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1.
PLoS One ; 15(7): e0235784, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658899

RESUMO

Soft tissue is composed of cells surrounded by an extracellular matrix that is made up of a diverse array of intricately organized proteins. These distinct components work in concert to maintain homeostasis and respond to tissue damage. During tissue repair, extracellular matrix proteins and their degradation products are known to influence physiological processes such as angiogenesis and inflammation. In this study we developed a discovery platform using a decellularized extracellular matrix biomaterial to identify new chemotrophic factors derived from the extracellular matrix. An in vitro culture of RAW.264 macrophage cells with the biomaterial ovine forestomach matrix led to the identification of a novel ~12 kDa chemotactic factor, termed 'MayDay', derived from the N-terminal 31-188 sequence of decorin. The recombinant MayDay protein was shown to be a chemotactic agent for mesenchymal stromal cells in vitro and in vivo. We hypothesize that the macrophage-induced cleavage of decorin, via MMP-12, leads to the release of the chemotactic molecule MayDay, that in turn recruits cells to the site of damaged tissue.


Assuntos
Fatores Quimiotáticos/farmacologia , Decorina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Fatores Quimiotáticos/química , Quimiotaxia/efeitos dos fármacos , Decorina/química , Células-Tronco Mesenquimais/citologia , Camundongos , Fragmentos de Peptídeos/química , Células RAW 264.7 , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Ovinos
2.
Clin Chim Acta ; 491: 1-7, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30629950

RESUMO

The extracellular matrix (ECM) prevents invasion of tumour cells and possesses an intrinsic mechanism to down-regulate signalling processes that promote cancer proliferation. Small Leucine Rich Proteoglycans (SLRPs) are ubiquitous ECM components involved in matrix structural organization and as such can potentially regulate cancer cell multiplication, angiogenesis and migration. Decorin, a class I SLRP that modulates collagen fibrillogenesis, also functions as a natural pan-tyrosine kinase inhibitor to reduce tumour growth. In fact, decreased decorin expression has been associated with tumour aggressiveness and lower survival. In contrast, biglycan, another class I SLRP, was highly expressed in cancer and was associated with metastatic activity and lower survival. Tissue expression of lumican, a class II SLRP, was associated with clinical outcome and appears tumour specific. Recently, decorin, biglycan and lumican were found to be potential biomarkers in bladder cancer. This review updates our current understanding on the molecular interplay and significance of decorin, biglycan and lumican expression in cancer.


Assuntos
Biglicano/química , Biglicano/metabolismo , Decorina/química , Decorina/metabolismo , Leucina , Lumicana/química , Lumicana/metabolismo , Neoplasias/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/patologia
3.
J Am Chem Soc ; 139(46): 16986-16995, 2017 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29111696

RESUMO

Glycomics represents one of the last frontiers and most challenging in omic analysis. Glycosylation occurs in the endoplasmic reticulum and the Golgi organelle and its control is neither well-understood nor predictable based on proteomic or genomic analysis. One of the most structurally complex classes of glycoconjugates is the proteoglycans (PGs) and their glycosaminoglycan (GAG) side chains. Previously, our laboratory solved the structure of the chondroitin sulfate chain of the bikunin PG. The current study examines the much more complex structure of the dermatan sulfate GAG chain of decorin PG. By utilizing sophisticated separation methods followed by compositional analysis, domain mapping, and tandem mass spectrometry coupled with analysis by a modified genetic algorithm approach, the structural motif for the decorin dermatan sulfate chain was determined. This represents the second example of a GAG with a prominent structural motif, suggesting that the structural variability of this class of glycoconjugates is somewhat simpler than had been expected.


