Integrated Metabolomics and Proteomics of Symptomatic and Early Presymptomatic States of Colitis.

Colitis has a multifactorial pathogenesis with a strong cross-talk among microbiota, hypoxia, and tissue metabolism. Here, we aimed to characterize the molecular signature of the disease in symptomatic and presymptomatic stages of the inflammatory process at the tissue and fecal level. The study is based on two different murine models for colitis, and HR-MAS NMR on "intact" colon tissues and LC-MS/MS on colon tissue extracts were used to derive untargeted metabolomics and proteomics information, respectively. Solution NMR was used to derive metabolomic profiles of the fecal extracts. By combining metabolomic and proteomic analyses of the tissues, we found increased anaerobic glycolysis, accompanied by an altered citric acid cycle and oxidative phosphorylation in inflamed colons; these changes associate with inflammation-induced hypoxia taking place in colon tissues. Different colitis states were also characterized by significantly different metabolomic profiles of fecal extracts, attributable to both the dysbiosis characteristic of colitis as well as the dysregulated tissue metabolism. Strong and distinctive tissue and fecal metabolomic signatures can be detected before the onset of symptoms. Therefore, untargeted metabolomics of tissues and fecal extracts provides a comprehensive picture of the changes accompanying the disease onset already at preclinical stages, highlighting the diagnostic potential of global metabolomics for inflammatory diseases.

Proteolytic Vesicles Derived from Salmonella enterica Serovar Typhimurium-Infected Macrophages: Enhancing MMP-9-Mediated Invasion and EV Accumulation.

Proteolysis of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a crucial role in the immune response to bacterial infections. Here we report the secretion of MMPs associated with proteolytic extracellular vesicles (EVs) released by macrophages in response to Salmonella enterica serovar Typhimurium infection. Specifically, we used global proteomics, in vitro, and in vivo approaches to investigate the composition and function of these proteolytic EVs. Using a model of S. Typhimurium infection in murine macrophages, we isolated and characterized a population of small EVs. Bulk proteomics analysis revealed significant changes in protein cargo of naïve and S. Typhimurium-infected macrophage-derived EVs, including the upregulation of MMP-9. The increased levels of MMP-9 observed in immune cells exposed to S. Typhimurium were found to be regulated by the toll-like receptor 4 (TLR-4)-mediated response to bacterial lipopolysaccharide. Macrophage-derived EV-associated MMP-9 enhanced the macrophage invasion through Matrigel as selective inhibition of MMP-9 reduced macrophage invasion. Systemic administration of fluorescently labeled EVs into immunocompromised mice demonstrated that EV-associated MMP activity facilitated increased accumulation of EVs in spleen and liver tissues. This study suggests that macrophages secrete proteolytic EVs to enhance invasion and ECM remodeling during bacterial infections, shedding light on an essential aspect of the immune response.

Tumor-reactive antibodies evolve from non-binding and autoreactive precursors.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Conformation-Specific Inhibitory Anti-MMP-7 Monoclonal Antibody Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Chemotherapeutic Cell Kill.

Matrix metalloproteases (MMPs) undergo post-translational modifications including pro-domain shedding. The activated forms of these enzymes are effective drug targets, but generating potent biological inhibitors against them remains challenging. We report the generation of anti-MMP-7 inhibitory monoclonal antibody (GSM-192), using an alternating immunization strategy with an active site mimicry antigen and the activated enzyme. Our protocol yielded highly selective anti-MMP-7 monoclonal antibody, which specifically inhibits MMP-7's enzyme activity with high affinity (IC50 = 132 ± 10 nM). The atomic model of the MMP-7-GSM-192 Fab complex exhibited antibody binding to unique epitopes at the rim of the enzyme active site, sterically preventing entry of substrates into the catalytic cleft. In human PDAC biopsies, tissue staining with GSM-192 showed characteristic spatial distribution of activated MMP-7. Treatment with GSM-192 in vitro induced apoptosis via stabilization of cell surface Fas ligand and retarded cell migration. Co-treatment with GSM-192 and chemotherapeutics, gemcitabine and oxaliplatin elicited a synergistic effect. Our data illustrate the advantage of precisely targeting catalytic MMP-7 mediated disease specific activity.

Distinct extracellular-matrix remodeling events precede symptoms of inflammation.

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases.

Correction: Cardiac remodeling secondary to chronic volume overload is attenuated by a novel MMP9/2 blocking antibody.

[This corrects the article DOI: 10.1371/journal.pone.0231202.].

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates.

The bell-shaped members of the Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in an aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the 'somersaulting' locomotion in Hydra We applied atomic force microscopy to measure the local mechanical properties of Hydra's body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersaulting primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix polymerization impairs the 'somersault' movement. These results provide a mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

Different Inhibitors of Aß42-Induced Toxicity Have Distinct Metal-Ion Dependency.

