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1.
Mater Sci Eng C Mater Biol Appl ; 107: 110212, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761208

RESUMO

A critical challenge to the development of tissue engineering small-diameter vascular grafts is to achieve rapid endothelialization and long-term anticoagulation. It is necessary to graft both adhesion and antithrombus factors onto the surface of polycaprolactone without burst release to promote endothelial cell affinity and antithrombogenicity. A bionic structure with a nanocoating that allows a biologically responsive, long-term release was employed in this work to enable the grafting of various bioactive molecules such as gelatin, polylysine, and heparin. This approach involved orienting the biomimetic vascular structures; the self-assembly grafting of gelatin, polylysine, and heparin nanoparticles; and genipin crosslinking to form a multiphase crosslinked nanocoating. In this biologically inspired design, vascular endothelialization and long-term anticoagulation were successfully induced through a matrix metallopeptidase 2 regulative mechanism by delivering both adhesion and antithrombus factors with a responsive, long-term release without burst release. The method provided a simple and effective approach for delivering dual factors for tissue engineering small-diameter vascular grafts.


Assuntos
Materiais Biomiméticos/química , Nanotecnologia , Materiais Biomiméticos/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/citologia , Prótese Vascular , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Gelatina/química , Heparina/química , Heparina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Nanofibras/química , Poliésteres/química , Polilisina/química , Propriedades de Superfície
2.
Int J Mol Sci ; 20(20)2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31618886

RESUMO

The role of metalloproteinases (MMPs) on the migration and invasion of cancer cells has been correlated with tumor aggressiveness, namely with the up-regulation of MMP-2 and 9. Herein, two pyridine-containing macrocyclic compounds, [15]pyN5 and [16]pyN5, were synthesized, chemically characterized and evaluated as potential MMP inhibitors for breast cancer therapy using 3D and 2D cellular models. [15]pyN5 and [16]pyN5 (5-20 µM) showed a marked inhibition of MMPs activity (100% at concentrations ≥ 7.5 µM) when compared to ARP-100, a known MMP inhibitor. The inhibitory activity of [15]pyN5 and [16]pyN5 was further supported through in silico docking studies using Goldscore and ChemPLP scoring functions. Moreover, although no significant differences were observed in the invasion studies in the presence of all MMPs inhibitors, cell migration was significantly inhibited by both pyridine-containing macrocycles at concentrations above 5 µM in 2D cells (p < 0.05). In spheroids, the same effect was observed, but only with [16]pyN5 at 20 µM and ARP-100 at 40 µM. Overall, [15]pyN5 and [16]pyN5 led to impaired breast cancer cell migration and revealed to be potential inhibitors of MMPs 2 and 9.


Assuntos
Compostos Macrocíclicos/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Piridinas/farmacologia , Sítios de Ligação , Catálise , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Compostos Macrocíclicos/química , Metaloproteinase 2 da Matriz/química , Metaloproteinase 9 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Piridinas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Zinco/química
3.
Nanoscale ; 11(39): 18426-18435, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576881

RESUMO

This work explored the application of matrix metalloproteinase 2-targeted superparamagnetic nanoprobes for magnetic resonance imaging (MRI), near infrared (NIR) fluorescence imaging and photodynamic therapy of tumors. PEG, PAMAM (G5) and matrix metalloproteinase 2 (MMP2) were attached to the surface of carboxylated Fe3O4 nanoparticles (NPs) using a chemical coupling method and then finally loaded with the photosensitizer chlorin e6 (Ce6). In vitro and in vivo experiments demonstrated that the Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes exhibited excellent stability, precise tumor targeting and biocompatibility. Furthermore, the fluorescence properties of Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes were analogous to Ce6 and could be employed for fluorescence imaging. Meanwhile, the Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes have also been shown to be effective as contrast agents for T2-weighted MRI. The target molecule MMP2 enhanced the tumor targeting ability of Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes. Additionally, the Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes significantly inhibited tumor growth compared with PBS and free Ce6. This work will inspire greater enthusiasm for the construction of multifunctional magnetic nanoplatforms for biomedical applications.


