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1.
Gene ; 707: 151-171, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31075413

RESUMO

Collagen alpha-1(III) chain, also known as the alpha 1 chain of type III collagen, is a protein that in humans is encoded by the COL3A1 gene. Three alpha 1 chains are required to form the type III collagen molecule which has a long triple-helical domain. Type III collagen, an extracellular matrix protein, is synthesized by cells as a pre-procollagen. It is found as a major structural component in hollow organs such as large blood vessels, uterus and bowel. Other functions of type III collagen include interaction with platelets in the blood clotting cascade and it is also an important signaling molecule in wound healing. Mutations in the COL3A1 gene cause the vascular type of Ehlers-Danlos syndrome (vEDS; OMIM 130050). It is the most serious form of EDS, since patients often die suddenly due to a rupture of large arteries. Inactivation of the murine Col3a1 gene leads to a shorter life span in homozygous mutant mice. The mice die prematurely from a rupture of major arteries mimicking the human vEDS phenotype. The biochemical and cellular effects of COL3A1 mutations have been studied extensively. Most of the glycine mutations lead to the synthesis of type III collagen with reduced thermal stability, which is more susceptible for proteinases. Intracellular accumulation of this normally secreted protein is also found. Ultrastructural analyses have demonstrated dilated rough endoplasmic reticulum and changes in the diameter of collagen fibers. Other clinical conditions associated with type III collagen are several types of fibroses in which increased amounts of type III collagen accumulate in the target organs.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Síndrome de Ehlers-Danlos/genética , Animais , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/mortalidade , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Camundongos , Mutação , Fenótipo , Conformação Proteica , Estabilidade Proteica , Distribuição Tecidual
2.
Biochim Biophys Acta Mol Cell Res ; 1866(11): 118458, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-30880148

RESUMO

The discoidin domain receptors, DDR1 and DDR2, are a subfamily of receptor tyrosine kinases that are activated upon binding to collagen. DDR-collagen interactions play an important role in cell proliferation and migration. Over the past few decades, synthetic peptides and recombinant collagen have been developed as tools to study the biophysical characteristics of collagen and various protein-collagen interactions. Herein we review how these techniques have been used to understand DDR-collagen interactions. Using synthetic collagen-like peptides, the GVM-GFO motif has been found to be the major binding site on collagens II and III for DDR1 and DDR2. An X-ray co-crystal structure of the DDR2 DS domain bound to a synthetic collagen-like peptide containing the GVM-GFO motif further provides molecular details of the DDR-collagen interactions. Recombinant collagen has also been used to provide further validation of the GVM-GFO binding motif. Although GVM-GFO has been defined as the minimal binding site, in synthetic peptide studies at least two triplets N-terminal to the essential GVM-GFO binding motif in collagen III sequence are needed for DDR2 activation at high peptide concentrations.


Assuntos
Colágeno/química , Receptores com Domínio Discoidina/química , Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , Animais , Sequência de Bases , Sítios de Ligação , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo II/química , Colágeno Tipo III/química , Cristalografia por Raios X , Receptor com Domínio Discoidina 1/química , Receptor com Domínio Discoidina 2/química , Receptores com Domínio Discoidina/metabolismo , Humanos , Modelos Moleculares , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica
3.
Acta Biomater ; 87: 97-107, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30708064

RESUMO

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Voice disorders affect about 1/3rd of the US population and significantly reduce quality of life. Patients with vocal fold fibrosis have few treatment options. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. Various studies have used collagen or thiolated hyaluronan (HA) with gelatin as potential tissue engineering therapies. However, there is room for improvement in providing cells with more relevant biological cues that mimic the native tissue microenvironment and sustain regeneration. The present study introduces the use of type I collagen and type III collagen along with thiolated HA as a natural biomimetic hydrogel for vocal fold tissue engineering applications.


