Hervé Kempf

CLONING AND EXPRESSION PATTERN OF EPAS1 IN THE CHICKEN EMBRYO: COLOCALIZATION WITH TYROSINE HYDROXYLASE

EPAS1 is a hypoxia‐inducible transcription factor, highly expressed in vasculature and recently shown to be necessary for catecholamine production during embryogenesis. We report here the cloning and detailed expression pattern of this factor in the chicken embryo. We show that chicken EPAS1 presents an overall identity of 76% with the human sequence and that it is strongly expressed in the blood vessel wall, mostly in endothelial cells, but also in vascular smooth muscle cells. Moreover, we report non‐vascular expression sites: liver, kidney, and, quite interestingly, cells of the sympathetic nervous system where EPAS1 is coexpressed with one of its putative target genes, the tyrosine hydroxylase. EPAS1 could therefore represent the link between the vascular system and the sympathetic nervous system, both sensitive to hypoxia.

Coexpression of endothelial PAS protein 1 with essential angiogenic factors suggests its involvement in human vascular development.

Endothelial PAS protein 1 (EPAS1) is a bHLH‐PAS transcription factor involved in cellular response to hypoxia. Its precise role in angiogenesis is unclear, but several genes essential to vascular development, including those encoding vascular endothelial growth factor (VEGF), its receptor VEGFR‐2 and Tie2, are thought to be targets of EPAS1. To investigate whether this transcription factor and its putative targets were expressed concomitantly, we performed in situ hybridization on serial adjacent sections of human embryos at gestational ages of 3 to 6 weeks. We studied expression of the genes encoding EPAS1, VEGF, VEGFR‐1, and ‐2, Tie2, and its ligands, angiopoietin (Ang) 1 and 2. We also compared these expression profiles with that of hypoxia‐inducible factor 1α (HIF1α). EPAS1 transcripts were detected in several types of endothelial cell: in blood vessels walls, the endocardium, the glomeruli of the mesonephros, and the sinusoids of the liver. In these endothelial cells, expression of EPAS1 systematically or partly coincided with Tie2 and the VEGF receptors expression. There was also some overlap between the sites of synthesis of EPAS1 and VEGF mRNAs, principally in hepatocytes and sympathetic ganglion cells. In addition, we found that EPAS1 and HIF1α transcripts were often colocalized, suggesting a functional redundancy of these two transcription factors during development. These observations are consistent with transactivation by EPAS1 of the expression of its putative target genes during embryogenesis, suggesting that this transcription factor is involved in human angiogenesis. They provide evidence that EPAS1 is involved in the regulation of vascular maturation, remodeling, or stabilization rather than in the early steps of embryonic angiogenesis.

Molecular cloning, expression and tissue distribution of a chicken angiotensin II receptor

A cDNA encoding a chicken angiotensin II receptor from adrenal gland was isolated to serve as a molecular tool to study the role of AngII in avian embryonic development. This cDNA, sharing a high homology with another avian receptor (turkey), encodes a protein of 359 amino acids with 75% sequence identity with the mammalian type 1 receptor. Transient xpression has revealed pharmacological properties distinct from mammalian receptors and a functional coupling leading to the increase in inositol phosphate production. The AngII receptor mRNA is expressed in classical target organs for AngII (adrenal gland, heart, kidney) and, interestingly, in endothelial cells where it may mediate the peculiar vasorelaxation effect of AngII in the chicken.

Angiopoietin 2 Alters Pancreatic Vascularization in Diabetic Conditions

Aims/hypothesis Islet vascularization, by controlling beta-cell mass expansion in response to increased insulin demand, is implicated in the progression to glucose intolerance and type 2 diabetes. We investigated how hyperglycaemia impairs expansion and differentiation of the growing pancreas. We have grafted xenogenic (avian) embryonic pancreas in severe combined immuno-deficient (SCID) mouse and analyzed endocrine and endothelial development in hyperglycaemic compared to normoglycaemic conditions. Methods 14 dpi chicken pancreases were grafted under the kidney capsule of normoglycaemic or hyperglycaemic, streptozotocin-induced, SCID mice and analyzed two weeks later. Vascularization was analyzed both quantitatively and qualitatively using either in situ hybridization with both mouse- and chick-specific RNA probes for VEGFR2 or immunohistochemistry with an antibody to nestin, a marker of endothelial cells that is specific for murine cells. To inhibit angiopoietin 2 (Ang2), SCID mice were treated with 4 mg/kg IP L1–10 twice/week. Results In normoglycaemic condition, chicken-derived endocrine and exocrine cells developed well and intragraft vessels were lined with mouse endothelial cells. When pancreases were grafted in hyperglycaemic mice, growth and differentiation of the graft were altered and we observed endothelial discontinuities, large blood-filled spaces. Vessel density was decreased. These major vascular anomalies were associated with strong over-expression of chick-Ang2. To explore the possibility that Ang2 over-expression could be a key step in vascular disorganization induced by hyperglycaemia, we treated mice with L1–10, an Ang-2 specific inhibitor. Inhibition of Ang2 improved vascularization and beta-cell density. Conclusions This work highlighted an important role of Ang2 in pancreatic vascular defects induced by hyperglycaemia.