Assuntos
Decorina/química , Dermatan Sulfato/química , Algoritmos , Animais , Decorina/isolamento & purificação , Dermatan Sulfato/isolamento & purificação , Suínos
4.
Biomaterials ; 134: 154-165, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28463693

RESUMO

Skin scarring after deep dermal injuries is a major clinical problem due to the current therapies limited to established scars with poor understanding of healing mechanisms. From investigation of aberrations within the extracellular matrix involved in pathophysiologic scarring, it was revealed that one of the main factors responsible for impaired healing is abnormal collagen reorganization. Here, inspired by the fundamental roles of decorin, a collagen-targeting proteoglycan, in collagen remodeling, we created a scar-preventive collagen-targeting glue consisting of a newly designed collagen-binding mussel adhesive protein and a specific glycosaminoglycan. The collagen-targeting glue specifically bound to type I collagen in a dose-dependent manner and regulated the rate and the degree of fibrillogenesis. In a rat skin excisional model, the collagen-targeting glue successfully accelerated initial wound regeneration as defined by effective reepithelialization, neovascularization, and rapid collagen synthesis. Moreover, the improved dermal collagen architecture was demonstrated by uniform size of collagen fibrils, their regular packing, and a restoration of healthy tissue component. Collectively, our natural healing-inspired collagen-targeting glue may be a promising therapeutic option for improving the healing rate with high-quality and effective scar inhibition.


Assuntos
Colágeno/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Adesivos Teciduais/química , Adesivos Teciduais/uso terapêutico , Cicatrização/efeitos dos fármacos , Animais , Colágeno Tipo I/química , Colágeno Tipo I/uso terapêutico , Decorina/química , Decorina/uso terapêutico , Eletroforese em Gel de Poliacrilamida , Feminino , Glicosaminoglicanos , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Células NIH 3T3 , Proteínas/química , Proteínas/uso terapêutico , Proteoglicanas/química , Proteoglicanas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Pele/efeitos dos fármacos , Pele/metabolismo
5.
J Biomed Mater Res A ; 105(8): 2241-2251, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28426898

RESUMO

Polymer scaffolds are used as an alternative to support tissue regeneration when it does not occur on its own. Cell response on polymer scaffolds is determined by factors such as polymer composition, topology, and the presence of other molecules. We evaluated the cellular response of murine skeletal muscle myoblasts on aligned or unaligned fibers obtained by electrospinning poly(ε-caprolactone) (PCL), and blends with poly(lactic-co-glycolic acid) (PLGA) or decorin, a proteoglycan known to regulate myogenesis. The results showed that aligned PCL fibers with higher content of PLGA promote cell growth and improve the quality of differentiation with PLGA scaffolds having the highest confluence at over 68% of coverage per field of view for myoblasts and more than 7% of coverage for myotubes. At the same time, the addition of decorin greatly improves the quantity and quality of differentiated cells in terms of cell fusion, myotube length and thickness, being 71, 10, and 51% greater than without the protein, respectively. Interestingly, our results suggest that at certain concentrations, the effect of decorin on myoblast differentiation exceeds the topological effect of fiber alignment. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2241-2251, 2017.


Assuntos
Mioblastos/citologia , Poliésteres/química , Tecidos Suporte/química , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Decorina/química , Ácido Láctico/química , Camundongos , Desenvolvimento Muscular , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico
6.
Colloids Surf B Biointerfaces ; 155: 17-24, 2017 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-28390241

RESUMO

Orthopedic implants, using materials such as titanium, are extensively used in clinical surgeries. Despite its popularity, titanium is still inadequate to reliable osseointegration due to aseptic loosing. Fibrous encapsulation on the titanium implant interface prevents osseointegration and leads to the loosing of orthopedic implant. In this study, decorin was loaded on titanium surface by polydopamine film to examine fibrous encapsulation inhibition and bone growth acceleration. The coating of decorin was evaluated by X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. Quantitative analysis showed increased decorin coating on titanium surface when decorin in the loading solution increases. To test the effect of decorin modification, fibroblast and osteoblast cultures were utilized in vitro. The results showed that the functions of fibroblasts (proliferation, migration and collagen synthesis) were significantly attenuated on the decorin-modified surfaces and this anti-fibrous effect could be due to fibrotic gene suppression by decorin. In contrast, osteoblastic activities, such as calcium deposition and alkaline phosphatase (ALP) activity, were enhanced by the modified decorin. These results suggest that decorin coating on titanium surface inhibited proliferation and function of fibroblasts and improved that of osteoblasts. Therefore, this study is potentially useful for enhancing orthopedic implant.


Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Decorina/farmacologia , Osteoblastos/efeitos dos fármacos , Titânio/farmacologia , Actinas/genética , Actinas/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Cálcio/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Decorina/química , Di-Hidroxifenilalanina/química , Expressão Gênica , Camundongos , Células NIH 3T3 , Osteoblastos/citologia , Osteoblastos/metabolismo , Próteses e Implantes , Propriedades de Superfície , Titânio/química , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
7.
J Negat Results Biomed ; 16(1): 7, 2017 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-28412940

RESUMO

BACKGROUND: In vitro studies suggest that the multiple functions of decorin are related to both its core protein and its dermatan sulfate chain. To determine the contribution of the dermatan sulfate chain to the functional properties of decorin in vivo, a mutant mouse whose decorin lacked a dermatan sulfate chain was generated. RESULTS: Homozygous mice expressing only the decorin core protein developed and grew in a similar manner to wild type mice. In both embryonic and postnatal mice, all connective tissues studied, including cartilage, skin and cornea, appeared to be normal upon histological examination, and their collagen fibrils were of normal diameter and organization. In addition, abdominal skin wounds healed in an identical manner in the mutant and wild type mice. CONCLUSIONS: The absence of a dermatan sulfate chain on decorin does not appear to overtly influence its functional properties in vivo.


Assuntos
Decorina/metabolismo , Dermatan Sulfato/metabolismo , Desenvolvimento Embrionário , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Sequência de Bases , Cartilagem/patologia , Cartilagem/ultraestrutura , Decorina/química , Decorina/genética , Técnicas de Introdução de Genes , Homozigoto , Camundongos Endogâmicos C57BL , Cicatrização
8.
J Pharm Pharmacol ; 69(6): 633-641, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28266029

RESUMO

OBJECTIVES: Decorin (DCN) is a negative regulatory factor for the growth of cancer cells and can inhibit the proliferation, metastasis of cancer cells and angiogenesis in cancer tissues. The aims of this study were to prepare the nanoparticles consisting of DCN and poly lactic-co-glycolic acid (PLGA) modified by anti-alpha fetoprotein (AFP) monoclonal antibody (mAb) and to examine the conventional physical properties, the in-vitro release of DCN and the targeting effect of these nanoparticles on HepG2 cells. KEY FINDINGS: The encapsulated plasmid was slowly and steadily released from the nanoparticles. The targeted PLGA nanoparticles were initiatively taken in HepG2 cells high-efficiently. According to the results of RT-PCR, DCN gene in AFPmAb-PLGA-rhDCN nanoparticles can be expressed in HepG2 cells successfully. These nanoparticles significantly inhibited the proliferation of HepG2 cells and induced apoptosis. The mRNA expression of Bcl-2 gene in the AFPmAb-PLGA-rhDCN-treated groups appeared significantly to decrease and the caspase-3 gene had the opposite trend as compared with that of control group (P < 0.01). CONCLUSION: These studies revealed that these nanoparticles were capable of specifically targeting the HepG2 cells and inhibiting the proliferation and they induce apoptosis of HepG2 cells in vitro, which was in a dose- and time-dependent manner.


Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Decorina/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , alfa-Fetoproteínas/farmacologia , Anticorpos Monoclonais/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Genes bcl-2/genética , Células Hep G2 , Humanos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , RNA Mensageiro/genética , alfa-Fetoproteínas/química
9.
Biosci Rep ; 37(3)2017 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-28356486

RESUMO

In a recent publication in Bioscience Reports "Contaminants in commercial preparations of 'purified' small leucine-rich proteoglycans may distort mechanistic studies", Brown et al. identified by mass spectrometry and immunoblotting that certain commercial preparations of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, in fact, contained a mix of several proteoglycans that also included fibromodulin and aggrecan. The preparations were thus not suitable to study specific activities of decorin or biglycan. Decorin and biglycan are widely studied SLRPs that are considered to have highly multi-functional effects on cells. Decorin is of interest as a transforming growth factor-ß antagonist and is also finding use in tissue engineering materials. This Commentary discusses Brown et al.'s findings and general issues raised for researchers who work with commercially sourced purified proteoglycans.


