Laura Saieva

Role of exosomes released by chronic myelogenous leukemia cells in angiogenesis

Our study is designed to assess if exosomes released from chronic myelogenous leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM‐1 and VCAM‐1 cell adhesion molecules and interleukin‐8 expression. The stimulation of cell‐cell adhesion molecules was paralleled by a dose‐dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE‐cadherin and β‐catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.

Type 3 innate lymphoid cells producing IL-17 and IL-22 are expanded in the gut, in the peripheral blood, synovial fluid and bone marrow of patients with ankylosing spondylitis

Background The aim of the study was to better characterise the immunological origin and the behaviour of interleukin (IL)-23-responsive innate lymphoid cells (ILCs) in the gut, synovial fluid (SF) and bone marrow (BM) of patients with ankylosing spondylitis (AS). Methods ILC1, ILC2 and ILC3 cells were determined and characterised by confocal microscopy and flow cytometry in ileal and BM biopsies, in peripheral blood (PB) and SF mononuclear cells obtained from patients with AS and controls. Mucosal vascular addressin cell adhesion molecule 1 (MADCAM-1), IL-7, IL-15 and aggregates of lymphoid tissue inducer cells (LTi) were evaluated by immunohistochemistry. The in vitro ability of epithelial cells in driving the differentiation of ILC3 and the effect of tumour necrosis factor inhibitors (TNFi) on the frequency of ILC3 and the expression of MADCAM1 were also assessed. Results ILC3 characterised as Lyn−RORc−Tbet+ NKp44+ cells were significantly expanded in the gut, SF and BM of patients with AS compared with controls, produced high levels of IL-17 and IL-22 and expressed α4β7. MADcAM1 was overexpressed in BM and ileal high endothelial venules. IL-7 was significantly increased in AS gut, especially in the context of Paneth cells, and accompanied by the presence of aggregates of c-kit/IL-7R+ cells (LTi). In in vitro experiments, epithelial cells from patients with AS actively induced differentiation of ILC3 from LTi. TNFi efficacy was accompanied by a significant decrease in the percentage of intestinal and circulating ILC3 and in the expression of MADCAM1. Conclusions Gut-derived IL-17+ and IL-22+ILC3 are expanded in the peripheral blood, SF and inflamed BM of patients with AS, suggesting the presence of an active homing axis between the gut and the inflamed sacroiliac joints.

Exosome-mediated crosstalk between chronic myelogenous leukemia cells and human bone marrow stromal cells triggers an interleukin 8-dependent survival of leukemia cells.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by the Bcr-Abl oncoprotein with constitutive tyrosine kinase activity. Exosomes are nanovesicles released by cancer cells that are involved in cell-to-cell communication thus potentially affecting cancer progression. It is well known that bone marrow stromal microenvironment contributes to disease progression through the establishment of a bi-directional crosstalk with cancer cells. Our hypothesis is that exosomes could have a functional role in this crosstalk. Interleukin-8 (IL 8) is a proinflammatory chemokine that activates multiple signalling pathways downstream of two receptors (CXCR1 and CXCR2). We demonstrated that exosomes released from CML cells stimulate bone marrow stromal cells to produce IL 8 that, in turn, is able to modulate both in vitro and in vivo the leukemia cell malignant phenotype.

CD90+ liver cancer cells modulate endothelial cell phenotype through the release of exosomes containing H19 lncRNA

BackgroundCD90+ liver cancer cells have been described as cancer stem-cell-like (CSC), displaying aggressive and metastatic phenotype. Using two different in vitro models, already described as CD90+ liver cancer stem cells, our aim was to study their interaction with endothelial cells mediated by the release of exosomes.MethodsExosomes were isolated and characterized from both liver CD90+ cells and hepatoma cell lines. Endothelial cells were treated with exosomes, as well as transfected with a plasmid containing the full length sequence of the long non-coding RNA (lncRNA) H19. Molecular and functional analyses were done to characterize the endothelial phenotype after treatments.ResultsExosomes released by CD90+ cancer cells, but not by parental hepatoma cells, modulated endothelial cells, promoting angiogenic phenotype and cell-to-cell adhesion. LncRNA profiling revealed that CD90+ cells were enriched in lncRNA H19, and released this through exosomes. Experiments of gain and loss of function of H19 showed that this LncRNA plays an important role in the exosome-mediated phenotype of endothelial cells.ConclusionsOur data indicate a new exosome-mediated mechanism by which CSC-like CD90+ cells could influence their tumor microenvironment by promoting angiogenesis. Moreover, we suggest the lncRNA H19 as a putative therapeutic target in hepatocellular carcinoma.

