Search

Aortic dissection, characterized by a high immediate mortality, is primarily caused by excessive bleeding within the walls of the aorta or a severe tear within the intimal layer of the aorta. Inflammation, as well as oxidative stress and the degradation of extracellular matrix (ECM), are significant factors in the development and occurrence of aortic dissection. Matrix metalloproteinases (MMPs) are pivotal enzymes responsible for degrading the ECM. Inflammatory factors and oxidants can interact with MMPs, indicating the potential significance of MMPs in aortic dissection. A substantial body of evidence indicates that numerous MMPs are significantly upregulated in aortic dissection, playing a critical role in ECM degradation and the pathogenesis of aortic dissection. Furthermore, targeting these enzymes has demonstrated potential in facilitating ECM restoration and reducing the incidence of aortic dissection. This review initially provides a brief overview of MMP biology before delving into their expression patterns, regulatory mechanisms, and therapeutic applications in aortic dissection. A profound comprehension of the catabolic pathways associated with aortic dissection is imperative for the future development of potential preventive or therapeutic bio-interventions for aortic dissection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Intervertebral disc degeneration (IDD) is a spinal degenerative disease and one of the most important causes of musculoskeletal disability. Matrix metalloproteinase (MMP)-mediated extracellular matrix degradation is the core process of IDD. The regulators of MMPs in the intervertebral disc are still not fully known. In this study, using quantitative reverse transcription PCR, luciferase reporter assay, Western blotting, immunofluorescence, flow cytometry, and Cell Counting Kit-8 assay, we found that the miR-874-3p expression level was significantly decreased in IDD patients. MiR-874-3p could target and repress MMP2 and MMP3 expression in nucleus pulposus cells. These results could improve the understanding of IDD and provide a possible diagnostic marker and treatment candidate for IDD. The miR-874-3p/MMP2/MMP3 axis might also provide direction for future cancer and inflammation investigations., (Copyright © 2021. Published by Elsevier B.V.)

