Endothelial Cells Obtained from Patients Affected by Chronic Venous Disease Exhibit a Pro-Inflammatory Phenotype

The inflammatory properties of vein endothelium in relation to chronic venous disease (CVD) have been poorly investigated. Therefore, new insights on the characteristics of large vein endothelium would increase our knowledge of large vessel physiopathology. METHODOLOGY/PRINCIPAL FINDINGS: Surgical specimens of veins were obtained from the tertiary venous network (R3) and/or saphenous vein (SF) of patients affected by CVD and from control individuals. Highly purified venous endothelial cell (VEC) cultures obtained from CVD patients were characterized for morphological, phenotypic and functional properties compared to control VEC. An increase of CD31/PECAM-1, CD146 and ICAM-1 surface levels was documented at flow cytometry in pathological VEC with respect to normal controls. Of note, the strongest expression of these pro-inflammatory markers was observed in VEC obtained from patients with more advanced disease. Similarly, spontaneous cell proliferation and resistance to starvation was higher in pathological than in normal VEC, while the migratory response of VEC showed an opposite trend, being significantly lower in VEC obtained from pathological specimens. In addition, in keeping with a higher baseline transcriptional activity of NF-kB, the release of the pro-inflammatory cytokines osteoprotegerin (OPG) and vascular endothelial growth factor (VEGF) was higher in pathological VEC cultures with respect to control VEC. Interestingly, there was a systemic correlation to these in vitro data, as demonstrated by higher serum OPG and VEGF levels in CVD patients with respect to normal healthy controls. CONCLUSION/SIGNIFICANCE: Taken together, these data indicate that large vein endothelial cells obtained from CVD patients exhibit a pro-inflammatory phenotype, which might significantly contribute to systemic inflammation in CVD patients

Changes in expression profiles of internal jugular vein wall and plasma protein levels in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory, demyelinating and degenerative disorder of the central nervous system (CNS). Several observations support interactions between vascular and neurodegenerative mechanisms in multiple sclerosis (MS). To investigate the contribution of the extracranial venous compartment, we analysed expression profiles of internal jugular vein (IJV), which drains blood from CNS, and related plasma protein levels. METHODS: We studied a group of MS patients (n = 19), screened by echo-color Doppler and magnetic resonance venography, who underwent surgical reconstruction of IJV for chronic cerebrospinal venous insufficiency (CCSVI). Microarray-based transcriptome analysis was conducted on specimens of IJV wall from MS patients and from subjects undergoing carotid endarterectomy, as controls. Protein levels were determined by multiplex assay in: i) jugular and peripheral plasma from 17 MS/CCSVI patients; ii) peripheral plasma from 60 progressive MS patients, after repeated sampling and iii) healthy individuals. RESULTS: Of the differentially expressed genes (≥ 2 fold-change, multiple testing correction, P < 0.05), the immune-related CD86 (8.5 fold-change, P = 0.002) emerged among the up regulated genes (N = 409). Several genes encoding HOX transcription factors and histones potentially regulated by blood flow, were overexpressed. Smooth muscle contraction and cell adhesion processes emerged among down regulated genes (N = 515), including the neuronal cell adhesion L1CAM as top scorer (5 fold-change, P = 5 × 10- 4). Repeated measurements in jugular/peripheral plasma and overtime in peripheral plasma showed conserved individual plasma patterns for immune-inflammatory (CCL13, CCL18) and adhesion (NCAM1, VAP1, SELL) proteins, despite significant variations overtime (SELL P < 0.0001). Both age and MS disease phenotypes were determinants of VAP1 plasma levels. Data supported cerebral related-mechanisms regulating ANGPT1 levels, which were remarkably lower in jugular plasma and correlated in repeated assays but not between jugular/peripheral compartments. CONCLUSIONS: This study provides for the first time expression patterns of the IJV wall, suggesting signatures of altered vascular mRNA profiles in MS disease also independently from CCSVI. The combined transcriptome-protein analysis provides intriguing links between IJV wall transcript alteration and plasma protein expression, thus highlighting proteins of interest for MS pathophysiology

Nutlin-3 downregulates the expression of the Oncogene TCL1 in primary B Chronic Lymphocytic Leukemic cells.

