RESUMEN
Collagen posttranslational processing is crucial for its proper assembly and function. Disruption of collagen processing leads to tissue development and structure disorders like osteogenesis imperfecta (OI). OI-related collagen processing machinery includes prolyl 3-hydroxylase 1 (P3H1), peptidyl-prolyl cis-trans isomerase B (PPIB), and cartilage-associated protein (CRTAP), with their structural organization and mechanism unclear. We determine cryo-EM structures of the P3H1/CRTAP/PPIB complex. The active sites of P3H1 and PPIB form a face-to-face bifunctional reaction center, indicating a coupled modification mechanism. The structure of the P3H1/CRTAP/PPIB/collagen peptide complex reveals multiple binding sites, suggesting a substrate interacting zone. Unexpectedly, a dual-ternary complex is observed, and the balance between ternary and dual-ternary states can be altered by mutations in the P3H1/PPIB active site and the addition of PPIB inhibitors. These findings provide insights into the structural basis of collagen processing by P3H1/CRTAP/PPIB and the molecular pathology of collagen-related disorders.
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Colágeno , Microscopía por Crioelectrón , Ciclofilinas , Proteínas de la Matriz Extracelular , Humanos , Colágeno/metabolismo , Colágeno/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Ciclofilinas/metabolismo , Ciclofilinas/química , Ciclofilinas/genética , Dominio Catalítico , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Procesamiento Proteico-Postraduccional , Sitios de Unión , Unión Proteica , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Modelos Moleculares , Mutación , Osteogénesis Imperfecta/metabolismo , Osteogénesis Imperfecta/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/química , Glicoproteínas de Membrana , Proteoglicanos , Chaperonas Moleculares , Prolil HidroxilasasRESUMEN
Extracellular matrix (ECM) proteins provide anchorage and structural strength to cells and tissues in the body and, thus, are fundamental molecular components for processes of cell proliferation, growth, and function. Atomic force microscopy (AFM) has increasingly become a valuable approach for studying biological molecules such as ECM proteins at the level of individual molecules. Operational modes of AFM can be used to acquire the measurements of the physical, electronic, and mechanical properties of samples, as well as for viewing the intricate details of the surface chemistry of samples. Investigations of the morphology and properties of biomolecules at the nanoscale can be useful for understanding the interactions between ECM proteins and biological molecules such as cells, DNA, and other proteins. Methods for preparing protein samples for AFM studies require only basic steps, such as the immersion of a substrate in a dilute solution or protein, or the deposition of liquid droplets of protein suspensions on a flat, clean surface. Protocols of nanolithography have been used to define the arrangement of proteins for AFM studies. Using AFM, mechanical and force measurements with tips that are coated with ECM proteins can be captured in ambient or aqueous environments. In this review, representative examples of AFM studies are described for molecular-level investigations of the structure, surface assembly, protein-cell interactions, and mechanical properties of ECM proteins (collagen, elastin, fibronectin, and laminin). Methods used for sample preparation as well as characterization with modes of AFM will be discussed.
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Proteínas de la Matriz Extracelular , Microscopía de Fuerza Atómica , Microscopía de Fuerza Atómica/métodos , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Humanos , AnimalesRESUMEN
Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.
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Proteínas de Unión al Calcio , Humanos , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Estabilidad Proteica , Dominios ProteicosRESUMEN
Electrochemical immunosensors, surpassing conventional diagnostics, exhibit significant potential for cancer biomarker detection. However, achieving a delicate balance between signal sensitivity and operational stability, especially at the heterostructure interface, is crucial for practical immunosensors. Herein, porous carbon (PC) integration with Ti3C2Tx-MXene (MX) and gold nanoparticles (Au NPs) constructs a versatile immunosensing platform for detecting extracellular matrix protein-1 (ECM1), a breast cancer-associated biomarker. The inclusion of PC provided robust structural support, enhancing electrolytic diffusion with an expansive surface area while synergistically facilitating charge transfer with Ti3C2Tx. The biosensor optimized with 1.0 mg PC demonstrates a robust electrochemical redox response to the surface-bound thionine (th) redox probe, utilizing an inhibition-based strategy for ECM1 detection. The robust antibody-antigen interactions across the PC-integrated Ti3C2Tx-Au NPs platform (MX-Au-C-1) enabled robust ECM1 detection within 0.1-7.5 nM, with a low limit of detection (LOD) of 0.012 nM. The constructed biosensor shows improved operational stability with a 98.6 % current retention over 1 h, surpassing MXene-integrated (MX-Au) and pristine Au NPs (63.2 % and 44.3 %, respectively) electrodes. Moreover, the successful adaptation of the artificial neural network (ANN) model for predictive analysis of the generated DPV data further validates the accuracy of the biosensor, promising its future application in AI-powered remote health monitoring.
