Update February 2018

Our vasculature plays diverse and critical roles in homeostasis and disease. In recent decades, the use of zebrafish has driven our understanding of vascular development into new areas, identifying new genes and mechanisms controlling vessel formation and allowing unprecedented observation of the cellular and molecular events that shape the developing vasculature. Here, we highlight key mechanisms controlling formation of the zebrafish vasculature and investigate how knowledge from this highly tractable model system has informed our understanding of vascular disease in humans.

The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS.

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4–25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFbeta in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFbeta Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.

Zebrafish, an amenable small teleost fish with a complex mammal-like circulatory system, is being increasingly used for drug screening and toxicity studies. It combines the biological complexity of in vivo models with a higher-throughput screening capability compared with other available animal models. Externally growing, transparent embryos, displaying well-defined blood and lymphatic vessels, allow the inexpensive, rapid, and automatable evaluation of drug candidates that are able to inhibit neovascularisation. Here, we briefly review zebrafish as a model for the screening of anti(lymph)angiogenic drugs, with emphasis on the advantages and limitations of the different zebrafish-based in vivo assays.

Kaposi's sarcoma (KS) is an endothelial tumor causally linked to Kaposi's sarcoma herpesvirus (KSHV) infection. At early stages of KS, inflammation and aberrant neoangiogenesis are predominant, while at late stages the disease is characterized by the proliferation of KSHV-infected spindle cells (SC). Since KSHV infection modifies the endothelial cell (EC) identity, the origin of SCs remains elusive. Yet, pieces of evidence indicate the lymphatic origin. KSHV-infected ECs display increased proliferative, angiogenic and migratory capacities which account for KS oncogenesis. Here we propose a model in which KSHV reprograms the EC identity, induces DNA damage and establishes a dysregulated gene expression program involving interplay of latent and lytic genes allowing continuous reinfection of ECs attracted to the tumor by the secretion of virus-induced cellular factors.

Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens.

Renal fibrosis is the final common pathway of chronic kidney diseases. Lymphatic vessel (LV) proliferation is found in human renal diseases and other fibrotic diseases, suggesting that lymphangiogenesis is associated with the progression or suppression of kidney diseases. However, the purpose of LV proliferation is not completely understood. We investigated the effect of vascular endothelial growth factor (VEGF)-C on lymphangiogenesis, inflammation, and fibrosis in the mouse kidney using the unilateral ureteral obstruction (UUO) model. In UUO mice, significant proliferation of LVs was accompanied by tubulointerstitial nephritis and fibrosis. We continuously administered recombinant human VEGF-C to UUO model mice using an osmotic pump (UUO+VEGF-C group). Lymphangiogenesis was significantly induced in the UUO+VEGF-C group compared with the vehicle group, despite similar numbers of capillaries in both groups. The number of infiltrating macrophages, and levels of inflammatory cytokines and transforming growth factor-beta1 were reduced in the UUO+VEGF-C group compared with the vehicle group. Renal fibrosis was consequently attenuated in the UUO+VEGF-C group. In cultured lymphatic endothelial cells, administration of VEGF-C increased the activity and proliferation of lymphatic endothelial cells (LECs) and expression of adhesion molecules such as vascular cell adhesion molecule-1. These findings suggest that induction of lymphangiogenesis ameliorates inflammation and fibrosis in the renal interstitium. Enhancement of the VEGF-C signaling pathway in LECs may be a therapeutic strategy for renal fibrosis.