Assuntos
Nanopartículas de Magnetita , Neoplasias Experimentais , Imagem Óptica , Fotoquimioterapia , Fármacos Fotossensibilizantes , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/uso terapêutico , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/tratamento farmacológico , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461891

RESUMO

Matrix metaloproteinase-2 (MMP-2) is an extracellular Zn2+ protease specific to type I and IV collagens. Its expression is associated with several inflammatory, degenerative, and malignant diseases. Conformational properties, domain movements, and interactions between MMP-2 and its associated metal ions were characterized using a 1.0 µs molecular dynamics simulation. Dihedral principle component analysis revealed ten families of conformations with the greatest degree of variability occurring in the link region connecting the catalytic and hemopexin domains. Dynamic cross-correlation analysis indicated domain movements corresponding to the opening and closing of the hemopexin domain in relation to the fibronectin and catalytic domains facilitated by the link region. Interaction energies were calculated using the molecular mechanics Poisson Boltzman surface area-interaction entropy (MMPBSA-IE) analysis method and revealed strong binding energies for the catalytic Zn2+ ion 1, Ca2+ ion 1, and Ca2+ ion 3 with significant conformational stability at the binding sites of Zn2+ ion 1 and Ca2+ ion 1. Ca2+ ion 2 diffuses freely away from its crystallographically defined binding site. Zn2+ ion 2 plays a minor role in conformational stability of the catalytic domain while Ca2+ ion 3 is strongly attracted to the highly electronegative sidechains of the Asp residues around the central ß-sheet core of the hemopexin domain; however, the interacting residue sidechain carboxyl groups are outside of Ca2+ ion 3's coordination sphere.


Assuntos
Metaloproteinase 2 da Matriz/química , Simulação de Dinâmica Molecular , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Zinco/química , Zinco/metabolismo
5.
Biochimie ; 166: 223-232, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31362036

RESUMO

The 72-kDa type IV collagenase or gelatinase A is the second member of the matrix metalloproteinase family, MMP-2. Since the discovery of its first two substrates within components of the extracellular matrix, denatured interstitial type I collagen and native type IV collagen, the roles and various levels of regulation of MMP-2 have been intensively studied, mainly in vitro. Its (over)expression in most if not all tumors was considered a hallmark of cancer aggressiveness and boosted investigations aiming at its inhibition. Unfortunately, the enthusiasm subsided like a soufflé after clinical trial failures, mostly because of insufficient knowledge of in vivo MMP-2 activities and detrimental side effects of broad-spectrum MMP inhibition. Nowadays, MMP-2 remains a major topic of interest in research, the second in the MMP family after MMP-9. This review presents a broad overview of the major features of this protease. This knowledge is crucial to identify diagnostic or therapeutic strategies focusing on MMP-2. In this sense, recent publications and clinical trials underline the potential value of measuring circulating or tissular MMP-2 levels as diagnostic or prognostic tools, or as a useful secondary outcome for therapies against other primary targets. Direct MMP-2 inhibition has benefited from substantial progress in the design of more specific inhibitors but their in vivo application remains challenging but certainly worth the efforts it receives.


Assuntos
Metaloproteinase 2 da Matriz , Inibidores de Metaloproteinases de Matriz/farmacologia , Neoplasias/enzimologia , Biomarcadores Tumorais/química , Biomarcadores Tumorais/fisiologia , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Humanos , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/fisiologia , Células Tumorais Cultivadas
6.
Biochim Biophys Acta Mol Basis Dis ; 1865(9): 2441-2450, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31175931

