Decreased lymphatic HIF-2α accentuates lymphatic remodeling in lymphedema.

Pathologic lymphatic remodeling in lymphedema evolves during periods of tissue inflammation and hypoxia through poorly defined processes. In human and mouse lymphedema, there is a significant increase of hypoxia inducible factor 1 α (HIF-1α), but a reduction of HIF-2α protein expression in lymphatic endothelial cells (LECs). We questioned whether dysregulated expression of these transcription factors contributes to disease pathogenesis and found that LEC-specific deletion of Hif2α exacerbated lymphedema pathology. Even without lymphatic vascular injury, the loss of LEC-specific Hif2α caused anatomic pathology and a functional decline in fetal and adult mice. These findings suggest that HIF-2α is an important mediator of lymphatic health. HIF-2α promoted protective phosphorylated TIE2 (p-TIE2) signaling in LECs, a process also replicated by upregulating TIE2 signaling through adenovirus-mediated angiopoietin-1 (Angpt1) gene therapy. Our study suggests that HIF-2α normally promotes healthy lymphatic homeostasis and raises the exciting possibility that restoring HIF-2α pathways in lymphedema could mitigate long-term pathology and disability.

Non-canonical WNT-signaling controls differentiation of lymphatics and extension lymphangiogenesis via RAC and JNK signaling.

Development of lymphatics takes place during embryogenesis, wound healing, inflammation, and cancer. We previously showed that Wnt5a is an essential regulator of lymphatic development in the dermis of mice, however, the mechanisms of action remained unclear. Here, whole-mount immunostaining shows that embryonic day (ED) 18.5 Wnt5a-null mice possess non-functional, cyst-like and often blood-filled lymphatics, in contrast to slender, interconnected lymphatic networks of Wnt5a+/- and wild-type (wt) mice. We then compared lymphatic endothelial cell (LEC) proliferation during ED 12.5, 14.5, 16.5 and 18.5 between Wnt5a-/-, Wnt5a+/- and wt-mice. We did not observe any differences, clearly showing that Wnt5a acts independently of proliferation. Transmission electron microscopy revealed multiple defects of LECs in Wnt5a-null mice, such as malformed inter-endothelial junctions, ruffled cell membrane, intra-luminal bulging of nuclei and cytoplasmic processes. Application of WNT5A protein to ex vivo cultures of dorsal thoracic dermis from ED 15.5 Wnt5a-null mice induced flow-independent development of slender, elongated lymphatic networks after 2 days, in contrast to controls showing an immature lymphatic plexus. Reversely, the application of the WNT-secretion inhibitor LGK974 on ED 15.5 wt-mouse dermis significantly prevented lymphatic network elongation. Correspondingly, tube formation assays with human dermal LECs in vitro revealed increased tube length after WNT5A application. To study the intracellular signaling of WNT5A we used LEC scratch assays. Thereby, inhibition of autocrine WNTs suppressed horizontal migration, whereas application of WNT5A to inhibitor-treated LECs promoted migration. Inhibition of the RHO-GTPase RAC, or the c-Jun N-terminal kinase JNK significantly reduced migration, whereas inhibitors of the protein kinase ROCK did not. WNT5A induced transient phosphorylation of JNK in LECs, which could be inhibited by RAC- and JNK-inhibitors. Our data show that WNT5A induces formation of elongated lymphatic networks through proliferation-independent WNT-signaling via RAC and JNK. Non-canonical WNT-signaling is a major mechanism of extension lymphangiogenesis, and also controls differentiation of lymphatics.

Lymphatic Anatomy.