Peptidylarginine deiminase (PAD) family members have a vital role in maintaining the stability of the extracellular matrix (ECM) during remodelling in several heart diseases. PAD-mediated deamination, or citrullination, has been studied in different physiological and pathological conditions in the body. However, the role of PAD isoforms has not been fully studied in cardiovascular system. Citrullination is a post-translational modification that involves conversion of peptidyl-based arginine to peptidyl-based citrulline by PAD family members in a calcium-dependent manner. Upregulation of PADs have been observed in various cardiovascular diseases, including venous thrombosis, cardiac fibrosis, heart failure, atherosclerosis, coronary heart disease and acute inflammation. In this review, experimental aspects of in vivo and in vitro studies related to the roles PAD isoforms in cardiovascular diseases including mechanisms, pathophysiological and therapeutic properties are discussed. Pharmacological strategies for targeting PAD family proteins in cardiac diseases have not yet been studied. Furthermore, the role played by PAD family members in the remodelling process during the progression of cardiovascular diseases is not fully understood., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors and Editor-in-Chief. The corresponding author contacted the journal and stated: “…we have obtained different results from this paper (onecut2 targeted regulation of Smurf2 / GSK-3 β part), and repeated experiments cannot fully verify this result”. The authors requested retraction of the article. Concern was also raised about the integrity of an image in Figure 1H, which appears to also be found in another publication, as detailed here: https://pubpeer.com/publications/FBECF0BCB952DCC563AA5C4D760B32 and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. The journal requested the corresponding author comment on these concerns and provide the raw data. The authors were unable to satisfactorily fulfill this request. The Editor-in-Chief assessed the case and decided to retract the article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Peyronie's disease (PD) is a fibrotic disorder of the penile tunica albuginea, characterised by the formation of a localised fibrous plaque that can lead to deformity and erectile dysfunction. Nonsurgical therapeutic options for PD are limited in efficacy and safety. Myofibroblasts are key cells in the pathogenesis of PD, and inhibition of myofibroblast transformation has been suggested as a therapeutic option., Objective: To identify potential drugs using a novel phenotypic assay and then to test them using in vitro and in vivo models of PD., Design, Setting, and Participants: We have developed and validated a phenotypic screening assay that measures myofibroblast transformation, by which we tested 21 compounds that were suggested to be efficacious in treating PD. The successful hits from this assay were further tested using in vitro and in vivo models of PD., Results and Limitations: The new assay was able to detect transforming growth factor-β1-induced myofibroblast transformation. Using this assay, phosphodiesterase type 5 inhibitors (PDE5i) and selective oestrogen receptor modulators (SERMs) were identified to significantly inhibit myofibroblast transformation. A PDE5i (vardenafil) and an SERM (tamoxifen) inhibited myofibroblast transformation, collagen gel contraction, and extracellular matrix production in a synergistic fashion. In a rat model of PD, the antifibrotic effect of the combination of vardenafil and tamoxifen was greater than that of each drug alone. This study is limited by not providing a molecular mechanism for the proposed synergy., Conclusions: This is the first demonstration of a synergistic activity between a PDE5i and an SERM discovered through a phenotypic screening approach. Future clinical trials using a combination of these drugs should be considered during the active phase of PD, given the early evidence of benefit in both in vitro and in vivo models., Patient Summary: This report suggests that the combination of a phosphodiesterase type 5 inhibitor and a selective oestrogen receptor modulator may be efficacious in treating Peyronie's disease in its active phase., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: Human tissue kallikrein 15 (KLK15) is the last cloned member of the KLK-related gene family. Despite being implicated in multiple cancers, its pathophysiological role remains unknown. We aimed to biochemically characterize KLK15 and preliminarily study its role in cancer., Design & Methods: Recombinant KLK15 protein was produced, purified to homogeneity and quantified by mass spectrometry (parallel reaction monitoring analysis). We profiled the enzymatic activity of KLK15 using fluorogenic peptide substrates, and performed kinetic analysis to discover the cleavage sites. As KLK15 has mainly been associated with prostate cancer, we used a degradomic approach and subsequent KEGG pathway analysis to identify a number of putative protein substrates in the KLK15-treated prostate cancer cell line PC3., Results: We discovered trypsin-like activity in KLK15, finding that it cleaves preferentially after arginine (R). The enzymatic activity of KLK15 was regulated by different factors such as pH, cations and serine protease inhibitors. Notably, we revealed that KLK15 most likely interacts with the extracellular matrix (ECM) receptor group., Conclusion: To our knowledge, this is the first study that experimentally verifies the trypsin-like activity of KLK15. We show here for the first time that KLK15 may be able to cleave many ECM components, similar to several members of the KLK family. Thus the protease could potentially be linked to tumorigenesis by promoting metastasis via this mechanism., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Neospora caninum is one of the most efficient transplacentally transmitted pathogens in cattle and is a cause of abortion in this domestic species. The invasion and proliferation of Neospora caninum in the placenta and its dissemination to the foetus are crucial events in the outcome of an infection. In the bovine placenta, the placentomes are formed by maternal caruncles, which are delimited by a maternal epithelium and foetal cotyledons, which are delimited by an epithelial layer named the trophoblast. These epithelia form a physical barrier against foetal infection. Furthermore, trophoblast cells act as an innate immune defence at the foetal-maternal interface. Neospora caninum invades and proliferates in trophoblast cells in vitro, but it is unknown whether host cell modulation events, which affect the immune response and other processes in the trophoblast, occur. In this work, we investigated the transcriptomic modulation by Neospora caninum infection in the bovine trophoblast cell line F3. In addition, two Neospora caninum isolates with marked differences in virulence, Nc-Spain1H and the Nc-Spain7, were used in this study to investigate the influence of these isolates in F3 modulation. The results showed a clear influence on extracellular matrix reorganisation, cholesterol biosynthesis and the transcription factor AP-1 network. Interestingly, although differences in the transcriptome profiles induced by each isolate were observed, specific isolate-modulated processes were not identified, suggesting very similar regulation in both isolates. Differential expression of the N. caninum genes between both isolates was also investigated. Genes involved in host cell attachment and invasion (SAG-related and microneme proteins), glideosome, rhoptries, metabolic processes, cell cycle and stress response were differentially expressed between the isolates, which could explain their variability. This study provides a global view of Neospora caninum interactions with bovine trophoblast cells and of the intra-specific differences between two Neospora caninum isolates with biological differences., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Heme peroxidases are the most abundant type of peroxidase catalyzing a H 2 O 2 -dependent oxidation of a wide variety of substrates. They are involved in numerous processes like the innate immune response, hormone and prostaglandin synthesis and crosslinking of proteins within extracellular matrixes (ECM) as well as molecules within the cuticle and chorion of arthropods and nematodes. In the present study, a Lepeophtheirus salmonis heme peroxidase (LsHPX) 1 was characterized. Amino acids in the active site of heme peroxidases were conserved, and the predicted protein sequence showed the highest similarity to genes annotated as chorion peroxidases and genes suggested to be involved in cuticle hardening or adhesion. LsHPX1 exhibited a dynamic expression during ontogenesis and during the nauplius molting cycle. Transcripts were localized to muscle cells near the muscle-tendon junction, in nerve tissue especially at neuromuscular junctions, subcuticular epithelium, subepithelial cells facing the hemolymph, exocrine glands within the subepithelial tissue and in isolated cells within the testis. Knock-down of LsHPX1 in nauplius larvae decreased the swimming activity of emerging copepodids. Histological analysis of knock-down animals revealed increased spacing between myofibers and changes in subepithelial and exocrine gland tissue. Considering these results, the potential role of LsHPX1 in crosslinking molecules of salmon louse ECMs is discussed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Age, gender and systemic diseases all influence cardiac function and remodelling. In cats, age and gender associated myocardial remodelling and the effect of systemic diseases on the myocardium have so far not been studied. The aim of the study was therefore to investigate whether relevant cytokines and extracellular matrix (ECM) remodelling enzymes are expressed in the myocardium of cats with non-cardiac diseases and whether transcription levels are influenced by age and gender., Methods: The study was performed on myocardial samples from 26 cats aged between 2 and 19 years that had died with non-cardiac diseases. Seventeen cats were female (2 entire) and nine were male (1 entire). Of these, nine cats were diagnosed with diseases unlikely to affect the myocardium (control cats). The remaining 17 cats suffered from diseases with likely systemic effects. All hearts were assessed for any pathological changes, and the myocardium was analysed for interleukin (IL)-1, -2, -4, -6, -18, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, matrix metalloproteinase (MMP)-2, -3, -13, tissue inhibitor of MMP (TIMP)-1, -2 and -3 transcriptions using quantitative RT-PCR assays., Results: Despite the absence of any histological evidence of myocardial damage, inflammation and fibrosis, the myocardium of all the cats was found to constitutively transcribe cytokines and ECM remodelling enzymes, with generally higher mRNA concentrations in the atria than in the ventricles. The young and male cats exhibited higher transcription levels throughout the myocardium in comparison to the older and female cats. Furthermore, age-associated transcription pattern differed between male and female cats., Conclusion: The constitutive transcription of ECM remodelling enzymes suggests continuous myocardial remodelling throughout the entire life of a cat. The myocardium of young and male cats appears to be in a pro-inflammatory state, whereas in older and female cats the myocardium exhibits a reduced inflammatory reaction to systemic disease. Age-associated cardiac remodelling seems to be influenced by non-hormonal factors in male and female cats., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The anterior cruciate ligament (ACL) is known to have a poor self-healing ability. In contrast, the medial collateral ligament (MCL) can heal relatively well and restore the joint function. Transforming growth factor-beta1 (TGF-β1) is considered to be an important chemical mediator in the wound healing of the ligaments. While the role of TGF-β1-induced expressions of the lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs), which respectively facilitate the extracellular matrix (ECM) repair and degradation, is poorly understood. In this study, we used equibiaxial stretch chamber to mimic mechanical injury of ACL and MCL fibroblasts, and aimed to determine the intrinsic differences between ACL and MCL by characterizing the differential expressions of LOXs and MMPs in response to TGF-β1 after mechanical injury. By using semi-quantitative PCR, quantitative real-time PCR, western blot and zymography, we found TGF-β1 induced injured MCL to express more LOXs than injured ACL (up to 1.85-fold in LOX, 2.21-fold in LOXL-1, 1.71-fold in LOXL-2, 2.52-fold in LOXL-3 and 3.32-fold in LOXL-4). Meanwhile, TGF-β1 induced injured ACL to express more MMPs than injured MCL fibroblasts (up to 2.33-fold in MMP-1, 2.45-fold in MMP-2, 1.89-fold in MMP-3 and 1.50-fold in MMP-12). The further protein results were coincident with the gene expressions above. The different expressions of LOXs and MMPs inferred the intrinsic differences between ACL and MCL, and the intrinsic differences could help to explain their differential healing abilities., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

