Update September 2016

VEGF-C/VEGFR-3 signaling plays a central role in lymphatic development, regulating the budding of lymphatic progenitor cells from embryonic veins and maintaining the expression of PROX1 during later developmental stages. However, how VEGFR-3 activation translates into target gene expression is still not completely understood. We used cap analysis of gene expression (CAGE) RNA sequencing to characterize the transcriptional changes invoked by VEGF-C in LECs and to identify the transcription factors (TFs) involved. We found that MAFB, a TF involved in differentiation of various cell types, is rapidly induced and activated by VEGF-C. MAFB induced expression of PROX1 as well as other TFs and markers of differentiated LECs, indicating a role in the maintenance of the mature LEC phenotype. Correspondingly, E14.5 Mafb(−/−) embryos showed impaired lymphatic patterning in the skin. This suggests that MAFB is an important TF involved in lymphangiogenesis.

Lymphangiogenesis plays a crucial role during development, in cancer metastasis and in inflammation. Activation of VEGFR-3 (also known as FLT4) by VEGF-C is one of the main drivers of lymphangiogenesis, but the transcriptional events downstream of VEGFR-3 activation are largely unknown. Recently, we identified a wave of immediate early transcription factors that are upregulated in human lymphatic endothelial cells (LECs) within the first 30 to 80 min after VEGFR-3 activation. Expression of these transcription factors must be regulated by additional pre-existing transcription factors that are rapidly activated by VEGFR-3 signaling. Using transcription factor activity analysis, we identified the homeobox transcription factor HOXD10 to be specifically activated at early time points after VEGFR-3 stimulation, and to regulate expression of immediate early transcription factors, including NR4A1. Gain- and loss-of-function studies revealed that HOXD10 is involved in LECs migration and formation of cord-like structures. Furthermore, HOXD10 regulates expression of VE-cadherin, claudin-5 and NOS3 (also known as e-NOS), and promotes lymphatic endothelial permeability. Taken together, these results reveal an important and unanticipated role of HOXD10 in the regulation of VEGFR-3 signaling in lymphatic endothelial cells, and in the control of lymphangiogenesis and permeability.

BACKGROUND: The lymphatic vasculature regulates tissue physiology and immunity throughout life. The self renewal mechanism that maintains the lymphatic vasculature during conditions of homeostasis is unknown. The purpose of this study was to investigate the cellular mechanism of lymphatic endothelial cell (LEC) self renewal and lymphatic vessel maintenance. METHODS: Inductive genetic techniques were used to label LECs with tandem dimer tomato (tdT) in adult mice. Two types of studies were performed, those with high dose inductive conditions to label nearly all the lymphatic vessels and studies with low dose inductive conditions to stochastically label individual clones or small populations of LECs. We coupled image guidance techniques and live fluorescence microscopy imaging with lineage tracing to track the fate of entire tdT(+) cutaneous lymphatic vessels or the behavior of individual or small populations of LECs over 11 months. We tracked the fate of 110 LEC clones and 80 small LEC populations (clusters of 2–7 cells) over 11 months and analyzed their behavior using quantitative techniques. RESULTS: The results of the high dose inductive studies showed that the lymphatic vessels remained tdT(+) over 11 months, suggesting passage and expression of the tdT transgene from LEC precursors to progenies, an intrinsic model of self- renewal. Interestingly, the morphology of tdT(+) lymphatic vasculature appeared relatively stable without significant remodeling during this time period. By following the behavior of labeled LEC clones or small populations of LECs individually over 11 months, we identified diverse LEC fates of proliferation, quiescence, and extinction. Quantitative analysis of this data revealed that the average lymphatic endothelial clone or small population remained stable in size despite diverse individual fates. CONCLUSION: The results of these studies support a mechanism of invariant asymmetry to self renew the lymphatic vasculature during homeostasis. These original findings raise important questions related to the plasticity and self renewal properties that maintain the lymphatic vasculature during life.

Immune privilege of the central nervous system (CNS) has been ascribed to the presence of a blood-brain barrier and the lack of lymphatic vessels within the CNS parenchyma. However, immune reactions occur within the CNS and it is clear that the CNS has a unique relationship with the immune system. Recent developments in high-resolution imaging techniques have prompted a reassessment of the relationships between the CNS and the immune system. This review will take these developments into account in describing our present understanding of the anatomical connections of the CNS fluid drainage pathways towards regional lymph nodes and our current concept of immune cell trafficking into the CNS during immunosurveillance and neuroinflammation. Cerebrospinal fluid (CSF) and interstitial fluid are the two major components that drain from the CNS to regional lymph nodes. CSF drains via lymphatic vessels and appears to carry antigen-presenting cells. Interstitial fluid from the CNS parenchyma, on the other hand, drains to lymph nodes via narrow and restricted basement membrane pathways within the walls of cerebral capillaries and arteries that do not allow traffic of antigen-presenting cells. Lymphocytes targeting the CNS enter by a two-step process entailing receptor-mediated crossing of vascular endothelium and enzyme-mediated penetration of the glia limitans that covers the CNS. The contribution of the pathways into and out of the CNS as initiators or contributors to neurological disorders, such as multiple sclerosis and Alzheimer's disease, will be discussed. Furthermore, we propose a clear nomenclature allowing improved precision when describing the CNS-specific communication pathways with the immune system.

