Lymphatic Pathways of the Thorax: Predictable Patterns of Spread.

OBJECTIVE. This article reviews thoracic lymphatic pathways and tributaries, discusses lymphatic anatomic variants and their clinical implications, and emphasizes common patterns of thoracic lymphadenopathy from extrapulmonary malignancies. CONCLUSION. Recognition of common patterns and pathways of thoracic lymphatic drainage can help identify the site of tumor origin and allow a more focused examination of disease extent, both of which are important for disease prognosis and management.

MicroRNA-126a Directs Lymphangiogenesis Through Interacting With Chemokine and Flt4 Signaling in Zebrafish.

OBJECTIVE: MicroRNA-126 (miR-126) is an endothelium-enriched miRNA and functions in vascular integrity and angiogenesis. The application of miRNA as potential biomarker and therapy target has been widely investigated in various pathological processes. However, its role in lymphatic diseases had not been widely explored. We aimed to reveal the role of miR-126 in lymphangiogenesis and the regulatory signaling pathways for potential targets of therapy. APPROACH AND RESULTS: Loss-of-function studies using morpholino oligonucleotides and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system showed that silencing of miR-126a severely affected the formation of parachordal lymphangioblasts and thoracic duct in zebrafish embryos, although their development in miR-126b knockdown embryos was normal. Expression analyses by in situ hybridization and immunofluorescence indicated that miR-126a was expressed in lymphatic vessels, as well as in blood vessels. Time-lapse confocal imaging assay further revealed that knockdown of miR-126a blocked both lymphangiogenic sprouts budding from the posterior cardinal vein and lymphangioblasts extension along horizontal myoseptum. Bioinformatics analysis and in vivo report assay identified that miR-126a upregulated Cxcl12a by targeting its 5' untranslated region. Moreover, loss- and gain-of-function studies revealed that Cxcl12a signaling acted downstream of miR-126a during parachordal lymphangioblast extension, whereby Flt4 signaling acts as a cooperator of miR-126a, allowing it to modulate lymphangiogenic sprout formation. CONCLUSIONS: These findings demonstrate that miR-126a directs lymphatic endothelial cell sprouting and extension by interacting with Cxcl12a-mediated chemokine signaling and Vegfc-Flt4 signal axis. Our results suggest that these key regulators of lymphangiogenesis may be involved in lymphatic pathogenesis of cardiovascular diseases.

The thoracic duct: clinical importance, anatomic variation, imaging, and embolization.

UNLABELLED: The thoracic duct is the body's largest lymphatic conduit, draining upwards of 75 % of lymphatic fluid and extending from the cisterna chyli to the left jugulovenous angle. While a typical course has been described, it is estimated that it is present in only 40-60% of patients, often complicating already challenging interventional procedures. The lengthy course predisposes the thoracic duct to injury from a variety of iatrogenic disruptions, as well as spontaneous benign and malignant lymphatic obstructions and idiopathic causes. Disruption of the thoracic duct frequently results in chylothoraces, which subsequently cause an immunocompromised state, contribute to nutritional depletion, and impair respiratory function. Although conservative dietary treatments exist, the majority of thoracic duct disruptions require embolization in the interventional suite. This article provides a comprehensive review of the clinical importance of the thoracic duct, relevant anatomic variants, imaging, and embolization techniques for both diagnostic and interventional radiologists as well as for the general medical practitioner. KEY POINTS: • Describe clinical importance, embryologic origin, and typical course of the thoracic duct. • Depict common/lesser-known thoracic duct anatomic variants and discuss their clinical significance. • Outline the common causes of thoracic duct injury and indications for embolization. • Review the thoracic duct embolization procedure including both pedal and intranodal approaches. • Present and illustrate the success rates and complications associated with the procedure.

Junb controls lymphatic vascular development in zebrafish via miR-182.

