Replicating landmine blast loading in cellular in vitro models.

Trauma arising from landmines and improvised explosive devices promotes heterotopic ossification, the formation of extra-skeletal bone in non-osseous tissue. To date, experimental platforms that can replicate the loading parameter space relevant to improvised explosive device and landmine blast wave exposure have not been available to study the effects of such non-physiological mechanical loading on cells. Here, we present the design and calibration of three distinct in vitro experimental loading platforms that allow us to replicate the spectrum of loading conditions recorded in near-field blast wave exposure. We subjected cells in suspension or in a three-dimensional hydrogel to strain rates up to 6000 s-1 and pressure levels up to 45 MPa. Our results highlight that cellular activation is regulated in a non-linear fashion-not by a single mechanical parameter, it is the combined action of the applied mechanical pressure, rate of loading and loading impulse, along with the extracellular environment used to convey the pressure waves. Finally, our research indicates that PO MSCs are finely tuned to respond to mechanical stimuli that fall within defined ranges of loading.

CXCR4, the master regulator of neutrophil trafficking in homeostasis and disease.

BACKGROUND: Chemokines play a critical role in orchestrating the distribution and trafficking of neutrophils in homeostasis and disease. RESULTS: The CXCR4/CXCL12 chemokine axis has been identified as a central regulator of these processes. CONCLUSION: In this review, we focus on the role of CXCR4/CXCL12 chemokine axis in regulating neutrophil release from the bone marrow and the trafficking of senescent neutrophils back to the bone marrow for clearance under homeostasis and disease. We also discuss the role of CXCR4 in fine-tuning neutrophil responses in the context of inflammation.

Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma.

Myofibroblast accumulation, subepithelial fibrosis, and vascular remodeling are complicating features of chronic asthma, but the mechanisms are not clear. Platelet-derived growth factors (PDGFs) regulate the fate and function of various mesenchymal cells and have been implicated as mediators of lung fibrosis. However, it is not known whether PDGF-BB signaling via PDGFRß, which is critical for the recruitment of pericytes to blood vessels, plays a role in airway remodeling in chronic asthma. In the present study, we used a selective PDGFRß inhibitor (CP-673451) to investigate the role of PDGFRß signaling in the development of airway remodeling and lung dysfunction in an established mouse model of house dust mite-induced chronic allergic asthma. Unexpectedly, we found that pharmacological inhibition of PDGFRß signaling in the context of chronic aeroallergen exposure led to exacerbated lung dysfunction and airway smooth muscle thickening. Further studies revealed that the inflammatory response to aeroallergen challenge in mice was associated with decreased PDGF-BB expression and the loss of pericytes from the airway microvasculature. In parallel, cells positive for pericyte markers accumulated in the subepithelial region of chronically inflamed airways. This process was exacerbated in animals treated with CP-673451. The results indicate that perturbed PDGF-BB/PDGFRß signaling and pericyte accumulation in the airway wall may contribute to airway remodeling in chronic allergic asthma.

A role for Rab27 in neutrophil chemotaxis and lung recruitment.

BACKGROUND: Neutrophils are a critical part of the innate immune system. Their ability to migrate into infected or injured tissues precedes their role in microbial killing and clearance. We have previously shown that Rab27a can promote neutrophil migration by facilitating uropod release through protease secretion from primary granule exocytosis at the cell rear. Rab27b has been implicated in primary granule exocytosis but its role in neutrophil migration has not been investigated. RESULTS: Here we found Rab27b to be expressed in bone marrow derived neutrophils and Rab27b knockout (Rab27b KO) along with Rab27a/b double knockout (Rab27DKO) neutrophils exhibited impaired transwell migration in vitro in response to chemokines MIP-2 and LTB4. Interestingly, no additional defect in migration was observed in Rab27DKO neutrophils compared with Rab27b KO neutrophils. In vivo, Rab27DKO mice displayed severe impairment in neutrophil recruitment to the lungs in a MIP-2 dependent model but not in an LPS dependent model. CONCLUSIONS: These data taken together implicate Rab27b in the regulation of neutrophil chemotaxis, likely through the regulation of primary granule exocytosis.

Rab27a-mediated protease release regulates neutrophil recruitment by allowing uropod detachment.