Purpose: Corneal neovascularization, in particular lymphangiogenesis, is a limiting factor in corneal transplant survival. Novel treatment approaches focus on (selective) inhibition and regression of lymphatic vessels. Imaging clinically invisible corneal lymphatic vessels is a prerequisite for these strategies. Using a murine model, this study investigates whether corneal lymphatic vessels can be imaged using microscopic optical coherence tomography (mOCT). Methods: Corneal neovascularization was induced by intrastromal placement of 11.0 nylon sutures in one eye of BALB/c mice. After 2 weeks, cross-sectional images and volumes of the corneas with a 0.5 mm lateral and axial field of view were acquired using a custom-built mOCT system enabling a resolution of 1 mum at a B-scan rate of 165/s. Three of the six animals received an additional intrastromal injection of India ink 24 hours before the measurement to stain the corneal lymphatic system in vivo. Immunohistochemistry using CD31 and LYVE-1 was used to validate the mOCT findings. Results: Using mOCT, lymphatic vessels were visible as dark vessel-like structures with the lumen lacking a hyperreflective wall and mostly lacking cells. However, individual, slowly moving particles, which most likely are immune cells, occasionally could be observed inside the lumen. In lymphatic vessels of ink-stained corneas, hyperreflection and shadowing underneath was observed. Ink-filled lymphatic vessels were colocalized in consecutive corneal flat mounts of the same specimen. Conclusions: Corneal lymphatic vessels can be imaged using mOCT. This novel approach opens new options for noninvasive clinical imaging of corneal lymphatic vessels for diagnostic and therapeutic indications.

Purpose: Pathologic corneal (lymph) angiogenesis is a known risk factor for immune-mediated allograft rejections after corneal transplantation. However, there is no established treatment to regress pre-existing pathological corneal blood and lymphatic vessels. This study assessed the possibility to regress both vessel types by photodynamic therapy (PDT) after intravenous (i.v.) verteporfin injection, the influence of timing of PDT after verteporfin injection, and the effect on graft survival in high-risk keratoplasty. Methods: BALB/c mice were used for suture-induced inflammatory corneal neovascularization to induce combined hem- and lymphangiogenesis. The treated group received PDT 3 minutes, 1 hour, and 24 hours after an i.v. verteporfin injection (control group: phosphate buffered saline). Corneal flatmounts were excised 3 days, 1 week, and 2 weeks after corneal PDT and stained with cluster of differentiation 31 (CD31) and lymphatic vessel endothelial hyaluronan receptor 1 antibodies (LYVE-1) to quantify hem- and lymphangiogenesis. Graft survival rates were compared between high-risk recipients with and without preoperative PDT. Results: Corneal blood vessels were significantly reduced when PDT was performed 3 minutes after i.v. verteporfin injection, whereas lymphatic vessels showed no significant difference. Both blood and lymphatic vessels were regressed when PDT was performed 1 hour or 24 hours after i.v. verteporfin application. Long-term allograft survival increased significantly in PDT-pretreated eyes when compared with controls. Conclusions: PDT after i.v. verteporfin injection can selectively regress pre-existing corneal blood vessels or both blood and lymphatic vessels depending on the timing of PDT after verteporfin injection. The pretreatment of recipients with PDT and i.v. verteporfin might be a promising new method to improve graft survival in high-risk eyes.

Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box-GATA-ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

Antigens derived from viral infection or vaccination can persist within a host for many weeks after resolution of the infection or vaccine responses. We previously identified lymphatic endothelial cells (LEC) as the repository for this antigen archival, yet LECs are unable to present their archived antigens to CD8(+) T cells, and instead transfer their antigens to CD11c(+) antigen-presenting cells (APC). Here we show that the exchange of archived antigens between LECs and APCs is mediated by migratory dendritic cells (DC). After vaccination, both migratory basic leucine zipper ATF-like transcription factor 3 (BatF3)-dependent and BatF3-independent DCs are responsible for antigen exchange and cross-presentation. However, exchange of archived viral antigens is mediated only by BatF3-dependent migratory DCs potentially acquiring apoptotic LECs. In conclusion, LEC-archived antigens are exchanged with migratory DCs, both directly and through LEC apoptosis, to cross-present archived antigens to circulating T cells.