RESUMO

Although macrophage migration inhibitory factor (MIF) is known to have antioxidant property, the role of MIF in cardiac fibrosis has not been well understood. We found that MIF was markedly increased in angiotension II (Ang-II)-infused mouse myocardium. Myocardial function was impaired and cardiac fibrosis was aggravated in Mif-knockout (Mif-KO) mice. Functionally, overexpression of MIF and MIF protein could inhibit the expression of fibrosis-associated collagen (Col) 1a1, COL3A1 and α-SMA, and Smad3 activation in mouse cardiac fibroblasts (CFs). Consistently, MIF deficiency could exacerbate the expression of COL1A1, COL3A1 and α-SMA, and Smad3 activation in Ang-II-treated CFs. Interestingly, microRNA-29b-3p (miR-29b-3p) and microRNA-29c-3p (miR-29c-3p) were down-regulated in the myocardium of Ang-II-infused Mif-KO mice but upregulated in CFs with MIF overexpression or by treatment with MIF protein. MiR-29b-3p and miR-29c-3p could suppress the expression of COL1A1, COL3A1 and α-SMA in CFs through targeting the pro-fibrosis genes of transforming growth factor beta-2 (Tgfb2) and matrix metallopeptidase 2 (Mmp2). We further demonstrated that Mif inhibited reactive oxygen species (ROS) generation and Smad3 activation, and rescued the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Smad3 inhibitors, SIS3 and Naringenin, and Smad3 siRNA could reverse the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Taken together, our data demonstrated that the Smad3-miR-29b/miR-29c axis mediates the inhibitory effect of macrophage migration inhibitory factor on cardiac fibrosis.


Assuntos
Fatores Inibidores da Migração de Macrófagos/metabolismo , MicroRNAs/metabolismo , Proteína Smad3/metabolismo , Regiões 3' não Traduzidas , Animais , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Cardiomegalia/patologia , Cardiomegalia/veterinária , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/química , MicroRNAs/genética , Miocárdio/citologia , Miocárdio/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta2/química , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Regulação para Cima
7.
Biomater Sci ; 7(7): 2920-2933, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31090763

RESUMO

Stem cell-derived exosomes have been recognized as a potential therapy for cardiovascular disease. However, the low retention rate of exosomes after transplantation in vivo remains a major challenge in clinical applications. The aim of this study is to investigate whether human umbilical cord mesenchymal stem cell derived exosomes (UMSC-Exo) encapsulated in functional peptide hydrogels could increase the retention and stability of exosomes and improve heart function in a rat myocardial infarction model. Our results demonstrated that the PA-GHRPS peptide protected H9C2 cells from H2O2-induced oxidative stress. The gelatinization ability of PA-GHRPS can be enhanced by peptide NapFF. Therefore, these two peptides were mixed to form the PGN hydrogel, which was used to encapsulate exosomes. Our data showed that the PGN hydrogel was able to encapsulate exosomes effectively and ensured a stable and sustained release of exosomes. The exosome/PGN hydrogel mixture was injected into the infarcted border zone of rat hearts. Compared to the exosome treatment alone, the mixture improved the myocardial function by reducing inflammation, fibrosis and apoptosis, and by promoting angiogenesis. The strategy used in this study provided a practical and effective method to harness exosomes for myocardial regeneration.


Assuntos
Exossomos/metabolismo , Coração/efeitos dos fármacos , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Peptídeos/química , Peptídeos/farmacologia , Cordão Umbilical/citologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Cápsulas , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Exossomos/química , Coração/fisiologia , Coração/fisiopatologia , Humanos , Peróxido de Hidrogênio/farmacologia , Metaloproteinase 2 da Matriz/química , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos
8.
J Nucl Med ; 60(10): 1474-1482, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30954944

RESUMO

Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111In-radiolabeled PA-L1, 111In-PA-WTK563C, or 111In-LFE687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1-mediated delivery of 111In-LFE687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LFE687A Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Radioisótopos de Índio/química , Neoplasias/diagnóstico por imagem , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Transporte Biológico , Linhagem Celular Tumoral , Humanos , Cinética , Células MCF-7 , Metaloproteinase 14 da Matriz/química , Metaloproteinase 2 da Matriz/química , Metaloproteinases da Matriz/metabolismo , Camundongos , Mutação , Transplante de Neoplasias
9.
Bioorg Med Chem ; 27(9): 1891-1902, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30926311

RESUMO

MMP2 and MMP9, also called gelatinases, play a primary role in the angiogenic switch, as a fundamental step of tumor progression, and show high degree of structural similarity. Clinically successful gelatinase inhibitors need to be highly selective as opposite effects have been found for the two enzymes, and the S1' subsite is the major driver to attain selective and potent inhibitors. The synthesis of d-proline-derived hydroxamic acids containing diverse appendages at the amino group, varying in length and decoration allowed to give insight on the MMP2/MMP9 selectivity around the S1' subsite, resulting in the identification of sub-nanomolar compounds with high selectivity up to 730. Molecular docking studies revealed the existence of an additional hydrophobic channel at the bottom of S1' subsite for MMP2 enzyme useful to drive selectivity towards such gelatinase.


