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1.
Bull Acad Natl Med ; 198(2): 339-48; discussion 348-9, 2014 Feb.
Article in French | MEDLINE | ID: mdl-26263708

ABSTRACT

Renal tubular dysgenesis is a severe disease characterized by the absence of differentiated proximal tubules, leading to fetal anuria and persistent oligohydramnios. The absence of amniotic fluid results in a series of malformations, including facial dysmorphia, limb deformation and also lung hypoplasia, leading to respiratory distress at birth. The disease is linked to mutations in the AGT, REN ACE andAGTR1 genes that compose the renin-angiotensin system (RAS). The absence of functional RAS leads to fetal and neonatal hypotension, renal hypoperfusion, and tubular dysgenesis. The use of cellular models expressing these mutations has advanced our understanding of the structure-function relationship of RAS proteins, notably by showing that defective misfolded proteins undergo either intracellular accumulation and retention, or rapid degradation. Moreover, these studies confirm that ACE has to be inserted in the plasma membrane to be active.


Subject(s)
Kidney Tubules, Proximal/abnormalities , Mutation , Renin-Angiotensin System/genetics , Urogenital Abnormalities/genetics , Animals , Female , Fetus/abnormalities , Genetic Association Studies , Humans , Pregnancy
2.
Hum Mol Genet ; 23(6): 1479-91, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-24163131

ABSTRACT

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges).


Subject(s)
Fetal Death/genetics , Kidney Tubules, Proximal/abnormalities , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Urogenital Abnormalities/genetics , Animals , CHO Cells , Cricetulus , Crystallography, X-Ray , Female , HEK293 Cells , Humans , Infant, Newborn , Male , Models, Molecular , Mutation, Missense , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/chemistry , Protein Conformation , Protein Structure, Secondary , Protein Transport
3.
Hypertension ; 60(2): 411-8, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22710644

ABSTRACT

Brain renin-angiotensin system hyperactivity has been implicated in the development and maintenance of hypertension. We reported previously in the brain that aminopeptidase A and aminopeptidase N are involved in the metabolism of angiotensin II and angiotensin III, respectively. By using in vivo specific and selective aminopeptidase A and aminopeptidase N inhibitors, we showed that angiotensin III is one of the main effector peptides of the brain renin-angiotensin system, exerting a tonic stimulatory control more than blood pressure in hypertensive rats. Aminopeptidase A, the enzyme generating brain angiotensin III, thus represents a potential target for the treatment of hypertension. We demonstrated here the antihypertensive effects of RB150, a prodrug of the specific and selective aminopeptidase A inhibitor, EC33, in spontaneously hypertensive rats, a model of human essential hypertension. Oral administration of RB150 in conscious spontaneously hypertensive rats inhibited brain aminopeptidase A activity, demonstrating the central bioavailability of RB150 and its ability to generate EC33 into the brain. Oral RB150 treatment dose-dependently reduced blood pressure in spontaneously hypertensive rats with an ED(50) of 30 mg/kg, lasting for several hours. This decrease in blood pressure is partly attributed to a decrease in sympathetic tone, reducing vascular resistance. This treatment did not modify systemic renin-angiotensin system activity. Concomitant oral administration of RB150 with a systemic renin-angiotensin system blocker, enalapril, potentiated the RB150-induced blood pressure decrease achieved in <2 hours. Thus, RB150 may be the prototype of a new class of centrally active antihypertensive agents that might be used in combination with classic systemic renin-angiotensin system blockers to improve blood pressure control.


Subject(s)
Antihypertensive Agents/therapeutic use , Disulfides/therapeutic use , Enzyme Inhibitors/therapeutic use , Glutamyl Aminopeptidase/antagonists & inhibitors , Hypertension/drug therapy , Hypertension/physiopathology , Sulfonic Acids/therapeutic use , Administration, Oral , Animals , Antihypertensive Agents/administration & dosage , Antihypertensive Agents/pharmacology , Blood Pressure/drug effects , Blood Pressure/physiology , Brain/metabolism , Disease Models, Animal , Disulfides/administration & dosage , Disulfides/pharmacology , Dose-Response Relationship, Drug , Enalapril/administration & dosage , Enalapril/pharmacology , Enalapril/therapeutic use , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Glutamyl Aminopeptidase/metabolism , Heart Rate/drug effects , Heart Rate/physiology , Male , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/physiology , Sulfonic Acids/administration & dosage , Sulfonic Acids/pharmacology
4.
Cell Immunol ; 276(1-2): 114-21, 2012.
Article in English | MEDLINE | ID: mdl-22578600

