Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus.

We have recently described two kindreds presenting thoracic aortic aneurysm and/or aortic dissection (TAAD) and patent ductus arteriosus (PDA) and mapped the disease locus to 16p12.2-p13.13 (ref. 3). We now demonstrate that the disease is caused by mutations in the MYH11 gene affecting the C-terminal coiled-coil region of the smooth muscle myosin heavy chain, a specific contractile protein of smooth muscle cells (SMC). All individuals bearing the heterozygous mutations, even if asymptomatic, showed marked aortic stiffness. Examination of pathological aortas showed large areas of medial degeneration with very low SMC content. Abnormal immunological recognition of SM-MHC and the colocalization of wild-type and mutant rod proteins in SMC, in conjunction with differences in their coimmunoprecipitation capacities, strongly suggest a dominant-negative effect. Human MYH11 gene mutations provide the first example of a direct change in a specific SMC protein leading to an inherited arterial disease.

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.

Dominant renin gene mutations associated with early-onset hyperuricemia, anemia, and chronic kidney failure.

Through linkage analysis and candidate gene sequencing, we identified three unrelated families with the autosomal-dominant inheritance of early onset anemia, hypouricosuric hyperuricemia, progressive kidney failure, and mutations resulting either in the deletion (p.Leu16del) or the amino acid exchange (p.Leu16Arg) of a single leucine residue in the signal sequence of renin. Both mutations decrease signal sequence hydrophobicity and are predicted by bioinformatic analyses to damage targeting and cotranslational translocation of preprorenin into the endoplasmic reticulum (ER). Transfection and in vitro studies confirmed that both mutations affect ER translocation and processing of nascent preprorenin, resulting either in reduced (p.Leu16del) or abolished (p.Leu16Arg) prorenin and renin biosynthesis and secretion. Expression of renin and other components of the renin-angiotensin system was decreased accordingly in kidney biopsy specimens from affected individuals. Cells stably expressing the p.Leu16del protein showed activated ER stress, unfolded protein response, and reduced growth rate. It is likely that expression of the mutant proteins has a dominant toxic effect gradually reducing the viability of renin-expressing cells. This alters the intrarenal renin-angiotensin system and the juxtaglomerular apparatus functionality and leads to nephron dropout and progressive kidney failure. Our findings provide insight into the functionality of renin-angiotensin system and stress the importance of renin analysis in families and individuals with early onset hyperuricemia, anemia, and progressive kidney failure.

Expression of dual angiogenic/neurogenic growth factors in human primary brain tumors.

Brain tumors, benign or malignant, are characterized by a very high degree of vascularization. Recent accumulating evidence suggests that during development the neuronal wiring follows the same routes as the vasculature and that these two systems may share some of the same factors for guidance. Thus, expression of dual angiogenic/neurogenic growth factors was evaluated by in situ hybridization in human primary brain tumors of three different types, i.e., astrocytomas, oligodendrogliomas, and ependymomas, of increasing grades, in relation with the grade and type of the tumor. For this evaluation we selected vascular endothelial growth factor (VEGF-A) and its receptors VEGF-R1 and VEGF-R2 and the neuropilins 1 and 2 (NRP-1 and NRP-2), which have proangiogenic properties, platelet-derived growth factor (PDGF) receptor-beta (PDGF-Rß), which is required for the functional maturation of blood vessels, the ephrins and their Eph receptors, angiotensinogen (AGT) and thrombospondin-2 (TSP-2), which have potential antiangiogenic properties, and netrin-1 (Net-1), which regulates vascular architecture. We show that the expression of the VEGF-NRP system, PDGF-Rß, TSP-2, AGT, and Net-1 are differentially regulated, either increased or decreased, in relation with the type and grade of the tumor, whereas regulation of the ephrinB system does not seem to be relevant in these human brain tumors.

Enhanced expression of ephrins and thrombospondins in the dermis of patients with early diffuse systemic sclerosis: potential contribution to perturbed angiogenesis and fibrosis.

