VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.

BACKGROUND: The adherens protein VE-cadherin (vascular endothelial 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 spatiotemporal functions of VE-cadherin in cardiac lymphatics and mechanistically elucidate how VE-cadherin loss influences prolymphangiogenic signaling pathways, such as adrenomedullin and VEGF (vascular endothelial growth factor)-C/VEGFR3 (vascular endothelial growth factor receptor 3) signaling. METHODS: Cdh5flox/flox;Prox1CreERT2 mice were used to delete VE-cadherin in lymphatic endothelial cells across life stages, including embryonic, postnatal, and adult. Lymphatic architecture and function was characterized using immunostaining and functional lymphangiography. To evaluate the impact of temporal and functional regression of cardiac lymphatics in Cdh5flox/flox;Prox1CreERT2 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 knockdown of VE-cadherin in cultured lymphatic endothelial cells. 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, Cdh5flox/flox;Prox1CreERT2 mice exhibited preserved cardiac function, both at baseline and following myocardial infarction, compared with 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 noncanonically transactivated by adrenomedullin signaling, impairing downstream processes such as cellular proliferation. CONCLUSIONS: VE-cadherin is an essential scaffolding protein to maintain prolymphangiogenic 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.

TGF-ß superfamily co-receptors in cancer.

Transforming growth factor-ß (TGF-ß) superfamily signaling via their cognate receptors is frequently modified by TGF-ß superfamily co-receptors. Signaling through SMAD-mediated pathways may be enhanced or depressed depending on the specific co-receptor and cell context. This dynamic effect on signaling is further modified by the release of many of the co-receptors from the membrane to generate soluble forms that are often antagonistic to the membrane-bound receptors. The co-receptors discussed here include TßRIII (betaglycan), endoglin, BAMBI, CD109, SCUBE proteins, neuropilins, Cripto-1, MuSK, and RGMs. Dysregulation of these co-receptors can lead to altered TGF-ß superfamily signaling that contributes to the pathophysiology of many cancers through regulation of growth, metastatic potential, and the tumor microenvironment. Here we describe the role of several TGF-ß superfamily co-receptors on TGF-ß superfamily signaling and the impact on cellular and physiological functions with a particular focus on cancer, including a discussion on recent pharmacological advances and potential clinical applications targeting these co-receptors.

Characterization of Antibodies against Receptor Activity-Modifying Protein 1 (RAMP1): A Cautionary Tale.

Calcitonin gene-related peptide (CGRP) is a key component of migraine pathophysiology, yielding effective migraine therapeutics. CGRP receptors contain a core accessory protein subunit: receptor activity-modifying protein 1 (RAMP1). Understanding of RAMP1 expression is incomplete, partly due to the challenges in identifying specific and validated antibody tools. We profiled antibodies for immunodetection of RAMP1 using Western blotting, immunocytochemistry and immunohistochemistry, including using RAMP1 knockout mouse tissue. Most antibodies could detect RAMP1 in Western blotting and immunocytochemistry using transfected cells. Two antibodies (844, ab256575) could detect a RAMP1-like band in Western blots of rodent brain but not RAMP1 knockout mice. However, cross-reactivity with other proteins was evident for all antibodies. This cross-reactivity prevented clear conclusions about RAMP1 anatomical localization, as each antibody detected a distinct pattern of immunoreactivity in rodent brain. We cannot confidently attribute immunoreactivity produced by RAMP1 antibodies (including 844) to the presence of RAMP1 protein in immunohistochemical applications in brain tissue. RAMP1 expression in brain and other tissues therefore needs to be revisited using RAMP1 antibodies that have been comprehensively validated using multiple strategies to establish multiple lines of convincing evidence. As RAMP1 is important for other GPCR/ligand pairings, our results have broader significance beyond the CGRP field.

cSrc is necessary for epididymal development and is incorporated into sperm during epididymal transit.

Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.

Yin-yang1 is required in the mammalian oocyte for follicle expansion.