Oligomers of amyloid ß-protein (Aß) are thought to be the proximal toxic agents initiating the neuropathologic process in Alzheimer's disease (AD). Therefore, targeting the self-assembly and oligomerization of Aß has been an important strategy for designing AD therapeutics. In parallel, research into the metallobiology of AD has shown that Zn2+ can strongly modulate the aggregation of Aß in vitro and both promote and inhibit the neurotoxicity of Aß, depending on the experimental conditions. Thus, successful inhibitors of Aß self-assembly may have to inhibit the toxicity not only of Aß oligomers themselves but also of Aß-Zn2+ complexes. However, there has been relatively little research investigating the effects of Aß self-assembly and toxicity inhibitors in the presence of Zn2+. Our group has characterized previously a series of Aß42 C-terminal fragments (CTFs), some of which have been shown to inhibit Aß oligomerization and neurotoxicity. Here, we asked whether three CTFs shown to be potent inhibitors of Aß42 toxicity maintained their activity in the presence of Zn2+. Biophysical analysis showed that the CTFs had different effects on oligomer, ß-sheet, and fibril formation by Aß42-Zn2+ complexes. However, cell viability experiments in differentiated PC-12 cells incubated with Aß42-Zn2+ complexes in the absence or presence of these CTFs showed that the CTFs completely lost their inhibitory activity in the presence of Zn2+ even when applied at 10-fold excess relative to Aß42. In light of these results, we tested another inhibitor, the molecular tweezer CLR01, which coincidentally had been shown to have a high affinity for Zn2+, suggesting that it could disrupt both Aß42 oligomerization and Aß42-Zn2+ complexation. Indeed, we found that CLR01 effectively inhibited the toxicity of Aß42-Zn2+ complexes. Moreover, it did so at a lower concentration than needed for inhibiting the toxicity of Aß42 alone. In agreement with these results, CLR01 inhibited ß-sheet and fibril formation in Aß42-Zn2+ complexes. Our data suggest that, for the development of efficient therapeutic agents, inhibitors of Aß self-assembly and toxicity should be examined in the presence of relevant metal ions and that molecular tweezers may be particularly attractive candidates for therapy development.

LOXL2 Inhibition Paves the Way for Macrophage-Mediated Collagen Degradation in Liver Fibrosis.

Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM) proteins and enzymes, especially fibrillary collagens, and represents a major cause of morbidity and mortality worldwide. Lysyl oxidases (LOXs) drive covalent crosslinking of collagen fibers, thereby promoting stabilization and accumulation of liver fibrosis while limiting its resolution. Here we show in a carbon tetrachloride (CCl4)-induced liver fibrosis murine model that treatment with a novel anti-lysyl oxidase like 2 (LOXL2) neutralizing antibody, which targets extracellular LOXL2, significantly improves fibrosis resolution. LOXL2 inhibition following the onset of fibrosis accelerated and augmented collagen degradation. This was accompanied by increased localization of reparative monocyte-derived macrophages (MoMFs) in the proximity of fibrotic fibers and their representation in the liver. These cells secreted collagenolytic matrix metalloproteinases (MMPs) and, in particular, the membrane-bound MT1-MMP (MMP-14) collagenase. Inducible and selective ablation of infiltrating MoMFs negated the increased "on-fiber" accumulation of MMP-14-expressing MoMFs and the accelerated collagenolytic activity observed in the anti-LOXL2-treated mice. Many studies of liver fibrosis focus on preventing the progression of the fibrotic process. In contrast, the therapeutic mechanism of LOXL2 inhibition presented herein aims at reversing existing fibrosis and facilitating endogenous liver regeneration by paving the way for collagenolytic macrophages.

Cardiac remodeling secondary to chronic volume overload is attenuated by a novel MMP9/2 blocking antibody.

