.


Assuntos
Movimento Celular/fisiologia , Matriz Extracelular/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Esquizofrenia/patologia , Adolescente , Adulto , Estudos de Casos e Controles , Linhagem Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Estudos de Coortes , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Proteínas da Matriz Extracelular/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Olfatória/patologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Adulto Jovem
12.
Biochem Biophys Res Commun ; 485(3): 621-626, 2017 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-28161637

RESUMO

Ameloblastin (Ambn) and enamelin (Enam) play a pivotal role in enamel mineralization. Previous studies have demonstrated that these enamel-related gene products also affect bone growth and remodeling; however, the underlying mechanisms have not been elucidated. In the present study, we examined the effects of Ambn and Enam on the receptor activator of nuclear factor kappa-B ligand (RANKL) expression induced with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone (DEX) on mouse bone marrow stromal cell line ST2 cells. We then verified the effect of Ambn and Enam on osteoclastogenesis. We found that pretreatment with recombinant human Ambn (rhAmbn) and recombinant human Enam (rhEnam) remarkably suppressed RANKL mRNA and protein expression induced with 1,25(OH)2D3 and DEX. Interestingly, rhAmbn and rhEnam attenuated the phosphorylation of mitogen-activated protein kinases (MAPK), including ERK1/2, JNK, and p38 in ST2 cells stimulated with 1,25(OH)2D3 and DEX. Moreover, pretreatment with specific inhibitors of ERK1/2 and p38, but not JNK, blocked RANKL mRNA and protein expression. Cell co-culture results showed that rhAmbn and rhEnam downregulated mouse bone marrow cell differentiation into osteoclasts induced with 1,25(OH)2D3 and DEX-stimulated ST2 cells. These results suggest that Ambn and Enam may indirectly suppress RANKL-induced osteoclastogenesis via downregulation of p38 and ERK1/2 MAPK signaling pathways in bone marrow stromal cells.


Assuntos
Proteínas do Esmalte Dentário/farmacologia , Proteínas da Matriz Extracelular/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Ligante RANK/metabolismo , Animais , Western Blotting , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Calcitriol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Proteínas do Esmalte Dentário/genética , Dexametasona/farmacologia , Proteínas da Matriz Extracelular/genética , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Ligante RANK/genética , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vitaminas/farmacologia
13.
Hum Mutat ; 38(4): 439-450, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28074631

RESUMO

Mutations in genes encoding proteins of the smooth muscle cell (SMC) contractile apparatus contribute to familial aortic aneurysms. To investigate the pathogenicity of these mutations, SMC are required. We demonstrate a novel method to generate SMC-like cells from human dermal fibroblasts by transdifferentiation to study the effect of variants in genes encoding proteins of the SMC contractile apparatus (ACTA2 and MYH11) in patients with aortic aneurysms. Dermal fibroblasts from seven healthy donors and cells from seven patients with MYH11 or ACTA2 variants were transdifferentiated into SMC-like cells within a 2-week duration using 5 ng/ml TGFß1 on a scaffold containing collagen and elastin. The induced SMC were comparable to primary human aortic SMC in mRNA expression of SMC markers which was confirmed on the protein level by immunofluorescence quantification analysis and Western blotting. In patients with MYH11 or ACTA2 variants, the effect of intronic variants on splicing was demonstrated on the mRNA level in the induced SMC, allowing classification into pathogenic or nonpathogenic variants. In conclusion, direct conversion of human dermal fibroblasts into SMC-like cells is a highly efficient method to investigate the pathogenicity of variants in proteins of the SMC contractile apparatus.


