Maresin 1 resolves aged-associated macrophage inflammation to improve bone regeneration.

Inflammaging is associated with poor tissue regeneration observed in advanced age. Specifically, protracted inflammation after acute injury has been associated with decreased bone fracture healing and increased rates of nonunion in elderly patients. Here, we investigated the efficacy of using Maresin 1 (MaR1), an omega-3 fatty acid-derived pro-resolving agent, to resolve inflammation after tibial fracture injury and subsequently improving aged bone healing. Aged (24-month-old mice) underwent tibial fracture surgery and were either treated with vehicle or MaR1 3 days after injury. Fracture calluses were harvested 7 days, 14 days, 21 days, and 28 days after injury to investigate inflammatory response, cartilage development, bone deposition, and mechanical integrity, respectively. Healing bones from MaR1-treated mice displayed decreased cartilage formation and increased bone deposition which resulted in increased structural stiffness and increased force to fracture in the later stages of repair. In the early stages, MaR1 treatment decreased the number of pro-inflammatory macrophages within the fracture callus and decreased the level of inflammatory biomarkers in circulation. In tissue culture models, MaR1 treatment of bone marrow-derived macrophages from aged mice protected cells form a pro-inflammatory phenotype and induced an anti-inflammatory fate. Furthermore, the secretome of MaR1-treated bone marrow-derived macrophages was identified as osteoinductive, enhancing osteoblast differentiation of bone marrow stromal cells. Our findings here identify resolution of inflammation, and MaR1 itself, to be a point of intervention to improve aged bone healing.

Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of bone repair in mice.

The pace of repair declines with age and, while exposure to a young circulation can rejuvenate fracture repair, the cell types and factors responsible for rejuvenation are unknown. Here we report that young macrophage cells produce factors that promote osteoblast differentiation of old bone marrow stromal cells. Heterochronic parabiosis exploiting young mice in which macrophages can be depleted and fractionated bone marrow transplantation experiments show that young macrophages rejuvenate fracture repair, and old macrophage cells slow healing in young mice. Proteomic analysis of the secretomes identify differential proteins secreted between old and young macrophages, such as low-density lipoprotein receptor-related protein 1 (Lrp1). Lrp1 is produced by young cells, and depleting Lrp1 abrogates the ability to rejuvenate fracture repair, while treating old mice with recombinant Lrp1 improves fracture healing. Macrophages and proteins they secrete orchestrate the fracture repair process, and young cells produce proteins that rejuvenate fracture repair in mice.

The Role of the Immune Cells in Fracture Healing.

PURPOSE OF REVIEW: Bone fracture healing is a complex physiological process relying on numerous cell types and signals. Inflammatory factors secreted by immune cells help to control recruitment, proliferation, differentiation, and activation of hematopoietic and mesenchymal cells. Within this review we will discuss the functional role of immune cells as it pertains to bone fracture healing. In doing so, we will outline the cytokines secreted and their effects within the healing fracture callus. RECENT FINDINGS: Macrophages have been found to play an important role in fracture healing. These immune cells signal to other cells of the fracture callus, modulating bone healing. Cytokines and cellular signals within fracture healing continue to be studied. The findings from this work have helped to reinforce the importance of osteoimmunity in bone fracture healing. Owing to these efforts, immunomodulation is emerging as a potential therapeutic target to improve bone fracture healing.

Exposure to a youthful circulaton rejuvenates bone repair through modulation of ß-catenin.

The capacity for tissues to repair and regenerate diminishes with age. We sought to determine the age-dependent contribution of native mesenchymal cells and circulating factors on in vivo bone repair. Here we show that exposure to youthful circulation by heterochronic parabiosis reverses the aged fracture repair phenotype and the diminished osteoblastic differentiation capacity of old animals. This rejuvenation effect is recapitulated by engraftment of young haematopoietic cells into old animals. During rejuvenation, ß-catenin signalling, a pathway important in osteoblast differentiation, is modulated in the early repair process and required for rejuvenation of the aged phenotype. Temporal reduction of ß-catenin signalling during early fracture repair improves bone healing in old mice. Our data indicate that young haematopoietic cells have the capacity to rejuvenate bone repair and this is mediated at least in part through ß-catenin, raising the possibility that agents that modulate ß-catenin can improve the pace or quality of fracture repair in the ageing population.

Modulation of type II TGF-ß receptor degradation by integrin-linked kinase.

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-ß (TGF-ß) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-ß and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-ß receptor stimulation. We now show that ILK associates with type II TGF-ß receptors (TßRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TßRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-ß receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TßRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TßRII away from degradative pathways during their differentiation into myofibroblasts.

Macrophages promote osteoblastic differentiation in-vivo: implications in fracture repair and bone homeostasis.

