Ceramide 1-Phosphate Protects Endothelial Colony-Forming Cells From Apoptosis and Increases Vasculogenesis In Vitro and In Vivo.

OBJECTIVE: Ceramide 1-phosphate (C1P) is a bioactive sphingolipid highly augmented in damaged tissues. Because of its abilities to stimulate migration of murine bone marrow-derived progenitor cells, it has been suggested that C1P might be involved in tissue regeneration. In the present study, we aimed to investigate whether C1P regulates survival and angiogenic activity of human progenitor cells with great therapeutic potential in regenerative medicine such as endothelial colony-orming cells (ECFCs). Approach and Results: C1P protected ECFC from TNFα (tumor necrosis factor-α)-induced and monosodium urate crystal-induced death and acted as a potent chemoattractant factor through the activation of ERK1/2 (extracellular signal-regulated kinases 1 and 2) and AKT pathways. C1P treatment enhanced ECFC adhesion to collagen type I, an effect that was prevented by ß1 integrin blockade, and to mature endothelial cells, which was mediated by the E-selectin/CD44 axis. ECFC proliferation and cord-like structure formation were also increased by C1P, as well as vascularization of gel plug implants loaded or not with ECFC. In a murine model of hindlimb ischemia, local administration of C1P alone promoted blood perfusion and reduced necrosis in the ischemic muscle. Additionally, the beneficial effects of ECFC infusion after ischemia were amplified by C1P pretreatment, resulting in a further and significant enhancement of leg reperfusion and muscle repair. CONCLUSIONS: Our findings suggest that C1P may have therapeutic relevance in ischemic disorders, improving tissue repair by itself, or priming ECFC angiogenic responses such as chemotaxis, adhesion, proliferation, and tubule formation, which result in a better outcome of ECFC-based therapy.

Human Plasmacytoid Dendritic Cells Elicited Different Responses after Infection with Pathogenic and Nonpathogenic Junin Virus Strains.

The arenavirus Junin virus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. We characterized the JUNV infection of human peripheral blood-derived plasmacytoid dendritic cells (hpDC), demonstrating that hpDC are susceptible to infection with the C#1 strain (attenuated) and even more susceptible to infection with the P (virulent) JUNV strain. However, hpDC elicited different responses in terms of viability, activation, maturation, and cytokine expression after infection with both JUNV strains.

Acidic preconditioning improves the proangiogenic responses of endothelial colony forming cells.

OBJECTIVE: Acidosis is present in several pathological conditions where vasculogenesis takes place including ischemia, tumor growth and wound healing. We have previously demonstrated that acidosis induces human CD34+ cell apoptosis. Considering that endothelial colony-forming cells (ECFC) are a subpopulation of CD34+ cells and key players in vasculogenesis, in the present study we investigated the effect of acidosis on the survival and functionality of ECFC. APPROACH AND RESULTS: Endothelial colony-forming cells obtained by differentiation of human cord blood CD34+ cells in endothelial growth medium-2 for 14-21 days were exposed at pH 7.4, 7.0 or 6.6. We found that acidosis failed to induce ECFC apoptosis and, although an early reduction in proliferation, chemotaxis, wound healing and capillary-like tubule formation was observed, once the medium pH was restored to 7.4, ECFC proliferation and tubulogenesis were augmented. Stromal cell derived factor-1 (SDF1)-driven migration and chemokine receptor type 4 surface expression were also increased. The maximal proangiogenic effect exerted by acidic preconditioning was observed after 6 h at pH 6.6. Furthermore, preconditioned ECFC showed an increased ability to promote tissue revascularization in a murine model of hind limb ischemia. Immunoblotting assays showed that acidosis activated AKT and ERK1/2 and inhibited p38 pathways. Proliferation rises triggered by acidic preconditioning were no longer observed after AKT or ERK1/2 inhibition, whereas p38 suppression not only mimicked but also potentiated the effect of acidosis on ECFC tubule formation abilities. CONCLUSIONS: These results demonstrate that acidic preconditioning greatly increases ECFC-mediated angiogenesis in vitro including ECFC proliferation, tubulogenesis and SDF1-driven chemotaxis and is a positive regulator of microvessel formation in vivo.