Neutrophil migration is vital for immunity and precedes effector functions such as pathogen killing. Here, we report that this process is regulated by the Rab27a GTPase, a protein known to control granule exocytosis. Rab27a-deficient (Rab27a KO) neutrophils exhibit migration defects in vitro and in vivo, and live-cell microscopy suggests that delayed uropod detachment causes the migratory defect. Surface expression of CD11b, a key adhesion molecule, is increased in chemokine-stimulated Rab27a KO neutrophils compared with the control, suggesting a turnover delay caused by a defect in elastase secretion from azurophilic granules at the rear of bone marrow polymorphonuclear leukocytes (BM-PMNs). We suggest that Rab27a-dependent protease secretion regulates neutrophil migration through proteolysis-dependent de-adhesion of uropods, a mechanism that could be conserved in cell migration and invasion.

VEGFR1 stimulates a CXCR4-dependent translocation of megakaryocytes to the vascular niche, enhancing platelet production in mice.

It has previously been reported that VEGF-A stimulates megakaryocyte (MK) maturation in vitro. Here we show that treatment of mice with the isoform VEGF-A(165) resulted in a significant increase in circulating numbers of platelets. Using specific VEGFR1 and VEGFR2 blocking mAbs and selective VEGFR1 and 2 agonists, PlGF-2 and VEGF-E, respectively, we show directly that stimulation of VEGFR1, but not VEGFR2, increases circulating platelet numbers in vivo. Using flow cytometric analysis of harvested MKs, we show that while PlGF does not change the absolute numbers of MKs present in the bone marrow and the spleen, it increases both their maturation and cell-surface expression of CXCR4 in the bone marrow. Histology of the bone marrow revealed a redistribution of MKs from the endosteal to the vascular niche in response to both VEGF-A(165) and PlGF-2 treatment in vivo. Antagonism of CXCR4 suppressed both the VEGFR1-stimulated redistribution of megakyocytes within the bone marrow compartment and the VEGF-A(165)-induced thrombocytosis. In conclusion, we define a novel proinflammatory VEGFR1-mediated pathway that stimulates the maturation and up-regulation of CXCR4 on megakaryocytes, leading to their redistribution within the bone marrow environment, thereby enhancing platelet production in vivo.

3D printed hybrid scaffolds do not induce adverse inflammation in mice and direct human BM-MSC chondrogenesis in vitro.

Biomaterials that can improve the healing of articular cartilage lesions are needed. To address this unmet need, we developed novel 3D printed silica/poly(tetrahydrofuran)/poly(ε-caprolactone) (SiO2/PTHF/PCL-diCOOH) hybrid scaffolds. Our aim was to carry out essential studies to advance this medical device towards functional validation in pre-clinical trials. First, we show that the chemical composition, microarchitecture and mechanical properties of these scaffolds were not affected by sterilisation with gamma irradiation. To evaluate the systemic and local immunogenic reactivity of the sterilised 3D printed hybrid scaffolds, they were implanted subcutaneously into Balb/c mice. The scaffolds did not trigger a systemic inflammatory response over one week of implantation. The interaction between the host immune system and the implanted scaffold elicited a local physiological reaction with infiltration of mononuclear cells without any signs of a chronic inflammatory response. Then, we investigated how these 3D printed hybrid scaffolds direct chondrogenesis in vitro. Human bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs) seeded within the 3D printed hybrid scaffolds were cultured under normoxic or hypoxic conditions, with or without chondrogenic supplements. Chondrogenic differentiation assessed by both gene expression and protein production analyses showed that 3D printed hybrid scaffolds support hBM-MSC chondrogenesis. Articular cartilage-specific extracellular matrix deposition within these scaffolds was enhanced under hypoxic conditions (1.7 or 3.7 fold increase in the median of aggrecan production in basal or chondrogenic differentiation media). Our findings show that 3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffolds have the potential to support the regeneration of cartilage tissue.

Neutrophil kinetics in health and disease.

Neutrophils play a key role in the elimination of pathogens. They are remarkably short-lived with a circulating half life of 6-8h and hence are produced at a rate of 5x10(10)-10x10(10) cells/day. Tight regulation of these cells is vital because they have significant histotoxic capacity and are widely implicated in tissue injury. This review outlines our current understanding of how neutrophils are released from the bone marrow; in particular, the role of the CXC chemokine receptor 4/stromal-derived factor 1 axis, the relative size and role of the freely circulating and marginated (i.e. slowly transiting) pools within the vascular compartment, and the events that result in the uptake and removal of circulating neutrophils. We also review current understanding of how systemic stress and inflammation affect this finely balanced system.