ABSTRACT

Plasmacytoid dendritic cells (PDCs) from human umbilical cord blood (UCB) produce lower amounts of IFN-α upon TLR stimulation compared with adult counterparts. This difference may play a role in the low graft-versus-host disease rate after UCB transplantation and in the impaired immune response of the neonate to pathogens. Comparing UCB PDC to their adults counterparts, we found that they exhibited a mature surface phenotype and a normal antigen uptake. They upregulated costimulatory molecules upon activation, although with delayed kinetics. Protein, but not ARN, levels of TLR-9, MyD88, IRAK1 and IRF-7, involved in the TLR-9 signaling pathway were reduced. The expression levels of miR-146a and miR-155, known to be involved in the post-transcriptional down-regulation of immune responses, were higher. These data point out a post-transcriptional down-regulation of the TLR-9/IRF-7 signaling pathway in UCB PDC.


Subject(s)
Dendritic Cells/immunology , Fetal Blood/immunology , MicroRNAs/genetics , RNA Interference , Signal Transduction , Toll-Like Receptors/immunology , Cell Differentiation , Dendritic Cells/cytology , Down-Regulation , Fetal Blood/cytology , Humans , Phenotype
5.
Dev Cell ; 20(1): 33-46, 2011 Jan 18.
Article in English | MEDLINE | ID: mdl-21238923

ABSTRACT

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.


Subject(s)
Blood Vessels/metabolism , Blood Vessels/pathology , Neovascularization, Pathologic/metabolism , Nerve Tissue Proteins/metabolism , Receptors, Cell Surface/metabolism , Receptors, Immunologic/metabolism , Animals , Antibodies, Blocking/pharmacology , Blood Vessels/drug effects , Blood Vessels/enzymology , Capillary Permeability/drug effects , Enzyme Activation/drug effects , Humans , Ligands , Mice , Models, Biological , Netrin Receptors , Protein Binding/drug effects , Retinal Vessels/drug effects , Retinal Vessels/metabolism , Retinal Vessels/pathology , Signal Transduction/drug effects , Sus scrofa , Vascular Endothelial Growth Factor A/metabolism , src-Family Kinases/metabolism
6.
Hum Mol Genet ; 20(2): 301-11, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21036942

ABSTRACT

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized by persistent fetal anuria and perinatal death. During the systematic screening of mutations of the different genes of the renin-angiotensin system associated with RTD, two missense mutations in the renin gene were previously identified, the first affects one of the two catalytic aspartates (D38N) of renin, and the second, S69Y, is located upstream of the 'flap', a mobile ß-hairpin structure which covers the substrate-binding site of renin. Here we report a novel renin mutation leading to the duplication of the tyrosine residue Y15dup, homologous to Y9 in some other aspartyl proteases, which seems to play a crucial role along the activation pathway. The biochemical and cellular mechanisms underlying renin inactivation were investigated. We expressed prorenin constructs harboring the identified point mutations in two established cell lines, able (AtT-20 cells) or unable (CHO cells) to process prorenin to renin and we evaluated the cellular localization of renin mutants and their functional properties. All three mutants were misfolded to different levels, were enzymatically inactive and exhibited abnormal intracellular trafficking. We suggest a misfolding of Y15dup renin, a partial misfolding of D38N prorenin and a misfolding of S69Y prorenin leading to complete absence of secretion. The structural consequences of the renin mutations were estimated by molecular modeling, which suggested some important structural alterations. This is the first characterization of the mechanisms underlying loss of renin function in RTD.