JUNB, a subunit of the AP-1 transcription factor complex, mediates gene regulation in response to a plethora of extracellular stimuli. Previously, JUNB was shown to act as a critical positive regulator of blood vessel development and homeostasis as well as a negative regulator of proliferation, inflammation and tumour growth. Here, we demonstrate that the oncogenic miR-182 is a novel JUNB target. Loss-of-function studies by morpholino-mediated knockdown and the CRISPR/Cas9 technology identify a novel function for both JUNB and its target miR-182 in lymphatic vascular development in zebrafish. Furthermore, we show that miR-182 attenuates foxo1 expression indicating that strictly balanced Foxo1 levels are required for proper lymphatic vascular development in zebrafish. In conclusion, our findings uncover with the Junb/miR-182/Foxo1 regulatory axis a novel key player in governing lymphatic vascular morphogenesis in zebrafish.

Essential role of Apelin signaling during lymphatic development in zebrafish.

OBJECTIVE: Apelin and its cognate receptor Aplnr/Apj are essential for diverse biological processes. However, the function of Apelin signaling in lymphatic development remains to be identified, despite the preferential expression of Apelin and Aplnr within developing blood and lymphatic endothelial cells in vertebrates. In this report, we aim to delineate the functions of Apelin signaling during lymphatic development. APPROACH AND RESULTS: We investigated the functions of Apelin signaling during lymphatic development using zebrafish embryos and found that attenuation of Apelin signaling substantially decreased the formation of the parachordal vessel and the number of lymphatic endothelial cells within the developing thoracic duct, indicating an essential role of Apelin signaling during the early phase of lymphatic development. Mechanistically, we found that abrogation of Apelin signaling selectively attenuates lymphatic endothelial serine-threonine kinase Akt 1/2 phosphorylation without affecting the phosphorylation status of extracellular signal-regulated kinase 1/2. Moreover, lymphatic abnormalities caused by the reduction of Apelin signaling were significantly exacerbated by the concomitant partial inhibition of serine-threonine kinase Akt/protein kinase B signaling. Apelin and vascular endothelial growth factor-C (VEGF-C) signaling provide a nonredundant activation of serine-threonine kinase Akt/protein kinase B during lymphatic development because overexpression of VEGF-C or apelin was unable to rescue the lymphatic defects caused by the lack of Apelin or VEGF-C, respectively. CONCLUSIONS: Taken together, our data present compelling evidence suggesting that Apelin signaling regulates lymphatic development by promoting serine-threonine kinase Akt/protein kinase B activity in a VEGF-C/VEGF receptor 3-independent manner during zebrafish embryogenesis.

HOXC9 regulates formation of parachordal lymphangioplasts and the thoracic duct in zebrafish via stabilin 2.

HOXC9 belongs to the family of homeobox transcription factors, which are regulators of body patterning and development. HOXC9 acts as a negative regulator on blood endothelial cells but its function on lymphatic vessel development has not been studied. The hyaluronan receptor homologs stabilin 1 and stabilin 2 are expressed in endothelial cells but their role in vascular development is poorly understood. This study was aimed at investigating the function of HOXC9, stabilin 2 and stabilin 1 in lymphatic vessel development in zebrafish and in endothelial cells. Morpholino-based expression silencing of HOXC9 repressed parachordal lymphangioblast assembly and thoracic duct formation in zebrafish. HOXC9 positively regulated stabilin 2 expression in zebrafish and in HUVECs and expression silencing of stabilin 2 phenocopied the HOXC9 morphant vascular phenotype. This effect could be compensated by HOXC9 mRNA injection in stabilin 2 morphant zebrafish embryos. Stabilin 1 also regulated parachordal lymphangioblast and thoracic duct formation in zebrafish but acts independently of HOXC9. On a cellular level stabilin 1 and stabilin 2 regulated endothelial cell migration and in-gel sprouting angiogenesis in endothelial cells. HOXC9 was identified as novel transcriptional regulator of parachordal lymphangioblast assembly and thoracic duct formation in zebrafish that acts via stabilin 2. Stabilin 1, which acts independently of HOXC9, has a similar function in zebrafish and both receptors control important cellular processes in endothelial cells.

The RAS guanyl nucleotide-releasing protein RasGRP1 is involved in lymphatic development in zebrafish.