Macrophages are activated in inflammation and during early phases of repair processes. Interestingly, they are also present in bone during development, but their function during this process is unclear. Here, we explore the function of macrophages in bone development, growth, and repair using transgenic mice to constitutively or conditionally deplete macrophages. Depletion of macrophages led to early skeletal growth retardation and progressive osteoporosis. By 3 months of age, macrophage-deficient mice displayed a 25% reduction in bone mineral density and a 70% reduction in the number of trabecular bone compared to control littermates. Despite depletion of macrophages, functional osteoclasts were still present in bones, lining trabecular bone and the endosteal surface of the cortical bone. Furthermore, ablation of macrophages led to a 60% reduction in the number of bone marrow mesenchymal progenitor cells and a decrease in the ability of these cells to differentiate to osteoblasts. When macrophages were depleted during fracture repair, bone union was impaired. Calluses from macrophage-deficient animals were smaller, and contained less bone and more fibrotic tissue deposition. Taken together, this shows that macrophages are crucial for maintaining bone homeostasis and promoting fracture repair by enhancing the differentiation of mesenchymal progenitors.

Fibroblasts from phenotypically normal palmar fascia exhibit molecular profiles highly similar to fibroblasts from active disease in Dupuytren's Contracture.

BACKGROUND: Dupuytren's contracture (DC) is a fibroproliferative disorder characterized by the progressive development of a scar-like collagen-rich cord that affects the palmar fascia of the hand and leads to digital flexion contractures. DC is most commonly treated by surgical resection of the diseased tissue, but has a high reported recurrence rate ranging from 27% to 80%. We sought to determine if the transcriptomic profiles of fibroblasts derived from DC-affected palmar fascia, adjacent phenotypically normal palmar fascia, and non-DC palmar fascial tissues might provide mechanistic clues to understanding the puzzle of disease predisposition and recurrence in DC. METHODS: To achieve this, total RNA was obtained from fibroblasts derived from primary DC-affected palmar fascia, patient-matched unaffected palmar fascia, and palmar fascia from non-DC patients undergoing carpal tunnel release (6 patients in each group). These cells were grown on a type-1 collagen substrate (to better mimic their in vivo environments). Microarray analyses were subsequently performed using Illumina BeadChip arrays to compare the transcriptomic profiles of these three cell populations. Data were analyzed using Significance Analysis of Microarrays (SAM v3.02), hierarchical clustering, concordance mapping and Venn diagram. RESULTS: We found that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected fascia of DC patients exhibited a much greater overlap than fibroblasts derived from the palmar fascia of patients undergoing carpal tunnel release. Quantitative real time RT-PCR confirmed the differential expression of select genes validating the microarray data analyses. These data are consistent with the hypothesis that predisposition and recurrence in DC may stem, at least in part, from intrinsic similarities in the basal gene expression of diseased and phenotypically unaffected palmar fascia fibroblasts. These data also demonstrate that a collagen-rich environment differentially alters gene expression in these cells. In addition, Ingenuity pathway analysis of the specific biological pathways that differentiate DC-derived cells from carpal tunnel-derived cells has identified the potential involvement of microRNAs in this fibroproliferative disorder. CONCLUSIONS: These data show that the transcriptomic profiles of DC-disease fibroblasts and fibroblasts from unaffected palmar fascia in DC patients are highly similar, and differ significantly from the transcriptomic profiles of fibroblasts from the palmar fascia of patients undergoing carpal tunnel release.

Integrin-linked kinase is required for TGF-ß1 induction of dermal myofibroblast differentiation.

Cutaneous repair after injury requires activation of resident dermal fibroblasts and their transition to myofibroblasts. The key stimuli for myofibroblast formation are activation of transforming growth factor-ß (TGF-ß) receptors and mechanotransduction mediated by integrins and associated proteins. We investigated the role of integrin-linked kinase (ILK) in TGF-ß1 induction of dermal fibroblast transition to myofibroblasts. ILK-deficient fibroblasts treated with TGF-ß1 exhibited attenuation of Smad 2 and 3 phosphorylation, accompanied by impaired transcriptional activation of Smad targets, such as α-smooth muscle actin. These alterations were not limited to Smad-associated TGF-ß1 responses, as stimulation of noncanonical mitogen-activated protein kinase pathways by this growth factor was also diminished in the absence of ILK. ILK-deficient fibroblasts exhibited abnormalities in the actin cytoskeleton, and did not form supermature focal adhesions or contractile F-actin stress fibers, indicating a severe impairment in their capacity to differentiate into myofibroblasts. These defects extended to the inability of cells to contract extracellular matrices when embedded in collagen lattices. We conclude that ILK is necessary to transduce signals implicated in the transition of dermal fibroblasts to myofibroblasts originating from matrix substrates and TGF-ß1.

Molecular mechanisms and treatment strategies for Dupuytren's disease.