Dysbiosis and zonulin upregulation alter gut epithelial and vascular barriers in patients with ankylosing spondylitis

Background Dysbiosis has been recently demonstrated in patients with ankylosing spondylitis (AS) but its implications in the modulation of intestinal immune responses have never been studied. The aim of this study was to investigate the role of ileal bacteria in modulating local and systemic immune responses in AS. Methods Ileal biopsies were obtained from 50 HLA-B27+ patients with AS and 20 normal subjects. Silver stain was used to visualise bacteria. Ileal expression of tight and adherens junction proteins was investigated by TaqMan real-time (RT)-PCR and immunohistochemistry. Serum levels of lipopolysaccharide (LPS), LPS-binding protein (LPS-BP), intestinal fatty acid-BP (iFABP) and zonulin were assayed by ELISA. Monocyte immunological functions were studied in in vitro experiments. In addition the effects of antibiotics on tight junctions in human leukocyte antigen (HLA)-B27 transgenic (TG) rats were assessed. Results Adherent and invasive bacteria were observed in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation. Impairment of the gut vascular barrier (GVB) was also present in AS, accompanied by significant upregulation of zonulin, and associated with high serum levels of LPS, LPS-BP, iFABP and zonulin. In in vitro studies zonulin altered endothelial tight junctions while its epithelial release was modulated by isolated AS ileal bacteria. AS circulating monocytes displayed an anergic phenotype partially restored by ex vivo stimulation with LPS+sCD14 and their stimulation with recombinant zonulin induced a clear M2 phenotype. Antibiotics restored tight junction function in HLA-B27 TG rats. Conclusions Bacterial ileitis, increased zonulin expression and damaged intestinal mucosal barrier and GVB, characterises the gut of patients with AS and are associated with increased blood levels of zonulin, and bacterial products. Bacterial products and zonulin influence monocyte behaviour.

Curcumin modulates chronic myelogenous leukemia exosomes composition and affects angiogenic phenotype, via exosomal miR-21

Tumor derived exosomes are vesicles which contain proteins and microRNAs that mediate cell-cell communication and are involved in angiogenesis and tumor progression. Curcumin derived from the plant Curcuma longa, shows anticancer effects. Exosomes released by CML cells treated with Curcumin contain a high amount of miR-21 that is shuttled into the endothelial cells in a biologically active form. The treatment of HUVECs with CML Curcu-exosomes reduced RhoB expression and negatively modulated endothelial cells motility. We showed that the addition of CML control exosomes to HUVECs caused an increase in IL8 and VCAM1 levels, but Curcu-exosomes reversed these effects thus attenuating their angiogenic properties. This antiangiogenic effect was confirmed with in vitro and in vivo vascular network formation assays. SWATH analysis of the proteomic profile of Curcu-exosomes revealed that Curcumin treatment deeply changes their molecular properties, in particular, Curcumin induces a release of exosomes depleted in pro-angiogenic proteins and enriched in proteins endowed with anti-angiogenic activity. Among the proteins differential expressed we focused on MARCKS, since it was the most modulated protein and a target of miR-21. Taken together our data indicated that also Curcumin attenuates the exosomes ability to promote the angiogenic phenotype and to modulate the endothelial barrier organization.