Assuntos
Materiais Biomiméticos/química , Colágeno Tipo III/química , Colágeno Tipo I/química , Fibroblastos/metabolismo , Ácido Hialurônico/química , Hidrogéis/química , Engenharia Tecidual , Prega Vocal/metabolismo , Linhagem Celular Transformada , Fibroblastos/citologia , Humanos
4.
Biochem Biophys Res Commun ; 508(4): 1018-1023, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30545625

RESUMO

Collagen is one of the most abundant and important proteins in the human body. Human collagen type III (hCOL3A1) belongs to the fibril-forming collagens and is widely distributed in extensible connective tissue like skin, internal organs, or the vascular system. It plays key roles in wound healing, collagen fibrillogenesis, and normal cardiovascular development in human. The charged residues are considered to be an important characteristic of hCOL3A1, especially for collagen binding and recognition. Here we found that a triple helix fragment of hCOL3A1, Gly489-Gly510, contained multiple charged residues, as well as representative Glu-Lys-Gly and Glu-Arg-Gly charged triplets. We solved the crystal structure of this new fragment to a high-resolution of 1.50 Šand identified some important conformations of this new triple-helix region, including strong hydrogen bonds in interchain and interhelical interactions in addition to obvious flexible bending for the triple helix. We also found that the synthetic collagen peptides around this region exhibited potent activities through integrin-mediated peptide-membrane interaction. We then developed a method to produce a recombinant protein consisting of 16 tandem repeats of the triple-helix fragment of hCOL3A1 with strong activity without cytotoxicity. These results provide a strong base for further functional studies of human collagen type III and the method developed in this study can be applied to produce hCOL3A1-derived proteins or other tandem-repeat proteins with membrane adhesion activity.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo III/metabolismo , Fibroblastos/citologia , Células 3T3 , Sequência de Aminoácidos , Animais , Adesão Celular , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Integrinas/metabolismo , Camundongos , Peptídeos/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química
5.
Biomed Mater ; 14(1): 015007, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30421723

RESUMO

Reconstituted fibrillary collagen is one of the most advantageous biomaterials for biomedical applications. The objective of the research project described in this paper was to evaluate whether riboflavin-induced photo-crosslinking could be used as a non-toxic alternative to glutaraldehyde (GA)-crosslinking for the preparation of wet spun collagen filaments. Collagen filaments were produced on a laboratory wet spinning line and crosslinked with GA or riboflavin with and without UV exposure. Based on mechanical and thermal analyses, it was concluded that the combination of riboflavin and UV light leads to crosslinked collagen filaments having improved mechanical and thermal properties. Furthermore, riboflavin-crosslinked filaments exhibited a higher cytocompatibility for human mesenchymal stem cells compared to GA-crosslinked filaments.


Assuntos
Materiais Biocompatíveis/química , Colágeno Tipo III/química , Colágeno Tipo I/química , Reagentes para Ligações Cruzadas/química , Glutaral/química , Riboflavina/química , Proliferação de Células , Citoesqueleto/metabolismo , Colágenos Fibrilares , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Microscopia Confocal , Estresse Mecânico , Resistência à Tração , Engenharia Tecidual , Raios Ultravioleta
6.
Biophys J ; 115(8): 1457-1469, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30269884

RESUMO

The predominant structural protein in vertebrates is collagen, which plays a key role in extracellular matrix and connective tissue mechanics. Despite its prevalence and physical importance in biology, the mechanical properties of molecular collagen are far from established. The flexibility of its triple helix is unresolved, with descriptions from different experimental techniques ranging from flexible to semirigid. Furthermore, it is unknown how collagen type (homo- versus heterotrimeric) and source (tissue derived versus recombinant) influence flexibility. Using SmarTrace, a chain-tracing algorithm we devised, we performed statistical analysis of collagen conformations collected with atomic force microscopy to determine the protein's mechanical properties. Our results show that types I, II, and III collagens-the key fibrillar varieties-exhibit similar molecular flexibilities. However, collagen conformations are strongly modulated by salt, transitioning from compact to extended as KCl concentration increases in both neutral and acidic pH. Although analysis with a standard worm-like chain model suggests that the persistence length of collagen can attain a wide range of values within the literature range, closer inspection reveals that this modulation of collagen's conformational behavior is not due to changes in flexibility but rather arises from the induction of curvature (either intrinsic or induced by interactions with the mica surface). By modifying standard polymer theory to include innate curvature, we show that collagen behaves as an equilibrated curved worm-like chain in two dimensions. Analysis within the curved worm-like chain model shows that collagen's curvature depends strongly on pH and salt, whereas its persistence length does not. Thus, we find that triple-helical collagen is well described as semiflexible irrespective of source, type, pH, and salt environment. These results demonstrate that collagen is more flexible than its conventional description as a rigid rod, which may have implications for its cellular processing and secretion.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo II/química , Colágeno Tipo I/química , Meio Ambiente , Matriz Extracelular/química , Conformação Proteica , Algoritmos , Animais , Elasticidade , Humanos , Modelos Moleculares , Ratos
7.
Kaohsiung J Med Sci ; 34(4): 223-230, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29655411