OBJECTIVE: Monoclonal antibody derivatives are promising drugs for the treatment of various diseases due to their high matrix metalloproteinases (MMP) active site specificity. We studied the effects of a novel antibody, SDS3, which specifically recognizes the mature active site of MMP9/2 during ventricular remodeling progression in a mouse model of chronic volume overload (VO). METHODS: VO was induced by creating an aortocaval fistula (ACF) in 10- to 12-week-old C57BL male mice. The VO-induced mice were treated with either vehicle control (PBS) or with SDS3 twice weekly by intraperitoneal (ip) injection. The relative changes in cardiac parameters between baseline (day 1) and end-point (day 30), were evaluated by echocardiography. The effects of SDS3 treatment on cardiac fibrosis, cardiomyocyte volume, and cardiac inflammation were tested by cardiac staining with Masson's trichrome, wheat Germ Agglutinin (WGA), and CD45, respectively. Serum levels of TNFα and IL-6 with and without SDS3 treatment were tested by ELISA. RESULTS: SDS3 significantly reduced cardiac dilatation, left ventricular (LV) mass, and cardiomyocyte hypertrophy compared to the vehicle treated animals. The antibody also reduced the heart-to-body weight ratio of the ACF animals to values comparable to those of the controls. Interestingly, the SDS3 group underwent significant reduction of cardiac inflammation and pro-inflammatory cytokine production, indicating a regulatory role for MMP9/2 in tissue remodeling, possibly by tumor necrosis factor alpha (TNFα) activation. In addition, significant changes in the expression of proteins related to mitochondrial function were observed in ACF animals, these changes were reversed following treatment with SDS3. CONCLUSION: The data suggest that MMP9/2 blockage with SDS3 attenuates myocardial remodeling associated with chronic VO by three potential pathways: downregulating the extracellular matrix proteolytic cleavage, reducing the cardiac inflammatory responses, and preserving the cardiac mitochondrial structure and function.

The ECM path of senescence in aging: components and modifiers.

The extracellular matrix (ECM) is a key noncellular component in all organs and tissues. It is composed of a large number of proteins including collagens, glycoproteins (GP), and ECM-associated proteins, which show diversity of biochemical and biophysical functions. The ECM is dynamic both in normal physiology of tissues and under pathological conditions. One cellular phenomenon associated with changes in both ECM components expression and in ECM remodeling enzymes secretion is cellular senescence. It represents a stable state form of cell cycle arrest induced in proliferating cells by various forms of stress. Short-term induction of senescence is essential for tumor suppression and tissue repair. However, long-term presence of senescent cells in tissues may have a detrimental role in promoting tissue damage and aging. Up to date, there is insufficient knowledge about the interplay between the ECM and senescence cells. Since changes in the ECM occur in many physiological and pathological conditions in which senescent cells are present, a better understanding of ECM-senescence interactions is necessary. Here, we will review the functions of the different ECM components and will discuss the current knowledge about their regulation in senescent cells and their influence on the senescence state.

MMP9 modulates the metastatic cascade and immune landscape for breast cancer anti-metastatic therapy.

Metastasis, the main cause of cancer-related death, has traditionally been viewed as a late-occurring process during cancer progression. Using the MMTV-PyMT luminal B breast cancer model, we demonstrate that the lung metastatic niche is established early during tumorigenesis. We found that matrix metalloproteinase 9 (MMP9) is an important component of the metastatic niche early in tumorigenesis and promotes circulating tumor cells to colonize the lungs. Blocking active MMP9, using a monoclonal antibody specific to the active form of gelatinases, inhibited endogenous and experimental lung metastases in the MMTV-PyMT model. Mechanistically, inhibiting MMP9 attenuated migration, invasion, and colony formation and promoted CD8+ T cell infiltration and activation. Interestingly, primary tumor burden was unaffected, suggesting that inhibiting active MMP9 is primarily effective during the early metastatic cascade. These findings suggest that the early metastatic circuit can be disrupted by inhibiting active MMP9 and warrant further studies of MMP9-targeted anti-metastatic breast cancer therapy.

Exosomes as a storehouse of tissue remodeling proteases and mediators of cancer progression.

Rapidly increasing scientific reports of exosomes and their biological effects have improved our understanding of their cellular sources and their cell-to-cell communication. These nano-sized vesicles act as potent carriers of regulatory bio-macromolecules and can induce regulatory functions by delivering them from its source to recipient cells. The details of their communication network are less understood. Recent studies have shown that apart from delivering its cargo to the cells, it can directly act on extracellular matrix (ECM) proteins and growth factors and can induce various remodeling events. More importantly, exosomes carry many surface-bound proteases, which can cleave different ECM proteins and carbohydrates and can shed cell surface receptors. These local extracellular events can modulate signaling cascades, which consequently influences the whole tissue and organ. This review aims to highlight the critical roles of exosomal proteases and their mechanistic insights within the cellular and extracellular environment.

Next generation matrix metalloproteinase inhibitors - Novel strategies bring new prospects.

