Lymphatic Distribution of Etanercept Following Intravenous and Subcutaneous Delivery to Rats.

PURPOSE: The purpose of this work was to investigate the role of the lymphatic system in the pharmacokinetics of etanercept, a fusion protein. METHODS: Etanercept 1 mg/kg was administered intravenously (IV) and subcutaneously (SC) to thoracic lymph duct-cannulated and sham-operated control rats. Blood and lymph samples were obtained for up to 6 days. RESULTS: Model-based SC bioavailability of etanercept was 65.2% in the control group. In lymph-cannulated rats, etanercept concentration in the lymph was consistently lower than in serum following IV dosing; and the concentration in the lymph was significantly higher than in serum after SC injection. The absorption occurred predominantly through the lymphatic pathway (82.7%), and only 17.3% by direct uptake into the central compartment (blood pathway). Lymphatic cannulation reduced the area under the serum concentration-time curve by 28% in IV group and by 91% in SC group. A mechanistic pharmacokinetic model that combined dual absorption pathways with redistribution of the systemically available protein drug into lymph was developed. The model successfully captured serum and lymph data in all groups simultaneously, and all parameters were estimated with sufficient precision. CONCLUSIONS: Lymphatic system was shown to play an essential role in systemic disposition and SC absorption of etanercept.

Effects of Long-Term High-Fat Diet and Its Reversal on Lipids and Lipoproteins Composition in Thoracic Duct Lymph in Pigs.

BACKGROUND This study was carried out to evaluate the effects of a long-term high-fat diet on lipids and lipoproteins composition in thoracic duct lymph in pigs. MATERIAL AND METHODS We examined lymph taken from the thoracic duct from 24 female white sharp-ear pigs, divided into 3 experimental groups fed different diets for 12 months: (a) the control group, fed the standard balanced diet; (b) the HFD group, fed an unbalanced, high-fat diet, and (c) the reversal diet group (RD), fed an unbalanced, high-fat diet for 9 months and then a standard balanced diet for 3 months. RESULTS Lymph analysis after 12 months of fixed diets revealed significantly higher concentration of proteins in the HFD group in comparison to the control and RD groups. Examination of lymph lipoproteins fractions showed that the high-fat diet in the HFD group in comparison to control group caused an increase in cholesterol, phospholipids, and proteins content within HDL and chylomicrons. There were also more proteins within HDL in the HFD group in comparison to the RD group and more triglycerides within chylomicrons in the HFD group in comparison to the control group. CONCLUSIONS A long-term high-fat diet resulted in changed structure of HDL and chylomicrons in the thoracic duct lymph. Alterations in HDL composition suggest that a high-fat diet enhances reverses cholesterol transport. Changes in chylomicrons structure show the adaptation to more intense transport of dietary fat from the intestine to the liver under the influence of a high-fat diet. Reversal to a standard balanced diet had the opposite effects.

Quantification of Angiogenesis and Lymphangiogenesis in the Dual ex vivo Aortic and Thoracic Duct Assay.

BACKGROUND: Lymphatic vessel formation (lymphangiogenesis) plays important roles in cancer metastasis, organ rejection, and lymphedema, but the underlying molecular events remain unclear. Furthermore, despite significant overlap in the molecular families involved in angiogenesis and lymphangiogenesis, little is known about the crosstalk between these processes. The ex vivo aortic ring assay and lymphatic ring assay have enabled detailed studies of vessel sprouting, but harvesting and imaging clear thoracic duct samples remain challenging. Here we present a modified ex vivo dual aortic ring and thoracic duct assay using tissues from dual fluorescence reporter Prox1- GFP/Flt1-DsRed (PGFD) mice, which permit simultaneous visualization of blood and lymphatic endothelial cells. OBJECTIVE: To characterize the concurrent sprouting of intrinsically fluorescent blood and lymphatic vessels from harvested aorta and thoracic duct samples. METHODS: Dual aorta and thoracic duct specimens were harvested from PGFD mice, grown in six types of endothelial cell growth media (one control, five that each lack a specific growth factor), and visualized by confocal fluorescence microscopy. Linear mixed models were used to compare the extent of vessel growth and sprouting over a 28-day period. RESULTS: Angiogenesis occurred prior to lymphangiogenesis in our assay. The control medium generally induced superior growth of both vessel types compared with the different modified media formulations. The greatest decrease in lymphangiogenesis was observed in vascular endothelial growth factor-C (VEGF-C)-devoid medium, suggesting the importance of VEGF-C in lymphangiogenesis. CONCLUSION: The modified ex vivo dual aortic ring and thoracic duct assay represents a powerful tool for studying angiogenesis and lymphangiogenesis in concert.

