The inflammatory cytokine TNFα cooperates with Ras in elevating metastasis and turns WT-Ras to a tumor-promoting entity in MCF-7 cells.

BACKGROUND: In the present study we determined the relative contribution of two processes to breast cancer progression: (1) Intrinsic events, such as activation of the Ras pathway and down-regulation of p53; (2) The inflammatory cytokines TNFα and IL-1ß, shown in our published studies to be highly expressed in tumors of >80% of breast cancer patients with recurrent disease. METHODS: Using MCF-7 human breast tumor cells originally expressing WT-Ras and WT-p53, we determined the impact of the above-mentioned elements and cooperativity between them on the expression of CXCL8 (ELISA, qRT-PCR), a member of a "cancer-related chemokine cluster" that we have previously identified. Then, we determined the mechanisms involved (Ras-binding-domain assays, Western blot, luciferase), and tested the impact of Ras + TNFα on angiogenicity (chorioallantoic membrane assays) and on tumor growth at the mammary fat pad of mice and on metastasis, in vivo. RESULTS: Using RasG12V that recapitulates multiple stimulations induced by receptor tyrosine kinases, we found that RasG12V alone induced CXCL8 expression at the mRNA and protein levels, whereas down-regulation of p53 did not. TNFα and IL-1ß potently induced CXCL8 expression and synergized with RasG12V, together leading to amplified CXCL8 expression. Testing the impact of WT-Ras, which is the common form in breast cancer patients, we found that WT-Ras was not active in promoting CXCL8; however, TNFα has induced the activation of WT-Ras: joining these two elements has led to cooperative induction of CXCL8 expression, via the activation of MEK, NF-κB and AP-1. Importantly, TNFα has led to increased expression of WT-Ras in an active GTP-bound form, with properties similar to those of RasG12V. Jointly, TNFα + Ras activities have given rise to increased angiogenesis and to elevated tumor cell dissemination to lymph nodes. CONCLUSIONS: TNFα cooperates with Ras in promoting the metastatic phenotype of MCF-7 breast tumor cells, and turns WT-Ras into a tumor-supporting entity. Thus, in breast cancer patients the cytokine may rescue the pro-cancerous potential of WT-Ras, and together these two elements may lead to a more aggressive disease. These findings have clinical relevance, suggesting that we need to consider new therapeutic regimens that inhibit Ras and TNFα, in breast cancer patients.

Perturbed actin cap as a new personalized biomarker in primary fibroblasts of Huntington's disease patients.

Primary fibroblasts from patient's skin biopsies are directly isolated without any alteration in the genome, retaining in culture conditions their endogenous cellular characteristics and biochemical properties. The aim of this study was to identify a distinctive cell phenotype for potential drug evaluation in fibroblasts from Huntington's Disease (HD) patients, using image-based high content analysis. We show that HD fibroblasts have a distinctive nuclear morphology associated with a nuclear actin cap deficiency. This in turn affects cell motility in a similar manner to fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients used as known actin cap deficient cells. Moreover, treatment of the HD cells with either Latrunculin B, used to disrupt actin cap formation, or the antioxidant agent Mitoquinone, used to improve mitochondrial activity, show expected opposite effects on actin cap associated morphological features and cell motility. Deep data analysis allows strong cluster classification within HD cells according to patients' disease severity score which is distinct from HGPS and matching controls supporting that actin cap is a biomarker in HD patients' cells correlated with HD severity status that could be modulated by pharmacological agents as tool for personalized drug evaluation.

The involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in atherosclerosis.

