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Abstract
Our understanding of proteolysis of ECM and cell surface molecules has expanded dramatically. We now know that the most significant proteolytic events are confined to the pericellular environment. With the discovery of new proteinases with new functions come new avenues for exploration in areas of development and disease. The synergy between adhesion, ECM, and proteolysis has been advanced by discovery of the intimate relationships between proteinases and signal transduction, particularly as effectors of epithelial–stromal interactions. Understanding the mechanisms by which pericellular proteinases are regulated and activated, the nature of their molecular targets, and how adhesion and proteolysis are integrated will provide exciting avenues for investigation over the next few years.
... Thus, the overexpressed enzymes can be used as a stimulus for the design of drug carriers that respond to tumor microenvironment. [28][29][30][31] In multicellular organisms, the extracellular matrix (ECM) is a non-cellular component composed of various macromolecules and minerals present around the cells. The ECM is mainly composed of four kinds of substances, namely collagen, non-collagen, elastin, proteoglycan, and aminoglycan. ...
... The ECM is mainly composed of four kinds of substances, namely collagen, non-collagen, elastin, proteoglycan, and aminoglycan. [28] ECM plays a physical role in supporting cells, holding water, and connecting cells to form tissues and organs. It also plays an integral biological role in cells' growth, death, polarity, shape, migration, and metabolic activities. ...
Hydrogel‐based drug delivery systems (DDSs) can leverage therapeutically beneficial outcomes in cancer therapy. In this domain, polyethylene glycol (PEG) has become increasingly popular as a biomedical polymer and has found clinical use. Owing to their excellent biocompatibility, facile modifiability, and high drug encapsulation rate, PEG hydrogels have shown great promise as drug delivery platforms. Here, the progress in emerging novel designs of PEG‐hydrogels as DDSs for anti‐cancer therapy is reviewed and discussed, focusing on underpinning multiscale release mechanisms categorized under stimuli‐responsive and non‐responsive drug release. The responsive drug delivery approaches are discussed, and the underpinning release mechanisms are elucidated, covering the systems functioning based on either exogenous stimuli‐response, such as photo‐ and magnetic‐sensitive PEG hydrogels, or endogenous stimuli‐response, such as enzyme‐, pH‐, reduction‐, and temperature‐sensitive PEG hydrogels. Special attention is paid to the commercial potential of PEG‐based hydrogels in cancer therapy, highlighting the limitations that need to be addressed in future research for their clinical translation.
... Plasmin is also able to convert pro-MMP 1, 3 and 9 into their active forms. The plasminogen activators therefore play a role in tumour cell invasion and metastasis (Werb Z 1997), and uPA has been shown to be an independent prognostic marker in both breast carcinoma and colorectal carcinoma (Duffy MJ 1996). ...
... Cleavage of plasminogen by MMP 2,3,7,9 and 12 generates angiostatin (Dong Z 1997, Cornelius LA 1998, Gorrin Rivas MJ 2000, and MMP 3,9, 12, 13 and 20 might be involved in the generation of endostatin, a C-terminal fragment of the basement membrane collagen type XVIII (Ferreras M 2000). Angiostatin and endostatin reduce endothelial cell proliferation (O'Reilly MS 1994, 1997 and endostatin may inhibit endothelial-cell invasion by acting as an inhibitor of MMP 14 and MMP 2 (Kim YM 2000). MMP 12 may also inhibit tumour angiogenesis by cleavage and shedding of cell surface bound urokinase-type plasminogen-activator receptor, which is required for endothelial cell invasion into fibrin (Koolwijk P 2001). ...
p>The expression pattern of MMPs (Collagenases MMPs 1,8,13; Gelantinases MMPs 2,9; Stromelysins MMPs 3,7) and TIMPs (TIMPs 1 and 2) was examined in fresh tissue and paraffin sections from 30 patients undergoing hepatic resection for colorectal carcinoma metastases and 3 patients with benign liver lesions, using semiquantitative RT-PCR and immunohistochemistry. It was also examined, using immunohistochemistry, in the paraffin sections of the primary tumours giving rise to these liver metastases.
MMP 1, MMP 9, MMP 3 and MMP 7 mRNAs were highly expressed in colorectal liver metastases. Immunohistochemistry confirmed these findings, MMPs 1 and 9 being expressed by tumour stromal cells, whereas MMPs 3 and 7 were expressed by adeenocarcinoma and stromal cells. TIMP 1 and TIMP 2 mRNA was expressed by both colorectal carcinoma metastases and distal liver. The expression of TIMP 1 and 2 mRNA in the adjacent liver was markedly lower than in both the metastasis and distal liver. In the benign lesions TIMP 1 and 2 mRNA expression appeared equal throughout. All the primary tumours MMP 1,8,2,9,3,7 and TIMP 1 and 2. Tumour stromal cells expressed MMP 1 (fibroblasts and macrophages), MMP 8 (fibroblasts), MMP 2 (fibroblasts), MMP 9 (macrophages). MMPs 3 and 7 were expressed by both adenocarcinoma cells and tumour stromal macrophages. TIMP 1 was expressed by macrophages in the tumour stroma and TIMP 2 by fibroblasts.
High expression of MMPs 1,9,3 and 7 in the CRC metastasis and down regulation of TIMP 1 and 2 in the immediate adjacent liver tissue may facilitate local growth of hepatic metastatic disease. Further studies are required to elucidate the mechanisms involved in this complex relationship between metastasis liver, MMP and TIMP.</p
... Aberrant plasmin activity was detected in the OA joint (29,47), but until this report, the function of activated plasmin in OA pathogenesis remained unclear. Previous reports described a role for plasmin in degrading ECM components of cartilage and lubricin (36,48,49) , and in activating MMPs (37,(50)(51)(52). Guided by these observations, we found that plasmin is pathogenic in the development of post-traumatic OA, and that it promotes disease progression through several mechanisms. ...
Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3′-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.
... Similar to the observations made during initial secretome profiling, terms related to proteolysis represented the top 5 molecular function terms ( Figures 1B, 2C). This is reflective of the broad functions of proteases which include extracellular matrix assembly and remodeling and aligns with the top GO cellular component terms (69). Likewise, protease-related functions were represented in biological process GO terms; interestingly, immune cell functions were also represented ( Figure 1B). ...
Background
Lung cancer is the leading cause of cancer related death worldwide, mainly due to the late stage of disease at the time of diagnosis. Non-invasive biomarkers are needed to supplement existing screening methods to enable earlier detection and increased patient survival. This is critical to EGFR-driven lung adenocarcinoma as it commonly occurs in individuals who have never smoked and do not qualify for current screening protocols.
Methods
In this study, we performed mass spectrometry analysis of the secretome of cultured lung cells representing different stages of mutant EGFR driven transformation, from normal to fully malignant. Identified secreted proteins specific to the malignant state were validated using orthogonal methods and their clinical activity assessed in lung adenocarcinoma patient cohorts.
Results
We quantified 1020 secreted proteins, which were compared for differential expression between stages of transformation. We validated differentially expressed proteins at the transcriptional level in clinical tumor specimens, association with patient survival, and absolute concentration to yield three biomarker candidates: MDK, GDF15, and SPINT2. These candidates were validated using ELISA and increased levels were associated with poor patient survival specifically in EGFR mutant lung adenocarcinoma patients.
Conclusions
Our study provides insight into changes in secreted proteins during EGFR driven lung adenocarcinoma transformation that may play a role in the processes that promote tumor progression. The specific candidates identified can harnessed for biomarker use to identify high risk individuals for early detection screening programs and disease management for this molecular subgroup of lung adenocarcinoma patients.
... cells and ECM has been demonstrated to play a key role in epithelium formation, as well as disease occurrence 6 . Indeed, ECM (especially collagens) is essential for controlling cell behavior during tissue formation in many organ systems [7][8][9][10] ; and change of ECM assembly and disassembly properties is a critical feature for airway epithelial development, while imbalance of ECM organization can lead to abnormal epithelial remodeling 6,11,12 . However, the mechanisms of how epithelial progenitor cells interact with ECM during airway development remain unclear, which is partially due to the lack of a biomimetic model that can recapitulate the development of progenitor cells within ECM in vitro. ...
Extracellular matrix (ECM) assembly/disassembly is a critical regulator for airway epithelial development and remodeling. Airway organoid is widely used in respiratory research, yet there is limited study to indicate the roles and mechanisms of ECM organization in epithelial growth and differentiation by using in vitro organoid system. Moreover, most of current Matrigel-based airway organoids are in basal-out orientation where accessing the apical surface is challenging. We present a human apical-out airway organoid using a biochemically defined hybrid hydrogel system. During human nasal epithelial progenitor cells (hNEPCs) differentiation, the gel gradually degrade, leading to the organoid apical surfaces facing outward. The expression and activity of ECM-degrading enzymes, matrix metalloproteinases (MMP7, MMP9, MMP10 and MMP13) increases during organoid differentiation, where inhibition of MMPs significantly suppresses the normal ciliation, resulting in increased goblet cell proportion. Moreover, a decrease of MMPs is found in goblet cell hyperplastic epithelium in inflammatory mucosa. This system reveals essential roles of epithelial-derived MMPs on epithelial cell fate determination, and provides an applicable platform enabling further study for ECM in regulating airway development in health and diseases.
... MMPs are also integral to wound healing in association with angiogenesis. MMPs such as MMP-9, MMP-2 have varied roles in the pathogenesis of secondary mechanisms of injury and recovery after spinal cord injury [14,15]. MMPs tend to have a unique temporal profile in the injured spinal cord, with some MMPs causing cord damage in the early injury phase while some promoting recovery in the late injury [16]. ...
Background:
Spinal cord injury (SCI) can result in lifelong disability. Currently, the literature suggests that biomarkers are helpful in prognosticating SCI, but there is no specific biomarker to date. This is the first study that predicted the prognosis dynamically using biomarkers.
Aim:
To elucidate the role of biomarkers in prognosticating acute traumatic SCI.
Methods:
Blood samples were obtained from 35 patients of acute traumatic SCI at presentation, immediate post-op, and at 6 weeks. At 6 months follow-up, patients were divided into two groups, i.e, improved and non-improved based on the improvement in the ASIA grade compared to presentation. A non-parametric test was used for comparing mean NSE, MMP-2, S100-B, and NF serum levels at presentation, immediate post-op, and 6 weeks post-op follow-up between the two groups.
Results:
There was a significant difference (p = 0.03) in the NF values at presentation between the two groups. The difference of NSE values at 6 weeks was also significant (p = 0.016) between the two groups. S-100B levels were also significantly different between both groups at presentation (p=0.016), and at the immediate post-op stage (p=0.007). MMP-2 levels neither displayed any specific trend nor any significant difference between the two groups.
Conclusion:
Higher NF values at presentation, and higher S-100B levels at presentation and immediate post-operative period correlated with poor outcome. Also, increased NSE values after surgery are indicative of no improvement. These levels can be used at various stages to predict the prognosis. However, further studies are required on this topic extensively to know the exact cut-off values of these markers to predict the prognosis accurately.
Clinical trials registry number:
REF/2020/01/030616.