Alleviation of Metabolic Syndrome with Dietary Egg White Protein.

Abdominal fat accumulation causes metabolic syndrome, which is a cluster of metabolic abnormalities such as dyslipidemia, glucose intolerance, insulin resistance or hyperinsulinemia, and hypertension, leading to the development of diabetes and cardiovascular disease. Diets are known to contribute to the development or prevention of metabolic syndrome. Several studies have reported that the quality of dietary proteins may be an important modulator of the risk of this syndrome. We investigated the effects of consuming egg white protein (EWP) or lactic-fermented egg white (LE), an easy-to-consume form of egg white, on the development of metabolic syndrome in animal models and humans. In comparison with casein, dietary EWP decreased lymphatic lipid transport in thoracic lymph duct-cannulated rats. In an in vitro experiment, EWP pepsin hydrolysate decreased the cholesterol micellar solubility and cholesterol transfer rate from micelles to oil phase, and increased water-holding capacity, settling volume in water, and relative viscosity compared with casein pepsin hydrolysate. The daily consumption of LE for 8 weeks reduced serum total cholesterol and LDL cholesterol levels in men with mild hypercholesterolemia. Furthermore, dietary EWP reduced the body fat mass of rats by increasing the body protein mass and accelerating hepatic ß-oxidation. The daily consumption of LE for 12 weeks reduced the visceral fat area and improved the ratio of the visceral to subcutaneous fat area. Taken together, these results indicated that dietary EWP and LE would be useful for preventing or alleviating metabolic syndrome.

Inhibition of Niemann-Pick C1-Like 1 by Ezetimibe Reduces Dietary 5ß,6ß-Epoxycholesterol Absorption in Rats.

PURPOSE: Oxycholesterols (OCs) are produced from cholesterol by oxidation of the steroidal backbone and side-chain. OCs are present in blood and evidence suggests their involvement in disease development and progression. However, limited information is available regarding the absorption mechanisms and relative absorption rates of dietary OCs. Although ezetimibe is known to inhibit intestinal cholesterol absorption via Niemann-Pick C1-Like 1 (NPC1L1), whether it also inhibits dietary OC absorption is unclear. METHODS: We investigated the effects of ezetimibe on OC absorption in rats fed an OC-rich diet containing 10 different OCs. We collected lymphatic fluid using permanent cannulation of the thoracic duct and quantified OC levels. RESULTS: Ezetimibe treatment significantly reduced the apparent absorption of 5ß,6ß-epoxycholesterol (5,6ß-epoxy) and its levels in the proximal intestinal mucosa in OC-fed rats. Using in silico analyses, the binding energy of NPC1L1 N-terminal domain (NPC1L1-NTD) and 5,6ß-epoxy was found to be similar to that of NPC1L1-NTD and cholesterol, suggesting that polar uncharged amino acids located in the steroidal part of 5,6ß-epoxy were involved. CONCLUSION: Our results indicate that ezetimibe-mediated inhibition of dietary OC absorption varies depending on the specific OC, and only the absorption of 5,6ß-epoxy is significantly reduced.

Identification of the Thoracic Duct Using Indocyanine Green During Cervical Lymphadenectomy.