Assuntos
Metaloproteinase 2 da Matriz/química , Inibidores de Metaloproteinases de Matriz/química , Prolina/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/farmacologia , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade
10.
Acta Biomater ; 89: 265-278, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851453

RESUMO

In the war against cancer, nanotechnology-based drug delivery systems may play a significant role by enhancing the efficacy of conventional therapies. Here, we tried to address some major limitations plaguing anticancer drugs, namely, poor water solubility and off-target toxicity. The systems we propose are cross-linked polyelectrolyte nanocapsules based on an oil-core and a matrix metalloproteinase-2 (MMP-2)-cleavable shell. They can load hydrophobic drugs, prevent their systemic leakage, and release their payload upon an endogenous stimulus. Both the stability enhancement and the stimuli-responsive drug release mechanisms were achieved by cross-linking the nanocapsule shell with an MMP-2-sensitive peptide. On the basis of this strategy, the system maintained its stability in PBS up to one month. Further, when tested on 3D tumor and healthy spheroid models, the nanocapsules were able to disrupt their integrity preferentially in the tumor-like microenvironment. The high level of MMP-2 enzymes expressed by tumor spheroids, indeed, catalyzed the disassembly of the nanocapsules, which ultimately leads to drug release. Therefore, this device holds great potential as a smart system that allows for the safe transport of hydrophobic drugs and for a spatially controlled release upon an endogenous stimulus coming from the very nature of the tumor itself. STATEMENT OF SIGNIFICANCE: The performance of nanoparticle-based therapeutic approaches is often hindered by some intrinsic limitations typically including laborious drug loading methods, synthetic routes of preparation and stability issues. In this work, we implemented for the first time a smart drug delivery strategy into oil-core multilayer nanocapsules, which represent an ideal family of nanocarriers. To this aim, we developed a robust method enabling the use of soft matters like oil-core nanocapsules combined with a microenvironmentally triggered release mechanism. The efficacy of nanocapsules was tested on tumor and healthy spheroids. Results clearly demonstrate their selective drug release, triggered by a stimulus intrinsically present in tumor microenvironment. We believe this study is of particular interest for cancer research and paves the way for the use of these robust stimuli-responsive nanocapsules in vivo.


Assuntos
Metaloproteinase 2 da Matriz/química , Nanocápsulas , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Peptídeos , Polieletrólitos , Microambiente Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Humanos , Nanocápsulas/química , Nanocápsulas/uso terapêutico , Neoplasias/enzimologia , Neoplasias/patologia , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologia , Polieletrólitos/química , Polieletrólitos/farmacocinética , Polieletrólitos/farmacologia
11.
ACS Chem Biol ; 14(4): 775-783, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30807095

RESUMO

Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using self-assembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self-assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pretreated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment.


Assuntos
Transplante de Células , Metaloproteinase 2 da Matriz , Sindecanas , Animais , Membrana Celular/metabolismo , Movimento Celular , Sobrevivência Celular , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Multimerização Proteica , Sindecanas/química , Sindecanas/metabolismo
12.
Adv Healthc Mater ; 8(3): e1801545, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30620448

RESUMO

Nanomedicine is a promising, noninvasive approach to reduce atherosclerotic plaque burden. However, drug delivery is limited without the ability of nanocarriers to sense and respond to the diseased microenvironment. In this study, nanomaterials are developed from peptide amphiphiles (PAs) that respond to the increased levels of matrix metalloproteinases 2 and 9 (MMP2/9) or reactive oxygen species (ROS) found within the atherosclerotic niche. A pro-resolving therapeutic, Ac2-26, derived from annexin-A1 protein, is tethered to PAs using peptide linkages that cleave in response to MMP2/9 or ROS. By adjusting the molar ratios and processing conditions, the Ac2-26 PA can be co-assembled with a PA containing an apolipoprotein A1-mimetic peptide to create a targeted, therapeutic nanofiber (ApoA1-Ac226 PA). The ApoA1-Ac2-26 PAs demonstrate release of Ac2-26 within 24 h after treatment with MMP2 or ROS. The niche-responsive ApoA1-Ac2-26 PAs are cytocompatible and reduce macrophage activation from interferon gamma and lipopolysaccharide treatment, evidenced by decreased nitric oxide production. Interestingly, the linkage chemistry of ApoA1-Ac2-26 PAs significantly affects macrophage uptake and retention. Taken together, these findings demonstrate the potential of PAs to serve as an atheroma niche-responsive nanocarrier system to modulate the inflammatory microenvironment, with implications for atherosclerosis treatment.