Subject(s)
Point Mutation , Protein Transport , Renin/genetics , Renin/metabolism , Urogenital Abnormalities/genetics , Urogenital Abnormalities/pathology , Amino Acid Sequence , Animals , CHO Cells , Cell Line , Cricetinae , Cricetulus , Female , Humans , Kidney Tubules, Proximal/abnormalities , Kidney Tubules, Proximal/pathology , Models, Molecular , Molecular Sequence Data , Pregnancy , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Renin/analysis , Renin/chemistry , Sequence Alignment
7.
Thorax ; 65(11): 963-70, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20965933

ABSTRACT

BACKGROUND: Factors involved in the regulation of muscle mass in chronic obstructive pulmonary disease (COPD) are still poorly understood. Comparing the signalisation involved in muscle mass regulation between two muscles with different levels of activation within the same subjects is an interesting strategy to tease out the impact of local (muscle activity) versus systemic factors in the regulation of muscle mass. A study was undertaken to measure and compare the protein levels of p-AKT, AKT, Atrogin-1, p-p70S6K, p-4E-BP1, p-GSK3ß as well as the mRNA expression of Atrogin-1, MuRF1 and FoxO-1 in the quadriceps and the diaphragm of 12 patients with COPD and 7 controls with normal lung function. METHODS: Diaphragm biopsies were obtained during thoracic surgery and quadriceps samples were obtained from needle biopsies. Protein content and mRNA expression were measured by western blot and quantitative PCR, respectively. RESULTS: Increased mRNA expressions of Atrogin-1, MuRF1 and FoxO-1 were found in the quadriceps compared with the diaphragm only in patients with COPD. The quadriceps/diaphragm ratio for MuRF1 was higher in COPD. The protein level of p-p70S6K was decreased in the quadriceps compared with the diaphragm in patients with COPD. The quadriceps/diaphragm ratios of p-p70S6K and p-GSK3ß were lower in patients with COPD than in controls. CONCLUSIONS: These results indicate a greater susceptibility to a catabolic/anabolic imbalance favouring muscle atrophy in the quadriceps compared with the diaphragm in patients with COPD. The balance between the atrophy and hypertrophy signalling is inhomogeneous between respiratory and lower limb muscles, suggesting that local factors are likely to be involved in the regulation of muscle mass in COPD.


Subject(s)
Diaphragm/pathology , Muscular Atrophy/etiology , Pulmonary Disease, Chronic Obstructive/complications , Quadriceps Muscle/pathology , Aged , Biopsy , Diaphragm/metabolism , Female , Forced Expiratory Volume/physiology , Gene Expression Regulation , Humans , Hypertrophy/etiology , Hypertrophy/pathology , Male , Middle Aged , Muscle Proteins/biosynthesis , Muscle Proteins/genetics , Muscular Atrophy/metabolism , Muscular Atrophy/pathology , Muscular Atrophy/physiopathology , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/physiopathology , Quadriceps Muscle/metabolism , RNA, Messenger/genetics , Vital Capacity/physiology
8.
J Immunother ; 33(4): 382-90, 2010 May.
Article in English | MEDLINE | ID: mdl-20386468

ABSTRACT

Lymphoid differentiation and activation critically depend on cytokine stimulation and the interleukin-7 (IL-7) signaling in particular. Although it has been demonstrated that IL-7 may play a role in natural killer (NK) cell maturation, the effect of IL-7 stimulation on mature human NK cells has not been studied. We, therefore, investigated the expression and functional activity of IL-7Ralpha on mature NK populations from adult blood. In this article, we demonstrate that IL-7Ralpha is specifically expressed in the CD56bright noncytotoxic cytokine-producing NK subset. Importantly, this expression is thymus independent, contrary to what is observed in mice. In addition, we show that IL-7Ralpha is expressed at higher levels on NKG2A+CD56bright NK cells. In contrast to IL-15 stimulation, IL-7 does not increase NK cell cytotoxicity, interferon-gamma production, or the expression of activation markers, indicating that these cytokines play different functions in NK homeostasis and activation. However, IL-7 promotes the survival of the CD56bright NK subset and inhibits apoptosis by increasing BCL2 expression. These data should be taken into account when considering the clinical use of IL-7, particularly after stem cell transplantation.


