ABSTRACT
The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.
Subject(s)
Calcitonin Gene-Related Peptide , Macrophages , Neutrophils , Nociceptors , Wound Healing , Animals , Mice , Autocrine Communication , Calcitonin Gene-Related Peptide/metabolism , Calcitonin Gene-Related Peptide/pharmacology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Efferocytosis , Macrophages/cytology , Macrophages/metabolism , Monocytes/cytology , Monocytes/metabolism , Muscle, Skeletal , NAV1.8 Voltage-Gated Sodium Channel/deficiency , NAV1.8 Voltage-Gated Sodium Channel/genetics , NAV1.8 Voltage-Gated Sodium Channel/metabolism , Neutrophils/cytology , Neutrophils/metabolism , Nociceptors/metabolism , Paracrine Communication , Peripheral Nervous System Diseases/complications , Receptor Activity-Modifying Protein 1/metabolism , Regeneration/drug effects , Skin , Thrombospondin 1/metabolism , Wound Healing/drug effects , Wound Healing/immunology , Humans , Male , FemaleABSTRACT
Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
Subject(s)
Macrophages/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/injuries , Myoblasts/cytology , Nicotinamide Phosphoribosyltransferase/metabolism , Stem Cell Niche , Zebrafish/metabolism , Animals , Cell Proliferation , Disease Models, Animal , Humans , Macrophages/cytology , Male , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myoblasts/metabolism , Nicotinamide Phosphoribosyltransferase/genetics , PAX7 Transcription Factor/metabolism , RNA-Seq , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Regeneration/physiology , Single-Cell Analysis , Zebrafish/immunologyABSTRACT
Electrospun nanofibrous scaffolds containing natural substances with wound healing properties such as Emu oil (EO) may have a great potential for increasing the efficiency of stem cell-based skin bioengineering. For this purpose, EO blended PCL/PEG electrospun nanofibrous mats were successfully fabricated and characterized using FE-SEM, FTIR and Universal Testing Machine. The efficiency of the scaffolds in supporting the adherence, cytoprotection, proliferation and differentiation of adipose tissue-derived stem cells (ADSCs) to keratinocyte was evaluated. GC/MS and HPLC were used to determine the composition of pure EO, which revealed to be mainly fatty acids and carotenoids. FE-SEM and cell proliferation assays showed that adhesion and proliferation of ADSCs on EO-PCL/PEG nanofibers was significantly higher than on PCL/PEG nanofibers. Additionally, EO-PCL/PEG nanofibers with free radical scavenging properties conferred a cytoprotective effect against cell-damaging free radicals, while the ability to support cell adhesion and growth was maintained or even improved. Immunostaining of ADSCs on EO-PCL/PEG nanofibers confirmed the change in morphology of ADSCs from spindle to polygonal shape suggesting their differentiation toward an epidermal linage. Moreover, the expression levels of the keratin 10, filaggrin, and involucrin that are involved in epidermal differentiation were upregulated in a stage-specific manner. This preliminary study shows that EO-PCL/PEG nanofibers could be a good candidate for the fabrication of wound dressings and skin bioengineered substitutes with ADSCs.
Subject(s)
Adipose Tissue/drug effects , Cell Proliferation/drug effects , Cytoprotection/drug effects , Oils/pharmacology , Stem Cells/drug effects , Adipose Tissue/cytology , Cell Adhesion/drug effects , Cell Differentiation/drug effects , Cell Proliferation/physiology , Cytoprotection/physiology , Filaggrin Proteins , Humans , Mesenchymal Stem Cells/cytology , Nanofibers , Skin/pathology , Stem Cells/cytologyABSTRACT
Netrin 1 was initially identified as an axon guidance factor, and recent studies indicate that it inhibits chemokine-directed monocyte migration. Despite its importance as a neuroimmune guidance cue, the role of netrin 1 in osteoclasts is largely unknown. Here we detected high netrin 1 levels in the synovial fluid of rheumatoid arthritis patients. Netrin 1 is potently expressed in osteoblasts and synovial fibroblasts, and IL-17 robustly enhances netrin 1 expression in these cells. The binding of netrin 1 to its receptor UNC5b on osteoclasts resulted in activation of SHP1, which inhibited VAV3 phosphorylation and RAC1 activation. This significantly impaired the actin polymerization and fusion, but not the differentiation of osteoclast. Strikingly, netrin 1 treatment prevented bone erosion in an autoimmune arthritis model and age-related bone destruction. Therefore, the netrin 1-UNC5b axis is a novel therapeutic target for bone-destructive diseases.
