Extracellular vesicles (EVs), mainly exosomes and microvesicles, are bilayer lipids containing biologically active information, including nucleic acids and proteins. They are involved in cell communication and signalling, mediating many biological functions including cell growth, migration and proliferation. Recently, EVs have received great attention in the field of tissue engineering and regenerative medicine. Many in vivo and in vitro studies have attempted to evaluate the chondrogenesis potential of these microstructures and their roles in cartilage regeneration. EVs derived from mesenchymal stem cells (MSCs) or chondrocytes have been found to induce chondrocyte proliferation and chondrogenic differentiation of stem cells in vitro. Preclinical studies have shown that exosomes derived from MSCs have promising results in cartilage repair and in cell-free therapy of osteoarthritis. This review will focus on the in vitro and in vivo chondrogenesis and cartilage regeneration of EVs as well as their potential in the treatment of osteoarthritis. 相似文献
This paper presents a framework for modelling biological tissues based on discrete particles. Cell components (e.g. cell membranes, cell cytoskeleton, cell nucleus) and extracellular matrix (e.g. collagen) are represented using collections of particles. Simple particle to particle interaction laws are used to simulate and control complex physical interaction types (e.g. cell-cell adhesion via cadherins, integrin basement membrane attachment, cytoskeletal mechanical properties). Particles may be given the capacity to change their properties and behaviours in response to changes in the cellular microenvironment (e.g., in response to cell-cell signalling or mechanical loadings). Each particle is in effect an ‘agent’, meaning that the agent can sense local environmental information and respond according to pre-determined or stochastic events. The behaviour of the proposed framework is exemplified through several biological problems of ongoing interest. These examples illustrate how the modelling framework allows enormous flexibility for representing the mechanical behaviour of different tissues, and we argue this is a more intuitive approach than perhaps offered by traditional continuum methods. Because of this flexibility, we believe the discrete modelling framework provides an avenue for biologists and bioengineers to explore the behaviour of tissue systems in a computational laboratory. 相似文献
Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.The cartilage of the mammalian skeletal system has two distinct roles. The epiphyseal cartilage of the growth plate drives endochondral bone growth, and the hyaline cartilage at the weight-bearing surfaces of bones facilitates joint articulation. In both environments, chondrocyte-regulated production, assembly, and turnover of the extracellular matrix (ECM)1 are essential for the tissue to withstand compressive forces and respond to mechanical loading. The major structural constituents of cartilage ECM are the heterotypic collagen II/IX/XI fibrils and proteoglycan-glycosaminoglycan networks of aggrecan and hyaluronan. Loss of joint function in osteoarthritis (OA) is strongly associated with net loss of aggrecan and collagen breakdown caused by an imbalance of ECM homeostasis (1). In addition, many inherited human chondrodysplasias involve disruption of cartilage matrix assembly or cell-matrix interactions, resulting in abnormal skeletal development and in some cases early onset cartilage degeneration (2, 3).The alterations in chondrocyte metabolism that occur during OA are complex and remain poorly understood (4). An early response to loss or fragmentation of ECM components is attempted tissue repair through secretion of anabolic factors, cell proliferation, and matrix remodeling (5). However, the resulting product is a fibrocartilage that does not recapitulate the composition or precise architecture of the original hyaline articular cartilage. This limited capacity of cartilage for regeneration has driven research into cartilage tissue engineering (6). Production of authentic hyaline cartilage in vitro remains challenging due to the dedifferentiation of primary chondrocytes upon removal from their three-dimensional matrix environment (7). However, improved “neocartilage” culture systems have been developed through evaluation of suitable chondroprogenitor or chondrocyte subpopulations and optimization of exogenous support matrices and growth factors (8, 9). The therapeutic target of neocartilage culture is autologous tissue repair. However, there is fundamental value in using neocartilage systems to elucidate mechanisms of protein integration into the ECM and the role of specific protein interactions during cartilage maturation.Cartilage profiling by 2-DE and mass spectrometry-based proteomics is generating important new insight into mechanisms of cartilage degeneration in vitro and in vivo (10). For example, anabolic factors with potential roles in cartilage repair, including connective tissue growth factor and inhibin βA (activin), were identified in the secretome of human OA cartilage explants (11). Comparison of cartilage protein extracts from normal donors and OA patients revealed significantly increased levels of the serine protease Htra1 in patient cartilage (12) and that Htra1-mediated proteolysis of aggrecan may significantly contribute to OA pathology (13). Targeted analysis of the chondrocyte mitochondrial proteome highlighted OA-related changes in energy production and protection against reactive oxygen species (14). Obtaining sufficient chondrocytes from human donors for proteomics unfortunately requires expansion of the cell population with potential loss of the chondrocyte phenotype during prolonged culture. Other drawbacks encountered with human samples