ABSTRACT
The release of growth factors, cytokines and extracellular matrix modifiers by activated platelets is an important step in the process of healthy wound healing. Extracellular vesicles (EVs) released by activated platelets carry this bioactive cargo in an enriched form, and may therefore represent a potential therapeutic for the treatment of delayed wound healing, such as chronic wounds. While EVs show great promise in regenerative medicine, their production at clinical scale remains a critical challenge and their tolerability in humans is still to be fully established. In this work, we demonstrate that Ligand-based Exosome Affinity Purification (LEAP) chromatography can successfully isolate platelet EVs (pEVs) of clinical grade from activated platelets, which retain the regenerative properties of the parent cell. LEAP-isolated pEVs display the expected biophysical features of EV populations and transport essential proteins in wound healing processes, including insulin growth factor (IGF) and transforming growth factor beta (TGF-ß). In vitro studies show that pEVs induce proliferation and migration of dermal fibroblasts and increase dermal endothelial cells' angiogenic potential, demonstrating their wound healing potential. pEV treatment activates the ERK and Akt signalling pathways within recipient cells. In a first-in-human, double-blind, placebo-controlled, phase I clinical trial of healthy volunteer adults, designed primarily to assess safety in the context of wound healing, we demonstrate that injections of LEAP-purified pEVs in formulation buffer are safe and well tolerated (Plexoval II study, ACTRN12620000944932). As a secondary objective, biological activity in the context of wound healing rate was assessed. In this cohort of healthy participants, in which the wound bed would not be expected to be deficient in the bioactive cargo that pEVs carry, all wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed at the single dose tested. The outcomes of this study evidence that pEVs manufactured through the LEAP process can be injected safely in humans as a potential wound healing treatment, and warrant further study in clinical trials designed expressly to assess therapeutic efficacy in patients with delayed or disrupted wound healing.
Subject(s)
Extracellular Vesicles , Hematopoietic Stem Cell Transplantation , Adult , Humans , Blood Platelets/metabolism , Endothelial Cells , Extracellular Vesicles/metabolism , Wound Healing/physiologyABSTRACT
Ribonuclease 5, also known as angiogenin, is a stable and abundant ribonuclease in milk whey protein, which is able to regulate several cellular functions, including capillary formation, neuron survival, and epithelial cell growth. Ribonuclease 5 is important for protein synthesis directly stimulating rRNA synthesis in the nucleolus. Here, we show that biologically active RNase5 can be purified from bovine milk. Furthermore, we show that milk-derived RNase5 directly stimulates muscle cell differentiation in vitro, inducing C2C12 cell differentiation and myogenesis. When supplemented into the diet of healthy adult mice, milk-derived RNase5 preparations promoted muscle weight gain and grip strength. Collectively, these data indicate that milk-derived RNase5 preparations exhibit a novel role in skeletal muscle cell function.
Subject(s)
Cell Differentiation/drug effects , Cell Proliferation/drug effects , Milk/enzymology , Muscle Development/drug effects , Muscle, Skeletal/growth & development , Ribonuclease, Pancreatic/pharmacology , Animal Feed , Animals , Cattle , Cell Line , Dietary Supplements , Mice , Milk/chemistry , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effectsABSTRACT
Matrix metalloproteinase (MMP)-8 and MMP-9 play several roles in inflammation, including degradation of extracellular matrix (ECM) components and regulation of cytokine activity. To determine the roles of MMP-8 and MMP-9 in a neutrophil-dependent inflammatory response, we used a murine model of corneal inflammation in which LPS is injected into the corneal stroma. In contrast to wild-type mice, we found that i) lipopolysaccharide (LPS)-injected CXCR2(-/-) corneas had impaired neutrophil infiltration and did not express either MMP-8 or MMP-9; ii) neutrophil migration through the central cornea was impaired in Mmp8(-/-), but not Mmp9(-/-), mice; iii) neutrophil migration was inhibited in collagenase-resistant mice; iv) the chemotactic Pro-Gly-Pro (PGP) tripeptide that binds CXCR2 was decreased in CXCR2(-/-) mice; v) PGP production was impaired in Mmp8(-/-) corneas; and vi) neutralizing anti-PGP antibody did not inhibit neutrophil infiltration in Mmp8(-/-) mice. We found no effects of MMP-8 on LPS-induced CXC chemokine (LIX, or CXCL5)-induced neutrophil recruitment or on LPS-induced CXC chemokine production. Together, these studies indicate that neutrophils contribute to the production of both MMP-8 and MMP-9 in LPS-injected corneas and that MMP-8 regulates neutrophil migration through the dense collagenous ECM of the corneal stroma by generating chemotactic PGP during inflammation.
