Cardiac sympathetic overdrive, M2 macrophage activation and fibroblast heterogeneity are associated with cardiac remodeling in a chronic pressure overload rat model of HFpEF.

Heart failure with preserved ejection fraction (HFpEF) is a multifaceted pathogenesis disease and the exact mechanisms driving HFpEF have not been completely elucidated. Pressure overload hypertrophy (POH) related fibroblasts and M2 macrophages in HFpEF myocardium have been recently identified and are now of great interest. Sympathetic overdrive has also been implicated in HFpEF. This study is designed to dynamically observe the potential roles of aforementioned mechanisms in pathological remodeling and cardiac dysfunction in chronic PO rats. Surgical constriction of the abdominal aorta was used for induction of HFpEF. Echocardiography, electrocardiogram, hemodynamic measurement, hematoxylin and eosin staining, Masson staining, immunohistochemistry and immunofluorescence were performed to assess the changes in heart dysfunction, cardiac remodeling and driving mechanisms at different time points (2, 18, 24 weeks). The PO induced HFpEF model was well established, which was confirmed by the persistent increase in carotid artery systolic and diastolic blood pressure, and left ventricle hypertrophy at the corresponding postoperative stage. Meanwhile, PO hypertrophy gradually developed into HFpEF, associated with QT and QTc intervals prolongation, normal systolic (EF was maintained at >50%) but impaired diastolic function (increasing LVEDP and LV -dP/dtmin, abnormal E/A ratio), increased myocytes size, and observed relatively slight inflammatory infiltration but robust reactive fibrosis. IHC staining further confirmed that macrophages (CD68) but not neutrophils (MPO) or T cells (CD3) accounted for a predominant proportion of infiltrating cells. Mechanistically, we found that the infiltrating macrophages in the heart expressed high levels of CD206 which was simultaneously adjacent to POH fibroblasts appeared to overexpression of α-SMA in PO rats at late stages. Interestingly, we distinguished two different POHF sub-populations during PO induced HFpEF development, according to non overlapping signals of α-SMA and PDGFRα/ß proteins. Additionally, PO led to a pronounced exaggeration in sympathetic fibers at all time points. These findings suggest that the establishing model here begins with cardiac sympathetic overdrive, subsequently along with immune cells especially M2 macrophage accumulation and fibroblast heterogeneity at later stages is associated with the development of cardiac maladaptive remodeling and diastolic dysfunction thus further progression to HFpEF.

First transcriptomic insight into the reprogramming of human macrophages by levan-type fructans.

Based on stimuli in the biological milieu, macrophages can undergo classical activation into the M1 pro-inflammatory (anti-cancer) phenotype or to the alternatively activated M2 anti-inflammatory one. Drug-free biomaterials have emerged as a new therapeutic strategy to modulate macrophage phenotype. Among them, polysaccharides polarize macrophages to M1 or M2 phenotypes based on the surface receptors they bind. Levan, a fructan, has been proposed as a novel biomaterial though its interaction with macrophages has been scarcely explored. In this study, we investigate the interaction of non-hydrolyzed and hydrolyzed Halomonas levan and its sulfated derivative with human macrophages in vitro. Viability studies show that these levans are cell compatible. In addition, RNA-sequencing analysis reveals the upregulation of pro-inflammatory pathways. These results are in good agreement with real time-quantitative polymerase chain reaction that indicates higher expression levels of C-X-C Motif Chemokine Ligand 8 and interleukin-6 genes and the M2-to-M1 reprogramming of these cells upon levan treatment. Finally, cytokine release studies confirm that hydrolyzed levans increase the secretion of pro-inflammatory cytokines and reprogram IL-4-polarized macrophages to the M1 state. Overall findings indicate that Halomonas levans trigger a classical macrophage activation and pave the way for their application in therapeutic interventions requiring a pro-inflammatory phenotype.

Amphiphilic galactomannan nanoparticles trigger the alternative activation of murine macrophages.