Subject(s)
Arthritis, Rheumatoid/drug therapy , Bone Resorption/prevention & control , Nerve Growth Factors/pharmacology , Osteoclasts/metabolism , Synovial Membrane/metabolism , Tumor Suppressor Proteins/pharmacology , Animals , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Bone Resorption/genetics , Bone Resorption/metabolism , Bone Resorption/pathology , Disease Models, Animal , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Male , Mice , Mice, Inbred ICR , Mice, Mutant Strains , Nerve Growth Factors/biosynthesis , Nerve Growth Factors/genetics , Netrin Receptors , Netrin-1 , Neuropeptides/genetics , Neuropeptides/metabolism , Osteoclasts/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Proto-Oncogene Proteins c-vav/genetics , Proto-Oncogene Proteins c-vav/metabolism , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Synovial Membrane/pathology , Tumor Suppressor Proteins/biosynthesis , Tumor Suppressor Proteins/genetics , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolismABSTRACT
The netrin family of proteins are involved in axon guidance during central nervous system development. In vertebrates, two membrane bound forms and five secreted forms of netrin have been reported. In addition to their critical role in neural morphogenesis, a growing number of reports suggest that netrin family proteins also play a role in inflammatory conditions, angiogenesis, and tumorigenesis. In these processes, Unc5 and DCC family proteins serve as receptors of netrin proteins. Recently, it was reported that some netrin family proteins may be involved in the pathogenesis of skeletal diseases including osteoporosis and arthritis. For example, administration of secreted netrin family proteins such as netrin 1 and netrin 4 has prophylactic potential in pathogenic bone degradation in mice. However, netrin 1 blocking antibody also protects mice from inflammatory bone destruction. Therefore, netrin family proteins are involved in the regulation of bone homeostasis, but their bona fide roles in the skeletal system remain controversial. In this review, we discuss the osteo-innate-immune functions of the netrin family of proteins, and summarize their therapeutic potential.
Subject(s)
Arthritis/drug therapy , Arthritis/pathology , Bone and Bones/drug effects , Bone and Bones/pathology , Netrins/therapeutic use , Osteoporosis/drug therapy , Osteoporosis/pathology , Animals , Arthritis/immunology , Bone and Bones/immunology , Humans , Inflammation/drug therapy , Inflammation/immunology , Inflammation/pathology , Netrins/immunology , Osteoclasts/drug effects , Osteoclasts/immunology , Osteoclasts/pathology , Osteoporosis/immunologyABSTRACT
Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF164 was fused to a sequence derived from α2-plasmin inhibitor (α2-PI1-8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An α2-PI1-8-fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-α2-PI1-8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-α2-PI1-8-VEGF164 allowed exquisitely dose-dependent angiogenesis: concentrations ≥25 µg/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01-5.0 µg/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-α2-PI1-8-VEGF164 was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.
Subject(s)
Fibrin/pharmacokinetics , Gene Transfer Techniques , Ischemia/therapy , Neovascularization, Physiologic/physiology , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/pharmacokinetics , Animals , Female , Gels/pharmacokinetics , Genetic Therapy/methods , Hindlimb , Human Umbilical Vein Endothelial Cells , Humans , Mice , Mice, Inbred Strains , Mice, SCID , Muscle, Skeletal/blood supply , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacokinetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival.
