Suppression of the fibrotic encapsulation of silicone implants by inhibiting the mechanical activation of pro-fibrotic TGF-ß.

The fibrotic encapsulation of implants involves the mechanical activation of myofibroblasts and of pro-fibrotic transforming growth factor beta 1 (TGF-ß1). Here, we show that both softening of the implant surfaces and inhibition of the activation of TGF-ß1 reduce the fibrotic encapsulation of subcutaneous silicone implants in mice. Conventionally stiff silicones (elastic modulus, ~2 MPa) coated with a soft silicone layer (elastic modulus, ~2 kPa) reduced collagen deposition as well as myofibroblast activation without affecting the numbers of macrophages and their polarization states. Instead, fibroblasts around stiff implants exhibited enhanced intracellular stress, increased the recruitment of αv and ß1 integrins, and activated TGF-ß1 signalling. In vitro, the recruitment of αv integrin to focal adhesions and the activation of ß1 integrin and of TGF-ß were higher in myofibroblasts grown on latency-associated peptide (LAP)-coated stiff silicones than on soft silicones. Antagonizing αv integrin binding to LAP through the small-molecule inhibitor CWHM-12 suppressed active TGF-ß signalling, myofibroblast activation and the fibrotic encapsulation of stiff subcutaneous implants in mice.

Tumor cell MT1-MMP is dispensable for osteosarcoma tumor growth, bone degradation and lung metastasis.

The membrane-anchored matrix metalloprotease MT1-MMP is a potent collagenolytic enzyme with a well-established role in extracellular matrix turnover and cellular invasion into collagen-rich tissues. MT1-MMP is highly expressed in various types of cancer and has been demonstrated to be directly involved in several stages of tumor progression, including primary tumor growth, angiogenesis, invasion and metastasis. Osteosarcoma is the most common type of primary bone cancer. This disease is characterized by invasive tumor growth, leading to extensive bone destruction, and metastasis to the lungs. The tumor cells in human osteosarcoma display a strong expression of MT1-MMP, but the role of MT1-MMP in osteosarcoma progression is currently unknown. In this study, we investigated the role of MT1-MMP during various stages of osteosarcoma development. We utilized an optimized orthotopic murine osteosarcoma model and human osteosarcoma cells in which the MT1-MMP gene was knocked out using CRISPR/Cas9. We observed a strong expression of MT1-MMP in wildtype cells of both primary tumors and lung metastases, but, surprisingly, MT1-MMP deficiency did not affect primary tumor growth, bone degradation or the formation and growth of lung metastases. We therefore propose that, unlike findings reported in other cancers, tumor-expressed MT1-MMP is dispensable for all stages of osteosarcoma progression.

Cellular uptake of collagens and implications for immune cell regulation in disease.

As the dominant constituent of the extracellular matrix (ECM), collagens of different types are critical for the structural properties of tissues and make up scaffolds for cellular adhesion and migration. Importantly, collagens also directly modulate the phenotypic state of cells by transmitting signals that influence proliferation, differentiation, polarization, survival, and more, to cells of mesenchymal, epithelial, or endothelial origin. Recently, the potential of collagens to provide immune regulatory signals has also been demonstrated, and it is believed that pathological changes in the ECM shape immune cell phenotype. Collagens are themselves heavily regulated by a multitude of structural modulations or by catabolic pathways. One of these pathways involves a cellular uptake of collagens or soluble collagen-like defense collagens of the innate immune system mediated by endocytic collagen receptors. This cellular uptake is followed by the degradation of collagens in lysosomes. The potential of this pathway to regulate collagens in pathological conditions is evident from the increased extracellular accumulation of both collagens and collagen-like defense collagens following endocytic collagen receptor ablation. Here, we review how endocytic collagen receptors regulate collagen turnover during physiological conditions and in pathological conditions, such as fibrosis and cancer. Furthermore, we highlight the potential of collagens to regulate immune cells and discuss how endocytic collagen receptors can directly regulate immune cell activity in pathological conditions or do it indirectly by altering the extracellular milieu. Finally, we discuss the potential collagen receptors utilized by immune cells to directly detect ECM-related changes in the tissues which they encounter.

