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Nitric oxide (NO), a potent vasodilator produced by endothelial cells, is thought to be the endothelium-dependent relaxing factor (EDRF) which mediates vascular relaxation in response to acetylcholine, bradykinin and substance P in many vascular beds. NO has been implicated in the regulation of blood pressure and regional blood flow, and also affects vascular smooth-muscle proliferation and inhibits platelet aggregation and leukocyte adhesion. Abnormalities in endothelial production of NO occur in atherosclerosis, diabetes and hypertension. Pharmacological blockade of NO production with arginine analogues such as L-nitroarginine (L-NA) or L-N-arginine methyl ester affects multiple isoforms of nitric oxide synthase (NOS), and so cannot distinguish their physiological roles. To study the role of endothelial NOS (eNOS) in vascular function, we disrupted the gene encoding eNOS in mice. Endothelium-derived relaxing factor activity, as assayed by acetylcholine-induced relaxation, is absent, and the eNOS mutant mice are hypertensive. Thus eNOS mediates basal vasodilation. Responses to NOS blockade in the mutant mice suggest that non-endothelial isoforms of NOS may be involved in maintaining blood pressure.

Bone morphogenetic protein (BMP) signals coordinate developmental patterning and have essential physiological roles in mature organisms. Here we describe the first known small-molecule inhibitor of BMP signaling-dorsomorphin, which we identified in a screen for compounds that perturb dorsoventral axis formation in zebrafish. We found that dorsomorphin selectively inhibits the BMP type I receptors ALK2, ALK3 and ALK6 and thus blocks BMP-mediated SMAD1/5/8 phosphorylation, target gene transcription and osteogenic differentiation. Using dorsomorphin, we examined the role of BMP signaling in iron homeostasis. In vitro, dorsomorphin inhibited BMP-, hemojuvelin-and interleukin 6-stimulated expression of the systemic iron regulator hepcidin, which suggests that BMP receptors regulate hepcidin induction by all of these stimuli. In vivo, systemic challenge with iron rapidly induced SMAD1/5/8 phosphorylation and hepcidin expression in the liver, whereas treatment with dorsomorphin blocked SMAD1/5/8 phosphorylation, normalized hepcidin expression and increased serum iron levels. These findings suggest an essential physiological role for hepatic BMP signaling in iron-hepcidin homeostasis.Signals mediated by BMP ligands serve diverse roles throughout the life of vertebrates. During embryogenesis, the dorsoventral axis is established by BMP signaling gradients formed by the coordinated expression of ligands, receptors, co-receptors and soluble antagonists 1-3 . Excess BMP signaling causes ventralization (an expansion of ventral structures at the expense of dorsal structures) whereas diminished BMP signaling causes dorsalization (an expansion of dorsal structures at the expense of ventral structures) 1,3,4 . BMPs are key regulators of gastrulation,

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues 1-4 and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2)5. Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I Correspondence should be addressed to P.B.Y. (pbyu@partners.org). 8 Present address:

SUMMARY Lung stem cells are instructed to produce lineage-specific progeny through unknown factors in their microenvironment. We used clonal three-dimensional (3D) co-cultures of endothelial cells and distal lung stem cells, bronchioalveolar stem cells (BASCs), to probe the instructive mechanisms. Single BASCs had bronchiolar and alveolar differentiation potential in lung endothelial cell co-cultures. Gain and loss of function experiments showed BMP4-Bmpr1a signaling triggers calcineurin/NFATc1-dependent expression of Thrombospondin-1 (Tsp1) in lung endothelial cells to drive alveolar lineage-specific BASC differentiation. Tsp1-null mice exhibited defective alveolar injury repair, confirming a crucial role for the BMP4-NFATc1-TSP1 axis in lung epithelial differentiation and regeneration in vivo. Discovery of this pathway points to methods to direct the derivation of specific lung epithelial lineages from multipotent cells. These findings elucidate a pathway that may be a critical target in lung diseases and provide new tools to understand the mechanisms of respiratory diseases at the single cell level.

Blood pressure is a heritable trait, but no common genetic variants contributing to blood pressure in humans have been definitively established. Natriuretic peptides (NP) have blood pressure-lowering properties. Genotyping SNPs at the NPPA/NPPB locus in 14,743 individuals of European ancestry identified associations of plasma atrial natriuretic peptide with rs5068 (P=8×10−70), rs198358 (P=8×10−30), and rs632793 (P=2×10−10), and of plasma B-type natriuretic peptide with rs5068 (P=3×10−12), rs198358 (P=1×10−25), and rs632793 (P=2×10−68). In 29,717 individuals, the alleles of rs5068 and rs198358 related to increased circulating NP concentrations were associated with lower systolic (P=2×10−6 and 6×10−5, respectively) and diastolic blood pressure (P=1×10−6 and 5×10−5), and reduced odds of hypertension (odds ratio 0.85, 95% confidence interval, 0.79–0.92, P=4×10−5; odds ratio 0.90, 95% confidence interval, 0.85–0.95, P=2×10−4, respectively). Common genetic variants related to circulating NP concentrations contribute to inter-individual variation in blood pressure and hypertension.

A structure-activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo [1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo [1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g. plasma t 1/2 = 1.6 h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.Bone morphogenetic proteins (BMPs) are a group of > 25 protein ligands that comprise a subset of the transforming growth factor β (TGF-β) family. BMPs modulate a multitude of biological processes, including bone and cartilage formation during embryogenesis. 1a However, they are also intimately involved with numerous nonosteogenic developmental and physiological processes throughout adulthood as well as several pathological conditions. BMPs bind to two classes of cell surface bone morphogenetic protein receptors (BMPR-I and BMPRII). 1a The BMPR-I receptor class consists of three receptor types, activin receptor-like kinase-2 (ALK-2 or ActR-IA), ALK-3 (BMPR-IA) and ALK-6 (BMPR-IB). The BMPR-II receptor class is comprised of three receptor types, BMPR-II, ActR-IIA and ActR-IIB. Binding of BMPs results in the formation of heterotetrameric complexes containing two type I and two *To whom correspondence should be addressed: Phone: 617-768-8640, Fax: 617-768-8606, E-mail: gcuny@rics.bwh.harvard.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Recently, dorsomorphin, 1, 7a, 11, 12 was discovered as an inhibitor of SMAD 1/5/8 phosphorylation by BMP type 1 receptors (ALK2, 3, and 6) utilizing a phenotypic screen to identify compounds that perturb embryonic dorsoventral axis formation. Furthermore, this inhibition was shown to decrease BMP-regulated hepatic hepcidin gene transcription, leading to increased iron levels in vivo. 7a However, 1 only demonstrated moderate inhibition of SMAD 1/5/8 phosphorylation by BMPR-I with an IC50 ~ 0.5 µM and lacks metabolic stability. Herein, we describe the results of a structu...

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