Molecular mechanisms of environmental pollutant-induced cartilage damage: from developmental disorders to osteoarthritis.

The objective of the present study was to review the molecular mechanisms of the adverse effects of environmental pollutants on chondrocytes and extracellular matrix (ECM). Existing data demonstrate that both heavy metals, including cadmium (Cd), lead (Pb), and arsenic (As), as well as organic pollutants, including polychlorinated dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCB), bisphenol A, phthalates, polycyclic aromatic hydrocarbons (PAH), pesticides, and certain other organic pollutants that target cartilage ontogeny and functioning. Overall, environmental pollutants reduce chondrocyte viability through the induction apoptosis, senescence, and inflammatory response, resulting in cell death and impaired ECM production. The effects of organic pollutants on chondrocyte development and viability were shown to be mediated by binding to the aryl hydrocarbon receptor (AhR) signaling and modulation of non-coding RNA expression. Adverse effects of pollutant exposures were observed in articular and growth plate chondrocytes. These mechanisms also damage chondrocyte precursors and subsequently hinder cartilage development. In addition, pollutant exposure was shown to impair chondrogenesis by inhibiting the expression of Sox9 and other regulators. Along with altered Runx2 signaling, these effects also contribute to impaired chondrocyte hypertrophy and chondrocyte-to-osteoblast trans-differentiation, resulting in altered endochondral ossification. Several organic pollutants including PCDD/Fs, PCBs and PAHs, were shown to induce transgenerational adverse effects on cartilage development and the resulting skeletal deformities. Despite of epidemiological evidence linking human environmental pollutant exposure to osteoarthritis or other cartilage pathologies, the data on the molecular mechanisms of adverse effects of environmental pollutant exposure on cartilage tissue were obtained from studies in laboratory rodents, fish, or cell cultures and should be carefully extrapolated to humans, although they clearly demonstrate that cartilage should be considered a putative target for environmental pollutant toxicity.

Novel Gadolinium-Free Ultrasmall Nanostructured Positive Contrast for Magnetic Resonance Angiography and Imaging.

Nanostructured contrast agents are promising alternatives to Gd3+-based chelates in magnetic resonance (MR) imaging techniques. A novel ultrasmall paramagnetic nanoparticle (UPN) was strategically designed to maximize the number of exposed paramagnetic sites and r1 while minimizing r2, by decorating 3 nm titanium dioxide nanoparticles with suitable amounts of iron oxide. Its relaxometric parameters are comparable to those of gadoteric acid (GA) in agar phantoms, and the r2/r1 ratio of 1.38 at 3 T is close to the ideal unitary value. The strong and prolonged contrast enhancement of UPN before renal excretion was confirmed by T1-weighted MR images of Wistar rats after intravenous bolus injection. Those results associated with good biocompatibility indicate its high potential as an alternative blood-pool contrast agent to the GA gold standard for MR angiography, especially for patients with severe renal impairment.

Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters.

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

Neutrophils induce proangiogenic T cells with a regulatory phenotype in pregnancy.

Although neutrophils are known to be fundamental in controlling innate immune responses, their role in regulating adaptive immunity is just starting to be appreciated. We report that human neutrophils exposed to pregnancy hormones progesterone and estriol promote the establishment of maternal tolerance through the induction of a population of CD4+ T cells displaying a GARP+CD127loFOXP3+ phenotype following antigen activation. Neutrophil-induced T (niT) cells produce IL-10, IL-17, and VEGF and promote vessel growth in vitro. Neutrophil depletion during murine pregnancy leads to abnormal development of the fetal-maternal unit and reduced empbryo development, with placental architecture displaying poor trophoblast invasion and spiral artery development in the maternal decidua, accompanied by significantly attenuated niT cell numbers in draining lymph nodes. Using CD45 congenic cells, we show that induction of niT cells and their regulatory function occurs via transfer of apoptotic neutrophil-derived proteins, including forkhead box protein 1 (FOXO1), to T cells. Unlike in women with healthy pregnancies, neutrophils from blood and placental samples of preeclamptic women fail to induce niT cells as a direct consequence of their inability to transfer FOXO1 to T cells. Finally, neutrophil-selective FOXO1 knockdown leads to defective placentation and compromised embryo development, similar to that resulting from neutrophil depletion. These data define a nonredundant function of neutrophil-T cell interactions in the regulation of vascularization at the maternal-fetal interface.