Effects of tetrahydrouridine on pharmacokinetics and pharmacodynamics of oral decitabine.

The deoxycytidine analog decitabine (DAC) can deplete DNA methyl-transferase 1 (DNMT1) and thereby modify cellular epigenetics, gene expression, and differentiation. However, a barrier to efficacious and accessible DNMT1-targeted therapy is cytidine deaminase, an enzyme highly expressed in the intestine and liver that rapidly metabolizes DAC into inactive uridine counterparts, severely limiting exposure time and oral bioavailability. In the present study, the effects of tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase, on the pharmacokinetics and pharmacodynamics of oral DAC were evaluated in mice and nonhuman primates. Oral administration of THU before oral DAC extended DAC absorption time and widened the concentration-time profile, increasing the exposure time for S-phase-specific depletion of DNMT1 without the high peak DAC levels that can cause DNA damage and cytotoxicity. THU also decreased interindividual variability in pharmacokinetics seen with DAC alone. One potential clinical application of DNMT1-targeted therapy is to increase fetal hemoglobin and treat hemoglobinopathy. Oral THU-DAC at a dose that would produce peak DAC concentrations of less than 0.2µM administered 2×/wk for 8 weeks to nonhuman primates was not myelotoxic, hypomethylated DNA in the γ-globin gene promoter, and produced large cumulative increases in fetal hemoglobin. Combining oral THU with oral DAC changes DAC pharmacology in a manner that may facilitate accessible noncytotoxic DNMT1-targeted therapy.

Regulation of neutrophil extracellular trap formation by anti-inflammatory drugs.

The formation of neutrophil extracellular traps (NETs) is a newly described phenomenon that increases the bacteria-killing ability and the inflammatory response of neutrophils. Because NET generation occurs in an inflammatory microenvironment, we examined its regulation by anti-inflammatory drugs. Treatment of neutrophils with dexamethasone had no effect, but acetylsalicylic acid (ASA) treatment prevented NET formation. NETosis was also abrogated by the presence of BAY 11-7082 [(E)-3-[4-methylphenylsulfonyl]-2-propenenitrile] and Ro 106-9920 [6-(phenylsulfinyl)tetrazolo[1,5-b]pyridazine], two structurally unrelated nuclear factor-κB (NF-κB) inhibitors. The decrease in NET formation mediated by ASA, BAY-11-7082, and Ro 106-9920 was correlated with a significant reduction in the phosphorylation of NF-κB p65 subunit, indicating that the activation of this transcription factor is a relevant signaling pathway involved in the generation of DNA traps. The inhibitory effect of these drugs was also observed when NET generation was induced under acidic or hyperthermic conditions, two stress signals of the inflammatory microenvironment. In a mouse peritonitis model, while pretreatment of animals with ASA or BAY 11-7082 resulted in a marked suppression of NET formation along with increased bacteremia, dexamethasone had no effect. Our results show that NETs have an important role in the local control of infection and that ASA and NF-κB blockade could be useful therapies to avoid undesired effect of persistent neutrophil activation.

Binding of galectin-1 to αIIbß3 integrin triggers "outside-in" signals, stimulates platelet activation, and controls primary hemostasis.

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin-1 (Gal-1), a ß-galactoside-binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal-1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti-α(IIb) subunit ofα(IIb)ß(3) integrin or platelets from patients with Glanzmann's thrombasthenia syndrome (α(IIb)ß(3) deficiency), identified this integrin as a functional Gal-1 receptor in platelets. Binding of Gal-1 to platelets triggered the phosphorylation of ß(3)-integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA(2)) release, and Ca(2+) mobilization. Not only soluble but also immobilized Gal-1 promoted platelet activation. Gal-1-deficient (Lgals1(-/-)) mice showed increased bleeding time (P<0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1(-/-) platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of α(IIb)ß(3) was also observed in Lgals1(-/-) platelets, indicating a failure in the "outside-in" signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin-integrin interactions, for regulating platelet activation.

The synthetic phospholipid C8-C1P determines pro-angiogenic and pro-reparative features in human macrophages restraining the proinflammatory M1-like phenotype.