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Biomarcadores de Tumor , Técnicas Biosensibles , Neoplasias de la Mama , Carbono , Oro , Aprendizaje Automático , Nanopartículas del Metal , Titanio , Oro/química , Humanos , Neoplasias de la Mama/diagnóstico , Biomarcadores de Tumor/análisis , Carbono/química , Titanio/química , Porosidad , Nanopartículas del Metal/química , Técnicas Biosensibles/métodos , Femenino , Inmunoensayo/métodos , Técnicas Electroquímicas/métodos , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/análisis , Límite de DetecciónRESUMEN
The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies. Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.
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Proteolisis , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/inmunología , Animales , Humanos , Camélidos del Nuevo Mundo , Procolágeno/metabolismo , Procolágeno/química , Dominios Proteicos , Modelos Moleculares , Colágenos Fibrilares/metabolismo , Colágenos Fibrilares/química , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Cristalografía por Rayos XRESUMEN
In cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells that move through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECM protein types on leader cells and migrating clusters is unknown. To address this, we investigated the response of leader cells and migrating clusters upon moving from one ECM protein to another using a photoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since they are abundant in two distinct regions in living tissues, namely basement membrane and connective tissue. Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areas was achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled the deposition of the second ECM protein in the new irradiated regions, resulting in region-selective heterogeneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation from laminin into laminin compared to those migrating from laminin into collagen were observed. The formation of focal adhesion and actin structures for cells within the same cluster in the ECM proteins responded according to the underlying ECM protein type. Finally, integrin ß1 was crucial for the appearance of leader cells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin, integrin ß1 was not responsible. This highlights the correlation between leader cells in collective migration and the biochemical signals that arise from underlying extracellular matrix proteins.
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Movimiento Celular , Proteínas de la Matriz Extracelular , Laminina , Laminina/química , Laminina/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Animales , Integrina beta1/metabolismo , Integrina beta1/química , Ratones , Polietilenglicoles/química , Humanos , Fenotipo , Matriz Extracelular/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/químicaRESUMEN
Microfibril-associated glycoprotein 4 (MFAP4) is a 36-kDa extracellular matrix glycoprotein with critical roles in organ fibrosis, chronic obstructive pulmonary disease, and cardiovascular disorders, including aortic aneurysms. MFAP4 multimerises and interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. Structural details of MFAP4 and its potential interfaces for these interactions are unknown. Here, we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer, where two sets of homodimers constitute the top and bottom halves of each octamer. Each homodimer is linked together by an intermolecular disulphide bond. A C34S missense mutation prevents disulphide-bond formation between monomers but does not prevent octamer assembly. The atomic model, built into the 3.55 Å cryo-EM map, suggests that salt-bridge interactions mediate homodimer assembly, while non-polar residues form the interface between octamer halves. In the absence of calcium, an MFAP4 octamer dissociates into two tetramers. Binding studies with fibrillin-1, tropoelastin, LTBP4, and small fibulins show that MFAP4 has multiple surfaces for protein-protein interactions, most of which depend upon MFAP4 octamer assembly. The C34S mutation does not affect these protein interactions or cell interactions. MFAP4 assemblies with fibrillin-1 abrogate MFAP4 interactions with cells.