Endogenous annexin A1 (AnxA1) modulates early-phase gestation and offspring sex-ratio skewing.

Annexin A1 (AnxA1) is a glucocorticoid-regulated anti-inflammatory protein secreted by phagocytes and other specialised cells. In the endocrine system, AnxA1 controls secretion of steroid hormones and it is abundantly expressed in the testis, ovaries, placenta and seminal fluid, yet its potential modulation of fertility has not been described. Here, we observed that AnxA1 knockout (KO) mice delivered a higher number of pups, with a higher percentage of female offsprings. This profile was not dependent on the male features, as sperm from KO male mice did not present functional alterations, and had an equal proportion of Y and X chromosomes, comparable to wild type (WT) male mice. Furthermore, mismatched matings of male WT mice with female KO yielded a higher percentage of female pups per litter, a phenomenon which was not observed when male KO mice mated with female WT animals. Indeed, AnxA1 KO female mice displayed several differences in parameters related to gestation including (i) an arrested estrous cycle at proestrus phase; (ii) increased sites of implantation; (iii) reduced pre- and post-implantation losses; (iv) exacerbated features of the inflammatory reaction in the uterine fluid during implantation phase; and (v) enhanced plasma progesterone in the beginning of pregnancy. In summary, herein we highlight that AnxA1 pathway as a novel determinant of fundamental non-redundant regulatory functions during early pregnancy.

Cytotoxic effects of a novel maleimide derivative on epithelial and tumor cells.

Novel N-triazolyl maleimide derivatives were synthesized by azide-alkyne Huisgen cycloaddition (1,3-dipolar cycloaddition) and tested for cytotoxicity against a cell line derived from human melanomas SK-Mel-28 and SK-Mel-103, and human umbilical vein endothelial cell lines (HUVEC). The 4l was chose to be biologically tested due to incorporation of benzyl triazolic to the nitrogen of maleimide has not been tested before, and due the satisfactory yield. The analysis of cell metabolism, using the MTT method, showed that the compound 4l impaired cell metabolism in HUVEC only in high concentration (100µM). A lower concentration of compound 4l, whether in association or not with paclitaxel, was required to cause toxicity in both SK-Mel-28 and SK-Mel-103 cells in comparison with HUVEC cells. Moreover, the ability of 4l to cause cell death was evaluated by flow cytometry, and the data obtained highlighted the apoptotic action of 4l and paclitaxel co-treatment on Sk-Mel-28 cells only, which corroborated the greater efficacy of maleimide compounds against cancer cells. Together, our data provide promising data on the selectivity of maleimide compounds to cancer cells, and suggest that novel maleimide-substituted compounds may be synthesized and tested on different cancer cell lines, as primary or co-adjuvant agents of cancer cell toxicity.

Chronic exposure of diesel exhaust particles induces alveolar enlargement in mice.

BACKGROUND: Diesel exhaust particles (DEPs) are deposited into the respiratory tract and are thought to be a risk factor for the development of diseases of the respiratory system. In healthy individuals, the timing and mechanisms of respiratory tract injuries caused by chronic exposure to air pollution remain to be clarified. METHODS: We evaluated the effects of chronic exposure to DEP at doses below those found in a typical bus corridor in Sao Paulo (150 µg/m3). Male BALB/c mice were divided into mice receiving a nasal instillation: saline (saline; n = 30) and 30 µg/10 µL of DEP (DEP; n = 30). Nasal instillations were performed five days a week, over a period of 90 days. Bronchoalveolar lavage (BAL) was performed, and the concentrations of interleukin (IL)-4, IL-10, IL-13 and interferon-gamma (INF-γ) were determined by ELISA-immunoassay. Assessment of respiratory mechanics was performed. The gene expression of Muc5ac in lung was evaluated by RT-PCR. The presence of IL-13, MAC2+ macrophages, CD3+, CD4+, CD8+ T cells and CD20+ B cells in tissues was analysed by immunohistochemistry. Bronchial thickness and the collagen/elastic fibers density were evaluated by morphometry. We measured the mean linear intercept (Lm), a measure of alveolar distension, and the mean airspace diameter (D0) and statistical distribution (D2). RESULTS: DEP decreased IFN-γ levels in BAL (p = 0.03), but did not significantly alter IL-4, IL-10 and IL-13 levels. MAC2+ macrophage, CD4+ T cell and CD20+ B cell numbers were not altered; however, numbers of CD3+ T cells (p ≤ 0.001) and CD8+ T cells (p ≤ 0.001) increased in the parenchyma. Although IL-13 (p = 0.008) expression decreased in the bronchiolar epithelium, Muc5ac gene expression was not altered in the lung of DEP-exposed animals. Although respiratory mechanics, elastic and collagen density were not modified, the mean linear intercept (Lm) was increased in the DEP-exposed animals (p ≤ 0.001), and the index D2 was statistically different (p = 0.038) from the control animals. CONCLUSION: Our data suggest that nasal instillation of low doses of DEP over a period of 90 days results in alveolar enlargement in the pulmonary parenchyma of healthy mice.