Monocytes (Mo) are highly plastic myeloid cells that differentiate into macrophages after extravasation, playing a pivotal role in the resolution of inflammation and regeneration of injured tissues. Wound-infiltrated monocytes/macrophages are more pro-inflammatory at early time points, while showing anti-inflammatory/pro-reparative phenotypes at later phases, with highly dynamic switching depending on the wound environment. Chronic wounds are often arrested in the inflammatory phase with hampered inflammatory/repair phenotype transition. Promoting the tissue repair program switching represents a promising strategy to revert chronic inflammatory wounds, one of the major public health loads. We found that the synthetic lipid C8-C1P primes human CD14+ monocytes, restraining the inflammatory activation markers (HLA-DR, CD44, and CD80) and IL-6 when challenged with LPS, and preventing apoptosis by inducing BCL-2. We also observed increased pseudo-tubule formation of human endothelial-colony-forming cells (ECFCs) when stimulated with the C1P-macrophages secretome. Moreover, C8-C1P-primed monocytes skew differentiation toward pro-resolutive-like macrophages, even in the presence of inflammatory PAMPs and DAMPs by increasing anti-inflammatory and pro-angiogenic gene expression patterns. All these results indicate that C8-C1P could restrain M1 skewing and promote the program of tissue repair and pro-angiogenic macrophage.

Epigenetic regulation by decitabine of melanoma differentiation in vitro and in vivo.

Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells. The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine), which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation were examined. Treatment of human and murine melanoma cells in vitro with concentrations of decitabine that did not cause apoptosis inhibited proliferation accompanied by cellular differentiation. A decrease in promoter methylation, and increase in expression of the melanocyte late-differentiation driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors (CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with differentiation. Effects were independent of the TP53, p16/CDKN2A and also the BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic, characterized by diminished ability of decitabine to deplete DNMT1. Treatment of murine melanoma models in vivo with intermittent, low-dose decitabine, administered sub-cutaneously to limit high peak drug levels that cause cytotoxicity and increase exposure time for DNMT1 depletion, and with tetrahydrouridine to decrease decitabine metabolism and further increase exposure time, inhibited tumor growth and increased molecular and tumor stromal factors implicated in melanocyte differentiation. Modification of decitabine dose, schedule and formulation for differentiation rather than cytotoxic objectives inhibits the growth of melanoma cells in vitro and in vivo.

Identification of galectins as novel regulators of platelet signaling and function.

Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis. However, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Platelets can be activated by soluble molecules including thrombin, TXA2 , adenosine diphosphate (ADP), and serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor and collagen. In this article, we review recent advances on the role of galectins in platelet physiology. By acting in either soluble or immobilized form, these glycan-binding proteins trigger platelet activation through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer.

The low viability of human CD34+ cells under acidic conditions is improved by exposure to thrombopoietin, stem cell factor, interleukin-3, or increased cyclic adenosine monophosphate levels.

BACKGROUND: Transplanted hematopoietic progenitor cells (CD34+) have shown great promise in regenerative medicine. However, the therapeutic potential of transplanted cells is limited by their poor viability. It is well known that the microenvironment in which progenitors reside substantially affects their behavior. Because extracellular acidosis is a common feature of injured tissues or the tumor microenvironment and is a critical regulator of cell survival and activation, we evaluated the impact of acidosis on CD34+ cell biology. STUDY DESIGN AND METHODS: Apoptosis was evaluated by fluorescence microscopy and binding of annexin V, hypodiploid cells, Bcl-xL expression, active caspase-3, and mitochondrial membrane potential was determined by flow cytometry. Colony-forming units were studied by clonogenic assays, and cell cycle was evaluated by flow cytometry. RESULTS: Exposure of CD34+ cells to low pH (7.0-6.5) caused intracellular acidification, decreased cell proliferation, and triggered apoptosis via the mitochondrial pathway. Whereas exposure to thrombopoietin (TPO), stem cell factor (SCF), interleukin (IL)-3 or increases in cyclic adenosine monophosphate (cAMP) levels prevented CD34+ cell death induced by acidic conditions, granulocyte-macrophage-colony-stimulating factor, FMS-like tyrosine kinase 3-ligand, erythropoietin, and vascular endothelial growth factor had no effect. Despite their cytoprotective effect, CD34+ cell expansion triggered by TPO, SCF, or IL-3 was significantly impaired at low pH. However, a cocktail of these three cytokines synergistically supported proliferation, cell cycle progression, and colony formation. DISCUSSION: Our findings indicate that an acidic milieu is deleterious for CD34+ cells and that a combination of certain cytokines and cAMP donors may improve cell viability and function. These data may be useful to develop new therapeutic strategies or to optimize protocols for regenerative medicine.