Recent development of new lymphatic imaging and intervention techniques, such as intranodal lymphangiogram, dynamic contrast enhanced magnetic resonance lymphangiography and lymphatic embolization, have resulted in the resurgence of interest in the lymphatic anatomy. The lymphatic system is a continuous maze of interlacing vessels and lymph nodes and is extremely complex and variable. This presents a significant challenge for interpretation of imaging and performance of interventions on this system. There is an embryological reason for this complexity and variability; the lymphatic system sprouts off of primordia from several locations in the body, which later fuse together at different stages of development of the embryo. The lymphatic system can be divided in three distinct parts: soft tissue lymphatics, intestinal lymphatics, and liver lymphatics. Liver and intestinal lymphatics generate approximately 80% of the body lymph and are functionally the most important parts of the lymphatic system. However, their normal anatomy and pathological changes are relatively unknown. In this chapter we will explore the anatomy of these three systems relevant to lymphatic imaging and interventions.

Characterisation of Development and Electrophysiological Mechanisms Underlying Rhythmicity of the Avian Lymph Heart.

Despite significant advances in tissue engineering such as the use of scaffolds, bioreactors and pluripotent stem cells, effective cardiac tissue engineering for therapeutic purposes has remained a largely intractable challenge. For this area to capitalise on such advances, a novel approach may be to unravel the physiological mechanisms underlying the development of tissues that exhibit rhythmic contraction yet do not originate from the cardiac lineage. Considerable attention has been focused on the physiology of the avian lymph heart, a discrete organ with skeletal muscle origins yet which displays pacemaker properties normally only found in the heart. A functional lymph heart is essential for avian survival and growth in ovo. The histological nature of the lymph heart is similar to skeletal muscle although molecular and bioelectrical characterisation during development to assess mechanisms that contribute towards lymph heart contractile rhythmicity have not been undertaken. A better understanding of these processes may provide exploitable insights for therapeutic rhythmically contractile tissue engineering approaches in this area of significant unmet clinical need. Here, using molecular and electrophysiological approaches, we describe the molecular development of the lymph heart to understand how this skeletal muscle becomes fully functional during discrete in ovo stages of development. Our results show that the lymph heart does not follow the normal transitional programme of myogenesis as documented in most skeletal muscle, but instead develops through a concurrent programme of precursor expansion, commitment to myogenesis and functional differentiation which offers a mechanistic explanation for its rapid development. Extracellular electrophysiological field potential recordings revealed that the peak-to-peak amplitude of electrically evoked local field potentials elicited from isolated lymph heart were significantly reduced by treatment with carbachol; an effect that could be fully reversed by atropine. Moreover, nifedipine and cyclopiazonic acid both significantly reduced peak-to-peak local field potential amplitude. Optical recordings of lymph heart showed that the organ's rhythmicity can be blocked by the HCN channel blocker, ZD7288; an effect also associated with a significant reduction in peak-to-peak local field potential amplitude. Additionally, we also show that isoforms of HCN channels are expressed in avian lymph heart. These results demonstrate that cholinergic signalling and L-type Ca2+ channels are important in excitation and contraction coupling, while HCN channels contribute to maintenance of lymph heart rhythmicity.

Extravascular endothelial and hematopoietic islands form through multiple pathways in midgestation mouse embryos.

The de novo generation of hematopoietic cells occurs during midgestation when a population of endothelial cells called hemogenic endothelium transitions into hematopoietic progenitors and stem cells. In mammalian embryos, the newly formed hematopoietic cells form clusters in the lumens of the major arteries in the embryo proper and in the vascular plexus of the yolk sac. Small clusters of hematopoietic cells that are independent of the vasculature (referred to here as extravascular islands) were shown to form in the mesentery during vascular remodeling of the vitelline artery. Using three-dimensional imaging of whole mouse embryos we demonstrate that extravascular budding of hematopoietic clusters is a more widespread phenomenon that occurs from the vitelline and the umbilical arteries both proximal to the embryo proper and distal in the extraembryonic yolk sac and placenta. Furthermore, we show that there are several mechanisms by which hematopoietic clusters leave the arteries, including vascular remodeling and extrusion. Lastly, we provide static images suggesting that extravascular islands contribute to the formation of new blood vessels. Thus, extravascular islands may represent a novel mechanism of vasculogenesis whereby established vessels contribute endothelial and hematopoietic cells to developing vascular beds.

Development of the lymphatic system: new questions and paradigms.

