Yousef P. Barbin

EDB fibronectin and angiogenesis -- a novel mechanistic pathway

Extra domain-B containing fibronectin (EDB+ FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB+ FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-β1, and ET-1 upregulated EDB+ FN expression. Treatment of cells exposed to high glucose with TGF-β1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB+ FN expression. In order to elucidate the functional significance of EDB+ FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB+ FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB+ FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB+ FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.

Endothelin-1 promotes migration and induces elevation of [Ca2+]i and phosphorylation of MAP kinase of a human extravillous trophoblast cell line.

A highly proliferative, migratory and invasive subpopulation of human placental trophoblasts, known as extravillous trophoblasts (EVT), invades the uterus and its vasculature, to establish an adequate exchange of key molecules between the maternal and fetal circulation. Our earlier studies provided evidence for a positive regulation of migration/invasion of EVT by an autocrine factor IGFII and a paracrine, decidua-derived factor IGFBP1. The present study examined the role played by endothelin (ET)-1, also produced at the fetal-maternal interface, and its receptor subtypes ET(A) and ET(B) in the regulation of human EVT cell functions. We utilized an in vitro propagated EVT cell line (HTR-8/SVneo) which exhibits the phenotypic and functional characteristics of EVT in situ. Reverse transcription-PCR with primers specific for prepro-ET-1, ET(A) and ET(B) cDNAs demonstrated the expression of all these genes in HTR-8/SVneo cells. While proliferation was not influenced, migration of these cells through porous Transwell membranes was stimulated by exogenous ET-1. ET-1 also induced biphasic elevation of cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) consisting of an initial transient followed by a sustained plateau, as measured by spectrofluorimetry. The dependence of the Ca(2+) response on phospholipase C (PLC) was demonstrated by its abrogation in the presence of PLC inhibitor U73122. Furthermore, ET-1 treatment of EVT cells rapidly stimulated phosphorylation of MAP kinase (ERK1/2). By using ET receptor antagonists and agonists, it was shown that both ET(A) and ET(B) receptors were responsible for the effects of ET-1 on migration, [Ca(2+)](i) and MAPK phosphorylation. Thus, ET-1 may represent an autocrine/paracrine mediator of invasive trophoblast function.

Contributions of endothelin-1 and sodium hydrogen exchanger-1 in the diabetic myocardium.

Endothelin‐1 (ET‐1) and sodium hydrogen exchanger‐1 (NHE‐1) are important mediators of several disease processes affecting the heart, especially relating to myocardial ischemia. There is evidence that their actions may be interrelated. Their contributions to diabetic heart disease have not been extensively documented. Accordingly, the aim of this study was to investigate the interactive roles of ET‐1 and NHE‐1 in the pathogenesis of diabetic cardiomyopathy, a significant cause of morbidity in diabetic patients.

Pro-oxidant Role of Heme Oxygenase in Mediating Glucose-induced Endothelial Cell Damage

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5 mmol/l) and high (25 mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 μmol/l), HO antagonist (SnPPIX, 10 μmol/l), and NO synthase blocker (l-NAME, 200 μmol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2′-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.

Endothelin-mediated remodeling in aortas of diabetic rats

Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelinA (ETA) receptor, the predominant receptor on smooth muscle cells, in diabetes‐induced vascular hypertrophy and remodeling.

ED-B FIBRONECTIN IN NON–SMALL CELL LUNG CARCINOMA

Fibronectin (FN), a matrix glycoprotein, has been shown to undergo alternative splicing exclusively during organogenesis and tumorigenesis. One such splice variant, extradomain-B (ED-B) FN, is normally absent in normal adult tissues and is proposed to be a marker of tumoral angiogenesis. The present study was aimed at elucidating whether ED-B FN is expressed in non–small cell lung carcinomas and whether such aberrant expression correlates with tumoral angiogenesis. Frozen tissues from 28 non–small cell lung carcinomas (consisting of both squamous cell carcinomas and adenocarcinomas) along with paired normal tissue samples were collected from the tissue bank collection of the Department of Pathology, London Health Sciences Center, Canada. Frozen tissue specimens were subjected to RNA extraction and real time reverse transcriptase–polymerase chain reaction (RT-PCR) with respect to total and ED-B FN isoform expression. In addition, paraffin-embedded tissue sections from the same cases were collected for histological analysis using ED-B FN antibody. Tumor tissues were further stained with CD34 antibody and analyzed semiquantitatively for tumor microvessel density. The results demonstrate up-regulation of ED-B FN mRNA levels in lung tumor tissues as compared to paired normal tissues. Furthermore, ED-B FN expression was localized specifically to tumor cells and was found to correlate with tumor microvessel density. These findings provide evidence of possible involvement of ED-B FN in pulmonary tumoral angiogenesis. Furthermore, ED-B FN may potentially be used as a diagnostic marker and a target for antiangiogenic therapy.

Glucose-induced regulation of novel iron transporters in vascular endothelial cell dysfunction

Increased iron indices have been associated with the development of diabetes and its complications. In the present study, we have investigated the glucose-induced alteration of iron transporters, divalent metal transporter-1 (DMT-1), iron regulated transporter protein-1 (IREG-1), and transferrin receptor (TfR), in endothelial cell iron accumulation and oxidative stress. Cells were exposed to high glucose levels and subjected to gene expression, protein expression, iron measurement and assessment of oxidative stress. Our results show, for the first time, expression of DMT-1 and IREG-1 in vascular endothelial cells. Our data further indicates upregulation of DMT-1 and IREG-1 mRNA and protein in response to high levels of glucose. TfR, however, exhibited a modest decrease in response to high levels of glucose. Increased expression of DMT-1 and IREG-1 was associated with iron accumulation and oxidative stress. Furthermore, our results show differential expression of iron transporters with treatment of high glucose-exposed cells with two different iron chelators. In conclusion, our study suggests that glucose-induced alteration of iron transporters may arbitrate iron accumulation and oxidative stress in endothelial cells.