The molecular basis of the lymphatic development remains largely unknown. Using zebrafish as a model, we discovered a novel role for the Ras guanine-releasing protein 1 (RasGRP1), a protein involved in Ras activation in lymphangiogenesis. Secondary lymphatic sprouts from the posterior cardinal vein give rise to thoracic duct which is the first lymphatic vessel in zebrafish. Knockdown of rasgrp1 by injecting morpholino in zebrafish embryos impaired formation of thoracic duct accompanied by pericardial and truck edema, whereas blood vessel development of the embryos was largely unaffected. In rasgrp1-knockdown embryos, the number of sprouts producing the string of parachordal lymphangioblast cells was reduced. Meanwhile the total number of the secondary sprouts was not changed. As a result, the number of venous intersegmental vessels was increased, whereas the number of lymphatic vessel was reduced at a later stage. The lymphatic developmental defects caused by rasgrp1 knockdown could be rescued by ectopic expression of a constitutively active HRas. Further analysis revealed that RasGRP1 knockdown could synergize with flt4/vegfr3 knockdown to induce defects in lymphangiogenesis. Taken together, this finding demonstrates a critical role for RasGRP1 in lymphatic development in zebrafish.

Anatomy of the thoracic duct.

The thoracic duct is a major anatomic structure of the upper part of the abdomen, chest, and the lower part of the neck. This article reviews the embryology, anatomy, and multiple variations of the thoracic duct. Proper knowledge of this anatomy should ease understanding the pathophysiology of diseases involving the lymph channels and also prevent injury to the duct during major procedures in which the duct or its tributaries can be involved.

Role of synectin in lymphatic development in zebrafish and frogs.

The molecular basis of lymphangiogenesis remains incompletely characterized. Here, we document a novel role for the PDZ domain-containing scaffold protein synectin in lymphangiogenesis using genetic studies in zebrafish and tadpoles. In zebrafish, the thoracic duct arises from parachordal lymphangioblast cells, which in turn derive from secondary lymphangiogenic sprouts from the posterior cardinal vein. Morpholino knockdown of synectin in zebrafish impaired formation of the thoracic duct, due to selective defects in lymphangiogenic but not angiogenic sprouting. Synectin genetically interacted with Vegfr3 and neuropilin-2a in regulating lymphangiogenesis. Silencing of synectin in tadpoles caused lymphatic defects due to an underdevelopment and impaired migration of Prox-1(+) lymphatic endothelial cells. Molecular analysis further revealed that synectin regulated Sox18-induced expression of Prox-1 and vascular endothelial growth factor C-induced migration of lymphatic endothelial cells in vitro. These findings reveal a novel role for synectin in lymphatic development.

Role of delta-like-4/Notch in the formation and wiring of the lymphatic network in zebrafish.

OBJECTIVE: To study whether Notch signaling, which regulates cell fate decisions and vessel morphogenesis, controls lymphatic development. METHODS AND RESULTS: In zebrafish embryos, sprouts from the axial vein have lymphangiogenic potential because they give rise to the first lymphatics. Knockdown of delta-like-4 (Dll4) or its receptors Notch-1b or Notch-6 in zebrafish impaired lymphangiogenesis. Dll4/Notch silencing reduced the number of sprouts producing the string of parchordal lymphangioblasts; instead, sprouts connecting to the intersomitic vessels were formed. At a later phase, Notch silencing impaired navigation of lymphatic intersomitic vessels along their arterial templates. CONCLUSIONS: These studies imply critical roles for Notch signaling in the formation and wiring of the lymphatic network.

[An anatomical variation: the aberrant termination of the thoracic duct]. / Una variación anatómica: la desembocadura aberrante del conducto torácico.

In a male cadaver dissected at the Department of Morphology, University del Valle, Call (Colombia), a rarely described anatomical variation was found. It consisted of an aberrant termination or drainage of the thoracic lymph duct. Normally, this duct ascends in the thorax behind the esophagus, gradually diverges towards the left side of the neck and ends in the left jugulo-subclavian confluent--either in the internal jugular vein or in the subclavian vein. In the case of this cadaver, the thoracic duct diverged towards the right side of the neck to end in the right internal jugular vein. The present work describes the embryonic origin of the duct and offers a possible explanation for the anatomical variation encountered.

LPA1 is essential for lymphatic vessel development in zebrafish.