The lymphatic system is a blind-ended network of vessels that plays important roles in mediating tissue fluid homeostasis, intestinal lipid absorption and the immune response. A profound understanding of the development of lymphatic vessels, as well as of the molecular cues governing their formation and morphogenesis, might prove essential for our ability to treat lymphatic-related diseases. The embryonic origins of lymphatic vessels have been debated for over a century, with a model claiming a venous origin for the lymphatic endothelium being predominant. However, recent studies have provided new insights into the origins of lymphatic vessels. Here, we review the molecular mechanisms controlling lymphatic specification and sprouting, and we discuss exciting findings that shed new light on previously uncharacterized sources of lymphatic endothelial cells.

Histology Atlas of the Developing Mouse Hepatobiliary Hemolymphatic Vascular System with Emphasis on Embryonic Days 11.5-18.5 and Early Postnatal Development.

A critical event in embryo development is the proper formation of the vascular system, of which the hepatobiliary system plays a pivotal role. This has led researchers to use transgenic mice to identify the critical steps involved in developmental disorders associated with the hepatobiliary vascular system. Vascular development is dependent upon normal vasculogenesis, angiogenesis, and the transformation of vessels into their adult counterparts. Any alteration in vascular development has the potential to cause deformities or embryonic death. Numerous publications describe specific stages of vascular development relating to various organs, but a single resource detailing the stage-by-stage development of the vasculature pertaining to the hepatobiliary system has not been available. This comprehensive histology atlas provides hematoxylin & eosin and immunohistochemical-stained sections of the developing mouse blood and lymphatic vasculature with emphasis on the hepatobiliary system between embryonic days (E) 11.5-18.5 and the early postnatal period. Additionally, this atlas includes a 3-dimensional video representation of the E18.5 mouse venous vasculature. One of the most noteworthy findings of this atlas is the identification of the portal sinus within the mouse, which has been erroneously misinterpreted as the ductus venosus in previous publications. Although the primary purpose of this atlas is to identify normal hepatobiliary vascular development, potential embryonic abnormalities are also described.

Lymphangiogenesis and Lymphangiogenic Growth Factors.

Lymphedema is a progressive disease caused by damage to the lymphatic network. Recent development in the fields of preclinical growth factor research and lymphedema microsurgery promise new hope for lymphedema patients. In this article, we review the latest results on basic research and highlight the role of specific growth factors in normal lymphatic development and several disease states. Lymph node transfer, a new promising method in reconstructive lymphatic microsurgery, is also dependent on the lymphatic vascular regrowth and lymphangiogenic growth factors. We discuss the scientific basis of lymph node transfer and therapeutic potential of lymphangiogenic growth factors in the treatment of lymphedema.

A novel CCBE1 mutation leading to a mild form of hennekam syndrome: case report and review of the literature.

BACKGROUND: Mutations in CCBE1 have been found to be responsible for a subset of families with autosomal recessive Hennekam syndrome. Hennekam syndrome is defined as the combination of generalized lymphatic dysplasia (ie. lymphedema and lymphangiectasia), variable intellectual disability and characteristic dysmorphic features. The patient we describe here has a lymphatic dysplasia without intellectual disability or dysmorphism caused by mutation in CCBE1, highlighting the phenotypic variability that can be seen with abnormalities in this gene. CASE PRESENTATION: Our patient is a 5 week old child of Pakistani descent who presented to our center with generalized edema, ascites, and hypoalbuminemia. She was diagnosed with a protein losing enteropathy secondary to segmental primary intestinal lymphangiectasia. As the generalized edema resolved, it became clear that she had mild persistent lymphedema in her hands and feet. No other abnormalities were noted on examination and development was unremarkable at 27 months of age. Given the suspected genetic etiology and the consanguinity in the family, we used a combination of SNP genotyping and exome sequencing to identify the underlying cause of her disease. We identified several large stretches of homozygosity in the patient that allowed us to sort the variants found in the patient's exome to identify p.C98W in CCBE1 as the likely pathogenic variant. CONCLUSIONS: CCBE1 mutation analysis should be considered in all patients with unexplained lymphatic dysplasia even without the other features of classic Hennekam syndrome.