OBJECTIVES: Herein, we determined the significance of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in atherosclerotic vascular disease. BACKGROUND: Inflammation is associated with the pathogenesis of atherosclerosis. The TNF-related apoptosis-inducing ligand/APO-2L, a member of the TNF superfamily, has a role in apoptosis induction and is recognized for its immunomodulatory properties. METHODS: Stable and vulnerable atherosclerotic human plaques and aortas from atherosclerotic mice were assayed for the presence of TRAIL, and its inducibility was assayed by immunoblot and real-time polymerase chain reaction on peripheral mononuclear cells incubated with oxidized low-density lipoprotein (oxLDL). Enzyme-linked immunosorbent assay was used for the determination of soluble TRAIL levels in atherosclerotic patients. RESULTS: Tumor necrosis factor-related apoptosis-inducing ligand is present in stable atherosclerotic lesions, is increased in vulnerable plaques, and is found to colocalize with CD3 cells and oxLDL. The TNF-related apoptosis-inducing ligand messenger ribonucleic acid (mRNA) and protein expression was up-regulated in peripheral blood mononuclear cells after incubation with oxLDL. Serum levels of soluble TRAIL but not TNF-alpha or Fas-ligand were reduced significantly in patients with unstable angina as compared with patients with stable atherosclerotic disease and healthy subjects. A negative correlation was demonstrated between soluble TRAIL and C-reactive protein levels but not with levels of mRNA of TRAIL in peripheral blood mononuclear cells. CONCLUSIONS: Tumor necrosis factor-related apoptosis-inducing ligand is expressed in plaque-infiltrating CD3 cells and induced by oxLDL, whereas levels of soluble TRAIL are reduced in patients with acute coronary syndromes and negatively correlate with C-reactive protein levels. These results support a possible role for TRAIL in atherosclerosis.

Transfer of endothelial progenitor and bone marrow cells influences atherosclerotic plaque size and composition in apolipoprotein E knockout mice.

OBJECTIVE: Recent clinical trials use cell therapy with bone marrow (BM) cells or endothelial progenitor cells (EPCs) for ischemic syndromes. We explored the effect of BM cell- or spleen cell-derived EPC transfer on plaque size and stability markers in the apolipoprotein E knockout (apoE KO) mouse model. METHODS AND RESULTS: ApoE KO mice aged 10 weeks served as recipients. Labeled BM cells and spleen cell-derived EPCs from age-matched apoE KO mice were injected intravenously to 2 groups of recipient mice each. Additional mice served as controls receiving saline. Both protocols were repeated 3 times at 2 weekly intervals. On killing, plaque size and character were studied, lipid profile analyzed, and serum and aortic cytokines assayed. Spleen cell-derived cells contained a significantly larger number of endothelial cell precursors. Labeled EPCs and BM cells were found abundantly in the spleens, yet also in the lesions of the recipient mice. Aortic sinus lesion size was significantly increased in mice receiving BM cells (n=10) in the EPC-treated group (n=10) compared with controls (n=10; a 54% and a 34% increase in aortic sinus plaque area, respectively). Mice receiving EPCs exhibited plaques with larger lipid cores and thinner fibrous caps and a higher number of infiltrating CD3 cells. RT-PCR analysis of aortas revealed reduced expression of mRNA for interleukin-10 (IL-10) in both cell transfer groups. Higher serum concentrations of IL-6 and monocyte chemoattractant protein-1 were found in sera from BM recipients, whereas lower IL-10 levels were found in mice transfused with spleen-derived EPCs. CONCLUSIONS: Transfer of BM cells and EPCs may result in an increase in atherosclerotic lesion size, whereas EPC transfer could also potentially influence plaque stability.

Circulating erythropoietin levels and prognosis in patients with congestive heart failure: comparison with neurohormonal and inflammatory markers.

BACKGROUND: Considerable morbidity and mortality are still associated with congestive heart failure (CHF) syndromes, despite improvement in therapy. Activation of neurohormonal, inflammatory, and oxidative mechanisms has been shown to contribute to the significant morbidity and mortality. Erythropoietin (EPO) is a cytokine known to regulate erythroid proliferation, attenuate apoptosis and oxidative stress, and promote angiogenesis. We prospectively evaluated the predictive value of baseline EPO, N-terminal pro-B-type natriuretic peptide, and C-reactive protein levels in patients with clinically controlled chronic CHF. METHODS: One hundred eighty-eight outpatients from a CHF clinic had baseline assessment of EPO, N-terminal pro-B-type natriuretic peptide, and C-reactive protein levels and a complete clinical data profile. These patients were followed up for 24 months for any hospitalization due to CHF or mortality. RESULTS: Circulating EPO levels were higher in CHF patients and increased in subjects with higher New York Heart Association scores. Levels of EPO (at a cutoff of 23 mU/mL) and N-terminal pro-B-type natriuretic peptide (cutoff at the median of 1556 pg/mL) were found to be strong predictors of mortality and CHF hospitalization, whereas C-reactive protein levels (cutoff of 10 mg/L) predicted CHF hospitalizations but not mortality. Left ventricular ejection fraction was found to be a predictor of mortality but not of CHF hospitalizations. Serum levels of EPO were significantly correlated with N-terminal pro-B-type natriuretic peptide and C-reactive protein levels but not with left ventricular ejection fraction. CONCLUSION: If confirmed in large-scale clinical studies, determination of circulating EPO levels may aid in predicting morbidity and mortality in patients with clinically controlled congestive CHF.