During inflammation, myeloperoxidase (MPO) released by circulating leukocytes accumulates within the subendothelial matrix by binding to and transcytosing the vascular endothelium. Oxidative reactions catalyzed by subendothelial-localized MPO are implicated as a cause of endothelial dysfunction in vascular disease. While the subendothelial matrix is a key target for MPO-derived oxidants during disease, the implications of this damage for endothelial morphology and signaling are largely unknown. We found that endothelial-transcytosed MPO produced hypochlorous acid (HOCl) that reacted locally with the subendothelial matrix and induced covalent cross-linking of the adhesive matrix protein fibronectin. Real-time biosensor and live cell imaging studies revealed that HOCl-mediated matrix oxidation triggered rapid membrane retraction from the substratum and adjacent cells (de-adhesion). De-adhesion was linked with the alteration of Tyr-118 phosphorylation of paxillin, a key adhesion-dependent signaling process, as well as Rho kinase-dependent myosin light chain-2 phosphorylation. De-adhesion dynamics were dependent on the contractile state of cells, with myosin II inhibition with blebbistatin attenuating the rate of membrane retraction. Rho kinase inhibition with Y-27632 also conferred protection, but not during the initial phase of membrane retraction, which was driven by pre-existing actomyosin tensile stress. Notably, diversion of MPO from HOCl production by thiocyanate or nitrite attenuated de-adhesion and associated signaling responses, despite the latter substrate supporting MPO-catalyzed fibronectin nitration. These data show that subendothelial-localized MPO employs a novel "outside-in" mode of redox signaling, involving HOCl-mediated matrix oxidation. These MPO-catalyzed oxidative events are likely to play a previously unrecognized role in altering endothelial integrity and signaling during inflammatory vascular disorders., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15 mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart., (Published by Elsevier Inc.)

