Endoglin is required in Pax3-derived cells for embryonic blood vessel formation.

Mutations in endoglin, a TGFß/BMP coreceptor, are causal for hereditary hemorrhagic telangiectasia (HHT). Endoglin-null (Eng-/-) mouse embryos die at embryonic day (E)10.5-11.5 due to defects in angiogenesis. In part, this is due to an absence of vascular smooth muscle cell differentiation and vessel investment. Prior studies from our lab and others have shown the importance of endoglin expression in embryonic development in both endothelial cells and neural crest stem cells. These studies support the hypothesis that endoglin may play cell-autonomous roles in endothelial and vascular smooth muscle cell precursors. However, the requirement for endoglin in vascular cell precursors remains poorly defined. Our objective was to specifically delete endoglin in neural crest- and somite-derived Pax3-positive vascular precursors to understand the impact on somite progenitor cell contribution to embryonic vascular development. Pax3Cre mice were crossed with Eng+/- mice to obtain compound mutant Pax3(Cre/+);Eng+/- mice. These mice were then crossed with homozygous endoglin LoxP-mutated (Eng(LoxP/LoxP)) mice to conditionally delete the endoglin gene in specific lineages that contribute to endothelial and smooth muscle constituents of developing embryonic vessels. Pax3(Cre/+);Eng(LoxP/)(-) mice showed a variety of vascular defects at E10.5, and none of these mice survived past E12.5. Embryos analyzed at E10.5 showed malformations suggestive of misdirection of the intersomitic vessels. The dorsal aorta showed significant dilation with associated vascular smooth muscle cells exhibiting disorganization and enhanced expression of smooth muscle differentiation proteins, including smooth muscle actin. These results demonstrate a requirement for endoglin in descendants of Pax3-expressing vascular cell precursors, and thus provides new insight into the cellular basis underlying adult vascular diseases such as HHT.

The Nrarp gene encodes an ankyrin-repeat protein that is transcriptionally regulated by the notch signaling pathway.

We have identified a gene encoding a novel protein that is transcriptionally regulated by the Notch signaling pathway in mammals. This gene, named Nrarp (for Notch-regulated ankyrin-repeat protein), encodes a 114 amino acid protein that has a unique amino-terminus and a carboxy-terminal domain containing two ankyrin-repeat motifs. A Xenopus homolog of the Nrarp gene was previously identified in a large-scale in situ hybridization screen of randomly isolated cDNA clones. We demonstrate that in T-cell and myoblast cell lines expression of the Nrarp gene is induced by the intracellular domain of the Notch1 protein, and that this induction is mediated by a CBF1/Su(H)/Lag-1 (CSL)-dependent pathway. During mouse embryogenesis, the Nrarp gene is expressed in several tissues in which cellular differentiation is regulated by the Notch signaling pathway. Expression of the Nrarp gene is downregulated in Notch1 null mutant mouse embryos, indicating that expression of the Nrarp gene is regulated by the Notch pathway in vivo. Thus, Nrarp transcript levels are regulated by the level of Notch1 signaling in both cultured cell lines and mouse embryos. During somitogenesis, the Nrarp gene is expressed in a pattern that suggests that Nrarp expression may play a role in the formation of somites, and Nrarp expression in the paraxial mesoderm is altered in several Notch pathway mutants that exhibit defects in somite formation. These observations demonstrate that the Nrarp gene is an evolutionarily conserved transcriptional target of the Notch signaling pathway.

Notch signaling is essential for vascular morphogenesis in mice.

The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generated Notch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the related Notch1 gene. Embryos homozygous for mutations of both the Notch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. Both Notch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.

A novel angiotensin analog with subnanomolar affinity for angiotensin-converting enzyme.