Annexin A1 regulates neutrophil clearance by macrophages in the mouse bone marrow.

Under homeostatic conditions, a proportion of senescent CXCR4(hi) neutrophils home from the circulation back to the bone marrow, where they are phagocytosed by bone marrow macrophages. In this study, we have identified an unexpected role for the anti-inflammatory molecule annexin A1 (AnxA1) as a critical regulator of this process. We first observed that AnxA1(-/-) mice have significantly increased neutrophil numbers in their bone marrow while having normal levels of GM and G colony-forming units, monocytes, and macrophages. Although AnxA1(-/-) mice have more neutrophils in the bone marrow, a greater proportion of these cells are senescent, as determined by their higher levels of CXCR4 expression and annexin V binding. Consequently, bone marrow neutrophils from AnxA1(-/-) mice exhibit a reduced migratory capacity in vitro. Studies conducted in vitro also show that expression of AnxA1 is required for bone marrow macrophages, but not peritoneal macrophages, to phagocytose apoptotic neutrophils. Moreover, in vivo experiments indicate a defect in clearance of wild-type neutrophils in the bone marrow of AnxA1(-/-) mice. Thus, we conclude that expression of AnxA1 by resident macrophages is a critical determinant for neutrophil clearance in the bone marrow.

Marginalising dyslexic researchers is bad for science.

Communication in the sciences is often based on text, which places researchers with dyslexia at a disadvantage. However, this means that science is missing out on the original insights and specific strengths in exploration that dyslexic researchers bring to their disciplines. Here we discuss how the scientific community can address the challenges that dyslexic researchers face, and how science stands to benefit as a result. We discuss this in the context of a new theoretical framework proposing the existence of complementary learning strategies that could play a key role in scientific progress, particularly with regard to accelerating innovation.

Drug-based mobilisation of mesenchymal stem/stromal cells improves cardiac function post myocardial infarction.

There is an unmet need for treatments that prevent the progressive cardiac dysfunction following myocardial infarction. Mesenchymal stem/stromal cells (MSCs) are under investigation for cardiac repair; however, culture expansion prior to transplantation is hindering their homing and reparative abilities. Pharmacological mobilisation could be an alternative to MSC transplantation. Here, we report that endogenous MSCs mobilise into the circulation at day 5 post myocardial infarction in male Lewis rats. This mobilisation can be significantly increased by using a combination of the FDA-approved drugs mirabegron (ß3-adrenoceptor agonist) and AMD3100 (CXCR4 antagonist). Blinded cardiac magnetic resonance imaging analysis showed the treated group to have increased left ventricular ejection fraction and decreased end systolic volume at 5 weeks post myocardial infarction. The mobilised group had a significant decrease in plasma IL-6 and TNF-α levels, a decrease in interstitial fibrosis, and an increase in the border zone blood vessel density. Conditioned medium from blood-derived MSCs supported angiogenesis in vitro, as shown by tube formation and wound healing assays. Our data suggest a novel pharmacological strategy that enhances myocardial infarction-induced MSC mobilisation and improves cardiac function after myocardial infarction.

3D printed hybrid scaffolds for bone regeneration using calcium methoxyethoxide as a calcium source.

Introduction: Hybrids consist of inorganic and organic co-networks that are indistinguishable above the nanoscale, which can lead to unprecedented combinations of properties, such as high toughness and controlled degradation. Methods: We present 3D printed bioactive hybrid scaffolds for bone regeneration, produced by incorporating calcium into our "Bouncy Bioglass", using calcium methoxyethoxide (CME) as the calcium precursor. SiO2-CaOCME/PTHF/PCL-diCOOH hybrid "inks" for additive manufacturing (Direct Ink Writing) were optimised for synergy of mechanical properties and open interconnected pore channels. Results and Discussion: Adding calcium improved printability. Changing calcium content (5, 10, 20, 30, and 40 mol.%) of the SiO2-CaOCME/PTHF/PCL-diCOOH hybrids affected printability and mechanical properties of the lattice-like scaffolds. Hybrids containing 30 mol.% calcium in the inorganic network (70S30CCME-CL) printed with 500 µm channels and 100 µm strut size achieved the highest strength (0.90 ± 0.23 MPa) and modulus of toughness (0.22 ± 0.04 MPa). These values were higher than Ca-free SiO2/PTHF/PCL-diCOOH hybrids (0.36 ± 0.14 MPa strength and 0.06 ± 0.01 MPa toughness modulus). Over a period of 90 days of immersion in simulated body fluid (SBF), the 70S30CCME-CL hybrids also kept a stable strain to failure (~30 %) and formed hydroxycarbonate apatite within three days. The extracts released by the 70S30CCME-CL hybrids in growth medium did not cause cytotoxic effects on human bone marrow stromal cells over 24 h of culture.