The lymphatic vasculature is essential for maintaining interstitial fluid homeostasis, and dysfunctional lymphangiogenesis contributes to various pathological processes, including inflammatory disease and tumor metastasis. Mutations in FOXC2 are dominantly associated with late-onset lymphedema; however, the precise role of FOXC2 and a closely related factor, FOXC1, in the lymphatic system remains largely unknown. Here we identified a molecular cascade by which FOXC1 and FOXC2 regulate ERK signaling in lymphatic vessel growth. In mice, lymphatic endothelial cell-specific (LEC-specific) deletion of Foxc1, Foxc2, or both resulted in increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis. Compared with LECs from control animals, LECs from mice lacking both Foxc1 and Foxc2 exhibited aberrant expression of Ras regulators, and embryos with LEC-specific deletion of Foxc1 and Foxc2, alone or in combination, exhibited ERK hyperactivation. Pharmacological ERK inhibition in utero abolished the abnormally enlarged lymphatic vessels in FOXC-deficient embryos. Together, these results identify FOXC1 and FOXC2 as essential regulators of lymphangiogenesis and indicate a new potential mechanistic basis for lymphatic-associated diseases.

Full-thickness skin defects can be covered with dermal skin substitutes in combination with split-thickness skin grafts. However, slow vascularization of the matrices bears the risk of wound infection and extends the hospitalization of patients. To overcome these problems, we herein introduce a promising vascularization strategy. Green fluorescent protein (GFP)+ adipose tissue-derived microvascular fragments (ad-MVF) were isolated from epididymal fat pads of C57BL/6-Tg(CAG-EGFP)1Osb/J mice. ad-MVF were seeded on collagen-glycosaminoglycan matrices which were implanted into full-thickness skin defects in the dorsal skinfold chamber of wild-type C57BL/6 mice. Non-seeded matrices served as controls. Vascularization, lymphangiogenesis and integration of the implants were studied using intravital fluorescence microscopy, histology and immunohistochemistry over 14 days. ad-MVF rapidly reassembled into microvascular networks within the implants which developed interconnections to the host microvasculature. Accordingly, vascularization of the implants was markedly accelerated, as indicated by a significantly higher microvessel density when compared to controls. Moreover, dense lymphatic networks originating from the GFP+ ad-MVF developed within the implants. This was associated with an improved implant integration. Hence, seeding ad-MVF on collagen-glycosaminoglycan matrices represents a potential strategy to reduce morbidity and hospitalization of patients undergoing the treatment of full-thickness skin defects.

Herpes simplex virus type 1 (HSV-1) infection of the cornea induces vascular endothelial growth factor (VEGF)-A-dependent lymphangiogenesis. However, the extent to which HSV-1-induced corneal lymphangiogenesis impacts the adaptive immune response has not been characterized. Here, we used floxed VEGF-A mice to study the importance of newly created corneal lymphatic vessels in the host adaptive immune response to infection. Whereas the mice infected with the parental virus (strain SC16) exhibited robust corneal lymphangiogenesis, mice that received the recombinant virus (SC16 ICP0-Cre) that expresses Cre recombinase under the control of infected cell protein 0 (ICP0), an HSV-1 immediate early gene, showed a significant reduction in lymphangiogenesis. There was no difference in virus recovered from the cornea of mice infected with SC16 vs SC16 ICP0-Cre. However, viral loads were significantly elevated in the trigeminal ganglia (TG) of mice with reduced corneal lymphangiogenesis. The increase in viral titer correlated with a significant loss of HSV-1-specific CD8+ T cells that traffic to the TG of mice infected with the recombinant virus. Intrastromal delivery of size exclusion dye (FITC-dextran) revealed a time-dependent defect in the ability of the lymphatic vessels in SC16 ICP0-Cre infected mice to transport soluble antigen from the cornea to the draining lymph nodes. We interpret these results to suggest that the newly created lymphatic vessels in the cornea driven by HSV-1 infection are critical in the delivery of soluble viral antigen to the draining lymph node and subsequent development of the CD8+ T cell response to HSV-1.Immunology and Cell Biology accepted article preview online, 31 August 2016. doi:10.1038/icb.2016.80.

Secondary lymphedema is a common disorder associated with acquired functional impairment of the lymphatic system. The goal of this study was to evaluate the therapeutic efficacy of aligned nanofibrillar collagen scaffolds (BioBridge) positioned across the area of lymphatic obstruction in guiding lymphatic regeneration. In a porcine model of acquired lymphedema, animals were treated with BioBridge scaffolds, alone or in conjunction with autologous lymph node transfer as a source of endogenous lymphatic growth factor. They were compared with a surgical control group and a second control group in which the implanted BioBridge was supplemented with exogenous vascular endothelial growth factor-C (VEGF-C). Three months after implantation, immunofluorescence staining of lymphatic vessels demonstrated a significant increase in lymphatic collectors within close proximity to the scaffolds. To quantify the functional impact of scaffold implantation, bioimpedance was used as an early indicator of extracellular fluid accumulation. In comparison to the levels prior to implantation, the bioimpedance ratio was significantly improved only in the experimental BioBridge recipients with or without lymph node transfer, suggesting restoration of functional lymphatic drainage. These results further correlated with quantifiable lymphatic collectors, as visualized by contrast-enhanced computed tomography. They demonstrate the therapeutic potential of BioBridge scaffolds in secondary lymphedema.