... Controlling the cellular and tissue function through various methodologies have been explored from time immemorial [1][2][3][4][5]. Such control over cells and tissues are required in order to treat various ailments that humans face and to facilitate the healing and regeneration of various tissues and organs such as skin, bone, heart, liver and so on, to provide a better quality of life. ...
Sensing the mechanical properties of the substrates or the matrix by the cells and the tissues, the subsequent downstream responses at the cellular, nuclear and epigenetic levels and the outcomes are beginning to get unraveled more recently. There have been various instances where researchers have established the underlying connection between the cellular mechanosignalling pathways and cellular physiology, cellular differentiation, and also tissue pathology. It has been now accepted that mechanosignalling, alone or in combination with classical pathways, could play a significant role in fate determination, development, and organization of cells and tissues. Furthermore, as mechanobiology is gaining traction, so do the various techniques to ponder and gain insights into the still unraveled pathways. This review would briefly discuss some of the interesting works wherein it has been shown that specific alteration of the mechanical properties of the substrates would lead to fate determination of stem cells into various differentiated cells such as osteoblasts, adipocytes, tenocytes, cardiomyocytes, and neurons, and how these properties are being utilized for the development of organoids. This review would also cover various techniques that have been developed and employed to explore the effects of mechanosignalling, including imaging of mechanosensing proteins, atomic force microscopy (AFM), quartz crystal microbalance with dissipation measurements (QCMD), traction force microscopy (TFM), microdevice arrays, Spatio-temporal image analysis, optical tweezer force measurements, mechanoscanning ion conductance microscopy (mSICM), acoustofluidic interferometric device (AID) and so forth. This review would provide insights to the researchers who work on exploiting various mechanical properties of substrates to control the cellular and tissue functions for tissue engineering and regenerative applications, and also will shed light on the advancements of various techniques that could be utilized to unravel the unknown in the field of cellular mechanobiology.
Graphical Abstract
... The extracellular matrices (ECMs) are complex networks of biomolecules surrounding cells, providing mechanical supports and biochemical cues to cells, and affecting their morphology, movement, and fate [ 1 , 2 ]. Biochemically, proteins and glycosaminoglycans (GAG) are the major constituents [1] . Mechanically, ECM is viscoelastic material mainly composed of soft polymer network and stiff collagen fibril. ...
Extracellular matrix (ECM) provides various types of direct interactions with cells and a dynamic environment, which can be remodeled through different assembly/degradation mechanisms to adapt to different biological processes. Herein, through introducing polyphosphate-modified hyaluronic acid and bioactive glass (BG) nano-fibril into a self-assembled hydrogel system with peptide-polymer conjugate, we can realize many new ECM-like functions in a synthetic polymer network. The hydrogel network formation is mediated by coacervation, followed by a gradual transition of peptide structure from α-helix to β-sheet. The ECM-like hydrogels can be degraded through a number of orthogonal mechanisms, including treatments with protease, hyaluronidase, alkaline phosphatase, and calcium ion. As 2D coating, the ECM-like hydrogels can be used to modify the planar surface to promote the adhesion of mesenchymal stromal cells, or to coat the cell surface in a layer-by-layer fashion to shield the interaction with the substrate. As ECM-like hydrogels for 3D cell culture, the system is compatible with injection and cell encapsulation. Upon incorporating fragmented electrospun bioactive glass nano-fibril into the hydrogels, the synergetic effects of soft hydrogel and stiff reinforcement nanofibers on recapitulating ECM functions result in reduced cell circularity in 3D. Finally, by injecting the ECM-like hydrogels into mice, gradual degradations over a time period of one month and high biocompatibility have been shown in vivo. The contribution of complex network dynamics and hierarchical structures to cell-biomatrix interaction can be investigated multi-dimensionally, as many mechanisms are orthogonal to each other and can be regulated individually. STATEMENT OF SIGNIFICANCE: A list of native ECM features has attracted the most interest and attention in the research of synthetic biomaterials. In this research, we have described a simple ECM-like hydrogel system in which the complex and elegant functions of native ECM can be recapitulated in a chemically defined synthetic system. The ECM-like hydrogel systems were developed to avoid undesired features of biological substances (e.g., ethical concerns, batch-to-batch variation, immunogenicity, and potential risk of contamination), as well as gaining new functions to facilitate bioengineering applications (e.g., 3D cell culture, injection, and high stability). To this end, we have developed an ECM-like hydrogel system and provide evidence that this purely synthetic biomaterial is a promising candidate for cell bioengineering applications.
... Взаимодействие «клетка-клетка» и «клетка-внеклеточный матрикс» (ВКМ) определяет морфогенез, путь миграции клеток, развитие или потерю тканеспецифичных функций, что имеет первоочередное значение при компенсации и адаптации ткани к патогенным факторам [16]. Помимо этого, эти процессы влекут за собой изменение гомеостаза, способствуют развитию иммунных заболеваний и опухолевых новообразований [17,18]. ...
Introduction. Osteopathic correction (OC) is aimed to eliminate somatic dysfunctions, which are reversible structural and functional disorders of tissue mobility. Adaptation mechanisms of the organism imply structural
The aim of the study is to investigate the fundamental possibility of using functional (not nosological) autoimmunological indicators to evaluate the results of osteopathic correction.
Materials and methods. The prospective study was conducted on the basis of the Department of Osteopathy of the Mechnikov NWSMU and the Institute of Osteopathy (Saint-Petersburg) in 2020–2021. 10 young and middle-aged people (20–52 years old) were examined. Patients underwent osteopathic correction in the amount of 2–3 sessions. Patients were examined according to the algorithms of osteopathic diagnostics; the ratio of antibody titers to 24 autoantigens of various body tissues and organs was evaluated using the ELI-Viscero-Test-24 method before and after the OC courses.
Results. The relative content of autoantibodies to the main connective tissue protein collagen significantly ( p =0,037) increased from a median value of 6 % ( Q 1– Q 3 2–9 %) to 11 % ( Q 1– Q 3 2–22 %). The other autoimmunological indicators varied in different directions.
Conclusion. It is likely that OC triggers the processes of connective tissue restructuring, which are reflected in an increase in the indicators of auto-AT to collagen. Changes in other autoimmunological indicators require more detailed studies on a larger sample.
... Metabolism of ECM Proteolysis of ECM components during physiological and pathological processes is accomplished by the assembly, structural remodeling, correction of overexpression, and release of bioactive fragments and growth factors. [17] In the process of ECM degradation, matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM), and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS) play important roles, [18][19][20] all of which belong to the superfamily of metzincin proteases, a family of multidomain zinc-dependent endo-proteases. Among them, the main enzymes to degrade ECM are MMPs, [21] which can degrade most of the important protein components of ECM, including collagen, fibronectin, elastin, and laminin. ...
Infertility has become a worldwide issue, and many patients cannot benefit from assisted reproductive technologies (ART). The extracellular matrix (ECM) is critical in tissue organization and remodeling. The female reproductive system plays an important role in menstruation, pregnancy, and ovulation and may influence fertility. In addition, ECM has a wide variety of components, good biological properties, and extensive application experience as a biomaterial. In-depth research on the ECM in the female reproductive system and the development and application of ECM-derived materials may provide new ideas for solving infertility problems. This review aimed to summarize the regulation and changes of ECM in the uterus and ovary, and to discuss the progress of research on ECM-derived materials in reproductive tissue engineering. An extensive search in PubMed and Embase was conducted using keywords including extracellular matrix, uterus, ovary, tissue engineering, and material. We are devoted to combining research on ECM-derived materials with clinical practice and intend to provide ideas for solving clinical problems in reproductive medicine.
... Extracellular proteolysis has major functional consequences for remodeling cell surface proteins and the extracellular matrix (ECM). [12][13][14] Alterations in proteolytic systems underlie multiple pathological conditions, including neurodegenerative disorders 15,16 , inflammatory diseases 17 , and cancers 18,19 . Blocking pericellular proteolysis will provide insights into proteolysis-regulated (which was not certified by peer review) is the author/funder. ...
Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type (WT) POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro and catalytic domains for four different matrix metalloproteases (MMPs), such as MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impairs cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2, that is known to transform it from a tumor suppressor to an oncoprotein. We believe the EDS method greatly facilitates the generation antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.
Significance
We have developed a highly efficient platform to facilitate the directed selection in vitro of antibodies to a wide range of functional epitopes on proteins. This method uses a bioinformatic program to guide mutations in the local site of interest to create a decoy antigen that can effectively remove antibodies not binding the site of interest by negative selection, followed by positive selection with the WT antigen to identify antibodies to the epitope of interest. We demonstrate the generality and versatility of this method by successfully producing functional antibodies to block specific proteolytically sensitive epitopes on five different proteins including enzymes important in cancer. The epitope-directed selection (EDS) approach greatly facilitates the identification of binders to specific sites of interest on proteins to probe function and as potential immunotherapeutics.
... However, DPP4 participates with other cell surface proteases in malignant transformation and cancer progression by facilitating invasion [35][36][37], as was first suggested by Werb [38]. In this sense, the knowledge acquired in recent years about cancer stem cells (CSCs) is remarkable, as is the fact that subsets of CSCs expressing CD26 have been implicated in metastases of many cancers [13,31,39], including haematological (leukemic stem cells or LSCs, reviewed in [34]). ...
This Special Issue presents new knowledge on the complex behaviour of dipeptidyl peptidase 4 (DPP4, EC 3 [...]
... Among their many roles, proteases activate enzymes by removing inhibitory domains, release cytokines, initiate or repress signal transduction, and modulate cell adhesion. 8 As a result, aberrant proteolysis contributes to many pathological states including inflammatory diseases and most cancers. 9 Interrogating the biological roles of extracellular proteases requires knowledge of cleavage events, and many of these remain either ill-defined or uncharacterized. ...
Proteolytic cleavage of cell surface proteins triggers critical processes including cell-cell interactions, receptor activation, and shedding of signaling proteins. Consequently, dysregulated extracellular proteases contribute to malignant cell phenotypes including most cancers. To understand these effects, methods are needed that identify proteolyzed membrane proteins within diverse cellular contexts. Herein we report a proteomic approach, called cell surface N-terminomics, to broadly identify precise cleavage sites (neo-N-termini) on the surface of living cells. First, we functionalized the engineered peptide ligase, called stabiligase, with an N-terminal nucleophile that enables covalent attachment to naturally occurring glycans. Upon the addition of a biotinylated peptide ester, glycan-tethered stabiligase efficiently tags extracellular neo-N-termini for proteomic analysis. To demonstrate the versatility of this approach, we identified and characterized 1532 extracellular neo-N-termini across a panel of different cell types including primary immune cells. The vast majority of cleavages were not identified by previous proteomic studies. Lastly, we demonstrated that single oncogenes, KRAS(G12V) and HER2, induce extracellular proteolytic remodeling of proteins involved in cancerous cell growth, invasion, and migration. Cell surface N-terminomics is a generalizable platform that can reveal proteolyzed, neoepitopes to target using immunotherapies.
... Plasmin can also activate PAR2, thereby modulating endothelial function (47). Finally, plasmin activates several matrix metalloproteases (48)(49)(50), which in turn, may impact on inflammation, hemostasis and tissue remodeling (51). Not surprisingly, plasmin has direct effects on complement activation, which will be further discussed below. ...