Assuntos
Anexina A1 , Apolipoproteína A-I , Aterosclerose , Portadores de Fármacos , Imunoterapia , Nanofibras , Peptídeos , Placa Aterosclerótica , Animais , Anexina A1/química , Anexina A1/farmacologia , Apolipoproteína A-I/química , Apolipoproteína A-I/farmacologia , Aterosclerose/imunologia , Aterosclerose/patologia , Aterosclerose/terapia , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/farmacologia , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/farmacologia , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/farmacologia , Camundongos , Nanofibras/química , Nanofibras/uso terapêutico , Peptídeos/química , Peptídeos/farmacologia , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Placa Aterosclerótica/terapia
13.
Hum Mutat ; 40(5): 539-551, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30668888

RESUMO

Sorsby fundus dystrophy (SFD) is a macular degeneration caused by mutations in TIMP3, the majority of which introduce a novel cysteine. However, the exact molecular mechanisms underlying SFD remain unknown. We aimed to provide novel insights into the functional consequences of a distinct N-terminal mutation. Haplotype reconstruction in three SFD families revealed that the identified c.113C>G, p.(Ser38Cys) mutation is a founder in Belgian and northern French families with a late-onset SFD phenotype. Functional consequences of the p.(Ser38Cys) mutation were investigated by high-resolution Western blot analysis of wild type and mutant TIMP3 using patient fibroblasts and in vitro generated proteins, and by molecular modeling of TIMP3 and its interaction partners. We could not confirm a previous hypothesis on dimerization of mutant TIMP3 proteins. However, we identified aberrant intramolecular disulfide bonding. Our data provide evidence for disruption of the established Cys36-Cys143 disulfide bond and formation of a novel Cys36-Cys38 bond, possibly associated with increased glycosylation of the protein. In conclusion, we propose a novel pathogenetic mechanism underlying the p.(Ser38Cys) TIMP3 founder mutation involving intramolecular disulfide bonding. These results provide new insights into the pathogenesis of SFD and other retinopathies linked to mutations in TIMP3, such as age-related macular degeneration.


Assuntos
Efeito Fundador , Degeneração Macular/diagnóstico , Degeneração Macular/genética , Mutação , Domínios e Motivos de Interação entre Proteínas , Inibidor Tecidual de Metaloproteinase-3/química , Inibidor Tecidual de Metaloproteinase-3/genética , Idoso , Dissulfetos , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Haplótipos , Humanos , Masculino , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Peso Molecular , Linhagem , Conformação Proteica , Relação Estrutura-Atividade , Inibidor Tecidual de Metaloproteinase-3/metabolismo
14.
Fish Shellfish Immunol ; 84: 404-413, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30316944