OBJECTIVE: To determine the skin and fibroblast expression of ephrins (EphB4 and EphrinB2) and thrombospondins (TSPs: TSP1 and TSP2) in patients with SSc. METHODS: All experiments were performed in skin sections and dermal fibroblasts issued from control and clinically involved/non-involved SSc skin biopsies. Dermal fibroblasts were stimulated with hypoxia or TGF-ß, or treated with TGF-ß-neutralizing antibodies. Ephrin and TSP mRNA levels were assessed in skin tissue and dermal fibroblasts by in situ hybridization and quantitative RT-PCR, respectively, and protein levels were assessed by immunohistochemistry and western blots, respectively. RESULTS: Enhanced ephrin and TSP mRNA and protein levels were observed in clinically involved SSc skin. EphrinB2, TSP1 and TSP2 mRNA and protein levels were also up-regulated in non-involved SSc skin. Similar mRNA and protein levels of ephrinB2 and EphB4 were detected in unstimulated and stimulated control and SSc dermal fibroblasts. TSP1 and TSP2 mRNA and protein levels were significantly increased in fibroblasts issued from involved and non-involved SSc skin. This up-regulation was not modified by hypoxic exposure, but was markedly reduced by the addition of TGF-ß-neutralizing antibodies. Stimulation of healthy fibroblasts with TGF-ß significantly increased TSP1 and TSP2 mRNA and protein levels. CONCLUSION: EphB4 and EphrinB2 are up-regulated in clinically involved skin of SSc patients, suggesting their participation in SSc-perturbed angiogenesis. TSP1 and TSP2 are up-regulated in both clinically involved and non-involved SSc skin and are constitutively overexpressed in a TGF-ß-dependent and hypoxia-independent manner in SSc dermal fibroblasts, suggesting their potential early contribution in SSc pathogenesis.

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.

Bone morphogenetic protein-9 is a circulating vascular quiescence factor.

Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-beta receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC(50) (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

Effect of apelin on glomerular hemodynamic function in the rat kidney.

Apelin is a vasoactive peptide identified as the endogenous ligand of an orphan G protein-coupled receptor called APJ. Apelin and its receptor have been found in the brain and the cardiovascular system. Here we show that the apelin receptor mRNA is highly expressed in the glomeruli while its level of expression is lower in all nephron segments including collecting ducts that express vasopressin V2 receptors. Intravenous injection of apelin 17 into lactating rats induced a significant diuresis. Apelin receptor mRNA was also found in endothelial and vascular smooth muscle cells of glomerular arterioles. Apelin administration caused vasorelaxation of angiotensin II-preconstricted efferent and afferent arterioles as shown by an increase in their diameter. Activation of endothelial apelin receptors caused release of nitric oxide which inhibited angiotensin II-induced rise in intracellular calcium. In addition, it appears that apelin had a direct receptor-mediated vasoconstrictive effect on vascular smooth muscle. These results show that apelin has complex effects on the pre- and post glomerular microvasculature regulating renal hemodynamics. Its role on tubular function (if any) remains to be determined.

Angiogenesis and diabetes: different responses to pro-angiogenic factors in the chorioallantoic membrane assay.

Hyperglycemia induces defects in angiogenesis without alteration in the expression of major vascular growth factors in the chicken chorioallantoic membrane (CAM) model. A direct negative effect of hyperglycemia on angiogenesis may participate in failures of "therapeutic angiogenesis" trials. Here, we tested the hypothesis that the response to pro-angiogenic molecules such as angiotensin-converting enzyme (ACE), endothelin-1 (ET-1), and vascular endothelial growth factor-A (VEGF) is altered by hyperglycemia. Transfected (Chinese hamster ovary [CHO] or human embryonic kidney [HEK]) cells overexpressing ACE, ET-1, or VEGF were deposed onto the CAM of hyperglycemic or control embryos. The proangiogenic effect was evaluated 3 d later by angiography and histological analyses. Gene expression in response to these factors was assessed by in situ hybridization. Only VEGF overexpression evoked a proangiogenic response in the CAM from hyperglycemic embryos, upregulating the expression of endogenous VEGF, VEGF-R2, and Tie-2, all of them related to activation of endothelial cells. In conclusion, in a model where hyperglycemia does not alter the major vascular growth factor expression, the negative effect of diabetes on capillary density was overcome only by VEGF overexpression, whereas responses to other vasoactive peptides were practically abolished under hyperglycemic conditions.