Subject(s)
Interleukin-7/pharmacology , Killer Cells, Natural/drug effects , Lymphocyte Subsets/drug effects , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Receptors, Interleukin-7/metabolism , Animals , Apoptosis/drug effects , CD56 Antigen/biosynthesis , Cell Survival/drug effects , Cells, Cultured , Cytotoxicity, Immunologic/drug effects , Humans , Interferon-gamma/metabolism , Interleukin-15/pharmacology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Killer Cells, Natural/pathology , Lymphocyte Activation/drug effects , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Lymphocyte Subsets/pathology , Mice , NK Cell Lectin-Like Receptor Subfamily C/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Receptors, Interleukin-7/genetics , Thymus Gland/metabolism , Thymus Gland/pathology
9.
COPD ; 5(2): 75-84, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18415806

ABSTRACT

Peripheral muscle wasting is a feature of chronic obstructive pulmonary disease (COPD). Potent therapeutic strategies are needed to improve peripheral muscle mass in these patients. We hypothesized that the evaluation of the mRNA expression profile of quadriceps muscle could be useful in identifying key biochemical pathways involved in the wasting process. We monitored mRNA expression profile of quadriceps muscle in four patients with COPD with muscle atrophy (age: 71.3 +/- 2.1 years, mean SD; FEV(1) 28.3 +/- 10.8 % predicted) and four control subjects (age: 66.5 +/- 1.3 years) using HuU95v2 gene chips. Fifty-seven mRNAs transcripts (0.5%) were found to be differentially expressed in muscles of COPD patients (i.e., p < 0.01). Among them, forkhead box O -1 and -3 and insulin-like growth factor-1 expressions being significantly elevated in COPD subjects. Concomitantly, a significant reduction in mRNA expression of two myofilament proteins was observed. Energy production appears to be impaired as indicated by the significant rise in nicotinamide N-methyltransferase mRNA expression. This study provides for the first time evidence that genes are selectively expressed in limb muscles of COPD patients and further research need to focus on their functional roles in the pathogenesis of muscle dysfunction.


Subject(s)
Muscular Atrophy/metabolism , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Quadriceps Muscle/metabolism , RNA, Messenger/metabolism , Aged , Forced Expiratory Volume , Humans , Male , Microarray Analysis , Middle Aged , Muscular Atrophy/etiology , Oligonucleotide Array Sequence Analysis , Polymerase Chain Reaction , Pulmonary Disease, Chronic Obstructive/complications , Pulmonary Disease, Chronic Obstructive/physiopathology , Vital Capacity
10.
Clin Exp Pharmacol Physiol ; 35(4): 431-3, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18307735

ABSTRACT

1. In mice, inactivation of any of the components of the renin-angiotensin system (i.e. renin, angiotensin-converting enzyme, angiotensinogen and AT1 receptor) is dispensable for survival at birth. Animals can survive although they are more sensitive to salt depletion than the wild type mice. 2. Renal tubular dysgenesis (RTD) is a human disease consisting of severe abnormalities of renal tubular development and resulting in profound anuria and perinatal death. 3. Familial RTD is an autosomal recessive disease due to genetic defects in any of the constituents of the renin system. 4. Complete gene inactivation of the renin system in RTD leads to neonatal anuria and death. Proximal tubules are almost absent; renal artery hyperplasia is found in all cases of RTD. An intense stimulation of renin gene expression is noted in the kidney of patients with mutations affecting angiotensinogen, angiotensin-converting enzyme and AT1 receptor. 5. The more severe phenotype in humans than in mice devoid of a functional renin system may be attributable to the difference in nephrogenesis between mice and humans. In mice, nephrogenesis is completed 2 weeks after birth, whereas in humans it is completed before birth, at 38 weeks of gestation.


Subject(s)
Renin-Angiotensin System/physiology , Animals , Gene Expression Regulation , Humans , Infant, Newborn , Mice , Renin/genetics , Renin/metabolism , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/genetics
11.
Hypertension ; 51(2): 267-74, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18158355

ABSTRACT

Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polypeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration.