In 1969, Dr. Oscar Ratnoff, a pioneer in delineating the mechanisms by which coagulation is activated and complement is regulated, wrote, “In the study of biological processes, the accumulation of information is often accelerated by a narrow point of view. The fastest way to investigate the body’s defenses against injury is to look individually at such isolated questions as how the blood clots or how complement works. We must constantly remind ourselves that such distinctions are man-made. In life, as in the legal cliché, the devices through which the body protects itself form a seamless web, unwrinkled by our artificialities.” Our aim in this review, is to highlight the critical molecular and cellular interactions between coagulation and complement, and how these two major component proteolytic pathways contribute to the seamless web of innate mechanisms that the body uses to protect itself from injury, invading pathogens and foreign surfaces.
... q/"MMP acrzvi(y -The cell surface of many cells is the location of many events that mediate cell behaviour. Many of these events are created or cancelled via pericellular proteolysis (Werb 1997). SpeciGc means exist that facilitate the colocalisation of MMPs with their substrate. ...
p>The aim of this study was to determine the effect of TIMP-1 and TIMP-2 on HSC survival and determine whether this effect was through effects on inhibition of MMP activity. In vivo studies of experimental liver fibrosis demonstrated a correlation between resolution of fibrosis, falling, TIMP-1 mRNA, loss of HSC and transient increase in collagenolytic activity. This data suggested that TIMP-1 was likely to have an effect promoting HSC survival. In vitro studies of culture activated HSC demonstrated that neither TIMP-1 nor TIMP-2 regulated HSC proliferation suggesting that the pro survival effect was likely to be via inhibition of apoptosis. Incubation with TIMP-1 or TIMP-2 significantly reduced apoptosis of HSC induced by a number of stimuli in a dose-dependent manner. Neutralising antibodies to TIMP-1 and TIMP-2 increased HSC apoptosis compared non-immune IgG control. Whilst both a synthetic selective inhibitor of MMP-2 and a broad spectrum synthetic MMP inhibitor reduced HSC apoptosis, a non-functional mutated TIMP-1 (T2G mutant) had no effect indicating that the inhibition of apoptosis by TIMP-1 was via inhibition of MMP activity. MMP-2 like TIMP-1 is expressed by activated HSC. Addition of recombinant active MMP-2 to HSC resulted in significantly enhanced apoptosis and was associated with cleavage of N-cadherin which could be reduced by co-incubation with recombinant TIMP-1 but not by the non-functional T2G mutant TIMP-1. Furthermore, selective MMP-2 inhibitors protected N-cadherin from cleavage. Active MMP-2 cleaves intact N-cadherin into similar sized fragments in vitro. Blockade of N-cadherin binding with blocking antibody or HAVDI blocking peptide promoted HSC apoptosis, suggesting N-cadherin is a potential target mechanism for the regulation of survival and apoptosis of HSC via active MMP-2 mediated cleavage. Finally, to determine if effects on apoptosis by TIMP-1 were through changes in cell-matrix interactions, HSC plated onto a mutated collagen that is resistant to degradation by MMP-2 were more resistant to apoptosis induced by active MMP-2 than HSCs plated onto normal wild type collagen-1.</p
... A 2 nd TIMP binding site (not specific for TIMP-2) becomes available on cleavage of the pro-fragment during enzyme activation and is responsible for the MMPs deactivation. Reproduced from Yu AE et al, 1997.Section 1.13 The Transmembrane Matrix Metalloproteinases (MT -MMPs)These are the membrane bound MMPs (MT-MMPs), which have been shown to play an important role, in the activation ofMMPs(Sato H et aI, 1994;Werb Z, 1997; Knauper V et aI, 1996). At present seven MT-MMPs have been identified, with the most widely studied being MT1 -MMP (MMP-14), which activates MMP-2 and 13(Sato H et aI, 1994, Knauper V et aI, 1996). ...
p>We have investigated the hypothesis that a desmoplastic reaction (DR), characterised by the deposition of collagens I and III produced by activated stromal cells, offers Colorectal Cancer (CRC) liver metastases a growth and survival advantage.
Immunohistochemical staining of liver specimens obtained at resection for CRC, demonstrated increased deposition of fibrillar collagens and alterations in collagen IV distribution within the tumour as part of a DR. In addition, the deposited fibrillar collagens were closely associated with increased numbers of activated hepatic stellate cells/myofibroblasts. Β1 integrins were highly expressed by both cancer and stromal cells throughout the tumour stroma. However, in poorly differentiated areas of the CRC metastases β1 integrins appeared to be down-regulated, with αv integrin (especially αbβ5) expression upregulated.
Clonogenic (survival) and PARP cleavage (apoptosis) assays, showed that collagen I compared to collagen IV significantly increased the survival and reduced the rate of cellular apoptosis for CRC treated with chemotherapy (5-Fluorouracil). The adhesion and proliferation of CRC cells on collagens I and IV was significantly reduced in a dose dependent manner, after incubation with β1 integrin neutralising antibodies (5-10μg/ml), compared to IgG controls. In contrast, αvβ3 and αvβ5 neutralising antibodies (5-20μg.ml), had no influence on the CRC cell adhesion, but significantly reduced the rate of proliferation of the CRC cell lines on collagens, especially for the highly metastatic KM12SM cell line.
These results support a role for the desmoplastic reaction in supporting CRC metastases, mediated via β1 and αv integrins. As CRC adopt a more aggressive malignant phenotype, matrix turnover reveals specific binding epitopes which upon ligation by αv integrins plays a key growth regulatory role.</p
... The study of growth regulation by extracellular matrix is potentially complicated by a number of factors that have been controlled for where possible. Peri-cellular proteolysis is a process whereby proteases expressed by cells lead to matrix remodelling around cells (Liotta and Kohn, 2001;Werb, 1997). This may have 2 consequences. ...
p>Pancreatic cancer (PC) is characterised by a profound desmoplastic reaction, which contains fibrillar collagen (collagen type I and III).
Histological examination of PCs using immunohistochemistry demonstrated gross distortion of structure of the normal pancreas. The glandular structures of the normal pancrease (surrounding by type IV collagen) were altered in PC, with wide bands of fibrotic stroma (containing type I collagen) separating the malignant glands. Numerous myofibroblastic pancreatic stellate cells (PSC) were identified throughout the desmoplastic reaction, often closely associated with cancer cells.
Potential interaction between PSC and cancer cells were tested in vitro using primary cultures of human PSC and pancreatic cancer cell lines (MIA PaCa-2, Panc-1 and AsPC-1). Conditioned media from each PC cell line stimulated proliferation of PSC. PSC secreted collagen in excess of the cancer cells. Furthermore conditioned media from AsPC-1 cells increased PSC procollagen 1 gene expression and collagen secretion (which correlated with TGFβ<sub>1</sub> expression). PSC expressed tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) at high levels (compared to matrix metalloprotease-2) thus promoting the accumulation of extracellular matrix.
There is a detrimental interaction between pancreatic cancer cells and PSC that stimulates formation of the desmoplastic reaction abundant in type I collagen. In turn, type I collagen promotes growth of malignant cells in pancreatic cancer. This contributes to the understanding of the origin of the desmoplastic reaction by defining the interaction between pancreatic cancer cells and primary cultures of human PSC. Moreover, it demonstrates that the consequence of these interactions is deleterious to the host.</p
... The SP one turned out to be the most appropriate scaffold for OS cell adhesion, spreading and proliferation (Figs. 2 and 3) in a 3D culture manner of multi-layer cells ( Figure S7). As there were not significant differences in surface characterization among three scaffolds before cell incubation ( Figure S8), the diverse results were possibly due to the large micropores between the microfibers that induced rapid diffusion of nutrients [76] in the case of MP or LP. Then surface roughness, hydrophilicity and biological activity could be further improved by modifying the surface with a PDA layer without significant changes of the mechanical and structural properties (Fig. 4), in agreement with previous publication of our group [77] and Chen et al. [34]. ...
The pronounced influence of extracellular matrix (ECM) on bone tumor progression and metastasis have gained increased attention, which is difficult to reflect in current in vitro methods including 2D cell culture and 3D tumor spheroids. Thus, novel osteosarcoma (OS) models are highly requested to recreate in vivo complexity of bone niche in an in vitro prototype. Herein a type of poly(L-lactide) scaffold-based OS model was established by 3D-printing technique to obtain similar mechanical and structural properties as native cortical bones where OS occurred. The cultured OS cells exhibited significant differences in morphology, proliferation, cytoskeleton reorganization and energy expenditure, comparing with traditional 2D monolayers and 3D spheroids. Moreover, the 3D scaffold-based OS model revealed a remarkable potential of ECM remodeling in the expression of ECM components, recognizing receptors and growth factors. The transcriptomic analysis was then utilized to unravel distinct signaling pathways and molecules in the 3D scaffold-based OS model. Further investigation of comparison with clinical trials suggested that the predictive biomarkers found in patient biopsy samples revealed similar expression patterns in the 3D scaffold-based OS model. The proposed OS model highlighted the feasibility of recapitulating tumor niche, and it would have broad applicability for mechanistic studies of OS development, targeted therapy and drug screening.
... There are many different types of collagen and corresponding communication receptors in the maternal-fetal interface, including collagen type I, III, IV [18]. Collagen and other ECM components hydrolysis can regulate the assembly of ECM, correct the excess expression of proteins in ECM and reshape its structure, and play the role of releasing biologically active fragments and growth factors in the process of growth, morphological change, tissue repair and pathological change [19]. Matrix metalloproteinases (MMPs), A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) are zinc-dependent endopeptidases that play a critical role in the destruction of extracellular matrix proteins [20]. ...
Extracellular matrix (ECM) is characterized as widespread, abundant, and pluripotent. Among ECM members, collagen is widely accepted as one of the most prominent components for its essential structural property that can provide a scaffold for other components of ECM and the rich biological functions, which has been extensively used in tissue engineering. Emerging evidence has shown that the balance of ECM degradation and remodeling is vital to regulations of maternal–fetal interface including menstrual cycling, decidualization, embryo implantation and pregnancy maintenance. Moreover, disorders in these events may eventually lead to failure of pregnancy. Although the improvement of assisted conception and embryo culture technologies bring hope to many infertile couples, some unfavorable outcomes, such as recurrent implantation failure (RIF), recurrent pregnancy loss (RPL) or recurrent miscarriage (RM), keep troubling the clinicians and patients. Recently, in vitro three-dimensional (3D) model mimicking the microenvironment of the maternal–fetal interface is developed to investigate the physiological and pathological conditions of conception and pregnancy. The progress of this technology is based on clarifying the role of ECM in the endometrium and the interaction between endometrium and conceptus. Focusing on collagen, the present review summarized the degradation and regulation of ECM and its role in normal menstruation, endometrium receptivity and unsatisfying events occurring in infertility treatments, as well as the application in therapeutic approaches to improve pregnancy outcomes. More investigations about ECM focusing on the maternal–fetal interface interaction with mesenchymal stem cells or local immunoregulation may inspire new thoughts and advancements in the clinical application of infertility treatments.