Angiotensinogen impairs angiogenesis in the chick chorioallantoic membrane.

Angiotensinogen shares with other members of the serine protease inhibitor (serpin) family antiangiogenic properties. Angiotensinogen inhibits in vitro endothelial cell proliferation, and is antiangiogenic in ovo in the chick chorioallantoic membrane assay. The cellular mode of action of angiotensinogen has been studied by applying purified human angiotensinogen or Chinese hamster ovary cells producing recombinant angiotensinogen onto the developing chorioallantoic membrane. Vessel density of the control and angiotensinogen-treated areas was quantitated by using Sambucus nigra lectin, a specific endothelial cell marker. After 48 h of angiotensinogen treatment by either applying purified angiotensinogen or angiotensinogen-producing Chinese hamster ovary cells, there was a 70% decrease in mesodermic vessel density in comparison to the control sections. Angiotensinogen treatment induced a strong decrease in endothelial cell proliferation of the chorioallantoic membrane vasculature, as shown by incorporation of bromo-deoxyuridine. Two days after local angiotensinogen treatment, increased apoptosis of endothelial cells of mesodermal blood vessels was detected by transferase-mediated deoxyuridine triphosphate nick end labeling assay. As assessed by in situ hybridization, the gene expression pattern of the main vascular growth factors and their receptors was not altered by angiotensinogen. Angiotensinogen, therefore, impairs angiogenesis without altering the expression level of vascular growth factors through the induction of apoptosis and decreased endothelial cell proliferation.

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.

Anti-angiogenic properties of myo-inositol trispyrophosphate in ovo and growth reduction of implanted glioma.

We investigate here the anti-angiogenic properties of the synthetic compound myo-inositol trispyrophosphate (ITPP). By increasing oxy-haemoglobin dissociation, ITPP has the potential to counteract the effects of hypoxia, a critical regulator of angiogenesis and cancer progression. ITPP inhibited angiogenesis of the chorioallantoic membrane (CAM), as analyzed with an original program dedicated to automated quantification of angiogenesis in this model. ITPP also markedly reduced tumor progression and angiogenesis in an experimental model of U87 glioma cell nodules grafted onto the CAM. These results point out the potential of ITPP for the development of a new class of anti-angiogenic and anti-cancer compounds.

Multiple promoters in the WNK1 gene: one controls expression of a kidney-specific kinase-defective isoform.

WNK1 is a serine-threonine kinase, the expression of which is affected in pseudohypoaldosteronism type II, a Mendelian form of arterial hypertension. We characterized human WNK1 transcripts to determine the molecular mechanisms governing WNK1 expression. We report the presence of two promoters generating two WNK1 isoforms with a complete kinase domain. Further variations are achieved by the use of two polyadenylation sites and tissue-specific splicing. We also determined the structure of a kidney-specific isoform regulated by a third promoter and starting at a novel exon. This transcript is kinase defective and has a predominant expression in the kidney compared to the other WNK1 isoforms, with, furthermore, a highly restricted expression profile in the distal convoluted tubule. We confirmed that the ubiquitous and kidney-specific promoters are functional in several cells lines and identified core promoters and regulatory elements. In particular, a strong enhancer element upstream from the kidney-specific exon seems specific to renal epithelial cells. Thus, control of human WNK1 gene expression of kinase-active or -deficient isoforms is mediated predominantly through the use of multiple transcription initiation sites and tissue-specific regulatory elements.

The hematopoietic system: a new niche for the renin-angiotensin system.