RESUMO

Temporomandibular joint (TMJ) is one of the most complex joints of the human body. Due to its unique movement, in terms of combination of rotation and translator movement, disc of the joint plays an important role to maintain its normal function. In order to sustain the normal function of the TMJ, disc must be kept in proper position as well as maintain normal shape in all circumstances. Once the disc is not any more in its normal position during function of the joint, disturbance of the joint can be occurred which will lead to subsequent distortion of the disc. Shape of the disc can be influenced by many factors i.e.: abnormal function or composition of the disc itself. Etiology of the internal derangement of the disc remains controversial. Multifactorial theory has been postulated in most of previous manuscripts. Disc is composed of mainly extracellular matrix. Abnormal proportion of collagen type I & III may also leads to joint hypermobility which may be also a predisposing factor of this disorder. Thus it can be recognized as local manifestation of a systemic disorder. Different treatment modalities with from conservative treatment to surgical intervention distinct success rate have been reported. Recently treatment with extracellular matrix injection becomes more and more popular to strengthen the joint itself. Since multifactorial in character, the best solution of the treatment modalities should be aimed to resolve possible etiology from different aspects. Team work may be indication to reach satisfied results.


Assuntos
Artroscopia/métodos , Modalidades de Fisioterapia , Transtornos da Articulação Temporomandibular/fisiopatologia , Transtornos da Articulação Temporomandibular/terapia , Articulação Temporomandibular/fisiopatologia , Artrocentese , Colágeno Tipo I/química , Colágeno Tipo I/metabolismo , Colágeno Tipo III/química , Colágeno Tipo III/metabolismo , Matriz Extracelular/química , Matriz Extracelular/patologia , Matriz Extracelular/transplante , Humanos , Ácido Hialurônico/uso terapêutico , Equipamentos Ortopédicos , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Articulação Temporomandibular/anormalidades , Articulação Temporomandibular/metabolismo , Articulação Temporomandibular/cirurgia , Transtornos da Articulação Temporomandibular/metabolismo , Transtornos da Articulação Temporomandibular/patologia
8.
Matrix Biol ; 70: 72-83, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29551664

RESUMO

Type III collagen is a major fibrillar collagen consisting of three identical α1(III)-chains that is particularly present in tissues exhibiting elastic properties, such as the skin and the arterial wall. Heterozygous mutations in the COL3A1 gene result in vascular Ehlers-Danlos syndrome (vEDS), a severe, life-threatening disorder, characterized by thin, translucent skin and propensity to arterial, intestinal and uterine rupture. Most human vEDS cases result from a missense mutation substituting a crucial glycine residue in the triple helical domain of the α1(III)-chains. The mechanisms by which these mutant type III collagen molecules cause dermal and vascular fragility are not well understood. We generated a transgenic mouse line expressing mutant type III collagen, containing a typical helical glycine substitution (p.(Gly182Ser)). This Col3a1Tg-G182S mouse line displays a phenotype recapitulating characteristics of human vEDS patients with signs of dermal and vascular fragility. The Col3a1Tg-G182S mice develop severe transdermal skin wounds, resulting in early demise at 13-14weeks of age. We found that this phenotype was associated with a reduced total collagen content and an abnormal collagen III:I ratio, leading to the production of severely malformed collagen fibrils in the extracellular matrix of dermal and arterial tissues. These results indicate that expression of the glycine substitution in the α1(III)-chain disturbs formation of heterotypic type III:I collagen fibrils, and thereby demonstrate a key role for type III collagen in collagen fibrillogenesis in dermal and arterial tissues.