Assuntos
Leucina/química , Proteoglicanas/sangue , Agrecanas/química , Biglicano/química , Decorina/química , Fibromodulina/química , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta/antagonistas & inibidores
10.
Glycoconj J ; 34(3): 277-283, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27614617

RESUMO

Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)-1 â†’ 3-ß-D-GalNAc(4-OSO3)-1]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)-1 â†’ 4-α-D-GlcN(SO3)-1 (6-O-SO3)-1]n and [4-α-L-IdoA-(2-O-SO3)-1 â†’ 4-α-D-GlcN(SO3)-1]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.


Assuntos
Estruturas Animais/fisiologia , Dermatan Sulfato/química , Dissacarídeos/química , Filogenia , Urocordados/fisiologia , Estruturas Animais/anatomia & histologia , Estruturas Animais/química , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Colágeno/química , Decorina/química , Dermatan Sulfato/isolamento & purificação , Dissacarídeos/isolamento & purificação , Matriz Extracelular/química , Matriz Extracelular/fisiologia , Hemolinfa/química , Hemolinfa/fisiologia , Urocordados/anatomia & histologia , Urocordados/química , Urocordados/classificação
11.
Circulation ; 134(11): 817-32, 2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27559042

RESUMO

BACKGROUND: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). METHODS: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. RESULTS: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. CONCLUSIONS: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.


Assuntos
Fibrilação Atrial/metabolismo , Decorina , Miostatina/antagonistas & inibidores , Peptídeos , Animais , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Decorina/química , Decorina/metabolismo , Decorina/farmacologia , Feminino , Células HEK293 , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Camundongos , Camundongos Mutantes , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miostatina/metabolismo , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Proteômica
12.
PLoS One ; 11(6): e0157603, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27315119

RESUMO

Cartilage injury and degenerative tissue progression remain poorly understood by the medical community. Therefore, various tissue engineering strategies aim to recover areas of damaged cartilage by using non-traditional approaches. To this end, the use of biomimetic scaffolds for recreating the complex in vivo cartilage microenvironment has become of increasing interest in the field. In the present study, we report the development of two novel biomaterials for cartilage tissue engineering (CTE) with bioactive motifs, aiming to emulate the native cartilage extracellular matrix (ECM). We employed a simple mixture of the self-assembling peptide RAD16-I with either Chondroitin Sulfate (CS) or Decorin molecules, taking advantage of the versatility of RAD16-I. After evaluating the structural stability of the bi-component scaffolds at a physiological pH, we characterized these materials using two different in vitro assessments: re-differentiation of human articular chondrocytes (AC) and induction of human adipose derived stem cells (ADSC) to a chondrogenic commitment. Interestingly, differences in cellular morphology and viability were observed between cell types and culture conditions (control and chondrogenic). In addition, both cell types underwent a chondrogenic commitment under inductive media conditions, and this did not occur under control conditions. Remarkably, the synthesis of important ECM constituents of mature cartilage, such as type II collagen and proteoglycans, was confirmed by gene and protein expression analyses and toluidine blue staining. Furthermore, the viscoelastic behavior of ADSC constructs after 4 weeks of culture was more similar to that of native articular cartilage than to that of AC constructs. Altogether, this comparative study between two cell types demonstrates the versatility of our novel biomaterials and suggests a potential 3D culture system suitable for promoting chondrogenic differentiation.


Assuntos
Cartilagem Articular/metabolismo , Sulfatos de Condroitina/uso terapêutico , Decorina/química , Engenharia Tecidual , Tecidos Suporte/química , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/lesões , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Sulfatos de Condroitina/química , Decorina/uso terapêutico , Matriz Extracelular/efeitos dos fármacos , Humanos , Oligopeptídeos/metabolismo , Oligopeptídeos/uso terapêutico
13.
J Control Release ; 231: 2-16, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-26951927

