Receptor-activity–modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein–coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

Background: The adherens protein VE-cadherin has diverse roles in organ-specific lymphatic vessels. However, its physiological role in cardiac lymphatics and its interaction with lymphangiogenic factors, has not been fully explored. We sought to determine the spatio-temporal functions of VE-cadherin in cardiac lymphatics and mechanistically elucidate how VE-cadherin loss influences pro-lymphangiogenic signaling pathways, such as adrenomedullin (AM) and VEGF-C/VEGFR3 signaling. Methods: Cdh5 flox/flox ;Prox1CreER T2 mice were used to delete VE-cadherin in lymphatic endothelial cells (LECs) across life stages, including embryonic, postnatal and adult. Lymphatic architecture and function was characterized utilizing immunostaining and functional lymphangiography. To evaluate the impact of temporal and functional regression of cardiac lymphatics in Cdh5 flox/flox ;Prox1CreER T2 mice, left anterior descending artery ligation was performed and cardiac function and repair after myocardial infarction was evaluated by echocardiography and histology. Cellular effects of VE-cadherin deletion on lymphatic signaling pathways were assessed by knock-down of VE-cadherin in cultured LECs. Results: Embryonic deletion of VE-cadherin produced edematous embryos with dilated cardiac lymphatics with significantly altered vessel tip morphology. Postnatal deletion of VE-cadherin caused complete disassembly of cardiac lymphatics. Adult deletion caused a temporal regression of the quiescent epicardial lymphatic network which correlated with significant dermal and cardiac lymphatic dysfunction, as measured by fluorescent and quantum dot lymphangiography, respectively. Surprisingly, despite regression of cardiac lymphatics, Cdh5 flox/flox ;Prox1CreER T2 mice exhibited preserved cardiac function, both at baseline and following myocardial infarction, compared to control mice. Mechanistically, loss of VE-cadherin leads to aberrant cellular internalization of VEGFR3, precluding the ability of VEGFR3 to be either canonically activated by VEGF-C or non-canonically transactivated by AM signaling, impairing downstream processes such as cellular proliferation. Conclusions: VE-cadherin is an essential scaffolding protein to maintain pro-lymphangiogenic signaling nodes at the plasma membrane, which are required for the development and adult maintenance of cardiac lymphatics, but not for cardiac function basally or after injury.

The heart is imbued with a vast lymphatic network which is responsible for fluid homeostasis and immune cell trafficking. Disturbances in the forces that regulate microvascular fluid movement can result in myocardial edema, which has pro-fibrotic and pro-inflammatory consequences and contributes to cardiovascular dysfunction. This review explores the complex relationship between cardiac lymphatics, myocardial edema and cardiac disease. It covers the revised paradigm of microvascular forces and fluid movement around the capillary, as well as the arsenal of preclinical tools and animal models used to model myocardial edema and cardiac disease. Clinical studies of myocardial edema and their prognostic significance are examined in parallel to the recent elegant animal studies discerning the pathophysiological role and therapeutic potential of cardiac lymphatics in different cardiovascular disease models. This review highlights the outstanding questions of interest to both basic scientists and clinicians regarding the roles of cardiac lymphatics in health and disease.

Numerous studies have focused on the molecular signaling pathways that govern the development and growth of lymphatics in the hopes of elucidating promising druggable targets. G protein-coupled receptors (GPCRs) are currently the largest family of membrane receptors targeted by FDA-approved drugs, but there remain many unexplored receptors, including orphan GPCRs with no known biological ligand or physiological function. Thus, we sought to illuminate the cadre of GPCRs expressed at high levels in lymphatic endothelial cells and identified four orphan receptors: GPRC5B, AGDRF5/GPR116, FZD8 and GPR61. Compared to blood endothelial cells, GPRC5B is the most abundant GPCR expressed in cultured human lymphatic endothelial cells (LECs), and in situ RNAscope shows high mRNA levels in lymphatics of mice. Using genetic engineering approaches in both zebrafish and mice, we characterized the function of GPRC5B in lymphatic development. Morphant gprc5b zebrafish exhibited failure of thoracic duct formation, and Gprc5b−/− mice suffered from embryonic hydrops fetalis and hemorrhage associated with subcutaneous edema and blood-filled lymphatic vessels. Compared to Gprc5+/+ littermate controls, Gprc5b−/− embryos exhibited attenuated developmental lymphangiogenesis. During the postnatal period, ~30% of Gprc5b−/− mice were growth-restricted or died prior to weaning, with associated attenuation of postnatal cardiac lymphatic growth. In cultured human primary LECs, expression of GPRC5B is required to maintain cell proliferation and viability. Collectively, we identify a novel role for the lymphatic-enriched orphan GPRC5B receptor in lymphangiogenesis of fish, mice and human cells. Elucidating the roles of orphan GPCRs in lymphatics provides new avenues for discovery of druggable targets to treat lymphatic-related conditions such as lymphedema and cancer.