Lysophosphatidic acid (LPA) has long been implicated in regulating vascular development via endothelial cell-expressed G protein-coupled receptors. However, because of a lack of notable vascular defects reported in LPA receptor knockout mouse studies, the regulation of vasculature by LPA receptors in vivo is still uncertain. Using zebrafish as a model, we studied the gene expression patterns and functions of an LPA receptor, LPA(1), during embryonic development, in particular, vascular formation. Whole-mount in situ hybridization experiments revealed that zebrafish lpa(1) (zlpa(1)) was ubiquitously expressed early in development, and its expression domains were later localized to the head region and the vicinity of the dorsal aorta. The expression of zlpa(1) surrounding the dorsal aorta suggests its role in vasculature development. Knocking down of zLPA(1) by injecting morpholino (MO) oligonucleotides at 0.625-1.25 ng per embryo resulted in the absence of thoracic duct and edema in pericardial sac and trunk in a dose-dependent manner. These zlpa(1)-MO-resulted defects could be specifically rescued by ectopic expression of zlpa(1). In addition, overexpression of vegf-c, a well-known lymphangiogenic factor, also partially ameliorated the inhibition of thoracic duct development. Taken together, these results demonstrate that LPA(1) is necessary for lymphatic vessel formation during embryonic development in zebrafish.

An avian model for studies of embryonic lymphangiogenesis.

Embryonic development of lymphatics (lymphangiogenesis) in recent years has rarely been studied experimentally. Using an avian model, we showed that both intra- and extra-embryonic blood vessels of chick and quail embryos are accompanied by lymphatics. The lymphatics of the chorioallantoic membrane (CAM) are drained by lymphatic trunks of the umbilicus and are connected to the posterior lymph hearts. Intra-embryonic lymphatics are drained via paired thoracic ducts into the jugulo-subclavian junction. The lymphatic endothelial cells are characterized by the expression of Vascular Endothelial Growth Factor Receptors (VEGFR) -2 and -3. Application of VEGF-C, the ligand of these two receptors, on the differentiated CAM, induces proliferation of lymphatic endothelial cells and formation of huge lymphatic sinuses. These lymphatics derive from pre-existing lymphatic endothelial cells, whereas, in early embryos lymphangioblasts are present in the mesenchyme. This phenomenon can be demonstrated by interspecific grafting experiments between chick and quail embryos. Together with the early lymph sacs, the lymphangioblasts form the embryonic lymphatic system. Our studies demonstrate the importance of lymphangioblasts and lymphangiogenic growth factors in embryonic lymphangiogenesis.

[Variants in the organization of the cervical portion of the human thoracic duct in prenatal ontogeny]. / Varianty organizatsii sheinoi chasti grudnogo protoka cheloveka v prenatal'nom periode ontogeneza.

Using macro- and microscopic methods stages of formation, forms of spatial and temporal organization and definitive variants of cervical part of human thoracic duct were studied in 49 5-8 wks old embryos, 120 8.5-36 wks old embryo and 10 human newborns. Variability of structure of thoracic duct cervical part is a reflection of parameters characterizing magistralization of jugular prevertebral lymphatic plexus: its depth (maximum, medium, minimum), topographic variant (medial, lateral, combined) and length (small, moderate, significant). With the aspect genetic 3 types of its organization were distinguished: jugular (51%), subjugular (11.3%) and combined (37.7%). In prematurely born children and newborns jugular type of the duct cervical part is the most favourable for the drainage.

[Features of morphogenesis of the initial region of the rat thoracic duct in prenatal ontogenesis]. / Osobennosti morfogeneza nachal'nogo otdela grudnogo protoka v prenatal'nom ontogeneze krysy.

In embryogenesis initial region of thoracic duct and its roots is formed in rat at later stages than in man. Their morphogenesis and topography and small number of lumbar lymph nodes correlate with species-specific peculiarities of organogenesis in rat.

[The development of the thoracic duct lymphangions in the prenatal period of human ontogeny]. / Razvitie limfangionov grudnogo protoka v prenatal'nom periode ontogeneza cheloveka.

