RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

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.

Orphan G-Protein Coupled Receptor GPRC5B Is Critical for Lymphatic Development.

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.

BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development.

Arteries and veins acquire distinct molecular identities prior to the onset of embryonic blood circulation, and their specification is crucial for vascular development. The transcription factor COUP-TFII currently functions at the top of a signaling pathway governing venous fate. It promotes venous identity by inhibiting Notch signaling and subsequent arterialization of endothelial cells, yet nothing is known about what regulates COUP-TFII expression in veins. We now report that the chromatin-remodeling enzyme BRG1 promotes COUP-TFII expression in venous endothelial cells during murine embryonic development. Conditional deletion of Brg1 from vascular endothelial cells resulted in downregulated COUP-TFII expression and aberrant expression of arterial markers on veins. BRG1 promotes COUP-TFII expression by binding conserved regulatory elements within the COUP-TFII promoter and remodeling chromatin to make the promoter accessible to transcriptional machinery. This study provides the first description of a factor promoting COUP-TFII expression in vascular endothelium and highlights a novel role for chromatin remodeling in venous specification.

The chromatin-remodeling enzyme BRG1 modulates vascular Wnt signaling at two levels.

The ATP-dependent chromatin-remodeling enzyme brahma-related gene 1 (BRG1) regulates transcription of specific target genes during embryonic and postnatal development. Deletion of Brg1 from embryonic blood vessels results in yolk sac vascular remodeling defects. We now report that misregulation of the canonical Wnt signaling pathway underlies many Brg1 mutant vascular phenotypes. Brg1 deletion resulted in down-regulation of several Wnt receptors of the frizzled family, degradation of the intracellular Wnt signaling molecule ß-catenin, and an overall decrease in Wnt signaling in endothelial cells. Pharmacological stabilization of ß-catenin significantly rescued Brg1 mutant vessel morphology and transcription of Wnt target genes. Our data demonstrate that BRG1 impacts the canonical Wnt pathway at two different levels in vascular endothelium: through transcriptional regulation of both Wnt receptor genes and Wnt target genes. These findings establish an epigenetic mechanism for the modulation of Wnt signaling during embryonic vascular development.

Chromatin-remodeling complex specificity and embryonic vascular development.

Vascular development is a dynamic process that relies on the coordinated expression of numerous genes, but the factors that regulate gene expression during blood vessel development are not well defined. ATP-dependent chromatin-remodeling complexes are gaining attention for their specific temporal and spatial effects on gene expression during vascular development. Genetic mutations in chromatin-remodeling complex subunits are revealing roles for the complexes in vascular signaling pathways at discrete developmental time points. Phenotypic analysis of these models at various stages of vascular development will continue to expand our understanding of how chromatin remodeling impacts new blood vessel growth. Such research could also provide novel therapeutic targets for the treatment of vascular pathologies.

Dermal Lymphatic Capillaries Do Not Obey Murray's Law.

Lymphatic vessels serve as a major conduit for the transport of interstitial fluid, immune cells, lipids and drugs. Therefore, increased knowledge about their development and function is relevant to clinical issues ranging from chronic inflammation and edema, to cancer metastasis to targeted drug delivery. Murray's Law is a widely-applied branching rule upheld in diverse circulatory systems including leaf venation, sponge canals, and various human organs for optimal fluid transport. Considering the unique and diverse functions of lymphatic fluid transport, we specifically address the branching of developing lymphatic capillaries, and the flow of lymph through these vessels. Using an empirically-generated dataset from wild type and genetic lymphatic insufficiency mouse models we confirmed that branching blood capillaries consistently follow Murray's Law. However surprisingly, we found that the optimization law for lymphatic vessels follows a different pattern, namely a Murray's Law exponent of ~1.45. In this case, the daughter vessels are smaller relative to the parent than would be predicted by the hypothesized radius-cubed law for impermeable vessels. By implementing a computational fluid dynamics model, we further examined the extent to which the assumptions of Murray's Law were violated. We found that the flow profiles were predominantly parabolic and reasonably followed the assumptions of Murray's Law. These data suggest an alternate hypothesis for optimization of the branching structure of the lymphatic system, which may have bearing on the unique physiological functions of lymphatics compared to the blood vascular system. Thus, it may be the case that the lymphatic branching structure is optimized to enhance lymph mixing, particle exchange, or immune cell transport, which are particularly germane to the use of lymphatics as drug delivery routes.

Author Correction: Notch signaling pathway is a potential therapeutic target for extracranial vascular malformations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Calcitonin-Receptor-Like Receptor Signaling Governs Intestinal Lymphatic Innervation and Lipid Uptake.

