Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP–Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia

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

Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta

The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia

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

Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta

The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia

Adrenomedullin signaling is necessary for murine lymphatic vascular development

The lymphatic vascular system mediates fluid homeostasis, immune defense, and tumor metastasis. Only a handful of genes are known to affect the development of the lymphatic vasculature, and even fewer represent therapeutic targets for lymphatic diseases. Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through the calcitonin receptor–like receptor (calcrl) when the receptor is associated with a receptor activity–modifying protein (RAMP2). Here we report on the involvement of these genes in lymphangiogenesis. AM-, calcrl-, or RAMP2-null mice died mid-gestation after development of interstitial lymphedema. This conserved phenotype provided in vivo evidence that these components were required for AM signaling during embryogenesis. A conditional knockout line with loss of calcrl in endothelial cells confirmed an essential role for AM signaling in vascular development. Loss of AM signaling resulted in abnormal jugular lymphatic vessels due to reduction in lymphatic endothelial cell proliferation. Furthermore, AM caused enhanced activation of ERK signaling in human lymphatic versus blood endothelial cells, likely due to induction of CALCRL gene expression by the lymphatic transcriptional regulator Prox1. Collectively, our studies identify a class of genes involved in lymphangiogenesis that represent a pharmacologically tractable system for the treatment of lymphedema or inhibition of tumor metastasis