The role of the renin-angiotensin system was previously thought to be restricted to the cardiovascular system. It now appears that this system also has important functions in other tissues. Hematopoiesis can be affected by inhibitors of the renin system in patients and in various experimental models. The renin system, particularly angiotensin II, has a role in different stages of hematopoiesis, notably during the first wave in the chick embryo (primitive hematopoiesis) and in the human adult (definitive hematopoiesis). In addition, the renin-angiotensin system in mice is involved in reconstitutive hematopoiesis following experimental irradiation; inhibition of this system improved the hematopoietic recovery in this situation. The clinical relevance and therapeutic applications of these findings offer a new area of clinical research. In this article, we review the evidence for a role for the renin system in the control of hematopoiesis at its different stages.

Steroidogenesis in aldosterone-producing adenoma revisited by transcriptome analysis.

CONTEXT: Primary aldosteronism (PAL) is the most frequent cause of secondary arterial hypertension. In PAL, aldosterone production is chronic, excessive, and autonomous. OBJECTIVE: The objective of this study was to identify the angiotensin-II independent alterations of steroidogenesis responsible for PAL. DESIGN: Genomewide gene expression was compared in two tissues differentiated for aldosterone production, both nonstimulated by circulating angiotensin II and differing in their autonomy to produce aldosterone: aldosterone-producing adenoma (APA) and its adjacent dissected zona glomerulosa (ZG). SETTING: The setting of this study was the Comete Network. PATIENTS: Patients with APA were studied. INTERVENTION: Transcriptome comparison was made of one APA and its adjacent ZG by serial analysis of gene expression; validation by in situ hybridization was performed for 19 genes in 11 samples. OUTCOME: The study outcome was genes differentially expressed in APA and adjacent ZG. RESULTS: Activation of steroidogenesis in PAL is restricted to the overexpression of the enzymes producing aldosterone-specific steroids, aldosterone synthase and also 21-hydroxylase, suggesting that upstream precursor production is not limiting. Increased expression of high-density lipoprotein receptor, adrenodoxin and P450 oxidoreductase suggests that these systems provide cholesterol and electrons to the mitochondrial steroidogenic enzymes. As for acute stimulation of aldosterone production, an activation of calcium signaling is suggested by concordant overexpression of calcium-binding proteins or effectors. Calcium activation may result from an abnormal activity of G(q) protein-coupled receptors. This calcium activation may be the starting point of the other gene expression changes observed in APA. Finally, other differentially expressed genes include three genes encoding unidentified proteins. CONCLUSION: This work provides an original and integrated view of the mechanisms of aldosterone production in PAL.

Hyperglycemia-induced defects in angiogenesis in the chicken chorioallantoic membrane model.

Diabetes is associated with abnormal angiogenesis. Increased angiogenesis contributes to severe forms of diabetic retinopathy, but angiogenesis is decreased in response to myocardial ischemia in diabetic patients. We evaluated the direct effect of hyperglycemia on angiogenesis in the chicken chorioallantoic membrane assay, a model of active neoangiogenesis. Hyperglycemia, lasting up to 7 days, was induced in 7-day-old chick embryos by a single intravitellus glucose injection. Control embryos received either water (volumic control) or mannitol (osmotic control). Hyperglycemia decreased angiogenesis in this model from the 5th day on. The pattern and expression level of the main vascular growth factors' genes were not altered by hyperglycemia, as assessed by in situ hybridization and semiquantitative RT-PCR. As early as 2 days after hyperglycemia was induced, an increased apoptosis of endothelial cells and pericytes was detected by transferase-mediated deoxyuridine triphosphate nick-end labeling assay and electron microscopy. In the meantime, endothelial cell proliferation was decreased, as assessed by incorporation of bromo-deoxyuridine. Hyperglycemia can therefore impair angiogenesis without altering the expression level of vascular growth factors through induction of apoptosis and decreased proliferation of endothelial cells.

Expression of endothelins in human cardiogenesis.