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Microscopía por Crioelectrón , Proteínas de la Matriz Extracelular , Fibrilina-1 , Microfibrillas , Tropoelastina , Humanos , Adipoquinas , Calcio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Fibrilina-1/metabolismo , Fibrilina-1/genética , Fibrilina-1/química , Glicoproteínas , Células HEK293 , Microfibrillas/metabolismo , Microfibrillas/química , Microfibrillas/ultraestructura , Modelos Moleculares , Mutación Missense , Unión Proteica , Multimerización de Proteína , Tropoelastina/metabolismo , Tropoelastina/química , Tropoelastina/genéticaRESUMEN
The extracellular matrix (ECM) proteome represents an important component of the tissue microenvironment that controls chemical flux and induces cell signaling through encoded structure. The analysis of the ECM represents an analytical challenge through high levels of post-translational modifications, protease-resistant structures, and crosslinked, insoluble proteins. This review provides a comprehensive overview of the analytical challenges involved in addressing the complexities of spatially profiling the extracellular matrix proteome. A synopsis of the process of synthesizing the ECM structure, detailing inherent chemical complexity, is included to present the scope of the analytical challenge. Current chromatographic and spatial techniques addressing these challenges are detailed. Capabilities for multimodal multiplexing with cellular populations are discussed with a perspective on developing a holistic view of disease processes that includes both the cellular and extracellular microenvironment.
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Proteínas de la Matriz Extracelular , Proteoma , Proteínas de la Matriz Extracelular/química , Proteoma/metabolismo , Proteómica/métodos , Matriz Extracelular/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Extracellular matrix (ECM) remodeling is accompanied by the continuous synthesis and degradation of the ECM components. This dynamic process plays an important role in guiding cell adhesion, migration, proliferation, and differentiation, as well as in tissue development, body repair, and maintenance of homeostasis. Nanomaterials, due to their photoelectric and catalytic properties and special structure, have garnered much attention in biomedical fields for use in processes such as tissue engineering and disease treatment. Nanomaterials can reshape the cell microenvironment by changing the synthesis and degradation of ECM-related proteins, thereby indirectly changing the behavior of the surrounding cells. This review focuses on the regulatory role of nanomaterials in the process of cell synthesis of different ECM-related proteins and extracellular protease. We discuss influencing factors and possible related mechanisms of nanomaterials in ECM remodeling, which may provide different insights into the design and development of nanomaterials for the treatment of ECM disorder-related diseases.
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Matriz Extracelular , Nanoestructuras , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Ingeniería de Tejidos , Adhesión CelularRESUMEN
The collagen IV network plays a crucial role in providing structural support and mechanical integrity to the basement membrane and surrounding tissues. A key aspect of this network is the formation of intra- and inter-collagen fibril crosslinks. One particular crosslink, an inter-residue sulfilimine bond, has been found, so far, to be unique to collagen IV. More specifically, these crosslinks are primarily formed between methionine and lysine or hydroxylysine residues and can occur within a single collagen fibril or between different collagen fibrils. Due to its significance as the major crosslink in the collagen IV network, the sulfilimine bond plays critical roles in tissue development and various human diseases. While the proposed reaction mechanism for sulfilimine bond formation is supported by experimental evidence, the precise nature of this bond remained uncertain until computational studies were conducted. The process involves the reaction of hypohalous acids (e.g., HOBr, HOCl), produced by a peroxidasin enzyme in the basement membrane, with the sidechain sulfur of methionine or sidechain nitrogen of lysine/hydroxylysine residues in collagen IV, to form halosulfonium or haloamine intermediates, respectively. The halosulfonium/haloamine then reacts with the sidechain amine/sulfide of the lysine (or hydroxylysine) or methionine respectively, eventually resulting in the formation of the sulfilimine (MetSîNLys/Hyl) crosslink. The sulfilimine product formed not only plays a crucial role in physiological processes but also finds applications in various industrial and pharmaceutical contexts. In this review, we provide a comprehensive summary of existing studies, including our own research, aimed at understanding the reaction mechanism, protonation states, characteristic nature, and dynamic behavior of the sulfilimine bond in collagen IV. The goal is to offer readers an overview of this critically important biochemical bond.