Subject(s)
Adaptor Proteins, Signal Transducing/deficiency , Bone Density/physiology , Osteoclasts/metabolism , Osteoporosis/physiopathology , Adaptor Proteins, Signal Transducing/genetics , Animals , Bone Density/genetics , CSK Tyrosine-Protein Kinase , Cell Cycle Proteins , Cell Differentiation/genetics , Cell Survival/genetics , Cells, Cultured , HSP90 Heat-Shock Proteins , Immunoblotting , Mice, Knockout , Molecular Chaperones , Myeloid Cells/cytology , Myeloid Cells/metabolism , Osteoclasts/cytology , Osteoporosis/genetics , Osteoporosis/metabolism , src-Family Kinases/metabolismABSTRACT
By binding growth factors (GFs), the ECM tightly regulates their activity. We recently reported that the heparin-binding domain II of fibronectin acts as a promiscuous high-affinity GF-binding domain. Here we hypothesized that fibrin, the provisional ECM during tissue repair, also could be highly promiscuous in its GF-binding capacity. Using multiple affinity-based assays, we found that fibrin(ogen) and its heparin-binding domain bind several GFs from the PDGF/VEGF and FGF families and some GFs from the TGF-ß and neurotrophin families. Overall, we identified 15 unique binding interactions. The GF binding ability of fibrinogen caused prolonged retention of many of the identified GFs within fibrin. Thus, based on the promiscuous and high-affinity interactions in fibrin, GF binding may be one of fibrin's main physiological functions, and these interactions may potentially play an important and ubiquitous role during tissue repair. To prove this role in a gain-of-function model, we incorporated the heparin-binding domain of fibrin into a synthetic fibrin-mimetic matrix. In vivo, the multifunctional synthetic matrix could fully mimic the effect of fibrin in a diabetic mouse model of impaired wound healing, demonstrating the benefits of generating a hybrid biomaterial consisting of a synthetic polymeric scaffold and recombinant bioactive ECM domains. The reproduction of GF-ECM interactions with a fibrin-mimetic matrix could be clinically useful, and has the significant benefit of a more straightforward regulatory path associated with chemical synthesis rather than human sourcing.
Subject(s)
Biomimetic Materials/pharmacology , Extracellular Matrix , Fibrin/pharmacology , Fibrinogen/pharmacology , Intercellular Signaling Peptides and Proteins/pharmacology , Wound Healing/drug effects , Animals , Biomimetic Materials/chemistry , Disease Models, Animal , Fibrin/chemistry , Fibrinogen/chemistry , Human Umbilical Vein Endothelial Cells , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Male , Mice , Mice, Mutant Strains , Protein BindingABSTRACT
With a view toward reduction of graft loss, we explored pancreatic islet transplantation within fibrin matrices rendered pro-angiogenic by incorporation of minimal doses of vascular endothelial growth factor-A165 and platelet-derived growth factor-BB presented complexed to a fibrin-bound integrin-binding fibronectin domain. Engineered matrices allowed for extended release of pro-angiogenic factors and for their synergistic signaling with extracellular matrix-binding domains in the post-transplant period. Aprotinin addition delayed matrix degradation and prolonged pro-angiogenic factor availability within the graft. Both subcutaneous (SC) and epididymal fat pad (EFP) sites were evaluated. We show that in the SC site, diabetes reversal in mice transplanted with 1,000 IEQ of syngeneic islets was not observed for islets transplanted alone, while engineered matrices resulted in a diabetes median reversal time (MDRT) of 38 days. In the EFP site, the MDRT with 250 IEQ of syngeneic islets within the engineered matrices was 24 days versus 86 days for islets transplanted alone. Improved function of engineered grafts was associated with enhanced and earlier (by day 7) angiogenesis. Our findings show that by engineering the transplant site to promote prompt re-vascularization, engraftment and long-term function of islet grafts can be improved in relevant extrahepatic sites.
Subject(s)
Fibrin/chemistry , Islets of Langerhans Transplantation , Islets of Langerhans/drug effects , Vascular Endothelial Growth Factor A/pharmacology , Animals , Becaplermin , Cell Proliferation/drug effects , Humans , Hydrogels/chemistry , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins c-sis/chemistry , Proto-Oncogene Proteins c-sis/deficiency , Proto-Oncogene Proteins c-sis/pharmacology , Vascular Endothelial Growth Factor A/chemistryABSTRACT
The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)--a key ECM crosslinking enzyme--is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.