Anti-inflammatory mechanisms of the annexin A1 protein and its mimetic peptide Ac2-26 in models of ocular inflammation in vivo and in vitro.

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1(-/-) mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach.

Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1.

OBJECTIVE AND DESIGN: Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. METHODS: We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. RESULTS: LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. CONCLUSION: ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Retinal toxicity of heavy metals and its involvement in retinal pathology.

The objective of the present review is to discuss epidemiological evidence demonstrating the association between toxic metal (Cd, Pb, Hg, As, Sn, Ti, Tl) exposure and retinal pathology, along with the potential underlying molecular mechanisms. Epidemiological studies demonstrate that Cd, and to a lesser extent Pb exposure, are associated with age-related macular degeneration (AMD), while the existing evidence on the levels of these metals in patients with diabetic retinopathy is scarce. Epidemiological data on the association between other toxic metals and metalloids including mercury (Hg) and arsenic (As), are limited. Clinical reports and laboratory in vivo studies have shown structural alterations in different layers of retina following metal exposure. Examination of retina samples demonstrate that toxic metals can accumulate in the retina, and the rate of accumulation appears to increase with age. Experimental studies in vivo and in vitro studies in APRE-19 and D407 cells demonstrate that toxic metal exposure may cause retinal damage through oxidative stress, apoptosis, DNA damage, mitochondrial dysfunction, endoplasmic reticulum stress, impaired retinogenesis, and retinal inflammation. However, further epidemiological as well as laboratory studies are required for understanding the underlying molecular mechanisms and identifying of the potential therapeutic targets and estimation of the dose-response effects.

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases.

Introduction: Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology. Methods: To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut. Results: The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 µg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis. Discussion: The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results. Conclusion: Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy.

Synthesis and preliminary biological evaluation of a compound library of triazolylcyclitols.

A small library of compounds was prepared by a combination of toluene dioxygenase (TDO)-catalyzed enzymatic dihydroxylation and copper(I)-catalyzed Hüisgen cycloaddition. Some compounds were obtained by coupling an alkyne and a conduritol derivative, while more complex structures were obtained by a double Hüisgen reaction of a dialkyne and two molecules of the cyclitol. The compounds were fully characterized and subjected to preliminary biological screening.

Immunotoxic mechanisms of cigarette smoke and heat-not-burn tobacco vapor on Jurkat T cell functions.

Cigarette smoke (CS) affects immune functions, leading to severe outcomes in smokers. Robust evidence addresses the immunotoxic effects of combustible tobacco products. As heat-not-burn tobacco products (HNBT) vaporize lower levels of combustible products, we here compared the effects of cigarette smoke (CS) and HNBT vapor on Jurkat T cells. Cells were exposed to air, conventional cigarettes or heatsticks of HNBT for 30 min and were stimulated or not with phorbol myristate acetate (PMA). Cell viability, proliferation, reactive oxygen species (ROS) production, 8-OHdG, MAP-kinases and nuclear factor κB (NFκB) activation and metallothionein expression (MTs) were assessed by flow cytometry; nitric oxide (NO) and cytokine levels were measured by Griess reaction and ELISA, respectively. Levels of metals in the exposure chambers were quantified by inductively coupled plasma mass spectrometry. MT expressions were quantified by immunohistochemistry in the lungs and liver of C57Bl/6 mice exposed to CS, HNBT or air (1 h, twice a day for five days: via inhalation). While both CS and HBNT exposures increased cell death, CS led to a higher number of necrotic cells, increased the production of ROS, NO, inflammatory cytokines and MTs when compared to HNBT-exposed cells, and led to a higher expression of MTs in mice. CS released higher amounts of metals. CS and HNBT exposures decreased PMA-induced interleukin-2 (IL-2) secretion and impaired Jurkat proliferation, effects also seen in cells exposed to nicotine. Although HNBT vapor does not activate T cells as CS does, exposure to both HNBT and CS suppressed proliferation and IL-2 release, a pivotal cytokine involved with T cell proliferation and tolerance, and this effect may be related to nicotine content in both products.