Graphical abstract
... SERPINE1 inhibits MMPs, thereby limiting the degradation of collagen and other ECM molecules, as well as repressing plasmin, which converts the inactive pro-MMP to active MMP. 272 In patients with AAA, the 4G allele of the SERPINE1 SNP was underrepresented, causing the effect of lower levels of SERPINE1 to inhibit MMPs. 273,274 The only study to date examining this SNP in an intracranial aneurysm population was conducted by Ruigrok and colleagues who demonstrated an association of this SNP, in 382 Dutch Caucasian patients. ...
Introduction: Intracranial aneurysms affect 2-5% of the general population.1 A tiny proportion of these rupture leading to aneurysmal subarachnoid haemorrhage (SAH), which can be fatal instantly or lead to life-changing neurological morbidity in a young group of stroke survivors. Understanding the pathophysiological mechanisms underlying intracranial aneurysms, and being able to predict which patients have a higher risk of rupture has led to intensive research efforts, via epidemiological, molecular and genetic studies. Our aim was to determine the association of candidate genes with intracranial aneurysms in a large UK Caucasian population. Methods: We performed a case-control genetic association study of single nucleotide polymorphisms (SNPs) in over 1600 patients with intracranial aneurysms from a UK-wide Genetic and Observational Subarachnoid Haemorrhage (GOSH) study and 1500 controls from the Wellcome cohort,2 utilising a candidate-gene approach. We conducted a literature review and performed a meta-analysis of the existing candidate gene studies to better determine which genes would be suitable for analysis in our cohort. We also performed a new meta-analysis using our data for each SNP examined to determine if our genetic associations with intracranial aneurysms were robust. Results: We examined 22 SNPs related to vascular endothelial integrity, the extracellular matrix, and inflammation. Two SNPs showed associations with intracranial aneurysms in our UK cohort: the D allele of ACE Insertion/Deletion SNP (associated with vascular endothelial function; OR 1.14 [1.02-1.28], p=0.02) and the MMP-2 C>T rs243865 SNP (associated with extracellular matrix integrity; OR 1.18 [1.04 – 1.33], p=0.012). Conclusions: We found associations with intracranial aneurysms for the D allele of the ACE I/D SNP, and a potentially functionally significant MMP-2 SNP. Both genes have plausible connections to IA pathophysiology (endothelial function and extracellular matrix integrity, respectively), and could potentially predispose patient to aneurysm rupture as demonstrated by sub-group analysis.
... The proteolysis occurring in tissues relates not only to the extracellular matrix per se but also concerns the so-called ectodomain shedding, i.e., proteolytic cleavage of cell surface proteins. Modification, degradation, and changes in the activity of these proteins are one of the mechanisms of the cell's response to changes in microenvironment conditions [177,178]. Enzymes of the ADAM (a disintegrin and metalloproteases) family, also known as adamalysins, are mainly involved in this process. They have various functions, primarily engaged in intercellular interactions and signal transduction [19, 179,180]. ...
The existence of orderly structures, such as tissues and organs is made possible by cell adhesion, i.e., the process by which cells attach to neighbouring cells and a supporting substance in the form of the extracellular matrix. The extracellular matrix is a three-dimensional structure composed of collagens, elastin, and various proteoglycans and glycoproteins. It is a storehouse for multiple signalling factors. Cells are informed of their correct connection to the matrix via receptors. Tissue disruption often prevents the natural reconstitution of the matrix. The use of appropriate implants is then required. This review is a compilation of crucial information on the structural and functional features of the extracellular matrix and the complex mechanisms of cell–cell connectivity. The possibilities of regenerating damaged tissues using an artificial matrix substitute are described, detailing the host response to the implant. An important issue is the surface properties of such an implant and the possibilities of their modification.
... To invade the surrounding healthy tissue, a tumour must overcome the defences developed by the body to maintain homeostatic control. An important barrier to tumour invasion is the ECM, which is a strong scaffold of proteins that holds tissue cells in place and initiates signalling pathways for cellular processes such as migration, differentiation and proliferation [6,7]. The healthy cells encased by the ECM form another barrier to invasion by creating a competitive environment for the tumour cells. ...
In this paper, we carry out a travelling-wave analysis of a model of tumour invasion with degenerate, cross-dependent diffusion. We consider two types of invasive fronts of tumour tissue into extracellular matrix (ECM), which represents healthy tissue. These types differ according to whether the density of ECM far ahead of the wave front is maximal or not. In the former case, we use a shooting argument to prove that there exists a unique travelling-wave solution for any positive propagation speed. In the latter case, we further develop this argument to prove that there exists a unique travelling-wave solution for any propagation speed greater than or equal to a strictly positive minimal wave speed. Using a combination of analytical and numerical results, we conjecture that the minimal wave speed depends monotonically on the degradation rate of ECM by tumour cells and the ECM density far ahead of the front.
... Two such limitations include absence of reactive ligands on PCL scaffolds, which leads to ineffective cell adhesion and down-stream cellular events, [7] and the large micropores between the scaffold microfibers that cause rapid diffusion of nutrients. [10] In order to solve these problems, peptide molecules or extracellular matrix (ECM) proteins are widely used in PCL surface modification by covalent or noncovalent interactions. [11][12][13] Yet, single noncovalent bonding methods usually lead to a quick bulk release of the peptides due to the relatively weak interactions, while covalent interaction methods involve complex chemical reactions along with nondegradable by-products and may also cause decreased diffusion rate of the peptides. ...
Clinically, cartilage damage is frequently accompanied with subchondral bone injuries caused by disease or trauma. However, the construction of biomimetic scaffolds to support both cartilage and subchondral bone regeneration remains a great challenge. Herein, a novel strategy is adopted to realize the simultaneous repair of osteochondral defects by employing a self‐assembling peptide hydrogel (SAPH) FEFEFKFK (F, phenylalanine; E, glutamic acid; K, lysine) to coat onto 3D‐printed polycaprolactone (PCL) scaffolds. Results show that the SAPH‐coated PCL scaffolds exhibit highly improved hydrophilicity and biomimetic extracellular matrix (ECM) structures compared to PCL scaffolds. In vitro experiments demonstrate that the SAPH‐coated PCL scaffolds promote the proliferation and osteogenic differentiation of rabbit bone mesenchymal stem cells (rBMSCs) and maintain the chondrocyte phenotypes. Furthermore, 3% SAPH‐coated PCL scaffolds significantly induce simultaneous regeneration of cartilage and subchondral bone after 8‐ and 12‐week implantation in vivo, respectively. Mechanistically, by virtue of the enhanced deposition of ECM in SAPH‐coated PCL scaffolds, SAPH with increased stiffness facilitates and remodels the microenvironment around osteochondral defects, which may favor simultaneous dual tissue regeneration. These findings indicate that the 3% SAPH provides efficient and reliable modification on PCL scaffolds and SAPH‐coated PCL scaffolds appear to be a promising biomaterial for osteochondral defect repair.
... In native tissue, the crosslinking of ECM proteins has been processed by enzyme-mediated reactions. 3 The enzyme in the living system is a protein that mainly acts as a natural catalyst. Chemical reactions in the living system depend on enzyme activity. ...
Hydrogel system based on enzyme‐mediated mild crosslinking reaction has been a promising approach in tissue engineering. Inspired by skin melanin synthesis and marine mussel adhesion, tyrosinase‐mediated hydrogel crosslinking has been exploited as cell‐friendly reactions and explicit reaction mechanisms. Hydrogel prepared by tyrosinase exhibits appealing properties as a dynamic scaffold for cell delivery and as a bioink for 3D bioprinting. Recapitulating the structure of the native extracellular matrix (ECM), innovative tyrosinase‐mediated hydrogel crosslinking has now shifted to the field of translational medicine. Biomimetic hydrogel with in situ tyrosinase crosslinking can be efficiently and easily applicable to the disease model for therapeutic purposes. In this review, we will discuss a broad range of tyrosinase‐mediated tissue engineering from the specific mechanism of tyrosinase reaction and designing a strategy for tyrosinase‐mediated hydrogel crosslinking to dynamic applications in tissue engineering. In addition, we report the current challenges and future perspectives for the translational applications.
... Previous studies have demonstrated that TGF-β1 mediated tumor cell EMT and metastasis through the MAPK pathway [15]. Furthermore, tumor cells can migrate and invade target organs by expressing matrix metalloproteinases (MMPs), such as MMP-2, to degrade the extracellular matrix (ECM) proteins [16]. These studies may reveal that clarifying the relationship between EMT and MAPK signaling pathways may play a crucial role in PTC metastasis. ...
Background:
Peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligands have been widely shown to correlate with epithelial-mesenchymal transition (EMT) and cancer progression. Lobeglitazone (LGZ) is a novel ligand of PPAR-γ; and its role in EMT and metastasis in papillary thyroid carcinoma (PTC) is poorly understood. We aimed to investigate the role of LGZ in metastatic behavior of PTC cells.
Methods:
Half maximal inhibitory concentration (IC50) values of LGZ in BRAF-mutated PTC cell lines (BCPAP and K1) were determined using MTT assay. Rosiglitazone (RGZ), the PPAR-γ ligand was used as a positive control. The protein expression of PPAR-γ, cell-surface proteins (E-cadherin, N-cadherin), cytoskeletal protein (Vimentin), transcription factor (Snail), p38 mitogenactivated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 pathway, and matrix metalloproteinase (MMP)-2 expression were measured using Western blotting. Changes in E-cadherin expression were also determined using immunocytochemistry. Cell migration and invasion were analyzed using wound healing and Matrigel invasion assays.
Results:
Treatment with LGZ or RGZ significantly inhibited transforming growth factor-beta1 (TGF-β1)-induced EMT-associated processes such as fibroblast-like morphological changes, EMT-related protein expression, and increased cell migration and invasion in BCPAP and K1 cells. LGZ restored TGF-β1-induced loss of E-cadherin, as observed using immunocytochemistry. Furthermore, LGZ and RGZ suppressed TGF-β1-induced MMP-2 expression and phosphorylation of p38 MAPK, but not ERK1/2. Although there was no change in PPAR-γ expression after treatment with LGZ or RGZ, the effect of downstream processes mediated by LGZ was hampered by GW9662, a PPAR-γ antagonist.
Conclusion:
LGZ inhibits TGF-β1-induced EMT, migration, and invasion through the p38 MAPK signaling pathway in a PPAR-γ-dependent manner in PTC cells.
... One limitation of PCL scaffolds is the absence of a bioactive component, which leads to ineffective cell adhesion and growth and stimulation of downstream cellular events [8]. Another limitation comes from the limited resolution, which makes it difficult to construct cell-friendly nanostructures for nutrient retention alone [9]. ...