Why increased nuchal translucency is associated with congenital heart disease: a systematic review on genetic mechanisms.

This overview provides insight into the underlying genetic mechanism of the high incidence of cardiac defects in fetuses with increased nuchal translucency (NT). Nuchal edema, the morphological equivalent of increased NT, is likely to result from abnormal lymphatic development and is strongly related to cardiac defects. The underlying genetic pathways are, however, unknown. This study aims to present a systematic overview of genes involved in both cardiac and lymphatic development in mouse embryos. A search of PubMed and the Mammalian Phenotype Browser was performed. Fifteen candidate genes involved in both cardiac and lymphatic development were identified: Adrenomedullin; Chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TFII); Cyp51; Ephrin-B2; Forkhead box protein C2 (Foxc2); Nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1); Neurofibromatosis type 1 (Nf1); Phosphoinositide 3-kinase encoding isoform p110α (Pik3ca); Podoplanin; Prospero-related homeobox 1 (Prox1); T-box 1 (Tbx1); Tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains 1 (Tie1); vascular endothelial growth factor (Vegf)-A; Vegf receptor-3 (Vegfr-3); and Vascular endothelial zinc finger 1 (Vezf1). Mutations in all but one gene (Pik3ca) resulted in both a cardiac defect and nuchal edema. Candidate genes - mainly encoding for endothelium - are involved in both cardiac and lymphatic development. Alterations in candidate genes are associated with the strong relation between increased NT and cardiac defects.

Tie1 is required for lymphatic valve and collecting vessel development.

Tie1 is a receptor tyrosine kinase with broad expression in embryonic endothelium. Reduction of Tie1 levels in mouse embryos with a hypomorphic Tie1 allele resulted in abnormal lymphatic patterning and architecture, decreased lymphatic draining efficiency, and ultimately, embryonic demise. Here we report that Tie1 is present uniformly throughout the lymphatics and from late embryonic/early postnatal stages, becomes more restricted to lymphatic valve regions. To investigate later events of lymphatic development, we employed Cre-loxP recombination utilizing a floxed Tie1 allele and an Nfatc1Cre line, to provide loxP excision predominantly in lymphatic endothelium and developing valves. Interestingly, unlike the early prenatal defects previously described by ubiquitous endothelial deletion, excision of Tie1 with Nfatc1Cre resulted in abnormal lymphatic defects in postnatal mice and was characterized by agenesis of lymphatic valves and a deficiency of collecting lymphatic vessels. Attenuation of Tie1 signaling in lymphatic endothelium prevented initiation of lymphatic valve specification by Prox1 high expression lymphatic endothelial cells that is associated with the onset of turbulent flow in the lymphatic circulation. Our findings reveal a fundamental role for Tie1 signaling during lymphatic vessel remodeling and valve morphogenesis and implicate it as a candidate gene involved in primary lymphedema.

The left-right Pitx2 pathway drives organ-specific arterial and lymphatic development in the intestine.

The dorsal mesentery (DM) is the major conduit for blood and lymphatic vessels in the gut. The mechanisms underlying their morphogenesis are challenging to study and remain unknown. Here we show that arteriogenesis in the DM begins during gut rotation and proceeds strictly on the left side, dependent on the Pitx2 target gene Cxcl12. Although competent Cxcr4-positive angioblasts are present on the right, they fail to form vessels and progressively emigrate. Surprisingly, gut lymphatics also initiate in the left DM and arise only after-and dependent on-arteriogenesis, implicating arteries as drivers of gut lymphangiogenesis. Our data begin to unravel the origin of two distinct vascular systems and demonstrate how early left-right molecular asymmetries are translated into organ-specific vascular patterns. We propose a dual origin of gut lymphangiogenesis in which prior arterial growth is required to initiate local lymphatics that only subsequently connect to the vascular system.

Essential role of the zinc finger transcription factor Casz1 for mammalian cardiac morphogenesis and development.