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Proteínas de la Matriz Extracelular , Iminas , Peroxidasa , Humanos , Peroxidasa/química , Proteínas de la Matriz Extracelular/química , Lisina , Hidroxilisina , Colágeno Tipo IV/química , Metionina/químicaRESUMEN
Small leucine rich proteoglycans (SLRPs) are the largest family of proteoglycans, with 18 members that are subdivided into five classes. SLRPs are small in size and can be present in tissues as glycosylated and non-glycosylated proteins, and the most studied SLRPs include decorin, biglycan, lumican, keratocan and fibromodulin. SLRPs specifically bind to collagen fibrils, regulating collagen fibrillogenesis and the biomechanical properties of tissues, and are expressed at particularly high levels in fibrous tissues, such as the cornea. However, SLRPs are also very active components of the ECM, interacting with numerous growth factors, cytokines and cell surface receptors. Therefore, SLRPs regulate major cellular processes and have a central role in major fundamental biological processes, such as maintaining corneal homeostasis and transparency and regulating corneal wound healing. Over the years, mutations and/or altered expression of SLRPs have been associated with various corneal diseases, such as congenital stromal corneal dystrophy and cornea plana. Recently, there has been great interest in harnessing the various functions of SLRPs for therapeutic purposes. In this comprehensive review, we describe the structural features and the related functions of SLRPs, and how these affect the therapeutic potential of SLRPs, with special emphasis on the use of SLRPs for treating ocular surface pathologies.
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Proteoglicanos Tipo Condroitín Sulfato , Proteínas de la Matriz Extracelular , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/metabolismo , Proteoglicanos Pequeños Ricos en Leucina , Decorina , Sulfato de Queratano/metabolismo , Colágeno , BiologíaRESUMEN
The ankle-link complex (ALC) consists of USH2A, WHRN, PDZD7, and ADGRV1 and plays an important role in hair cell development. At present, its architectural organization and signaling role remain unclear. By establishing Adgrv1 Y6236fsX1 mutant mice as a model of the deafness-associated human Y6244fsX1 mutation, the authors show here that the Y6236fsX1 mutation disrupts the interaction between adhesion G protein-coupled receptor V subfamily member 1 (ADGRV1) and other ALC components, resulting in stereocilia disorganization and mechanoelectrical transduction (MET) deficits. Importantly, ADGRV1 inhibits WHRN phosphorylation through regional cAMP-PKA signaling, which in turn regulates the ubiquitination and stability of USH2A via local signaling compartmentalization, whereas ADGRV1 Y6236fsX1 does not. Yeast two-hybrid screening identified the E3 ligase WDSUB1 that binds to WHRN and regulates the ubiquitination of USH2A in a WHRN phosphorylation-dependent manner. Further FlAsH-BRET assay, NMR spectrometry, and mutagenesis analysis provided insights into the architectural organization of ALC and interaction motifs at single-residue resolution. In conclusion, the present data suggest that ALC organization and accompanying local signal transduction play important roles in regulating the stability of the ALC.
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Sordera , Animales , Humanos , Ratones , Proteínas Portadoras/genética , Sordera/genética , Sordera/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Mutación/genética , FosforilaciónRESUMEN
BACKGROUND: Retinitis pigmentosa (RP) is an inherited disorder that causes progressive loss of vision. This study aimed to describe the possible causative variants of the USH2A gene in Korean RP families and their associated phenotypes. MATERIALS AND METHODS: We recruited 94 RP families (220 subjects, including 94 probands and 126 family members) in a Korean cohort, and analyzed USH2A gene variants through whole-exome sequencing. The pathogenicity of the variants was classified according to American College of Medical Genetics and Genomics and Association for Molecular Pathology guidelines. RESULTS: We found 14 USH2A disease-causing variants, including 5 novel variants. Disease causing variants were identified in 10 probands with RP, accounting for 10.6% (10/94) of the Korean RPs in the cohort. To visually represent the structural changes induced by novel variants, we modeled the three-dimensional structures of the wild-type and mutant proteins. CONCLUSIONS: This study expands the spectrum of USH2A variants and provides information for future therapeutic strategies for RP.
