[Show abstract][Hide abstract] ABSTRACT: Although angiogenesis is viewed as a fundamental component of inflammatory bowel disease (IBD) pathogenesis, we presently lack a thorough knowledge of the cell type(s) involved in its induction and maintenance in the inflamed intestinal mucosa. This study aimed to determine whether platelet (PLT) adhesion to inflamed intestinal endothelial cells of human origin may favour angiogenesis. Unstimulated or thrombin-activated human PLT were overlaid on resting or tumour necrosis factor (TNF)-α-treated human intestinal microvascular endothelial cells (HIMEC), in the presence or absence of blocking antibodies to either vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, integrin α(v)β(3) , tissue factor (TF) or fractalkine (FKN). PLT adhesion to HIMEC was evaluated by fluorescence microscopy, and release of angiogenic factors (VEGF and soluble CD40L) was measured by ELISA. A matrigel tubule formation assay was used to estimate PLT capacity to induce angiogenesis after co-culturing with HIMEC. TNF-α up-regulated ICAM-1, α(v)β(3) and FKN expression on HIMEC. When thrombin-activated PLT were co-cultured with unstimulated HIMEC, PLT adhesion increased significantly, and this response was further enhanced by HIMEC activation with TNF-α. PLT adhesion to HIMEC was VCAM-1 and TF independent but ICAM-1, FKN and integrin α(v)β(3) dependent. VEGF and sCD40L were undetectable in HIMEC cultures either before or after TNF-α stimulation. By contrast, VEGF and sCD40L release significantly increased when resting or activated PLT were co-cultured with TNF-α-pre-treated HIMEC. These effects were much more pronounced when PLT were derived from IBD patients. Importantly, thrombin-activated PLT promoted tubule formation in HIMEC, a functional estimate of their angiogenic potential. In conclusion, PLT adhesion to TNF-α-pre-treated HIMEC is mediated by ICAM-1, FKN and α(v)β(3) , and is associated with VEGF and sCD40L release. These findings suggest that inflamed HIMEC may recruit PLT which, upon release of pro-angiogenic factors, actively contribute to inflammation-induced angiogenesis.
Full-text · Article · Feb 2010 · Journal of Cellular and Molecular Medicine
[Show abstract][Hide abstract] ABSTRACT: Anthracycline-based chemotherapy represents a milestone in the treatment of breast cancer. We previously demonstrated in an in vitro model that cyclin E overexpression is associated with increased expression of manganese superoxide dismutase (MnSOD) and resistance to doxorubicin. In the present study, immunohistochemical expression of cyclin E and MnSOD was evaluated in 134 early breast cancer patients receiving adjuvant epirubicin-based chemotherapy regimens containing epirubicin. Both parameters were correlated with the available clinicopathological parameters and with the outcome of patients. Overexpression of cyclin E and MnSOD was detected in 46 (34.3%) and 56 (41.8%) patients, respectively, and expression levels of the two proteins were related. Disease-free and alive patients displayed a lower mean percentage of cyclin E-expressing cells than relapsed and dead patients, respectively. Kaplan-Meier survival analysis demonstrated a significant separation between high versus low cyclin E-expressing tumors in terms of overall survival (P = 0.038 by log-rank). Similar results were obtained considering the subset of node-negative patients separately. No significant relationship with patient outcome was observed for MnSOD expression levels. At multivariate analysis cyclin E failed to demonstrate an independent prognostic value. In conclusion, the results of the present study support previous evidence that increased cyclin E expression is associated with higher MnSOD expression levels and poorer outcome, at least as evaluated in terms of overall survival. Further studies are warranted to evaluate the usefulness of cyclin E as a prognostic marker to identify breast cancer patients at higher risk of death from the disease when treated with adjuvant anthracycline-based therapy.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a novel component in inflammatory bowel disease (IBD) pathogenesis. We have previously shown that immune-nonimmune interactions through the CD40-CD40-ligand (CD40L) pathway might sustain gut inflammation, although their effect on regulating inflammation-driven angiogenesis is unknown. The present study evaluated the role of the CD40-CD40L interaction in the promotion of immune-mediated angiogenesis in IBD.
Human nonimmune cells of colonic origin-namely, human intestinal fibroblasts (HIFs) and human intestinal microvascular endothelial cells (HIMECs)-were activated with either soluble CD40L (sCD40L), or CD40(+) D1.1 cells or CD40L-activated lamina propria T (LPT) cells before measuring pro-angiogenic cytokine release. Blocking antibodies to either CD40 or CD40L were used to disrupt the CD40-CD40L interaction. The dextran sodium sulphate (DSS) model of experimental colitis in CD40 and CD40L knockout mice was established to assess whether the CD40-CD40L pathway was implicated in controlling inflammation-driven angiogenesis in vivo.
