Angiogenin released from ABCB5+ stromal precursors improves healing of diabetic wounds by promoting angiogenesis

Severe angiopathy is a major driver for diabetes-associated secondary complications. Knowledge on the underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors at the edge of nonhealing diabetic wounds in a murine db/db model, closely mirroring human type 2 diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ stromal precursors. This compensates for the profoundly reduced angiogenin expression in nontreated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ stromal precursors before injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice, implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining stromal precursors–based therapies of nonhealing diabetic foot ulcers and other pathologies with impaired angiogenesis

Tissue Microenvironments Define and Get Reinforced by Macrophage Phenotypes in Homeostasis or during Inflammation, Repair and Fibrosis

Current macrophage phenotype classifications are based on distinct in vitro culture conditions that do not adequately mirror complex tissue environments. In vivo monocyte progenitors populate all tissues for immune surveillance which supports the maintenance of homeostasis as well as regaining homeostasis after injury. Here we propose to classify macrophage phenotypes according to prototypical tissue environments, e.g. as they occur during homeostasis as well as during the different phases of (dermal) wound healing. In tissue necrosis and/or infection, damage- and/or pathogen-associated molecular patterns induce proinflammatory macrophages by Toll-like receptors or inflammasomes. Such classically activated macrophages contribute to further tissue inflammation and damage. Apoptotic cells and antiinflammatory cytokines dominate in postinflammatory tissues which induce macrophages to produce more antiinflammatory mediators. Similarly, tumor-associated macrophages also confer immunosuppression in tumor stroma. Insufficient parenchymal healing despite abundant growth factors pushes macrophages to gain a profibrotic phenotype and promote fibrocyte recruitment which both enforce tissue scarring. Ischemic scars are largely devoid of cytokines and growth factors so that fibrolytic macrophages that predominantly secrete proteases digest the excess extracellular matrix. Together, macrophages stabilize their surrounding tissue microenvironments by adapting different phenotypes as feed-forward mechanisms to maintain tissue homeostasis or regain it following injury. Furthermore, macrophage heterogeneity in healthy or injured tissues mirrors spatial and temporal differences in microenvironments during the various stages of tissue injury and repair. Copyright (C) 2012 S. Karger AG, Base

Newly defined ATP-binding cassette subfamily B member 5 positive dermal mesenchymal stem cells promote healing of chronic iron-overload wounds via secretion of interleukin-1 receptor antagonist

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP‐binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+‐derived mesenchymal stem cells (MSCs) attenuated macrophage‐dominated inflammation and thereby accelerated healing of full‐thickness excisional wounds in the iron‐overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin‐1 receptor antagonist (IL‐1RA) secreted by ABCB5+‐derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti‐inflammatory M2 macrophages at the wound site. The beneficial anti‐inflammatory effect of IL‐1RA released from ABCB5+‐derived MSCs on human wound macrophages was conserved in humanized NOD‐scid IL2rγ null mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker‐enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans

Image based machine learning for identification of macrophage subsets

Macrophages play a crucial rule in orchestrating immune responses against pathogens and foreign materials. Macrophages have remarkable plasticity in response to environmental cues and are able to acquire a spectrum of activation status, best exemplified by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time consuming, require large numbers of cells and are resource intensive. In this study, we used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Fluorescent microscopy was used to assess cell morphology of different cell types which were stained for nucleus and actin distribution using DAPI and phalloidin respectively. By only analysing their morphology we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes with an average accuracy of 90%. Thus we suggest high-content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers

Syk-Mediated Translocation of PI3Kδ to the Leading Edge Controls Lamellipodium Formation and Migration of Leukocytes

The non-receptor tyrosine kinase Syk is mainly expressed in the hematopoietic system and plays an essential role in β2 integrin-mediated leukocyte activation. To elucidate the signaling pathway downstream of Syk during β2 integrin (CD11/CD18)-mediated migration and extravasation of polymorphonuclear neutrophils (PMN), we generated neutrophil-like differentiated HL-60 (dHL-60) cells expressing a fluorescently tagged Syk mutant lacking the tyrosine residue at the position 323 (Syk-Tyr323) that is known to be required for the binding of the regulatory subunit p85 of the phosphatidylinositol 3-kinase (PI3K) class IA. Syk-Tyr323 was found to be critical for the enrichment of the catalytic subunit p110δ of PI3K class IA as well as for the generation of PI3K products at the leading edge of the majority of polarized cells. In accordance, the translocation of PI3K p110δ to the leading edge was diminished in Syk deficient murine PMN. Moreover, the expression of EGFP-Syk Y323F interfered with proper cell polarization and it impaired efficient migration of dHL-60 cells. In agreement with a major role of β2 integrins in the recruitment of phagocytic cells to sites of lesion, mice with a Syk-deficient hematopoietic system demonstrated impaired PMN infiltration into the wounded tissue that was associated with prolonged cutaneous wound healing. These data imply a novel role of Syk via PI3K p110δ signaling for β2 integrin-mediated migration which is a prerequisite for efficient PMN recruitment in vivo

Signaling Mechanisms of Vav3, a Guanine Nucleotide Exchange Factor and Androgen Receptor Coactivator, in Physiology and Prostate Cancer Progression

