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Laurent Muller

Publications and source records attributed to Laurent Muller.

14 recordsLinked to original sources
PubMed2006

Extracellular matrix-bound angiopoietin-like 4 inhibits endothelial cell adhesion, migration, and sprouting and alters actin cytoskeleton.

Angiopoietin-like 4 (ANGPTL4) is a secreted protein that belongs to the angiopoietin family and is involved in angiogenesis and metabolism regulation. We previously reported the induction of angptl4 by hypoxia in endothelial cells and in human ischemic tissues from peripheral artery disease. We here observed in a mouse model of hindlimb ischemia that the mRNA upregulation in the vessels correlates with the accumulation of the full-length protein in ischemic tissues. We then investigated its functions in endothelial cells. In response to hypoxia, endogenous ANGPTL4 accumulates in the subendothelial extracellular matrix (ECM). Although the secreted protein undergoes proteolysis leading to truncated fragments present in the medium, only full-length ANGPTL4 interacts with the ECM. Competition and direct binding assays indicate that the strong interaction of ANGPTL4 with the ECM is heparin/heparan sulfate proteoglycan dependent. The balance between matrix-associated and soluble forms of ANGPTL4 points to the role of the ECM in the regulation of its bioavailability. The angiogenic function of the ECM-bound full-length protein was investigated using either the form associated with the conditioned ECM from ANGPTL4-transfected HEK293 cells or the purified immobilized protein. We show that matrix-associated and immobilized ANGPTL4 limit the formation of actin stress fibers and focal contacts in the adhering endothelial cells and inhibit their adhesion. Immobilized ANGPTL4 also decreases motility of endothelial cells and inhibits the sprouting and tube formation. Altogether, these findings show that hypoxic endothelial cells accumulate ANGPTL4 in the ECM, which in turn negatively regulates their angiogenic capacities through an autocrine pathway.

Actins↗
PubMed2006

Akt down-regulates ERK1/2 nuclear localization and angiotensin II-induced cell proliferation through PEA-15.

Angiotensin II (AngII) type 1 receptors (AT1) regulate cell growth through the extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K) pathways. ERK1/2 and Akt/protein kinase B, downstream of PI3K, are independently activated but both required for mediating AngII-induced proliferation when expressed at endogenous levels. We investigate the effect of an increase in the expression of wild-type Akt1 by using Chinese hamster ovary (CHO)-AT1 cells. Unexpectedly, Akt overexpression inhibits the AT1-mediated proliferation. This effect could be generated by a cross-talk between the PI3K and ERK1/2 pathways. A functional partner is the phosphoprotein enriched in astrocytes of 15 kDa (PEA-15), an Akt substrate known to bind ERK1/2 and to regulate their nuclear translocation. We report that Akt binds to PEA-15 and that Akt activation leads to PEA-15 stabilization, independently of PEA-15 interaction with ERK1/2. Akt cross-talk with PEA-15 does not affect ERK1/2 activation but decreases their nuclear activity as a result of the blockade of ERK1/2 nuclear accumulation. In response to AngII, PEA-15 overexpression displays the same functional consequences on ERK1/2 signaling as Akt overactivation. Thus, Akt overactivation prevents the nuclear translocation of ERK1/2 and the AngII-induced proliferation through interaction with and stabilization of endogenous PEA-15.

Angiotensin II↗
PubMed2006

Identification of a novel alternatively spliced variant endothelin converting enzyme-1 lacking a transmembrane domain.

Endothelin-converting enzyme (ECE)-1 cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (ECE-1a, 1b, 1c, and 1d) have been identified to date, they differ only in their amino terminus and share the catalytic domain located in the C-terminal end. In addition to full-length ECE-1 forms, we identified novel, alternatively spliced messenger RNAs (mRNAs) of ECE-1b, 1c, and 1d. These splice variants (SVs) lack exon 3', which codes for the transmembrane (TM) region and is present in full-length forms. SV mRNAs were highly expressed in endothelial cells (EC) derived from macrovascular and microvascular beds. Analyses of ECE-1d and its SV forms in stably transfected human embryonic kidney (HEK)-293 cells revealed that both proteins were recognized by antibodies to C-terminal ECE-1, but an antibody to the N-terminal only bound ECE-1d. The novel protein, designated ECE-1sv, has an apparent molecular weight of 75 kDa. ECE-1sv lacks the TM sequence (or signal peptide) and, therefore, is expected to remain cytosolic. Presence of ECE-1sv in different cellular compartments than the full-length forms of the ECE-1 may suggest a distinct physiologic role for these proteins.

Alternative Splicing↗
PubMed2005

Endothelin-converting enzyme-1, abundance of isoforms a-d and identification of a novel alternatively spliced variant lacking a transmembrane domain.

