Florence Pinet

Modulation of Human Colon Tumor-Stromal Interactions by the Endothelin System

Tumor neovascularization is considered to be a critical step in the development of a malignant tumor. Endothelin (ET)-1 is a powerful vasoconstrictor and mitogenic peptide that is produced by many cancer cell lines. The cellular distribution of the ET components was evaluated in human colon tumors and compared to normal colon. There was more of the ET components (preproET-1, endothelin-converting enzyme-1, and ETA and ETB receptors) in adenomas and adenocarcinomas than in the normal colon. There was overproduction of preproET-1 and endothelin-converting enzyme-1 in carcinoma cells and stromal vessels, suggesting that they are a local source of ET-1. ETA receptors were present in stromal myofibroblasts of neoplastic tissue, and there were large amounts of ETB receptors in the endothelium and myofibroblasts. There was also a redistribution of alpha-smooth muscle actin-positive cells in the vascular structures of tumors. An experimental rat model of induced colon cancer treated for 30 days with bosentan, a mixed antagonist of both ET receptors, confirmed the morphological changes observed during the tumor vascularization. Our data suggest that ET-1 and its receptor play a role in colon cancer progression, with ET-1 functioning as a negative modulator of the stromal response.

Regulation of Renin Secretion and Renin Synthesis by Second Messengers in Isolated Mouse Juxtaglomerular Cells

In this study we have characterized the synthesis and secretion of renin in short-term cultures of isolated mouse renal juxtaglomerular (JG) cells. Our findings suggest that those cultures can be util

Characterization of Precursor and Secreted Forms of Rat Angiotensinogen

Angiotensinogen precursors synthesized by rabbit reticulocyte lysate primed with rat liver RNA were compared with angiotensinogen secreted by rat hepatoma cells and rat hepatocytes using immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of glycosylation with tunicamycin permitted identification of the nonglycosylated form of secreted angiotensinogen. Whereas angiotensinogen secreted by hepatoma cells and hepatocytes showed electrophoretic heterogeneity (mol wt, 52-62 X 10(3], tunicamycin-treated cells secreted only a single angiotensinogen species [mol wt, 48.3 +/- 0.7 X 10(3) (mean +/- SD)], which could be cleaved by renin. Two putative angiotensinogen precursors were synthesized in the reticulocyte lysate: a major protein of 52.5 +/- 1.0 X 10(3) mol wt and a minor protein of 55.7 +/- 1.3 X 10(3) mol wt. Evidence that these proteins represent separate angiotensinogen precursors includes the following. 1) Both proteins were recognized by five different polyclonal antibodies and two monoclonal antibodies. 2) Both proteins increased in parallel in reticulocyte lysates primed with liver RNA from rats nephrectomized and given hormones that increase liver angiotensinogen production. 3) Both proteins were cleaved by renin to produce a single protein of 47.6 +/- 0.8 X 10(3) mol wt. 4) The des-angiotensin I-angiotensinogen generated by renin treatment of the lysate had an electrophoretic mobility identical to that of des-AI-angiotensinogen produced by renin treatment of nonglycosylated angiotensinogen secreted by tunicamycin-treated hepatoma cells and hepatocytes. These studies suggest that rat liver synthesizes two separate angiotensinogen precursors which may differ only in the size of their prepro sequence. The heterogeneity of secreted angiotensinogen can be fully accounted for by differences in N-glycosylation of asparagine residues of the molecule. Glycosylation of angiotensinogen is not essential for its synthesis, processing, and secretion or its hydrolysis by renin.

Effects of glucocorticoids and antiglucocorticoid on angiotensinogen production by hepatoma cells in culture

SummaryAngiotensinogen is synthesized in large amounts by Fao cells derived from the Reuber H35 rat hepatoma in a medium enriched with 5% fetal bovine serum (FBS). Treatment of FBS with dextran-coated charcoal removed endogenous steroids without modifying angiotensinogen production. This treatment allowed the study of the effects of steroids on angiotensinogen production. Hydrocortisone increased the angiotensinogen synthesis in a dosedependent manner. The antiglucocorticoid RU 38486 did not change the basal rate of angiotensinogen production but inhibited the stimulation by hydrocortisone. Similar results were obtained with dexamethasone.Angiotensinogen biosynthesis seems to be regulated by two distinct mechanisms: (a) glucocorticoid independent, controlling the basal rate of angiotensinogen production and (b) glucocorticoid dependent, mediating the increased rate of angiotensinogen production upon glucocorticoid treatment.