Urinary concentrating ability is central to mammalian water balance and depends on a medullary osmotic gradient generated by a countercurrent multiplication mechanism. Medullary hyperosmolarity is protected from washout by countercurrent exchange and efficient removal of interstitial fluid resorbed from the loop of Henle and collecting ducts. In most tissues, lymphatic vessels drain excess interstitial fluid back to the venous circulation. However, the renal medulla is devoid of classic lymphatics. Studies have suggested that the fenestrated ascending vasa recta (AVRs) drain the interstitial fluid in this location, but this function has not been conclusively shown. We report that late gestational deletion of the angiopoietin receptor endothelial tyrosine kinase 2 (Tie2) or both angiopoietin-1 and angiopoietin-2 prevents AVR formation in mice. The absence of AVR associated with rapid accumulation of fluid and cysts in the medullary interstitium, loss of medullary vascular bundles, and decreased urine concentrating ability. In transgenic reporter mice with normal angiopoietin-Tie2 signaling, medullary AVR exhibited an unusual hybrid endothelial phenotype, expressing lymphatic markers (prospero homeobox protein 1 and vascular endothelial growth factor receptor 3) as well as blood endothelial markers (CD34, endomucin, platelet endothelial cell adhesion molecule 1, and plasmalemmal vesicle-associated protein). Taken together, our data redefine the AVRs as Tie2 signaling-dependent specialized hybrid vessels and provide genetic evidence of the critical role of AVR in the countercurrent exchange mechanism and the structural integrity of the renal medulla.

Background Following convection from blood capillaries, plasma proteins are transported to loco-regional lymph nodes in two stages: first, uptake into peripheral lymphatics, and second, transport to nodes. Purpose To introduce a new parameter of lymphatic function that quantifies stage 2 - lymphatic drainage efficiency (LDE). Material and Methods Percentage injected activity (IIQ) in ilio-inguinal nodes 150 min following subcutaneous foot web-space injection of Tc-99 m-nanocolloid was measured in 102 patients undergoing lymphoscintigraphy using a method in which a standard is placed by image guidance over the nodes. Percentage activity leaving the injection depot by 150 min ( k) was measured in 60/102 patients. LDE (%) = 100 x (IIQ/ k). Abnormal lymphoscintigraphy was defined qualitatively as: (i) no activity in ilio-inguinal nodes at 45 min or negligible activity at 150 min (delay); (ii) lymph diversion through skin and/or deep system; and (iii) focal tracer accumulation suggesting cellulitis. Results Scintigraphy was bilaterally normal in 82 limbs, unilaterally normal in 40 limbs and abnormal in 82 limbs. IIQ correlated with k in bilaterally normal (r = 0.86; n = 52), unilaterally normal (r = 0.67; n = 27), and abnormal (r = 0.82; n = 41) limbs. IIQ, k, and LDE were significantly lower in unilaterally normal (9.3 +/- 5.4%, 13.8 +/- 7.1%, and 65 +/- 30%) compared with bilaterally normal limbs (15.4 +/- 8.4% [ P > 0.0001], 18.3 +/- 8.9% [ P = 0.025], and 84 +/- 30% [ P = 0.01]). LDE was lower in limbs displaying skin diversion and/or delay. Conclusion LDE is a new quantitative index that has potential value in clinical research but requires further clinical evaluation. Abnormal quantitative indices indicate that limbs unilaterally normal on lymphoscintigraphy are not functionally normal.

PURPOSE: Corneal lymphatic vessels are clinically invisible because of their thin walls and clear lymph fluid. There is no easy and established method for in vivo imaging of corneal lymphatic vessels so far. In this study, we present a novel approach to visualize corneal lymphatic vessels in vivo by injecting intrastromal fluorescein sodium. METHODS: Six- to eight-week-old female BALB/c mice were used in the mouse model of suture-induced corneal neovascularization. Two weeks after the suture placement, fluorescein sodium was injected intrastromally. The fluorescein, taken up by the presumed lymphatic vessels, was then tracked using a clinically used Spectralis HRA + OCT device. Immunohistochemistry staining with specific lymphatic marker LYVE-1 and pan-endothelial marker CD31 was used to confirm the indirect lymphangiography findings. RESULTS: By injecting fluorescein intrastromally, both corneal blood and lymphatic vessels were detected. While the lymphatic vessels were visible as bright vessel-like structures using HRA, the blood vessels appeared as dark networks. Fluorescein-labeled lymphatic vessels were colocalized with LYVE-1 in immunohistochemically stained sections of the same specimen. CONCLUSIONS: Corneal lymphatic vessels can be easily imaged in vivo in the murine model using intrastromal fluorescein injection.