CCL2/MCP-1 signaling drives extracellular matrix turnover by diverse macrophage subsets.

Macrophage plasticity, cellular origin, and phenotypic heterogeneity are perpetual challenges for studies addressing the biology of this pivotal immune cell in development, homeostasis, and tissue remodeling/repair. Consequently, a myriad of macrophage subtypes has been described in these contexts. To facilitate the identification of functional macrophage subtypes in vivo, here we used a flow cytometry-based assay that allows for detailed phenotyping of macrophages engaged in extracellular matrix (ECM) degradation. Of the five macrophage subtypes identified in the remodeling dermis by using this assay, collagen degradation was primarily executed by Ly6C - CCR2 + and Ly6C - CCR2 low macrophages via mannose receptor-dependent collagen endocytosis, while Ly6C + CCR2 + macrophages were the dominant fibrin-endocytosing cells. Unexpectedly, the CCL2/MCP1-CCR2 signaling axis was critical for both collagen and fibrin degradation, while collagen degradation was independent of IL-4Ra signaling. Furthermore, the cytokine GM-CSF selectively enhanced collagen degradation by Ly6C + CCR2 + macrophages. This study reveals distinct subsets of macrophages engaged in ECM turnover and identifies novel wound healing-associated functions for CCL2 and GM-CSF inflammatory cytokines.

Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities.

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1-5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1-5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1-5 was five times more effective than rMAP-1 and rC4BP1-5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

The collagen receptor uPARAP/Endo180 as a novel target for antibody-drug conjugate mediated treatment of mesenchymal and leukemic cancers.

A key task in developing the field of personalized cancer therapy is the identification of novel molecular targets that enable treatment of cancers not susceptible to other means of specific therapy. The collagen receptor uPARAP/Endo180 is overexpressed by malignant cells in several non-epithelial cancers, notably including sarcomas, glioblastomas and subsets of acute myeloid leukemia. In contrast, in healthy adult individuals, expression is restricted to minor subsets of mesenchymal cells. Functionally, uPARAP/Endo180 is a rapidly recycling endocytic receptor that delivers its cargo directly into the endosomal-lysosomal system, thus opening a potential route of entry into receptor-positive cells. This combination of specific expression and endocytic function appears well suited for targeting of uPARAP/Endo180-positive cancers by antibody-drug conjugate (ADC) mediated drug delivery. Therefore, we utilized a specific monoclonal antibody against uPARAP/Endo180, raised through immunization of a uPARAP/Endo180 knock-out mouse, which reacts with both the human and the murine receptor, to construct a uPARAP-directed ADC. This antibody was coupled to the highly toxic dolastatin derivative, monomethyl auristatin E, via a cathepsin-labile valine-citrulline linker. With this ADC, we show strong and receptor-dependent cytotoxicity in vitro in uPARAP/Endo180-positive cancer cell lines of sarcoma, glioblastoma and leukemic origin. Furthermore, we demonstrate the potency of the ADC in vivo in a xenograft mouse model with human uPARAP/Endo180-positive leukemic cells, obtaining a complete cure of all tested mice following intravenous ADC treatment with no sign of adverse effects. Our study identifies uPARAP/Endo180 as a promising target for novel therapy against several highly malignant cancer types.

Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction.

Discoidin domain receptor 1 (DDR1) is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA). Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

Biomaterial Encapsulation Is Enhanced in the Early Stages of the Foreign Body Reaction During Conditional Macrophage Depletion in Transgenic Macrophage Fas-Induced Apoptosis Mice.