Assuntos
Actinas/genética , Aneurisma Aórtico/genética , Transdiferenciação Celular/genética , Fibroblastos/metabolismo , Mutação , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Adulto , Idoso , Aneurisma Aórtico/patologia , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Derme/citologia , Proteínas da Matriz Extracelular/farmacologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Miócitos de Músculo Liso/citologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/farmacologia
14.
J Endod ; 43(2): 263-271, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28132711

RESUMO

INTRODUCTION: The present study investigated the in vitro effects of nephronectin (Npnt) on the proliferation, differentiation, and mineralization of a rat odontoblast-like cell line (MDPC-23 cells). METHODS: MDPC-23 cells were cultured on Npnt-coated polystyrene or in the presence of soluble Npnt. Cell proliferation was analyzed using a Cell Counting Kit-8 kit (Dojindo, Kumamoto, Japan). Alkaline phosphatase (ALP) activity was quantified using an ALP activity assay. A reverse-transcription polymerase chain reaction was performed to evaluate the messenger RNA (mRNA) expression level of odontogenic markers and integrin(s). Alizarin red staining was conducted to quantify the calcium deposition. RESULTS: Soluble Npnt had no adverse effect on the proliferation of MDPC-23 cells, but it exhibited concentration-dependent inhibitory activity toward differentiation. In contrast, coated Npnt promoted cell proliferation dramatically and significantly up-regulated the mRNA expression of odontogenesis-related genes; moreover, mRNA expression of integrin α1, α3, α5, ß1, and ß5 was found to be augmented. MDPC-23 cells cultured on Npnt-coated polystyrene displayed markedly higher ALP activity as early as day 3 after inoculation. In addition, mineralization was accelerated on Npnt-coated polystyrene. CONCLUSIONS: Npnt in its immobilized form enhanced the proliferation of MDPC-23 cells and induced this odontoblastic precursor cell line to differentiate into a mineralizing phenotype.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Odontoblastos/efeitos dos fármacos , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fenótipo , Poliestirenos , Ratos
15.
Stem Cells Dev ; 26(8): 599-607, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28095743

RESUMO

Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term self-renewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-week-old colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.


Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Células Secretoras de Insulina/citologia , Niacinamida/farmacologia , Pâncreas/citologia , Células Acinares/citologia , Células Acinares/metabolismo , Ativinas/farmacologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Exenatida , Proteínas da Matriz Extracelular/farmacologia , Feminino , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Peptídeos/farmacologia , Receptores Notch/genética , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Peçonhas/farmacologia
16.
Transplantation ; 101(2): e49-e56, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27764033

RESUMO

BACKGROUND: We have previously reported on a novel organ-specific immunomodifying therapy that provides protection from early allograft rejection in the absence of systemic immunosuppressive drugs. This novel therapy is a nanobarrier membrane called ImmunoCloak, consisting of a matrix of laminin, proteoglycans, fibronectin, and collagens. The membrane "immunocloaks" the luminal surfaces within the renal vasculature by covering the point of contact between donor vascular endothelial cells and the recipient's immune cells, without adversely affecting renal function. The resulting nonthrombogenic and nonimmunogenic apical surface significantly delays the onset of rejection fivefold over untreated controls. Currently, our focus is to elucidate the mechanisms of protection provided by placement of the membrane. METHODS: The mechanisms underlying the protective effect of the ImmunoCloak treatment was evaluated using human peripheral blood mononuclear cells and by testing for antigen presentation by cytokine/chemokine analysis using the Luminex platform, T cell allogeneic responses were measured by flow cytometry, and diapedesis was assessed using transwell plates. RESULTS: We now report that ImmunoCloak interrupts antigen presentation thereby preventing early T cell activation and interferes with diapedesis. There was significant inhibition in the synthesis of proinflammatory cytokines with a concordant blockade of T cell-mediated responses. The placement of the ImmunoCloak also significantly reduced leukocyte migration through the endothelial cell layer by 93%. CONCLUSIONS: Eliminating the need for nephrotoxic immunosuppressive drugs during the early posttransplant period could help to ameliorate the severity of delayed graft function and could provide a path to using more ischemically damaged renal allografts.