Regulation of the extracellular matrix by proteases and protease inhibitors is a fundamental biological process for normal growth, development and repair in the CNS. Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are the major extracellular-degrading enzymes. Two other enzyme families, a disintegrin and metalloproteinase (ADAM), and the serine proteases, plasminogen/plasminogen activator (P/PA) system, are also involved in extracellular matrix degradation. Normally, the highly integrated action of these enzyme families remodels all of the components of the matrix and performs essential functions at the cell surface involved in signaling, cell survival, and cell death. During the inflammatory response induced in infection, autoimmune reactions and hypoxia/ischemia, abnormal expression and activation of these proteases lead to breakdown of the extracellular matrix, resulting in the opening of the blood-brain barrier (BBB), preventing normal cell signaling, and eventually leading to cell death. There are several key MMPs and ADAMs that have been implicated in neuroinflammation: gelatinases A and B (MMP-2 and -9), stromelysin-1 (MMP-3), membrane-type MMP (MT1-MMP or MMP-14), and tumor necrosis factor-alpha converting enzyme (TACE). In addition, TIMP-3, which is bound to the cell surface, promotes cell death and impedes angiogenesis. Inhibitors of metalloproteinases are available, but balancing the beneficial and detrimental effects of these agents remains a challenge.

Mechanical loads are essential to normal bone growth, but excessive loads can lead to progressive deformities. In addition, growth plate extracellular matrix remodelling is essential to regulate the normal longitudinal bone growth process and to ensure physiological bone mineralization. In order to investigate the effects of static compression on growth plate extracellular matrix using an in vivo animal model, a loading device was used to precisely apply a compressive stress of 0.2 MPa for two weeks on the seventh caudal vertebra (Cd7) of rats during the pubertal growth spurt. Control, sham and loaded groups were studied. Growth modulation was quantified based on calcein labelling, and three matrix components (type II and X collagens, and aggrecan) were assessed using immunohistochemistry/safranin-O staining. As well, extracellular matrix components and enzymes (MMP-3 and -13, ADAMTS-4 and -5) were studied by qRT-PCR. Loading reduced Cd7 growth by 29% (p<0.05) and 15% (p=0.07) when compared to controls and shams respectively. No significant change could be observed in the mRNA expression of collagens and the proteolytic enzyme MMP-13. However, MMP-3 was significantly increased in the loaded group as compared to the control group (p<0.05). No change was observed in aggrecan and ADAMTS-4 and -5 expression. Low immunostaining for type II and X collagens was observed in 83% of the loaded rats as compared to the control rats. This in vivo study shows that, during pubertal growth spurt, two-week static compression reduced caudal vertebrae growth rates; this mechanical growth modulation occurred with decreased type II and X collagen proteins in the growth plate.