include the clinical heterogeneity of OA, lack of matched controls, and inherent genetic variation of human subjects (15). Alternatively, animal models that recapitulate hallmarks of progressive cartilage degeneration, such as aggrecan loss and articular surface fibrillation, are emerging as a powerful resource, particularly in mice lacking specific proteases or protease target sites (16, 17). The development of techniques for analysis of murine cartilage using proteomics has paved the way for differential analysis of normal and pathological or genetically targeted cartilage (18, 19).Label-free methods for relative peptide quantitation, such as ion intensity measurement and spectral counting, are emerging as reliable and cost-effective alternatives to chemical modification or isotopic peptide labeling (20). Combining orthogonal protein and/or peptide fractionation with high resolution HPLC-MS can achieve proteome-wide coverage (21). Because extensive sample fractionation can introduce redundancy and variation, improved sequence/proteome coverage must be balanced against the cost of additional sample handling and lengthy LC-MS runs (22).Here we describe a novel platform for analysis of mouse cartilage using solubility-based protein fractionation (19) combined with label-free quantitative tandem MS (LTQ-Orbitrap). Sequential extraction of 3-day postnatal (P3) mouse epiphyseal cartilage and 3-week neocartilage cultures revealed a marked transition from a high proportion of readily soluble components in P3 extracts to a greater proportion of poorly soluble proteins in neocartilage. Principal component analysis and hierarchical clustering were used to globally assess the inter-relationships between P3 cartilage and neocartilage NaCl and guanidine hydrochloride (GdnHCl) extracts. At a p value cutoff of 0.05, 403 proteins were classified as extract-specific, whereas 125 proteins were classified as tissue sample-specific. Many of the proteins significantly enriched in neocartilage were annotated by the terms cell adhesion, extracellular matrix, and cytoskeletal remodeling. Further statistical analysis identified a third important protein category in which protein solubility was altered between the P3 and neocartilage. Identification of proteins involved in neocartilage maturation has generated novel insight into the fundamental process of cartilage matrix development with potential for further analysis of engineered cartilaginous tissues with biomedical applications. 相似文献
During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease. 相似文献
Thyroid hormone (T3 or 3,5,3′-triiodothyronine) plays a causative role during amphibian metamorphosis. To investigate how T3 induces some cells to die and others to proliferate and differentiate during this process, we have chosen the model system of intestinal remodeling, which involves apoptotic degeneration of larval epithelial cells and proliferation and differentiation of other cells, such as the fibroblasts and adult epithelial cells, to form the adult intestine. We have established in vitro culture conditions for intestinal epithelial cells and fibroblasts. With this system, we show that T3 can enhance the proliferation of both cell types. However, T3 also concurrently induces larval epithelial apoptosis, which can be inhibited by the extracellular matrix (ECM). Our studies with known inhibitors of mammalian cell death reveal both similarities and differences between amphibian and mammalian cell death. These, together with gene expression analysis, reveal that T3 appears to simultaneously induce different pathways that lead to specific gene regulation, proliferation, and apoptotic degeneration of the epithelial cells. Thus, our data provide an important molecular and cellular basis for the differential responses of different cell types to the endogenous T3 during metamorphosis and support a role of ECM during frog metamorphosis. 相似文献
Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient''s blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor. 相似文献
Aerobacter aerogenes, Paracolobactrum aerogenoides, Spirillum serpens, and gelatinous strains of Chromobacterium violaceum produced an extracellular, ether-soluble, Ehrlich-positive substance when grown in media prepared with gelatin hydrolysate. The substance has been tentatively identified as pyrrole-2-carboxylic acid. Both hydroxy-l-proline and allo-d-hydroxyproline have been shown to be precursors of the material. Gelatinous strains of Chromobacterium violaceum, but not the other positive cultures, produced two ether-insoluble pyrroles as well, the precursors of which occur in gelatin hydrolysate but have not yet been identified. The property of pyrrole formation in bacteria and its possible use as an aid in identification of bacteria was discussed. 相似文献
Diabetic nephropathy (DN) is a serious complication in diabetes. Major typical
morphological changes are the result of changes in the extracellular matrix (ECM). Thus,
basement membranes are thickened and the glomerular mesangial matrix and the
tubulointerstitial space are expanded, due to increased amounts of ECM. One important ECM
component, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs in
basement membranes are decreased but increased in the mesangium and the tubulointerstitial
space. The amounts and structures of heparan sulfate chains are changed, and such changes
affect levels of growth factors regulating cell proliferation and ECM synthesis, with cell
attachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfate
structures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classes
also modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serine
proteases, such as plasminogen activator, as well as their corresponding inhibitors. The
levels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN.
Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesis
and turnover in DN. Whether ECM components can be used as markers for early kidney changes
is an important research topic, whereas at present, the clinical use remains to be
established. 相似文献
The design of artificial extracellular matrices has attracted much attention in tissue engineering as well as in cell biology
research. An immobilized recombinant epidermal growth factor (EGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated
as EGF-Fc) has been constructed. Mouse fibroblast Swiss 3T3 cells adhered both to EGF-Fc-coated and collagen-coated surfaces.
Phosphorylation of EGF receptor in A431 cells was induced by immobilized EGF-Fc as well as soluble EGF. Immobilized EGF-Fc
continuously activated mitogen-activated protein kinase (MAPK) in A431 cells whereas MAPK activation induced by soluble EGF
decreased rapidly with time. The cytoskeleton of A431 cells adhering onto immobilized EGF-Fc was filopodia whereas that of
the cells adhering onto collagen in the presence of soluble EGF was lammellipodia. 相似文献
The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.
The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell–matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets.The balance between hemostasis and thrombosis relies on a finely tuned adhesive response of blood platelets. Inadequate adhesion leads to bleeding, whereas excessive or inappropriate adhesion leads to thrombosis. Resting platelets are nonadhesive anuclear discs and do not interact with the vessel wall, but they have a plethora of receptors that sense activating signals (agonists) of various sorts. The activating signals include soluble factors such as thrombin, adenosine diphosphate (ADP), and epinephrine, all of which act on G-protein-coupled receptors (GPCRs) on the platelets. In addition, certain receptors for extracellular matrix (ECM) proteins (e.g., GPIb, GPVI, and some integrins) can also act as activating receptors. These diverse receptors trigger intracellular signaling pathways that activate (1) actin assembly leading to cell shape change and extension of filopodia; (2) exocytosis of secretory granules that release additional platelet agonists as well as adhesive ECM proteins; and (3) activation of additional cell-surface receptors such as the major platelet-specific integrin, αIIbβ3, that contribute further to the adhesion and aggregation of activated platelets. Thus, the interactions of platelet-ECM adhesion receptors with ECM proteins from the vessel wall, from the plasma, and from the platelets themselves, are central to both the initial adhesion and the subsequent activation and aggregation of platelets (Varga-Szabo et al. 2008). These adhesive interactions, together with coagulation (to which platelets also contribute), generate the fibrin clot, essentially a facultative ECM that forms the initial occlusion of the damaged vessel but also serves as a subsequent ECM substrate for wound healing. In this article, we will review what is known about the roles of ECM proteins and their receptors in platelet adhesion and aggregation, summarize the roles of the clot and provisional ECM in subsequent wound healing, point out various unanswered questions, and discuss briefly the contributions of the relevant cell–ECM interactions to disease and the potential for therapeutic interventions. 相似文献
Some diseases associated with a temporary deterioration in kidney function and/or development of proteinuria show an apparently complete functional remission once the initiating trigger is removed. While it was earlier thought that a transient impairment of kidney function is harmless, accumulating evidence now suggests that these patients are more prone to developing renal failure later in life. We therefore sought to investigate to what extent renal functional changes, inflammation and collagen deposition are reversible after cessation of disease induction, potentially explaining residual sensitivity to damage. Using a rat model of Angiotensin II (Ang II)-induced hypertensive renal disease we show the development of severe hypertension (212 ± 10.43 vs. 146 ± 1.4 mmHg, p<0.001) and proteinuria (51.4 ± 6.3 vs. 14.7 ± 2.0 mg/24h, p<0.01) with declined creatinine clearance (2.0 ± 0.5 vs. 4.9 ± 0.6 mL/min, p<0.001) to occur after 3 weeks of Ang II infusion. At the structural level, Ang II infusion resulted in interstitial inflammation (18.8 ± 4.8 vs. 3.6 ± 0.5 number of macrophages, p<0.001), renal interstitial collagen deposition and lymphangiogenesis (4.1 ± 0.4 vs. 2.2 ± 0.4 number of lymph vessels, p<0.01). Eight weeks after cessation of Ang II, all clinical parameters, pre-fibrotic changes such as myofibroblast transformation and increase in lymph vessel number (lymphangiogenesis) returned to control values. However, glomerular desmin expression, glomerular and periglomerular macrophages and interstitial collagens remained elevated. These dormant abnormalities indicate that after transient renal function decline, inflammation and collagen deposition may persist despite normalization of the initiating pathophysiological stimulus perhaps rendering the kidney more vulnerable to further damage. 相似文献