RESUMO

Matrix metalloproteinases (MMPs) contribute to both normal and pathological tissue remodeling. They act as regulatory molecules by working in enzyme cascades as well as processing matrix proteins, cytokines, growth factors and adhesion molecules to generate fragments with biological effects. So MMPs could play distrinct roles in the process of pathogen infection. In present study, we cloned a MMP-2 (LvMMP-2) gene from Litopenaeus vannamei. LvMMP-2, highly expressed in epidermis, located to endoplasmic reticulum in S2 cells. Results of real-time RT-PCR assay showed that LvMMP-2 was induced in shrimp hemocytes upon unfolded protein response or oxidative stress, but not via heat shock treatment. It is proved that the promoter activity of LvMMP-2 was enhanced by NF-E2-related factor 2 and AP-1 factor c-Jun. Further research showed that down-regulated LvMMP-2 contributing to oxidative stress injury, could reduce the cumulative mortality of shrimps under oxidative stress. Besides, our study also indicated that LvMMP-2 was accelerated by lipopolysaccharides injection. LvMMP-2 in S2 could increase the promoter activity of several antimicrobial peptide genes, and knocked-down expression of LvMMP-2 depressed the expression of penaeidin2 and ß-Defensin. Moreover, we showed that down-regulated LvMMP-2 suppressed the cumulative mortality of shrimp infected with white spot syndrome virus (WSSV) or with Vibrio alginolyticus. Collecting results suggested that LvMMP-2 involves in shrimp innate immune response, and also contributes to tissue injury caused by WSSV infection.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/imunologia , Penaeidae/genética , Penaeidae/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Metaloproteinase 2 da Matriz/química , Filogenia , Alinhamento de Sequência , Vibrio alginolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
15.
Cytokine ; 113: 340-346, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30352759

RESUMO

BACKGROUND: Inhibiting TNF-α is an effective therapy for inflammatory diseases such as rheumatoid arthritis. However, systemic, nondiscriminatory neutralization of TNF-α is associated with considerable adverse effects. METHODS: Here, we developed a trimeric chimeric TNF receptor by linking an N-terminal mouse Acrp30 trimerization domain and an MMP-2/9 substrate sequence to the mouse extracellular domain of TNF receptor 2 followed by a C-terminal mouse tetranectin coiled-coil domain (mouse Acrp-MMP-TNFR-Tn). RESULTS: Here, we show that the Acrp30 trimerization domain inhibited the binding activity of TNFR, possibly by closing the binding site of the trimeric receptor. Cleavage of the substrate sequence by MMP-9, an enzyme highly expressed in inflammatory sites, restored the binding activity of the mouse TNF receptor. We also constructed a recombinant human chimeric TNF receptor (human Acrp-MMP-TNFR-Tn) in which an MMP-13 substrate sequence was used to link the human Acrp and the human TNF receptor 2. Human Acrp-MMP-TNFR-Tn showed reduced binding activity, and MMP-13 digestion recovered its binding activity with TNF-α. CONCLUSION: Acrp-masked chimeric TNF receptors may be able to be used for inflammatory tissue-selective neutralization of TNF-α to reduce the adverse effects associated with systemic neutralization of TNF-α.


Assuntos
Adiponectina , Metaloproteinase 13 da Matriz , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Multimerização Proteica , Receptores Tipo II do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão , Fator de Necrose Tumoral alfa , Adiponectina/química , Adiponectina/genética , Adiponectina/metabolismo , Animais , Linhagem Celular , Humanos , Metaloproteinase 13 da Matriz/química , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Especificidade de Órgãos , Ligação Proteica , Domínios Proteicos , Receptores Tipo II do Fator de Necrose Tumoral/química , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo
16.
Biomacromolecules ; 20(1): 422-434, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30457842

RESUMO

The tissue environment is exceptionally complex, with well-controlled biochemical communication occurring between similar and dissimilar cells as well as between these cells and local extracellular matrices (ECM). To build an artificial ECM that can directly affect regional cell populations, a designer system should allow for controlled degradation, molecular release, and reorganization as related to local cellular function. (RADA)4 self-assembling peptide (SAP) hydrogels are excellent candidates for precisely tuned ECMs, or nanoscaffolds, with several beneficial qualities. They are a class of materials with uncomplicated fabrication and potentially allow for a diverse set of release strategies for many types of bioactive ligands. Enzyme-induced degradation and release of peptide sequences, synthesized within the SAP for on-demand cell signaling, could prove impactful to a plethora of human health applications. However, the degradation products and their release kinetics from these nanoscaffolds may greatly affect the overall system. To address this, enzyme kinetics in self-assembled hydrogels were studied by tethering matrix metalloproteinase 2 (MMP-2) cleavable peptide substrates of differing activities to the C-terminus of (RADA)4. High and low activity sequences, GPQG+IASQ (CP1) and GPQG+PAGQ (CP2), were respectively chosen for tunable release. When incubated with 5 nM MMP-2, over 3 days, both CP1 and CP2 sequences showed product formation values of ∼32% and ∼9% of the original substrate, respectively. On-demand product formation was found to be dependent upon both SAP composition and enzyme concentrations and could be tuned over the course of several days and weeks. Despite the fact that the self-assembling peptides are not directly cleavable by MMP-2, the CP1 and CP2 nanoscaffold morphology was visibly degraded by the protease. This degradation yielded a lower fractal dimensions for the matrix and suggested clearance of these materials may be possible over time.