Assuntos
Substituição de Aminoácidos , Artérias/metabolismo , Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/genética , Mutação , Pele/metabolismo , Animais , Artérias/patologia , Colágeno Tipo III/química , Colágeno Tipo III/deficiência , Modelos Animais de Doenças , Síndrome de Ehlers-Danlos/metabolismo , Síndrome de Ehlers-Danlos/mortalidade , Síndrome de Ehlers-Danlos/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Expressão Gênica , Glicina/química , Glicina/metabolismo , Heterozigoto , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Serina/química , Serina/metabolismo , Fatores Sexuais , Pele/patologia , Técnicas de Cultura de Tecidos
9.
Biophys J ; 114(3): 570-576, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29414702

RESUMO

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo III/metabolismo , Fenômenos Mecânicos , Proteólise , Tripsina/metabolismo , Bioensaio , Centrifugação , Humanos , Microscopia de Força Atômica , Nanotecnologia , Estabilidade Proteica
10.
PLoS One ; 13(1): e0191220, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29346445

RESUMO

Vascular Ehlers-Danlos syndrome (vEDS) is a dominantly inherited connective tissue disorder caused by mutations in the COL3A1 gene that encodes type III collagen (COLLIII), which is the major expressed collagen in blood vessels and hollow organs. The majority of disease-causing variants in COL3A1 are glycine substitutions and in-frame splice mutations in the triple helix domain that through a dominant negative effect are associated with the severe clinical spectrum potentially lethal of vEDS, characterized by fragility of soft connective tissues with arterial and organ ruptures. To shed lights into molecular mechanisms underlying vEDS, we performed gene expression profiling in cultured skin fibroblasts from three patients with different structural COL3A1 mutations. Transcriptome analysis revealed significant changes in the expression levels of several genes involved in maintenance of cell redox and endoplasmic reticulum (ER) homeostasis, COLLs folding and extracellular matrix (ECM) organization, formation of the proteasome complex, and cell cycle regulation. Protein analyses showed that aberrant COLLIII expression is associated with the disassembly of many structural ECM constituents, such as fibrillins, EMILINs, and elastin, as well as with the reduction of the proteoglycans perlecan, decorin, and versican, all playing an important role in the vascular system. Furthermore, the altered distribution of the ER marker protein disulfide isomerase PDI and the strong reduction of the COLLs-modifying enzyme FKBP22 are consistent with the disturbance of ER-related homeostasis and COLLs biosynthesis and post-translational modifications, indicated by microarray analysis. Our findings add new insights into the pathophysiology of this severe vascular disorder, since they provide a picture of the gene expression changes in vEDS skin fibroblasts and highlight that dominant negative mutations in COL3A1 also affect post-translational modifications and deposition into the ECM of several structural proteins crucial to the integrity of soft connective tissues.


Assuntos
Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/genética , Mutação , Substituição de Aminoácidos , Ciclo Celular/genética , Células Cultivadas , Colágeno Tipo III/química , Colágeno Tipo III/metabolismo , Síndrome de Ehlers-Danlos/etiologia , Síndrome de Ehlers-Danlos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Pele/metabolismo
11.
Biomed Mater Eng ; 29(1): 15-27, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29254070

RESUMO

BACKGROUND: The periodontal ligament (PDL), which maintains homeostasis in the periodontium, is a group of specialized connective tissue fibers attached to both the cementum and alveolar bone. Regeneration of periodontium with PDL cells has been investigated, and the chemical and molecular structures of scaffolds control the adhesion and differentiation of cells. Therefore, the development of adequate materials for PDL-derived cells is essential to regenerate the periodontium. OBJECTIVE: We evaluated the suitable passage time for PDL-derived cells and investigated the behaviors of PDL-derived cells grown on hydroxyapatite (HAp) scaffolds coated with type I and type III collagen. METHODS: PDL-derived cells were isolated with enzyme from the upper molars of male Wister rats. After characterization of HAp, type I collagen, and type III collagen, PDL-derived cells at passage 2 were seeded onto collagen-coated HAp. Cell adhesion, proliferative potential, and osteoconductivity were analyzed with immunostaining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, Alizarin S staining, and real-time polymerase chain reaction. RESULTS: Type I and III collagens were successfully coated on HAp. Gene expression analysis revealed that passage 2 was suitable for maintaining differentiation potential. Proliferative potential and cell adhesion were significantly higher on type III collagen than on HAp alone or type I collagen. In contrast, the osteoconductivity of type III collagen was significantly lower than those of HAp and type I collagen. CONCLUSION: PDL-derived cells on type I collagen differentiated into osteogenic cells and formed hard tissues. However, type III collagen enhanced the adhesion of PDL-derived cells and inhibited mineralization.