Poor distribution of antigen/adjuvant to target sites and inadequate induction of T cell responses remain major challenges in cancer immunotherapy because of the lack of appropriate delivery systems. Nanocarrier-based antigen delivery systems have emerged as an innovative strategy to improve vaccine efficacy. Here we present polymeric hybrid micelles (PHMs) as a simple and potent antigen/adjuvant co-delivery system with highly tunable properties. PHMs consist of two amphiphilic diblock copolymers, polycaprolactone-polyethylenimine (PCL-PEI) and polycaprolactone-polyethyleneglycol (PCL-PEG). PHMs with different proportions of cationic PCL-PEI were prepared and loaded with tyrosinase-related protein 2 (Trp2) peptide and adjuvant CpG oligodeoxynucleotide to generate the Trp2/PHM/CpG co-delivery system. Lymphatic and intracellular antigen delivery as a function of PCL-PEI proportion was evaluated in vitro and in vivo. PHMs containing 10% (w/w) PCL-PEI (Trp2/PHM10/CpG) showed the optimal balance of good distribution to lymph nodes, strong immunization effect after subcutaneous administration, and low toxicity to dendritic cells. In a mouse model of B16F10 melanoma, Trp2/PHM10/CpG showed significantly higher antigen-specific cytotoxic T lymphocyte activity and greater anticancer efficacy than Trp2/PHM0/CpG without PCL-PEI or a mixture of free Trp2 and CpG. These results provide new insights into how cationic segments affect the efficiency of antigen delivery by cationic nanocarriers. They also suggest that PHMs can serve as a structurally simple and highly tunable platform for co-delivery of antigen and adjuvant in cancer immunotherapy.

BACKGROUND: Secondary lymphedema commonly arises as a complication of cancer surgery and radiation treatment; however, the underlying mechanisms are poorly understood. Receptor activity-modifying protein 1 (RAMP1) forms a complex with calcitonin receptor-like receptor to generate the receptor for calcitonin gene-related peptide. The present study examined whether RAMP1 plays a role in increased lymphangiogenesis during secondary lymphedema. METHODS: A model of lymphedema was generated by surgical removal of pre-existing lymphatic vessels from the subcutaneous tissue on the tails of RAMP1-deficient (RAMP1-/-) mice and their wild-type (WT) counterparts. The maximum diameter of the tail, lymphangiogenesis, and macrophage recruitment were then examined. RESULTS: Compared with that in WT mice, lymphedema in the tails in RAMP1-/- mice was sustained, with suppressed lymphangiogenesis and reduced expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor 3 at the distal edge of the lesions. The newly formed lymphatic vessels in RAMP1-/- mice were dilated, with impaired lymphatic flow. RAMP1 was expressed by macrophages recruited into edematous tail tissues distal to the wound. The number of macrophages in RAMP1-/- mice was higher than that in WT mice. Expression of messenger RNA encoding M1 macrophage-related genes, including tumor necrosis factor-alpha and interleukin-1, was higher in RAMP1-/- mice than in WT mice, whereas expression of messenger RNA encoding M2 macrophage genes, including interleukin-10, was lower. CONCLUSIONS: RAMP1 signaling improves lymphedema and accelerates lymphangiogenesis associated with reduced recruitment of pro-inflammatory macrophages.

The lymphatic system contributes to body homeostasis by clearing fluid, lipids, plasma proteins, and immune cells from the interstitial space. Many studies have been performed to understand lymphatic function under normal conditions and during disease. Nevertheless, a further improvement in quantification of lymphatic behavior is needed. Here, we present advanced bright-field microscopy for in vivo imaging of lymph vessels and automated quantification of lymphatic function at temporal resolution of 2 ms. Full frame videos were compressed and recorded continuously at up to 540 frames per second. A new edge detection algorithm was used to monitor vessel diameter changes across multiple cross sections, while individual cells in the lymph vessels were tracked to estimate flow velocity. The system performance initially was verified in vitro using 10 mum microspheres as cell phantoms in 90-mum diameter tubes at flow velocities up to 4 cm/s. Using an in vivo rat model, we explored the mechanisms of lymphedema after surgical lymphadenectomy of mesentery. The system revealed a reduction of mesenteric lymph vessel contraction and flow rate. Thus, the described imaging system may be applicable to the study of lymphatic behavior during therapeutic and surgical interventions, and potentially during diseases of compromised lymph flow.