RESUMO

Adenovirus (Ad)-mediated cancer gene therapy has been proposed as a promising alternative to conventional therapy for cancer. However, success of systemically administered naked Ad has been limited due to the immunogenicity of Ad and the induction of hepatotoxicity caused by Ad's native tropism. In this study, we synthesized an epidermal growth factor receptor (EGFR)-specific therapeutic antibody (ErbB)-conjugated and PEGylated poly(amidoamine) (PAMAM) dendrimer (PPE) for complexation with Ad. Transduction of Ad was inhibited by complexation with PEGylated PAMAM (PP) dendrimer due to steric hindrance. However, PPE-complexed Ad selectively internalized into EGFR-positive cells with greater efficacy than either naked Ad or Ad complexed with PP. Systemically administered PPE-complexed oncolytic Ad elicited significantly reduced immunogenicity, nonspecific liver sequestration, and hepatotoxicity than naked Ad. Furthermore, PPE-complexed oncolytic Ad demonstrated prolonged blood retention time, enhanced intratumoral accumulation of Ad, and potent therapeutic efficacy in EGFR-positive orthotopic lung tumors in comparison with naked Ad. We conclude that ErbB-conjugated and PEGylated PAMAM dendrimer can efficiently mask Ad's capsid and retarget oncolytic Ad to be efficiently internalized into EGFR-positive tumor while attenuating toxicity induced by systemic administration of naked oncolytic Ad.


Assuntos
Adenoviridae/genética , Dendrímeros/química , Receptores ErbB/metabolismo , Neoplasias Pulmonares/terapia , Vírus Oncolíticos/genética , Adenoviridae/química , Animais , Linhagem Celular Tumoral , Cetuximab/química , Decorina/química , Receptores ErbB/imunologia , Regulação Neoplásica da Expressão Gênica , Genes erbB-1 , Terapia Genética , Humanos , Neoplasias Pulmonares/genética , Camundongos , Terapia Viral Oncolítica , Polietilenoglicóis/química , Ligação Proteica , Propriedades de Superfície , Distribuição Tecidual , Transdução Genética
14.
PLoS One ; 11(2): e0147948, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26828927

RESUMO

The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.


Assuntos
Colágeno/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Decorina/metabolismo , Condroitinases e Condroitim Liases/metabolismo , Córnea/patologia , Distrofias Hereditárias da Córnea/patologia , Decorina/química , Humanos , Imageamento Tridimensional , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Multimerização Proteica , Espalhamento a Baixo Ângulo , Homologia Estrutural de Proteína , Tomografia , Difração de Raios X
15.
Adv Drug Deliv Rev ; 97: 174-85, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26522384

RESUMO

Decorin is a prototypical small leucine-rich proteoglycan that epitomizes the multifunctional nature of this critical gene family. Soluble decorin engages multiple receptor tyrosine kinases within the target-rich environment of the tumor stroma and tumor parenchyma. Upon receptor binding, decorin initiates signaling pathways within endothelial cells downstream of VEGFR2 that ultimately culminate in a Peg3/Beclin 1/LC3-dependent autophagic program. Concomitant with autophagic induction, decorin blunts capillary morphogenesis and endothelial cell migration, thereby significantly compromising tumor angiogenesis. In parallel within the tumor proper, decorin binds multiple RTKs with high affinity, including Met, for a multitude of oncosuppressive functions including growth inhibition, tumor cell mitophagy, and angiostasis. Decorin is also pro-inflammatory by modulating macrophage function and cytokine secretion. Decorin suppresses tumorigenic growth, angiogenesis, and prevents metastatic lesions in a variety of in vitro and in vivo tumor models. Therefore, decorin would be an ideal therapeutic candidate for combating solid malignancies.


Assuntos
Decorina/metabolismo , Neoplasias/metabolismo , Animais , Autofagia , Decorina/química , Decorina/genética , Receptores ErbB/metabolismo , Terapia Genética , Humanos , Neoplasias/patologia , Neoplasias/terapia , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo
16.
Angew Chem Int Ed Engl ; 54(37): 10980-4, 2015 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-26216251

RESUMO

Mimicking the binding epitopes of protein-protein interactions by using small peptides is important for generating modular biomimetic systems. A strategy is described for the design of such bioactive peptides without accessible structural data for the targeted interaction, and the effect of incorporating such adhesion peptides in complex biomaterial systems is demonstrated. The highly repetitive structure of decorin was analyzed to identify peptides that are representative of the inner and outer surface, and it was shown that only peptides based on the inner surface of decorin bind to collagen. The peptide with the highest binding affinity for collagen I, LHERHLNNN, served to slow down the diffusion of a conjugated dye in a collagen gel, while its dimer could physically crosslink collagen, thereby enhancing the elastic modulus of the gel by one order of magnitude. These results show the potential of the identified peptides for the design of biomaterials for applications in regenerative medicine.


