Faeces-derived extracellular vesicles participate in the onset of barrier dysfunction leading to liver diseases.

The role of extracellular vesicles (EVs) from faeces (fEVs) and small circulating EVs (cEVs) in liver diseases such as non-alcoholic fatty diseases (NAFLD) and non-alcoholic steatohepatitis (NASH) has not been demonstrated. fEVs and cEVs of healthy donors, NAFLD and NASH patients were isolated and characterized. The effects of EVs were evaluated in intestinal, endothelial, Kupffer and stellate cells. Non-muscular myosin light chain kinase (nmMLCK) deficient mice were used in vivo. Bacterial origins of fEVs were analysed by 16s rDNA gene sequencing. fEVs and small cEVs were composed of prokaryotic and eukaryotic origins. Only NASH-fEVs exerted deleterious effects. NASH-fEVs increased intestinal permeability and reduced expression of tight junction proteins that were prevented by nmMLCK inhibition, increased endothelial cell permeability and inflammatory cytokines and chemokines requiring TLR4/lipopolysaccharide pathway. NASH-fEVs and NASH-cEVs activated profibrotic and proinflammatory proteins of hepatic stellate cells. Treatment with NASH-fEVs evoked an increase in intestinal permeability in wild type but not in nmMLCK deficient mice. Bacterial origins of fEVs were different between NAFLD and NASH patients and 16 amplicon sequence variants were differentially abundant. We demonstrate that fEVs actively participate in barrier dysfunctions leading to liver injuries underscoring the role of nmMLCK and lipopolysaccharide carried by fEVs.

Hospicells (ascites-derived stromal cells) promote tumorigenicity and angiogenesis.

The microenvironment is known to play a dominant role in cancer progression. Cells closely associated with tumoral cells, named hospicells, have been recently isolated from the ascites of ovarian cancer patients. Whilst these cells present no specific markers from known cell lineages, they do share some homology with bone marrow-derived or adipose tissue-derived human mesenchymal stem cells (CD9, CD10, CD29, CD146, CD166, HLA-1). We studied the role of hospicells in ovarian carcinoma progression. In vitro, these cells had no effect on the growth of human ovarian carcinoma cell lines OVCAR-3, SKOV-1 and IGROV-1. In vivo, their co-injection with adenocarcinoma cells enhanced tumor growth whatever the tumor model used (subcutaneous and intraperitoneally established xenografts in athymic mice). In addition, their injection increased the development of ascites in tumor-bearing mice. Fluorescent macroscopy revealed an association between hospicells and ovarian adenocarcinoma cells within the tumor mass. Tumors obtained by coinjection of hospicells and human ovarian adenocarcinoma cells presented an increased microvascularization indicating that the hospicells could promote tumorigenicity of ovarian tumor cells in vivovia their action on angiogenesis. This effect on angiogenesis could be attributed to the increased HIF1alpha and VEGF expression associated with the presence of the hospicells. Collectively, these data indicate a role for these ascite-derived stromal cells in promoting tumor growth by increasing angiogenesis.

Vascular endothelial growth factor receptor-1 (VEGFR-1) expression in human corneal fibroblast decreased with age.

PURPOSE: Mechanisms by which fibroblast networks between stromal lamellae are laid in the corneal stroma are far from clear. We have investigated the role of vascular endothelial growth factor receptors (VEGFRs) by in vitro studies in the human corneal network formation obtained from donors whose ages ranged from 19 to 89 years. METHODS: Corneal fibroblasts were prepared from cornea donations. The functional properties of these cells to form networks were analyzed using a semi solid matrix (substratum) of Matrigel. The presence of VEGF receptor-1 (VEGFR-1) and the functionality in these fibroblasts were investigated using immunofluorescence, molecular analysis (gene microarray, reverse transcription polymerase chain reaction [RT-PCR] and VEGFR siRNA transfections), and cell culture. RESULTS: Corneal fibroblasts from 61 donors were classified into two groups according to whether they formed (82%) a reticulum on Matrigel or not (18%). By RT-PCR and immunofluorescence analysis, we showed that corneal fibroblasts expressed VEGFR-1 (mRNA and protein). Further, cell culture analysis revealed that only the network (reticulum) forming corneal fibroblast expressed VEGFR-1 in contrast to non network-forming fibroblasts. Use of inhibitors such as VEGFR-1 siRNA transfection or neutralizing antibody (Avastin) indicated that VEGFR-1 was essential to the formation of the corneal network in vitro. CONCLUSIONS: The cell reticulum formation seemed to be directly related to the expression of VEGFR-1 in the corneal fibroblast, and this expression decreased with age. The decrease in VEGFR-1 expression is probably related to the diminution of autocrine functions, which may alter the overall tissular homeostasis. This may culminate in the gradual development of poor vision, which is observed in certain pathologies and in aging individuals.