The cornea is a transparent tissue devoid of blood and lymphatic vessels. However, various inflammatory conditions can cause hemangiogenesis and lymphangiogenesis in the cornea, compromising transparency and visual acuity. Mesenchymal stem/stromal cells (MSCs) have therapeutic potentials in a variety of diseases because of anti-inflammatory properties. Herein, we investigated the effects of MSCs on corneal angiogenesis using a model of suture-induced inflammatory corneal neovascularization. Data demonstrated that an intravenous administration of MSCs suppressed corneal inflammation and neovascularization, inhibiting both hemangiogenesis and lymphangiogenesis. MSCs reduced the levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, Tek, MRC1, and MRC2 in the cornea, which are expressed by pro-angiogenic macrophages. Moreover, the number of CD11b(+) monocytes/macrophages in the cornea, spleen, peripheral blood, and draining lymph nodes was decreased by MSCs. Depletion of circulating CD11b(+) monocytes by blocking antibodies replicated the effects of MSCs. Importantly, knockdown of tumor necrosis factor alpha (TNF-alpha)-stimulated gene/protein 6 (TSG-6) in MSCs abrogated the effects of MSCs in inhibiting corneal hemangiogenesis and lymphangiogenesis and monocyte/macrophage infiltration. Together, the results suggest that MSCs inhibit inflammatory neovascularization in the cornea by suppressing pro-angiogenic monocyte/macrophage recruitment in a TSG-6-dependent manner.

Blood vessels and lymphatic vessels are located in many tissues and organs throughout the body, and play important roles in a wide variety of prevalent diseases in humans. Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that can promote the remodeling of blood vessels and lymphatics in development and disease. Recent fundamental and translational studies have provided insight into the molecular mechanisms by which VEGF-D exerts its effects in human disease. Hence this protein is now of interest as a therapeutic and/or diagnostic target, or as a potential therapeutic agent, in a diversity of indications in cardiovascular medicine, cancer and the devastating pulmonary condition lymphangioleiomyomatosis. This has led to clinical trial programs to assess the effect of targeting VEGF-D signaling pathways, or delivering VEGF-D, in angina, cancer and ocular indications. This review summarizes our understanding of VEGF-D signaling in human disease, which is largely based on animal disease models and clinicopathological studies, and provides information about the outcomes of recent clinical trials testing agonists or antagonists of VEGF-D signaling.

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial-venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.

Recent reports have characterized functional lymphatic vessels, which drain both fluid and immune cells from the CSF to the deep cervical lymph nodes, lining the dural sinuses in mice. If conserved in the human brain these vessels could have profound implications for neuroinflammatory and neurodegenerative diseases. We provide evidence of the presence of lymphatic vessels in human dura obtained at autopsy, at the level of the superior sagittal sinus, in 4 individuals. Immunohistochemistry for the lymphatic vessel endothelial cell marker podoplanin revealed the widespread presence of multiple structures with a distinct lumen distributed throughout the superior sagittal sinus. These vessels provide a putative infrastructure for drainage of macromolecules from the brain parenchyma and represent an exciting avenue of exploration for involvement in the pathogenesis of neurodegenerative proteinopathies including Parkinson's disease.