This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys(11)), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of (125)I-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that (125)I-angiotensinogen 3-11(Lys(11)) bound a single high-affinity site in the presence of EDTA exhibiting a K(d) of 0.15 +/- 0.02 nM with a B(max) = 4295 +/- 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: (125)I-angiotensinogen 3-11(Lys(11)) >> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked (125)I-angiotensinogen 3-11(Lys(11)) specifically bound a protein of M(r) 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of (125)I-angiotensinogen 3-11(Lys(11)) and the ACE inhibitor, (125)I-351A, were also compared. These experiments demonstrated that the binding distributions of (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A binding. These results demonstrated that the binding site for (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A copurified. These experiments indicate that the novel angiotensin I analog, (125)I-angiotensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.

Angiotensin IV AT4-receptor system in the rat kidney.

Angiotensin IV, [[des-Asp1,Arg2]ANG II or ANG-(3-8)], has been shown to preferentially bind to a novel angiotensin binding site (AT4 receptor). The cellular location and function of this receptor in the rat kidney is unknown. Autoradiography localized AT4 receptors to the cell body and apical membrane of convoluted and straight proximal tubules in the cortex and outer stripe of the outer medulla. ANG IV (0.1 pM-1 microM) elicited a concentration-dependent decrease in transcellular Na+ transport (as measured by proximal tubule O2 consumption rates) in fresh suspensions of control or nystatin-stimulated (bypasses rate-limiting step of apical Na+ entry) rat proximal tubules. The inhibitory effect of 1 pM ANG IV was unaltered by either 1 microM losartan (AT1-receptor antagonist) or 1 microM PD-123319 (AT2-receptor antagonist) and yet was abolished by 1 microM divalinal-ANG IV (AT4-receptor antagonist) or ouabain pretreatment. These results demonstrate that the kidney AT4-receptor system is localized to the proximal tubule and suggests that one potential biological role of this system is in the regulation of Na+ transport by inhibiting a ouabain-sensitive component of Na(+)-K(+)-adenosinetriphosphatase activity in the rat.

Autoradiographic identification of kidney angiotensin IV binding sites and angiotensin IV-induced renal cortical blood flow changes in rats.

The present investigation initially determined that specific binding sites for the hexapeptide angiotensin IV (AngIV) are present in the rat kidney cortex and outer medulla but not in the inner medulla, using in vitro autoradiographic techniques. This binding site has been termed AT4, is distinct from the previously characterized AT1 and AT2 sites, and does not bind the specific AT1 receptor antagonist DuP753 or the AT2 receptor antagonist PD123177. Renal artery infusions of AngIV produced a dose-dependent increase in cortical blood flow without altering systemic blood pressure. In contrast, the infusion of angiotensin II (AngII) induced a dramatic decrease in cortical blood flow, accompanied by a significant elevation in systemic blood pressure. The infusion of [D-Val(1)]AngIV, an analog that does not bind at the AT4 receptor site, and the C-terminal truncated analogs AngIV (1-4) and AngIV (1-5) that possess lower affinity for this site, produced no change in cortical blood flow. The infusion of [Nle1]AngIV and [Lys1]AngIV, analogs that bind with high affinity at the AT4 receptor site, produced increases in cortical blood flow with no influence on blood pressure. Pretreatment with a specific AT4 receptor antagonist, Divalinal-AngIV, completely blocked AngIV-induced elevations in blood flow, but failed to influence AngII-induced decreases in blood flow, suggesting that these ligands are acting at different receptor sites. Pretreatment with the nitric oxide synthase inhibitor, NG-Monomethyl-L-Arginine, also blocked subsequent AngIV-induced increases in cortical blood flow. These data support the notion that AngIV exerts a unique influence upon renal hemodynamics via the AT4 receptor subtype, and suggest that AngIV-induced elevations in blood flow may be mediated by nitric oxide.

Characterization of the binding properties and physiological action of divalinal-angiotensin IV, a putative AT4 receptor antagonist.