High Expression of HLA-G in Ovarian Carcinomatosis: The Role of Interleukin-1ß.

The present study focuses on the influence of the tumor microenvironment on the expression of HLA-G in ovarian cancer and its impact on immune cells. We used carcinomatosis fluids (n = 16) collected from patients diagnosed with epithelial ovarian cancer, detected by an increase in CA125 levels. Our results indicate that HLA-G is expressed by 1) ascitic cell clusters, 2) stromal cells (hospicells) extracted from cancer cell clusters, and 3) cancer cell lines and tumor cells. The origin of HLA-G was linked to inflammatory cytokines present in the cancer microenvironment. In parallel, the ascitic fluid of patients with ovarian cancer contains soluble HLA-G (sHLA-G). The mesothelial cell layer and submesothelial tissues, as well as the immune cell infiltrate, do not secrete HLA-G. In contrast, sHLA-G is absorbed by peritoneal tissues along with mesothelial layers as well as immune cell infiltrates. We demonstrated that interleukin-1ß along with TGF-ß can be a major HLA-G-inducing factor that upregulates HLA-G expression through the NF-κB pathway. The level of HLA-G in ascites correlated positively with the expression of T regulatory (T-regs) cells, while it negatively correlated with the expression of natural killer and memory cells in tumor-infiltrating immune cells. In conclusion, the production of HLA-G is associated with the presence of inflammatory cytokines and is strongly correlated with microenvironment tolerant cells such as T-regs and diminution of NK and memory T cells.

A subset of bone marrow stromal cells regulate ATP-binding cassette gene expression via insulin-like growth factor-I in a leukemia cell line.

The importance of the insulin-like growth factor, IGF, as a signaling axis in cancer development, progression and metastasis is highlighted by its effects on cancer cells, notably proliferation and acquired resistance. The role of the microenvironment within which cancer cells evolve and which mediates this effect is far from clear. Here, the involvement of IGF-I in inducing multidrug resistance in a myeloid leukemia cell line, grown in the presence of bone marrow-derived stromal cells called 'Hospicells' (BMH), is demonstrated. We found that i) drug sensitive as well as resistant leukemia cells express IGF-I and its receptor IGF-IR. However, the resistant cells were found to secrete high levels of IGF-I. ii) Presence of exogenous IGF-I promoted cell proliferation, which decreased when an inhibitor of IGF-IR (picropodophyllin, PPP) was added. iii) BMH and IGF-I are both involved in the regulation of genes of the ATP binding cassette (ABC) related to resistance development (MDR1, MRP1, MRP2, MRP3 and BCRP). iv) The levels of ABC gene expression by leukemia cells were found to increase in the presence of increasing numbers of BMH. However, these levels decreased when IGF-IR was inhibited by addition of PPP. v) Co-culture of the drug-sensitive leukemia cells with BMH induced protection against the action of daunorubicin. This chemoresistance was amplified by the presence of IGF-I whereas it decreased when IGF-IR was inhibited. Our results underline the role of microenvironment in concert with the IGF-1 pathway in conferring drug resistance to leukemia cells.

Hospicells promote upregulation of the ATP-binding cassette genes by insulin-like growth factor-I via the JAK2/STAT3 signaling pathway in an ovarian cancer cell line.