BACKGROUND: Injury to the thoracic duct (TD) is the most common complication after a left lateral neck dissection, and it carries a high degree of morbidity. Currently, no routine diagnostic imaging is used to assist with TD identification intraoperatively. This report describes the first clinical experience with lymphangiography using indocyanine green (ICG) during lateral neck dissections. METHODS: In six patients undergoing left lateral neck dissection (levels 2-4) for either thyroid cancer or melanoma, 2.5-5 mg of ICG was injected in the dorsum of the left foot 15 min before imaging. Intraoperative imaging was performed with a hand-held near infrared (NIR) camera (Hamamatsu, PDE-Neo, Hamamatsu City, Japan). RESULTS: In five patients, the TD was visualized using NIR fluorescence, with a time of 15-90 min from injection to identification. Imaging was optimized by positioning the camera at the angle of the mandible and pointing into the space below the clavicle. No adverse reactions from the ICG injection occurred, and the time required for imaging was 5-10 min. No intraoperative TD injury was identified, and no chyle leak occurred postoperatively. For the one patient in whom the TD was not identified, it is unclear whether this was related to the timing of the injection or to duct obliteration from a prior dissection. CONCLUSION: This is the first described application of ICG lymphangiography to identify the thoracic duct during left lateral neck dissection. Identification of TD with ICG is technically feasible, simple to perform with NIR imaging, and safe, making it a potential important adjunct for the surgeon.

Polydom Is an Extracellular Matrix Protein Involved in Lymphatic Vessel Remodeling.

RATIONALE: Lymphatic vasculature constitutes a second vascular system essential for immune surveillance and tissue fluid homeostasis. Maturation of the hierarchical vascular structure, with a highly branched network of capillaries and ducts, is crucial for its function. Environmental cues mediate the remodeling process, but the mechanism that underlies this process is largely unknown. OBJECTIVE: Polydom (also called Svep1) is an extracellular matrix protein identified as a high-affinity ligand for integrin α9ß1. However, its physiological function is unclear. Here, we investigated the role of Polydom in lymphatic development. METHODS AND RESULTS: We generated Polydom-deficient mice. Polydom-/- mice showed severe edema and died immediately after birth because of respiratory failure. We found that although a primitive lymphatic plexus was formed, it failed to undergo remodeling in Polydom-/- embryos, including sprouting of new capillaries and formation of collecting lymphatic vessels. Impaired lymphatic development was also observed after knockdown/knockout of polydom in zebrafish. Polydom was deposited around lymphatic vessels, but secreted from surrounding mesenchymal cells. Expression of Foxc2 (forkhead box protein c2), a transcription factor involved in lymphatic remodeling, was decreased in Polydom-/- mice. Polydom bound to the lymphangiogenic factor Ang-2 (angiopoietin-2), which was found to upregulate Foxc2 expression in cultured lymphatic endothelial cells. Expressions of Tie1/Tie2 receptors for angiopoietins were also decreased in Polydom-/- mice. CONCLUSIONS: Polydom affects remodeling of lymphatic vessels in both mouse and zebrafish. Polydom deposited around lymphatic vessels seems to ensure Foxc2 upregulation in lymphatic endothelial cells, possibly via the Ang-2 and Tie1/Tie2 receptor system.

Levels of miRNA and Hormones in Thoracic Duct Lymph in Rats with Experimental Breast Cancer Induced by N-Methyl-N-Nitrosourea.

We studied hormone levels in the thoracic duct lymph and expression of miRNA involved in the pathogenesis of breast cancer induced in rats by intramammary injection of N-methyl-Nnitrosourea. The correlations between miRNA expression and hormone levels depended on the type of treatment.

Postnatal Deletion of Podoplanin in Lymphatic Endothelium Results in Blood Filling of the Lymphatic System and Impairs Dendritic Cell Migration to Lymph Nodes.

