Functional Crosstalk between PCSK9 Internalization and Pro-Inflammatory Activation in Human Macrophages: Role of Reactive Oxygen Species Release.

Atherosclerosis is a cardiovascular disease caused mainly by dyslipidemia and is characterized by the formation of an atheroma plaque and chronic inflammation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that induces the degradation of the LDL receptor (LDLR), which contributes to increased levels of LDL cholesterol and the progress of atherosclerosis. Given that macrophages are relevant components of the lipidic and inflammatory environment of atherosclerosis, we studied the effects of PCSK9 treatment on human macrophages. Our data show that human macrophages do not express PCSK9 but rapidly incorporate the circulating protein through the LDLR and also activate the pro-inflammatory TLR4 pathway. Both LDLR and TLR4 are internalized after incubation of macrophages with exogenous PCSK9. PCSK9 uptake increases the production of reactive oxygen species and reduces the expression of genes involved in lipid metabolism and cholesterol efflux, while enhancing the production of pro-inflammatory cytokines through a TLR4-dependent mechanism. Under these conditions, the viability of macrophages is compromised, leading to increased cell death. These results provide novel insights into the role of PCSK9 in the crosstalk of lipids and cholesterol metabolism through the LDLR and on the pro-inflammatory activation of macrophages through TLR4 signaling. These pathways are relevant in the outcome of atherosclerosis and highlight the relevance of PCSK9 as a therapeutic target for the treatment of cardiovascular diseases.

HIF-1α and PFKFB3 Mediate a Tight Relationship Between Proinflammatory Activation and Anerobic Metabolism in Atherosclerotic Macrophages.

OBJECTIVE: Although it is accepted that macrophage glycolysis is upregulated under hypoxic conditions, it is not known whether this is linked to a similar increase in macrophage proinflammatory activation and whether specific energy demands regulate cell viability in the atheromatous plaque. APPROACH AND RESULTS: We studied the interplay between macrophage energy metabolism, polarization, and viability in the context of atherosclerosis. Cultured human and murine macrophages and an in vivo murine model of atherosclerosis were used to evaluate the mechanisms underlying metabolic and inflammatory activity of macrophages in the different atherosclerotic conditions analyzed. We observed that macrophage energetics and inflammatory activation are closely and linearly related, resulting in dynamic calibration of glycolysis to keep pace with inflammatory activity. In addition, we show that macrophage glycolysis and proinflammatory activation mainly depend on hypoxia-inducible factor and on its impact on glucose uptake, and on the expression of hexokinase II and ubiquitous 6-phosphofructo-2-kinase. As a consequence, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Moreover, when macrophages' ability to increase glycolysis through 6-phosphofructo-2-kinase is experimentally attenuated, cell viability is reduced if subjected to proinflammatory or hypoxic conditions, but unaffected under control conditions. In addition to this, granulocyte-macrophage colony-stimulating factor enhances anerobic glycolysis while exerting a mild proinflammatory activation. CONCLUSIONS: These findings, in human and murine cells and in an animal model, show that hypoxia potentiates macrophage glycolytic flux in concert with a proportional upregulation of proinflammatory activity, in a manner that is dependent on both hypoxia-inducible factor -1α and 6-phosphofructo-2-kinase.

Metabolic signatures linked to macrophage polarization: from glucose metabolism to oxidative phosphorylation.

Macrophages are present in a large variety of locations, playing distinct functions that are determined by its developmental origin and by the nature of the activators of the microenvironment. Macrophage activation can be classified as pro-inflammatory (M1 polarization) or anti-inflammatory-pro-resolution-deactivation (M2), these profiles coexisting in the course of the immune response and playing a relevant functional role in the onset of inflammation (Figure 1). Several groups have analysed the metabolic aspects associated with macrophage activation to answer the question about what changes in the regulation of energy metabolism and biosynthesis of anabolic precursors accompany the different types of polarization and to what extent they are necessary for the expression of the activation phenotypes. The interest of these studies is to regulate macrophage function by altering their metabolic activity in a 'therapeutic way'.

Trabectedin modulates macrophage polarization in the tumor-microenvironment. Role of KV1.3 and KV1.5 channels.

Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor-associated macrophages (TAM) have a string of immunosuppressive functions and they are considered a therapeutic target in cancer. This study aimed to analyze the effects of trabectedin, an antitumor agent, on the tumor-microenvironment through the characterization of the electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but their treatment (16 h) with sub-cytotoxic concentrations of trabectedin increased their KV current due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv) exhibited an M2-like phenotype. TAMiv generated a small KV current and express high levels of M2 markers. K+ current from TAMs isolated from tumors generated in mice is a mixture of KV and KCa, and in TAM isolated from tumors generated in trabectedin-treated mice, the current is mostly driven by KCa. We conclude that the antitumor capacity of trabectedin is not only due to its effects on tumor cells, but also to the modulation of the tumor microenvironment, due, at least in part, to the modulation of the expression of different macrophage ion channels.

Immunometabolic actions of trabectedin and lurbinectedin on human macrophages: relevance for their anti-tumor activity.

In recent years, the central role of cell bioenergetics in regulating immune cell function and fate has been recognized, giving rise to the interest in immunometabolism, an area of research focused on the interaction between metabolic regulation and immune function. Thus, early metabolic changes associated with the polarization of macrophages into pro-inflammatory or pro-resolving cells under different stimuli have been characterized. Tumor-associated macrophages are among the most abundant cells in the tumor microenvironment; however, it exists an unmet need to study the effect of chemotherapeutics on macrophage immunometabolism. Here, we use a systems biology approach that integrates transcriptomics and metabolomics to unveil the immunometabolic effects of trabectedin (TRB) and lurbinectedin (LUR), two DNA-binding agents with proven antitumor activity. Our results show that TRB and LUR activate human macrophages toward a pro-inflammatory phenotype by inducing a specific metabolic rewiring program that includes ROS production, changes in the mitochondrial inner membrane potential, increased pentose phosphate pathway, lactate release, tricarboxylic acids (TCA) cycle, serine and methylglyoxal pathways in human macrophages. Glutamine, aspartate, histidine, and proline intracellular levels are also decreased, whereas oxygen consumption is reduced. The observed immunometabolic changes explain additional antitumor activities of these compounds and open new avenues to design therapeutic interventions that specifically target the immunometabolic landscape in the treatment of cancer.

Graphene Particles Interfere with Pro-Inflammatory Polarization of Human Macrophages: Functional and Electrophysiological Evidence.

The interaction of two types of fragmented graphene particles (30-160 nm) with human macrophages is studied. Since macrophages have significant phagocytic activity, the incorporation of graphene particles into cells has an effect on the response to functional polarization stimuli, favoring an anti-inflammatory profile. Incubation of macrophages with graphene foam particles, prepared by chemical vapor deposition, and commercially available graphene nanoplatelet particles does not affect cell viability when added at concentrations up to 100 µg mL-1 ; macrophages exhibit differential quantitative responses to each type of graphene particles. Although both materials elicit similar increases in the release of reactive oxygen species, the impact on the transcriptional regulation associated with the polarization profile is different; graphene nanoplatelets significantly modify this transcriptomic profile. Moreover, these graphene particles differentially affect the motility and phagocytosis of macrophages. After the incorporation of both graphene types into the macrophages, they exhibit specific responses in terms of the mitochondrial oxygen consumption and electrophysiological potassium currents at the cell plasma membrane. These data support the view that the physical structure of the graphene particles has an impact on human macrophage responses, paving the way for the development of new mechanisms to modulate the activity of the immune system.

Specific Effects of Trabectedin and Lurbinectedin on Human Macrophage Function and Fate-Novel Insights.

BACKGROUND: Tumor-associated macrophages (TAMs) play a crucial role in suppressing the immunosurveillance function of the immune system that prevents tumor growth. Indeed, macrophages can also be targeted by different chemotherapeutic agents improving the action over immune checkpoints to fight cancer. Here we describe the effect of trabectedin and lurbinectedin on human macrophage cell viability and function. METHODS: Blood monocytes from healthy donors were differentiated into macrophages and exposed to different stimuli promoting functional polarization and differentiation into tumor-associated macrophages. Cells were challenged with the chemotherapeutic drugs and the effects on cell viability and function were analyzed. RESULTS: Human macrophages exhibit at least two different profiles in response to these drugs. One-fourth of the blood donors assayed (164 individuals) were extremely sensitive to trabectedin and lurbinectedin, which promoted apoptotic cell death. Macrophages from other individuals retained viability but responded to the drugs increasing reactive oxygen production and showing a rapid intracellular calcium rise and a loss of mitochondrial oxygen consumption. Cell-membrane exposure of programmed-death ligand 1 (PD-L1) significantly decreased after treatment with therapeutic doses of these drugs, including changes in the gene expression profile of hypoxia-inducible factor 1 alpha (HIF-1α)-dependent genes, among other. CONCLUSIONS: The results provide evidence of additional onco-therapeutic actions for these drugs.