The immunoregulatory glycan-binding protein galectin-1 triggers human platelet activation.

Platelet activation is a critical process during inflammation, thrombosis, and cancer. Here, we show that galectin-1, an endogenous lectin with immunoregulatory properties, plays a key role in human platelet activation and function. Galectin-1 binds to human platelets in a carbohydrate-dependent manner and synergizes with ADP or thrombin to induce platelet aggregation and ATP release. Furthermore, galectin-1 induces F-actin polymerization, up-regulation of P-selectin, and GPIIIa expression; promotes shedding of microvesicles; and triggers conformational changes in GPIIb/IIIa. In addition, exposure to this lectin favors the generation of leukocyte-platelet aggregates. A further mechanistic analysis revealed the involvement of Ca(2+) and cyclic nucleotide-dependent pathways in galectin-1-mediated control of platelet activation. Finally, expression of endogenous galectin-1 in human platelets contributes to ADP-induced aggregation. Our study reveals a novel unrecognized role for galectin-1 in the control of platelet physiology with potential implications in thrombosis, inflammation, and metastasis.

Platelets Promote Macrophage Polarization toward Pro-inflammatory Phenotype and Increase Survival of Septic Mice.

We investigated the contribution of human platelets to macrophage effector properties in the presence of lipopolysaccharide (LPS), as well as the beneficial effects and time frame for platelet transfusion in septic animals. Our results show that platelets sequester both pro-(TNF-α/IL-6) and anti-(IL-10) inflammatory cytokines released by monocytes. Low LPS concentrations (0.01 ng/mL) induced M2 macrophage polarization by decreasing CD64 and augmenting CD206 and CD163 expression; yet, the presence of platelets skewed monocytes toward type 1 macrophage (M1) phenotype in a cell-contact-dependent manner by the glycoprotein Ib (GPIb)-CD11b axis. Accordingly, platelet-licensed macrophages showed increased TNF-α levels, bacterial phagocytic activity, and a reduced healing capability. Platelet transfusion increased inducible nitric oxide synthase (iNOS)+ macrophages, improving bacterial clearance and survival rates in septic mice up to 6 h post-infection, an effect that was abolished by CD11b and GPIb blockade. Our results demonstrate that platelets orchestrate macrophage effector responses, improving the clinical outcome of sepsis in a narrow but relevant time frame.

Activation of toll-like receptors 2 and 4 on CD34+ cells increases human megakaryo/thrombopoiesis induced by thrombopoietin.

BACKGROUND: Platelet Toll-like receptor (TLR)2/4 are key players in amplifying the host immune response; however, their role in human megakaryo/thrombopoiesis has not yet been defined. OBJECTIVES: We evaluated whether Pam3CSK4 or lipopolysaccharide (LPS), TLR2/4 ligands respectively, modulate human megakaryocyte development and platelet production. METHODS: CD34+ cells from human umbilical cord were stimulated with LPS or Pam3CSK4 with or without thrombopoietin (TPO). RESULTS: CD34+ cells and megakaryocytes express TLR2 and TLR4 at both RNA and protein level; however, direct stimulation of CD34+ cells with LPS or Pam3CSK4 had no effect on cell growth. Interestingly, both TLR ligands markedly increased TPO-induced CD34+ cell proliferation, megakaryocyte number and maturity, proplatelet and platelet production when added at day 0. In contrast, this synergism was not observed when TLR agonists were added 7 days after TPO addition. Interleukin-6 (IL-6) release was observed upon CD34+ or megakaryocyte stimulation with LPS or Pam3CSK4 but not with TPO and this effect was potentiated in combination with TPO. The increased proliferation and IL-6 production induced by TPO + LPS or Pam3CSK4 were suppressed by TLR2/4 or IL-6 neutralizing antibodies, as well as by PI3K/AKT and nuclear factor-κB inhibitors. Additionally, increased proplatelet and platelet production were associated with enhanced nuclear translocation of nuclear factor-E2. Finally, the supernatants of CD34+ cells stimulated with TPO+LPS-induced CFU-M colonies. CONCLUSIONS: Our data suggest that the activation of TLR2 and TLR4 in CD34+ cells and megakaryocytes in the presence of TPO may contribute to warrant platelet provision during infection episodes by an autocrine IL-6 loop triggered by PI3K/NF-κB axes.