OBJECTIVE: The lymphatic vascular system exerts major physiological functions in the transport of interstitial fluid from peripheral tissues back to the blood circulation and in the trafficking of immune cells to lymph nodes. Previous studies in global constitutive knockout mice for the lymphatic transmembrane molecule podoplanin reported perinatal lethality and a complex phenotype with lung abnormalities, cardiac defects, lymphedema, blood-filled lymphatic vessels, and lack of lymph node organization, reflecting the importance of podoplanin expression not only by the lymphatic endothelium but also by a variety of nonendothelial cell types. Therefore, we aimed to dissect the specific role of podoplanin expressed by adult lymphatic vessels. APPROACH AND RESULTS: We generated an inducible, lymphatic-specific podoplanin knockout mouse model (PdpnΔLEC) and induced gene deletion postnatally. PdpnΔLEC mice were viable, and their lymphatic vessels appeared morphologically normal with unaltered fluid drainage function. Intriguingly, PdpnΔLEC mice had blood-filled lymph nodes and vessels, most frequently in the neck and axillary region, and displayed a blood-filled thoracic duct, suggestive of retrograde filling of blood from the blood circulation into the lymphatic system. Histological and fluorescence-activated cell sorter analyses revealed normal lymph node organization with the presence of erythrocytes within lymph node lymphatic vessels but not surrounding high endothelial venules. Moreover, fluorescein isothiocyanate painting experiments revealed reduced dendritic cell migration to lymph nodes in PdpnΔLEC mice. CONCLUSIONS: These results reveal an important role of podoplanin expressed by lymphatic vessels in preventing postnatal blood filling of the lymphatic vascular system and in contributing to efficient dendritic cell migration to the lymph nodes.

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.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome.

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

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.

Apolipoprotein A-I Limits the Negative Effect of Tumor Necrosis Factor on Lymphangiogenesis.

OBJECTIVE: Lymphatic endothelial dysfunction underlies the pathogenesis of many chronic inflammatory disorders. The proinflammatory cytokine tumor necrosis factor (TNF) is known for its role in disrupting the function of the lymphatic vasculature. This study investigates the ability of apolipoprotein (apo) A-I, the principal apolipoprotein of high-density lipoproteins, to preserve the normal function of lymphatic endothelial cells treated with TNF. APPROACH AND RESULTS: TNF decreased the ability of lymphatic endothelial cells to form tube-like structures. Preincubation of lymphatic endothelial cells with apoA-I attenuated the TNF-mediated inhibition of tube formation in a concentration-dependent manner. In addition, apoA-I reversed the TNF-mediated suppression of lymphatic endothelial cell migration and lymphatic outgrowth in thoracic duct rings. ApoA-I also abrogated the negative effect of TNF on lymphatic neovascularization in an ATP-binding cassette transporter A1-dependent manner. At the molecular level, this involved downregulation of TNF receptor-1 and the conservation of prospero-related homeobox gene-1 expression, a master regulator of lymphangiogenesis. ApoA-I also re-established the normal phenotype of the lymphatic network in the diaphragms of human TNF transgenic mice. CONCLUSIONS: ApoA-I restores the neovascularization capacity of the lymphatic system during TNF-mediated inflammation. This study provides a proof-of-concept that high-density lipoprotein-based therapeutic strategies may attenuate chronic inflammation via its action on lymphatic vasculature.

Quantification of the passive and active biaxial mechanical behaviour and microstructural organization of rat thoracic ducts.

Mechanical loading conditions are likely to play a key role in passive and active (contractile) behaviour of lymphatic vessels. The development of a microstructurally motivated model of lymphatic tissue is necessary for quantification of mechanically mediated maladaptive remodelling in the lymphatic vasculature. Towards this end, we performed cylindrical biaxial testing of Sprague-Dawley rat thoracic ducts (n = 6) and constitutive modelling to characterize their mechanical behaviour. Spontaneous contraction was quantified at transmural pressures of 3, 6 and 9 cmH2O. Cyclic inflation in calcium-free saline was performed at fixed axial stretches between 1.30 and 1.60, while recording pressure, outer diameter and axial force. A microstructurally motivated four-fibre family constitutive model originally proposed by Holzapfel et al. (Holzapfel et al. 2000 J. Elast. 61, 1-48. (doi:10.1023/A:1010835316564)) was used to quantify the passive mechanical response, and the model of Rachev and Hayashi was used to quantify the active (contractile) mechanical response. The average error between data and theory was 8.9 ± 0.8% for passive data and 6.6 ± 2.6% and 6.8 ± 3.4% for the systolic and basal conditions, respectively, for active data. Multi-photon microscopy was performed to quantify vessel wall thickness (32.2 ± 1.60 µm) and elastin and collagen organization for three loading conditions. Elastin exhibited structural 'fibre families' oriented nearly circumferentially and axially. Sample-to-sample variation was observed in collagen fibre distributions, which were often non-axisymmetric, suggesting material asymmetry. In closure, this paper presents a microstructurally motivated model that accurately captures the biaxial active and passive mechanical behaviour in lymphatics and offers potential for future research to identify parameters contributing to mechanically mediated disease development.

