Genetic mouse models elucidate the roles of adrenomedullin in cardiovascular development and physiology

Adrenomedullin (AM) is a highly conserved, secreted 52 amino acid peptide that functions in physiological processes within the nervous, reproductive, and cardiovascular systems. AM is nearly ubiquitously expressed, but most highly expressed from the vasculature, lungs, and heart. During cardiovascular stresses including myocardial infarction, hypertension, renal failure and normal pregnancy, AM serum levels are elevated suggesting that AM functions as a cardioprotective factor. AM signals through a unique paradigm of G-protein coupled receptor (GPCR) signaling in which the affinity of calcitonin receptor-like receptor (CLR=protein, Calcrl=gene) for its ligands, AM or calcitonin gene-related peptide, is dictated by a family of single-pass transmembrane proteins called receptor activity modifying proteins (RAMPs). Ramp2-CLR and Ramp3-CLR specifically bind AM while RAMP1-CLR functions as the CGRP receptor. Research presented in this dissertation aims to further our understanding of AM signaling in mammalian cardiovascular physiology through the utilization of genetic mouse models and in vitro approaches. I demonstrate that gene-targeted knockout mice of AM, Calcrl, and Ramp2 are embryonic lethal from specific defects in lymphatic endothelial cell (LEC) proliferation resulting in generalized interstitial edema. Within this study, I present a model in which AM signaling components are enriched during LEC differentiation which, in the absence of AM signaling, explains the observed hypoplastic lymphatic vascular development. I also investigated whether a loss in LEC barrier function could have contributed to this phenotype. While I found no difference in the ultra structural features or expression of LEC junctional components in knockout mice, I show that in vivo and in vitro AM stabilizes the LEC barrier and can completely abrogate the highly permeabilizing actions of vascular endothelial growth factor A, supporting an important role of AM signaling in LEC barrier regulation. In a separate study, I generated and characterized a vascular smooth muscle cell-specific Calcrl deficient mouse. These mice survived to adulthood with no defects in the regulation of basal blood pressure or cardiovascular function which may be due to compensation by other hemodynamic mechanisms. Together, these studies have built a solid foundation that will one day benefit future clinical applications of AM in lymphatic and vascular smooth muscle pathologies

Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo

The lymphatic vascular system functions to maintain fluid homeostasis by removing fluid from the interstitial space and returning it to venous circulation. This process is dependent upon the maintenance and modulation of a semi-permeable barrier between lymphatic endothelial cells of the lymphatic capillaries. However, our understanding of the lymphatic endothelial barrier and the molecular mechanisms that govern its function remains limited. Adrenomedullin (AM) is a 52 amino acid secreted peptide which has a wide range of effects on cardiovascular physiology and is required for the normal development of the lymphatic vascular system. Here, we report that AM can also modulate lymphatic permeability in cultured dermal microlymphatic endothelial cells (HMVEC-dLy). AM stimulation caused a reorganization of the tight junction protein ZO-1 and the adherens protein VE-cadherin at the plasma membrane, effectively tightening the endothelial barrier. Stabilization of the lymphatic endothelial barrier by AM occurred independently of changes in junctional protein gene expression and AM−/− endothelial cells showed no differences in the gene expression of junctional proteins compared to wildtype endothelial cells. Nevertheless, local administration of AM in the mouse tail decreased the rate of lymph uptake from the interstitial space into the lymphatic capillaries. Together, these data reveal a previously unrecognized role for AM in controlling lymphatic endothelial permeability and lymphatic flow through reorganization of junctional proteins

Endothelial Restoration of Receptor Activity-Modifying Protein 2 Is Sufficient to Rescue Lethality, but Survivors Develop Dilated Cardiomyopathy.

RAMPs (receptor activity-modifying proteins) serve as oligomeric modulators for numerous G-protein-coupled receptors, yet elucidating the physiological relevance of these interactions remains complex. Ramp2 null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor signaling. We aimed to genetically rescue the Ramp2(-/-) lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with family B G-protein-coupled receptors in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2(-/-) mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2(-/-) Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking calcitonin receptor-like receptor. Yet, the hearts of Ramp2(-/-) Tg animals displayed dysregulation of family B G-protein-coupled receptors, including parathyroid hormone and glucagon receptors, as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional G-protein-coupled receptor pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/calcitonin receptor-like receptor signaling, in which future studies could elucidate novel and pharmacologically tractable pathways for treating cardiovascular diseases

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein–Induced Retinal AngiogenesisHighlights

OBJECTIVE: Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2, and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2. Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels

Decoy Receptor CXCR7 Modulates Adrenomedullin-Mediated Cardiac and Lymphatic Vascular Development

Atypical 7-transmembrane receptors, often called decoy receptors, act promiscuously as molecular sinks to regulate ligand bioavailability and consequently temper the signaling of canonical G protein-coupled receptor (GPCR) pathways. Loss of mammalian CXCR7, the most recently described decoy receptor, results in postnatal lethality due to aberrant cardiac development and myocyte hyperplasia. Here, we provide the molecular underpinning for this proliferative phenotype by demonstrating that the dosage and signaling of adrenomedullin (Adm = gene, AM = protein)—a mitogenic peptide-hormone required for normal cardiovascular development—is tightly controlled by CXCR7. To this end, Cxcr7−/− mice exhibit gain-of-function cardiac and lymphatic vascular phenotypes which can be reversed upon genetic depletion of adrenomedullin ligand. In addition to identifying a biological ligand accountable for the phenotypes of Cxcr7−/− mice, these results reveal a previously underappreciated role for decoy receptors as molecular rheostats in controlling the timing and extent of GPCR-mediated cardiac and vascular development

The Ras Activator RasGRP3 Mediates Diabetes-Induced Embryonic Defects and Affects Endothelial Cell Migration

Fetuses that develop in diabetic mothers have a higher incidence of birth defects that include cardiovascular defects, but the signaling pathways that mediate these developmental effects are poorly understood. It is reasonable to hypothesize that diabetic maternal effects are mediated by one or more pathways activated downstream of aberrant glucose metabolism, since poorly controlled maternal glucose levels correlate with the frequency and severity of the defects

Studies on the Mechanism of Catalytic Hydrogenations With Rhodium, Palladium, and Iridium

Abstract not availabl

Hydrops Fetalis, Cardiovascular Defects, and Embryonic Lethality in Mice Lacking the Calcitonin Receptor-Like Receptor Gene

Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl(−/−) pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl(−/−) embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl(−/−) embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM(−/−) phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl(−/−) and AM(−/−) embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development

Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein–Induced Retinal Angiogenesis

OBJECTIVE: Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. APPROACH AND RESULTS: Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. CONCLUSIONS: Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels

Bone morphogenetic protein 2 signaling negatively modulates lymphatic development in vertebrate embryos

RATIONALE: The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. OBJECTIVE: Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. METHODS AND RESULTS: BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell–derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development. CONCLUSIONS: Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development