An optimised protocol for platelet-rich plasma preparation to improve its angiogenic and regenerative properties.

Although platelet-rich plasma (PRP) is used as a source of growth factors in regenerative medicine, its effectiveness remains controversial, partially due to the absence of PRP preparation protocols based on the regenerative role of platelets. Here, we aimed to optimise the protocol by analysing PRP angiogenic and regenerative properties. Three optimising strategies were evaluated: dilution, 4 °C pre-incubation, and plasma cryoprecipitate supplementation. Following coagulation, PRP releasates (PRPr) were used to induce angiogenesis in vitro (HMEC-1 proliferation, migration, and tubule formation) and in vivo (chorioallantoic membrane), as well as regeneration of excisional wounds on mouse skin. Washed platelet releasates induced greater angiogenesis than PRPr due to the anti-angiogenic effect of plasma, which was decreased by diluting PRPr with saline. Angiogenesis was also improved by both PRP pre-incubation at 4 °C and cryoprecipitate supplementation. A combination of optimising variables exerted an additive effect, thereby increasing the angiogenic activity of PRPr from healthy donors and diabetic patients. Optimised PRPr induced faster and more efficient mouse skin wound repair compared to that induced by non-optimised PRPr. Acetylsalicylic acid inhibited angiogenesis and tissue regeneration mediated by PRPr; this inhibition was reversed following optimisation. Our findings indicate that PRP pre-incubation at 4 °C, PRPr dilution, and cryoprecipitate supplementation improve the angiogenic and regenerative properties of PRP compared to the obtained by current methods.

Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo.

BACKGROUND: We have previously demonstrated that acidic preconditioning of human endothelial colony-forming cells (ECFC) increased proliferation, migration, and tubulogenesis in vitro, and increased their regenerative potential in a murine model of hind limb ischemia without baseline disease. We now analyze whether this strategy is also effective under adverse conditions for vasculogenesis, such as the presence of ischemia-related toxic molecules or diabetes, one of the main target diseases for cell therapy due to their well-known healing impairments. METHODS: Cord blood-derived CD34+ cells were seeded in endothelial growth culture medium (EGM2) and ECFC colonies were obtained after 14-21 days. ECFC were exposed at pH 6.6 (preconditioned) or pH 7.4 (nonpreconditioned) for 6 h, and then pH was restored at 7.4. A model of type 2 diabetes induced by a high-fat and high-sucrose diet was developed in nude mice and hind limb ischemia was induced in these animals by femoral artery ligation. A P value < 0.05 was considered statistically significant (by one-way analysis of variance). RESULTS: We found that acidic preconditioning increased ECFC adhesion and the release of pro-angiogenic molecules, and protected ECFC from the cytotoxic effects of monosodium urate crystals, histones, and tumor necrosis factor (TNF)α, which induced necrosis, pyroptosis, and apoptosis, respectively. Noncytotoxic concentrations of high glucose, TNFα, or their combination reduced ECFC proliferation, stromal cell-derived factor (SDF)1-driven migration, and tubule formation on a basement membrane matrix, whereas almost no inhibition was observed in preconditioned ECFC. In type 2 diabetic mice, intravenous administration of preconditioned ECFC significantly induced blood flow recovery at the ischemic limb as measured by Doppler, compared with the phosphate-buffered saline (PBS) and nonpreconditioned ECFC groups. Moreover, the histologic analysis of gastrocnemius muscles showed an increased vascular density and reduced signs of inflammation in the animals receiving preconditioned ECFC. CONCLUSIONS: Acidic preconditioning improved ECFC survival and angiogenic activity in the presence of proinflammatory and damage signals present in the ischemic milieu, even under high glucose conditions, and increased their therapeutic potential for postischemia tissue regeneration in a murine model of type 2 diabetes. Collectively, our data suggest that acidic preconditioning of ECFC is a simple and inexpensive strategy to improve the effectiveness of cell transplantation in diabetes, where tissue repair is highly compromised.