Endothelin-converting enzyme-1 (ECE-1) cleaves big endothelins, as well as bradykinin and beta-amyloid peptide. Several isoforms of ECE-1 (a-d) have been identified to date; they differ only in their NH(2) terminus but share the catalytic domain located in the COOH-terminal end. Using quantitative PCR, we found ECE-1d to be the most abundant type in several endothelial cells (EC) types. In addition to full-length ECE-1 forms we have identified novel, alternatively spliced mRNAs of ECE-1 b-d. These splice variants (SVs) lack exon 3', which codes for the transmembrane region and is present in full-length forms. SVs mRNA were highly expressed in EC derived from macro and microvascular beds but much less so in other, non-endothelial cells expressing ECE-1, which suggests that the splicing mechanism is cell-specific. Analyses of ECE-1d and its SV form in stably transfected HEK-293 cells revealed that both proteins were recognized by anti COOH-terminal ECE-1 antibodies, but anti NH(2)-terminal antibodies only bound ECE-1d. The novel protein, designated ECE-1 sv, has an apparent molecular mass of 75 kDa; by using site-directed mutagenesis its start site was identified in a region common to all ECE-1 forms suggesting that ECE-1 b-d SV mRNAs are translated into the same protein. In agreement with the findings demonstrating common COOH terminus for ECE-1sv and ECE-1d, both exhibited a similar catalytic activity. However, immunofluorescence staining and differential centrifugation revealed a distinct intracellular localization for these two proteins. The presence of ECE-1sv in different cellular compartments than full-length forms of the enzyme may suggest a distinct physiological role for these proteins.

Alternative Splicing↗
PubMed2005

PAR-1 activation on human late endothelial progenitor cells enhances angiogenesis in vitro with upregulation of the SDF-1/CXCR4 system.

OBJECTIVE: The importance of PAR-1 in blood vessel development has been demonstrated in knockout mice. As endothelial progenitor cells (EPCs) are involved in postnatal vasculogenesis, we examined whether they express PAR-1 and whether stimulation by the peptide SFLLRN modulates their angiogenic properties. METHODS AND RESULTS: EPC expanded from human CD34+ cord blood cells expressed PAR-1. PAR-1 activation induced EPC proliferation in a concentration-dependent manner far more potently than that of human umbilical vein endothelial cells. PAR-1 activation also enhanced actin reorganization, promoting both spontaneous migration in a Boyden chamber assay and migration toward SDF-1 and VEGF. As shown by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), EPC stimulation by SFLLRN significantly enhanced the mRNA expression of SDF-1 and its receptor CXCR-4. PAR-1 activation also increased CXCR4 expression on EPC and induced SDF-1 secretion, leading to autocrine stimulation. PAR-1 stimulation by SFLLRN also increased the formation of capillary-like structures by EPC in Matrigel, and this effect was abrogated by anti-CXCR-4, anti-SDF-1, and MEK inhibitor pretreatment. CONCLUSIONS: Human EPCs express functional PAR-1. PAR-1 activation promotes cell proliferation and CXCR4-dependent migration and differentiation, leading to a proangiogenic effect.

Actin Cytoskeleton↗
PubMed2004

The C-terminal domain of aminopeptidase A is an intramolecular chaperone required for the correct folding, cell surface expression, and activity of this monozinc aminopeptidase.

Aminopeptidase A (APA, EC 3.4.11.7) is a type II integral membrane glycoprotein responsible for the conversion of angiotensin II to angiotensin III in the brain. Previous site-directed mutagenesis studies and the recent molecular modeling of the APA zinc metallopeptidase domain have shown that all the amino acids involved in catalysis are located between residues 200 and 500. The APA ectodomain is cleaved in the kidney into an N-terminal fragment corresponding to the zinc metallopeptidase domain, and a C-terminal fragment of unknown function. We investigated the function of this C-terminal domain, by expressing truncated APAs in Chinese hamster ovary and AtT-20 cells. Deletion of the C-terminal domain abolished the maturation and enzymatic activity of the N-terminal domain, which was retained in the endoplasmic reticulum as an unfolded protein bound to calnexin. Expression in trans of the C-terminal domain resulted in association of the N- and C-terminal domains soon after biosynthesis, allowing folding rescue, maturation, cell surface expression, and activity of the N-terminal zinc metallopeptidase domain. We also show that the C-terminal domain is not required for the catalytic activity of APA but is essential for its activation. Moreover, we show that the C-terminal domain of aminopeptidase N (EC 3.4.11.2, APN) also promotes maturation and cell surface expression of the N-terminal domain of APN, suggesting a common role of the C-terminal domain in the monozinc aminopeptidase family. Our data provide the first demonstration that the C-terminal domain of an eukaryotic exopeptidase acts as an intramolecular chaperone.

Aminopeptidases↗
PubMed2004

N-linked glycosylation of the human bradykinin B2 receptor is required for optimal cell-surface expression and coupling.

To investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHO-K1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cell-surface to a much greater extent than the non-glycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function.

Animals↗
PubMed2003

Hemodynamic and cardiac electrophysiologic effects of lidocaine-bupivacaine mixture in anesthetized and ventilated piglets.