Subject(s)
Cell Engineering/methods , Factor XIIIa/chemistry , Factor XIIIa/metabolism , Hydrogels/chemistry , Light , Mesenchymal Stem Cells/cytology , Amino Acid Sequence , Animals , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Humans , Mesenchymal Stem Cells/metabolism , Mice , Microtechnology , Photolysis , Polyethylene Glycols/chemistry , RabbitsABSTRACT
Graphene and graphene oxide (GO), due to their unique chemical and physical properties, possess biochemical characteristics that can trigger intercellular signals promoting tissue regeneration. Clinical applications of thin GO-derived sheets have inspired the development of various tissue regeneration and repair approaches. In this study, we demonstrate that ultrathin sheets of plasma-functionalized and reduced GO, with the oxygen content ranging from 3.2 % to 22 % and the nitrogen content from 0 % to 8.3 %, retain their essential mechanical and molecular integrity, and exhibit robust potential for regenerating bone tissue and blood vessels across multiple cellular and animal models. Initially, we observed the growth of blood vessels and bone tissue in vitro using these functionalized GO sheets on human adipose-derived mesenchymal stem cells and umbilical vein endothelial cells. Remarkably, our study indicates a 2.5-fold increase in mineralization and two-fold increase in tubule formation even in media lacking osteogenic and angiogenic supplements. Subsequently, we observed the initiation, conduction, and formation of bone and blood vessels in a rat tibial osteotomy model, evident from a marked 4-fold increase in the volume of low radio-opacity bone tissue and a significant elevation in connectivity density, all without the use of stem cells or growth factors. Finally, we validated these findings in a mouse critical-size calvarial defect model (33 % higher healing rate) and a rat skin lesion model (up to 2.5-fold increase in the number of blood vessels, and 35 % increase in blood vessels diameter). This study elucidates the pro-osteogenic and pro-angiogenic properties of both pristine and plasma-treated GO ultrathin films. These properties suggest their significant potential for clinical applications, and as valuable biomaterials for investigating fundamental aspects of bone and blood vessel regeneration.
Subject(s)
Bone Regeneration , Graphite , Human Umbilical Vein Endothelial Cells , Mesenchymal Stem Cells , Animals , Graphite/chemistry , Humans , Rats , Bone Regeneration/drug effects , Bone Regeneration/physiology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Osteogenesis/drug effects , Osteogenesis/physiology , Mice , Blood Vessels , Rats, Sprague-Dawley , Bone and Bones/blood supply , Bone and Bones/drug effects , Plasma Gases/pharmacology , Plasma Gases/chemistry , Tibia/blood supply , Neovascularization, Physiologic/drug effects , Tissue Engineering/methodsABSTRACT
Scaffolds for bone defect treatment should ideally support vascularization and promote bone formation, to facilitate the translation into biomedical device applications. This study presents a novel approach utilizing 3D-printed water-dissolvable polyvinyl alcohol (PVA) sacrificial molds to engineer polymerized High Internal Phase Emulsion (polyHIPE) scaffolds with microchannels and distinct multiscale porosity. Two sacrificial mold variants (250 µm and 500 µm) were generated using fused deposition modeling, filled with HIPE, and subsequently dissolved to create polyHIPE scaffolds containing microchannels. In vitro assessments demonstrated significant enhancement in cell infiltration, proliferation, and osteogenic differentiation, underscoring the favorable impact of microchannels on cell behavior. High loading efficiency and controlled release of the osteogenic factor BMP-2 were achieved, with microchannels facilitating release of the growth factor. Evaluation in a mouse critical-size calvarial defect model revealed enhanced vascularization and bone formation in microchanneled scaffolds containing BMP-2. This study not only introduces an accessible method for creating multiscale porosity in polyHIPE scaffolds but also emphasizes its capability to enhance cellular infiltration, controlled growth factor release, and in vivo performance. The findings suggest promising applications in bone tissue engineering and regenerative medicine, and are expected to facilitate the translation of this type of biomaterial scaffold. STATEMENT OF SIGNIFICANCE: This study holds significance in the realm of biomaterial scaffold design for bone tissue engineering and regeneration. We demonstrate a novel method to introduce controlled multiscale porosity and microchannels into polyHIPE scaffolds, by utilizing 3D-printed water-dissolvable PVA molds. The strategy offers new possibilities for improving cellular infiltration, achieving controlled release of growth factors, and enhancing vascularization and bone formation outcomes. This microchannel approach not only marks a substantial stride in scaffold design but also demonstrates its tangible impact on enhancing osteogenic cell differentiation and fostering robust bone formation in vivo. The findings emphasize the potential of this methodology for bone regeneration applications, showcasing an interesting advancement in the quest for effective and innovative biomaterial scaffolds to regenerate bone defects.