Extracellular superoxide dismutase (EC-SOD) contributes only a small fraction to total SOD activity in the heart but is strategically located to scavenge free radicals in the extracellular compartment. EC-SOD expression is decreased in myocardial-infarction (MI)-induced heart failure, but whether EC-SOD can abrogate oxidative stress or modify MI-induced ventricular remodeling has not been previously studied. Consequently, the effects of EC-SOD gene deficiency (EC-SOD KO) on left ventricular (LV) oxidative stress, hypertrophy, and fibrosis were studied in EC-SOD KO and wild-type mice under control conditions, and at 4 and 8 weeks after permanent coronary artery ligation. EC-SOD KO had no detectable effect on LV function in normal hearts but caused small but significant increases of LV fibrosis. At 8 weeks after MI, EC-SOD KO mice developed significantly more LV hypertrophy (LV mass increased 1.64-fold in KO mice compared to 1.35-fold in wild-type mice; p<0.01) and more fibrosis and myocyte hypertrophy which was more prominent in the peri-infarct region than in the remote myocardium. EC-SOD KO mice had greater increases of nitrotyrosine in the peri-infarct myocardium, and this was associated with a greater reduction of LV ejection fraction, a greater decrease of sarcoplasmic or endoplasmic reticulum calcium2+ ATPase, and a greater increase of atrial natriuretic peptide in the peri-infarct zone compared to wild-type mice. EC-SOD KO was associated with more increases of phosphorylated p38 (p-p38(Thr180/Tyr182)), p42/44 extracellular signal-regulated kinase (p-Erk(Thr202/Tyr204)), and c-Jun N-terminal kinase (p-JNK(Thr183/Tyr185)) both under control conditions and after MI, indicating that EC-SOD KO increases activation of mitogen-activated protein kinase signaling pathways. These findings demonstrate that EC-SOD plays an important role in protecting the heart against oxidative stress and infarction-induced ventricular hypertrophy.

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In particular, the gelatinases MMP-2 and MMP-9 contribute to disruption of the blood-brain barrier and hemorrhagic transformation following ischemic injury. In addition to extracellular matrix degradation, MMPs may directly regulate neuronal cell death through mechanisms that are not completely understood. Here we describe the spatio-temporal distribution of activated MMP-2 and MMP-9 in the brain of rats subjected to 2 h middle cerebral artery occlusion (MCAo) followed by different periods of reperfusion (15 min, 2 h, 6 h and 22 h). By in situ zymography we have observed that gelatinases become activated 15 min and 2 h after the beginning of reperfusion in the ischemic core and penumbra, respectively. In situ zymography signal broadly co-localized with NeuN-positive cells, thus suggesting that proteolysis mainly occurs in neurons. Gelatinolytic activity was mainly detected in cell nuclei, marginally appearing in the cytosol only at later stages following the insult; we did not detect variations in gelatinolysis in the extracellular matrix. Finally, we report that pharmacological inhibition of MMPs by N-[(2R)-2-(hydroxamidocarbonyl-methyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001) significantly reduces brain infarct volume induced by transient MCAo. Taken together our data underscore the crucial role of gelatinases during the early stages of reperfusion and further extend previous observations documenting the detrimental role of these enzymes in the pathophysiology of brain ischemia.