Chronic allograft fibrosis is the major cause of graft loss in kidney transplantation. Progression can only be reduced by inhibition of the renin-angiotensin system (RAS). We tested the hypothesis that the protection provided by ACE inhibition also decreases capillary rarefaction, lymphangiogenesis and podocyte injury in allograft fibrosis. Fisher kidneys were transplanted into bilaterally nephrectomized Lewis rats treated with enalapril (60mg/kg/day) (ACEi) or vehicle. Proteinuria, blood urea nitrogen and plasma creatinine were regularly assessed and grafts were harvested for morphological and immunohistological analysis at various times up to 32 weeks. In the vehicle group many new lymphatic capillaries and severe and diffuse mononuclear infiltration of allografts were observed already 1 week after transplantation. Lymphangiogenesis increased till week 4 by which time inflammatory infiltration became focal. Lymphatic capillaries were often located at sites of inflammation. Progressive interstitial fibrosis, glomerulosclerosis, capillary rarefaction and proteinuria appeared later, at weeks 4–12. The number of lymphatic capillary cross sections strongly correlated with interstitial fibrosis score. Podoplanin immunostaining, a marker of healthy podocytes, disappeared from inflamed or sclerotic glomerular areas. ACEi protected from lymphangiogenesis and associated inflammation, preserved glomerular podoplanin protein expression, reduced glomerulosclerosis, proteinuria, tubulointerstitial fibrosis and blood capillary rarefaction at 32 weeks. In conclusion, ACE inhibition considerably decreased and/or delayed both glomerulosclerosis and tubulointerstitial injury. Prevention of glomerular podoplanin loss and proteinuria could be attributed to the known intra-glomerular pressure lowering effects of ACE inhibition. Reduction of lymphangiogenesis could contribute to amelioration of tubulointerstitial fibrosis and inflammatory infiltration after ACE inhibition.

Traditional liposomes degrade into lower-order micelles when PEGylated to even minor degrees (6–7 mol%), and thus can offer only limited steric exclusion against opsonization during in vivo delivery. In this work, we present for the first time a liposome coated exclusively by PEGylated phospholipids, utilizing lipid-coated calcium phosphate (CaP) cores of diverse sizes (10–15nm, 30–40nm) as well as varying PEG chain lengths (350–5000 Da). Such LCP particles exhibit a PEG chain length-dependent circulation longevity and robust immune evasion, while facilitating both strong accumulation within solid tumors upon intravenous injection and a more rapid and extensive lymphatic drainage upon subcutaneous administration. Further, these fully-PEGylated liposomes remain amenable to active targeting strategies which facilitate improved degrees of focused distribution and nanoparticle uptake, represent an outer leaflet lipid surface density commensurate with the formation of a lipid bilayer, and avoid use of scale-limited physical resuspension methods. We expect such improved delivery properties to translate into improved therapeutic safety and efficacy for a variety of systemic and lymphatic diseases.

Vascular endothelial growth factor D (VEGF-D) promotes the lymph node metastasis of cancer by inducing the growth of lymphatic vasculature, but its specific roles in tumorigenesis have not been elucidated. We monitored the effects of VEGF-D in cutaneous squamous cell carcinoma (cSCC) by subjecting transgenic mice overexpressing VEGF-D in the skin (K14-mVEGF-D) and VEGF-D knockout mice to a chemical skin carcinogenesis protocol involving 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate treatments. In K14-mVEGF-D mice, tumor lymphangiogenesis was significantly increased and the frequency of lymph node metastasis was elevated in comparison with controls. Most notably, the papillomas regressed more often in K14-mVEGF-D mice than in littermate controls, resulting in a delay in tumor incidence and a remarkable reduction in the total tumor number. Skin tumor growth and metastasis were not obviously affected in the absence of VEGF-D; however, the knockout mice showed a trend for reduced lymphangiogenesis in skin tumors and in the untreated skin. Interestingly, K14-mVEGF-D mice showed an altered immune response in skin tumors. This consisted of the reduced accumulation of macrophages, mast cells, and CD4(+) T-cells and an increase of cytotoxic CD8(+) T-cells. Cytokine profiling by flow cytometry and quantitative real time PCR revealed that elevated VEGF-D expression results in an attenuated Th2 response and promotes M1/Th1 and Th17 polarization in the early stage of skin carcinogenesis, leading to an anti-tumoral immune environment and the regression of primary tumors. Our data suggest that VEGF-D may be beneficial in early-stage tumors since it suppresses the pro-tumorigenic inflammation, while at later stages VEGF-D-induced tumor lymphatics provide a route for metastasis.