Macrophages are highly plastic phagocytic cells that can exist in distinct phenotypes and play key roles in physiological and pathological pathways. They can be classically activated to the pro-inflammatory M1 phenotype or alternatively activated to an M2 anti-inflammatory one by various stimuli in the biological milieu. Different biomaterials polarize macrophages to M1 or M2 phenotypes and emerged as a very promising strategy to modulate their activation and performance. In this work, we investigate the ability of drug-free amphiphilic nanoparticles (hydrodynamic diameter of ~130 nm) produced by the self-assembly of a graft copolymer of hydrolyzed galactomannan, a natural polysaccharide of galactose and mannose, that was hydrophobized in the side-chain with poly(methyl methacrylate) blocks and that can encapsulate hydrophobic drugs, to trigger macrophage polarization. The compatibility and uptake of the nanoparticles are demonstrated in the murine macrophage cell line RAW264.7 by a metabolic assay, confocal laser scanning fluorescence microscopy (CLSFM) and imaging flow cytometry in the absence and the presence of endocytosis inhibitors. Results indicate that they are internalized by both clathrin- and caveolin-mediated endocytosis. The ability of these drug-free nanoparticles to polarize these cells to the M2-like phenotype and to switch an M1 to an M2 phenotype is confirmed by the downregulation of the M1 marker cluster of differentiation 80 (CD80), and upregulation of M2 markers CD163 and CD206, as measured by flow cytometry and CLSFM. In addition, we preliminarily assess the effect of the nanoparticles on wound healing by tracking the closure of an artificial wound of RAW264.7 macrophages in a scratch assay. Findings indicate a faster closure of the wound in the presence of the nanoparticles with respect to untreated cells. Finally, a migration assay utilizing a macrophage/fibroblast co-culture model in vitro demonstrates that M2 polarization increases fibroblast migration by 24-fold with respect to untreated cells. These findings demonstrate the ability of this nanotechnology platform to interfere and change the macrophages phenotype in vitro and represent robust evidence for the investigation of their therapeutic performance alone or in combination with an encapsulated hydrophobic drug in wound models in vivo.

LC3-Associated Phagocytosis (LAP): A Potentially Influential Mediator of Efferocytosis-Related Tumor Progression and Aggressiveness.

One aim of cancer therapies is to induce apoptosis of tumor cells. Efficient removal of the apoptotic cells requires coordinated efforts between the processes of efferocytosis and LC3-associated phagocytosis (LAP). However, this activity has also been shown to produce anti-inflammatory and immunosuppressive signals that can be utilized by live tumor cells to evade immune defense mechanisms, resulting in tumor progression and aggressiveness. In the absence of LAP, mice exhibit suppressed tumor growth during efferocytosis, while LAP-sufficient mice show enhanced tumor progression. Little is known about how LAP or its regulators directly affect efferocytosis, tumor growth and treatment responses, and identifying the mechanisms involved has the potential to lead to the discovery of novel approaches to target cancer cells. Also incompletely understood is the direct effect of apoptotic cancer cells on LAP. This is particularly important as induction of apoptosis by current cytotoxic cancer therapies can potentially stimulate LAP following efferocytosis. Herein, we highlight the current understanding of the role of LAP and its relationship with efferocytosis in the tumor microenvironment with a view to presenting novel therapeutic strategies.

Increased Vascular Adhesion Protein 1 (VAP-1) Levels are Associated with Alternative M2 Macrophage Activation and Poor Prognosis for Human Gliomas.