The Rho GTPase guanine nucleotide exchange factor (GEF) Vav3 is the third member of the Vavfamily of GEFS and is activated by tyrosine phosphorylation. Through stimulation of Rho GTPaseactivity, Vav3 promotes cell migration, invasion, and other cellular processes. Work from our laboratory first established that Vav3 is upregulated in models of castration-resistant prostate cancer progression and enhances androgen receptor as well as androgen receptor splice variant activity. Recent analysis of clinical specimens supports Vav3 as a potential biomarker of aggressive prostate cancer. Consistent with a role in promoting castration-­resistant disease, Vav3 is a versatile enhancer of androgen receptor by both ligand-dependent and ligand-independent mechanisms and as such impacts established pathways of androgen receptor reactivation in advanced prostate cancer. Distinct Vav3 domains and mechanisms participate in ligand-dependent and -independent androgen receptor coactivation. To provide a physiologic context, we review Vav3 actions elucidated by gene knockout studies. This chapter describes the pervasive role of Vav3 in progression of prostate cancer to castration resistance. We discuss the mechanisms by which prostate cancer cells exploit Vav3 signaling to promote androgen receptor activity under different hormonal milieus, which are relevant to clinical prostate cancer. Lastly, we review the data on the emerging role for Vav3 in other cancers ranging from leukemias to gliomas.https://nsuworks.nova.edu/hpd_medsci_faculty_books/1002/thumbnail.jp

An Agent-Based Model of a Hepatic Inflammatory Response to Salmonella: A Computational Study under a Large Set of Experimental Data

Citation: Shi, Z. Z., Chapes, S. K., Ben-Arieh, D., & Wu, C. H. (2016). An Agent-Based Model of a Hepatic Inflammatory Response to Salmonella: A Computational Study under a Large Set of Experimental Data. Plos One, 11(8), 39. doi:10.1371/journal.pone.0161131We present an agent-based model (ABM) to simulate a hepatic inflammatory response (HIR) in a mouse infected by Salmonella that sometimes progressed to problematic proportions, known as "sepsis". Based on over 200 published studies, this ABM describes interactions among 21 cells or cytokines and incorporates 226 experimental data sets and/or data estimates from those reports to simulate a mouse HIR in silico. Our simulated results reproduced dynamic patterns of HIR reported in the literature. As shown in vivo, our model also demonstrated that sepsis was highly related to the initial Salmonella dose and the presence of components of the adaptive immune system. We determined that high mobility group box-1, C-reactive protein, and the interleukin-10: tumor necrosis factor-a ratio, and CD4+ T cell: CD8+ T cell ratio, all recognized as biomarkers during HIR, significantly correlated with outcomes of HIR. During therapy-directed silico simulations, our results demonstrated that anti-agent intervention impacted the survival rates of septic individuals in a time-dependent manner. By specifying the infected species, source of infection, and site of infection, this ABM enabled us to reproduce the kinetics of several essential indicators during a HIR, observe distinct dynamic patterns that are manifested during HIR, and allowed us to test proposed therapy-directed treatments. Although limitation still exists, this ABM is a step forward because it links underlying biological processes to computational simulation and was validated through a series of comparisons between the simulated results and experimental studies

Stimulation of bovine monocyte-derived macrophages with lipopolysaccharide, interferon-ɣ, Interleukin-4 or Interleukin-13 does not induce detectable changes in nitric oxide or arginase activity

Background: Bacterial lipopolysaccharide and interferon-γ stimulation of rodent macrophages in vitro induces up-regulation of inducible nitric oxide synthase, whereas interleukin-4 stimulation results in increased activity of arginase-1. Thus different stimulants result in differing macrophage phenotypes, appropriate for responses to a range of pathogens. The current study was conducted in order to determine whether bovine macrophages derived from monocytes and spleen respond similarly. Results: Lipopolysaccharide and interferon-γ did not induce detectable increases in nitric oxide production by bovine monocyte-derived or splenic macrophages in vitro. Similarly, interleukin-4 and interleukin-13 did not affect arginase activity. However, changes in transcription of genes coding for these products were detected. Conclusion: Differences between macrophage activation patterns exist between cattle and other species and these differences may occur during the post-transcription phase

Efficient discovery of anti-inflammatory small-molecule combinations using evolutionary computing

The control of biochemical fluxes is distributed, and to perturb complex intracellular networks effectively it is often necessary to modulate several steps simultaneously. However, the number of possible permutations leads to a combinatorial explosion in the number of experiments that would have to be performed in a complete analysis. We used a multiobjective evolutionary algorithm to optimize reagent combinations from a dynamic chemical library of 33 compounds with established or predicted targets in the regulatory network controlling IL-1β expression. The evolutionary algorithm converged on excellent solutions within 11 generations, during which we studied just 550 combinations out of the potential search space of ~9 billion. The top five reagents with the greatest contribution to combinatorial effects throughout the evolutionary algorithm were then optimized pairwise. A p38 MAPK inhibitor together with either an inhibitor of IκB kinase or a chelator of poorly liganded iron yielded synergistic inhibition of macrophage IL-1β expression. Evolutionary searches provide a powerful and general approach to the discovery of new combinations of pharmacological agents with therapeutic indices potentially greater than those of single drugs