First-pass hepatic metabolism can significantly limit oral drug bioavailability. Drug transport from the intestine through the lymphatic system, rather than the portal vein, circumvents first-pass metabolism. However, the majority of drugs do not have the requisite physicochemical properties to facilitate lymphatic access. Herein, we describe a prodrug strategy that promotes selective transport through the intestinal lymph vessels and subsequent release of drug in the systemic circulation, thereby enhancing oral bioavailability. Using testosterone (TST) as a model high first-pass drug, glyceride-mimetic prodrugs incorporating self-immolative (SI) spacers, resulted in remarkable increases (up to 90-fold) in TST plasma exposure when compared to the current commercial product testosterone undecanoate (TU). This approach opens new opportunities for the effective development of drugs where oral delivery is limited by first-pass metabolism and provides a new avenue to enhance drug targeting to intestinal lymphoid tissue.

ETHNOPHARMACOLOGICAL RELEVANCE: Lymphatic system plays an important role in maintaining the fluid homeostasis and normal immune responses, anatomic or functional obstruction of which leads to lymphedema, and treatments for therapeutic lymphangiogenesis are efficiency for secondary lymphedema. Total saponins of panaxnotoginseng (PNS) are a mixture isolated from Panaxnotoginseng (Burkill) F.H.Chen, which has been used as traditional Chinese medicine in China for treatment of cardio- and cerebro-vascular diseases. The aim of this study was to determine the effect and mechanism of PNS on lymphangiogenesis. METHODS: The Tg (fli1:egfp; gata1:dsred) transgenic zebrafish embryos were treated with different concentrations of PNS (10, 50, 100muM) for 48hours with or without the 6hours pretreatment of the 30muM Vascular endothelial growth factors receptor (VEGFR)-3 kinase inhibitor, followed with morphological observation and lympangiogenesis of thoracic duct assessment. The effect of PNS on cell viability, migration, tube formation and Vascular endothelial growth factors (VEGF)-C mRNA and protein expression of lymphatic endothelial cells (LECs) were determined. The role of phosphatidylinositol-3 (PI-3)-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2 pathways, c-Jun N-terminal kinase (JNK) and P38 mitogen activated protein kinases (MAPK) signaling in PNS-induced VEGF-C expression of LECs by using pharmacological agents to block each signal. RESULTS: PNS promotes lymphangiogenesis of thoracic duct in zebrafish with or without VEGFR3 Kinase inhibitor pre-impairment. PNS promotes proliferation, migration and tube formation of LECs. The tube formation induced by PNS could be blocked by VEGFR3 Kinase inhibitor. PNS induce VEGF-C expression of LEC, which could be blocked by ERK1/2, PI3K and P38MAPK signaling inhibitors. CONCLUSION: PNS activates lymphangiogenesis both in vivo and in vitro by up-regulating VEGF-C expression and activation of ERK1/2, PI3K and P38MAPK signaling. These findings provide a novel insight into the role of PNS in lymphangiogenesis and suggest that it might be an attractive and suitable therapeutic agent for treating secondary lymphedema or other lymphatic system impairment related disease.

BACKGROUND: HIV infection persists for a longer time in AIDS patient compared to many other viral diseases. This is mainly because the HIV resides maximally in lymphatic system mainly the lymph nodes. Most of the present anti-HIV drugs have very poor bioavailability at lymphatic tissue. Hence, pharmaceutical scientists have made many efforts to formulate anti-HIV drugs for targeting lymphatic system. The exploration of nanoparticulate drug delivery systems have been popularly investigated for lymphatic targeting and for improving therapeutic efficacy. METHODS: An electronic search was undertaken to review the recent publications and patents from the available resources on nanoformulations of anti-HIV drugs for lymphatic delivery. RESULTS: Various carrier systems such as liposomes, polymeric nanoparticles, solid-lipid nanoparticles, nanostructured lipid carriers, polymeric micelles, dendrimers, and nanocrystals have been tried for lymphatic targeting. These nanoparticles are widely studied as passive targeting carriers for lymphatic systems. There is dearth of active targeting for anti-HIV drugs. The studies on surface modified nanoparticles have shown promising results for lymphatic targeting. CONCLUSION: One of the reasons for low success rate in targeting the lymphatic tissue is poor-understanding of pharmacokinetic interactions of novel delivery systems in disease pathology. Apart from this, there are several hurdles in biological screening models and clinical trials. These issues should never be neglected in developing newer targeted delivery systems for treatment of AIDS.

Lymphatic vessels play important roles in fluid drainage and in immune responses, as well as in pathological processes including cancer progression and inflammation. While the molecular regulation of the earliest lymphatic vessel differentiation and development has been investigated in much detail, less is known about the control and timing of lymphatic vessel maturation in different organs, which often occurs postnatally. We investigated the time course of lymphatic vessel development on the pleural side of the diaphragmatic muscle in mice, the so-called submesothelial initial diaphragmatic lymphatic plexus. We found that this lymphatic network develops largely after birth and that it can serve as a reliable and easily quantifiable model to study physiological lymphangiogenesis in vivo. Lymphangiogenic growth in this tissue was highly dependent on vascular endothelial growth factor receptor (VEGFR)-3 signaling, whereas VEGFR-1 and -2 signaling was dispensable. During diaphragm development, macrophages appeared first in a linearly arranged pattern, followed by ingrowth of lymphatic vessels along these patterned lines. Surprisingly, ablation of macrophages in colony-stimulating factor-1 receptor (Csf1r)-deficient mice and by treatment with a CSF-1R-blocking antibody did not inhibit the general lymphatic vessel development in the diaphragm but specifically promoted branch formation of lymphatic sprouts. In agreement with these findings, incubation of cultured lymphatic endothelial cells with conditioned medium from P7 diaphragmatic macrophages significantly reduced LEC sprouting. These results indicate that the postnatal diaphragm provides a suitable model for studies of physiological lymphangiogenic growth and maturation, and for the identification of modulators of lymphatic vessel growth.