Macrophages are pivotal cells during the foreign body reaction (FBR), as they orchestrate the proinflammatory microenvironment inside and around biomaterials by secretion of inflammatory mediators. Furthermore, they are responsible for the degradation of biomaterials and are thought to instruct the fibroblasts that generate a fibrous capsule around implanted biomaterials. In this study, we investigated the events during the FBR when macrophages are not present. Hexamethylenediisocyanate crosslinked collagen scaffolds were implanted in "Macrophage Fas-Induced Apoptosis" mice, which allow "on demand" macrophage depletion. We observed that macrophage depletion completely inhibited inflammatory ingrowth into the scaffolds and resulted in an increased capsule size. Quantitative polymerase chain reaction analysis revealed decreased expression levels of proinflammatory mediators such as TNFα and IL1ß, and increased expression levels of collagens and fibroblast-stimulating growth factors such as EGF, FGF1, FGF2, and TGFα. Our results indicate that macrophages are indeed crucial for the generation of a proinflammatory microenvironment inside implanted biomaterials, leading to inflammatory ingrowth. In contrast, macrophages do not appear to be important for the generation of a fibrous capsule around implanted biomaterials. In fact, our data suggest that the macrophages present in the capsule might instruct the surrounding fibroblasts to produce less fibroblast-stimulating factors and less collagens.

Ultrastructural aspects of foreign body giant cells generated on different substrates.

Implantation of biomaterials into the body, e.g. for tissue engineering purposes, induces a material-dependent inflammatory response called the foreign body reaction (FBR). A hallmark feature of this response is the formation of large multinucleated cells: foreign body giant cells (FBGCs). Biomaterials like cross-linked and non-cross-linked collagen often induce the formation of FBGCs. It is unknown whether different biomaterials result in the formation of different FBGCs. To investigate this, we implanted cross-linked and non-cross-linked dermal sheep collagen subcutaneously in mice. After 21 days the implanted material was collected and prepared for ultrastructural analysis. More FBGCs formed on and between implants of cross-linked collagen compared to non-cross-linked material. The ultrastructural aspects of the FBGCs present on the two types of implants proved to be similar. On both materials, they formed long slender protrusions on the basolateral membrane, they were very rich in mitochondria, contained numerous nuclei, and showed signs of the presence of a clear zone facing the implanted material. Similar clear zones, that resemble osteoclastic features, were also seen in FBGCs generated in vitro on bone slices, but these cells did not form a ruffled border. However, similarities in ultrastructure such as the occurrence of slender protrusions and high mitochondrion content were also found in the FBGCs generated in vitro. These data indicate that FBGCs formed on different substrates share many morphological characteristics. The formation of long finger-like protrusions seemed typical for the FBGCs, in vivo as well as in vitro, however the function of these structures needs further analysis.

3,4-Dihydroxy-L-Phenylalanine as a Novel Covalent Linker of Extracellular Matrix Proteins to Polyacrylamide Hydrogels with a Tunable Stiffness.

Cells acquire mechanical information from their surrounding and convert this into biochemical activity. The concept and mechanism behind this cellular mechanosensing and mechanotransduction are often studied by means of two-dimensional hydrogels. Polyacrylamide hydrogels (PAAMs) offer chemical, mechanical, and optical advantages but due to their inert surface do not allow protein and cell adherence. Several cross-linkers have been used to functionalize the surface of PAAMs with extracellular matrix (ECM) proteins to enable cell culture. However, the most commonly used cross-linkers are either unstable, expensive, or laborious and often show heterogeneous coating or require PAAM modification. Here, we introduce 3,4-dihydroxy-l-phenylalanine (L-DOPA) as a novel cross-linker that can functionalize PAAMs with ECM without the above-mentioned disadvantages. A homogenous collagen type I and fibronectin coating was observed after L-DOPA functionalization. Fibroblasts responded to differences in PAAMs' stiffness; morphology, cell area, and protein localization were all affected as expected, in accordance with literature where other cross-linkers were used. In conclusion, L-DOPA can be used as a cross-linker between PAAMs and ECM and represents a novel, straightforward, nonlaborious, and robust method to functionalize PAAMs for cell culture to study cell mechanosensing.

Mechanical control of cardiac myofibroblasts.