Regenerating the meniscus remains challenging because of its avascular, aneural, and alymphatic nature. Three-dimensional (3D) printing technology provides a promising strategy to fabricate biomimetic meniscal scaffolds with an anisotropic architecture, a proper biomechanical microenvironment, and bioactive components. Herein, 3D printing technology is adopted by coencapsulating chemokines (platelet-derived growth factor-BB, PDGF-BB) and small chondroinductive molecules (kartogenin, KGN) within biomimetic polycaprolactone (PCL)/hydrogel composite scaffolds. The incorporated PDGF-BB is expected to promote endogenous stem cell homing, and KGN in poly(lactic-co-glycolic) acid (PLGA) microspheres is employed to target the chondrogenesis of resident mesenchymal stem cells (MSCs). First, we chose basic bioinks composed of gelatin methacrylamide (GelMA) and hyaluronic acid methacrylate (HAMA) and then incorporated four concentrations (0%, 0.5%, 1.0%, and 2.0%) of meniscal extracellular matrix (MECM) into the bioink to systematically study the superiority of these combinations and identify the optimally printable bioink. Next, we investigated the scaffold morphology and drug release profile. The effects of releasing the drugs in a sequentially controlled manner from the composite scaffolds on the fate of MSCs were also evaluated. The biofabricated scaffolds, with and without dual drug loading, were further studied in a rabbit model established with a critical-size medial meniscectomy. We found that meniscal scaffolds containing both drugs had combinational advantages in enhancing cell migration and synergistically promoted MSC chondrogenic differentiation. The dual drug-loaded scaffolds also significantly promoted in vivo neomeniscal regeneration 3 and 6 months after implantation in terms of histological and immunological phenotypes. The results presented herein reveal that this 3D-printed dual drug-releasing meniscal scaffold possesses the potential to act as an off-the-shelf product for the clinical treatment of meniscal injury and related joint degenerative diseases.
... Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that control the degradation of a wide spectrum of extracellular matrix-associated (ECM) and cell surface-associated proteins. MMPs (MMP1-28) are widely expressed during tumor progression and metastasis [4,5]. The influence of MMP7 and MMP9 on RCC metastasis has been previously observed. ...
The occurrence of metastasis is a serious risk for renal cell carcinoma (RCC) patients. In order to develop novel therapeutic approaches to control the progression of metastatic RCC, it is of urgent need to understand the molecular mechanisms underlying RCC metastasis and identify prognostic markers of metastatic risk. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been known to be closely associated with extracellular matrix (ECM) turnover, which plays a highly active role in tumor metastasis. Recent studies have shown that immunophilin FK-506-binding protein 51 (FKBP51) may be important for the regulation of ECM function, and exert effects on the invasion and migration of tumor cells. However, the mechanisms underlying these activities remain unclear. The present study detected the role of FKBP51 in clear cell renal cell carcinoma (ccRCC), the most common subtype of RCC, and found that FKBP51 significantly promotes ccRCC invasion and migration by binding with the TIMP3, connecting TIMP3 with Beclin1 complex and increasing autophagic degradation of TIMP3. Given the important roles that TIMPs/MMPs play in ECM regulation and remodeling, our findings will provide new perspective for future investigation of the regulation of metastasis of kidney cancer and other types of cancer.
... [13] Typically, the activity of MMPs is tightly regulated by inhibitors like tissue inhibitors of metalloproteinases (TIMPs) and α2-macroglobulin which are endogenous tissue-specific and circulating inhibitors, respectively. [14] Based on the sequence homology and substrate specificity, [15] MMPs can be grouped as (a) collagenases -MMP-1, MMP-8, MMP-13, and MMP-18, (b) gelatinases -MMP-2 Periodontitis can be defined as "an inflammation of the periodontal tissues resulting in clinical attachment loss, alveolar bone loss, and periodontal pocketing." [18] Various studies over the decade have shown an association between levels of MMPs and periodontal tissue destruction. ...
Background:
Matrix metalloproteinases (MMPs) are a group of host-derived zinc-dependent enzymes which mediates the destruction of the extracellular matrix. In periodontitis, there is excess production of MMPs associated with periodontal tissue destruction. The aim of this study was to estimate the level MMP-9 in both active and latent form in gingival tissue (GT) samples collected from periodontitis patients with different rates of progression and compare it with healthy individuals.
Materials and methods:
Sixty patients were selected and divided into three groups, 20 each: Group A (slowly/moderately progressing periodontitis), Group B (rapidly progressing periodontitis), and Group C (clinical periodontal health). Plaque index, gingival index, periodontal probing depth (PPD), and clinical attachment level were recorded. GT samples were collected from all 60 patients and MMP-9 expressions were measured using gelatin zymography and western blotting.
Results:
Levels of active MMP-9 (aMMP-9) and latent MMP-9 (lMMP-9) were significantly high in both Group A (GA) (aMMP-9: 2.05 arbitrary unit [AU]/lMMP-9: 2.54 AU) and Group B (GB) (aMMP-9: 1.32 AU/lMMP-9: 1.74 AU) when compared to that of Group C (GC) (aMMP-9: 0.93/lMMP-9: 1.08 AU). In GA, levels of aMMP-9 showed a significant correlation with PPD values. No other correlations were found.
Conclusion:
The levels of aMMP-9 and lMMP-9 were increased in both the types of periodontitis when compared with periodontally healthy individuals. A significant correlation was found between PPD and activities of aMMP-9 in slowly/moderately progressing periodontitis patients. However, further studies are required to confirm these findings.
... Cell invasion is a complex process that requires controlled degradation of the extracellular matrix (ECM) that is achieved by proteases such as serine proteases and matrix metalloproteinases (MMPs) [1,2]. The MMPs are large family of zinc-dependent proteases that are produced as inactive latent forms and converted into their active forms by removal of N-terminal propeptides [3]. ...
Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.
... To invade the surrounding healthy tissue, a tumour must overcome the defenses developed by the body to maintain homeostatic control. An important barrier to tumour invasion is the ECM, a strong scaffold of proteins that holds tissue cells in place and initiates signalling pathways for cellular processes such as migration, differentiation and proliferation [33,34]. The healthy cells encased by the ECM form another barrier to invasion by creating a competitive environment for the tumour cells. ...
In this paper, we carry out a travelling-wave analysis of a model of tumour invasion with degenerate, cross-dependent diffusion. We consider two types of invasive fronts of tumour tissue into extracellular matrix (ECM), which represents healthy tissue. These types differ according to whether the density of ECM far ahead of the wave front is maximal or not. In the former case, we use a shooting argument to prove that there exists a unique travelling wave solution for any positive propagation speed. In the latter case, we further develop this argument to prove that there exists a unique travelling wave solution for any propagation speed greater than or equal to a strictly positive minimal wave speed. Using a combination of analytical and numerical results, we conjecture that the minimal wave speed depends monotonically on the degradation rate of ECM by tumour cells and the ECM density far ahead of the front.
... As a member of zinc endopeptidases, matrix metalloproteases (MMPs) are well-known of degrading extracellular matrix such as collagen, fibronectin, and laminin [5,6], are necessary for rebuilding of wound healing, tissue morphogenesis and extracellular matrix [7]. However, it is detrimental that the over-expression and excessive proteolytic activity of MMPs result in various pathological conditions including the disruption of blood brain barrier or BSCB integrity following SCI and ischemic brain injury [8,9]. ...
As a serious trauma of the neurological system, spinal cord injury (SCI) results in permanent disability, gives rise to immediate vascular damage and a wide range of matters that induce the breakage of blood spinal cord barrier (BSCB). SCI activates the expression of MMP-2/9, which are considered to accelerate the disruption of BSCB. Recent research shows that Dl-3-n-butylphthalide (NBP) exerted protective effects on blood spinal cord barrier in animals after SCI, but the underlying molecular mechanism of NBP on the BSCB undergoing SCI is unknown. Here, our research show that NBP inhibited the expression of MMP-2/9, then improved the permeability of BSCB following SCI. After the T9 level of spinal cord performed with a moderate injury, NBP was managed by intragastric administration and further performed once a day. NBP remarkably improved the permeability of BSCB and junction proteins degration, then promoted locomotion recovery. The protective effect of NBP on BSCB destruction is related to the regulation of MMP-2/9 induced by SCI. Moreover, NBP obviously inhibited the MMP-2/9 expression and junction proteins degradation in microvascular endothelial cells. In conclusion, our results indicate that MMP-2/9 are relevant to the breakdown of BSCB, NBP impairs BSCB destruction through inhibiting MMP-2/9 and promotes functional recovery subjected to SCI. NBP is likely to become a new nominee as a therapeutic to treat SCI via a transigent BSCB.
... The degradation of the ECM proteins such as fibronectin has been shown to affect the apoptotic program of ECs and epithelial cells. [27][28][29][30] Another important element in EC survival is the adherens junction, in which VE-cadherin anchors the cytoskeleton of neighboring cells via or ␥-catenin. [31][32][33] Both the degradation of VE-cadherin and -catenin have been observed during apoptosis, implicating their role in apoptosis. ...
Background— The forkhead factor, FOXO3a, is known to induce apoptosis in endothelial cells (ECs). However, its effects on extracellular matrices (ECM), which are important in EC survival, remained unknown. Here, we evaluated the role of FOXO3a on EC-ECM interaction. Methods and Results— Constitutively active FOXO3a was transduced to human umbilical vein endothelial cells by adenoviral vector (Ad-TM-FOXO3a). Ad-TM-FOXO3a transfection led to dehiscence of ECs from fibronectin-coated plates, resulting in anoikis, which was significantly reversed by matrix metalloproteinase (MMP) inhibitor, GM6001. FOXO3a increased the expression of MMP-3 (stromelysin-1) but decreased the expression of tissue inhibitors of metalloproteinases-1 (TIMP-1), which was associated with increased MMP enzymatic activity in zymography. Pathophysiologic conditions such as serum starvation or heat shock also induced activation of endogenous FOXO3a, leading to activation of MMP-3 and apoptosis, which was reversed by GM6001. Delivery of Ad-TM-FOXO3a to the intraluminal surface in vivo led to EC denudation, disrupted vascular integrity, and impaired endothelium-dependent vasorelaxation. Conclusion— Activation of MMPs and possible ECM disruption represent novel mechanisms of FOXO3a-mediated apoptosis in ECs.
... This finding was consistent with a study on human DP spheres [9]. ECM serves an important role in tissue and organ morphogenesis and in the maintenance of cell and tissue structure and function [65]. In natural hair development, ECM is produced far before hair follicle morphogenesis by pre-follicular tissue. ...
The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer (Cervus nippon) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.
... This inactive form enables the spatial and temporary activation of TGF-β for diverse physiological needs. Several TGF-β activators have been identified to date, most of which are markers of extracellular matrix (ECM) disorders, such as MMP, 70 plasmin, 71 and thrombospondin-1 (TSP1), 72 as well as an appropriate pH. 73 TGF-β1 is abundant in the bone matrix, cartilage and skin, where it regulates the physical activities of these organs or tissues. ...
Osteoarthritis comprises several joint disorders characterized by articular cartilage degeneration and persistent pain, causing disability and economic burden. The incidence of osteoarthritis is rapidly increasing worldwide due to aging and obesity trends. Basic and clinical research on osteoarthritis has been carried out for decades, but many questions remain unanswered. The exact role of subchondral bone during the initiation and progression osteoarthritis remains unclear. Accumulating evidence shows that subchondral bone lesions, including bone marrow edema and angiogenesis, develop earlier than cartilage degeneration. Clinical interventions targeting subchondral bone have shown therapeutic potential, while others targeting cartilage have yielded disappointing results. Abnormal subchondral bone remodeling, angiogenesis and sensory nerve innervation contribute directly or indirectly to cartilage destruction and pain. This review is about bone-cartilage crosstalk, the subchondral microenvironment and the critical role of both in osteoarthritis progression. It also provides an update on the pathogenesis of and interventions for osteoarthritis and future research targeting subchondral bone.
Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here, we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these, we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro- and catalytic domains for three different matrix metalloproteases (MMPs), MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impair cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2 (EphA2), that is known to transform it from a tumor suppressor to an oncoprotein. We believe that the EDS method greatly facilitates the generation of antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications.
The extracellular matrix (ECM) is a highly complex structure through which biochemical and mechanical signals are transmitted. In processes of cell migration, the ECM also acts as a scaffold, providing structural support to cells as well as points of potential attachment. Although the ECM is a well-studied structure, its role in many biological processes remains difficult to investigate comprehensively due to its complexity and structural variation within an organism. In tandem with experiments, mathematical models are helpful in refining and testing hypotheses, generating predictions, and exploring conditions outside the scope of experiments. Such models can be combined and calibrated with in vivo and in vitro data to identify critical cell-ECM interactions that drive developmental and homeostatic processes, or the progression of diseases. In this review, we focus on mathematical and computational models of the ECM in processes such as cell migration including cancer metastasis, and in tissue structure and morphogenesis. By highlighting the predictive power of these models, we aim to help bridge the gap between experimental and computational approaches to studying the ECM and to provide guidance on selecting an appropriate model framework to complement corresponding experimental studies.
Previous work showed that matrix metalloproteinase-7 (MMP-7) regulates colon cancer activities through an interaction with syndecan-2 (SDC-2) and SDC-2-derived peptide that disrupts this interaction exhibits anticancer activity in colon cancer. Here, to identify potential anticancer agents, a library of 1379 FDA-approved drugs that interact with the MMP-7 pro-domain were virtually screened by protein-ligand docking score analysis using the GalaxyDock3 program. Among five candidates selected based on their structures and total energy values for interacting with the MMP-7 pro-domain, the known mechanistic target of rapamycin kinase (mTOR) inhibitor, everolimus, showed the highest binding affinity and strongest ability to disrupt the interaction of the MMP-7 pro-domain with the SDC-2 extracellular domain in vitro. Everolimus treatment of the HCT116 human colon cancer cell line did not affect the mRNA expression levels of MMP-7 and SDC-2, but reduced the adhesion of cells to MMP-7 pro-domain-coated plates and the cell-surface localization of MMP-7. Thus, everolimus appears to inhibit to the interaction between MMP-7 and SDC-2. Everolimus treatment of HCT116 cells also reduced their gelatin-degradation activity and anticancer activities, including colony formation. Interestingly, cells treated with sirolimus, another mTOR inhibitor, triggered less gelatin-degradation activity, suggesting that this inhibitory effect of everolimus was not due to inhibition of the mTOR pathway. Consistently, everolimus inhibited the colony-forming ability of mTOR-resistant HT29 cells. Together, these data suggest that, in addition to inhibiting mTOR signaling, everolimus exerts anticancer activity by interfering with the interaction of MMP-7 and SDC-2, and could be a useful therapeutic anticancer drug for colon cancer.
Glaucoma is a heterogeneous group of progressive diseases that leads to irreversible blindness. Secondary glaucoma refers to glaucoma caused by a known underlying condition. Pseudoexfoliation and pigment dispersion syndromes are common causes of secondary glaucoma. Their respective deposits may obstruct the trabecular meshwork, leading to aqueous humor outflow resistance, ocular hypertension, and optic neuropathy. There are no disease‐specific interventions available for either. Pseudoexfoliation syndrome is characterized by fibrillar deposits (pseudoexfoliative material) on anterior segment structures. Over a decade of multiomics analyses taken together with the current knowledge on pseudoexfoliative glaucoma warrant a re‐think of mechanistic possibilities. We propose that the presence of nucleation centers (e.g., vitamin D binding protein), crosslinking enzymes (e.g., transglutaminase 2), aberrant extracellular matrix, flawed endocytosis, and abnormal aqueous‐blood barrier contribute to the formation of proteolytically resistant pseudoexfoliative material. Pigment dispersion syndrome is characterized by abnormal iridolenticular contact that disrupts iris pigment epithelium and liberates melanin granules. Iris melanogenesis is aberrant in this condition. Cytotoxic melanogenesis intermediates leak out of melanosomes and cause iris melanocyte and pigment epithelium cell death. Targeting melanogenesis can likely decrease the risk of pigmentary glaucoma. Skin and melanoma research provides insights into potential therapeutics. We propose that specific prostanoid agonists and fenofibrates may reduce melanogenesis by inhibiting cholesterol internalization and de novo synthesis. Additionally, melatonin is a potent melanogenesis suppressor, antioxidant, and hypotensive agent, rendering it a valuable agent for pigmentary glaucoma. In pseudoexfoliative glaucoma, where environmental insults drive pseudoexfoliative material formation, melatonin's antioxidant and hypotensive properties may offer adjunct therapeutic benefits.
This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology
Organoid is an emerging frontier technology in the field of life science., in which pluripotent stem cells or tissue‐derived differentiated/progenitor cells form 3D structures according to their multi‐directional differentiation potential and self‐assembly ability. Nowadays, although various types of organoids have been widely investigated, their construction is still complicated in operation, uncertain in yield, and poor in reproducibility for the structure and function of native organs. Constructing a biomimetic microenvironment for stem cell proliferation and differentiation in vitro has been recognized as a key to driving this field. This review reviews the recent development of engineered biomimetic microenvironments for organoids. Firstly, we summarize the composition of the matrix for organoid culture. Then, strategies for engineering the microenvironment from biophysical, biochemical, and cellular perspectives have been discussed in detail. Subsequently, the newly developed monitoring technologies are also reviewed. Finally, a brief conclusion and outlook are presented for the inspiration of future research.
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The extracellular matrix (ECM), composed of interlinked proteins outside of cells, is an important component of the human body that helps maintain tissue architecture and cellular homeostasis. As people age, the ECM undergoes changes that can lead to age-related morbidity and mortality. Despite its importance, ECM aging remains understudied in the field of geroscience. In this review, we discuss the core concepts of ECM integrity, outline the age-related challenges and subsequent pathologies and diseases, summarize diagnostic methods detecting a faulty ECM, and provide strategies targeting ECM homeostasis. To conceptualize this, we built a technology research tree to hierarchically visualize possible research sequences for studying ECM aging. This strategic framework will hopefully facilitate the development of future research on interventions to restore ECM integrity, which could potentially lead to the development of new drugs or therapeutic interventions promoting health during aging.
In this paper a rapid, selective, and ultrasensitive protocol for the detection of the active form of matrix metalloproteinase-1 (MMP-1), which is a novel predictive and prognostic biomarker, was presented, which might strengthen the current predictive systems. The biosensor construction procedure was extremely simple, economical, and time-saving, as it involved only the chemisorption step of the voltammetrically active receptor (tripeptide (Cys-Gly-Ile) labeled with methylene blue (MB) and the sealing thiol. The active form of MMP-1 was recognized based on its hydrolytic activity; as a consequence, the receptor fragment (-Ile-MB) was removed from the sensor surface. The biosensors constructed were characterized by a wide dynamic concentration response range (1.0 pg mL-1-1.0 μg mL-1) and a low detection limit (33 fg mL-1), especially the biosensor with voltammetric detection, without the amplification step. One of the important advantages of the proposed biosensors is that they can be directly used to analyze the content of the active form of MMP-1 in clinical samples without the dilution step and any other preparation step.
p>The aims of this study were to investigate MMP-3 production between individuals with different MMP-3 5A/6A genotypes: and to compare the association of the MMP-3 5A/6A genotype with susceptibility to CD and UC.
Myofibroblast cell lines were isolated from CD, UC patients and control subjects. The phenotype of these cells lines were characterised with immunohistochemistry. Cell lines were stimulated with TNF-α or IL-1β and the production of MMP-3 was measured by western blotting and ELISA.
Cell lines from CD, UC and control patients were myofibroblast-enriched populations of cells. Cell lines responded to stimulation with TNF-α by a dose-dependent rise in MMP-3 production. The 5A/6A polymorphism was analysed in 468 German sporadic inflammatory disease trios and 270 British and German multiplex IBD families using the transmission disequilibrium test (TDT). There was an overtransmission of the 5A allele to affected offspring (p=0.0012). There as an interaction between the MMP-3 gene 5A/6A polymorphism and the Caspase Activating Recruitment Domain (CARD) 15 gene, a well established gene for CD, such that overtransmission of the 5A allele was a significant in CARD15 carriers (p=0.0054) but not in non-carriers. In the CARD15 carriers, overtransmission of the 5A allele was associated with stenosis (p=0.0027), fistulising disease (p=0.007), previous surgical resection (p=0.0023), disease of the ileum (p=0.0001), and disease of the right colon (p=0.0015) in CD. The relationship of functional polymorphisms in promoters of the MMP-1,-3,-9 and -12 genes with IBD was also investigated in children and adults, and age-matched controls. No significant association was found between these polymorphisms and CD or UC, though the power of these studies was reduced by small sample numbers. In conclusion, the MMP-3 5A/6A polymorphism appears to be a genetic factor in CD.</p
p>In this study further investigations of the MMP-3 gene were undertaken in relation to atherosclerosis. Using the mutation scanning technique dideoxy fingerprinting (ddF), the promoter and coding regions of the MMP-3 gene were screened for unknown polymorphisms. Six new polymorphisms were identified using this method and confirmed by sequencing. Relative to transcription start site these were - 1986 T/C; - 1612 5A/6A; -1346 A/C; -709 A/G; -376G/C; and in exon 2, 802 A/G and 952 A/G. All six variants are single nucleotide polymorphisms (SNPs). Genotyping of these SNPs in unrelated individuals showed that they were in strong linkage disequilibrium with each other and with the previously identified 5A/6A polymorphism. The two commonest haplotypes were T-5A-A-A-G-A-A and C-6A-C-G-C-G-G. In a cohort of 913 patients with angiographically documented coronary artery disease (CAD), the 5A allele-containing genotypes were over-represented in patients with a history of MI (p=0.03). Furthermore, an association between the number of coronary arteries >50% stenosis and the 6A/6A genotype was observed (p=0.01).
To determine whether these polymorphisms affect MMP-3 promoter activity, transient transfection studies were performed using reporter genes for the two alleles of each individual polymorphism. Small but statistically significant differences of promoter activity were observed for each individual SNP. However, relative to the two commonest haplotypes, the common allele of three SNPs belonging to one haplotype conferred higher activity, whereas the common allele of the other two conferred lower activity.
These results suggest that individuals carrying the transcriptionally more active 5A allele are predisposed to the development of unstable plaques and CHD, whereas those carrying the transcriptionally less active 6A allele are more likely to develop stable but highly stenotic plaques.</p
Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.