Assuntos
Proteínas da Matriz Extracelular/farmacologia , Rejeição de Enxerto/prevenção & controle , Imunoterapia/métodos , Membranas Artificiais , Linfócitos T/efeitos dos fármacos , Aloenxertos , Apresentação do Antígeno/efeitos dos fármacos , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Colágeno/farmacologia , Fibronectinas/farmacologia , Rejeição de Enxerto/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Laminina/farmacologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Nanopartículas , Proteoglicanas/farmacologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Migração Transendotelial e Transepitelial/efeitos dos fármacos
17.
Int J Mol Sci ; 18(1)2016 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-28036084

RESUMO

After peripheral nerve injury, motor and sensory axons are able to regenerate but inaccuracy of target reinnervation leads to poor functional recovery. Extracellular matrix (ECM) components and neurotrophic factors (NTFs) exert their effect on different neuronal populations creating a suitable environment to promote axonal growth. Here, we assessed in vitro and in vivo the selective effects of combining different ECM components with NTFs on motor and sensory axons regeneration and target reinnervation. Organotypic cultures with collagen, laminin and nerve growth factor (NGF)/neurotrophin-3 (NT3) or collagen, fibronectin and brain-derived neurotrophic factor (BDNF) selectively enhanced sensory neurite outgrowth of DRG neurons and motor neurite outgrowth from spinal cord slices respectively. For in vivo studies, the rat sciatic nerve was transected and repaired with a silicone tube filled with a collagen and laminin matrix with NGF/NT3 encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres (MP) (LM + MP.NGF/NT3), or a collagen and fibronectin matrix with BDNF in PLGA MPs (FN + MP.BDNF). Retrograde labeling and functional tests showed that LM + MP.NGF/NT3 increased the number of regenerated sensory neurons and improved sensory functional recovery, whereas FN + MP.BDNF preferentially increased regenerated motoneurons and enhanced motor functional recovery. Therefore, combination of ECM molecules with NTFs may be a good approach to selectively enhance motor and sensory axons regeneration and promote appropriate target reinnervation.


Assuntos
Axônios/fisiologia , Proteínas da Matriz Extracelular/farmacologia , Neurônios Motores/fisiologia , Fatores de Crescimento Neural/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/tratamento farmacológico , Células Receptoras Sensoriais/fisiologia , Animais , Células Cultivadas , Proteínas da Matriz Extracelular/administração & dosagem , Proteínas da Matriz Extracelular/uso terapêutico , Feminino , Microesferas , Fatores de Crescimento Neural/administração & dosagem , Fatores de Crescimento Neural/uso terapêutico , Ratos , Ratos Sprague-Dawley
18.
PLoS One ; 11(10): e0164102, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27741237

RESUMO

OBJECTIVE: Therapeutic agents that are transformable via introducing cleavable linkage by locally enriched MMP-2 within inflamed synovium would enhance therapeutic efficacy on chronic inflammatory arthritis. Transforming growth factor-ß-inducible gene-h3 (ßig-h3), which consists of four fas-1 domains and an Arg-Gly-Asp (RGD) motif, intensifies inflammatory processes by facilitating adhesion and migration of fibroblast-like synoviocyte in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to investigate whether a MMP-2-cleavable peptide complex consisting of a fas-1 domain and an RGD peptide blocks the interaction between ßig-h3 and resident cells and leads to the amelioration of inflammatory arthritis. METHODS: We designed ßig-h3-derivatives, including the fourth fas-1 domain truncated for H1 and H2 sequences of mouse (MFK00) and MMP-2-cleavable peptide complex (MFK902). MMP-2 selectivity was examined by treatment with a series of proteases. MFK902 efficacy was determined by the adhesion and migration assay with NIH3T3 cells in vitro and collagen-induced arthritis (CIA) model using male DBA/1J mice in vivo. The mice were treated intraperitoneally with MFK902 at different dosages. RESULTS: MFK902 was specifically cleaved by active MMP-2 in a concentration-dependent manner, and ßig-h3-mediated adhesion and migration were more effectively inhibited by MFK902, compared with RGD or MFK00 peptides. The arthritis activity of murine CIA, measured by clinical arthritis index and incidence of arthritic paws, was significantly ameliorated after treatment with all dosages of MFK902 (1, 10, and 30 mg/kg). MFK902 ameliorated histopathologic deterioration and reduced the expression of inflammatory mediators simultaneously with improvement of clinical features. In addition, a favorable safety profile of MFK902 was demonstrated in vivo. CONCLUSION: The present study revealed that MMP-2-cleavable peptide complex based on ßig-h3 structure is a potent and safe therapeutic agent for chronic inflammatory arthritis, thus providing reliable evidence for a MMP-2-cleavable mechanism as a tissue-targeted strategy for treatment of RA.