Assuntos
Materiais Biocompatíveis , Moléculas de Adesão Celular/química , Colágeno Tipo I/química , Decorina/química , Peptídeos/química
17.
Biomacromolecules ; 16(3): 951-61, 2015 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-25645947

RESUMO

Proteoglycan spatiotemporal organization underpins extracellular matrix biology, but atomic scale glimpses of this microarchitecture are obscured by glycosaminoglycan size and complexity. To overcome this, multimicrosecond aqueous simulations of chondroitin and dermatan sulfates were abstracted into a prior coarse-grained model, which was extended to heterogeneous glycosaminoglycans and small leucine-rich proteoglycans. Exploration of relationships between sequence and shape led to hypotheses that proteoglycan size is dependent on glycosaminoglycan unit composition but independent of sequence permutation. Uronic acid conformational equilibria were modulated by adjacent hexosamine sulfonation and iduronic acid increased glycosaminoglycan chain volume and rigidity, while glucuronic acid imparted chain plasticity. Consequently, block copolymeric glycosaminoglycans contained microarchitectures capable of multivalent binding to growth factors and collagen, with potential for interactional synergy at greater chain number. The described atomic scale views of proteoglycans and heterogeneous glycosaminoglycans provide structural routes to understanding their fundamental signaling and mechanical biological roles and development of new biomaterials.


Assuntos
alfa-Globulinas/química , Sulfatos de Condroitina/química , Decorina/química , Dermatan Sulfato/química , Animais , Configuração de Carboidratos , Cartilagem/química , Bovinos , Humanos , Intestinos/química , Simulação de Dinâmica Molecular , Conformação Proteica , Tubarões , Sus scrofa , Traqueia/química
18.
Adv Exp Med Biol ; 802: 49-58, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24443020

RESUMO

Proteoglycans consist of a protein core to which at least one glycosaminoglycan chain is attached. They play important roles in the physiology and biomechanical function of tendons, ligaments and cardiovascular system through their involvement in regulation of assembly and maintenance of extracellular matrix, and as they participate in cell proliferation through their interactions with growth factors. They can be divided into two main groups of small and large proteoglycans. The small proteoglycans are also known as small leucine-rich proteoglycans (or SLRPs) which are encoded by 17 genes and are further subclassified into Classes I-V. Several members of Class I and II, such as decorin and biglycan from Class I, and Class II fibromodulin and lumican, are known to regulate collagen fibrillogenesis. Decorin limits the diameter of collagen fibrils during fibrillogenesis. The function of biglycan in fibrillogenesis is similar to that of decorin. Though biomechanical function of tendon is compromised in decorin-deficient mice, decorin can substitute for lack of biglycan in biglycan-deficient mice. New data also indicate an important role for biglycan in disorders of the cardiovascular system, including aortic valve stenosis and aortic dissection. Two members of the Class II of SLRPs, fibromodulin and lumican bind to the same site within the collagen molecule and can substitute for each other in fibromodulin- or lumican-deficient mice.Aggrecan and versican are the major representatives of the large proteoglycans. Though they are mainly found in the cartilage where they provide resilience and toughness, they are also present in tensile portions of tendons and, in slightly different biochemical form in fibrocartilage. Degradation with aggrecanase is responsible for the appearance of different forms of aggrecan and versican in different parts of the tendon where these cleaved forms play different roles. In addition, they are important components of the ventricularis of cardiac valves. Mutations in the gene for versican or in the gene for elastin (which binds to versican) lead to severe disruptions of normal developmental of the heart at least in mice.