Chronic myelogenous leukaemia exosomes modulate bone marrow microenvironment through activation of epidermal growth factor receptor

Chronic myelogenous leukaemia (CML) is a clonal myeloproliferative disorder. Recent evidence indicates that altered crosstalk between CML and mesenchymal stromal cells may affect leukaemia survival; moreover, vesicles released by both tumour and non‐tumour cells into the microenvironment provide a suitable niche for cancer cell growth and survival. We previously demonstrated that leukaemic and stromal cells establish an exosome‐mediated bidirectional crosstalk leading to the production of IL8 in stromal cells, thus sustaining the survival of CML cells. Human cell lines used are LAMA84 (CML cells), HS5 (stromal cells) and bone marrow primary stromal cells; gene expression and protein analysis were performed by real‐time PCR and Western blot. IL8 and MMP9 secretions were evaluated by ELISA. Exosomes were isolated from CML cells and blood samples of CML patients. Here, we show that LAMA84 and CML patients’ exosomes contain amphiregulin (AREG), thus activating epidermal growth factor receptor (EGFR) signalling in stromal cells. EGFR signalling increases the expression of SNAIL and its targets, MMP9 and IL8. We also demonstrated that pre‐treatment of HS5 with LAMA84 exosomes increases the expression of annexin A2 that promotes the adhesion of leukaemic cells to the stromal monolayer, finally supporting the growth and invasiveness of leukaemic cells. Leukaemic and stromal cells establish a bidirectional crosstalk: exosomes promote proliferation and survival of leukaemic cells, both in vitro and in vivo, by inducing IL8 secretion from stromal cells. We propose that this mechanism is activated by a ligand–receptor interaction between AREG, found in CML exosomes, and EGFR in bone marrow stromal cells.

Activated IL-22 pathway occurs in the muscle tissues of patients with polymyositis or dermatomyositis and is correlated with disease activity

OBJECTIVE The aim of this study was to assess the expression of IL-22, IL-22 receptor 1 (IL-22R1), IL-22 binding protein (IL-22BP) and p-STAT3 in muscle tissue from patients with PM and DM. METHODS Levels of IL-22, IL-22R1, IL-22BP and STAT3 mRNA were quantified by RT-PCR. The expression of IL-22, IL-22R1, IL-22BP and p-STAT3 was also analysed using immunohistochemistry. RESULTS Significant modulation of the IL-22 pathway was observed in inflammatory myopathic tissues. In particular, a significant overexpression of IL-22 at the protein but not the mRNA level was observed in PM/DM tissues and was correlated with myositis activity. IL-22R1 aberrant expression was also observed among infiltrating mononuclear cells and necrotic muscle cells. IL-22BP, which inhibits IL-22 signalling, was expressed only in some muscle fibres in PM/DM patients. CONCLUSION Our findings indicate that the IL-22 pathway is activated in inflammatory myopathic tissues and may be involved in the induction of muscle inflammatory processes and muscle necrosis.

IL‐17 polarization of MAIT cells is derived from the activation of two different pathways

MAIT cells are expanded in salivary glands of patients with Sjogrens syndrome and are IL-17 polarized. IL-7 and IL-23 induce IL-17 production activating two different pathways: IL-7 stimulation induces in fact a significant STAT3 and HIF1alpha upregulation, conversely, IL-23 stimulation significantly induces RORc overexpression in MAIT cells of patients with Sjogrens syndrome.

Label-free quantitative proteomic profiling of colon cancer cells identifies acetyl-CoA carboxylase alpha as antitumor target of Citrus limon-derived nanovesicles

We have previously isolated exosome-like nanoparticles from Citrus-limon juice, able to inhibit in vitro and in vivo tumor cell growth. In order to deeply understand the mechanism underlying nanovesicle effects, we performed a proteomic profile of treated colorectal cancer cells. Among the proteins differentially expressed after nanovesicle treatment, we found a significant downregulation of the Acetyl-CoA Carboxylase 1 (ACACA) and we demonstrated that silencing ACACA in cancer cells leads to a reduction of cell growth. Our study proved that the anti-tumor effects of Citrus-limon nanovesicles is partly mediated by lipid metabolism inhibition, in particular via ACACA downregulation. SIGNIFICANCE This study represents the attempt to achieve, by a proteomic approach, a better understanding of the role of lemon nanovesicles in affecting colorectal cancer cell growth.