Annexin A1/Formyl Peptide Receptor Pathway Controls Uterine Receptivity to the Blastocyst.

Embryo implantation into the uterine wall is a highly modulated, complex process. We previously demonstrated that Annexin A1 (AnxA1), which is a protein secreted by epithelial and inflammatory cells in the uterine microenvironment, controls embryo implantation in vivo. Here, we decipher the effects of recombinant AnxA1 in this phenomenon by using human trophoblast cell (BeWo) spheroids and uterine epithelial cells (Ishikawa; IK). AnxA1-treated IK cells demonstrated greater levels of spheroid adherence and upregulation of the tight junction molecules claudin-1 and zona occludens-1, as well as the glycoprotein mucin-1 (Muc-1). The latter effect of AnxA1 was not mediated through IL-6 secreted from IK cells, a known inducer of Muc-1 expression. Rather, these effects of AnxA1 involved activation of the formyl peptide receptors FPR1 and FPR2, as pharmacological blockade of FPR1 or FPR1/FPR2 abrogated such responses. The downstream actions of AnxA1 were mediated through the ERK1/2 phosphorylation pathway and F-actin polymerization in IK cells, as blockade of ERK1/2 phosphorylation reversed AnxA1-induced Muc-1 and claudin-1 expression. Moreover, FPR2 activation by AnxA1 induced vascular endothelial growth factor (VEGF) secretion by IK cells, and the supernatant of AnxA1-treated IK cells evoked angiogenesis in vitro. In conclusion, these data highlight the role of the AnxA1/FPR1/FPR2 pathway in uterine epithelial control of blastocyst implantation.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

Short-chain fatty acids stimulate the migration of neutrophils to inflammatory sites.

SCFAs (short-chain fatty acids) are produced by anaerobic bacterial fermentation. Increased concentrations of these fatty acids are observed in inflammatory conditions, such as periodontal disease, and at sites of anaerobic infection. In the present study, the effect of the SCFAs acetate, propionate and butyrate on neutrophil chemotaxis and migration was investigated. Experiments were carried out in rats and in vitro. The following parameters were measured: rolling, adherence, expression of adhesion molecules in neutrophils (L-selectin and beta2 integrin), transmigration, air pouch influx of neutrophils and production of cytokines [CINC-2alphabeta (cytokine-induced neutrophil chemoattractant-2alphabeta), IL-1beta (interleukin-1beta), MIP-1alpha (macrophage inflammatory protein-1alpha) and TNF-alpha (tumour necrosis factor-alpha)]. SCFAs induced in vivo neutrophil migration and increased the release of CINC-2alphabeta into the air pouch. These fatty acids increased the number of rolling and adhered cells as evaluated by intravital microscopy. SCFA treatment increased L-selectin expression on the neutrophil surface and L-selectin mRNA levels, but had no effect on the expression of beta2 integrin. Propionate and butyrate also increased in vitro transmigration of neutrophils. These results indicate that SCFAs produced by anaerobic bacteria raise neutrophil migration through increased L-selectin expression on neutrophils and CINC-2alphabeta release.

Lopap: a non-inflammatory and cytoprotective molecule in neutrophils and endothelial cells.