Lymphedema is a debilitating progressive condition that severely restricts quality of life and is frequently observed after cancer surgery. The mechanism underlying lymphedema development remains poorly understood, and no effective pharmacological means to prevent or alleviate the ailment is currently available. Using a mouse model of lymphedema, we show here that excessive generation of immature lymphatic vessels is essential for initial edema development and that this early process is also important for later development of lymphedema pathology. We found that CD4(+) T cells interact with macrophages to promote lymphangiogenesis, and that both lymphangiogenesis and edema were greatly reduced in macrophage-depleted mice, lymphocyte-deficient Rag2(?/?) mice or CD4(+) T-cell-deficient mice. Mechanistically, T helper type 1 and T helper type 17 cells activate lesional macrophages to produce vascular endothelial growth factor-C, which promotes lymphangiogenesis, and inhibition of this mechanism suppressed not only early lymphangiogenesis, but also later development of lymphedema. Finally, we show that atorvastatin suppresses excessive lymphangiogenesis and lymphedema by inhibiting T helper type 1 and T helper type 17 cell activation. These results demonstrate that the interaction between CD4(+) T cells and macrophages is a potential therapeutic target for prevention of lymphedema after surgery.

Leptin is a hormone produced by adipose tissue that regulates various physiological processes. Recent studies have shown that the level of circulating leptin is elevated in obese patients and have suggested a relationship between obesity and postoperative lymphedema. However, the mechanisms by which postoperative lymphedema develops in obese patients and the mechanisms by which leptin regulates lymphatic endothelial cell homeostasis such as tube formation and cell proliferation remain unknown. Here we report that leptin regulates tube formation and cell proliferation in human dermal lymphatic endothelial cells (HDLECs) by activation of the signal transducer and activator of transcription 3 pathway, which is downstream signaling of the leptin receptor. Additionally, we found that upregulation of suppressor of cytokine signaling 3 underlies the mechanisms by which a high dose of leptin inhibits cell proliferation and tube formation. Leptin also enhanced expression of the proinflammatory cytokine IL-6 in HDLECs. Interestingly, IL-6 rescues the compromised cell proliferation and tube formation caused by treatment with a high dose of leptin in an autocrine or paracrine manner. Taken together, our findings reveal a novel mechanism by which compromised HDLECs maintain their homeostasis during inflammation mediated by leptin and IL-6. Thus, regulating the level of leptin or IL-6 may be a viable strategy to reduce the incidence of postoperative lymphedema.

A combination of extrinsic (passive) and intrinsic (active) forces move lymph against a hydrostatic pressure gradient in most regions of the body. The effectiveness of the lymph pump system impacts not only interstitial fluid balance but other aspects of overall homeostasis. This review focuses on the mechanisms that regulate the intrinsic, active contractions of collecting lymphatic vessels in relation to their ability to actively transport lymph. Lymph propulsion requires not only robust contractions of lymphatic muscle cells, but contraction waves that are synchronized over the length of a lymphangion as well as properly functioning intraluminal valves. Normal lymphatic pump function is determined by the intrinsic properties of lymphatic muscle and the regulation of pumping by lymphatic preload, afterload, spontaneous contraction rate, contractility and neural influences. Lymphatic contractile dysfunction, barrier dysfunction and valve defects are common themes among pathologies that directly involve the lymphatic system, such as inherited and acquired forms of lymphoedema, and pathologies that indirectly involve the lymphatic system, such as inflammation, obesity and metabolic syndrome, and inflammatory bowel disease.

In the seventeenth century, opportunities to discover chyle came about through the revival of vivisection. Gaspare Aselli discovered chyle vessels in a living well-fed dog in 1622. He introduced the term 'lacteals' or milky veins. According to Aselli, the lacteals passed through a mesenteric gland which he called 'pancreas'. The 1627 edition of Aselli's booklet was the start of a 'lymphomania', which led to the dissection and vivisection of hundreds of animals, with the University of Leiden being the clear leader in this field. The prominent researchers in Leiden were Jacobus Sylvius and Johannes Walaeus, who performed diverse experiments to support Harvey's theories on systemic circulation, and to find out the correct anatomy and physiology of lacteals and mesenteric glands. Another centre of excellence was Padua, where Veslingius and Wirsung introduced the idea of the prominent role of the 'real pancreas', and its duct in the transformation of digested food into clear chyle. The idea of the transport of chyle to the liver was an additional support for Galen's theories regarding the function of the liver. Nevertheless, as time went on, there were fewer and fewer believers in Galenic doctrine.

