Tissue factor variants induce monocyte transformation and transdifferentiation into endothelial cell-like cells.

Essentials Monocytes (Mo) transdifferentiate into endothelial cell-like (ECL) cells. Mo induce tissue factor (TF) expression and secretion in microvascular endothelial cells (mECs). TF interacts with Mo in a paracrine fashion, inducing their transdifferentiation into ECL cells. TF generates a positive feedback crosstalk between Mo and mECs that promotes angiogenesis. SUMMARY: Background Monocytes (Mo) increase neovascularization by releasing proangiogenic mediators and/or transdifferentiating into endothelial cell-like (ECL) cells. Recently, we have reported that Mo-microvascular endothelial cells (mECs) crosstalk induces mEC-tissue factor (TF) expression and promotes angiogenesis. However, the effect of TF on Mo remains unknown. Objective Here, we analyzed whether TF might exert angiogenic effects by inducing transdifferentiation of Mo. Methods Full-length TF (flTF) and alternatively spliced TF (asTF) were overexpressed in mECs, and their supernatants were added to Mo cultures. CD16 positivity and expression of vascular endothelial cell (VEC) markers in Mo were analyzed by fluorescence activated cell sorting. The capacity to form tube-like structures were visualized in three-dimensional cultures. Results In mECs flTF and asTF expression and release were increased in cultures with Mo-conditioned media. TF variants induced expansion of a CD16+ Mo subset and Mo transdifferentiation into ECL-cells expressing VEC markers that can form new microvessels. CD16+ Mo exposed to TF showed an increased expression of VE-cadherin, von Willebrand factor (VWF) and eNOS. Mo cultured with supernatants obtained from TF-silenced mECs did not transdifferentiate to ECL-cells or expressed VEC markers. Blocking ß1-integrin in Mo significantly blocked the effects of the TF variants. Conclusions Mo induce mECs to express and release TF, which drives CD16- Mo to transform into CD16+ Mo and to transdifferentiate into ECL-cells that can form new microvessels. Our results reveal a TF-mediated positive feedback between mECs and Mo that stimulates Mo differentiation and induces angiogenesis.

Lipopolysaccharide downregulates CD91/low-density lipoprotein receptor-related protein 1 expression through SREBP-1 overexpression in human macrophages.

Sterol regulatory element-binding proteins (SREBPs) negatively modulate the expression of the CD91/low-density lipoprotein receptor-related protein (LRP1), a carrier and signaling receptor that mediates the endocytosis of more than 40 structurally and functionally distinct ligands. The aim of this work was to analyze whether lipopolysaccharide (LPS) can regulate LRP1 expression through SREBPs in human monocyte-derived macrophages (HMDM). LPS led to LRP1 mRNA and protein inhibition in a dose- and time-dependent manner. Concomitantly, a strong upregulation of SREBP-1 mRNA and SREBP-1 nuclear protein levels was observed in LPS-treated HMDM. The specific silencing of SREBP-1 efficiently prevented LRP1 reduction caused by LPS. SREBP-1 mRNA and nuclear protein levels remained high in HMDM treated with LPS unexposed or exposed to LDL. Native (nLDL) or aggregated LDL (agLDL) per se downregulated SREBP-2 expression levels and increased LRP1 expression. However, lipoproteins did not significantly alter the effect of LPS on SREBP-1 and LRP1 expression. Collectively, these data support that lipoproteins and LPS exert their modulatory effect on LRP1 expression through different SREBP isoforms, SREBP-2 and SREBP-1, respectively. These results highlight a crucial role of SREBP-1 as a mediator of the downregulatory effects of LPS on LRP1 expression in human macrophages, independently of the absence or presence of modified lipoproteins.

Tissue factor-Akt signaling triggers microvessel formation.

BACKGROUND: Tissue factor (TF) and its signaling mediators play a crucial role in angiogenesis. We have previously shown that TF-induced endothelial cell (EC) CCL2 release contributes to neovessel formation. OBJECTIVE: In this study, we have investigated the signaling pathways involved in TF-induced EC tube formation. METHODS: The human microvascular endothelial cell line (HMEC-1) cultured onto basement membrane-like gel (Matrigel) was used to study TF signaling pathways during neovessels formation. RESULTS: Inhibition of endogenous TF expression in ECs using siRNA resulted in inhibition of a stable tube-like structure formation in three-dimensional cultures, associated with a down-regulation of Akt activation, an increased phosphorylation of Raf at Ser(259) with a subsequent reduction of Raf kinase and a reduction of ERK1/2 phosphorylation ending up in Ets-1 transcription factor inhibition. Conversely, overexpression of TF resulted in an increase in tube formation and up-regulation of Akt protein. Moreover, immunoprecipitation of Akt and western blotting of the immunoprecipitates with anti-TF antibody revealed a direct interaction between TF and Akt. The effects of silencing TF were partially reversed by a PAR2 agonist that rescued tube formation, indicating that the TF-Akt pathway induces PAR2-independent effector signaling. Finally, enforced expression of Akt in TF-silenced ECs rescued tube formation in a Matrigel assay and induced Ets-1 phosphorylation. CONCLUSIONS: In EC, TF forms a complex with Akt activating Raf/ERK and Ets-1 signaling induces microvessel formation.