Fibroblasts produce and turn over collagenous extracellular matrix as part of the normal adaptive response to increased mechanical load in the heart, e.g. during prolonged exercise. However, chronic overload as a consequence of hypertension or myocardial injury trigger a repair program that culminates in the formation of myofibroblasts. Myofibroblasts are opportunistically activated from various precursor cells that all acquire a phenotype promoting excessive collagen secretion and contraction of the neo-matrix into stiff scar tissue. Stiff fibrotic tissue reduces heart distensibility, impedes pumping and valve function, contributes to diastolic and systolic dysfunction, and affects myocardial electrical transmission, potentially leading to arrhythmia and heart failure. Here, we discuss how mechanical factors, such as matrix stiffness and strain, are feeding back and cooperate with cytokine signals to drive myofibroblast activation. We elaborate on the importance of considering the mechanical boundary conditions in the heart to generate better cell culture models for mechanistic studies of cardiac fibroblast function. Elements of the force transmission and mechanoperception apparatus acting in myofibroblasts are presented as potential therapeutic targets to treat fibrosis.

Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

PURPOSE: The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. METHODS: A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor ß (TGF-ß [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. RESULTS: MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-ß1 and TGF-ß2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. CONCLUSIONS: Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

Macrophage phenotypes in the collagen-induced foreign body reaction in rats.

Implantation of biomaterials into the body elicits a material-dependent inflammatory response called the foreign body reaction (FBR). Macrophages play a pivotal role in the FBR by orchestrating the pro-inflammatory microenvironment around the biomaterials by secreting cytokines, chemokines and growth factors. When the biomaterial is porous or degradable, macrophages can migrate into the material and continue the generation of a pro-inflammatory microenvironment inside the materials. They also regulate the degradation of biomaterials by secreting proteolytic enzymes and by phagocytosis. We hypothesize that macrophages present in the different microenvironments of the FBR have different phenotypes. Fundamental knowledge of the phenotypes of macrophages and their dynamics during the FBR will contribute to our overall understanding of the mechanisms involved in the FBR, and may provide us with additional tools to modulate the FBR. To investigate the phenotype of macrophages in the FBR, we validated phenotype-specific markers for rat macrophages in vitro by stimulating them with IFNγ/LPS, IL4/IL13 or IL4/dexamethasone to induce classically activated macrophages (M1φ) or alternatively activated macrophages (M2φ). Gene expression analysis, Western blot and immunohistochemistry revealed that iNOS and CD206 are specifically expressed by M1φ and M2φ, respectively. Using these markers, we investigated the distribution of M1φ and M2φ in the FBR induced by subcutaneously implanted hexamethylenediisocyanate cross-linked dermal sheep collagen (HDSC) disks in AO rats. We found that part of the macrophages display an M2 phenotype, whereas the M1phenotype was not detected. Our data suggest that many macrophages in the FBR induced by HDSC do not fit into the classical M1 or M2 dichotomy.

The antitumor activity of hydrophobin SC3, a fungal protein.

The use of mushroom extracts has been common practice in traditional medicine for centuries, including the treatment of cancer. Proteins called hydrophobins are very abundant in mushrooms. Here, it was examined whether they have antitumor activity. Hydrophobin SC3 of Schizophyllum commune was injected daily intraperitoneally starting 1 day after tumor induction in two tumor mouse models (sarcoma and melanoma). SC3 reduced the size and weight of the melanoma significantly, but the sarcoma seemed not affected. However, microscopic analysis of the tumors 12 days after induction revealed a strong antitumor effect of SC3 on both tumors. The mitotic activity of the tumor decreased 1.6- (melanoma) to 2.3-fold (sarcoma), while the vital mass decreased 2.3- (melanoma) to 4.3-fold (sarcoma) compared to the control. Treatment did not cause any signs of toxicity. Behavior, animal growth, and weight of organs were similar to animals injected with vehicle, and no histological abnormalities were found in the organs. In vitro cell culture studies revealed no direct cytotoxic effect of SC3 towards sarcoma cells, while cytotoxic activity was observed towards melanoma cells at a high SC3 concentration. Daily treatment with SC3 did not result in detectable levels of anti-SC3 antibodies in the plasma. Instead, a cellular immune response was observed. Incubation of spleen cells with SC3 resulted in a 1.5- to 2.5-fold increase in interleukin-10 and TNF-α mRNA levels. In conclusion, the nontoxic fungal hydrophobin SC3 showed tumor-suppressive activity possibly via immunomodulation and may be of benefit as adjuvant in combination with chemotherapy and radiation.