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Retinitis Pigmentosa , Humanos , Secuenciación del Exoma , Mutación , Análisis Mutacional de ADN , Linaje , Retinitis Pigmentosa/genética , República de Corea/epidemiología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/químicaRESUMEN
Glycosaminoglycan (GAG) is a polysaccharide present on the cell surface as an extracellular matrix component, and is composed of repeating disaccharide units consisting of an amino sugar and uronic acid except in the case of the keratan sulfate. Sulfated GAGs, such as heparan sulfate, heparin, and chondroitin sulfate mediate signal transduction of growth factors, and their functions vary with the type and degree of sulfated modification. We have previously identified human and mouse cochlins as proteins that bind to sulfated GAGs. Here, we prepared a recombinant cochlin fused to human IgG-Fc or Protein A at the C-terminus as a detection and purification tag and investigated the ligand specificity of cochlin. We found that cochlin can be used as a specific probe for highly sulfated heparan sulfate and chondroitin sulfate E. We then used mutant analysis to identify the mechanism by which cochlin recognizes GAGs and developed a GAG detection system using cochlin. Interestingly, a mutant lacking the vWA2 domain bound to various types of GAGs. The N-terminal amino acid residues of cochlin contributed to its binding to heparin. Pathological specimens from human myocarditis patients were stained with a cochlin-Fc mutant. The results showed that both tryptase-positive and tryptase-negative mast cells were stained with this mutant. The identification of detailed modification patterns of GAGs is an important method to elucidate the molecular mechanisms of various diseases. The method developed for evaluating the expression of highly sulfated GAGs will help understand the biological and pathological importance of sulfated GAGs in the future.
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Sulfatos de Condroitina , Proteínas de la Matriz Extracelular , Heparitina Sulfato , Animales , Humanos , Ratones , Biomarcadores de Tumor/química , Proteínas de Unión al Calcio/química , Sulfatos de Condroitina/análisis , Heparitina Sulfato/análisis , Inmunohistoquímica/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Triptasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genéticaRESUMEN
Several tissue engineering approaches are based on the ability of mesenchymal cells to endogenously synthesize an extracellular matrix (ECM) in vitro, which can be seen as a form of biomaterial. Accordingly, the inter-donor variability of cell-assembled extracellular matrix (CAM) production is a key parameter to understand in order to progress towards clinical applications, especially for autologous strategies. In this study, CAMs were produced, under good manufacturing process conditions, from skin fibroblasts of 21 patients as part of a clinical trial to evaluate a tissue-engineered vascular graft. The inter-donor variability of CAM strength, thickness, hydroxyproline, and glycosaminoglycan was substantial (coefficient of variability of 33%, 19%, 24%, and 19%, respectively), but a significant correlation was observed between all four properties (Pearson r: 0.43 to 0.70; p-value ≤ 0.05). A CAM matrisome analysis, performed by mass spectrometry, revealed the presence of 70 ECM-related proteins. Our study shows that the relative abundance of 16 proteins (15 non-collagenous) correlated with CAM thickness. These proteins also correlated with CAM hydroxyproline content, as well as 21 other proteins that included fibrillar collagens and non-collagenous proteins. However, data demonstrated that only the relative abundance of type I collagen subunit alpha-1 was correlated to CAM strength. This study is the most extensive evaluation of CAM inter-donor variability to date and will help tissue engineers working with this type of biomaterial to design strategies that take into account this variability, especially for autologous tissue manufacturing.