Activation of cyclic AMP pathway prevents CD34(+) cell apoptosis.

OBJECTIVE: Although cAMP is involved in a number of physiologic functions, its role in hematopoietic cell fate decision remains poorly understood. We have recently demonstrated that in CD34(+)-derived megakaryocytes, cAMP-related agents prevent apoptosis. In this study we addressed the question of whether cAMP also regulates survival of their precursors, CD34(+) cells. METHODS: Apoptosis was evaluated by fluorescence microscopy, and detection of hypodiploid or annexin V(+) cells by flow cytometry. Mitochondrial membrane potential and bcl-xL or caspase-3 expression were assessed by flow cytometry. Colony-forming units were studied by clonogenic assays in methylcellulose. RESULTS: We found that two different cAMP analogs such as Dibutiril-cAMP and sp-5,6-DCl-BIMPS (BIMPS) promoted survival of human umbilical cord-derived CD34(+) cells by suppressing apoptosis induced by either nitric oxide (NO) or serum deprivation. Involvement of PKA and PI3K pathway was demonstrated by the ability of their specific inhibitors Rp-cAMP and Wortmannin or LY294002 respectively to reverse the antiapoptotic effect of BIMPS. Treatment of CD34(+) cell with BIMPS not only restrained the bcl-xL downregulation but also suppressed the loss of mitochondrial membrane potential and caspase-3 activation induced by serum starvation. While thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF) were not able to increase cAMP levels, the antiapoptotic activity exerted by these growth factors was blocked by inhibition of the adenylate cyclase and synergized by BIMPS. Cyclic AMP analogs suppressed the decreased colony formation in cells exposed to NO or serum deprivation. CONCLUSION: Altogether, our results strongly suggest that cAMP appears to be not only a key pathway controlling CD34(+) survival, but also a mediator of the TPO-, G-CSF- and SCF-mediated cytoprotection.

Downregulation of adaptor protein MyD88 compromises the angiogenic potential of B16 murine melanoma.

The mechanisms that link inflammatory responses to cancer development remain a subject of intense investigation, emphasizing the need to better understand the cellular and molecular pathways that create a tumor promoting microenvironment. The myeloid differentiation primary response protein MyD88 acts as a main adaptor molecule for the signaling cascades initiated from Toll-like receptors (TLRs) and the interleukin 1 receptor (IL-1R). MyD88 has been shown to contribute to tumorigenesis in many inflammation-associated cancer models. In this study, we sought to better define the role of MyD88 in neoplastic cells using a murine melanoma model. Herein, we have demonstrated that MyD88 expression is required to maintain the angiogenic switch that supports B16 melanoma growth. By knocking down MyD88 we reduced TLR-mediated NF-κB activation with no evident effects over cell proliferation and survival. In addition, MyD88 downregulation was associated with a decrease of HIF1α levels and its target gene VEGF, in correlation with an impaired capability to induce capillary sprouting and tube formation of endothelial cells. Melanomas developed from cells lacking MyD88 showed an enhanced secretion of chemoattractant ligands such as CCL2, CXCL10 and CXCL1 and have an improved infiltration of macrophages to the tumor site. Our results imply that cell-autonomous signaling through MyD88 is required to sustain tumor growth and underscore its function as an important positive modulator of tumor angiogenesis.

Schedule-Dependent Antiangiogenic and Cytotoxic Effects of Chemotherapy on Vascular Endothelial and Retinoblastoma Cells.