Interaction between tumor cells and their micro-environment has a crucial role in the development, progression and drug resistance of cancer. Our objective was to confirm the role of Hospicells, which are stromal cells from the cancer microenvironment, in drug resistance and tumor cell growth. We demonstrated that soluble factors secreted by Hospicells activate several genes and upregulate the JAK/STAT signaling pathway in ovarian cancer cell lines. Hospicells express all insulin-like growth factor (IGF) family as detected by gene array, RT-PCR, protein array and immunocytochemistry. While focusing attention on the microenvironment, we considered the role of IGF-I in proliferation and survival of ovarian cancer cells. Indeed, IGF-I is a major regulator of different stages of cancer development. We studied the effect of exogenously added IGF-I on the regulation of ATP-binding cassette (ABC) genes (MDR1, MRP1, MRP2, MRP3, MRP5 and BCRP) in the ovarian cancer cell line OVCAR3 and validated the results obtained using the IGF-IR antagonist picropodophyllin. IGF-I regulates the expression of ABC genes in OVCAR3 cells via the PI3-kinase, MEK and JAK2/STAT3 signaling pathways. The OVCAR3 cell line when co-cultured with Hospicells showed a marked degree of drug resistance. The drug resistance observed could be amplified with exogenous IGF-I. Addition of IGF-IR inhibitor, however, reduced the degree of resistance in these exposed cells. Cells that were treated with anticancer drugs and then exposed to IGF-I showed an increase in drug resistance and, thereby, an increase in cell survival. This observation indicates that drug resistance of OVCAR3 cells increases when there is synergy between OVCAR3 cells and Hospicells and it is amplified when IGF-I was exogenously added. In conclusion, inhibition of IGF-IR and targeting of the JAK2/STAT3 signaling pathway can be a target for ovarian cancer therapy.

Insulin growth factor promotes human corneal fibroblast network formation in vitro.

PURPOSE: Corneal fibroblast cell (CFC) reticulation is involved in the structural development of corneal stroma and in wound healing. In an earlier paper, it was reported that the expression of VEGFR-1 by CFCs is related to their reticulogenic properties in vitro and decreases with the age of the donors. The present study was focused on the nonreticulogenic corneal fibroblast population and explored whether these cells can be induced to form cell networks in vitro. METHODS: The network formation was analyzed using an array of signaling pathway inhibitors: wortmannin for PI3 kinase, U0126 for MEK-1/2 kinase, Rottlerin for PKC, farnesyl transferase inhibitor (FTI-277) for Ras, and picropodophyllin (PPP) for IGFR-1. Among the several growth factors studied, IGF seemed to be crucial to cell network formation. The presence of IGF signaling was demonstrated using gene array analysis and was confirmed by RT-PCR and immunocytochemistry and by cell network formation on reduced synthetic basement membrane arrays. The pleiotropic effect of IGF-1 on the cells was analyzed by protein cytokine array. RESULTS: The genesis of reticulation was found to occur via MEK-1/2 and IGFR pathways, since inhibitors of these signaling pathways reduced or prevented cell network formation. The addition of exogenous IGF-1 generated a cell network structure in corneal fibroblasts obtained from healthy donors, indicating the involvement of IGF-1. CONCLUSIONS: IGF signaling and the MEK-1/2 pathway are involved in the cell network formation of corneal fibroblast cells from aged donors.

Matrix metalloproteinase-9 is required for tubular network formation and migration of resistant breast cancer cells MCF-7 through PKC and ERK1/2 signalling pathways.

Matrix metalloproteinase-9 (MMP-9) strongly influences tumor development and metastasis. Using resistant (rMCF-7) and sensitive (sMCF-7) breast cancer lines we investigated the role of MMP-9 in cell migration (CM) and tubular network (TN) formation, two processes implied in tumor growth and metastasis. Our data demonstrate that MMP-9 which is critical for CM is necessary but not sufficient for TN formation and suggest a link between MDR1/P-gp and constitutive MMP-9. Both TN formation and CM are dependent on PKC and ERK1/2 pathways. This study reinforces the logic of combining therefore MMP inhibitors in cancer therapy, especially in patients with chemoresistance and invasion/metastasis.