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Matriz Extracelular , Fibroblastos , Materiales Biocompatibles/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Humanos , Hidroxiprolina , Ingeniería de Tejidos/métodosRESUMEN
BACKGROUND: Accumulating clinical and experimental evidence shows multiple biological effects of ginsenoside Rb1 (GRb1) in the treatment of aging related diseases such as osteoporosis (OP). Recently, GRb1 has attracted extensive attention as an anti-osteoporosis agent. Here, we sought to identify the mechanism by which GRb1 improves OP. METHODS: A dexamethasone (DEX)-induced rat model of OP was constructed and the rats were treated with GRb1 to examine its role in OP. We screened the action targets of GRb1 online and validated by performing functional experiments. The correlation between aryl hydrocarbon receptor (AHR) and proline/arginine-rich end leucine-rich repeat protein (PRELP) was identified through luciferase and chromatin immunoprecipitation assays. In the isolated osteoblasts from DEX-induced OP rats, the expression of osteogenic differentiation-associated genes, and nuclear factor-kappa B (NF-κB) pathway-related genes, mineralization, and number of calcium nodules were assessed. RESULTS: GRb1 enhanced the differentiation of osteoblasts, the mechanism of which was related to upregulation of AHR. AHR could promote the transcription of PRELP by binding to the PRELP promoter region and consequently caused its upregulation. Meanwhile, PRELP inhibited the activation of the NF-κB pathway, which underlay the promoting impact of AHR in the osteogenic differentiation. Additionally, GRb1 could ameliorate OP in DEX-induced rats via the AHR/PRELP/NF-κB axis. CONCLUSIONS: Our findings demonstrate that GRb1 might function as an effective candidate to prevent the progression of OP via regulation of the AHR/PRELP/NF-κB axis, revealing a new molecular mechanism underpinning the impact of GRb1 in the progression of OP and offering a theoretical contribution to the treatment of OP.
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Ginsenósidos , Osteoporosis , Animales , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/uso terapéutico , Ginsenósidos/farmacología , Ginsenósidos/uso terapéutico , FN-kappa B , Osteogénesis , Osteoporosis/inducido químicamente , Osteoporosis/tratamiento farmacológico , Osteoporosis/prevención & control , Ratas , Receptores de Hidrocarburo de ArilRESUMEN
Competent elastic fibers are critical to the function of the lung and right circulation. Murine models of elastopathies can aid in understanding the functional roles of the elastin and elastin-associated glycoproteins that constitute elastic fibers. Here, we quantify together lung and pulmonary arterial structure, function, and mechanics with right heart function in a mouse model deficient in the elastin-associated glycoprotein fibulin-5. Differences emerged as a function of genotype, sex, and arterial region. Specifically, functional studies revealed increased lung compliance in fibulin-5 deficiency consistent with a histologically observed increased alveolar disruption. Biaxial mechanical tests revealed that the primary branch pulmonary arteries exhibit decreased elastic energy storage capacity and wall stress despite only modest differences in circumferential and axial material stiffness in the fibulin-5 deficient mice. Histological quantifications confirm a lower elastic fiber content in the fibulin-5 deficient pulmonary arteries, with fragmented elastic laminae in the outer part of the wall - likely the reason for reduced energy storage. Ultrasound measurements confirm sex differences in compromised right ventricular function in the fibulin-5 deficient mice. These results reveal compromised right heart function, but opposite effects of elastic fiber dysfunction on the lung parenchyma (significantly increased compliance) and pulmonary arteries (trend toward decreased distensibility), and call for further probing of ventilation-perfusion relationships in pulmonary pathologies. Amongst many other models, fibulin-5 deficient mice can contribute to our understanding of the complex roles of elastin in pulmonary health and disease.
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Elastina , Proteínas de la Matriz Extracelular/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Proteínas de Unión al Calcio , Tejido Elástico , Elastina/metabolismo , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Femenino , Masculino , RatonesRESUMEN
The role of the extracellular matrix (ECM) remodeling in tumorigenesis and metastasis is becoming increasingly clear. Cancer development requires that tumor cells recruit a tumor microenvironment permissive for further tumor growth. This is a dynamic process that takes place by a cross-talk between tumor cells and ECM. As a consequence, molecules derived from the ECM changes associated to cancer are released into the bloodstream, representing potential biomarkers of tumor development. This article highlights the importance of developing and improving bioanalytical methods for the detection of ECM remodeling-derived components, as a step forward to translate the basic knowledge about cancer progression into the clinical practice.