Assuntos
Materiais Revestidos Biocompatíveis/química , Colágeno Tipo III/química , Durapatita/química , Ligamento Periodontal/citologia , Animais , Calcificação Fisiológica , Adesão Celular , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/química , Masculino , Osteogênese , Ligamento Periodontal/metabolismo , Ratos Wistar
12.
PLoS One ; 12(7): e0175582, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28704418

RESUMO

Collagen III is critical to the integrity of blood vessels and distensible organs, and in hemostasis. Examination of the human collagen III interactome reveals a nearly identical structural arrangement and charge distribution pattern as for collagen I, with cell interaction domains, fibrillogenesis and enzyme cleavage domains, several major ligand-binding regions, and intermolecular crosslink sites at the same sites. These similarities allow heterotypic fibril formation with, and substitution by, collagen I in embryonic development and wound healing. The collagen III fibril assumes a "flexi-rod" structure with flexible zones interspersed with rod-like domains, which is consistent with the molecule's prominence in young, pliable tissues and distensible organs. Collagen III has two major hemostasis domains, with binding motifs for von Willebrand factor, α2ß1 integrin, platelet binding octapeptide and glycoprotein VI, consistent with the bleeding tendency observed with COL3A1 disease-causing sequence variants.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo III/metabolismo , Hemostasia , Sequência de Aminoácidos , Sítios de Ligação , Colágeno Tipo III/genética , Humanos , Integrina alfa2beta1/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas , Estabilidade Proteica , Fator de von Willebrand/metabolismo
13.
Protein J ; 36(4): 322-331, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28589291

RESUMO

High-level expression of recombinant collagen by genetic engineering is urgently required. Recombinant collagen is different from natural collagen in its hydroxyproline (Hyp) content and thermal stability. To obtain hydroxylated collagen for applications in biomedicine and biomaterials, the human collagen α1(III) chain was co-expressed with the viral prolyl 4-hydroxylase A085R in Escherichia coli. Unlike previous reports using human prolyl 4-hydroxylase, this study examined the hydroxylation of full-length human collagen α1(III) chain (COL3A1) by viral prolyl 4-hydroxylase. The genes encoding these two proteins were controlled by different promoters, Ptac and PRPL, on a recombinant pKK223-3 plasmid. The sequencing results verified that the target genes were successfully inserted into the recombinant vector. Based on quantitative PCR, SDS-PAGE, and western blotting, successful expression by E. coli BL21(DE3) was detected at the mRNA and protein levels for both loci. Liquid chromatography-mass spectrometry (LC-MS/MS) results suggested that the highest Hyp yield was obtained when the two proteins were induced with 0.5 mM IPTG and heat-shock treatment at 50 °C, corresponding to high enzyme expression and low human collagen α1(III) chain expression levels. A biological activity analysis indicated that the recombinant collagen with the highest hydroxylation level supported the growth of baby hamster kidney cells, similar to observations for native collagen. The production of hydroxylated collagen in this study establishes a new method for collagen hydroxylation and provides a basis for the application of recombinant collagen expressed in E. coli.