Subject(s)
Angiotensin II/biosynthesis , Angiotensin I/metabolism , Catalytic Domain/genetics , Peptidyl-Dipeptidase A/metabolism , Angiotensin I/administration & dosage , Angiotensin I/pharmacology , Angiotensins/blood , Animals , Blood Pressure/drug effects , Bradykinin/blood , Hematocrit , Infusions, Intravenous , Kidney/physiology , Mice , Mice, Mutant Strains , Osmolar Concentration , Peptidyl-Dipeptidase A/genetics , Point Mutation , Renin/blood , Substrate Specificity , Zinc Fingers/genetics
12.
Eur J Pediatr ; 167(3): 311-6, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17443344

ABSTRACT

Renal tubular dysgenesis (RTD) is a clinical disorder either acquired during fetal development or inherited as an autosomal recessive condition. Inherited RTD is caused by mutations in the genes encoding the components of the renin-angiotensin system angiotensinogen, renin, angiotensin-converting enzyme and angiotensin II receptor type 1. Inherited RTD is characterized by early onset oligohydramnios, skull ossification defects, preterm birth and neonatal pulmonary and renal failure. The histological hallmark is the absence or poor development of proximal tubules. So far, all patients died either in utero or shortly after birth. We report the first patients with inherited RTD surviving the neonatal period and still being alive. Genetic and functional analysis of the renin-angiotensin system contributes to the diagnosis of RTD. In conclusion, the clinical diagnosis of inherited RTD is easily missed after birth without renal biopsy or information on affected family members. Genetic and functional analysis of the renin-angiotensin system contributes to correct diagnosis.


Subject(s)
Kidney Tubules/abnormalities , Renin-Angiotensin System/genetics , Adolescent , Adult , Child , Child, Preschool , Consanguinity , DNA Mutational Analysis , Female , Genes, Recessive , Humans , Infant , Infant, Newborn , Male , Oligohydramnios/genetics , Pregnancy
13.
Med Sci (Paris) ; 23(11): 975-9, 2007 Nov.
Article in French | MEDLINE | ID: mdl-18021710

ABSTRACT

The increased susceptibility of human newborns to infections is usually ascribed to the immaturity of the neonatal immune system. The neonatal immune system has never met microbial antigens, and thus the repertoire of its adaptative arm (T and B cells) is entirely pre-immune, or "naïve". However this neonatal pre-immune repertoire is similar to the adult pre-immune repertoire, and cord blood natural killer cells studies show that the innate immunity cells harbor the full killing machinery that characterize mature cells. Moreover, human neonates are able to show an adult-like allogeneic response. Taken together, several lines of evidence suggest that the neonatal immune system, although naïve, is fully mature. However, newborns display phenotypic and functional differences with adults in both adaptative and innate arms. Specific properties may explain these differences, as high number of regulatory T cells, low plasmacytoid dendritic cell response to stimuli and high IL-10 production. These properties are in line with the high susceptibility of newborns to infections and the low incidence of graft-versus-host-disease after cord blood transplantation. To explain these differences, we introduce a new model. Although naive, the neonatal immune system is mature, and these functional differences are due to a message originating from the placenta and aimed at inducing the foetus tolerance to its mother. Full understanding of the involved mechanisms will help to protect the newborn against infections and to improve cord blood transplantation outcome.


Subject(s)
Cord Blood Stem Cell Transplantation , Infections/immunology , Infections/therapy , Humans , Immune Tolerance , Infant, Newborn
14.
J Clin Invest ; 117(7): 1914-25, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17607364

ABSTRACT

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.


Subject(s)
Cardiovascular Diseases/metabolism , Cardiovascular Diseases/pathology , Hypertension/metabolism , Hypertension/mortality , Receptor, Angiotensin, Type 1/metabolism , Angiotensins/metabolism , Animals , Asparagine/genetics , Asparagine/metabolism , Blood Pressure , Cardiovascular Diseases/genetics , Cardiovascular Diseases/physiopathology , Disease Progression , Female , Fibrosis/metabolism , Fibrosis/pathology , Gene Expression Regulation , Hyperaldosteronism/complications , Hyperaldosteronism/metabolism , Hyperaldosteronism/pathology , Hypertension/genetics , Hypertension/physiopathology , Kidney/physiology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Receptor, Angiotensin, Type 1/genetics , Renin/blood , Signal Transduction
15.
Gene ; 399(1): 81-90, 2007 Sep 01.
Article in English | MEDLINE | ID: mdl-17597310