Assuntos
Metaloproteinase 2 da Matriz/química , Oligopeptídeos/química , Polimerização , Multimerização Proteica , Biocatálise , Humanos , Hidrogéis/química , Metaloproteinase 2 da Matriz/metabolismo , Proteólise , Polímeros Responsivos a Estímulos/química
17.
Nat Prod Res ; 33(12): 1765-1768, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29394875

RESUMO

Dysregulation of matrix metalloproteinases (MMPs) activity is known in many pathological conditions with which most of the conditions are related to elevate MMPs activities. Ficus deltoidea (FD) is a plant known for its therapeutic properties. In order to evaluate the therapeutic potential of FD leaf extract, we study the enzymatic inhibition properties of FD leaf extract and its major bioactive compounds (vitexin and isovitexin) on a panel of MMPs (MMP-2, MMP-8 and MMP-9) using experimental and computational approaches. FD leaf extract and its major bioactive compounds showed pronounced inhibition activity towards the MMPs tested. Computational docking analysis revealed that vitexin and isovitexin bind to the active site of the three tested MMPs. We also evaluated the cytotoxicity and cell migration inhibition activity of FD leaf extract in the endothelial EA.hy 926 cell line. Conclusively, this study provided additional information on the potential of FD leaf extract for therapeutical application.


Assuntos
Apigenina/farmacologia , Ficus/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Extratos Vegetais/farmacologia , Apigenina/química , Apigenina/metabolismo , Domínio Catalítico , Linhagem Celular , Movimento Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/química , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Extratos Vegetais/química , Folhas de Planta/química
18.
Bioconjug Chem ; 29(11): 3715-3725, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30277751

RESUMO

Matrix metalloproteinases (MMPs) are emerging as pivotal fine-tuners of cell function in tissue homeostasis and in various pathologies, in particular inflammation. In vivo monitoring of the activity of specific MMPs, therefore, provides high potential for assessing disease progression and tissue function, and manipulation of MMP activity in tissues and whole organisms may further provide a mode of controlling pathological processes. We describe here the synthesis of novel fluorinated and nonfluorinated analogues of a secondary sulfonamide-based lead structure, compound 2, and test their efficacy as in vivo inhibitors and tracers of the gelatinases, MMP-2 and MMP-9. Using a murine neuroinflammatory model, we show that compound 2 is a highly effective in vivo inhibitor of both MMP-2 and MMP-9 activity with little or no adverse effects even after long-term daily oral administration. A fluorescein-labeled derivative compound 17 shows direct binding to activated gelatinases surrounding inflammatory cuffs in the neuroinflammation model and to pancreatic ß-cells in the islets of Langerhans, colocalizing with MMP-2 and MMP-9 activity as detected using in situ zymography techniques. These results demonstrate that compound 2 derivatives have potential as in vivo imaging tools and for future development for specific MMP-2 versus MMP-9 probes. Our chemical modifications mainly target the residues directed toward the S1' and S2' pockets and, thereby, provide new information on the structure-activity relationships of this inhibitor type.


Assuntos
Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Linhagem Celular , Feminino , Halogenação , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/enzimologia , Células Secretoras de Insulina/metabolismo , Metaloproteinase 2 da Matriz/química , Metaloproteinase 9 da Matriz/química , Inibidores de Metaloproteinases de Matriz/efeitos adversos , Inibidores de Metaloproteinases de Matriz/síntese química , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Sulfonamidas/efeitos adversos , Sulfonamidas/síntese química
19.
J Nutr Sci Vitaminol (Tokyo) ; 64(4): 271-276, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30175790