Assuntos
Colágeno Tipo III/metabolismo , Escherichia coli/metabolismo , Plasmídeos/metabolismo , Prolil Hidroxilases/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo III/química , Colágeno Tipo III/genética , Colágeno Tipo III/farmacologia , Cricetinae , Escherichia coli/genética , Expressão Gênica , Humanos , Hidroxilação , Phycodnaviridae/química , Phycodnaviridae/enzimologia , Plasmídeos/química , Prolil Hidroxilases/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Transformação Bacteriana , Proteínas Virais/genética
14.
Sci Rep ; 7(1): 1392, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28469139

RESUMO

Tropocollagen types I and III were simultaneously fibrilized in vitro, and the differences between the geometric and mechanical properties of the heterotypic fibrils with different mixing ratios of tropocollagen III to I were investigated. Transmission electron microscopy was used to confirm the simultaneous presence of both tropocollagen types within the heterotypic fibrils. The incorporation of collagen III in I caused the fibrils to be thinner with a shorter D-banding than pure collagen I. Hertzian contact model was used to obtain the elastic moduli from atomic force microscope indentation testing using a force volume analysis. The results indicated that an increase in the percentage of tropocollagen III reduced the mechanical stiffness of the obtained fibrils. The mechanical stiffness of the collagen fibrils was found to be greater at higher loading frequencies. This observation might explain the dominance of collagen III over I in soft distensible organs such as human vocal folds.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo I/química , Tropocolágeno/química , Colágeno Tipo I/ultraestrutura , Colágeno Tipo III/ultraestrutura , Módulo de Elasticidade , Elasticidade , Técnicas In Vitro , Microscopia de Força Atômica , Tropocolágeno/ultraestrutura
15.
Aesthet Surg J ; 37(9): 1062-1068, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28510634

RESUMO

Background: Autologous collagen is an ideal soft tissue filler and may serve as a matrix for stem cell implantation and growth. Procurement of autologous collagen has been limited, though, secondary to a sufficient source. Liposuction is a widely performed and could be a source of autologous collagen. Objectives: The amount of collagen and its composition in liposuctioned fat remains unknown. The purpose of this research was to characterize an adipose-derived tissue-based product created using ultrasonic cavitation and cryo-grinding. This study evaluated the cellular and protein composition of the final product. Methods: Fat was obtained from individuals undergoing routine liposuction and was processed by a 2 step process to obtain only the connective tissue. The tissue was then evaluated by scanning electronic microscope, Western blot analysis, and flow cytometry. Results: Liposuctioned fat was obtained from 10 individuals with an average of 298 mL per subject. After processing an average of 1 mL of collagen matrix was obtained from each 100 mL of fat. Significant viable cell markers were present in descending order for adipocytes > CD90+ > CD105+ > CD45+ > CD19+ > CD144+ > CD34+. Western blot analysis showed collagen type II, III, IV, and other proteins. Scanning electronic microscope study showed a regular pattern of cross-linked, helical collagen. Additionally, vital staing demonstrated that the cells were still viable after processing. Conclusions: Collagen and cells can be easily obtained from liposuctioned fat by ultrasonic separation without alteration of the overall cellular composition of the tissue. Implantation results in new collagen and cellular growth. Collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat and may provide long term results. Level of Evidence: 5.


Assuntos
Adipócitos/citologia , Tecido Adiposo/química , Colágeno Tipo III/química , Colágeno Tipo II/química , Colágeno Tipo IV/química , Tecido Adiposo/citologia , Adulto , Western Blotting , Sobrevivência Celular , Colágeno Tipo II/isolamento & purificação , Colágeno Tipo III/isolamento & purificação , Colágeno Tipo IV/isolamento & purificação , Feminino , Citometria de Fluxo , Humanos , Lipectomia , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Células-Tronco/citologia
16.
Aesthet Surg J ; 37(9): 1069-1074, 2017 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-28510696

RESUMO

Background: In part 1 of this study it was shown that liposuctioned fat could be a sufficient source of autologous collagen for use as a filler or in reconstruction. The collagen composition in liposuctioned fat was shown to form a cross-linked helical matrix composed of types II, III, and IV. Additionally, viable adipocytes and fibroblasts among other cells were found. Objectives: The purpose of this research was to study the biology of this matrix after subsequent implantation compared to Juvederm (Allergan, Parsippany, NJ) common soft tissue filler. Methods: Fat was obtained from individuals undergoing routine liposuction and was processed by a two-step process to obtain a connective tissue matrix. The matrix was then cryo-frozen for a minimum of 4 weeks after which it was thawed and implanted in 46 nude mice. Juvederm Ultra was used as the control article and the animals followed for one year. Results: Liposuctioned fat was obtained from 10 individuals and processed as previously described. Mice were harvested at 3, 6, 9, and 12 months and histology obtained. There were no adverse effects from either article and the bio-reactivity rating was 0. The implanted collagen compared favorably to Juvederm at all stages and was found to be replaced by new collagen and fat. Conclusions: A collagen matrix with viable cells for autologous use can be obtained from liposuctioned fat which has been processed and cryo-frozen. The material lasts at least one year and is slowly replaced by new collagenand fat. Level of Evidence: 5.