BACKGROUND: The sensory blockade induced by a lidocaine-bupivacaine mixture combines the faster onset of lidocaine and the longer duration of bupivacaine. The current study compared the effects of large doses lidocaine (16 mg/kg), bupivacaine (4 mg/kg), and a mixture of 16 mg/kg lidocaine-4 mg/kg bupivacaine on hemodynamic and cardiac electrophysiologic parameters in anesthetized and ventilated piglets. METHODS: After carotid artery cannulation, a double micromanometer measured mean aortic pressure, left ventricular end diastolic pressure, and the first derivative of left ventricular pressure. Electrocardiogram recording and a bipolar electrode catheter measured RR, PQ, QRS, QT C, JT C, AH, and HV intervals. Lidocaine, bupivacaine, or the mixture was administered intravenously over 30 s, and studied parameters were measured throughout 30 min. RESULTS: Mean aortic pressure decreased in all groups ( P < 0.05). The first derivative of left ventricular pressure was decreased in all groups ( P < 0.001) but to a greater extent with the mixture compared with lidocaine ( P < 0.04). RR, QT C, and JT C intervals were similarly increased in all groups ( P < 0.05). In all groups, PQ, AH, HV, and QRS intervals were widened ( P < 0.001). The lengthening of PQ was greater with bupivacaine ( P < 0.02). The lengthening of AH was greater and delayed with bupivacaine compared with lidocaine ( P < 0.03). The lengthening of HV and the widening of QRS were greater and delayed with bupivacaine ( P < 0.01). The widening of QRS was greater with the mixture than with lidocaine ( P < 0.01). CONCLUSIONS: The alterations of ventricular conduction parameters are greater with 4 mg/kg bupivacaine than with a mixture of 16 mg/kg lidocaine-4 mg/kg bupivacaine, whereas the hemodynamic parameters are similarly altered.

Anesthesia↗
PubMed2003

[Effect of assisted ventilation and position on gastro-esophageal acid reflux after laparotomy].

AIMS: Gastro-esophageal reflux disease (GERD) is frequent in mechanically ventilated patients, often source of hemorragic or respiratory complications. The aim of this study was to determine promoting factors for acid reflux and to define the role of methods of ventilation and body position on reflux. METHODS: Fifteen patients with no history of GERD were included, following scheduled abdominal surgery. Reflux was measured in the proximal and distal esophagus by pH-metry. Events associated with reflux were detected by a videocamera linked to a videocassette recorder synchronized to the pH data logger. Position of the patients was randomized between supine and recumbent 30 degrees. Patients were first placed on mechanical ventilation then breathed spontaneously, determining 2 periods of equal duration during which provocative maneuvers were carried out. RESULTS: Acid reflux was absent in recumbent 30 degrees posture. Acid contact time and number of reflux in supine posture were 0.5 +/- 0.4% and 0.3 +/- 0.2/h respectively with mechanical ventilation and 4 +/- 2.5% and 1 +/- 0.5/h respectively with spontaneous breathing (P<0.05 vs position 30 degrees ) in distal esophagus. Twenty-five% of reflux episodes were associated with an event, mainly cough. CONCLUSION: Acid reflux is rare immediately following laparotomy. Seventy-five per cent of reflux occurred without any detectable event. Reflux was absent in semi recumbency. Mechanical ventilation did not facilitate reflux.

Aged↗
PubMed2002

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease.

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.

Acetylcysteine↗
PubMed2002

Evaluation of antimicrobial therapy management of 120 consecutive patients with secondary peritonitis.

OBJECTIVES: To evaluate antimicrobial therapy management of secondary peritonitis in a University Hospital. PATIENTS AND METHODS: All patients admitted to the intensive care unit of the University Hospital of Nîmes from 1 January 1997 to 31 July 1999 with a diagnosis of secondary peritonitis were retrospectively included. Patients' medical records were collected from the data recordings of the Department of Critical Care and Emergency and the Departments of Surgery. Acute Physiology and Chronic Health Evaluation II (APACHE II) was calculated for each patient at the time of admission. Antimicrobial treatment management before and after the diagnosis of peritonitis was studied. RESULTS: One hundred and twenty patients were included. Results concerning mortality, aetiology of peritonitis and microbiological data were in accordance with previous studies. APACHE II score (P = 0.005), age (P = 0.002), presence of Enterococcus in the peri-operative samples (P = 0.02) and period between diagnosis and surgery (P = 0.04) were predictive of death within 30 days after diagnosis of peritonitis. No significant difference was shown in the mortality rate in patients whose post-operative antibiotic treatment was changed following results of intra-operative peritoneal cultures versus patients having inappropriate treatment (P = 0.96). The same observations were noted for anti-enterococcal treatment. CONCLUSION: This study emphasizes the importance of prompt surgical treatment and shows the modest impact of adapting antibiotic treatment. The morbidity and mortality associated with the presence of Enterococcus, which was not influenced by antibiotic treatment, would seem to suggest the pro-inflammatory role of Enterococcus. However, prospective randomized studies are needed to evaluate the real contribution of enterococcal antibiotic coverage in this context.

Adult↗