The absorption of dietary fat requires complex neuroendocrine-mediated regulation of chylomicron trafficking through enterocytes and intestinal lymphatic vessels. Calcitonin-receptor-like receptor (Calcrl) is a G protein-coupled receptor that can bind either a lymphangiogenic ligand adrenomedullin, with coreceptor RAMP2, or the neuropeptide CGRP, with coreceptor RAMP1. The extent to which this common GPCR controls lipid absorption via lymphatics or enteric innervation remains unclear. We used conditional and inducible genetic deletion of Calcrl in lymphatics to elucidate the pathophysiological consequences of this receptor pathway under conditions of high-fat diet. Inefficient absorption of dietary fat coupled with altered lymphatic endothelial junctions in Calcrl fl/fl /Prox1-CreER T2 mice results in excessive, transcellular lipid accumulation and abnormal enterocyte chylomicron processing and failure to gain weight. Interestingly, Calcrl fl/fl /Prox1-CreER T2 animals show reduced and disorganized mucosal and submucosal innervation. Consistently, mice with genetic loss of the CGRP coreceptor RAMP1 also displayed mucosal and submucosal innervation deficits, substantiating the CGRP-biased function of Calcrl in the neurolymphocrine axis. Thus, the common Calcrl receptor is a critical regulator of lipid absorption through its cell-specific functions in neurolymphocrine crosstalk.

Lymphatic mimicry in maternal endothelial cells promotes placental spiral artery remodeling.

Molecular heterogeneity of endothelial cells underlies their highly specialized functions during changing physiological conditions within diverse vascular beds. For example, placental spiral arteries (SAs) undergo remarkable remodeling to meet the ever-growing demands of the fetus - a process which is deficient in preeclampsia. The extent to which maternal endothelial cells coordinate with immune cells and pregnancy hormones to promote SA remodeling remains largely unknown. Here we found that remodeled SAs expressed the lymphatic markers PROX1, LYVE1, and VEGFR3, mimicking lymphatic identity. Uterine natural killer (uNK) cells, which are required for SA remodeling and secrete VEGFC, were both sufficient and necessary for VEGFR3 activation in vitro and in mice lacking uNK cells, respectively. Using Flt4Chy/+ mice with kinase inactive VEGFR3 and Vegfcfl/fl Vav1-Cre mice, we demonstrated that SA remodeling required VEGFR3 signaling, and that disrupted maternal VEGFR3 signaling contributed to late-gestation fetal growth restriction. Collectively, we identified a novel instance of lymphatic mimicry by which maternal endothelial cells promote SA remodeling, furthering our understanding of the vascular heterogeneity employed for the mitigation of pregnancy complications such as fetal growth restriction and preeclampsia.

Notch signaling pathway is a potential therapeutic target for extracranial vascular malformations.

Notch expression has been shown to be aberrant in brain arteriovenous malformations (AVM), and targeting Notch has been suggested as an approach to their treatment. It is unclear whether extracranial vascular malformations follow the same patterning and Notch pathway defects. In this study, we examined human extracranial venous (VM) (n = 3), lymphatic (LM) (n = 10), and AV (n = 6) malformations, as well as sporadic brain AVMs (n = 3). In addition to showing that extracranial AVMs demonstrate interrupted elastin and that AVMs and LMs demonstrate abnormal α-smooth muscle actin just as brain AVMS do, our results demonstrate that NOTCH1, 2, 3 and 4 proteins are overexpressed to varying degrees in both the endothelial and mural lining of the malformed vessels in all types of malformations. We further show that two gamma secretase inhibitors (GSIs), DAPT (GSI-IX) and RO4929097, cause dose-dependent inhibition of Notch target gene expression (Hey1) and rate of migration of monolayer cultures of lymphatic endothelial cells (hLECs) and blood endothelial cells (HUVEC). GSIs also inhibit HUVEC network formation. hLECs are more sensitive to GSIs compared to HUVEC. GSIs have been found to be safe in clinical trials in patients with Alzheimer's disease or cancer. Our results provide further rationale to support testing of Notch inhibitors in patients with extracranial vascular malformations.

hCALCRL mutation causes autosomal recessive nonimmune hydrops fetalis with lymphatic dysplasia.

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.

Lymphatic deletion of calcitonin receptor-like receptor exacerbates intestinal inflammation.

Lymphatics play a critical role in maintaining gastrointestinal homeostasis and in the absorption of dietary lipids, yet their roles in intestinal inflammation remain elusive. Given the increasing prevalence of inflammatory bowel disease, we investigated whether lymphatic vessels contribute to, or may be causative of, disease progression. We generated a mouse model with temporal and spatial deletion of the key lymphangiogenic receptor for the adrenomedullin peptide, calcitonin receptor-like receptor (Calcrl), and found that the loss of lymphatic Calcrl was sufficient to induce intestinal lymphangiectasia, characterized by dilated lacteals and protein-losing enteropathy. Upon indomethacin challenge, Calcrlfl/fl/Prox1-CreERT2 mice demonstrated persistent inflammation and failure to recover and thrive. The epithelium and crypts of Calcrlfl/fl/Prox1-CreERT2 mice exhibited exacerbated hallmarks of disease progression, and the lacteals demonstrated an inability to absorb lipids. Furthermore, we identified Calcrl/adrenomedullin signaling as an essential upstream regulator of the Notch pathway, previously shown to be critical for intestinal lacteal maintenance and junctional integrity. In conclusion, lymphatic insufficiency and lymphangiectasia caused by loss of lymphatic Calcrl exacerbates intestinal recovery following mucosal injury and underscores the importance of lymphatic function in promoting recovery from intestinal inflammation.