The study was performed in 77 fetuses and 10 cadavers of newborns using the complex of methods, the basic one being the total preparation after A. V. Borisov. Thoracic duct lymphangions develop craniocaudad in 13-18 week fetuses. Maximal number of intervalvular segments (up to 35) was observed during the middle of intrauterine development. They achieve definitive number (15-20) by the moment of birth which correlates with active formation of muscular base. The wall of duct in fetus has constructions of capillary, postcapillary, non-muscular and muscular type vessel. Lymphangion is primitive in 13-28 week fetuses and definitive in 29-36 week ones. Local peculiarities of lymphangion myoarchitectonics were shown. The role of accessory beds of the duct in lymph passage was demonstrated.

[Variants in the definitive organization of the human thoracic duct from the positions of its preceding origin]. / Varianty definitivnoi organizatsii grudnogo protoka cheloveka s pozitsii predshestvuiushchego geneza.

The study was performed in 120 corpses of 8.5-36 weeks human fetuses using macro-microscopic methods complex. Thoracic duct definitive organization variants as monotrunk, transitional forms and plexus reflect strong, middle and slight magistralization extent of antevertebral and adjacent plexuses. Peculiarities of the duct regions positions are defined by topographical variants of adaptive reorganizations--left-side, right-side and bilateral ones.

Thoracic duct function in fetal, newborn, and adult sheep.

We measured thoracic duct lymph flow rate versus outflow pressure in 7 chronically catheterized adult sheep and in 6 newborn lambs and compared our results to data previously obtained from 10 fetal sheep. In fetal sheep the thoracic duct lymph flow rate was 34.5 +/- 17.2 ml/hr or 11.7 +/- 6.0 ml/kg/hr. Fetal thoracic duct lymph flow deviated from baseline between 8 and 12 torr outflow pressure and lymph stopped at 18 +/- 2.5 torr. In newborn lambs the thoracic duct lymph flow rate was 49.5 +/- 22.0 ml/hr or 7.4 +/- 2.5 ml/kg/hr. The range of outflow pressures over which newborn lymph flow deviated from baseline was between 15 and 18 torr and lymph flow stopped at 26.2 +/- 6.4 torr. Adult sheep thoracic duct lymph flow rate was 130 +/- 74 ml/hr or 2.3 +/- 1.3 ml/kg/hr. Adult lymph flow deviated from baseline between 25 and 35 torr and stopped at an outflow pressure of 41.7 +/- 6.7 torr. The ability of the thoracic duct to return lymph against an outflow pressure improves with maturation. However, lymph flow rate corrected for body weight is greatest in immature animals. The higher corrected lymph flow rate in conjunction with the decreased ability to pump against an outflow pressure may help account for immature animals predisposition for edema.

Thoracic duct lymph flow responses to hemorrhage in the ovine fetus.

Blood volume returns toward normal after hemorrhage much more rapidly in the fetus than in the adult due to a rapid entry of fluid and plasma proteins into the fetal circulation. One potential source of fetal fluids and plasma proteins is the lymphatic system, since basal lymph flow rate and interstitial protein concentration are high in the fetus. Furthermore, studies in adults suggest that lymph flow rate may increase following hemorrhage. To test the hypothesis that hemorrhage induces an increase in lymph flow in the fetus, 15 late-gestation ovine fetuses underwent left thoracic duct catheterization with low-resistance catheters and were studied 5 or more days after surgery at 134 +/- 1 (SE) days gestation. The protocol included three successive 30-min periods: control, hemorrhage, and recovery. During the first 5 min of the hemorrhagic period, 61 +/- 4 ml of fetal blood were removed. The blood was reinfused over the first 5 min of the recovery period. After the hemorrhage, fetal arterial pressure, venous pressure, and heart rate decreased (analysis of variance, P < 0.001). These variables significantly increased above basal levels following blood reinfusion. Fetal hematocrit (P < 0.001) and plasma protein concentration (P < 0.05) also decreased after the hemorrhage and returned to control values after the reinfusion. Fetal lymph flow rate was 0.55 +/- 0.06 (SE) ml/min before the hemorrhage and decreased by a maximum of 30.3 +/- 6.3% (P < 0.001) at 8 min after the end of the hemorrhage. Lymph flow rate was reduced by an average of 19.1 +/- 6.6% during the hemorrhagic period and returned to prehemorrhage levels following blood reinfusion.(ABSTRACT TRUNCATED AT 250 WORDS)