Absence of cell surface expression of human ACE leads to perinatal death.

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).

[Renal tubular dysgenesis and mutations in the renin-angiotensin system genes]. / Dysgénésie tubulaire rénale et mutations des gènes du système rénine angiotensine.

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.

Loss-of-function point mutations associated with renal tubular dysgenesis provide insights about renin function and cellular trafficking.

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.

Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells.

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.

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice.

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.

Atrophy and hypertrophy signalling of the quadriceps and diaphragm in COPD.

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.

Can we live without a functional renin-angiotensin system?

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.

Characterization of the first angiotensin-converting like enzyme in bacteria: Ancestor ACE is already active.

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.

[Neonatal immunology and cord blood transplantation]. / Immunologie néonatale et greffe de sang de cordon.

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.

Allowed and forbidden d-d transitions in poly(3,5-dimethylpyrazolyl)methane complexes of nickel(II).

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.

Characterization of the first non-insect invertebrate functional angiotensin-converting enzyme (ACE): leech TtACE resembles the N-domain of mammalian ACE.

Angiotensin-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood homoeostasis and reproduction in mammals. In vertebrates, both transmembrane and soluble ACE, containing one or two homologous active sites, have been characterized. So far, several ACEs from invertebrates have been cloned, but only in insects. They are soluble and display a single active site. Using biochemical procedures, an ACE-like activity was detected in our model, the leech, Theromyzon tessulatum. Annelida is the most distant phylum in which an ACE activity has been observed. To gain more insight into the leech enzyme, we have developed a PCR approach to characterize its mRNA. The approx. 2 kb cDNA has been predicted to encode a 616-amino-acid soluble enzyme containing a single active site, named TtACE (T. tessulatum ACE). Surprisingly, its primary sequence shows greater similarity to vertebrates than to invertebrates. Stable in vitro expression of TtACE in transfected Chinese-hamster ovary cells revealed that the leech enzyme is a functional metalloprotease. As in mammals, this 79 kDa glycosylated enzyme functions as a dipeptidyl carboxypeptidase capable of hydrolysing angiotensin I to angiotensin II. However, a weak chloride inhibitory effect and acetylated N-acetyl-SDKP (Ac SDAcKP) hydrolysis reveal that TtACE activity resembles that of the N-domain of mammalian ACE. In situ hybridization shows that its cellular distribution is restricted to epithelial midgut cells. Although the precise roles and endogenous substrates of TtACE remain to be identified, characterization of this ancestral peptidase will help to clarify its physiological roles in non-insect invertebrate species.

Catabolic/anabolic balance and muscle wasting in patients with COPD.

BACKGROUND: The mechanisms leading to muscle wasting in patients with COPD are still uncertain. This study was undertaken to evaluate the relationships among circulating levels of catabolic factors (ie, interleukin [IL]-6 and cortisol), anabolic factors (ie, bioavailable testosterone [Tbio], dehydroepiandrosterone sulfate [DHEAS], and insulin-like growth factor [IGF]-I), and mid-thigh muscle cross-sectional area (MTCSA) in patients with COPD. METHODS: Serum levels of the above factors were measured in 45 men with COPD (mean [+/- SEM] FEV(1), 43 +/- 3% predicted; mean age, 67 +/- 1 years) and 16 sedentary healthy men of similar age. MTCSA was quantified using CT scanning. Patients with COPD were subdivided into two groups according to the MTCSA (< 70 or >or= 70 cm(2)). RESULTS: There was a greater prevalence of hypogonadism (ie, Tbio, < 2 nmol/L) in patients with COPD compared to control subjects (22% vs 0%, respectively). Patients with an MTCSA of < 70 cm(2) had significantly reduced levels of DHEAS compared to those in healthy subjects (p < 0.01). IL-6 levels were significantly higher in both subgroups of COPD patients compared to those in control subjects (p < 0.005). The cortisol/DHEAS, IL-6/DHEAS, IL-6/Tbio, and IL-6/IGF-I ratios were significantly greater in COPD patients with an MTCSA of < 70 cm(2) compared to those in control subjects (p < 0.05). The cortisol/DHEAS and IL-6/DHEAS ratios were also significantly greater in COPD patients with an MTCSA of < 70 cm(2) than in COPD patients with an MTCSA of >or= 70 cm(2) (p < 0.05). In a stepwise multiple regression analysis, the IL-6/DHEAS ratio explained 20% of the variance in MTCSA (p < 0.005). CONCLUSION: Catabolic/anabolic disturbances were found in COPD patients leading to a shift toward catabolism and possibly to the development of peripheral muscle wasting.

Central antihypertensive effects of orally active aminopeptidase A inhibitors in spontaneously hypertensive rats.

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.

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B.

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.

IL-7 enhances survival of human CD56bright NK cells.

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.

Inherited renal tubular dysgenesis: the first patients surviving the neonatal period.

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.

Angiotensin-converting enzyme C-terminal catalytic domain is the main site of angiotensin I cleavage in vivo.

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.

Profiling of mRNA expression in quadriceps of patients with COPD and muscle wasting.

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.

Aminopeptidase N during the ontogeny of the chick.

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.