The multifaceted polycomb group gene Yin-Yang1 (Yy1) has been implicated in a variety of transcriptional regulatory roles both as an activator and silencer of gene expression. Here we examine the role of Yy1 during oocyte growth by conditional deletion of the locus in the growing oocyte. Our results indicate that YY1 is required for oocyte maturation and granulosa cell expansion. In mutant oocytes, we observe severely reduced expression of both Gdf9 and Bmp15, suggesting a mechanism underlying the failure of granulosa cell expansion. Consequently, we observe infertility, failure of estrus cycling, and altered reproductive hormone levels in mutant females. Additionally, we find that YY1-deficient oocytes exhibit altered levels of several oocyte-specific factors, including Pou5f1, Figla, Lhx8, Oosp1, and Sohlh2. These results document YY1's involvement in folliculogenesis and ovarian function in the mouse and indicate that YY1 is required specifically in the oocyte for oocyte-granulosa cell communication.

Cleavage and gastrulation in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus (Bate): a revised cell lineage and identification of a presumptive germ cell marker.

A previous study suggested that mesendoderm (ME) cell arrest occurred at the 64-cell stage and a ring of eight presumptive naupliar mesoderm cells or crown cells surrounded the blastopore in the Kuruma shrimp Penaeus (Marsupenaeus) japonicus. Since this varied from the pattern observed in other penaeoidean shrimp, cleavage and gastrulation was re-examined in P. japonicus using the nucleic acid stain Sytox Green and confocal microscopy. In contrast to the earlier study, cleavage and gastrulation followed the pattern observed in other penaeoidean shrimp. The ME cells arrested at the 32-cell stage, ingressed into the blastocoel, and resumed division after a three cell cycle delay. Nine naupliar mesoderm or crown cells surrounded the blastopore and their descendants invaginated during gastrulation. An intracellular body (ICB) was detected by Sytox Green and SYTO RNASelect staining to be segregated to one ME cell in P. japonicus, as described previously in Penaeus monodon. Staining of the ICB was eliminated by pre-treatment with RNase but not DNase. The ICB was also found in two other penaeoidean shrimp, Penaeus vannamei (Family Penaeidae) and Sicyonia ingentis (Family Sicyoniidae). The results support the hypothesis that the ICB is a germ granule found in the Dendrobranchiata.

Lymphatic Programing and Specialization in Hybrid Vessels.

Building on a large body of existing blood vascular research, advances in lymphatic research have helped kindle broader investigations into vascular diversity and endothelial plasticity. While the endothelium of blood and lymphatic vessels can be distinguished by a variety of molecular markers, the endothelia of uniquely diverse vascular beds can possess distinctly heterogeneous or hybrid expression patterns. These expression patterns can then provide further insight on the development of these vessels and how they perform their specialized function. In this review we examine five highly specialized hybrid vessel beds that adopt partial lymphatic programing for their specialized vascular functions: the high endothelial venules of secondary lymphoid organs, the liver sinusoid, the Schlemm's canal of the eye, the renal ascending vasa recta, and the remodeled placental spiral artery. We summarize the morphology and endothelial expression pattern of these vessels, compare them to each other, and interrogate their specialized functions within the broader blood and lymphatic vascular systems.

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.

E-Cigarette Exposure Delays Implantation and Causes Reduced Weight Gain in Female Offspring Exposed In Utero.

Electronic nicotine delivery system (e-cigarette) use is prevalent among pregnant women as a seemingly safe alternative to traditional tobacco use, known to result in fetal developmental abnormalities and impaired fertility of male offspring. However, little is known about the effects of e-cigarette use on fertility or pregnancy outcomes. A successful pregnancy is initiated by a multitude of dynamic molecular alterations in the uterus resulting in embryo implantation at day 4.5 in the mouse. We examined whether e-cigarette exposure impairs implantation and offspring health. Pregnant C57BL/6J mice were exposed five times a week to e-cigarette vapor or sham. After 4 months, e-cigarette exposed dams exhibited a significant delay in the onset of the first litter. Furthermore, exposure of new dams in early pregnancy significantly impaired embryo implantation, as evidenced by nearly complete absence of implantation sites in e-cigarette-exposed animals at day 5.5, despite exhibiting high levels of progesterone, an indicator of pregnancy. RNA microarray from day 4.5 pseudopregnant mice revealed significant changes in the integrin, chemokine, and JAK signaling pathways. Moreover, female offspring exposed to e-cigarettes in utero exhibited a significant weight reduction at 8.5 months, whereas males exhibited a slight but nonsignificant deficiency in fertility. Thus, e-cigarette exposure in mice impairs pregnancy initiation and fetal health, suggesting that e-cigarette use by reproductive-aged women or during pregnancy should be considered with caution.

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.

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.