Engagement of CD40 on HIFs promoted the release of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) and hepatocyte growth factor (HGF). LPT cells were potent inducers of pro-angiogenic cytokine secretion by HIFs. Supernatants from sCD40L-activated HIFs induced migration of HIMECs and tubule formation, both of which were inhibited by blocking antibodies to either VEGF, IL-8 or HGF. Both CD40- and CD40L-deficient mice were protected from DSS-induced colitis and displayed a significant impairment of gut inflammation-driven angiogenesis, as assessed by microvascular density.
The CD40-CD40L pathway appears to be crucially involved in regulating inflammation-driven angiogenesis, suggesting that strategies aimed at blocking CD40-CD40L interactions might be beneficial in acute and chronic intestinal injury.
[Show abstract][Hide abstract] ABSTRACT: Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.
Full-text · Article · Aug 2007 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a critical component of neoplastic and chronic inflammatory disorders, but whether angiogenesis also occurs in inflammatory bowel disease (IBD) has yet to be established. We assessed mucosal vascularization, expression of endothelial alphaVbeta3 integrin, angiogenic factors, and their bioactivity in Crohn's disease (CD) and ulcerative colitis (UC) mucosa.
Mucosal endothelium was immunostained for CD31 and factor VIII and quantified by digital morphometry. alphaVbeta3 expression was studied in vivo by confocal microscopy and in vitro by flow cytometric analysis of human intestinal microvascular endothelial cells (HIMECs). Vascular endothelial growth factor (VEGF), interleukin (IL)-8, and bFGF levels were measured in mucosal extracts and cells and angiogenic bioactivity shown by induction of HIMEC migration and the corneal and chorioallantoic membrane angiogenesis assays.
Microvessel density was increased in IBD mucosa. Endothelial alphaVbeta3 was strongly expressed in IBD but only sporadically in normal mucosa and was up-regulated in HIMECs by VEGF, tumor necrosis factor alpha, and bFGF. IBD mucosal extracts induced a significantly higher degree of HIMEC migration than control mucosa, and this response was mostly dependent on IL-8 and less on basic fibroblast growth factor or vascular endothelial growth factor. Compared with normal mucosa, IBD mucosal extracts induced a potent angiogenic response in both the corneal and chorioallantoic membrane assays.
These results provide morphological, phenotypic and functional evidence of potent angiogenic activity in both CD and UC mucosa, indicating that the local microvasculature undergoes an intense process of inflammation-dependent angiogenesis. Thus, angiogenesis appears to be an integral component of IBD pathogenesis, providing the practical and conceptual framework for anti-angiogenic therapies in IBD.
[Show abstract][Hide abstract] ABSTRACT: Abnormalities in the interactions of cells with the extracellular matrix (ECM) play an important role in the development and progression of many types of cancer and are a hallmark of malignant transformation. The dystroglycan (DG) complex is a transmembrane glycoprotein that forms a continuous link from the ECM to the actin cytoskeleton, providing structural integrity and perhaps transducing signal, in a manner similar to integrins. Deregulated expression of DG has been reported in a variety of human malignancies and related to tumor differentiation and aggressiveness. In breast cancer, reduced DG expression has been associated with patient survival and with loss of differentiation of tumor cells. Limited data are available on DG physiology in epithelial cells. In this study, we used the HC11 spontaneously immortalized murine mammary epithelial cells to study DG function(s) and regulation in normal cells. We found that expression of DG protein and mRNA is cell-cycle and cell-density regulated in these cells. Moreover, expression of both DG subunits increased upon lactogenic differentiation of the HC11 cells. The turnover of cell-surface-expressed DG was evaluated in the same cells and half-life of DG subunits was evaluated to be about 12 h. DG-specific small inhibitory RNAs were used to analyze the effects of a reduced expression of DG in these cells. Cells in which DG expression was suppressed were growth inhibited, accumulated in the S-phase of the cell cycle, failed to undergo lactogenic differentiation, and displayed an increase in the percentage of apoptotic cells. Moreover, changes were observed in the expression and/or activity of several molecules involved in cell growth control. These results demonstrate that DG expression is tightly regulated in normal mammary epithelial cells and support the hypothesis that DG is involved in several functions other than structural integrity in these cells. This finding provides new insight into the roles played by DG in epithelial cell physiology and will contribute to our understanding of its involvement in the process of epithelial cell transformation.
No preview · Article · May 2006 · Journal of Cellular Physiology
[Show abstract][Hide abstract] ABSTRACT: Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of inflammatory bowel disease (IBD). Although their etiology is still unknown, the pathogenic mechanisms underlying intestinal inflammation have made impressive progress in our understanding. In particular, the abnormalities underlying IBD pathogenesis are not restricted to those mediated by classical immune cells such as T and B lymphocytes, macrophages and dendritic cells, but also nonimmune cells. Interestingly, endothelium has become one of the major areas of investigation in gut inflammation.
No preview · Article · Jan 2006 · European review for medical and pharmacological sciences