A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) are major mediators in extracellular matrix (ECM) turnover and have gained increasing interest over the last years as major players in ECM remodeling during tissue homeostasis and the development of diseases. Although, ADAMTSs are recognized in playing important roles during tissue remodeling, and loss of function in various member of the ADAMTS family could be associated with the development of numerous diseases, limited knowledge is available about their specific substrates and mechanism of action. In this chapter, we will review current knowledge about ADAMTSs and their use as disease biomarkers.
The cell microenvironment plays a crucial role in regulating cell behavior and fate in physiological and pathological processes. As the fundamental component of the cell microenvironment, extracellular matrix (ECM) typically possesses complex ordered structures and provides essential physical and chemical cues to the cells. Hydrogels have attracted much attention in recapitulating the ECM. Compared to natural and synthetic polymer hydrogels, DNA hydrogels have unique programmable capability, which endows the material precise structural customization and tunable properties. This review focuses on recent advances in programmable DNA hydrogels as artificial extracellular matrix, particularly the pure DNA hydrogels. It introduces the classification, design, and assembly of DNA hydrogels, and then summarizes the state‐of‐the‐art achievements in cell encapsulation, cell culture, and tissue engineering with DNA hydrogels. Ultimately, the challenges and prospects for cellular applications of DNA hydrogels are delivered.
Among the many forms of epilepsy, one of the most studied is epilepsy of the temporal lobe (temporal lobe epilepsy) associated with the pathology of the limbic system, and especially the hippocampus. Sections of the limbic system are the source of epileptic seizures in this form of the disease, which is confirmed by electroencephalographic data, including those obtained using embedded electrodes [81], and the clinical effectiveness of surgery. Removal of certain parts of the medial temporal cortex, including part of the hippocampus, can heal or reduce the frequency and severity of seizures [92]. On the basis of structural changes, two main types of epilepsy of the temporal lobe are distinguished: 1) with the presence of a volumetric process (tumor, congenital pathology, blood vessel aneurysm, hemorrhage) affecting the limbic system; 2) without the presence of clearly verified volumetric changes in the medial temporal lobe [23]. In the latter case, the only structural manifestation of temporal lobe epilepsy is hippocampal sclerosis. The name reflects the most striking morphological manifestations of the disease - the loss of neurons primarily in the CA1 and CA3 zones of the horn of the ammonia and the development of replacement gliosis. Intravital brain imaging using functional positron emission tomography, magnetic resonance imaging, and magneto-encephalography confirms changes in the hippocampus in temporal lobe epilepsy, usually in the form of a decrease in its volume [60]. There is also a positive correlation between intravital structural and biochemical (in particular, the number of AMPA-A receptors and the intensity of absorption of F-fluoro-2-deoxy-D-glucose) changes in the sclerosed hippocampus and data from the study of surgical material [75].
Wound healing is a highly dynamic process and innovative therapeutic approaches are currently developed to address challenges of providing optimal wound care. In this study, phosphate-based glasses in the (CuO)x·(KPO3)79.5-x·(ZnO)20·(Ag2O)0.5 system (CuKPO3ZnAg), with different CuO/ KPO3 ratios were prepared by melt-quenching technique. Constant Cu concentrations were released from the samples during immersion in Simulated Body Fluid (SBF), while Zn concentrations were slightly decreased over time. Glass surface phosphatation leading to formation of Zn crystalline salts was revealed through spectroscopic techniques. This finding was supported by SEM images that illustrated new compound formation. Subsequent cytotoxicity evaluation on HaCaT Keratinocytes using the indirect MTT cell viability assay revealed a CuO concentration-dependent cytotoxicity profile and excellent biocompatibility at low CuO concentrations, in all CuKPO3ZnAg glasses. Furthermore, the (CuO)5·(KPO3)74.5·(ZnO)20·(Ag2O)0.5 sample (5CuKPO3ZnAg), demonstrated superior antibacterial potency against S. aureus (ATCC 25923) strain compared to amoxicillin and ciprofloxacin. In vivo full-thickness wound healing evaluation showed a significantly higher regenerative effect of the 5CuKPO3ZnAg sample, in terms of angiogenesis, collagen synthesis and re-epithelialization compared to non-treated wounds. These findings advance our understanding of the therapeutic perspectives of phosphate-based glasses, showing promising potential for wound-healing applications.
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Bufo gargarizans is one kind of economic animals with higher medicinal value in China. In this study, B. gargarizans (Bufo gargarizans) tadpoles were reared at three different water temperature (15, 22 and 29 °C) from Gosner stages 28–46. We investigated the effects of temperature on growth, development, survival, metamorphic duration, size and skeletal ossification at Gosner stage 40, 42, and 46, as well as thyroid tissue reached metamorphic climax (Gs42). Besides, we examined the transcription levels of endochondral ossification-related genes in hind limb at metamorphic climax (Gs42). Our results showed that the growth and development of tadpoles conform to the temperature-size rule (TSR). While warm temperature resulted in the decrease in body size and hind limb length, and shorten larval period, cold temperature led to increase in body size and hind limb length but prolonged larval period. Histological examinations revealed that warm and cold temperatures caused damage to thyroid tissue. Also, warm and cold temperatures inhibited the degree of ossification with the double staining methodology. Additionally, the real-time PCR results suggested that warm and cold temperatures significantly up-regulated Runx2, VEGF and VEGFR mRNA levels, and down-regulated TRβ, MMP9, MMP13 and Runx3 mRNA levels. The up-regulation of Dio2 level and down-regulation of Dio3 level were observed in warm temperature. TRα mRNA level was significantly increased in warm temperature, but decreased in cold temperature. Collectively, these observations demonstrated that warm and cold temperatures affected endochondral ossification in B. gargarizans tadpoles, which might influence their capacity to terrestrial locomotion.
Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.
The invasion of human malignant melanoma cells into the extracellular matrix (ECM) involves the accumulation of proteases at sites of ECM degradation where activation of matrix metalloproteases (MMP) occurs. Here, we show that when membrane type 1 MMP (MT-MMP) was overexpressed in RPMI7951 human melanoma cells, the cells made contact with the ECM, activated soluble and ECM-bound MMP-2, and degraded and invaded the ECM. Further experiments demonstrated the importance of localization of the MT-MMP to invadopodia. Overexpression of MT-MMP without invadopodial localization caused activation of soluble MMP-2, but did not facilitate ECM degradation or cell invasiveness. Up-regulation of endogenous MT-MMP with concanavalin A caused activation of MMP-2. However, concanavalin A treatment prevented invadopodial localization of MT-MMP and ECM degradation. Neither a truncated MT-MMP mutant lacking transmembrane (TM) and cytoplasmic domains (Delta TMMT-MMP), nor a chimeric MT-MMP containing the interleukin 2 receptor alpha chain (IL-2R) TM and cytoplasmic domains (Delta TMMT-MMP/TMIL-2R) were localized to invadopodia or exhibited ECM degradation. Furthermore, a chimera of the TM/cytoplasmic domain of MT-MMP (TMMT-MMP) with tissue inhibitor of MMP 1 (TIMP-1/TMMT-MMP) directed the TIMP-1 molecule to invadopodia. Thus, the MT-MMP TM/cytoplasmic domain mediates the spatial organization of MT-MMP into invadopodia and subsequent degradation of the ECM.
Matrix metalloproteinases (MMPs) classically have been implicated in basement membrane destruction associated with late-stage tumor cell invasion and metastasis. However, recent studies have demonstrated that one MMP family member, matrilysin, is expressed in a high percentage of early-stage human colorectal tumors. We analyzed matrilysin expression in benign intestinal tumors from mice heterozygous for the ApcMin allele (Min/+) and found that the mRNA was induced in the majority (88%) of these adenomas. Protein was detected in the tumor cells, where, surprisingly, it was predominantly immunolocalized to the lumenal surface of dysplastic glands rather than the basement membrane or extracellular matrix. To address the role of matrilysin in Min intestinal tumorigenesis, we generated Min/+ mice deficient in this MMP by gene targeting and homologous recombination. The absence of matrilysin resulted in a reduction in mean tumor multiplicity in Min/+ animals of approximately 60% and a significant decrease in the average tumor diameter. Based on these findings, we conclude that matrilysin is a suppressor of the Min phenotype, possibly by functioning in a capacity independent of matrix degradation. These results argue for the use of MMP inhibitors in the treatment and prevention of early-stage colon cancer.
There is a common polymorphism in the promoter sequence of the human stromelysin-1 gene, with one allele having a run of six adenosines (6A) and the other five adenosines (5A). We have previously reported, in a 3-year follow-up study of patients with coronary atherosclerosis, that those patients who are homozygous for the 6A allele show a more rapid progression of the disease. In this study, we have investigated whether the 5A/6A promoter polymorphism plays a role in the regulation of stromelysin-1 gene expression. In transient transfection experiments, a stromelysin-1 promoter construct with 6A at the polymorphic site was found to express less of the chloramphenicol acetyltransferase reporter gene than a construct containing 5A. Electrophoretic mobility shift assay and DNase I footprinting revealed the interaction of one or more nuclear protein(s) with the DNA sequence at the 5A/6A polymorphic site. The binding of one of the nucleoprotein factors was more readily detectable with an oligonucleotide probe corresponding to the 6A allele as compared with a probe corresponding to the 5A allele. Replacing the core binding sequence with a random DNA sequence abolished the interaction between the nuclear protein(s) and the probe and also increased reporter gene expression in transiently transfected cells. Thus, the common 5A/6A polymorphism of the human stromelysin-1 promoter appears to play an important role in regulating stromelysin-1 gene expression and may be involved in the progression of coronary heart disease.
Development of the metanephric kidney requires the concerted interaction of two tissues, the epithelium of the ureteric duct and the metanephric mesenchyme. Signals from the ureter induce the metanephric mesenchyme to condense and proliferate around the ureter tip, reciprocal signals from the mesenchyme induce the ureter tip to grow and to branch. Wnt genes encode secreted glycoproteins, which are candidate mediators of these signaling events. We have identified three Wnt genes with specific, non-overlapping expression patterns in the metanephric kidney, Wnt-4, Wnt-7b and Wnt-11. Wnt-4 is expressed in the condensing mesenchyme and the comma- and S-shaped bodies. Wnt-7b is expressed in the collecting duct epithelium from 13.5 days post coitum onward. Wnt-1l is first expressed in the nephric duct adjacent to the metanephric blastema prior to the outgrowth of the ureteric bud. Wnt-l1 expression in Danforth's short-tail mice suggests that signaling from the mesenchyme may regulate Wnt-ll activation. During metanephric development, Wnt-11 expression is confined to the tips of the branching ureter. Maintenance of this expression is independent of Wnt-4 signaling and mature mesenchymal elements in the kidney. Moreover, Wnt-ll expression is maintained in recombinants between ureter and lung mesenchyme suggesting that branching morphogenesis and maintenance of Wnt-ll expression are independent of metanephric mesenchyme-specific factors. Interference with proteoglycan synthesis leads to loss of Wnt-ll expression in the ureter tip. We suggest that Wnt-11 acts as an autocrine factor within the ureter epithelium and that its expression is regulated at least in part by proteoglycans.