Benzylamine and Thenylamine Derived Drugs Induce Apoptosis and Reduce Proliferation, Migration and Metastasis Formation in Melanoma Cells.

Melanomas are heterogeneous and aggressive tumors, and one of the worse in prognosis. Melanoma subtypes follow distinct pathways until terminal oncogenic transformation. Here, we have evaluated a series of molecules that exhibit potent cytotoxic effects over the murine and human melanoma cell lines B16F10 and MalMe-3M, respectively, both ex vivo and in animals carrying these melanoma cells. Ex vivo mechanistic studies on molecular targets involved in melanoma growth, migration and viability were evaluated in cultured cells treated with these drugs which exhibited potent proapoptotic and cytotoxic effects and reduced cell migration. These drugs altered the Wnt/ß-catenin pathway, which is important for the oncogenic phenotype of melanoma cells. In in vivo experiments, male C57BL/6 or nude mice were injected with melanoma cells that rapidly expanded in these animals and, in some cases were able to form metastasis in lungs. Treatment with anti-tumor drugs derived from benzylamine and 2-thiophenemethylamine (F10503LO1 and related compounds) significantly attenuated tumor growth, impaired cell migration, and reduced the metastatic activity. Several protocols of administration were applied, all of them leading to significant reduction in the tumor size and enhanced animal survival. Tumor cells carrying a luciferase transgene allowed a time-dependent study on the progression of the tumor. Molecular analysis of the pathways modified by F10503LO1 and related compounds defined the main relevant targets for tumor regression: the activation of pro-apoptotic and anti-proliferative routes. These data might provide the proof-of-principle and rationale for its further clinical evaluation.

Protection against gamma-radiation injury by protein tyrosine phosphatase 1B.

Protein tyrosine phosphatase 1B (PTP1B) is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs) from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic ß-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-ß signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant.

Beneficial Effect of Short Pretransplant Period of Hypothermic Pulsatile Perfusion of the Warm-Ischemic Kidney after Cold Storage: Experimental Study.

Warm ischemia (WI) produces a significant deleterious effect in potential kidney grafts. Hypothermic machine perfusion (HMP) seems to improve immediate graft function after transplant. Our aim was to analyze the effect of short pretransplant periods of pulsatile HMP on histology and renal injury in warm-ischemic kidneys. Twelve minipigs were used. WI was achieved in the right kidney by applying a vascular clamp for 45 min. After nephrectomy, autotransplant was performed following one of two strategies: cold storage of the kidneys or cold storage combined with perfusion in pulsatile HMP. The graft was removed early to study renal morphology, inflammation (fibrosis), and apoptosis. Proinflammatory activity and fibrosis were less pronounced after cold storage of the kidneys with HMP than after cold storage only. The use of HMP also decreased apoptosis compared with cold storage only. The detrimental effects on cells of an initial and prolonged period of WI seem to improve with a preservation protocol that includes a short period of pulsatile HMP after cold storage and immediately before the transplant, in comparison with cold storage only.

GM-CSF Enhances Macrophage Glycolytic Activity In Vitro and Improves Detection of Inflammation In Vivo.

UNLABELLED: (18)F-FDG accumulates in glycolytically active tissues and is known to concentrate in tissues that are rich in activated macrophages. In this study, we tested the hypotheses that human granulocyte-macrophage colony-stimulating factor (GM-CSF), a clinically used cytokine, increases macrophage glycolysis and deoxyglucose uptake in vitro and acutely enhances (18)F-FDG uptake within inflamed tissues such as atherosclerotic plaques in vivo. METHODS: In vitro experiments were conducted on human macrophages whereby inflammatory activation and uptake of radiolabeled 2-deoxyglucose was assessed before and after GM-CSF exposure. In vivo studies were performed on mice and New Zealand White rabbits to assess the effect of GM-CSF on (18)F-FDG uptake in normal versus inflamed arteries, using PET. RESULTS: Incubation of human macrophages with GM-CSF resulted in increased glycolysis and increased 2-deoxyglucose uptake (P < 0.05). This effect was attenuated by neutralizing antibodies against tumor necrosis factor-α or after silencing or inhibition of 6-phosphofructo-2-kinase. In vivo, in mice and in rabbits, intravenous GM-CSF administration resulted in a 70% and 73% increase (P < 0.01 for both), respectively, in arterial (18)F-FDG uptake in atherosclerotic animals but not in nonatherosclerotic controls. Histopathologic analysis demonstrated a significant correlation between in vivo (18)F-FDG uptake and macrophage staining (R = 0.75, P < 0.01). CONCLUSION: GM-CSF substantially augments glycolytic flux in vitro (via a mechanism dependent on ubiquitous type 6-phosphofructo-2-kinase and tumor necrosis factor-α) and increases (18)F-FDG uptake within inflamed atheroma in vivo. These findings demonstrate that GM-CSF can be used to enhance detection of inflammation. Further studies should explore the role of GM-CSF stimulation to enhance the detection of inflammatory foci in other disease states.

