The Multifunctional Adrenomedullin Peptide
; protein=AM) is a highly conserved multifunctional peptide that is implicated in a wide variety of physiological processes including angiogenesis and cardiovascular homeostasis [1
]. For over a decade, the association of ~2-fold elevations in plasma levels of AM peptide with a wide variety of cardiovascular disease conditions has prompted intense inquiry into understanding the functions and roles of AM in human disease (Fig.
). Moreover, the recent development of highly precise methods for the quantitation of midregional proadrenomedullin (MR-proADM) as a reliable surrogate of mature AM plasma levels [2
], has paved the way for the introduction of AM as a clinically useful biomarker for the staging of adverse cardiovascular events, including myocardial infarction, sepsis and community acquired pneumonia [3
]. While it is clear that AM can elicit powerful effects on vascular smooth muscle cells and thus acutely modulate vascular tone, numerous studies in the past 5 years have elucidated essential functions of AM on vascular endothelial cells. In the following sections we review the multi-faceted role of AM in endothelial cells during development, consider how perturbations in AM signaling may lead to vascular pathologies, and highlight recent discoveries regarding AM that have contributed in substantial ways to the broader field of vascular biology. Many of these discoveries have been unraveled through the use of sophisticated genetic animal models (Tables
), and so we have placed a special emphasis on describing the merits and shortcomings of these approaches and also highlighting current questions that are of predominant interest to the field today.
Fig. (1) Fold Change in Plasma Adrenomedullin Levels in a
Variety of Human Conditions. Bars indicate average fold change
in circulating AM levels in various disease categories or conditions
based on published human clinical data. The dashed horizontal line
at (more ...)
Gene Targeted Mouse Models for Studying Adrenomedullin Signaling
Vascular Assays for Studying Adrenomedullin Function
Adrenomedullin GPCR-Mediated Signaling in Endothelial Cells
G-protein coupled receptors (GPCRs) are widely expressed proteins that span the cell membrane 7 times and respond to a variety of stimuli including peptides, proteins, small organic compounds, lipids, amino acids, and cations. AM binds and signals through the GPCR calcitonin receptor-like receptor (gene=Calcrl
; protein=CLR). The discovery of a novel class of GPCR associated proteins called receptor activity-modifying proteins (gene=Ramp
; protein=RAMP) [7
] provided insight into how GPCRs signal. The RAMPs are single-pass transmembrane accessory proteins that regulate the translocation of GPCRs to the plasma membrane as well as provide ligand specificity to these receptors. The tissue specific and temporal expression pattern of RAMPs determines the responsiveness of GPCRs to particular ligands. For example, AM binds to the CLR receptor when CLR is associated with either RAMP2 or RAMP3. However, co-expression of CLR with RAMP1 changes the ligand specificity to another potent vasodilator called calcitonin gene-related peptide (CGRP), a related family member of the AM peptide. The ability of CLR to bind multiple ligands provides a unique mechanism by which the receptor can initiate a variety of signaling pathways. Since the AM receptor CLR and the 3 mammalian RAMPs are highly expressed in the vasculature, this cell signaling paradigm is being intensely investigated to determine how it can be exploited for the potential treatment of conditions such as pulmonary hypertension [8
], cardiovascular disorders [9
], and the inhibition of cancer metastasis [10
The binding of AM to its receptor CLR results in a myriad of downstream effects including modulation of endothelial cell survival, proliferation, and vessel permeability. For example, AM-induced proliferation and migration of lymphatic endothelial cells is mediated in part by cAMP and downstream MEK/ERK pathways [11
]. Similar results were shown using cultured human umbilical vein endothelial cells (HUVECs). AM-mediated induction of HUVEC proliferation and migration through activation of PKA, PI3K, and focal adhesion kinase were observed and then further substantiated in whole animal studies [12
]. AM induced the proliferation and migration of cultured HUVECs [12
] and numerous studies have shown a direct role for AM in endothelial growth and survival [14
Using in vitro
experiments, AM was found to regulate the permeability and migration of HUVECs [17
]. Previous studies indicated that adult Ramp2+/-
mice had increased vascular permeability and overexpression of Ramp2
in BECs resulted in reduced permeability [18
]. AM also reduces the permeability of HUVECs and pulmonary artery endothelial cells treated with permeabilizing agents including hydrogen peroxide and thrombin [19
]. AM has been shown to regulate the transport of molecules across the blood brain barrier in cerebral endothelial cells by modulating permeability [20
]. In cerebral endothelial cells, AM regulated various functions of the blood brain barrier including increasing transendothelial electrical resistance, reducing fluid-phase endocytosis, and reducing permeability for sodium fluorescein which indicate that the cerebral endothelial cell junctions are tightened by AM [20
]. Also in an in vivo
model, AM treatment reduced lung vascular permeability resulting from ventilator use in a mouse model where prolonged mechanical ventilation was administered [21
]. Overall, these data provide evidence for the role of AM as a junctional tightening factor to help regulate endothelial cell permeability.