Assuntos
Tecido Adiposo/transplante , Colágeno Tipo III/administração & dosagem , Colágeno Tipo II/administração & dosagem , Colágeno Tipo IV/administração & dosagem , Adipócitos/citologia , Tecido Adiposo/química , Adulto , Animais , Colágeno Tipo II/química , Colágeno Tipo III/química , Colágeno Tipo IV/química , Feminino , Fibroblastos/citologia , Humanos , Ácido Hialurônico/administração & dosagem , Lipectomia , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade
17.
J Biol Chem ; 292(22): 9273-9282, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28385890

RESUMO

Extracellular matrix proteins are biosynthesized in the rough endoplasmic reticulum (rER), and the triple-helical protein collagen is the most abundant extracellular matrix component in the human body. Many enzymes, molecular chaperones, and post-translational modifiers facilitate collagen biosynthesis. Collagen contains a large number of proline residues, so the cis/trans isomerization of proline peptide bonds is the rate-limiting step during triple-helix formation. Accordingly, the rER-resident peptidyl prolyl cis/trans isomerases (PPIases) play an important role in the zipper-like triple-helix formation in collagen. We previously described this process as "Ziploc-ing the structure" and now provide additional information on the activity of individual rER PPIases. We investigated the substrate preferences of these PPIases in vitro using type III collagen, the unhydroxylated quarter fragment of type III collagen, and synthetic peptides as substrates. We observed changes in activity of six rER-resident PPIases, cyclophilin B (encoded by the PPIB gene), FKBP13 (FKBP2), FKBP19 (FKBP11), FKBP22 (FKBP14), FKBP23 (FKBP7), and FKBP65 (FKBP10), due to posttranslational modifications of proline residues in the substrate. Cyclophilin B and FKBP13 exhibited much lower activity toward post-translationally modified substrates. In contrast, FKBP19, FKBP22, and FKBP65 showed increased activity toward hydroxyproline-containing peptide substrates. Moreover, FKBP22 showed a hydroxyproline-dependent effect by increasing the amount of refolded type III collagen in vitro and FKBP19 seems to interact with triple helical type I collagen. Therefore, we propose that hydroxyproline modulates the rate of Ziploc-ing of the triple helix of collagen in the rER.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo I/química , Ciclofilinas/química , Retículo Endoplasmático/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a Tacrolimo/química , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Ciclofilinas/genética , Ciclofilinas/metabolismo , Retículo Endoplasmático/genética , Humanos , Hidroxiprolina/química , Hidroxiprolina/metabolismo , Domínios Proteicos , Especificidade por Substrato , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo
18.
J Mater Sci Mater Med ; 28(4): 58, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28210969