Covalent attachment of synthetic and biological molecules to tissue surfaces can be used to enhance local drug delivery, reduce adhesions after surgery, and attach reconstructive biomaterials and tissue-engineered matrices to tissues. We present here a mild approach to coupling polymers to tissue surfaces through an enzyme catalyzed reaction between peptide modified polymer and native protein components of the tissue extracellular matrix (ECM). Tissue transglutaminase (tTG), a Ca2+-dependent enzyme that catalyzes the reaction between lysine and glutamine residues to form a epsilon(gamma-glutaminyl) lysine isopeptide bond, was incubated with cartilage in the presence of lysine (FKG-NH2) and glutamine (GQQQLG-NH2) peptides as well as peptide functionalized poly(ethylene glycol) (PEG). Immunohistochemistry was used to detect the presence of covalently bound PEG polymer at the tissue surface as well as to a depth of as much as 10 microm below the surface. Collagen II, fibronectin, osteopontin and osteonectin were found to react with the peptides and peptide modified PEG in the presence of tTG in solution, suggesting these cartilage ECM components as being substrates in the tissue reaction. The results illustrate the use of tTG as a simple, effective and biologically compatible method of coupling synthetic and biological molecules to cartilage and other tissues containing ECM proteins that are substrates of tTG.

The in vivo application of engineered matrices in human wound healing processes is often hampered by the slow rate of vascularization. Therefore much research is directed towards enhancing the angiogenic properties of such matrices. One approach for enhancing the vascularization is the incorporation of angiogenic growth factors. Recently, we and others have reported on immobilizing such factors into collagen matrices either by covalent attachment or by physical binding to covalently incorporated heparin. Especially the latter procedure has been shown to lead to substantial increase rates in vascularization in in vivo experiments. The increases have been proposed to depend on the sustained release of the incorporated angiogenic growth factors from the heparinized collagen matrices. In this paper, we report on investigations to study the release of vascular endothelial growth factor (VEGF) from collagen matrices under conditions which mimic potential in vivo situations. Relevant proteinase concentrations were deduced from in vitro experiments in which we evaluated the secretion of selected matrix metalloproteinases from fibroblasts in contact with collagen. The release of VEGF from non-modified, cross-linked and heparinized collagen matrices in the absence and in the presence of varying concentrations of proteinases was then determined by ELISA and liquid scintillation counting. The release behaviour appears to be controlled by both the presence of heparin and the levels of proteinases applied. Experiments with matrices containing radioactively labelled heparin suggest that VEGF release results from the consecutive and simultaneous release of three species of VEGF molecules that differ in their binding affinities to the differently modified collagen matrices. The species binding specifically to heparin most likely accounts for the previously observed increases in angiogenic potential between loading VEGF to non-heparinized and heparinized collagen matrices.

Dissociated glucocorticoids are synthetic ligands of the glucocorticoid receptor (GR) and which discriminate between transrepression and transactivation. These compounds were predicted to have large therapeutic benefits when compared to conventional glucocorticoids because of reduced side effects. In this study, we compared the transrepressive properties of different dissociated glucocorticoids on the interleukin-1beta (IL-1beta)-activated metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA) expression in rat mesangial cells (MC). Both proteinases regulate the turnover of extracellular matrix (ECM). We demonstrate that the GR agonist RU 24858, equipotent to dexamethasone (DEX), exhibited strong suppressive effects on the IL-1beta-induced MMP-9 and tPA mRNA levels concomitant with an inhibition of corresponding enzyme activities. In contrast, RU 24782 and RU 40066 exhibited weaker inhibitory activities on both proteinases. Mechanistically, the changes in MMP-9 expression level by different RUs were accompanied by an inhibition of cytokine-induced promoter activity indicating that the inhibition occurs on a transcriptional level. In parallel to the reduction in mRNA levels, we observed an attenuation of cytokine-induced DNA binding of nuclear factor kappa B (NF-kappaB) and reduced contents of the p65 subunit of NF-kappaB within cell nuclei. Along with these transrepressive activities RU 24858, RU 24782 and RU 40066 displayed similar transactivation potentials as indicated by induction of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) reporter gene and by induced expression level of plasminogen activator inhibitor 1 (PAI-1). Interestingly, the different RUs affected the expression of cAMP-induced tPA and inducible NO synthase with the same potency as the IL-1beta-induced protease expression thus indicating that these compounds equipotently modulate cytokine- and cAMP-driven gene expression.