Assuntos
Artrite Experimental/tratamento farmacológico , Proteínas da Matriz Extracelular/uso terapêutico , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Fator de Crescimento Transformador beta/uso terapêutico , Sequência de Aminoácidos , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Doença Crônica , Regulação para Baixo/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Microscopia de Fluorescência , Dados de Sequência Molecular , Células NIH 3T3 , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Índice de Gravidade de Doença , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
19.
Wound Repair Regen ; 24(6): 1030-1035, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27684720

RESUMO

Three-dimensional biomimetic scaffolds resembling the native extracellular matrix (ECM) are widely used in tissue engineering, however they often lack optimal bioactive cues needed for acceleration of cell proliferation, neovascularization, and tissue regeneration. In this study, the use of the ECM-related protein Olfactomedin-like 3 (Olfml3) demonstrates the importance and feasibility of fabricating efficient bioactive scaffolds without in vitro cell seeding prior to in vivo implantation. First, in vivo proangiogenic properties of Olfml3 were shown in a murine wound healing model by accelerated wound closure and a 1.4-fold increase in wound vascularity. Second, subcutaneous implantation of tubular scaffolds coated with recombinant Olfml3 resulted in enhanced cell in-growth and neovascularization compared with control scaffolds. Together, our data indicates the potential of Olfml3 to accelerate neovascularization during tissue regeneration by promoting endothelial cell proliferation and migration. This study provides a promising concept for the reconstruction of damaged tissue using affordable and effective bioactive scaffolds.


Assuntos
Antibacterianos/farmacologia , Materiais Biomiméticos , Proteínas da Matriz Extracelular/farmacologia , Matriz Extracelular/metabolismo , Glicoproteínas/farmacologia , Regeneração , Tecidos Suporte , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões/patologia , Animais , Materiais Biomiméticos/farmacologia , Modelos Animais de Doenças , Feminino , Camundongos , Medicina Regenerativa , Resistência à Tração , Engenharia Tecidual/métodos
20.
Life Sci ; 164: 23-30, 2016 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-27593572

RESUMO

AIMS: Kidney stone formation is a highly prevalent disease, affecting 8-10% of the human population worldwide. Proteins are the major constituents of human kidney stone's organic matrix and considered to play critical role in the pathogenesis of disease but their mechanism of modulation still needs to be explicated. Therefore, in this study we investigated the effect of human kidney stone matrix proteins on the calcium oxalate monohydrate (COM) mediated cellular injury. MAIN METHODS: The renal epithelial cells (MDCK) were exposed to 200µg/ml COM crystals to induce injury. The effect of proteins isolated from human kidney stone was studied on COM injured cells. The alterations in cell-crystal interactions were examined by phase contrast, polarizing, fluorescence and scanning electron microscopy. Moreover, its effect on the extent of COM induced cell injury, was quantified by flow cytometric analysis. KEY FINDINGS: Our study indicated the antilithiatic potential of human kidney stone proteins on COM injured MDCK cells. Flow cytometric analysis and fluorescence imaging ascertained that matrix proteins decreased the extent of apoptotic injury caused by COM crystals on MDCK cells. Moreover, the electron microscopic studies of MDCK cells revealed that matrix proteins caused significant dissolution of COM crystals, indicating cytoprotection against the impact of calcium oxalate injury. SIGNIFICANCE: The present study gives insights into the mechanism implied by urinary proteins to restrain the pathogenesis of kidney stone disease. This will provide a better understanding of the formation of kidney stones which can be useful for the proper management of the disease.


Assuntos
Oxalato de Cálcio/toxicidade , Células Epiteliais/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Cães , Células Epiteliais/citologia , Células Epiteliais/patologia , Citometria de Fluxo , Humanos , Células Madin Darby de Rim Canino/efeitos dos fármacos , Células Madin Darby de Rim Canino/ultraestrutura
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