Divalinal-Ang IV [V psi (CH2-NH2)YV psi (CH2-NH2)HPF] is being employed increasingly as a specific AT4 antagonist. This use, which necessitates a comprehensive physiological and pharmacological evaluation of Divalinal-Ang IV's functional and receptor binding characteristics in order to ensure its efficacy and specificity, was the stimulus for this study using bovine adrenal membranes. [125I]Ang IV and [125I]Divalinal-Ang IV were shown to bind with high affinity to a similar number of binding sites, suggesting that both bound the same receptor. This notion was verified by competition curves using [125I]Ang IV and [125I]Divalinal-Ang IV that indicated identical rank order affinities for several angiotensin-related peptides and 100% cross-displacement by Ang IV and Divalinal-Ang IV. Furthermore, an autoradiographic comparison of [125I]Ang IV and [125I]Divalinal-Ang IV in 20 microns sections of bovine adrenals revealed near identical binding distributions characterized by heavy binding in the glomerulosa layer and the medulla. Physiological studies in which test compounds were injected into the internal carotid of the rat and cerebral blood flor (CBF) was measured by laser Doppler flowmetry indicated that pretreatment with Divalinal-Ang IV, but not DuP 753 or PD123177, blocked the increased flow observed with Ang IV infusion. Conversely, DuP 753, but not Divalinal-Ang IV or PD123177, inhibited the decrease in flow witnessed with Ang II. Metabolic stability studies utilizing rat kidney homogenates as a peptidase source, demonstrated that the structural changes present in Divalinal-Ang IV greatly increased its resistance to metabolism as compared to Ang IV. Together, these studies show that Divalinal-Ang IV is a stable, efficacious and specific inhibitor of AT4 receptors.

Autoradiographic identification of brain angiotensin IV binding sites and differential c-Fos expression following intracerebroventricular injection of angiotensin II and IV in rats.

A unique angiotensin binding site specific for the hexapeptide, angiotensin II(3-8) (AngIV), has been previously reported by our laboratory in the guinea pig brain and is presently described in the rat brain. This angiotensin receptor subtype has been termed AT4 and is prominently distributed in cerebral cortex, piriform cortex, hippocampus, habenulae, colliculi, septum, periaqueductal gray, several thalamic nuclei, the arcuate nucleus of the hypothalamus and cerebellum. In the second part of the present investigation, separate groups of rats received i.c.v. injections of angiotensin II (AngII), AngIV or artificial cerebrospinal fluid (aCSF) and were euthanized 2 h later for the purpose of evaluating for brain c-Fos expression. After i.c.v.-injected AngIV, Fos-like immunoreactivity was present in the hippocampus and piriform cortex. This immunoreactivity was unaffected by i.c.v. pretreatment with the AT1 angiotensin receptor antagonist DuP 753 (losartan) or the AT2 receptor ligand PD123177 but was blocked by the AT4 angiotensin receptor antagonist, divalanal-AngIV. I.c.v. injection of AngII resulted in Fos-like immunoreactivity in the dorsal third and lateral ventricles, subfornical organ, lateral hypothalamus and amygdala. Pretreatment with losartan or PD123177 significantly interfered with this AngII-induced immunoreactivity while divalanal-AngIV did not. These results indicate that in both guinea pig and rat brains the AT4 receptor has a distribution different than that previously reported for AT1 and AT2 receptor subtypes. The c-Fos expression results suggest that different brain neuronal pathways are activated by i.c.v. injection of AngII and AngIV.

The angiotensin IV system: functional implications.