Maturation-dependent neointima formation in fowl aorta

Fowl show spontaneous elevation of blood pressure (BP) and neointimal plaque formation in the abdominal aorta at young ages. Maturation/age-dependent modulation of vascular lesions and a causal relationship between elevated BP and neointima formation, however, have not been clarified. We therefore intended to characterize, first, maturation/age-dependent neointimal plaque formation and vascular lesions and, second, their relationship to BP elevation. The BP measured in conscious domestic fowl, Gallus gallus, White Leghorn breed, DeKalb strain, via an indwelling catheter inserted into the ischiadic artery, increased with maturation in males; and at plateau level, BP (mmHg) was significantly (P<0.01) higher in males (194.0+/-4.6, n=11) than in females (169.3+/-3.1, n=10). Neointimal plaques consisting of neointimal cells and abundant extracellular matrix appeared initially in the distal segment of the abdominal aorta (lesion-prone area) of chicks as early as 6 weeks old. The area (size) of neointimal plaques right above the ischiadic bifurcation increased with maturation, whereas the plaque area became smaller with some degenerative changes in adult birds. In some birds, diffuse subendothelial hyperplasia and more extensive plaque formation at the branching points of the aorta were observed. The plaque area appears to be larger in birds, particularly in males that have higher BP (r=0.68). The width of aortic smooth muscle (SM) layers, measured in regions with no plaque, increased with age, whereas the number of cells per unit of area decreased, suggesting that hypertrophy of vascular SM occurs in response to exposure of the vascular wall to high BP. The number of cells was significantly (P<0.01) higher in the plaque than in underlying aortic SM layers or in layers with no plaque formation. Both neointimal plaques and underlying SM layers are immunohistochemically positive for alpha SM actin, suggesting that neointimal cells are modulated SM cells, whereas the staining with SM myosin heavy chain antibody is low in neointimal plaques. Furthermore, plasma arginine levels dropped in accordance with the time of neointimal plaque formation, whereas plasma cholesterol levels showed an age-dependent increase. The results suggest that spontaneous development of neointimal plaques may be a consequence of exposure to high BP and associated local hemodynamic changes.

Angiotensin receptor(s) in fowl.

The cloning of the avian Ang II receptor shows that it is molecularly close to the AT(1)-type mammalian receptor. However, pharmacological characterization in transfected cells shows that, even though the avian receptor is coupled to the phospholipase C, as is the AT(1), its profile of specificity towards antagonists appears different from that of the two angiotensin II mammalian receptor types. The fowl Ang II receptor mRNA is expressed in classical adult target organs for Ang II and, interestingly, also in endothelial cells, but not in vascular smooth muscle cells. In the endothelial cells, it may mediate the peculiar vasorelaxation effect of Ang II already reported in the chicken. The recent description of the expression pattern in the chick embryo shows that the avian Ang II receptor is expressed in many different mesenchymal tissues, a feature which is the signature of the AT(2) mammalian receptor. Altogether, these data imply that the avian Ang II receptor is an atypical receptor that cannot be readily classified as either of the two mammalian Ang II receptor types and, therefore, reinforce the evidence for another Ang II receptor in the avian class.

Expression of the chicken angiotensin II receptor: atypical pattern compared to its mammalian homologues

We have recently cloned and characterized pharmacologically a chicken angiotensin II receptor (cAT). To evaluate its putative role in developmental processes, we investigated its spatio-temporal distribution in the chicken embryo up to E14. The cAT mRNA is expressed in a developmental manner in the mesonephros and allantois, as well as in the heart, branchial arches or limbs. These results, the first to report the embryonic distribution of an angiotensin receptor in a non-mammalian species, show that its expression pattern does not correspond to either one of the two angiotensin receptor types in mammalian species.

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.

Fibroblast-growth factor 23 promotes terminal differentiation of ATDC5 cells

Objectives Fibroblast Growth Factor 23 (FGF23) is well documented as a crucial player in the systemic regulation of phosphate homeostasis. Moreover, loss-of-function experiments have revealed that FGF23 also has a phosphate-independent and local impact on skeletogenesis. Here, we used ATDC5 cell line to investigate the expression of FGF23 and the role it may play locally during the differentiation of these cells. Methods ATDC5 cells were differentiated in the presence of insulin, and treated with recombinant FGF23 (rFGF23), inorganic phosphate (Pi) and/or PD173074, an inhibitor of FGF receptors (FGFRs). The mRNA expressions of FGF23, FGFRs and markers of hypertophy, Col X and MMP13, were determined by qPCR analysis and FGF23 production was assessed by ELISA. FGFR activation was determined by immunoprecipitation and immunoblotting. Results FGF23 mRNA expression and production were increased during ATDC5 differentiation. At D28 in particular, rFGF23 stimulation increased hypertrophic markers expression, as Col X and MMP13, and mineralization. A synergic effect of Pi and rFGF23 stimulation was observed on these markers and on the mineralization process. The use of PD173074, a pan-FGFR inhibitor, decreased terminal differentiation of ATDC5 by preventing rFGF23 pro-hypertrophic effects. Conclusions Altogether, our results provide evidence that FGF23 plays an important role during differentiation of ATDC5 cell line, by promoting both hypertrophy and mineralization.

Response to: ‘Spontaneous hypertensive rat exhibits bone and meniscus phenotypes of osteoarthritis: is it an appropriate control for MetS-associated OA?’ by Chan and Wen

We thank Dr Chan and Dr Wen for their interest in our report1 and their resulting eLetter.2 We fully agree that, among the different components of metabolic syndrome (MetS), hypertension has very recently been brought out as a critical feature in the development of osteoarthritis (OA) in humans.3 4 In these reports using either data from the Framingham OA study3 or the Osteoarthritis Initiative study,4 it has been emphasised that high blood pressure (diastolic or systolic, respectively) was associated with increased incidence of radiographic knee OA. In order to further experimentally investigate the actual role of hypertension in OA onset and development, Chan and colleagues describe in a yet unpublished study the development of OA features in the spontaneous hypertensive rat (SHR) model, a widely characterised model of systemic hypertension.2 5 Although the spontaneous hypertensive heart failure (SHHF) rat strain we employed is deriving from the SHR strain6 and suffers high …