Genetic disruption of endothelin (ET) 1, endothelin-converting enzyme (ECE) 1, and endothelin receptor A (ET(A)) in "knockout" or mutant mouse models result in defects in branchial arch derived craniofacial tissues and in cardiac outflow and great vessel structures. Interestingly, certain types of human congenital cardiovascular malformations such as Catch 22 syndrome and type B interruption of the aortic arch strongly resemble defects seen in knockout animal models. To better address the exact involvement of the ET system in heart formation we explored the spatiotemporal pattern of expression of the components of the ET system during critical phases of cardiogenesis in the human embryo (3-6 weeks of development; Carnegie stages 10-17) by in situ hybridization. We detected high ET-1 mRNA expression in endocardial cells lining the heart outflow tract in the region where the future aortic valves will form. No hybridization signal corresponding to pre-pro-ET-3 was observed in the heart. At the same location, the underlying myocytes express ET(A) mRNA. Whereas a functional role of ET in the valve formation can be proposed because of the simultaneous presence of all the components of the endothelin system (ET-1/ECE-1/ET(A)), this seems not to be the case for the formation of the ventricular septum where endocardial cells do not express ET-1, and only a weak ET(A) hybridization signal was detected in the surrounding myocardium. An abnormal hemodynamism indirectly due to valve malformation may be the indirect cause of this septal defect. The results of this study suggest an important role for the ET system in the formation of certain anatomical structures of the developing human heart.

[Renin-secreting tumour: about a new diagnosed case during pregnancy]. / Tumeur à rénine. A propos d'un nouveau cas diagnostiqué au cours d'une grossesse.

A 29 year-old female patient developed severe arterial hypertension in the beginning of her second pregnancy. Investigations performed at 16 weeks of amenorrhea showed hypokaliemia in relation to severe hyperreninism: plasma active renin was 25 fold normal value, 94% as prorenin (prorenin representing 94% of total renin). Radiological investigations including ultrasonography and MRI disclosed an homogenous and avascular tumor in the right kidney. Its ablation confirmed renin tumor, and allowed recovery from HTA and continuation of pregnancy. This is the 75th reported case in the literature, enabling to make a new statement about diagnostic and therapeutic procedures, which are modified during pregnancy by contra-indication to X-rays and renin-angiotensin-aldosteron axis inhibitors.

2P1, a novel male mouse cDNA specifically expressed during meiosis.

We screened a mouse germinal cell expression library with a probe derived from Sob1, a human testis-specific cDNA, and identified 2P1, a new mouse cDNA. A database search revealed that 2P1 was 91% identical to ORF1 of E3-3, a rat gene probably involved in the regulation of alternative splicing. Sequencing showed that 2P1 has a destabilization motif in its 3'-untranslated region. Northern blotting showed strong gene expression in the testis and weak expression in the epididymis, with no signal detected in other tissues. RT-PCR analysis confirmed testis and epididymis expression. In situ hybridization revealed that 2P1 mRNA was absent in spermatogonia but expressed in spermatocytes. This last result was confirmed by RT-PCR of FACS isolated primary spermatocytes (pachytene stage). Using RT-PCR, purified spermatids were also shown to express 2P1.

A chicken model of pharmacologically-induced Hirschsprung disease reveals an unexpected role of glucocorticoids in enteric aganglionosis.

The enteric nervous system originates from neural crest cells that migrate in chains as they colonize the embryonic gut, eventually forming the myenteric and submucosal plexus. Failure of the neural crest cells to colonize the gut leads to aganglionosis in the terminal gut, a pathological condition called Hirschsprung disease (HSCR) in humans, also known as congenital megacolon or intestinal aganglionosis. One of the characteristics of the human HSCR is its variable penetrance, which may be attributable to the interaction between genetic factors, such as the endothelin-3/endothelin receptor B pathway, and non-genetic modulators, although the role of the latter has not well been established. We have created a novel HSCR model in the chick embryo allowing to test the ability of non-genetic modifiers to alter the HSCR phenotype. Chick embryos treated by phosphoramidon, which blocks the generation of endothelin-3, failed to develop enteric ganglia in the very distal bowel, characteristic of an HSCR-like phenotype. Administration of dexamethasone influenced the phenotype, suggesting that glucocorticoids may be environmental modulators of the penetrance of the aganglionosis in HSCR disease.