ABSTRACT

Angiotensin-converting enzyme (ACE) is a metallopeptidase that converts angiotensin I into angiotensin II. ACE is crucial in the control of cardiovascular and renal homeostasis and fertility in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two active sites, have been characterized. So far, only soluble, single domain ACEs from invertebrates have been cloned, and these have been implicated in reproduction in insects. Furthermore, an ACE-related carboxypeptidase was recently characterized in Leishmania, a unicellular eukaryote, suggesting the existence of ACE in more distant organisms. Interestingly, in silico databank analysis revealed that bacterial DNA sequences could encode putative ACE-like proteins, strikingly similar to vertebrates' enzymes. To gain more insight into the bacterial enzymes, we cloned the putative ACE from the phytopathogenic bacterium, Xanthomonas axonopodis pv. citri, named XcACE. The 2 kb open reading frame encodes a 672-amino-acid soluble protein containing a single active site. In vitro expression and biochemical characterization revealed that XcACE is a functional 72 kDa dipeptidyl-carboxypeptidase. As in mammals, this metalloprotease hydrolyses angiotensin I into angiotensin II. XcACE is sensitive to ACE inhibitors and chloride ions concentration. Variations in the active site residues, highlighted by structural modelling, can account for the different substrate selectivity and inhibition profile compared to human ACE. XcACE characterization demonstrates that ACE is an ancestral enzyme, provoking questions about its appearance and structure/activity specialisation during the course of evolution.


Subject(s)
Bacterial Proteins/chemistry , Peptidyl-Dipeptidase A/chemistry , Xanthomonas axonopodis/enzymology , Amino Acid Sequence , Angiotensin I/chemistry , Angiotensin II/chemistry , Angiotensin-Converting Enzyme Inhibitors/pharmacology , Bacterial Proteins/genetics , Cloning, Molecular , Computational Biology , Genome, Bacterial/genetics , Molecular Sequence Data , Peptidyl-Dipeptidase A/classification , Peptidyl-Dipeptidase A/genetics , Phylogeny , Protein Conformation , Sequence Homology, Amino Acid , Structure-Activity Relationship , Xanthomonas axonopodis/genetics
16.
Differentiation ; 74(2-3): 119-28, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16533310

ABSTRACT

Little is known about the production and function of metallopeptidases in embryonic development. One such enzyme, aminopeptidase N (APN), is present in several epithelia, the brain and angiogenic vessels in adults. APN promotes vascular growth and endothelial cell proliferation in physiological and pathological models of angiogenesis. However, its possible role in embryonic angiogenesis or other developmental processes is unknown. Its expression profile in the early phase of embryonic development has not been reported. We report here the expression of this enzyme during the early development of the chick embryo, using complementary techniques for monitoring APN mRNA, protein, and enzymatic activity. We detected APN in the embryo as early as gastrulation. In addition to the known sites of APN production identified in both adults and rat fetuses toward the end of gestation, APN was found in unexpected sites, such as the primitive streak, the dorsal folds of the neural tube, the somites, and the primordia of several organs. APN was present mostly in the cardiovascular compartment during the first 13 days of incubation, and in the hematopoietic compartment (yolk sac and aorta-gonad-mesonephros region) early in development. This study provides clues as to the possible role of APN in embryonic development.


Subject(s)
CD13 Antigens/metabolism , Embryonic Development , Animals , CD13 Antigens/analysis , CD13 Antigens/genetics , Chick Embryo , Gastrula/enzymology , In Situ Hybridization , Neovascularization, Physiologic , RNA, Messenger/analysis , RNA, Messenger/biosynthesis
17.
Photochem Photobiol ; 82(1): 57-63, 2006.
Article in English | MEDLINE | ID: mdl-16180948

ABSTRACT

Absorption spectra of four nickel(II) complexes with poly(pyrazolyl)methane ligands are presented in the NIR-VIS-UV region and the band system corresponding to the lowest-energy spin-allowed and spin-forbidden transitions is analyzed. A quantitative theoretical model involving coupled electronic states provides precise energies for the lowest-energy triplet and singlet excited states and allows comparisons between complexes with a variable number of nitrogen and oxygen ligator atoms. Singlet energies between 12,840 and 13,000 cm(-1) are determined for heteroleptic complexes. These energies are in an intermediate range between those for homoleptic complexes with either nitrogen or oxygen ligator atoms with singlet states at approximately 12,000 and 14,000 cm(-1), respectively. The new theoretical approach is compared to the traditional ligand-field parameters obtained from the maxima of the broad, spin-allowed absorption bands.