Chromosome 1p36 deletion syndrome is one of the most common terminal deletions observed in humans and is related to congenital heart disease (CHD). However, the 1p36 genes that contribute to heart disease have not been clearly delineated. Human CASZ1 gene localizes to 1p36 and encodes a zinc finger transcription factor. Casz1 is required for Xenopus heart ventral midline progenitor cell differentiation. Whether Casz1 plays a role during mammalian heart development is unknown. Our aim is to determine 1p36 gene CASZ1 function at regulating heart development in mammals. We generated a Casz1 knock-out mouse using Casz1-trapped embryonic stem cells. Casz1 deletion in mice resulted in abnormal heart development including hypoplasia of myocardium, ventricular septal defect, and disorganized morphology. Hypoplasia of myocardium was caused by decreased cardiomyocyte proliferation. Comparative genome-wide RNA transcriptome analysis of Casz1 depleted embryonic hearts identifies abnormal expression of genes that are critical for muscular system development and function, such as muscle contraction genes TNNI2, TNNT1, and CKM; contractile fiber gene ACTA1; and cardiac arrhythmia associated ion channel coding genes ABCC9 and CACNA1D. The transcriptional regulation of some of these genes by Casz1 was also found in cellular models. Our results showed that loss of Casz1 during mouse development led to heart defect including cardiac noncompaction and ventricular septal defect, which phenocopies 1p36 deletion syndrome related CHD. This suggests that CASZ1 is a novel 1p36 CHD gene and that the abnormal expression of cardiac morphogenesis and contraction genes induced by loss of Casz1 contributes to the heart defect.

Lymphatic function is required prenatally for lung inflation at birth.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

Alk3/Alk3b and Smad5 mediate BMP signaling during lymphatic development in zebrafish.

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.

Fusing VE-cadherin to α-catenin impairs fetal liver hematopoiesis and lymph but not blood vessel formation.

We have recently shown that genetic replacement of VE-cadherin by a VE-cadherin-α-catenin fusion construct strongly impairs opening of endothelial cell contacts during leukocyte extravasation and induction of vascular permeability in adult mice. Here we show that this mutation leads to lethality at midgestation on a clean C57BL/6 background. Investigating the reasons for embryonic lethality, we observed a lack of fetal liver hematopoiesis and severe lymphedema but no detectable defects in blood vessel formation and remodeling. As for the hematopoiesis defect, VE-cadherin-α-catenin affected neither the generation of hematopoietic stem and progenitor cells (HSPCs) from hemogenic endothelium nor their differentiation into multiple hematopoietic lineages. Instead, HSPCs accumulated in the fetal circulation, suggesting that their entry into the fetal liver was blocked. Edema formation was caused by disturbed lymphatic vessel development. Lymphatic progenitor cells of VE-cadherin-α-catenin-expressing embryos were able to leave the cardinal vein and migrate to the site of the first lymphatic vessel formation, yet subsequently, these cells failed to form large lumenized lymphatic vessels. Thus, stabilizing endothelial cell contacts by a covalent link between VE-cadherin and α-catenin affects recruitment of hematopoietic progenitors into the fetal liver and the development of lymph but not blood vessels.

Control of retinoid levels by CYP26B1 is important for lymphatic vascular development in the mouse embryo.

During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.

Dynamics of pleural fluid effusion and chylothorax in the fetus and newborn: role of the lymphatic system.

Pleural fluid effusion particularly chylothorax is a relatively rare occurrence in the newborn, but when it occurs it is often life-threatening. In this article, we describe and illustrate the morphologic features of the visceral and parietal pleura including pleural lymphatics and the physiology and pathophysiology of pleural fluid balance. The role and function of the lymphatic system in controlling the volume and composition of pleural liquid are detailed and a conceptual scheme presented. Finally, the crucial role of inadequate lymphatic drainage (either functional overload from an imbalance in Starling forces or mechanical insufficiency from lymphatic dysplasia) is emphasized.

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development.

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfc(um18) could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.