Enzymatic proteolysis of cell surface proteins and extracellular matrix (ECM) is critical for tissue homeostasis and cell signaling. These proteolytic activities are mediated predominantly by a family of proteases termed matrix metalloproteinases (MMPs). The growing evidence in recent years that ECM and non-ECM bioactive molecules (e.g., growth factors, cytokines, chemokines, on top of matrikines and matricryptins) have versatile functions redefines our view on the roles matrix remodeling enzymes play in many physiological and pathological processes, and underscores the notion that ECM proteolytic reaction mechanisms represent master switches in the regulation of critical biological processes and govern cell behavior. Accordingly, MMPs are not only responsible for direct degradation of ECM molecules but are also key modulators of cardinal bioactive factors. Many attempts were made to manipulate ECM degradation by targeting MMPs using small peptidic and organic inhibitors. However, due to the high structural homology shared by these enzymes, the majority of the developed compounds are broad-spectrum inhibitors affecting the proteolytic activity of various MMPs and other zinc-related proteases. These inhibitors, in many cases, failed as therapeutic agents, mainly due to the bilateral role of MMPs in pathological conditions such as cancer, in which MMPs have both pro- and anti-tumorigenic effects. Despite the important role of MMPs in many human diseases, none of the broad-range synthetic MMP inhibitors that were designed have successfully passed clinical trials. It appears that, designing highly selective MMP inhibitors that are also effective in vivo, is not trivial. The challenges related to designing selective and effective metalloprotease inhibitors, are associated in part with the aforesaid high structural homology and the dynamic nature of their protein scaffolds. Great progress was achieved in the last decade in understanding the biochemistry and biology of MMPs activity. This knowledge, combined with lessons from the past has drawn new "boundaries" for the development of the next-generation MMP inhibitors. These novel agents are currently designed to be highly specific, capable to discriminate between the homologous MMPs and ideally administered as a short-term topical treatment. In this review we discuss the latest progress in the fields of MMP inhibitors in terms of structure, function and their specific activity. The development of novel highly specific inhibitors targeting MMPs paves the path to study complex biological processes associated with ECM proteolysis in health and disease. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Dielectric Relaxation of Hydration Water in Native Collagen Fibrils.

The dielectric relaxation of hydrated collagen powders was studied over a wide temperature and frequency range. We revealed two mechanisms of dielectric relaxation in hydration water that are driven by the migration of ionic and orientation defects. At high water fractions in powders (h > 0.2), the hydration shell around the collagen triple helixes presents a spatial H-bonded network consisting of structural water bridges and cleft water channels. These two water phases provide the long-range paths for proton hopping and orientation defect migration. At low water fractions (h < 0.2) and in the hydrated collagen samples after the dehydrothermal treatment, the hydration shell presents localized individual water compartments not connected to one another. In these cases, the relaxation mechanism due to proton hopping either disappears or becomes inhibited by the orientation defect migration.

Extracellular Matrix Proteolysis by MT1-MMP Contributes to Influenza-Related Tissue Damage and Mortality.

Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival. We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.

Distinct biological events generated by ECM proteolysis by two homologous collagenases.

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

Tumor Cell Invasion Can Be Blocked by Modulators of Collagen Fibril Alignment That Control Assembly of the Extracellular Matrix.

Abnormal architectures of collagen fibers in the extracellular matrix (ECM) are hallmarks of many invasive diseases, including cancer. Targeting specific stages of collagen assembly in vivo presents a great challenge due to the involvement of various crosslinking enzymes in the multistep, hierarchical process of ECM build-up. Using advanced microscopic tools, we monitored stages of fibrillary collagen assembly in a native fibroblast-derived 3D matrix system and identified anti-lysyl oxidase-like 2 (LOXL2) antibodies that alter the natural alignment and width of endogenic fibrillary collagens without affecting ECM composition. The disrupted collagen morphologies interfered with the adhesion and invasion properties of human breast cancer cells. Treatment of mice bearing breast cancer xenografts with the inhibitory antibodies resulted in disruption of the tumorigenic collagen superstructure and in reduction of primary tumor growth. Our approach could serve as a general methodology to identify novel therapeutics targeting fibrillary protein organization to treat ECM-associated pathologies. Cancer Res; 76(14); 4249-58. ©2016 AACR.

Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo.

The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell-ECM and cell-cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.

Circular trimers of gelatinase B/matrix metalloproteinase-9 constitute a distinct population of functional enzyme molecules differentially regulated by tissue inhibitor of metalloproteinases-1.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) (EC 3.4.24.35) cleaves many substrates and is produced by most cell types as a zymogen, proMMP-9, in complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1). Natural proMMP-9 occurs as monomers, homomultimers and heterocomplexes, but our knowledge about the overall structure of proMMP-9 monomers and multimers is limited. We investigated biochemical, biophysical and functional characteristics of zymogen and activated forms of MMP-9 monomers and multimers. In contrast with a conventional notion of a dimeric nature of MMP-9 homomultimers, we demonstrate that these are reduction-sensitive trimers. Based on the information from electrophoresis, AFM and TEM, we generated a 3D structure model of the proMMP-9 trimer. Remarkably, the proMMP-9 trimers possessed a 50-fold higher affinity for TIMP-1 than the monomers. In vivo, this finding was reflected in a higher extent of TIMP-1 inhibition of angiogenesis induced by trimers compared with monomers. Our results show that proMMP-9 trimers constitute a novel structural and functional entity that is differentially regulated by TIMP-1.