Aside from the established role for platelets in regulating hemostasis and thrombosis, recent research has revealed a discrete role for platelets in the separation of the blood and lymphatic vascular systems. Platelets are activated by interaction with lymphatic endothelial cells at the lymphovenous junction, the site in the body where the lymphatic system drains into the blood vascular system, resulting in a platelet plug that, with the lymphovenous valve, prevents blood from entering the lymphatic circulation. This process, known as “lymphovenous hemostasis,” is mediated by activation of platelet CLEC-2 receptors by the transmembrane ligand podoplanin expressed by lymphatic endothelial cells. Lymphovenous hemostasis is required for normal lymph flow, and mice deficient in lymphovenous hemostasis exhibit lymphedema and sometimes chylothorax phenotypes indicative of lymphatic insufficiency. Unexpectedly, the loss of lymph flow in these mice causes defects in maturation of collecting lymphatic vessels and lymphatic valve formation, uncovering an important role for fluid flow in driving endothelial cell signaling during development of collecting lymphatics. This article summarizes the current understanding of lymphovenous hemostasis and its effect on lymphatic vessel maturation and synthesizes the outstanding questions in the field, with relationship to human disease.

Ginsenoside Rg1, extracted mainly from Panax ginseng, has been shown to exert strong pro-angiogenic activities in vivo. But it is unclear whether ginsenoside Rg1 could promote lung lymphangiogenesis to improve lymphatic transport of intrapulmonary silica in silicotic rats. Here we investigated the effect of ginsenoside Rg1 on lymphatic transport of silica during experimental silicosis, and found that ginsenoside Rg1 treatment significantly raised the silicon content in tracheobronchial lymph nodes and serum to reduce the silicon level in lung interstitium, meanwhile increased pulmonary lymphatic vessel density by enhancing the protein and mRNA expressions of vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3). The stimulative effect of ginsenoside Rg1 on lymphatic transport of silica was actively correlated with its pro-lymphangiogenic identity. And VEGFR-3 inhibitor SAR131675 blocked these above effects of ginsenoside Rg1. These findings suggest that ginsenoside Rg1 exhibits good protective effect against lung burden of silica during experimental silicosis through improving lymphatic transport of intrapulmonary silica, which is potentially associated with the activation of VEGF-C/VEGFR-3 signaling pathway.

BACKGROUND: Breast cancer-related lymphedema affects multiple aspects of patients' daily lives. The main aim of this study was to assess the impact of vascularized lymph node transfer on the quality of life in patients with lymphedema. METHODS: Between 2007 and 2012, 25 female patients with breast cancer-related lymphedema underwent vascularized lymph node transfer. In 22 cases, the patients underwent a simultaneous deep inferior epigastric artery perforator flap breast reconstruction based on the superficial circumflex iliac artery. The influence on quality of life was evaluated using the Upper Limb Lymphedema-27 questionnaire, which includes physical, psychological, and social dimensions. The authors also investigated risk factors for lymphedema, such as body mass index, smoking, age, and time between start of lymphedema and vascularized lymph node transfer, and their impact on quality of life. RESULTS: Twenty-one patients (84 percent) had an improvement of quality of life after vascularized lymph node transfer. The mean physical, psychological, and social scores were significantly improved postoperatively (p < 0.001). Risk factors for the development of lymphedema did not influence quality of life among patients with breast cancer-related lymphedema. Skin infections disappeared in 50 percent of the cases. Eleven patients (44 percent) discontinued compression therapy at a mean postoperative time interval of 29 months (range, 8 to 64 months). In the other patients (56 percent), the average frequency of compression therapy decreased from three sessions to one session per week. CONCLUSION: Vascularized lymph node transfer significantly improves quality of life among patients with breast cancer-related lymphedema. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

The truncated somatostatin receptor sst5TMD4 is associated with poor prognosis in breast cancer and increases breast cancer cell malignancy. Here, we examined the cellular/molecular mechanisms underlying this association, aiming to identify new molecular tools to improve diagnosis, prognosis or therapy. A gene expression array comparing sst5TMD4 stably-transfected MCF-7 cells and their controls (empty-plasmid) revealed the existence of profound alterations in the expression of genes involved in key tumoral processes, such as cell survival or angiogenesis. Moreover, sst5TMD4-overexpressing MCF-7 and MDA-MB-231 cells demonstrated increased expression/production of pro-angiogenic factors and enhanced capacity to form mammospheres. Consistently, sst5TMD4-expressing MCF-7 cells induced xenografted tumors with higher VEGF levels and elevated number of blood vessels. Importantly, sst5TMD4 was expressed in a subset of breast cancers, where it correlated with angiogenic markers, lymphatic metastasis, and reduced disease-free survival. These results, coupled to our previous data, support a relevant role of sst5TMD4 in the angiogenic process and reinforce the role of sst5TMD4 in breast cancer malignancy and metastatic potential, supporting its possible utility to develop new molecular biomarkers and drug therapies for these tumors.