Direct agonist/antagonist functions of dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) exhibits peak adrenal secretion in the fetus at term and around age 30 yr in the adult. Levels then progressively decline, which is associated with decreased levels of testosterone, dihydrotestosterone, and estrogen in peripheral tissues. DHEA supplementation in postmenopausal women increases bone formation and density, an effect mainly attributed to peripheral conversion to sex hormones. In this study, we tested DHEA for direct effects on the androgen (AR) and estrogen (ER) receptors. DHEA bound to AR with a Ki of 1 microM, which was associated with AR transcriptional antagonism on both the mouse mammary tumor virus and prostate-specific antigen promoters, much like the effects of bicalutamide. Unlike bicalutamide, DHEA stimulated, rather than inhibited, LNCaP cell growth, suggesting possible interaction with other hormone receptors. Indeed DHEA bound to ERalpha and ERbeta, with Ki values of 1.1 and 0.5 microM, respectively. Despite the similar binding affinities, DHEA showed preferential agonism of ERbeta with an EC50 of approximately 200 nm and maximal activation at 1 microM. With ERalpha we found 30-70% agonism at 5 microM, depending on the assay. Physiological levels of DHEA are approximately 30 nM and up to 90 nM in the prostate. DHEA at 30 nM is actually sufficient to activate ERbeta transcription to the same degree as estrogen at its circulating concentration, and additive effects are seen when the two were combined. Taken together, DHEA has the potential for physiologically relevant direct activation of ERbeta. With peak levels at term and age 30 yr, there is also a potential for antagonist effects on AR and partial agonism of ERalpha.

Bridged androstenediol analogs as ER-beta selective SERMs.

A series of bridged androstenediol derivatives was prepared. The bridged compounds exhibited reduced ER-beta selectivity relative to uncyclized analogs.

Androstene-3,5-dienes as ER-beta selective SERMs.

A series of androstene-3,5-diene derivatives were prepared. Despite lacking the C-3 hydroxyl previously believed necessary for ER activity, some of the analogs retained surprising affinity for ER-beta. For example, diene 4 retained excellent selectivity and potency as an ER-beta agonist and was more selective for ER-beta over the androgen receptor (AR).

Androstenediol analogs as ER-beta-selective SERMs.

A series of 19-substituted androstenediol derivatives was prepared. Some of the novel analogs were surprisingly potent and selective ligands for ER-beta.

Discovery of structurally diverse natural product antagonists of chemokine receptor CXCR3.

The chemokines (CXCL9, CXCL10 and CXCL11) and associated CXCR3 receptor are expressed during the inflammatory process from multiple sclerosis, atherosclerosis or organ transplantation resulting in the recruitment of lymphocytes leading to tissue damage. It is hypothesized that blocking of the ligand/CXCR3 receptor interaction has potential to provide opportunity for development of agents that would block tissue rejection. In this paper, four classes of natural product inhibitors (IC50 ranging 0.1-41 microM) have been described that block the CXCR3 receptor interaction of IP-10 ligand. These include a cyclic thiopeptide (duramycin), polyketide glycosides (roselipins), steroidal glycosides (hypoglausin A and dioscin) and a novel alkyl pyridinium alkaloid that were isolated by bioassay-guided fractionation of the organic extracts derived from actinomycete, fungal, plant and marine sources and discovered using 125I IP-10/CXCR3 binding assay. Duramycin was the most potent with an IC50 of 0.1 microM. Roselipins 2A, 2B and 1A showed IC50 values of 14.6, 23.5, and 41 microM, respectively. Diosgenin glycosides dioscin, hypoglaucin A and kallstroemin D exhibited IC50 values of 2.1, 0.47 and 3 microM, respectively. A novel cyclic 3-alkyl pyridinium salt isolated from a sponge displayed a binding IC50 of 0.67 microM.