Contrast enhanced photoacoustic detection of fibrillar collagen in the near infrared region-I.

Fibrillar collagen accumulation emerges as a promising biomarker in several diseases, such as desmoplastic tumors and unstable atherosclerotic plaque. Gold nanorods (GNRs) hold great potential as contrast agents in high-resolution, biomedically safe, and non-invasive photoacoustic imaging (PAI). This study presents the design and characterization of a specialized imaging tool which exploits GNR assisted targeted photoacoustic imaging that is tailored for the identification of fibrillar collagen. In addition to the photoacoustic characterization of collagen in the NIR 1 and 2 regions, we demonstrate the detailed steps of conjugating a decoy to GNRs. This study serves as a proof of concept, that demonstrates that conjugated collagenase-1 (MMP-1) generates a distinct and collagen-specific photoacoustic signal, facilitating real-time visualization in the wavelength range of 700-970 nm (NIR I). As most of the reported studies utilized the endogenous contrast of collagen in the NIR II wavelength that has major limitations to perform in vivo deep tissue imaging, the approach that we are proposing is unique and it highlights the promise of MMP-1 decoy-functionalized GNRs as novel contrast agents for photoacoustic imaging of collagen in the NIR 1 region. To our knowledge this is the first time functionalized GNRs are optimized for the detection of fibrillar collagen and utilized in the field of non-invasive photoacoustic imaging that can facilitate a better prognosis of desmoplastic tumors and broken atherosclerotic plaques.

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.

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.

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.

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.

Inhibition mechanism of membrane metalloprotease by an exosite-swiveling conformational antibody.

Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells.

Ovarian dendritic cells act as a double-edged pro-ovulatory and anti-inflammatory sword.

Ovulation and inflammation share common attributes, including immune cell invasion into the ovary. The present study aims at deciphering the role of dendritic cells (DCs) in ovulation and corpus luteum formation. Using a CD11c-EYFP transgenic mouse model, ovarian transplantation experiments, and fluorescence-activated cell sorting analyses, we demonstrate that CD11c-positive, F4/80-negative cells, representing DCs, are recruited to the ovary under gonadotropin regulation. By conditional ablation of these cells in CD11c-DTR transgenic mice, we revealed that they are essential for expansion of the cumulus-oocyte complex, release of the ovum from the ovarian follicle, formation of a functional corpus luteum, and enhanced lymphangiogenesis. These experiments were complemented by allogeneic DC transplantation after conditional ablation of CD11c-positive cells that rescued ovulation. The pro-ovulatory effects of these cells were mediated by up-regulation of ovulation-essential genes. Interestingly, we detected a remarkable anti-inflammatory capacity of ovarian DCs, which seemingly serves to restrict the ovulatory-associated inflammation. In addition to discovering the role of DCs in ovulation, this study implies the extended capabilities of these cells, beyond their classic immunologic role, which is relevant also to other biological systems.

Zinc-amyloid beta interactions on a millisecond time-scale stabilize non-fibrillar Alzheimer-related species.

The role of zinc, an essential element for normal brain function, in the pathology of Alzheimer's disease (AD) is poorly understood. On one hand, physiological and genetic evidence from transgenic mouse models supports its pathogenic role in promoting the deposition of the amyloid beta-protein (Abeta) in senile plaques. On the other hand, levels of extracellular ("free") zinc in the brain, as inferred by the levels of zinc in cerebrospinal fluid, were found to be too low for inducing Abeta aggregation. Remarkably, the release of transient high local concentrations of zinc during rapid synaptic events was reported. The role of such free zinc pulses in promoting Abeta aggregation has never been established. Using a range of time-resolved structural and spectroscopic techniques, we found that zinc, when introduced in millisecond pulses of micromolar concentrations, immediately interacts with Abeta 1-40 and promotes its aggregation. These interactions specifically stabilize non-fibrillar pathogenic related aggregate forms and prevent the formation of Abeta fibrils (more benign species) presumably by interfering with the self-assembly process of Abeta. These in vitro results strongly suggest a significant role for zinc pulses in Abeta pathology. We further propose that by interfering with Abeta self-assembly, which leads to insoluble, non-pathological fibrillar forms, zinc stabilizes transient, harmful amyloid forms. This report argues that zinc represents a class of molecular pathogens that effectively perturb the self-assembly of benign Abeta fibrils, and stabilize harmful non-fibrillar forms.