Assuntos
Aneurisma da Aorta Torácica/metabolismo , Estenose da Valva Aórtica/metabolismo , Matriz Extracelular/metabolismo , Ligamentos/metabolismo , Tendões/metabolismo , Agrecanas/química , Agrecanas/metabolismo , Animais , Aneurisma da Aorta Torácica/fisiopatologia , Estenose da Valva Aórtica/fisiopatologia , Biglicano/química , Biglicano/metabolismo , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/química , Colágeno/metabolismo , Decorina/química , Decorina/metabolismo , Matriz Extracelular/química , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Fibromodulina , Humanos , Sulfato de Ceratano/química , Sulfato de Ceratano/metabolismo , Ligamentos/química , Ligamentos/fisiopatologia , Lumicana , Camundongos , Ligação Proteica , Proteoglicanas/química , Proteoglicanas/metabolismo , Tendões/química , Tendões/fisiopatologia , Versicanas/química , Versicanas/metabolismo
19.
Vet Ophthalmol ; 17(3): 162-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-23718145

RESUMO

OBJECTIVE: To explore (i) the potential of polyethylenimine (PEI) nanoparticles as a vector for delivering genes into equine corneal fibroblasts (ECFs) using green fluorescent protein (GFP) marker gene, (ii) whether PEI nanoparticle-mediated decorin (DCN) gene therapy could be used to inhibit fibrosis in the equine cornea using an in vitro model. PROCEDURE: Polyethylenimine-DNA nanoparticles were prepared at nitrogen-to-phosphate (N-P) ratio of 15 by mixing 22 kDa linear PEI and a plasmid encoding either GFP or DCN. ECFs were generated from donor corneas as previously described. Initially, GFP was introduced into ECFs using PEI nanoparticles to confirm gene delivery, then DCN was introduced to evaluate for antifibrotic effects. GFP gene delivery was confirmed with real-time qPCR and ELISA. Changes in fibrosis after DCN therapy were quantified by measuring α-smooth muscle actin (αSMA) mRNA and protein levels with qPCR, immunostaining, and immunoblotting. Cytotoxicity was determined by evaluating cell morphology, cellular viability, and TUNEL assay. RESULTS: Polyethylenimine-green fluorescent protein-treated cultures showed 2.2 × 10(4) GFP plasmid copies/µg of cellular DNA and 2.1 pg of GFP/100 µL of lysate. PEI-DCN delivery significantly attenuated TGFß-induced transdifferentiation of fibroblasts to myofibroblasts (2-fold decrease of αSMA mRNA; P = 0.05) and significant inhibition of αSMA (49 ± 14.2%; P < 0.001) in immunocytochemical staining and immunoblotting were found. Furthermore, PEI-DNA nanoparticle delivery did not alter cellular phenotype at 24 h and cellular viability was maintained. CONCLUSIONS: Twenty-two kilo dalton Polyethylenimine nanoparticles are safe and effective for equine corneal gene therapy in vitro. PEI-mediated DCN gene delivery is effective at inhibiting TGFß-mediated fibrosis in this model.


Assuntos
Córnea/citologia , Decorina/metabolismo , Fibroblastos/efeitos dos fármacos , Técnicas de Transferência de Genes/veterinária , Cavalos , Polietilenoimina/farmacologia , Animais , Células Cultivadas , Decorina/química , Decorina/genética , Fibroblastos/citologia , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Nanopartículas , Polietilenoimina/química
20.
J Biol Chem ; 288(49): 35526-33, 2013 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-24169694

RESUMO

Decorin, the prototypical small leucine-rich proteoglycan, binds to collagen and thereby regulates collagen assembly into fibrils. The crystal structure of the decorin core protein revealed a tight dimer formed by the association of two monomers via their concave faces (Scott, P. G., McEwan, P. A., Dodd, C. M., Bergmann, E. M., Bishop, P. N., and Bella, J. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 15633-15638). Whether decorin binds collagen as a dimer has been controversial. Using analytical ultracentrifugation, we determined a dissociation constant of 1.37 ± 0.30 µm for the mouse decorin dimer. Dimerization could be abolished by engineering glycosylation sites into the dimer interface; other interface mutants remained dimeric. The monomeric mutants were as stable as wild-type decorin in thermal unfolding experiments. Mutations on the concave face of decorin abolished collagen binding regardless of whether the mutant proteins retained the ability to dimerize or not. We conclude that the concave face of decorin mediates collagen binding and that the dimer therefore must dissociate to bind collagen.


Assuntos
Colágeno/metabolismo , Decorina/química , Decorina/metabolismo , Animais , Cristalografia por Raios X , Decorina/genética , Glicosilação , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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