Erythropoietin promotes endothelial progenitor cell proliferative and adhesive properties in a PI 3-kinase-dependent manner.

OBJECTIVES: Patients with congestive heart failure (CHF) suffer considerable morbidity and mortality despite advances in therapy. Treatment with erythropoietin (Epo) has shown promise in CHF patients, yet its mechanisms of action remain elusive. Endothelial progenitor cells (EPC) contribute to postnatal angiogenesis and vasculogenesis, and Epo was shown to promote EPC mobilization. We explored the effect of chronic treatment with Epo on the numbers and functional properties of EPC in CHF patients. METHODS AND RESULTS: Twenty-eight patients with CHF treated with Epo for a mean period of 28 months were compared to a matched group (n = 28) with regard to the number of circulating hematopoietic and endothelial stem cells (either CD34+, CD34+/CD45+, CD34+/CD133+, CD34+/VEGF-R2+ or CD34+/CD133+/VEGF-R2+) as well as their proliferative and adhesive capacity. In vitro, Epo was added to cultured EPC from healthy subjects to test proliferation and adhesion. No differences were observed in circulating numbers of hematopoietic and endothelial stem cells between CHF patients chronically treated with Epo or untreated. EPC from Epo-treated patients exhibited enhanced proliferation as well as a trend towards adhesion to cultured endothelial cells prior to and following stimulation with TNF-alpha. Addition of Epo to EPC from healthy subjects dose-dependently increased their proliferation and adhesion to fibronectin, cultured endothelial cells, and cardiomyocytes. These effects were significantly reduced in the presence of phosphatidylinositol (PI) 3-kinase inhibitors. CONCLUSIONS: Chronic Epo treatment is associated with an increase in the adhesive and proliferative properties of circulating EPC in patients with CHF.

Temporal association between circulating proteolytic, inflammatory and neurohormonal markers in patients with coronary ectasia.

BACKGROUND: The prevalence of coronary ectasia (CE) appears to rise in recent years. However, the pathogenetic mechanisms that underlie this entity are not understood. We hypothesized that dysregulation of circulating matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) contributes to ectasia formation. We also evaluated the association of MMP with inflammatory or the neurohormonal markers. METHODS: We screened coronary angiographic procedures conducted in our hospital. Thirty-four patients with CEs were identified; 17 with a single ectasia and 17 with generalized ectasias. Two control groups; patients with atherosclerotic coronary disease (n=26) and patients with normal coronary arteries (n=27) were chosen randomly. Serum levels of MMP-2, MMP-3 and TIMP-1 were determined by ELISA. Pro B-type natriuretic peptide (proBNP) and high sensitivity C-reactive protein (hsCRP) serum levels were also measured. RESULTS: Serum levels of MMP-2, MMP-3, TIMP-1, proBNP and hsCRP did not differ between the three groups of patients. However, subgroup analysis demonstrated that MMP-3 level is significantly lower in patients with generalized CEs compared to those with single vessel ectasia. We also found a statistically significant correlation between proBNP and MMP-2/TIMP-1 (for MMP-2: r(2)=0.3, p=0.003 and for TIMP-1 r(2)=0.42, p<0.000l). By performing subgroup analysis we found that this correlation is only demonstrated in patients with CEs. No correlation was demonstrated between hsCRP and MMPs/TIMP. CONCLUSIONS: These observations suggest that MMP/TIMP imbalance is present in patients with generalized CE and may contribute to ectasia formation. This imbalance could be mediated by BNP.

Involvement of IKAP in peripheral target innervation and in specific JNK and NGF signaling in developing PNS neurons.

A splicing mutation in the ikbkap gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we attempted to elucidate the role of IKAP in PNS development in the chick embryo and found that IKAP is required for proper axonal outgrowth, branching, and peripheral target innervation. Moreover, we demonstrate that IKAP colocalizes with activated JNK (pJNK), dynein, and ß-tubulin at the axon terminals of dorsal root ganglia (DRG) neurons, and may be involved in transport of specific target derived signals required for transcription of JNK and NGF responsive genes in the nucleus. These results suggest the novel role of IKAP in neuronal transport and specific signaling mediated transcription, and provide, for the first time, the basis for a molecular mechanism behind the FD phenotype.