The brain renin-angiotensin system has been implicated in the central regulation of the cardiovascular system, body water balance, and cyclic regulation of reproductive hormones and behaviors. It also exerts some influence over the secretion of pituitary hormones. This system appears to be complete with the necessary precursors and enzymes for the formation and degradation of biologically active forms of angiotensins and several binding subtypes that are presumed to mediate these and other functions. Much information is now available on the AT1 site which preferentially binds angiotensin II (AngII), but also binds angiotensin III (AngIII), and appears to be responsible for mediating the above described classic angiotensin physiologies and behaviors. Less is known about the functional importance of the AT2 site which also binds AngII but preferentially binds AngIII. This site has been implicated in vascular growth and cerebral blood flow. Recently, an AT4 site has been discovered and characterized that preferentially binds AngII (3-8), a fragment of AngII referred to as angiotensin IV (AngIV). This AT4 site is prominent in cerebral cortex, hippocampus, basal ganglia, cerebellum, and spinal cord, as well as several peripheral tissues including kidney, bladder, heart, spleen, prostate, adrenals, and colon. The AT4 site may mediate memory acquisition and recall and the regulation of blood flow. The function(s) of the AT4 receptor subtype in peripheral tissues is currently unknown, although it does appear to be involved in kidney blood flow.

AT4 receptors: specificity and distribution.

The AT4 receptor specifically binds Angiotensin (Ang) IV and is distinct from the AT1 and AT2 receptors which bind Ang II and Ang III. The AT4 receptor in bovine adrenal cortex has a Kd of 0.74 +/- 0.14 nM and a Bmax of 3.82 +/- 1.12 pmol/mg prot. Competition curves demonstrated the following rank order of affinity: Ang IV >> Ang III >> d-Arg - Ang II >> Sar1,Ile8 - Ang II = Ang II = Ang II (1-7) >> DuP753 = CGP42112A = PD 123177. AT4 receptors were present in many tissues from several mammalian species including human and monkey. AT4 and AT1/AT2 receptors revealed a differential distribution in the rat kidney.

AT4 receptor binding characteristics: D-amino acid- and glycine-substituted peptides.

The ability of angiotensin IV (AIV) analogs to compete for [125I]AIV binding in heat-treated bovine adrenal membranes was examined. Angiotensin IV displayed a Ki of 2.63 +/- 0.12 nM. Peptides containing mono-substitutions with glycine or the corresponding D-amino acid in positions one, two, or three possessed K(i)s greater than 100 nM. Conversely, substitutions at positions four, five, and six produced peptides with Kis less than 8 nM. These data suggest that the N-terminal domains of the AIV peptide are critical for receptor binding, while the C-terminal domains play a less decisive role in receptor specificity.

Elucidation of a specific binding site for angiotensin II(3-8), angiotensin IV, in mammalian heart membranes.

Data are presented describing a new angiotensin binding site in rabbit and guinea pig heart, distinct from AT1 and AT2, that demonstrates high specificity and affinity for the hexapeptide fragment angiotensin II(3-8), which will be referred to here as angiotensin IV (AIV). Equilibrium binding in rabbit heart membranes was achieved in 2 hr at 37 degrees C and produced a calculated kinetic KD of .174 +/- .018 nM. Saturation equilibrium binding data for rabbit and guinea pig heart were best fit to a one-site model with Hill coefficients near unity. Guinea pig membranes exhibited a KD = 1.33 +/- .02 nM and a Bmax = 144 +/- 19 fmol/mg protein, and rabbit heart membranes had a KD = 1.70 +/- .50 nM and a Bmax = 731 +/- 163 fmol/mg protein. The binding site showed a high specificity for AIV, although it exhibited low affinity for angiotensin II, angiotensin III, Sar1,Ile8-angiotensin II, DuP 753, CGP42112A and PD123177. A large number of nonangiotensin-related peptides were unable to compete effectively for 125I-AIV binding. Deletions made from the C-terminal end of AIV caused a decrease in affinity: AIV > AII(3-7) >> AII(3-6) >> AII(3-5). Extension of the C-terminal end of AIV corresponding to the amino acids of human angiotensinogen caused little change in affinity. GTP gamma S had no effect on binding, suggesting non-G protein linkage. Binding was widely distributed throughout the heart; it was observed on cardiocytes and blood vessels as well as in the epicardium and the endocardium.