The pathogenesis of many anterior segment disorders and ocular complications following surgery are secondary to the wound healing response. The extent of clinical damage observed is closely related to the amount of scarring and tissue contraction. Matrix metalloproteinases (MMPs) are a family of enzymes that play a vital role in all stages of the wound healing process. They degrade all extracellular matrix components and also have the ability to synthesize collagen and extracellular matrix members, and are therefore important in the remodeling of a wound. Overexpression of MMPs results in excessive extracellular matrix degradation, leading to tissue destruction and loss of organ function. In the case of the anterior segment, this may mean the loss of visual function. This review focuses on the role MMPs have in the development of various anterior segment disorders. The importance of MMPs in the wound healing response and its potential modulation to manipulate the scarring response is being recognized, and current developments will be described.

The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is highly expressed in the extracellular matrix of lung tissue and is believed to protect the lung from oxidative damage that results in diseases such as pulmonary fibrosis. This study tests the hypothesis that proteolytic removal of the heparin-binding domain of EC-SOD results in clearance of the enzyme from the extracellular matrix of pulmonary tissues and leads to a loss of antioxidant protection. Using a polyclonal antibody to mouse EC-SOD, the immunodistribution of EC-SOD in normal and bleomycin-injured lungs was examined. EC-SOD labeling was strong in the matrix of vessels, airways, and alveolar surfaces and septa in control lungs. At 2 d post-treatment, a slight increase in EC-SOD staining was evident. In contrast, lungs examined 4 or 7 d post-treatment, showed an apparent loss of EC-SOD from the matrix and surface of alveolar septa. Notably, at 7 d post-treatment, the truncated form of EC-SOD was found in the bronchoalveolar lavage fluid of bleomycin-treated mice, suggesting that EC-SOD is being removed from the extracellular matrix through proteolysis. However, loss of EC-SOD through proteolysis did not correlate with a decrease in overall pulmonary EC-SOD activity. The negligible effect on EC-SOD activity may reflect the large influx of intensely staining inflammatory cells at day 7. These results indicate that injuries leading to pulmonary fibrosis have a significant effect on EC-SOD distribution due to proteolytic removal of the heparin-binding domain and may be important in enhancing pulmonary injuries by altering the oxidant/antioxidant balance in alveolar interstitial spaces.

One of the greatest biomedical breakthroughs of the twentieth century was the discovery of endothelium-derived relaxing factor and its identification as nitric oxide (NO). NO has received special attention ever since: besides its potent vasodilatory and vasoprotective effects, NO was identified as a key player in innate immunity and was found to act as an unconventional type of neurotransmitter. This article focuses on mechanisms of NO signalling that form the basis of functional cell responses to accommodate changes in the cellular microenvironment. Redox-based regulation of signal transduction and, on a more long-term scale, changes in gene expression will be exemplified by NO-modulation of matrix components and matrix-metabolizing enzymes. It seems to be a safe bet that ongoing analyses of NO signalling and gene expression will provide a wealth of promising therapeutic targets in human diseases.

Objectives: The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathologic oral processes such as periodontal tissue destruction, root caries, tumour invasion and temporomandibular joint disorders. The aim of this work was to test the effect of Zn, Cu, Sn and Hg ions on the activity of the major gingival gelatinolytic MMPs., Methods: Gingival explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers containing different metal ion concentrations. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation with specific antibodies. The eletrophoretic bands were scanned and the transmittance values were analyzed with the Sigmagel software (Sigma)., Results: ZnSO4 was a strong inhibitor of MMP-2 (I50 = 15 microM) and MMP-9 (I50 = 40 microM), whereas CuSO4, HgSO4 and SnCl2 showed less efficient inhibition potential., Significance: Our findings show that the activity of oral tissue MMPs may be modulated by metal ions present in the oral environment. Therefore, the accumulation of metals in connective tissue may interfere with the formation and resorption of the extracellular matrix components.