Current treatment of retinoblastoma involves using the maximum dose of chemotherapy that induces tumor control and is tolerated by patients. The impact of dose and schedule on the cytotoxicity of chemotherapy has not been studied. Our aim was to gain insight into the cytotoxic and antiangiogenic effect of the treatment scheme of chemotherapy used in retinoblastoma by means of different in vitro models and to evaluate potential effects on multi-drug resistance proteins. Two commercial and two patient-derived retinoblastoma cell types and two human vascular endothelial cell types were exposed to increasing concentrations of melphalan or topotecan in a conventional (single exposure) or metronomic (7-day continuous exposure) treatment scheme. The concentration of chemotherapy causing a 50% decrease in cell proliferation (IC50) was determined by MTT and induction of apoptosis was evaluated by flow cytometry. Expression of ABCB1, ABCG2 and ABCC1 after conventional or metronomic treatments was assessed by RT-qPCR. We also evaluated the in vivo response to conventional (0.6 mg/kg once a week for 2 weeks) and metronomic (5 days a week for 2 weeks) topotecan in a retinoblastoma xenograft model. Melphalan and topotecan were cytotoxic to both retinoblastoma and endothelial cells after conventional and metronomic treatments. A significant decrease in the IC50 (median, 13-fold; range: 3-23) was observed following metronomic chemotherapy treatment in retinoblastoma and endothelial cell types compared to conventional treatment (p<0.05). Metronomic topotecan or melphalan significantly inhibited in vitro tube formation in HUVEC and EPC compared to vehicle-treated cells (p<0.05). Both treatment schemes induced apoptosis and/or necrosis in all cell models. No significant difference was observed in the expression of ABCB1, ABCC1 or ABCG2 when comparing cells treated with melphalan or topotecan between treatment schedules at the IC50 or with control cells (p>0.05). In mice, continuous topotecan lead to significantly lower tumor volumes compared to conventional treatment after 14 days of treatment (p<0.05). Continuous exposure to melphalan or topotecan increased the chemosensitivity of retinoblastoma and endothelial cells to both chemotherapy agents with lower IC50 values compared to short-term treatment. These findings were validated in an in vivo model. None of the dosing modalities induced multidrug resistance mechanisms while apoptosis was the mechanism of cell death after both treatment schedules. Metronomic chemotherapy may be a valid option for retinoblastoma treatment allowing reductions of the daily dose.

Mediators and molecular pathways involved in the regulation of neutrophil extracellular trap formation mediated by activated platelets.

In addition to being key elements in hemostasis and thrombosis, platelets amplify neutrophil function. We aimed to gain further insight into the stimuli, mediators, molecular pathways, and regulation of neutrophil extracellular trap formation mediated by human platelets. Platelets stimulated by lipopolysaccharide, a wall component of gram-negative bacteria, Pam3-cysteine-serine-lysine 4, a mimetic of lipopeptide from gram-positive bacteria, Escherichia coli, Staphylococcus aureus, or physiologic platelet agonists promoting neutrophil extracellular trap formation and myeloperoxidase-associated DNA activity under static and flow conditions. Although P-selectin or glycoprotein IIb/IIIa were not involved, platelet glycoprotein Ib, neutrophil cluster of differentiation 18, and the release of von Willebrand factor and platelet factor 4 seemed to be critical for the formation of neutrophil extracellular traps. The secretion of these molecules depended on thromboxane A(2) production triggered by lipopolysaccharide or Pam3-cysteine-serine-lysine 4 but not on high concentrations of thrombin. Accordingly, aspirin selectively inhibited platelet-mediated neutrophil extracellular trap generation. Signaling through extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Src kinases, but not p38 or reduced nicotinamide adenine dinucleotide phosphate oxidase, was involved in platelet-triggered neutrophil extracellular trap release. Platelet-mediated neutrophil extracellular trap formation was inhibited by prostacyclin. Our results support a role for stimulated platelets in promoting neutrophil extracellular trap formation, reveal that an endothelium-derived molecule contributes to limiting neutrophil extracellular trap formation, and highlight platelet inhibition as a potential target for controlling neutrophil extracellular trap cell death.