Subject(s)
Collagen/metabolism , Corneal Stroma/metabolism , Keratitis/metabolism , Matrix Metalloproteinase 8/metabolism , Neutrophil Infiltration/physiology , Oligopeptides/biosynthesis , Proline/analogs & derivatives , Animals , Blotting, Western , Chemokines, CXC/biosynthesis , Corneal Stroma/immunology , Corneal Stroma/pathology , Electrophoresis, Polyacrylamide Gel , Female , Immunohistochemistry , Inflammation/metabolism , Inflammation/pathology , Keratitis/immunology , Keratitis/pathology , Lipopolysaccharides/toxicity , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Proline/biosynthesis , Receptors, Interleukin-8B/deficiencyABSTRACT
We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser(4)-Val(5) and Lys(79)-Arg(80). LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4 approximately 5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg(5)-Ser(6) and at Val(7)-Leu(8) in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.
Subject(s)
Chemotaxis/physiology , Lipopolysaccharides/pharmacology , Matrix Metalloproteinase 8/metabolism , Neutrophils/physiology , Animals , Cell Movement/drug effects , Cell Movement/physiology , Chemokines, CXC/drug effects , Chemokines, CXC/genetics , Chemokines, CXC/physiology , Chemotaxis/drug effects , Humans , Matrix Metalloproteinase 8/deficiency , Matrix Metalloproteinase 8/genetics , Mice , Mice, Knockout , Neutropenia/physiopathology , Neutrophils/drug effects , Wound HealingABSTRACT
Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.
Subject(s)
Cathepsin B/metabolism , Cathepsin D/metabolism , Chemokines, CC/physiology , Macrophage Inflammatory Proteins/physiology , Protein Processing, Post-Translational , Cathepsin B/physiology , Cathepsin D/physiology , Cell Line, Tumor , Cell Membrane/metabolism , Chemokine CCL20 , Chemokines, CC/antagonists & inhibitors , Chemokines, CC/biosynthesis , Chemokines, CC/metabolism , Glycosaminoglycans/metabolism , Humans , Hydrolysis , Interleukin-1/pharmacology , Ligands , Macrophage Inflammatory Proteins/antagonists & inhibitors , Macrophage Inflammatory Proteins/biosynthesis , Macrophage Inflammatory Proteins/metabolism , Melanoma/enzymology , Melanoma/immunology , Melanoma/metabolism , Neoplasm Proteins/biosynthesis , Peptide Fragments/metabolism , Peptide Fragments/physiology , Protein Binding , Protein Isoforms/metabolism , Protein Isoforms/physiology , Protein Processing, Post-Translational/immunology , Substrate Specificity , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.
Subject(s)
Breast Neoplasms/pathology , Enzyme Precursors/genetics , Gelatinases/genetics , Metalloendopeptidases/genetics , Neoplasm Metastasis/pathology , Animals , Brain Neoplasms/pathology , Brain Neoplasms/secondary , Cell Division/genetics , Cell Line, Tumor , Enzyme Precursors/metabolism , Female , Gelatinases/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Kidney Neoplasms/pathology , Kidney Neoplasms/secondary , Liver Neoplasms/pathology , Liver Neoplasms/secondary , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Metalloendopeptidases/metabolism , Mice , Mice, Nude , Neoplasm Invasiveness , Recombinant Proteins/metabolism , Transfection , Transplantation, HeterologousABSTRACT
Matrix metalloproteinases (MMPs) have fundamental roles in tumor progression, but most clinical trials with MMP inhibitors have not shown improvements in individuals with cancer. This may be partly because broad-range inhibitors also reduce host-protective antitumor properties of individual MMPs. We generated mice deficient in collagenase-2 (Mmp8), an MMP mainly produced by neutrophils in inflammatory reactions and detected in some malignant tumors. Loss of Mmp8 did not cause abnormalities during embryonic development or in adult mice. Contrary to previous studies with MMP-deficient mice, however, the absence of Mmp8 strongly increased the incidence of skin tumors in male Mmp8(-/-)mice. Female Mmp8(-/-)mice whose ovaries were removed or were treated with tamoxifen were also more susceptible to tumors compared with wild-type mice. Bone marrow transplantation experiments confirmed that Mmp8 supplied by neutrophils was sufficient to restore the natural protection against tumor development mediated by this protease in male mice. Histopathological analysis showed that mutant mice had abnormalities in the inflammatory response induced by carcinogens. Our study identifies a paradoxical protective role for Mmp8 in cancer and provides a genetic model to evaluate the molecular basis of gender differences in cancer susceptibility.