Mesenchymal stem cells.

Mesenchymal Stem cells (MSCs) are stromal cells that can be readily harvested from adult bone marrow and adipose tissue, but also umbilical cords. With respect to respiratory disease, the therapeutic potential of these cells lies in their paracrine effects which underlie their ability to enhance tissue regeneration and modulate immune responses. MSCs have been shown to be effective in a range of murine models of respiratory disease, and there are currently five clinical trials involving the administration of MSCs for respiratory diseases, including COPD and emphysema. This paper summarises the features of MSCs.

Interaction of monodispersed strontium containing bioactive glass nanoparticles with macrophages.

The cellular response of murine primary macrophages to monodisperse strontium containing bioactive glass nanoparticles (SrBGNPs), with diameters of 90 ± 10 nm and a composition (mol%) of 88.8 SiO2-1.8CaO-9.4SrO (9.4% Sr-BGNPs) was investigated for the first time. Macrophage response is critical as applications of bioactive nanoparticles will involve the nanoparticles circulating in the blood stream and macrophages will be the first cells to encounter the particles, as part of inflammatory response mechanisms. Macrophage viability and total DNA measurements were not decreased by particle concentrations of up to 250 µg/mL. The Sr-BGNPs were actively internalised by the macrophages via formation of endosome/lysosome-like vesicles bordered by a membrane inside the cells. The Sr-BGNPs degraded inside the cells, with the Ca and Sr maintained inside the silica network. When RAW264.7 cells were incubated with Sr-BGNPs, the cells were polarised towards the pro-regenerative M2 population rather than the pro-inflammatory M1 population. Sr-BGNPs are potential biocompatible vehicles for therapeutic cation delivery for applications in bone regeneration.

Deficiency of bone marrow beta3-integrin enhances non-functional neovascularization.

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

Bioglass/carbonate apatite/collagen composite scaffold dissolution products promote human osteoblast differentiation.

OssiMend® Bioactive (Collagen Matrix Inc., NJ) is a three-component porous composite bone graft device of 45S5 Bioglass/carbonate apatite/collagen. Our in vitro studies showed that conditioned media of the dissolution products of OssiMend Bioactive stimulated primary human osteoblasts to form mineralized bone-like nodules in vitro in one week, in basal culture media (no osteogenic supplements). Osteoblast differentiation was followed by gene expression analysis and a mineralization assay. In contrast, the dissolution products from commercial OssiMend (Bioglass-free carbonate apatite/collagen scaffolds), or from 45S5 Bioglass particulate alone, did not induce the mineralization of the extracellular matrix, but did induce osteoblast differentiation to mature osteoblasts, evidenced by the strong upregulation of BGLAP and IBSP mRNA levels. The calcium ions and soluble silicon species released from 45S5 Bioglass particles and additional phosphorus release from OssiMend mediated the osteostimulatory effects. Medium conditioned with OssiMend Bioactive dissolution had a much higher concentration of phosphorus and silicon than media conditioned with OssiMend and 45S5 Bioglass alone. While OssiMend and OssiMend Bioactive led to calcium precipitation in cell culture media, OssiMend Bioactive produced a higher concentration of soluble silicon than 45S5 Bioglass and higher dissolution of phosphorus than OssiMend. These in vitro results suggest that adding 45S5 Bioglass to OssiMend produces a synergistic osteostimulation effect on primary human osteoblasts. In summary, dissolution products of a Bioglass/carbonate apatite/collagen composite scaffold (OssiMend® Bioactive) stimulate human osteoblast differentiation and mineralization of extracellular matrix in vitro without any osteogenic supplements. The mineralization was faster than for dissolution products of ordinary Bioglass.

Lung Marginated and Splenic Murine Resident Neutrophils Constitute Pioneers in Tissue-Defense During Systemic E. coli Challenge.