Human macrophages primed with angiogenic factors show dynamic plasticity, irrespective of extracellular matrix components.

Macrophages are important in inflammation as well as in tissue repair processes. They can be activated by various stimuli and classified into two major groups: M1 (classically activated) or M2 (alternatively activated). Inflammation, angiogenesis and matrix remodeling play a major role in tissue repair. Here, we investigate the combined influence of a pro-angiogenic microenvironment and specific extracellular matrix (ECM) components or tissue culture polystyrene (TCPS) on the dynamics of human macrophage polarization. We established that human angiogenically primed macrophages cultured on different ECM components exhibit an M2-like polarization. These M2-like macrophages polarized to M1 and M2 macrophages with classical (LPS and IFNγ) stimuli and alternative (IL-4 and IL-13) stimuli respectively. Moreover, these M1 and M2 (primary) polarized macrophages rapidly underwent a secondary (re)polarization to M2 and M1 with conditioned media from M2 and M1 primary polarized macrophages respectively. In these initial priming and later (re)polarization processes the soluble factors had a dominant and orchestrating role, while the type of ECM (collagen I, fibronectin, versus tissue culture polystyrene) did not play a crucial role on the polarization of macrophages.

Endotoxin contamination delays the foreign body reaction.

Biomaterials are at continuous risk of bacterial contamination during production and application. In vivo, bacterial contamination of biomaterials delays the foreign body reaction (FBR). Endotoxins such as lipopolysaccharides (LPS), major constituents of the bacterial cell wall, are potent stimulators of the immune system in vitro and in vivo. In vitro, biomaterials contaminated with LPS induce the production of proinflammatory cytokines by adherent macrophages. This suggests that the presence of endotoxins on biomaterials will intensify the FBR. The effects of LPS on the course of the FBR have never been studied in vivo. In this study, the influence of LPS contamination on the FBR to subcutaneously implanted Puramatrix-loaded hexamethylenediisocyanate-crosslinked dermal sheep collagen (HDSC) disks was studied in rats. During the onset phase of the FBR, a massive influx of granulocytes was detected in LPS-contaminated disks, while their presence was prolonged. IL-10 production inside LPS-contaminated disks was increased at days 10 and 21. Macrophage densities were not affected by the presence of LPS. However, macrophage functionality was altered: giant cell formation and biomaterial degradation were delayed by LPS-contamination up to 21 days. On the basis of these results, we conclude that LPS delays the FBR. This finding indicates that endotoxin contamination has significant implications for the in vivo function of biomaterials and medical devices and emphasizes the importance of endotoxin testing.

The tissue response to photopolymerized PEG-p(HPMAm-lactate)-based hydrogels.

Hydrogels are three-dimensional networks of crosslinked hydrophilic polymers widely used for protein delivery and tissue engineering. To be eligible for in vivo applications, the hydrogels should not evoke an adverse tissue response. In this study the angiogenic and inflammatory responses in vivo after implantation of photopolymerized thermosensitive poly(hydroxypropyl methacrylamide lactate)-poly(ethyl copolymer hydrogels are investigated. Hydrogels consisting of polymers with different crosslink densities were subcutaneously implanted in Balb/c mice and histological evaluation of the tissue response was performed. The implants showed an acute and localized inflammatory reaction upon implantation, mainly characterized by a strong infiltration of granulocytes. The acute inflammatory reaction was followed by a milder chronic inflammation which was characterized by infiltration of macrophages and persistent but decreasing levels of granulocytes. The number of macrophages and blood vessels was associated with the biodegradation and resorption of the biomaterial and increased in time as the degradation of the materials progressed. The observed degradation rates in vivo correlated well with previously observed in vitro degradation rates, which suggests that hydrolysis is the main mechanism governing the degradation.