ABSTRACT
Regulatory T cells (Tregs) are key immune regulators that have shown promise in enhancing cardiac repair post-MI, although the mechanisms remain elusive. Here, we show that rapidly increasing Treg number in the circulation post-MI via systemic administration of exogenous Tregs improves cardiac function in male mice, by limiting cardiomyocyte death and reducing fibrosis. Mechanistically, exogenous Tregs quickly home to the infarcted heart and adopt an injury-specific transcriptome that mediates repair by modulating monocytes/macrophages. Specially, Tregs lead to a reduction in pro-inflammatory Ly6CHi CCR2+ monocytes/macrophages accompanied by a rapid shift of macrophages towards a pro-repair phenotype. Additionally, exogenous Treg-derived factors, including nidogen-1 and IL-10, along with a decrease in cardiac CD8+ T cell number, mediate the reduction of the pro-inflammatory monocyte/macrophage subset in the heart. Supporting the pivotal role of IL-10, exogenous Tregs knocked out for IL-10 lose their pro-repair capabilities. Together, this study highlights the beneficial use of a Treg-based therapeutic approach for cardiac repair with important mechanistic insights that could facilitate the development of novel immunotherapies for MI.
Subject(s)
Interleukin-10 , Macrophages , Mice, Inbred C57BL , Myocardial Infarction , T-Lymphocytes, Regulatory , Animals , Myocardial Infarction/immunology , Myocardial Infarction/genetics , Myocardial Infarction/pathology , T-Lymphocytes, Regulatory/immunology , Macrophages/immunology , Macrophages/metabolism , Male , Mice , Interleukin-10/metabolism , Interleukin-10/genetics , Phenotype , Myocardium/pathology , Myocardium/immunology , Myocardium/metabolism , Monocytes/immunology , Monocytes/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/immunology , Fibrosis , CD8-Positive T-Lymphocytes/immunology , Disease Models, Animal , Mice, KnockoutABSTRACT
Among therapeutic proteins, cytokines and growth factors have great potential for regenerative medicine applications. However, these molecules have encountered limited clinical success due to low effectiveness and major safety concerns, highlighting the need to develop better approaches that increase efficacy and safety. Promising approaches leverage how the extracellular matrix (ECM) controls the activity of these molecules during tissue healing. Using a protein motif screening strategy, we discovered that amphiregulin possesses an exceptionally strong binding motif for ECM components. We used this motif to confer the pro-regenerative therapeutics platelet-derived growth factor-BB (PDGF-BB) and interleukin-1 receptor antagonist (IL-1Ra) a very high affinity to the ECM. In mouse models, the approach considerably extended tissue retention of the engineered therapeutics and reduced leakage in the circulation. Prolonged retention and minimal systemic diffusion of engineered PDGF-BB abolished the tumour growth-promoting adverse effect that was observed with wild-type PDGF-BB. Moreover, engineered PDGF-BB was substantially more effective at promoting diabetic wound healing and regeneration after volumetric muscle loss, compared to wild-type PDGF-BB. Finally, while local or systemic delivery of wild-type IL-1Ra showed minor effects, intramyocardial delivery of engineered IL-1Ra enhanced cardiac repair after myocardial infarction by limiting cardiomyocyte death and fibrosis. This engineering strategy highlights the key importance of exploiting interactions between ECM and therapeutic proteins for developing effective and safer regenerative therapies.