The rapid response of neutrophils throughout the body to a systemic challenge is a critical first step in resolution of bacterial infection such as Escherichia coli (E. coli). Here we delineated the dynamics of this response, revealing novel insights into the molecular mechanisms using lung and spleen intravital microscopy and 3D ex vivo culture of living precision cut splenic slices in combination with fluorescent labelling of endogenous leukocytes. Within seconds after challenge, intravascular marginated neutrophils and lung endothelial cells (ECs) work cooperatively to capture pathogens. Neutrophils retained on lung ECs slow their velocity and aggregate in clusters that enlarge as circulating neutrophils carrying E. coli stop within the microvasculature. The absolute number of splenic neutrophils does not change following challenge; however, neutrophils increase their velocity, migrate to the marginal zone (MZ) and form clusters. Irrespective of their location all neutrophils capturing heat-inactivated E. coli take on an activated phenotype showing increasing surface CD11b. At a molecular level we show that neutralization of ICAM-1 results in splenic neutrophil redistribution to the MZ under homeostasis. Following challenge, splenic levels of CXCL12 and ICAM-1 are reduced allowing neutrophils to migrate to the MZ in a CD29-integrin dependent manner, where the enlargement of splenic neutrophil clusters is CXCR2-CXCL2 dependent. We show directly molecular mechanisms that allow tissue resident neutrophils to provide the first lines of antimicrobial defense by capturing circulating E. coli and forming clusters both in the microvessels of the lung and in the parenchyma of the spleen.

Biased action of the CXCR4-targeting drug plerixafor is essential for its superior hematopoietic stem cell mobilization.

Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.

CXCR2 mediates the recruitment of endothelial progenitor cells during allergic airways remodeling.

Airway remodeling is a central feature of asthma and includes the formation of new peribronchial blood vessels, which is termed angiogenesis. In a number of disease models, bone marrow-derived endothelial progenitor cells (EPCs) have been shown to contribute to the angiogenic response. In this study we set out to determine whether EPCs were recruited into the lungs in a model of allergic airways disease and to identify the factors regulating EPC trafficking in this model. We observed a significant increase in the number of peribronchial blood vessels at day 24, during the acute inflammatory phase of the model. This angiogenic response was associated with an increase in the quantity of EPCs recoverable from the lung. These EPCs formed colonies after 21 days in culture and were shown to express CD31, von Willebrand factor, and vascular endothelial growth factor (VEGF) receptor 2, but were negative for CD45 and CD14. The influx in EPCs was associated with a significant increase in the proangiogenic factors VEGF-A and the CXCR2 ligands, CXCL1 and CXCL2. However, we show directly that, while the CXCL1 and CXCL2 chemokines can recruit EPCs into the lungs of allergen-sensitized mice, VEGF-A was ineffective in this respect. Further, the blockade of CXCR2 significantly reduced EPC numbers in the lungs after allergen exposure and led to a decrease in the numbers of peribronchial blood vessels after allergen challenge with no effect on inflammation. The data presented here provide in vivo evidence that CXCR2 is critical for both EPC recruitment and the angiogenic response in this model of allergic inflammation of the airways.

The Secretive Life of Neutrophils Revealed by Intravital Microscopy.

Neutrophils are the most abundant circulating leukocyte within the blood stream and for many years the dogma has been that these cells migrate rapidly into tissues in response to injury or infection, forming the first line of host defense. While it has previously been documented that neutrophils marginate within the vascular beds of the lung and liver and are present in large numbers within the parenchyma of tissues, such as spleen, lymph nodes, and bone marrow (BM), the function of these tissue resident neutrophils under homeostasis, in response to pathogen invasion or injury has only recently been explored, revealing the unexpected role of these cells as immunoregulators or immune helpers and also unraveling their heterogeneity and plasticity. Neutrophils are highly motile cells and the use of intravital microscopy (IVM) to image cells within their environment with little manipulation has dramatically increased our understanding of the function, migratory behavior, and interaction of these short-lived cells with other innate and adaptive immune cells. Contrary to previous dogma, these studies have shown that marginated and tissue resident neutrophils are the first responders to pathogens and injury, critical in limiting the spread of infection and contributing to the orchestration of the subsequent immune response. The interplay of neutrophils, with other neutrophils, leukocytes, and stroma cells can also modulate and tune their early and late response in order to eradicate pathogens, minimize tissue damage, and, in certain circumstances, contribute to tissue repair. In this review, we will follow the extraordinary journey of neutrophils from their origin in the BM to their death, exploring their role as tissue resident cells in the lung, spleen, lymph nodes, and skin and outlining the importance of neutrophil subsets, their functions under homeostasis, and in response to infection. Finally, we will comment on how understanding these processes in greater detail at a molecular level can lead to development of new therapeutics.