PURPOSE: The oncogene TCL1 plays a key role in the development of B chronic lymphocytic leukemia (B-CLL), but it is not known whether TCL1 could be modulated by therapeutic approaches. EXPERIMENTAL DESIGN: B-CLL patient samples (n = 35) and B leukemic cell lines (EHEB, JVM2, JVM3, MEC1, MEC2, and BJAB) with different p53 status were exposed to Nutlin-3, a small-molecule inhibitor of the p53-MDM2 interaction. Modulations of the steady-state mRNA levels of TCL1 were analyzed by quantitative real-time PCR and Western blotting in both primary B-CLL samples and leukemic cell lines. In addition, transfection experiments with either p53 siRNA or with a TCL1 expression plasmid were carried out in the EHEB B-CLL cell line. RESULTS: Upon ex vivo treatment with Nutlin-3, TCL1 was significantly (P < 0.05) decreased in 23 of 28 B-CLL p53(wild-type). The functionality of the p53 pathway in the same leukemic cell samples was underscored by the concomitant ability of Nutlin-3 to significantly (P < 0.05) upregulate the p53 target gene MDM2 in the p53(wild-type) leukemic cells. The dependence of TCL1 downregulation by a functional p53 pathway was confirmed in a panel of B lymphoblastoid cell lines and by p53 knockdown experiments with p53 siRNA. The importance of TCL1 in promoting leukemic cell survival was underscored in transfection experiments, in which TCL1 overexpression significantly counteracted the Nutlin-3-mediated induction of apoptosis in EHEB. CONCLUSIONS: Our data indicate that the Nutlin-3 downregulates TCL1 mRNA and protein, which likely represents an important molecular determinant in the proapoptotic activity of Nutlin-3. Clin Cancer Re

Coagulation factor XII levels and intrinsic thrombin generation in multiple sclerosis

Background: Factor XII (FXII) activation initiates the intrinsic (contact) coagulation pathway. It has been recently suggested that FXII could act as an autoimmunity mediator in multiple sclerosis (MS). FXII depositions nearby dentritic cells were detected in the central nervous system of MS patients and increased FXII activity has been reported in plasma of relapsing remitting and secondary progressive MS patients. FXII inhibition has been proposed to treat MS. Objective: To investigate in MS patients multiple FXII-related variables, including the circulating amount of protein, its pro-coagulant function, and their variation over time. To explore kinetic activation features of FXII in thrombin generation (TG). Methods: In plasma from 74 MS patients and 49 healthy subjects (HS), FXII procoagulant activity (FXII:c) and FXII protein (FXII:Ag) levels were assessed. Their ratio (FXII:ratio) values were derived. Intrinsic TG was evaluated by different triggers. Results: Higher FXII:Ag levels (p = 0.003) and lower FXII:ratio (p &lt; 0.001) were detected in MS patients compared with HS. FXII variables were highly correlated over four time points, which supports investigation of FXII contribution to disease phenotype and progression. A significant difference over time was detected for FXII:c (p = 0.031). In patients selected for the lowest FXII:ratio, TG triggered by ellagic acid showed a trend in lower endogenous thrombin potential (ETP) in MS patients compared with HS (p = 0.042). Intrinsic triggering of TG by nucleic acid addition produced longer time parameters in patients than in HS and substantially increased ETP in MS patients (p = 0.004) and TG peak height in HS (p = 0.008). Coherently, lower FXII:ratio and longer lag time (p = 0.02) and time to peak (p = 0.007) point out a reduced response of FXII to activation in part of MS patients. Conclusion: In MS patients, factor-specific and modified global assays suggest the presence of increased FXII protein level and reduced function within the intrinsic coagulation pathway. These novel findings support further investigation by multiple approaches of FXII contribution to disease phenotype and progression