Dupuytren's disease (DD) is a common disease of the hand and is characterized by thickening of the palmar fascia and formation of tight collagenous disease cords. At present, the disease is incurable and the molecular pathophysiology of DD is unknown. Surgery remains the most commonly used treatment for DD, but this requires extensive postoperative therapy and is associated with high rates of recurrence. Over the past decades, more indepth exploration of the molecular basis of DD has raised the hopes of developing new treatment modalities. This paper reviews the clinical presentation and molecular pathophysiology of this disease, as well as current and emerging treatment. It also explores the implications of new findings in the laboratory for future treatment.

The potential roles of cell migration and extra-cellular matrix interactions in Dupuytren's disease progression and recurrence.

Dupuytren's disease is a pathological condition of the palmar fascia characterized by the formation of contractile disease cords that result in permanent finger contracture. This condition is believed to progress from a myofibroblast-rich nodule in the early clinical stages of the disease to a contractile disease cord spanning a portion of the fascia, leading to contracture of one or more digits. The mechanism(s) by which this disease progresses from a nodule to a collagenous disease cord are poorly understood. Here, we discuss two possible models of disease progression. Firstly, disease progression might be mediated by the proliferation and outward migration of disease cells from within the nodule to populate the adjacent palmar fascia, resulting in a disease cord containing contractile cells derived from the nodule itself. Alternatively, nodular cells may secrete disease-associated factors into the surrounding extra-cellular matrix, thereby altering its composition and triggering quiescent, phenotypically normal cells in the adjacent palmar fascia to take on a proliferative and contractile phenotype. Based on the available evidence and the current state of knowledge of myofibroblast biology, we hypothesize that extra-cellular matrix interactions resulting in conversion of adjacent palmar fascia cells to a disease phenotype is more likely than cell migration from the nodule. Understanding the mechanisms of Dupuytren's disease progression will assist in the development of effective therapeutic interventions to address the high clinical recurrence rate of this condition.

Periostin differentially induces proliferation, contraction and apoptosis of primary Dupuytren's disease and adjacent palmar fascia cells.

Dupuytren's disease, (DD), is a fibroproliferative condition of the palmar fascia in the hand, typically resulting in permanent contracture of one or more fingers. This fibromatosis is similar to scarring and other fibroses in displaying excess collagen secretion and contractile myofibroblast differentiation. In this report we expand on previous data demonstrating that POSTN mRNA, which encodes the extra-cellular matrix protein periostin, is up-regulated in Dupuytren's disease cord tissue relative to phenotypically normal palmar fascia. We demonstrate that the protein product of POSTN, periostin, is abundant in Dupuytren's disease cord tissue while little or no periostin immunoreactivity is evident in patient-matched control tissues. The relevance of periostin up-regulation in DD was assessed in primary cultures of cells derived from diseased and phenotypically unaffected palmar fascia from the same patients. These cells were grown in type-1 collagen-enriched culture conditions with or without periostin addition to more closely replicate the in vivo environment. Periostin was found to differentially regulate the apoptosis, proliferation, alpha smooth muscle actin expression and stressed Fibroblast Populated Collagen Lattice contraction of these cell types. We hypothesize that periostin, secreted by disease cord myofibroblasts into the extra-cellular matrix, promotes the transition of resident fibroblasts in the palmar fascia toward a myofibroblast phenotype, thereby promoting disease progression.

Type-1 Collagen differentially alters beta-catenin accumulation in primary Dupuytren's Disease cord and adjacent palmar fascia cells.

BACKGROUND: Dupuytren's Disease (DD) is a debilitating contractile fibrosis of the palmar fascia characterised by excess collagen deposition, contractile myofibroblast development, increased transforming growth factor-beta levels and beta-catenin accumulation. The aim of this study was to determine if a collagen-enriched environment, similar to in vivo conditions, altered beta-catenin accumulation by primary DD cells in the presence or absence of transforming growth factor-beta. METHODS: Primary DD and patient matched, phenotypically normal palmar fascia (PF) cells were cultured in the presence or absence of type-1 collagen and transforming growth factor-beta1. beta-catenin and alpha-smooth muscle actin levels were assessed by western immunoblotting and immunofluorescence microscopy. RESULTS: DD cells display a rapid depletion of cellular beta-catenin not evident in patient-matched PF cells. This effect was not evident in either cell type when cultured in the absence of type-1 collagen. Exogenous addition of transforming growth factor-beta1 to DD cells in collagen culture negates the loss of beta-catenin accumulation. Transforming growth factor-beta1-induced alpha-smooth muscle actin, a marker of myofibroblast differentiation, is attenuated by the inclusion of type-1 collagen in cultures of DD and PF cells. CONCLUSION: Our findings implicate type-1 collagen as a previously unrecognized regulator of beta-catenin accumulation and a modifier of TGF-beta1 signaling specifically in primary DD cells. These data have implications for current treatment modalities as well as the design of in vitro models for research into the molecular mechanisms of DD.