RESUMO

The less traumatic use of surgical adhesives rather than sutures for mesh fixation in hernia repair has started to gain popularity because they induce less host tissue damage and provoke less postoperative pain. This study examines the host tissue response to a new cyanoacrylate (CA) adhesive (n-octyl, OCA). Partial defects (3 × 5 cm) created in the rabbit anterior abdominal wall were repaired by mesh fixation using OCA, Glubran2®(n-butyl-CA), Ifabond®(n-hexyl-CA) or sutures. Samples were obtained at 14/90 days for morphology, collagens qRT-PCR/immunofluorescence and biomechanical studies. All meshes were successfully fixed. Seroma was detected mainly in the Glubran group at 14 days. Meshes fixed using all methods showed good host tissue incorporation. No signs of degradation of any of the adhesives were observed. At 14 days, collagen 1 and 3 mRNA expression levels were greater in the suture and OCA groups, and lower in Ifabond, with levels varying significantly in the latter group with respect to the others. By 90 days, expression levels had fallen in all groups, except for collagen 3 mRNA in Ifabond. Collagen I and III protein expression was marked in the suture and OCA groups at 90 days, but lower in Ifabond at both time points. Tensile strengths were similar across groups. Our findings indicate the similar behavior of the adhesives to sutures in terms of good tissue incorporation of the meshes and optimal repair zone strength. The lower seroma rate and similar collagenization to controls induced by OCA suggests its improved behavior over the other two glues. This article deals with a preclinical study to examine different aspects of the repair process in the host of three alkyl cyanoacrylates (n-butyl (GLUBRAN 2), n-hexyl (IFABOND), and n-octyl cyanoacrylate (EVOBOND)) compared to sutures (control), in the fixation of surgical meshes for hernia repair. It goes into detail about collagen deposition in the repair zone at short and medium term. The results obtained demonstrate lower seroma rate and similar collagenization to sutures induced by the n-octyl suggesting better behavior than the other two cyanoacrylates.


Assuntos
Colágeno Tipo III/química , Colágeno Tipo I/química , Cianoacrilatos/química , Hérnia Abdominal/cirurgia , Herniorrafia/métodos , Adesivos Teciduais , Animais , Fenômenos Biomecânicos , Masculino , Desenho de Prótese , RNA Mensageiro/metabolismo , Coelhos , Seroma/metabolismo , Resistência à Tração , Cicatrização
19.
Mol Med Rep ; 15(2): 936-940, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28035354

RESUMO

Aortopathy represents an important cause of mortality in industrialized countries, with a number of genes identified as predispose factors. It can be difficult to identify the genetic lesions underlying this disorder, particularly when the phenotype is atypical. The present study performed targeted next­generation sequencing of 428 genes associated with cardiovascular diseases in a family with aortopathy, the proband of which presented with abdominal aortic aneurysm rupture only, with tissue fragility noted in surgery. After targeted capture, sequencing and bioinformatics analysis, a missense mutation, p.A1259T, was identified in the collagen type III α1 (COL3A1) gene and co­segregated with the disease in the family. Crystal structure modeling revealed abnormal hydrogen bonds generated by the mutation, which likely affected the spatial structure of the procollagen C­propeptide. Mutations in the procollagen C­propeptide are rare and genotype­phenotype correlation may explain the atypical manifestations of affected individuals. The results of the present study suggested that targeted gene capture combined with next­generation sequencing can serve as a useful technique in the genetic diagnosis of aortopathy, particularly in the content of an atypical phenotype.


Assuntos
Colágeno Tipo III/genética , Síndrome de Ehlers-Danlos/genética , Mutação de Sentido Incorreto , Aneurisma da Aorta Abdominal/complicações , Aneurisma da Aorta Abdominal/genética , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , China , Colágeno Tipo III/química , Síndrome de Ehlers-Danlos/complicações , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Conformação Proteica
20.
Amino Acids ; 48(1): 169-81, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26306844

RESUMO

Cultural heritage parchments made from the reticular dermis of animals have been subject to studies of deterioration and conservation by amino acid analysis. The reticular dermis contains a varying mixture of collagen I and III (COL I and III). When dealing with the results of the amino acid analyses, till now the COL III content has not been taken into account. Based on the available amino acid sequences, we present a method for determining the amount of COL III in the reticular dermis of new and historical parchments calculated from the ratio of Ile/Val. We find COL III contents between 7 and 32 % in new parchments and between 0.2 and 40 % in the historical parchments. This is consistent with results in the literature. The varying content of COL III has a significant influence on the uncertainty of the amino acid analysis. Although we have not found a simple correlation between the COL III content and the degree of deterioration, our results show that this question must be taken into consideration in future studies of the chemical and physical deterioration of parchment measured by amino acid analysis and other analytical methods.


Assuntos
Aminoácidos/química , Colágeno Tipo III/química , Pele/química , Redação/história , Sequência de Aminoácidos , Animais , Bovinos , Colágeno Tipo I/química , História do Século XIX , História do Século XX , História do Século XXI , Dados de Sequência Molecular
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