7,322 research outputs found
Inhibitory antibodies designed for matrix metalloproteinase modulation
The family of matrix metalloproteinases (MMPs) consists of a set of biological targets that are involved in a multitude of severe pathogenic events such as different forms of cancers or arthritis. Modulation of the target class with small molecule drugs has not led to the anticipated success until present, as all clinical trials failed due to unacceptable side effects or a lack of therapeutic outcome. Monoclonal antibodies offer a tremendous therapeutic potential given their high target selectivity and good pharmacokinetic profiles. For the treatment of a variety of diseases there are already antibody therapies available and the number is increasing. Recently, several antibodies were developed for the selective inhibition of single MMPs that showed high potency and were therefore investigated in in vivo studies with promising results. In this review, we highlight the progress that has been achieved toward the design of inhibitory antibodies that successfully modulate MMP-9 and MMP-14
Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems
Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCysuPA(19-31)) directed against cysteine proteases and the urokinasetype plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCysuPA(19-31) or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OVMZ-6\#8). Additionally, OVMZ 6\#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vectortransfected control cells. Thus, these novel, compact, and smallsize inhibitors directed against up to three different tumorassociated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis
Multiple changes in gene expression in chronic human Achilles tendinopathy
Atlas™ cDNA cell interaction arrays (CLONTECH) were used to examine degenerate tissue from four patients with Achilles tendon disorders, in order to identify changes in expression of genes important in cell–cell and cell–matrix interactions. The greatest difference between normal (post-mortem) and degenerate tissue samples was in the level of MMP-3 (stromelysin) mRNA, which was down-regulated in all the degenerate samples. Quantitative RT-PCR assay of RNA extracted from paired ‘normal’ and degenerate Achilles tendon tissue samples taken from tendons during surgery mirrored the results of the arrays. Levels of MMP-3 mRNA were lower, whereas levels of type-I and type-III collagen mRNAs were significantly higher, in the degenerate compared to the ‘normal’ samples. Immunoblotting of proteins extracted from the same tendon samples showed that three of four normal tissue samples taken from individuals without apparent tendon disorder had much higher levels of MMP-3 protein than ‘normal’ or degenerate samples from patients with tendinosis. We suggest that MMP-3 may play an important role in the regulation of tendon extracellular matrix degradation and tissue remodelling
ASAP-SML: An Antibody Sequence Analysis Pipeline Using Statistical Testing and Machine Learning
Antibodies are capable of potently and specifically binding individual
antigens and, in some cases, disrupting their functions. The key challenge in
generating antibody-based inhibitors is the lack of fundamental information
relating sequences of antibodies to their unique properties as inhibitors. We
develop a pipeline, Antibody Sequence Analysis Pipeline using Statistical
testing and Machine Learning (ASAP-SML), to identify features that distinguish
one set of antibody sequences from antibody sequences in a reference set. The
pipeline extracts feature fingerprints from sequences. The fingerprints
represent germline, CDR canonical structure, isoelectric point and frequent
positional motifs. Machine learning and statistical significance testing
techniques are applied to antibody sequences and extracted feature fingerprints
to identify distinguishing feature values and combinations thereof. To
demonstrate how it works, we applied the pipeline on sets of antibody sequences
known to bind or inhibit the activities of matrix metalloproteinases (MMPs), a
family of zinc-dependent enzymes that promote cancer progression and undesired
inflammation under pathological conditions, against reference datasets that do
not bind or inhibit MMPs. ASAP-SML identifies features and combinations of
feature values found in the MMP-targeting sets that are distinct from those in
the reference sets
Overexpression of the vitronectin v10 subunit in patients with nonalcoholic steatohepatitis: Implications for noninvasive diagnosis of NASH
Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease
(NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause
of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of
liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL).
Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic
approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the
liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads
to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico
analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment
of the ECM prote
in vitro
nectin (VTN) was then selected as a promising circulating biomarker in
discriminating NASH. The analysis of sera of patients provided these major findings: the circulating
VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity
score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits.
In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive
discrimination of NASH from simple steatosi
Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration
Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies
Determination of the role and regulation of matrix metalloproteinase-25 during mouse secondary palate formation
Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression.
Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody.
Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development
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The role of MMP10 in non-small cell Lung cancer, and pharmacological evaluation of its potential as a target for therapeutic intervention. Investigation of the role of MMP10 in the tumour microenvironment of non-small cell lung cancer using gene, protein and mass spectrometry approaches to determine MMP10’s potential in drug development strategies
Non-Small Cell Lung Cancer (NSCLC), which accounts for 80% of all lung cancer cases, is associated with resistance to chemotherapy and poor prognosis. Exploitation of NSCLC-upregulated pathways that can either be targeted by novel therapeutics or used to improve the tumour-delivery of current chemotherapeutics are required. Among the matrix metalloproteinases (MMPs) that are essential for tumour development, MMP10 is a potential candidate as a therapeutic target based on its expression and contribution to NSCLC development. This research aims to explore the expression and functions of MMP10 in the tumour microenvironment of NSCLC and evaluate the potential of MMP10 as a target for therapeutic intervention. Herein, MMP10 expression at gene and protein levels were analysed in a panel of NSCLC cell lines using RT-PCR and Western blotting analysis. To determine MMP10 functional relevance, an in vitro angiogenesis assay using cell conditioned media was carried out. To identify specific peptide sequences for the design of prodrugs rationalised to be MMP10 activated, in vitro substrate cleavage studies were performed using a mass spectrometry approach to differentiate between MMP10 and the structurally similar MMP3. This study demonstrates that MMP10 is highly expressed in NSCLC and that high levels of MMP10 are associated with induction of angiogenesis, a crucial process supporting tumour growth. In addition to the achievement of having been able to differentiate between closely similar MMP3 and MMP10 through carefully monitoring the hydrolysis rate of compound 444259 (a known MMP substrate), data generated herein provides the basis for further studies to exploit MMP10 as a prodrug-activator.Full text was made available at the end of the embargo period, 12th Dec 201
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