Dietary egg white protein inhibits lymphatic lipid transport in thoracic lymph duct-cannulated rats.

Dietary egg white protein (EWP) decreases serum cholesterol levels. We previously showed that EWP decreased cholesterol absorption in the intestine. Rats subjected to permanent lymph duct cannulation were used to investigate the effects of dietary EWP on lipid transport. They were fed diets with 20% EWP and casein, and their lymph was collected to quantify lymphatic lipid levels. Dietary EWP decreased lymphatic cholesterol transport compared with casein. It was previously shown that EWP excluded cholesterol from bile acid micelles. Therefore, pepsin-hydrolyzed EWP and casein were prepared. EWP was not completely digested. Ovalbumin, which is the most abundant protein in EWP, showed resistance to digestion by pepsin. This study investigated the effects of EWP pepsin hydrolysate (EWP-ph) on cholesterol micellar solubility, cholesterol transfer from the micellar to the oil phase, water-holding capacity (WHC), settling volume in water (SV), and relative viscosity and compared them with the effects of casein pepsin hydrolysate (C-ph). EWP-ph significantly decreased the micellar solubility and transfer rate and increased the WHC, SV, and relative viscosity compared with C-ph. Moreover, the pepsin hydrolysate of ovalbumin, a major protein in EWP, played a role in decreasing cholesterol micellar solubility, leading to the inhibition of cholesterol absorption. In conclusion, dietary EWP decreased cholesterol intestinal absorption by exerting combined effects of these physicochemical properties in the gut.

Altered reactivity and nitric oxide signaling in the isolated thoracic duct from an ovine model of congenital heart disease with increased pulmonary blood flow.

We have previously shown decreased pulmonary lymph flow in our lamb model of chronically increased pulmonary blood flow, created by the in utero placement of an 8-mm aortopulmonary shunt. The purpose of this study was to test the hypothesis that abnormal lymphatic function in shunt lambs is due to impaired lymphatic endothelial nitric oxide (NO)-cGMP signaling resulting in increased lymphatic vascular constriction and/or impaired relaxation. Thoracic duct rings were isolated from 4-wk-old shunt (n = 7) and normal (n = 7) lambs to determine length-tension properties, vascular reactivity, and endothelial NO synthase protein. At baseline, shunt thoracic duct rings had 2.6-fold higher peak to peak tension and a 2-fold increase in the strength of contractions compared with normal rings (P < 0.05). In response to norepinephrine, shunt thoracic duct rings had a 2.4-fold increase in vascular tone compared with normal rings (P < 0.05) and impaired relaxation in response to the endothelium-dependent dilator acetylcholine (63% vs. 13%, P < 0.05). In vivo, inhaled NO (40 ppm) increased pulmonary lymph flow (normalized for resistance) ∼1.5-fold in both normal and shunt lambs (P < 0.05). Inhaled NO exposure increased bioavailable NO [nitrite/nitrate (NOx); ∼2.5-fold in normal lambs and ∼3.4-fold in shunt lambs] and cGMP (∼2.5-fold in both) in the pulmonary lymph effluent (P < 0.05). Chronic exposure to increased pulmonary blood flow is associated with pulmonary lymphatic endothelial injury that disrupts NO-cGMP signaling, leading to increased resting vasoconstriction, increased maximal strength of contraction, and impaired endothelium-dependent relaxation. Inhaled NO increases pulmonary lymph NOx and cGMP levels and pulmonary lymph flow in normal and shunt lambs. Therapies that augment NO-cGMP signaling within the lymphatic system may provide benefits, warranting further study.

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.