Schwann cells harbor the somatic NF1 mutation in neurofibromas: evidence of two different Schwann cell subpopulations.

Neurofibromas are one of the most characteristic features of neurofibromatosis type 1 (NF1), an inherited autosomal-dominant neurogenetic disorder affecting 1 in 3500 individuals worldwide. These benign tumors mainly consist of Schwann cells (SCs) and fibroblasts. Recent evidence demonstrates that somatic mutations at the NF1 gene are found in neurofibromas, but it has not been demonstrated whether SCs, fibroblasts and/or both cell types bear a somatic loss of NF1. We recently established a cell culture system that allows selective expansion of human SCs from neurofibromas. We cultured pure populations of SCs and fibroblasts derived from 10 neurofibromas with characterized NF1 mutations and found that SCs but not fibroblasts harbored a somatic mutation at the NF1 locus in all studied tumors. Furthermore, by culturing neurofibroma-derived SCs under different in vitro conditions we were able to obtain two genetically distinct SC subpopulations: NF1(-/-) and NF1(+/-). These data strongly support the idea that NF1 mutations in SCs, but not in fibroblasts, correlate to neurofibroma formation and demonstrate that only a portion of SCs in neurofibromas have mutations in both NF1 alleles.

Cytogenetic study on eleven cutaneous neoplasms and two pre-tumoral lesions from Xeroderma pigmentosum patients.

Eleven independent tumors (5 basa-cell carcinomas, 5 squamous-cell carcinomas and 1 malignant melanoma), 2 pretumoral lesions and one common nevus, developing in the skin of 10 unrelated XP patients were cytogenetically analyzed. No specific chromosomal changes were observed. Two features were relevant, however: emergence of several independent clones and over-involvement of telomeric and centromeric regions in the formation of chromosomal rearrangements. Jumping translocations were observed in 2 squamous-cell carcinomas involving telomeric and centromeric regions.

Jumping end-to-end dicentrics in a case of squamous cell carcinoma from a patient with xeroderma pigmentosum.

Cytogenetic studies of a skin squamous cell carcinoma from a xeroderma pigmentosum patient were performed at several passages. They show the existence of recurrent rearrangements: 53% were dicentrics, of which 67% were of the telomere-telomere type. The telomeric region of the long arm of chromosome 12 was the most involved (in 38% of dicentrics), followed by 22p. The origin of this type of jumping rearrangement and its possible role on cell proliferation are discussed.

Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum.

The distribution of spontaneous sister chromatid exchanges (SCEs) was studied in PHA-stimulated lymphocytes from 15 patients affected by xeroderma pigmentosum (XP). The study of unscheduled DNA synthesis (UDS) in twelve of these patients showed that seven were deficient and five proficient. The number of SCEs in XP patient cells was higher than in those of 19 controls, and the distributions of SCEs per cell were significantly different. However, the results varied when XP patients were considered in relation to their UDS: the group of XP patients with proficient UDS did not differ, whereas the group of XP patients with deficient UDS was very significantly different from controls. The group not tested for UDS was similar to the deficient UDS group. The possible relationship between the increase of SCEs and the type of DNA repair defect is discussed.

Jumping translocation of chromosome 14 in a skin squamous cell carcinoma from a xeroderma pigmentosum patient.

The cytogenetic study of a case of cutaneous squamous cell carcinoma developed in a child affected by xeroderma pigmentosum is described. In this paratetraploid tumor, virtually all mitoses had the following rearrangements: i(1q), i(1p), t(3q14q), del(9p), and der(19)t(8;19). In addition, there were several deletions of 1p and 1q. The del(9p) likely occurred as the first rearrangement. The distal segment of the short arm of chromosome #9 and the long arm of #19 and #22 were the most underrepresented and chromosome #6 the most overrepresented chromosome or chromosome segment. The most striking anomaly detected was a jumping translocation of chromosome #14, involved with chromosomes #1, #3, #5, #7, #9, #14, and #22. The breakage of chromosome #14 always occurred on the short arm.

Spontaneous chromosomal anomalies in lymphocytes from xeroderma pigmentosum. A study of ten patients.

A cytogenetic study of the lymphocytes from 6 classic and 4 variant forms of xeroderma pigmentosum is reported. This study performed on 978 R-banded metaphases shows that there is no specific chromosomal rearrangement in this disorder. In UDS-deficient forms, the rates of deletions, chromatid gaps and chromosome gaps are significantly increased. The preferential involvement of G-bands is discussed.