The mouse bone morphogenetic protein1 (Bmp1) gene encodes a secreted astacin metalloprotease that cleaves the COOH-propeptide of procollagen I, II and III. BMP-1 is also related to the product of the Drosophila patterning gene, tolloid (tld), which enhances the activity of the TGFbeta-related growth factor Decapentaplegic and promotes development of the dorsalmost amnioserosa. We have disrupted the mouse Bmp1 gene by deleting DNA sequences encoding the active site of the astacin-like protease domain common to all splice variants. Homozygous mutant embryos appear to have a normal skeleton, apart from reduced ossification of certain skull bones. However, they have a persistent herniation of the gut in the umbilical region and do not survive beyond birth. Analysis of the amnion of homozygous mutant embryos reveals the absence of the fold that normally tightly encloses the physiological hernia of the gut. At the electron microscopic level, the extracellular matrix of the amnion contains collagen fibrils with an abnormal morphology, consistent with the incorporation of partially processed procollagen molecules. Metabolical labelling and immunofluorescence studies also reveal abnormal processing and deposition of procollagen by homozygous mutant fibroblasts in culture.
Mice lacking the serine protease tissue plasminogen activator (tPA) are resistant to excitotoxin-mediated hippocampal neuronal degeneration. We have used genetic and cellular analyses to study the role of tPA in neuronal cell death. Mice deficient for the zymogen plasminogen, a known substrate for tPA, are also resistant to excitotoxins, implicating an extracellular proteolytic cascade in degeneration. The two known components of this cascade, tPA and plasminogen, are both synthesized in the mouse hippocampus. tPA mRNA and protein are present in neurons and microglia, whereas plasminogen mRNA and protein are found exclusively in neurons. tPA-deficient mice exhibit attenuated microglial activation as a reaction to neuronal injury. In contrast, the microglial response of plasminogen-deficient mice was comparable to that of wild-type mice, suggesting a tPA-mediated, plasminogen-independent pathway for activation of microglia. Infusion of inhibitors of the extracellular tPA/plasmin proteolytic cascade into the hippocampus protects neurons against excitotoxic injury, suggesting a novel strategy for intervening in neuronal degeneration.
Mast cell activation is a characteristic feature of chronic inflammation, a condition that may lead to fibrosis as a result of increased collagen synthesis by fibroblasts. We have investigated the potential of tryptase, the major protease of human mast cells, to stimulate collagen synthesis in the human lung fibroblast cell line MRC-5. Tryptase was isolated from human lung tissue by ion-exchange and affinity chromatography. At concentrations of 18 and 36 mU/ml, tryptase stimulated both an increase in cell numbers, and a fivefold increase in DNA synthesis as determined by methyl-[3H]thymidine incorporation. Similar concentrations of tryptase resulted in a 2.5-fold increase in collagen synthesis as determined both by incorporation of [3H]proline into collagen, and by assay of hydroxyproline concentrations in the supernatants. There was also a twofold increase in collagenolytic activity in the culture medium after tryptase treatment, indicating that the increase in collagen synthesis was not a consequence of decreased collagenase production. All of these actions of tryptase were reduced in the presence of the protease inhibitors leupeptin and benzamidine hydrochloride, indicating a requirement for an active catalytic site. SDS-PAGE and autoradiographic analysis of the [3H]collagen produced by the cells revealed it to be predominantly type I collagen. Our findings suggest that the release of tryptase from activated mast cells may provide a signal for abnormal fibrosis in inflammatory disease.
Thrombin is a coagulation protease that activates platelets, leukocytes, endothelial and mesenchymal cells at sites of vascular injury, acting partly through an unusual proteolytically activated G-protein-coupled receptor. Knockout of the gene encoding this receptor provided definitive evidence for a second thrombin receptor in mouse platelets and for tissue-specific roles for different thrombin receptors. We now report the cloning and characterization of a new human thrombin receptor, designated protease-activated receptor 3 (PAR3). PAR3 can mediate thrombin-triggered phosphoinositide hydrolysis and is expressed in a variety of tissues, including human bone marrow and mouse megakaryocytes, making it a candidate for the sought-after second platelet thrombin receptor. PAR3 provides a new tool for understanding thrombin signalling and a possible target for therapeutics designed selectively to block thrombotic, inflammatory and proliferative responses to thrombin.
We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti-MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the alpha2 integrin subunit but not by antibodies against the alpha1 or alpha3 subunits. We propose that interaction of the alpha2beta1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.
Structural changes in the extracellular matrix are necessary for cell migration during tissue remodeling and tumor invasion. Specific cleavage of laminin-5 (Ln-5) by matrix metalloprotease-2 (MMP2) was shown to induce migration of breast epithelial cells. MMP2 cleaved the Ln-5 gamma2 subunit at residue 587, exposing a putative cryptic promigratory site on Ln-5 that triggers cell motility. This altered form of Ln-5 is found in tumors and in tissues undergoing remodeling, but not in quiescent tissues. Cleavage of Ln-5 by MMP2 and the resulting activation of the Ln-5 cryptic site may provide new targets for modulation of tumor cell invasion and tissue remodeling.
The urokinase‐type plasminogen activator (u‐PA) system consists of the serine proteinases plasmin and u‐PA; the serpin inhibitors α2‐anti‐plasmin, PAI‐1 and PAI‐2; and the u‐PA receptor (u‐PAR). Two lines of evidence have strongly suggested an important and apparently causal role for the u‐PA system in cancer metastasis: results from experimental model systems with animal tumor metastasis and the finding that high levels of u‐PA, PAI‐1 and u‐PAR in many tumor types predict poor patient prognosis. We discuss here recent observations related to the molecular and cellular mechanisms underlying this role of the u‐PA system. Many findings suggest that the system does not support tumor metastasis by the unrestricted enzyme activity of u‐PA and plasmin. Rather, pericellular molecular and functional interactions between u‐PA, u‐PAR, PAI‐1, extracellular matrix proteins, integrins, endocytosis receptors and growth factors appear to allow temporal and spatial re‐organizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. Differential expression of components of the system by cancer and non‐cancer cells, regulated by paracrine mechanisms, appear to determine the involvement of the system in cancer cell–directed tissue remodeling. A detailed knowledge of these processes is necessary for utilization of the therapeutic potential of interfering with the action of the system in cancers. Int. J. Cancer 72:1–22, 1997. © 1997 Wiley‐Liss Inc.
Structural changes in the extracellular matrix are necessary for cell migration during tissue remodeling and tumor invasion.
Specific cleavage of laminin-5 (Ln-5) by matrix metalloprotease–2 (MMP2) was shown to induce migration of breast epithelial
cells. MMP2 cleaved the Ln-5 γ2 subunit at residue 587, exposing a putative cryptic promigratory site on Ln-5 that triggers
cell motility. This altered form of Ln-5 is found in tumors and in tissues undergoing remodeling, but not in quiescent tissues.
Cleavage of Ln-5 by MMP2 and the resulting activation of the Ln-5 cryptic site may provide new targets for modulation of tumor
cell invasion and tissue remodeling.
The urokinase-type plasminogen activator (u-PA) system consists of the serine proteinases plasmin and u-PA; the serpin inhibitors a2-anti-plasmin, PAI-1 and PAI-2; and the u-PA receptor (u-PAR). Two lines of evidence have strongly suggested an important and apparently causal role for the u-PA system in cancer metastasis: results from experimental model systems with animal tumor metastasis and the finding that high levels of u-PA, PAI-1 and u-PAR in many tumor types predict poor patient prognosis.Wediscuss here recent observations related to the molecular and cellular mecha- nisms underlying this role of the u-PA system. Many findings suggest that the system does not support tumor metastasis by the unrestricted enzyme activity of u-PA and plasmin. Rather, pericellular molecular and functional interactions between u-PA, u-PAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors and growth factors appear to allow temporal and spatial re-organizations of the system during cell migration and a selective degradation of extracel- lular matrix proteins during invasion. Differential expression of components of the system by cancer and non-cancer cells, regulated by paracrine mechanisms, appear to determine the involvement of the system in cancer cell–directed tissue remodeling. A detailed knowledge of these processes is necessary for utilization of the therapeutic potential of inter- fering with the action of the system in cancers. Int.
Plasmin(ogen) is an extracellular serine protease implicated in the activation of latent growth factors and procollagenase, degradation of extracellular matrix components, and fibrin clearance. Plasminogen (Plg) deficiency in mice results in high mortality, wasting, spontaneous gastrointestinal ulceration, rectal prolapse, and severe thrombosis. Furthermore, Plg-deficient mice display delayed wound healing following skin injury, a defect partly related to impaired keratinocyte migration. We generated mice deficient in Plg and fibrinogen (Fib) and show that removal of fibrin(ogen) from the extracellular environment alleviates the diverse spontaneous pathologies previously associated with Plg deficiency and corrects healing times. Mice deficient in Plg and Fib are phenotypically indistinguishable from Fib-deficient mice. These data suggest that the fundamental and possibly only essential physiological role of Plg is fibrinolysis.
Proteolytic remodeling of the extracellular matrix is an important aspect of the creation and progression of cancer. Matrix metalloproteinases are important at several points during multi-stage neoplastic progression in tumor cells and responding blood vessels, inflammatory cells and stroma.
The past year or two has seen great advances in the elucidation of significant roles for integrins in cancer cells. These include roles in signal transduction, gene expression, proliferation, apoptosis regulation, invasion and metastasis, and angiogenesis. In particular, integrin alphavbeta3 has been implicated in the neovascularization of tumors. In addition, this integrin has been shown to contribute to the survival, proliferation and metastatic phenotype of human melanoma.
Matrix metalloproteinases (MMP) comprise a family of structurally related proteinases that are believed to play a critical role in many physiological and pathological processes. Transgenic technology offers the possibility of determining whether MMPs contribute directly to these processes. For example, gain of function and loss of function models have confirmed causative roles of MMPs in the development of pulmonary emphysema and unexpectedly uncovered an MMP-dependent mechanism of inflammatory cell recruitment. Limitations of these techniques and powerful applications on the horizon are also presented as we embark on an era where controlled experiments can be performed in complex mammalian models.
The urokinase-type plasminogen activator (u-PA) system consists of the serine proteinases plasmin and u-PA; the serpin inhibitors alpha2-anti-plasmin, PAI-1 and PAI-2; and the u-PA receptor (u-PAR). Two lines of evidence have strongly suggested an important and apparently causal role for the u-PA system in cancer metastasis: results from experimental model systems with animal tumor metastasis and the finding that high levels of u-PA, PAI-1 and u-PAR in many tumor types predict poor patient prognosis. We discuss here recent observations related to the molecular and cellular mechanisms underlying this role of the u-PA system. Many findings suggest that the system does not support tumor metastasis by the unrestricted enzyme activity of u-PA and plasmin. Rather, pericellular molecular and functional interactions between u-PA, u-PAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors and growth factors appear to allow temporal and spatial re-organizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. Differential expression of components of the system by cancer and non-cancer cells, regulated by paracrine mechanisms, appear to determine the involvement of the system in cancer cell-directed tissue remodeling. A detailed knowledge of these processes is necessary for utilization of the therapeutic potential of interfering with the action of the system in cancers.
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