Glioma is characterized by a high heterogeneity in the brain tumor. Abundant tumor-associated macrophages (TAMs) exist as neoplastic tissues, implicating tumor plasticity and thus leading to therapeutic challenges. Vascular adhesion protein (VAP-1) potentially serves as a mediator for TAM immunity in tumor milieu. We previously demonstrated that VAP-1 could contribute to tumor malignancy, but its characteristics in TAM immunity of glioma progression are still unclear. This study explored the association of VAP-1 expression with TAM distribution as well as the resulting clinical significance and prognostic value in human gliomas. An in-depth analysis of AOC3 (VAP-1) gene expression was performed using 695 glioma samples derived from the cancer genome atlas (TCGA)-lower grade glioma and glioblastoma (GBMLGG) cohort. Bioinformatic analysis confirmed that VAP-1 expression is associated with poor prognosis of glioma patients (p = 0.0283). VAP-1 and TAM biomarkers (CD68, iNOS, and CD163) were evaluated by immunohistochemistry in 108 gliomas from Kaohsiung Medical University Hospital. VAP-1+ was expressed in 56 (51.85%) cases and this phenotype revealed a significant association with overall survival in Kaplan-Meier analysis (p < 0.0001). Immunohistochemical double staining showed that VAP-1 immunoreactivity was present around CD163+ M2 infiltration location, including aggressive lesions and neighboring neovasculature. We demonstrated that high VAP-1 expression levels positively correlated with CD163+ M2 activation and coexpression of these two proteins was associated with worse survival in gliomas (p < 0.0001). Multivariate analysis indicated that VAP-1 alone and co-expressed with CD163 were the significantly independent indicators (both p < 0.0001). Furthermore, VAP-1/CD163 coexpression exhibited excellent diagnostic accuracy in gliomas (AUC = 0.8008). In conclusion, VAP-1 and TAM CD163 M2 coexpression was found in glioma tissues belonging to a highly malignant subgroup that was associated with poor prognosis. These results implied VAP-1 abundance is closely linked to alternative M2 activation during glioma progression. From the aforementioned data, a reasonable inference is that VAP-1 combined with targeting M2 immunity might be an effective therapeutic target for human gliomas.

IL-4 type 1 receptor signaling up-regulates KCNN4 expression, and increases the KCa3.1 current and its contribution to migration of alternative-activated microglia.

The Ca(2+)-activated K(+) channel, KCa3.1 (KCNN4/IK1/SK4), contributes to "classical," pro-inflammatory activation of microglia, and KCa3.1 blockers have improved the outcome in several rodent models of CNS damage. For instance, blocking KCa3.1 with TRAM-34 rescued retinal ganglion neurons after optic nerve damage in vivo and, reduced p38 MAP kinase activation, production of reactive oxygen and nitrogen species, and neurotoxicity by microglia in vitro. In pursuing the therapeutic potential of KCa3.1 blockers, it is crucial to assess KCa3.1 contributions to other microglial functions and activation states, especially the IL-4-induced "alternative" activation state that can counteract pro-inflammatory states. We recently found that IL-4 increases microglia migration - a crucial function in the healthy and damaged CNS - and that KCa3.1 contributes to P2Y2 receptor-stimulated migration. Here, we discovered that KCa3.1 is greatly increased in alternative-activated rat microglia and then contributes to an enhanced migratory capacity. IL-4 up-regulated KCNN4 mRNA (by 6 h) and greatly increased the KCa3.1 current by 1 day, and this required de novo protein synthesis. The increase in current was sustained for at least 6 days. IL-4 increased microglial migration and this was reversed by blocking KCa3.1 with TRAM-34. A panel of inhibitors of signal-transduction mediators was used to analyze contributions of IL-4-related signaling pathways. Induction of KCNN4 mRNA and KCa3.1 current was mediated specifically through IL-4 binding to the type I receptor and, surprisingly, it required JAK3, Ras/MEK/ERK signaling and the transcription factor, activator protein-1, rather than JAK2, STAT6, or phosphatidylinositol 3-kinase.The same receptor subtype and pathway were required for the enhanced KCa3.1-dependent migration. In providing the first direct signaling link between an IL-4 receptor, expression and roles of an ion channel, this study also highlights the potential importance of KCa3.1 in alternative-activated microglia.