Although AM functions to promote endothelial cell growth and proliferation in both the blood and lymphatic vasculatures, Fritz-Six et al.
have shown that there is an enhanced effect of AM on lymphatic endothelial cells (LECs) as compared to blood endothelial cells (BECs) [22
]. This biological distinction in AM function is based upon the finding that LECs are enriched in the expression of AM and its receptor components, Calcrl
]. This increase in Calcrl
expression is mediated in part by induction of the transcriptional regulator of lymphatic specification, Prox1
]. It is therefore not surprising that loss of any component of the AM signaling axis (Adm
, or Ramp2
) results in embryonic lethality associated with profound lymphatic vascular defects [22
]. Furthermore, several in vitro
and in vivo
experiments reveal that AM controls lymphatic permeability and flow through reorganization of junctional proteins ZO-1 and an adherens protein VE-Cadherin, independent of changes in junctional protein gene expression [25
]. Administration of AM to a monolayer of LECs resulted in tightening of the lymphatic endothelial barrier by reorganization of a tight junction protein at the plasma membrane to form continuous cell-cell contacts. Through the use of in vivo
tail microlymphography, local administration of AM in a SvEv129/6 mouse tail resulted in decreased velocity of lymph uptake from the interstitial space and movement through the lymphatic dermal capillaries in the tail [25
]. Thus, it becomes critically important to consider the pleiotropic effects of AM not just on blood endothelial cells, but also on neighboring lymphatic vessels—a dynamic that may ultimately help resolve the complex functions of AM peptide in cardiovascular disease, tumor progression and inflammation.
While activation of GPCRs typically leads to induction of classical second messenger signaling systems, it is now appreciated that more complex levels of regulation exist [26
]. Therefore, it is not surprising that pathway cross-talk is one mechanism through which AM modulates certain endothelial cell functions. For example, Yurugi-Kobayashi et al.
describe a novel embryonic stem cell differentiation system to study mechanisms of arterial-venous specification. They demonstrated that coordinated signaling of AM/cAMP, VEGF, and Notch induces arterial endothelial cell differentiation from vascular progenitors [28
]. Furthermore, GPCR-induced transactivation of receptor tyrosine kinases is another mechanism that allows interaction between signaling molecules. Evidence exists that AM and VEGF pathways are likely to interact in endothelial cells. Although an earlier study claimed that AM-induced capillary tube formation in HUVECs was independent of VEGF activation [14
], a more recent study by Guidolin et al.
demonstrated that VEGFR2 inactivation inhibited AM-mediated angiogenesis in HUVECs [29
]. This latter finding suggests that the pro-angiogenic effects of AM require transactivation of the receptor tyrosine kinase VEGFR2. Although controversy still exists regarding the degree of cooperation between pathways, it is certainly intriguing to consider that regulation of endothelial cell biology may very likely involve coordination of multiple signaling molecules. We now must begin to unravel these complexities and elucidate whether these interactions occur differentially in blood and lymphatic endothelial cells and identify the intermediate molecular players involved in pathway cross-talk in the vasculature.