19.
Hypertension ; 46(5): 1169-74, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16203874

ABSTRACT

Human epidemiological studies have shown that low birth weight is associated with hypertension in adulthood. Rodent models of intrauterine growth retardation (IUGR) support these findings because offspring from undernourished dams develop hypertension. Angiotensin-converting enzyme 2 (ACE2) is a newly described renin-angiotensin system (RAS) component that competes with ACE for angiotensin peptide hydrolysis and therefore may modulate blood pressure. However, ACE2 potential participation in hypertension programming remains unknown, although RAS alterations were reported in IUGR models. Hence, we first investigated the tissue distribution of ACE2 and ACE in the rat and then whether hypertension programming differentially affects both enzymes. Using multiplex RT-PCR and in situ hybridization, we show that ACE2 mRNA is widely expressed and coregionalized with ACE. Moreover, tissues involved in blood pressure homeostasis (lung, heart, and kidney) express high levels of both enzymes. Enzymatic assays reveal that ACE2 and ACE are coactive in these tissues. Adult (4-month-old) offspring from 70% food-restricted dams throughout gestation (FR30 rats) present mild hypertension, impaired renal morphology, as well as elevated plasma angiotensin II and aldosterone, suggesting alterations of the systemic RAS. In FR30 rats, we show that ACE2 and ACE activities are increased only in the lung, whereas their mRNA expression is not significantly altered, showing that the enzymes display tissue-specific sensitivity to programming. Our results indicate that ACE2 and ACE are coexpressed in numerous rat tissues and that their increased activity in the lung of FR30 rats may participate in hypertension programming.


Subject(s)
Carboxypeptidases/metabolism , Hypertension/enzymology , Hypertension/etiology , Lung/enzymology , Malnutrition/complications , Peptidyl-Dipeptidase A/metabolism , Pregnancy Complications , Aldosterone/blood , Angiotensin II/blood , Angiotensin-Converting Enzyme 2 , Animals , Animals, Newborn/growth & development , Animals, Newborn/metabolism , Blood Pressure , Corticosterone/blood , Disease Susceptibility , Electrolytes/urine , Female , Hypertension/pathology , Hypertension/physiopathology , Kidney/metabolism , Kidney/pathology , Male , Malnutrition/blood , Pregnancy , Pregnancy Complications/blood , Rats , Rats, Inbred WKY , Renin/blood , Renin/metabolism , Tissue Distribution
20.
Int J Cancer ; 116(3): 378-84, 2005 Sep 01.
Article in English | MEDLINE | ID: mdl-15800942

ABSTRACT

Like most extracellular matrix (ECM) components, fibronectin (Fn) is proteolyzed generating specific activities. Fibronectin proteinase (Fn-proteinase) represents such a cryptic activity located in the gelatin-binding domain (GBD) of Fn and displays a zinc metalloproteinase activity. The migration-stimulating factor (MSF) is a truncated Fn isoform generated by alternative mRNA splicing and corresponds to the N-terminal part of Fn that comprises the GBD. We show that several human mammary epithelial cells express MSF and constitutively produce Fn-proteinase activity. Furthermore, recombinant MSF produced by HEK-293 and MCF-7 cells possesses a constitutive Fn-proteinase activity. Mutating the putative zinc-binding motif, HEXXH, of the protein abolishes its activity thereby demonstrating its specificity. Using PCR, we showed that MSF is barely expressed in normal breast tissues, whereas its expression is significantly increased in tumors. Furthermore, an association between MSF expression and invasive capacity is observed in various breast adenocarcinoma cell lines. Indeed, when stably transfected in non-invasive MCF-7 cells, MSF promotes cell migration in a mechanism mostly dependent on its Fn-proteinase activity. In summary, our study shows that: (i) MSF displays constitutive Fn-proteinase activity; (ii) MSF expression is induced in human breast cancer; and (iii) MSF confers pro-migratory activity that depends mostly on its Fn-proteinase activity. These results suggest that MSF may be involved in tumor progression.


Subject(s)
Aminopeptidases/physiology , Breast Neoplasms/pathology , Cell Movement , Cytokines/pharmacology , Fibronectins/physiology , Neoplasm Invasiveness/physiopathology , Alternative Splicing , Catalysis , Humans , Polymerase Chain Reaction , Protein Isoforms , Tumor Cells, Cultured
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