Interest in lipases from microorganisms, animals, and plants has greatly increased in the past decade due to their applications in biotransformations and organic syntheses. We are reporting the purification and characterization of two lipases from the fungus, Ophiostoma piliferum, a saprophytic organism commonly found on wood. A major and a minor lipase have been co-purified by hydrophobic interaction chromatography on octyl sepharose FF, followed by ion exchange chromatography on Q sepharose FF. The lipases bound very tightly to octyl sepharose resulting in greater than 100-fold purification in this one step. The major lipase has a molecular weight of approximately 60 kDa, a pI of 3.79, and is glycosylated as determined by PAS staining. The minor lipase, which composes 10% of the total protein, has a pI of 3.6, and molecular weight of approximately 52 kDa and did not stain with the PAS reagent. Deglycosylation of the major lipase produced two proteins of lower molecular weight, a 55 kDa protein and a 52 kDa protein. The deglycosylated protein at 52 kDa co-migrates with the minor lipase on SDS-PAGE gels. N-terminal amino acid sequencing of the major and minor lipases indicated both lipases have the same N-termini and MALDI-TOF mass spectral analysis showed similar peptide patterns. Available data indicate that the lipases are derived from the same protein and appear to differ in their post-translational modification as evidenced by their pIs and molecular weight difference. The pH rate profile and thermal stability were determined for the purified O. piliferum lipase and were consistent with a mesophilic lipase. In aqueous solution, the lipases exhibited a higher rate of hydrolysis for p-nitrophenylbutyrate (C4) than for p-nitrophenylstearate (C18), which is an unexpected result.

An approach utilizing robotics (automation) for the rapid and reliable determination of protease inhibitor concentration in rat plasma samples is described. The bioassay protocol using an immobilized peptide substrate allows high sample throughput, compatible with parallel synthesis/SAR development strategy.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs), is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumour cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

Studies suggest that the interplay between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinases (TIMPs) is an important mediator of tumour invasion and metastasis. Using immunohistochemistry, 40 specimens of colorectal cancer were examined for the presence of TIMP-1 and the MMPs, stromelysin, gelatinases A and B and interstitial collagenase. Neither enzyme nor TIMP-1 was detected in histologically normal mucosa. Within malignant tissue, stromelysin and gelatinase A were conspicuously absent in tumor cells but were immunolocalized to the extracellular matrix and for gelatinase A also to peritumoural fibroblast-like cells. Gelatinase B was confined to polymorphonuclear leucocytes. Interstitial collagenase was not identified. TIMP-1 was present in only three of the 40 tumours within the malignant stroma. These observations suggest that the mesenchymal elements of colorectal carcinomas, by acting as a source of MMPs and TIMPs, may modulate tumour invasion.

1. Pig synovial fibroblasts in culture were studied to determine if they were an easily reproducible model system for studying the actions of cytokines and growth factors on human synovial cells. The biochemical analyses were conducted by activity assays, enzymography and Northern blot. 2. Human recombinant interleukin-1 alpha, basic fibroblast growth factor and transforming growth factor-beta 1 were studied in combinations because of their known involvement in controlling tissue remodelling. 3. The response of pig fibroblasts to these agents, in terms of the expression of matrix metalloproteinases (collagenase, gelatinase and stromelysin) and their inhibitors (TIMPs), show that they behave similarly enough to human cells for use when supplies of human primary cells are unavailable.

An inhibitory effect of alkaline phosphatase (LP) activity on short and long term fetal rat calvaria cell cultures was recorded with both acidic fibroblast growth factor (aFGF) and epidermal growth factor (EGF) at a concentration of 30 ng ml-1. This inhibition was well correlated with the nodule number on long term culture, except for EGF treatment in subconfluent cell culture.

1. A neutral thiol protease was purified from the culture filtrate of newly excysted metacercariae of Paragonimus westermani to homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a monomeric form with mol. wt 22,000. 2. It expressed activity on t-butyloxycarbonyl-valyl-leucyl-lysyl-4-methyl-coumaryl-7-amide in the presence of cysteine at an optimal pH of 7.5, and also the activity was significantly affected by thiol protease inhibitors, indicating that the enzyme belongs to a neutral thiol protease family. 3. The enzyme hydrolyzed protein substrates, azocoll, casein and fluorescein isothiocyanate-labeled collagen, and showed low specificity toward hemoglobin, but no activity with elastin Congo Red and bovine serum albumin. 4. Catalytic property on fluorogenic substrates demonstrated that the enzyme cleaved preferentially the carboxylic side of the basic residue in N-substituted peptides.