Intravenous chemotherapy has poor access to metastatic lymph nodes (LNs) and is limited by short-lived drug concentrations. Here, we describe the administration of chemotherapy via the lymphatic network as a new concept for the prevention and treatment of metastatic LNs. A metastatic LN can be treated by the injection of drugs into an upstream LN, either the sentinel LN (SLN) or another upstream LN. In a mouse model, tumor cells were inoculated into the subiliac LN (SiLN) to induce metastasis to the proper axillary LN (PALN). Two routes were used for drug delivery to the PALN, namely from the SiLN and from the accessory axillary LN (AALN). We found that tumor masses were formed in lymphatic vessels between the SiLN and PALN. The flow of fluorescent solution injected into the SiLN towards the PALN decreased with tumor mass formation. Delivery from the AALN (free of metastatic tumor cells) to the PALN was identified as an alternative route. Intranodal injection can deliver high concentrations of drugs to secondary metastatic LNs. The study advocates a new concept for the prevention and treatment of metastatic lymph nodes whereby drugs injected into upstream lymph nodes can reach metastatic lymph nodes via the lymphatic network.

Precondition for tumor lymphatic metastasis is that tumor cells induce formation of original and newborn lymphatic vessels and invade surrounding lymphatic vessels in tumor stroma, while some pathway-related molecules play an important role in mechanisms associated with proliferation and migration of lymphatic endothelial cells (LECs) and tumor cells. In lymphangiogenesis and lymphatic metastasis, the pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, such as Furin-like enzyme, CNTN1, Prox1, LYVE-1, Podoplanin, SOX18, SDF1 and CXCR4, are direct constitutors as a portion of VEGFC/D-VEGFR3/NRP2 axis, and their biological activities rely on this ligand-receptor system. These axis-related signal molecules could gradually produce waterfall-like cascading effects, mediate differentiation and maturation of LECs, remodel original and neonatal lymphatic vessels, as well as ultimately promote tumor cell chemotaxis, migration, invasion and metastasis to lymphoid tracts. This review summarizes the structure and function features of pathway-related molecules of VEGFC/D-VEGFR3/NRP2 axis, the expression changes of these molecules in different anatomic organs or histopathologic types or development stages of various tumors, the characteristics of transduction, implementation, integration of signal networks, the interactive effects on biological behaviors between tumor cells and lymphatic endothelial cells, and their molecular mechanisms and significances in tumor lymphangiogenesis and lymphatic metastasis.

BACKGROUND: In lymphedema, excess adipose tissue occurs with progression of the disease because of chronic lymph stasis, impeding lymphatic flow. Recently, liposuction has been used as a less-invasive procedure to remove this excess tissue. Given the existing poor lymph drainage in patients with lymphatic diseases, extra caution should be taken to avoid damaging lymphatic vessels during liposuction. We developed a new technique (Fibro-Lipo-Lymph-Aspiration with a Lymph Vessel Sparing Procedure [FLLA-LVSP]) to improve chronic swelling in patients with advanced lymphedema. The FLLA-LSVP highlights the superficial lymphatic pathways in the treated limb. This visibility allows surgeons to avoid these pathways, while removing the maximum amount of excess tissue. METHOD: One hundred forty-six patients with primary or secondary lymphedema that had already been treated by lymphatic microsurgery, in Genoa, Italy, were included in this retrospective study. All patients had residual fibrotic/adipose tissue, resistant to conservative treatments. Indocyanine green fluorescent dye and Blue Patent Violet dye were injected laterally/medially to the main superficial veins at the wrist/ankle of the limb to be treated. Using a photodynamic camera, the superficial lymphatic network was made visible and sketched onto the skin in indelible ink. After the microlymphography, the excess adipose tissue was carefully aspirated. Preoperative and postoperative excess limb volume was calculated using circumferential measurements and the formula of a frustum. RESULTS: For the upper limb, 0.80 L, on average, and 2.42 L for the lower limb were removed with the FLLA-LVSP. For the upper limb, there was an average presurgery excess volume of 20.19%, which reduced to 2.68% after the FLLA-LVSP (Z score = −6.90, P < 0.001). Similarly, for the lower limb, there was an average presurgery excess limb volume of 21.24% and a reduction to 2.64% postoperatively (Z score = −3.57, P < 0.01). Immediate postoperative microlymphography and Blue Patent Violet test confirmed no lymphatic complications. No episodes of postoperative infection occurred. CONCLUSIONS: The FLLA-LVSP is efficient. An entire leg can be completed within 90 minutes. Recovery time is short, and cosmetic results are immediate. More importantly, the removal of excess tissue is completed without further damage to lymphatic vessels. When used after microsurgery, FLLA-LVSP offers the possibility of removing almost all obstacles to lymphatic flow.