Lopap (Lonomia obliqua prothrombin activator protease) is a member of the lipocalin family isolated from the extract of L obliqua bristles. Lopap displays serine protease-like activities, including coagulation disturbance, cytokine secretion and antiapoptotic activity in human cultured endothelial cells. Here, we have investigated the effects of the recombinant protein (rLopap) on the inflammatory and apoptotic processes of neutrophils and endothelial cells from male Wistar rats. We found that rLopap did not induce in vivo leukocyte-endothelial interactions in the microvasculature, initial steps of leukocyte recruitment during inflammation. Incubation of rLopap with neutrophils or endothelial cells prevented apoptosis evoked by serum deprivation and induced nitric oxide (NO) production in both cell types, and increased the expression of ICAM-1 by endothelial cells. Simultaneous incubation of endothelial cells or neutrophils with rLopap and N omega-nitro-L-arginine methyl ester (L-NAME), a non-specific inhibitor of NO synthases, inhibited NO production and impaired the protection on apoptosis. Differently, incubation of endothelial cells with monoclonal antibody anti ICAM-1 did not change the protection on apoptosis evoked by rLopap. Together, these results indicate that rLopap does not display inflammatory properties in vivo but inhibits apoptosis of neutrophils and endothelial cells depending, at least in part, on NO production.

Extracellular annexin-A1 promotes myeloid/granulocytic differentiation of hematopoietic stem/progenitor cells via the Ca2+/MAPK signalling transduction pathway.

Annexin A1 (AnxA1) modulates neutrophil life span and bone marrow/blood cell trafficking thorough activation of formyl-peptide receptors (FPRs). Here, we investigated the effect of exogenous AnxA1 on haematopoiesis in the mouse. Treatment of C57BL/6 mice with recombinant AnxA1 (rAnxA1) reduced the granulocyte-macrophage progenitor (GMP) population in the bone marrow, enhanced the number of mature granulocytes Gr-1+Mac-1+ in the bone marrow as well as peripheral granulocytic neutrophils and increased expression of mitotic cyclin B1 on hematopoietic stem cells (HSCs)/progenitor cells (Lin-Sca-1+c-Kit+: LSK). These effects were abolished by simultaneous treatment with Boc-2, an FPR pan-antagonist. In in vitro studies, rAnxA1 reduced both HSC (LSKCD90lowFLK-2-) and GMP populations while enhancing mature cells (Gr1+Mac1+). Moreover, rAnxA1 induced LSK cell proliferation (Ki67+), increasing the percentage of cells in the S/G2/M cell cycle phases and reducing Notch-1 expression. Simultaneous treatment with WRW4, a selective FPR2 antagonist, reversed the in vitro effects elicited by rAnxA1. Treatment of LSK cells with rAnxA1 led to phosphorylation of PCLγ2, PKC, RAS, MEK, and ERK1/2 with increased expression of NFAT2. In long-term bone marrow cultures, rAnxA1 did not alter the percentage of LSK cells but enhanced the Gr-1+Mac-1+ population; treatment with a PLC (U73122), but not with a PKC (GF109203), inhibitor reduced rAnxA1-induced phosphorylation of ERK1/2 and Elk1. Therefore, we identify here rAnxA1 as an inducer of HSC/progenitor cell differentiation, favouring differentiation of the myeloid/granulocytic lineage, via Ca2+/MAPK signalling transduction pathways.

In vivo blockade of Ca(+2)-dependent nitric oxide synthases impairs expressions of L-selectin and PECAM-1.

Interactions of leukocytes with endothelium play a role for the immune system modulated by endogenous agents, such as glucocorticoids and nitric oxide (NO). Glucocorticoids inhibit leukocyte-endothelial interactions whereas the role of NO is still controversial. In this study, the activity of Ca(+2)-dependent nitric oxide synthases was in vivo blocked in male Wistar rats by given l-NAME, 20mgkg(-1) for 14 days dissolved in drinking water and expression of adhesion molecules involved in leukocyte-endothelial interactions was investigated. Expressions of L-selectin and PECAM-1 in peripheral leukocytes and PECAM-1 in endothelial cells were reduced by l-NAME treatment. Only L-selectin expression was controlled at transcriptional levels. These effects were not dependent on endogenous glucocorticoids, as corticosterone levels were not altered in l-NAME-treated rats. Our results show that NO, produced at physiological levels, controls expression of constitutive adhesion molecules expressions in cell membranes by different mechanisms of action.