The Proteome and Secretome of Human Arterial Smooth Muscle Cell

Smooth muscle cells (SMCs) play a crucial role in cardiovascular diseases. Proteomic analysis using two-dimensional gel electrophoresis (2DE) associated with mass spectrometry allows characterization of the proteome and secretome of human smooth muscle. The presence of a distinct SMC population in the arterial wall implies that under normal conditions, SMCs are phenotypically heterogeneous. Intracellular and secreted proteins from a primary culture of SMCs obtained from patients undergoing coronary bypass surgery were analyzed using 2DE in order to determine their specific features. The 2D reference maps show that SMCs are involved in a wide range of biological functions. They could constitute a useful tool for a wide range of investigators involved in vascular biology, allowing them to investigate SMC protein changes associated with cardiovascular disorders or environmental stimuli.

Proteomic analysis in cardiovascular diseases.

1 Cardiovascular diseases are a major cause of morbidity and mortality in western countries. The molecular mechanisms responsible for heart dysfunction are still largely unknown, except in cases of genetic defects or alteration of genes and proteins. 2 The publication of genome sequences from humans and other species has demonstrated the complexity of biology, including the finding that one gene does not encode for only one protein but for several, due to mRNA splicing and post‐translational modifications. 3 Proteomic analysis can provide an overall understanding of changes in the levels of protein expression. Differential proteomics is a powerful tool for improving our understanding of integrated biochemical responses. The main techniques used are two‐dimensional electrophoresis (2D‐gel) and Surface‐Enhanced Laser Desorption/Ionization Time of Flight (SELDI‐TOF) to separate proteins associated with mass spectrometry. Bioinformatic tools make it possible to compare protein profiles obtained from diverse biological samples. 4 The combination of these approaches has proved to be particularly interesting for studying cardiovascular diseases and thereby improving our understanding of the mechanisms involved and identifying new biochemical factors and biomarkers involved in these diseases.

Renin secretion from malignant pulmonary metastatic tumour cells of vascular origin.

1. A pulmonary chemodectoma/glomangiosarcoma that had metastasized from the thigh was studied after removal from a 22 year old Algerian patient with hypertension, high plasma prorenin and signs of secondary aldosteronism.

Proteomic Analysis of Plasma of Patients with Left Ventricular Remodeling After Myocardial Infarction: Usefulness of SELDI-TOF

SELDI-TOF and depletion of major blood proteins is one of the most promising approaches for accessing low-abundance biomarkers. The use of combinatorial peptide ligand library (CPLL) for selecting the low-abundance proteins and of liquid-phase isoelectric focusing for purifying the corresponding proteins was tested in plasma or serum from patients with myocardial infarction (MI). Here, we describe the SELDI profiling of CPLL-treated plasma to select low-abundance proteins in plasma and the strategy for purification and mass spectrometry identification. This approach shows the potential to select and identify candidate biomarkers in patients with left ventricular remodeling after MI.

Expression and Implication of Clusterin in Left Ventricular Remodeling After Myocardial Infarction

Background: Left ventricular remodeling (LVR) after myocardial infarction is associated with an increased risk of heart failure and death. In spite of a modern therapeutic approach, LVR remains relatively frequent and difficult to predict in clinical practice. Our aim was to identify new biomarkers of LVR and understand their involvement in its development. Methods and Results: Proteomic analysis of plasma from the REVE-2 study (Remodelage Ventriculaire)—a study dedicated to the analysis of LVR which included 246 patients after a first anterior myocardial infarction—identified increased plasma levels of CLU (clusterin) in patients with high LVR. We used a rat model of myocardial infarction to analyze CLU expression in the LV and found a significant increase that was correlated with LVR parameters. We found increased CLU expression and secretion in primary cultures of rat neonate cardiomyocytes hypertrophied by isoproterenol. Silencing of CLU in hypertrophied neonate cardiomyocytes induced a significant decrease in cell size, ANP (atrial natriuretic peptide), and BNP (B-type natriuretic peptide) expression, associated with a decreased ERK (extracellular signal-regulated kinase) 1/2 activity, suggesting a prohypertrophic role of CLU. We then confirmed a significant increase of both intracellular p-CLU (precursor form of CLU) and m-CLU (mature form of CLU) in failing human hearts. Finally, the circulating levels of CLU (secreted form) were increased in patients with chronic heart failure who died from cardiovascular cause during a 3-year follow-up (n=99) compared with survivors (n=99). Conclusions: Our results show for the first time that plasma CLU levels are associated with LVR post–myocardial infarction, have in part a cardiac origin, and are a predictor of early death in heart failure patients.