Lymphedema following breast cancer surgery is considered to be mainly due to the mechanical injury from surgery. Recent research identified that inflammation-infection and obesity may be the important predictors for lymphedema. The purpose of this exploratory research was to prospectively examine phenotype of arm lymphedema defined by limb volume and lymphedema symptoms in relation to inflammatory genes in women treated for breast cancer. A prospective, descriptive and repeated-measure design using candidate gene association method was used to enroll 140 women at pre-surgery and followed at 4–8 weeks and 12 months post-surgery. Arm lymphedema was determined by a perometer measurement of >/ = 5% limb volume increase from baseline of pre-surgery. Lymphedema symptom phenotype was evaluated using a reliable and valid instrument. Saliva samples were collected for DNA extraction. Genes known for inflammation were evaluated, including lymphatic specific growth factors (VEGF-C & VEGF-D), cytokines (IL1-a, IL-4, IL6, IL8, IL10, & IL13), and tumor necrosis factor-a (TNF-a). No significant associations were found between arm lymphedema phenotype and any inflammatory genetic variations. IL1-a rs17561 was marginally associated with symptom count phenotype of >/ = 8 symptoms. IL-4 rs2070874 was significantly associated with phenotype of impaired limb mobility and fluid accumulation. Phenotype of fluid accumulation was significantly associated with IL6 rs1800795, IL4 rs2243250 and IL4 rs2070874. Phenotype of discomfort was significantly associated with VEGF-C rs3775203 and IL13 rs1800925. Precision assessment of heterogeneity of lymphedema phenotype and understanding the biological mechanism of each phenotype through the exploration of inherited genetic susceptibility is essential for finding a cure. Further exploration of investigative intervention in the context of genotype and gene expressions would advance our understanding of heterogeneity of lymphedema phenotype.

BACKGROUND AND OBJECTIVE: Yellow nail syndrome (YNS) is a rare and poorly described disease process. In this case-control study, clinical features and findings on HRCT were compared with idiopathic bronchiectasis (IBx). METHODS: A review of all patients attending an adult bronchiectasis clinic between 2007 and 2013 identified 25 YNS patients. IBx patients were matched in a 2:1 ratio for age, duration of symptoms and gender. RESULTS: Median age of onset was 53 years. There were 12 male and 23 Caucasian YNS patients. Respiratory manifestations included chronic productive cough (100%), chronic rhinosinusitis (88%), pleural effusions (20%) and lymphoedema (12%). Chest symptoms preceded yellow nails in the majority (68%). Abnormal nails persisted at follow-up in 23 of 25 patients but improved in 14. In both disorders, there was symmetrical, predominantly lower lobe bronchiectasis on HRCT. Extent (P = 0.04), severity (P = 0.03) and bronchial wall thickness (P = 0.05) scores were lower in YNS, with less upper and middle lobe disease. Multivariate analysis showed an independent association with increased mucus plugging in YNS. There was a similar prevalence of Pseudomonas aeruginosa infection and mild lung function abnormalities. CONCLUSION: Bronchiectasis in YNS is less severe than IBx but is associated with increased mucus plugging, onset is in middle age and there is no female predominance. Treatment targeted at improved secretion clearance may improve both chest and nail symptoms, with consideration of long-term macrolide antibiotics.

The intrinsic lymphatic pump is critical to proper lymph transport and is impaired in models of the metabolic syndrome (MetSyn). Lymphatic contractile inhibition under inflammatory conditions has been linked with elevated nitric oxide (NO) production by activated myeloid-derived cells. We utilized a high fructose-fed rat model of MetSyn to test our hypothesis that inhibition of the MLV pump function in MetSyn animals was dependent on NO and was associated with altered macrophage recruitment and polarization within the mesenteric lymphatic vessels (MLV). MetSyn resulted in a greater accumulation of M1-skewed (CD163+ MHCII+) macrophages that were observed both within the perivascular adipose tissue and invested along the lymphatic vessels in MetSyn rats when compared to control rats. LECs and LMCs basally express the macrophage maturation polarization cytokines monocyte colony-stimulating factor and dramatically up regulate the M1 promoting cytokine granulocyte/monocyte colony-stimulating factor in response to lipopolysaccharide stimulation. MetSyn MLVs exhibited altered phasic contraction frequency. Incubation of MetSyn MLVs with LNAME or glibenclamide had a partial restoration of lymphatic contraction frequency. The data presented here provide the first evidence for a correlation between alterations in macrophage status and lymphatic dysfunction that is partially mediated by NO and KATP channel in MetSyn rats. This article is protected by copyright. All rights reserved.

Lymphatic malformations (LMs) are congenital lymphatic lesions that impose significant and costly morbidities on affected patients. Treatment options are limited due to incomplete understanding of LM pathobiology. Expression of an activated beta2-adrenergic receptor has been described in LM tissue, suggesting that this pathway may contribute to the clinical manifestations of LM. We hypothesized that propranolol, a beta-adrenergic receptor antagonist, might improve symptoms of patients with LMs and lymphatic anomalies. A retrospective chart review of patients treated with propranolol as an adjunct therapy was conducted; analyses included demographic characteristics, clinical features, and response to propranolol. Three patients with cystic and noncystic LMs displayed clinical improvement at a minimum dose of 0.7 mg/kg/d, whereas symptomatic relapses were observed when propranolol doses dropped below this threshold. Two patients with Klippel-Trenaunay syndrome demonstrated partial clinical responses with reduced edema. The fetus of a mother treated with propranolol from a gestational age of 35 weeks through delivery displayed arrested growth of a cervicofacial LM. Our retrospective review suggests that propranolol improved symptoms in a subset of patients with lymphatic anomalies. Propranolol treatment may also limit the growth of congenital LMs in utero.