ABSTRACT
Skin wound healing is a highly coordinated process involving multiple tissue-resident and recruited cell types. Cells within the wound microenvironment respond to key secreted factors such as pro-proliferative growth factors and immunomodulatory cytokines to repair the skin and promptly restore its essential barrier role. Therefore, recombinant growth factors and cytokines are promising therapeutics for skin wounds, in particular for large acute wounds such as burns, or wounds associated with underlying pathologies such as nonhealing chronic and diabetic wounds. However, translation of growth factors and cytokines into clinically effective treatments has been limited. Short half-life, poor stability, rapid diffusion, uncontrolled signaling, and systemic side effects are currently the key challenges to developing efficient growth factor- and cytokine-based therapies. To overcome these limitations, novel delivery systems have been developed to improve the regenerative potential of recombinant growth factors and cytokines. In this review, we discuss biomaterial and protein engineering strategies used to optimize the delivery of growth factor and cytokine therapeutics for skin wound treatment.
Subject(s)
Cytokines , Wound Healing , Biocompatible Materials , Intercellular Signaling Peptides and Proteins , SkinABSTRACT
Numerous components of the immune system, including inflammatory mediators, immune cells and cytokines, have a profound modulatory effect on the homeostatic regulation and regenerative activity of endogenous stem cells and progenitor cells. Thus, understanding how the immune system interacts with stem/progenitor cells could build the foundation to design novel and more effective regenerative therapies. Indeed, utilizing and controlling immune system components may be one of the most effective approaches to promote tissue regeneration. In this review, we first summarize the effects of various immune cell types on endogenous stem/progenitor cells, focusing on the tissue healing context. Then, we present interesting regenerative strategies that control or mimic the effect of immune components on stem/progenitor cells, in order to enhance the regenerative capacity of endogenous and transplanted stem cells. We highlight the potential clinical translation of such approaches for multiple tissues and organ systems, as these novel regenerative strategies could considerably improve or eventually substitute stem cell-based therapies. Overall, harnessing the power of the cross-talk between the immune system and stem/progenitor cells holds great potential for the development of novel and effective regenerative therapies.
Subject(s)
Inflammation Mediators , Stem Cell Transplantation , Cytokines , Regenerative Medicine , Wound HealingABSTRACT
Nociceptors can fine-tune local or systemic immunity, but the mechanisms of nociceptive modulation in endotoxic death remain largely unknown. Here, we identified C-type lectin Reg3γ as a nociceptor-enriched hormone that protects the host from endotoxic death. During endotoxemia, nociceptor-derived Reg3γ penetrates the brain and suppresses the expression of microglial indoleamine dioxygenase 1, a critical enzyme of the kynurenine pathway, via the Extl3-Bcl10 axis. Endotoxin-administered nociceptor-null mice and nociceptor-specific Reg3γ-deficient mice exhibit a high mortality rate accompanied by decreased brain HK1 phosphorylation and ATP production despite normal peripheral inflammation. Such metabolic arrest is only observed in the brain, and aberrant production of brain quinolinic acid, a neurotoxic metabolite of the kynurenine pathway, causes HK1 suppression. Strikingly, the central administration of Reg3γ protects mice from endotoxic death by enhancing brain ATP production. By identifying nociceptor-derived Reg3γ as a microglia-targeted hormone, this study provides insights into the understanding of tolerance to endotoxic death.