Subject(s)
Genetic Predisposition to Disease , Matrix Metalloproteinase 8/physiology , Skin Neoplasms/enzymology , Animals , Blotting, Western , Female , Fluorescent Antibody Technique , Genotype , Male , Matrix Metalloproteinase 8/genetics , Mice , Mice, Knockout , Sex Factors , Skin Neoplasms/geneticsABSTRACT
A tissue inhibitor of metalloproteinases-2 (TIMP-2)-independent mechanism for generating the first activational cleavage of pro-matrix metalloproteinase-2 (MMP-2) was identified in membrane type-1 MMP (MT1-MMP)-transfected MCF-7 cells and confirmed in TIMP-2-deficient fibroblasts. In contrast, the second MMP-2-activational step was found to be TIMP-2 dependent in both systems. MMP-2 hemopexin C-terminal domain was found to be critical for the first step processing, confirming a need for membrane tethering. We propose that the intermediate species of MMP-2 forms the well-established trimolecular complex (MT1-MMP/TIMP-2/MMP-2) for further TIMP-2-dependent autocatalytic cleavage to the fully active species. This alternate mechanism may supplement the traditional TIMP-2-mediated first step mechanism.
Subject(s)
Enzyme Precursors/metabolism , Gelatinases/metabolism , Matrix Metalloproteinase 2/metabolism , Metalloendopeptidases/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Animals , Binding Sites , Breast Neoplasms/metabolism , Catalytic Domain , Cell Line , Enzyme Activation , Enzyme Precursors/chemistry , Fibroblasts/cytology , Fibroblasts/metabolism , Gelatinases/chemistry , Humans , Matrix Metalloproteinase 14 , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , Mice , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tissue Inhibitor of Metalloproteinase-2/chemistry , Tissue Inhibitor of Metalloproteinase-2/genetics , Transfection , Tumor Cells, CulturedABSTRACT
Orthotopic or intracardiac injection of human breast cancer cell lines into immunocompromised mice allows study of the molecular basis of breast cancer metastasis. We have established a quantitative real-time PCR approach to analyze metastatic spread of human breast cancer cells inoculated into nude mice via these routes. We employed MDA-MB-231 human breast cancer cells genetically tagged with a bacterial beta-galactosidase (Lac-Z) retroviral vector, enabling their detection by TaqMan real-time PCR. PCR detection was linear, specific, more sensitive than conventional PCR, and could be used to directly quantitative metastatic burden in bone and soft organs. Attesting to the sensitivity and specificity of the PCR detection strategy, as few as several hundred metastatic MDA-MB-231 cells were detectable in 100 microns segments of paraffin-embedded lung tissue, and only in samples adjacent to sections that scored positive by histological detection. Moreover, the measured real-time PCR metastatic burden in the bone environment (mouse hind-limbs, n = 48) displayed a high correlation to the degree of osteolytic damage observed by high resolution X-ray analysis (r2 = 0.972). Such a direct linear relationship to tumor burden and bone damage substantiates the so-called 'vicious cycle' hypothesis in which metastatic tumor cells promote the release of factors from the bone which continue to stimulate the tumor cells. The technique provides a useful tool for molecular and cellular analysis of human breast cancer metastasis to bone and soft organs, can easily be extended to other cell/marker/organ systems, and should also find application in preclinical assessment of anti-metastatic modalities.