RESUMO

Abdominal aortic aneurysm (AAA) is a vascular disease characterized by chronic inflammation in the infrarenal aorta. Epidemiologic data have clearly linked tobacco smoking to aneurysm formation and a faster rate of expansion. It suggested that nicotine, one of the main ingredients of tobacco, has been suggested to be associated with AAA development and rupture. In the condition where no established drugs are available; therefore, an effective approach to prevent the vascular damage from nicotine consumption may be the use of dietary functional food factors. However, little is known about the relationship between dietary components and AAA. In this study, we estimated the effect of dietary deoxyribonucleic acid (DNA) on the vascular wall. After habituation for 5 d, the mice were divided into four groups: control diet and distilled water group (C), DNA-Na diet and distilled water group (DNA), control diet and 0.5 mg/mL nicotine solution group (C-Nic), DNA-Na diet, and 0.5 mg/mL nicotine solution group (DNA-Nic). The dietary DNA attenuated the degradation of elastin fibers induced by nicotine administration. The areas stained positive for MMP-2 in the DNA-Nic group were significantly suppressed compared to C-Nic mice. These data suggest that the dietary DNA may prevent the weakening of the aortic wall via inhibition of the MMP-2-dependent pathway. In conclusion, we have revealed the protective effect of dietary DNA on the vascular pathology of nicotine-administrated mice. A nucleic acid-rich diet might be useful for people who consume nicotine via smoking, chewing tobacco, or nicotine patches.


Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/prevenção & controle , DNA/uso terapêutico , Suplementos Nutricionais , Modelos Animais de Doenças , Elastina/metabolismo , Endotélio Vascular/metabolismo , Túnica Adventícia/efeitos dos fármacos , Túnica Adventícia/imunologia , Túnica Adventícia/metabolismo , Túnica Adventícia/patologia , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Antioxidantes/uso terapêutico , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/imunologia , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Fármacos Cardiovasculares/uso terapêutico , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Imuno-Histoquímica , Masculino , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Nicotina/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Proteólise/efeitos dos fármacos
20.
Am J Vet Res ; 79(9): 986-994, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30153058

RESUMO

OBJECTIVE To investigate the effect of lipopolysaccharide (LPS) on type VII collagen- cleaving matrix metalloproteinases (MMPs) in the lamellar tissue of extracorporeally perfused equine limbs. SAMPLE 10 right forelimbs and 3 left forelimbs collected from 10 adult horses after slaughter at a licensed abattoir. PROCEDURES Extracorporeal perfusion of the isolated equine limbs was performed for 10 hours under physiologic conditions (control-perfused limbs; n = 5) and with the addition of 80 ng of LPS/L of perfusate (LPS-perfused limbs; 5). Lamellar tissue specimens were then collected from the dorsal aspect of the hooves. Additionally, corresponding control specimens were collected from the 3 nonperfused left forelimbs. Immunohistochemical analysis was performed on paraffin-embedded tissue blocks with antibodies against total (latent and active) MMP-1, MMP-2, MMP-8, and MMP-9 as well as antibody against active MMP-9. Intensity of immunohistochemical staining was scored, and stain distribution in the lamellar tissue was noted. RESULTS Staining intensity of total and active MMP-9 was significantly increased in LPS-perfused versus control-perfused limbs. No such difference was identified for MMP-1, MMP-2, and MMP-8. CONCLUSIONS AND CLINICAL RELEVANCE Of the 4 MMPs that are capable of degrading type VII collagen, MMP-9 was the only one for which production increased in the lamellar tissue of isolated equine limbs perfused with versus without a clinically relevant concentration of LPS. These results suggested that MMP-9 may be involved in initiation of pathological changes in lamellar tissue in endotoxin-induced laminitis, whereas MMP-1, MMP-2, and MMP-8 may be less relevant.


Assuntos
Colágeno Tipo VII/química , Endotoxinas/química , Metaloproteinases da Matriz/química , Animais , Extremidades , Casco e Garras , Cavalos , Imuno-Histoquímica , Lipopolissacarídeos , Metaloproteinase 1 da Matriz/química , Metaloproteinase 2 da Matriz/química , Metaloproteinase 8 da Matriz/química , Metaloproteinase 9 da Matriz/química , Perfusão/veterinária
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