Subject(s)
Kynurenine , Microglia , Pancreatitis-Associated Proteins/metabolism , Adenosine Triphosphate/metabolism , Animals , Endotoxins/metabolism , Hormones/metabolism , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Kynurenine/metabolism , Mice , Microglia/metabolism , N-Acetylglucosaminyltransferases/metabolism , Nociceptors/metabolismABSTRACT
It has recently been shown that some growth factors (GFs) have strong interactions with nonproteoglycan extracellular matrix proteins. Relevant here, the 12th-14th type three repeats of fibronectin (FN III12-14) have been shown to bind insulin-like growth factor binding-protein-3, fibroblast growth factor (FGF)-2, and vascular endothelial growth factor (VEGF)-A with high affinity. Since FN III12-14 is known to bind GFs from different families, we hypothesized that this domain could be highly promiscuous in its GF-binding capacity. We used biochemical approaches and surface plasmon resonance to investigate such interactions with recombinant FN III12-14. We found that FN III12-14 binds most of the GFs from the platelet-derived growth factor (PDGF)/VEGF and FGF families and some GFs from the transforming growth factor-ß and neurotrophin families, with K(D) values in the nanomolar range, without inhibiting GF activity. Overall, 25 new binding interactions were identified. In a clinically relevant fibrin matrix, a fibrin-binding variant of FN III12-14 was highly effective as a GF delivery system. For instance, in matrices functionalized with FN III12-14, PDGF-BB-induced sprouting of human smooth muscle cell spheroids was greatly enhanced. We show that FN III12-14 is a highly promiscuous ligand for GFs and also holds great potential in clinical healing applications.
Subject(s)
Fibronectins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix/metabolism , Fibronectins/chemistry , Fibronectins/genetics , Fibronectins/pharmacology , Heparin/pharmacology , Humans , Insulin-Like Growth Factor Binding Proteins/metabolism , Phosphorylation/drug effects , Platelet-Derived Growth Factor/metabolism , Protein Binding/drug effects , Tissue Engineering , Transforming Growth Factor beta/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Chronic wounds are a major clinical problem where wound closure is prevented by pathologic factors, including immune dysregulation. To design efficient immunotherapies, an understanding of the key molecular pathways by which immunity impairs wound healing is needed. Interleukin-1 (IL-1) plays a central role in regulating the immune response to tissue injury through IL-1 receptor (IL-1R1). Generating a knockout mouse model, we demonstrate that the IL-1-IL-1R1 axis delays wound closure in diabetic conditions. We used a protein engineering approach to deliver IL-1 receptor antagonist (IL-1Ra) in a localised and sustained manner through binding extracellular matrix components. We demonstrate that matrix-binding IL-1Ra improves wound healing in diabetic mice by re-establishing a pro-healing microenvironment characterised by lower levels of pro-inflammatory cells, cytokines and senescent fibroblasts, and higher levels of anti-inflammatory cytokines and growth factors. Engineered IL-1Ra has translational potential for chronic wounds and other inflammatory conditions where IL-1R1 signalling should be dampened.
Subject(s)
Diabetes Mellitus, Experimental/physiopathology , Interleukin 1 Receptor Antagonist Protein/genetics , Wound Healing/physiology , Animals , Interleukin 1 Receptor Antagonist Protein/metabolism , Male , Mice , Mice, KnockoutABSTRACT
Growth factors can stimulate tissue regeneration, but the side effects and low effectiveness associated with suboptimal delivery systems have impeded their use in translational regenerative medicine. Physiologically, growth factor interactions with the extracellular matrix control their bioavailability and spatiotemporal cellular signalling. Growth factor signalling is also controlled at the cell surface level via binding to heparan sulfate proteoglycans, such as syndecans. Here we show that vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor-BB (PDGF-BB) that were engineered to have a syndecan-binding sequence trigger sustained low-intensity signalling (tonic signalling) and reduce the desensitization of growth factor receptors. We also show in mouse models that tonic signalling leads to superior morphogenetic activity, with syndecan-binding growth factors inducing greater bone regeneration and wound repair than wild-type growth factors, as well as reduced tumour growth (associated with PDGF-BB delivery) and vascular permeability (triggered by VEGF-A). Tonic signalling via syndecan binding may also enhance the regenerative capacity of other growth factors.