BACKGROUND: The status of sentinel lymph node (SLN) is one of the most predictive prognostic factors in patients with clinically localized malignant melanomas (MMs). However, since the positive SLN metastatic rate is as low as 20%, it is desirable to minimize SLN biopsy performance with imaging. By dynamic lymphoscintigraphy, we have proposed the lymphatic transit rate (LTR), the value that the distance between the primary lesion and SLN is divided by scintigraphic saturation time. LTR represents the scintigraphic saturation velocity and can be used for evaluation of metastasis of skin cancers. PURPOSE: To evaluate LTR as a predictive parameter for the nodal metastasis in limb MMs. METHODS: Dynamic lymphoscintigraphy data from 36 lymph nodes in 36 patients with primary MM on the limb were analyzed. The initial sites of the MMs were the lower limb in 24 patients and the upper limb in 12 patients. Histopathologically, nodal metastasis was found in 10 patients. RESULTS: In the lower limb MM, the mean LTRs were 3.49cm/min in histologically non-metastatic SLNs and 4.49cm/min in histologically metastatic SLNs (P = 0.0056). In the upper limb MM, the mean LTRs were 2.59cm/min in non-metastatic SLNs and 3.94cm/min in metastatic SLNs (P = 0.0162). Thus, significantly higher LTRs were obtained in the metastatic SLNs. All SLNs with LTR <4.0cm/min in the lower limb MM and those with LTR <3.0cm/min in the upper limb MM were non-metastatic. CONCLUSION: LTR is a useful predictive indicator for nodal metastasis and SLN biopsy performance in MMs.

Extracellular vesicles (EVs) are key mediators of intercellular communication that have been ignored for decades. Tumour cells benefit from the secretion of vesicles as they can influence the behaviour of neighbouring tumour cells within the tumour microenvironment. Several studies have shown that extracellular vesicles play an active role in pre-metastatic niche formation and importantly, they are involved in the metastatic organotropism of different tumour types. Tumour-derived EVs carry and transfer molecules to recipient cells, modifying their behaviour through a process defined as “EV-driven education”. EVs favour metastasis to sentinel lymph nodes and distal organs by reinforcing angiogenesis, inflammation and lymphangiogenesis. Hence, in this review we will summarize the main mechanisms by which tumour-derived EVs regulate lymph node and distal organ metastasis. Moreover, since some cancers metastasize through the lymphatic system, we will discuss recent discoveries about the presence and function of tumour EVs in the lymph. Finally, we will address the potential value of tumour EVs as prognostic biomarkers in liquid biopsies, specially blood and lymphatic fluid, and the use of these tools as early detectors of metastases.

PURPOSE: To validate usefulness of magnetic resonance (MR) lymphangiography for evaluation of peripheral lymphedema in upper extremities by comparison with lymphoscintigraphy. MATERIALS AND METHODS: This prospective study had institutional review board approval and written informed consent was obtained from all patients. Initially, protocol of MR lymphangiography for upper extremity was established in seven healthy volunteers with 3.0T fat-saturated three-dimensional gradient-echo MR after gadobutrol injection. Then six patients with unilateral lymphedema of the upper extremities were examined with MR lymphangiography and lymphoscintigraphy, and the results were correlated with each other. Four categories were defined to scale the quality of drainage. Results of both techniques were separately evaluated by two radiologists and a nuclear physician. We evaluated sensitivity, specificity and correlation of both techniques. RESULTS: MR lymphangiography showed sensitivities of 100% for all four categories, while lymphoscintigraphy yielded a sensitivity of 83.3% for delineation of lymph vessels and 100% for the other three categories. Specificity of MR lymphangiography was 85.7% for delay of drainage and 100% for other three categories, while lymphoscintigraphy showed specificity of 66.7% for pattern of lymphatic drainage and 100% for other three categories. Delay and pattern of drainage was same in 83.3% and non-visualization of axillary LNs was indistinguishably noted in all patients on both techniques. Anatomic level of enhanced lymph vessel was identical in 66.7% of the patients. CONCLUSION: MR lymphangiography showed better performance for depiction of lymph vessels. MR lymphangiography and lymphoscintigraphy yielded same results in all or most patients for evaluation of axillary lymph nodes enhancement and lymphatic drainage in upper extremity.

OBJECTIVE: Several methods are used to evaluate arm volumes. The most commonly used methods are water displacement and the circumferential method (CM), but these techniques have some limitation in application in clinical settings and accuracy. Recently, the laser scanner three-dimensional (LS3D) method was successfully proposed as a valid method for volume measurements of the upper limb in healthy individuals. The aim of the study was to compare, in terms of intraobserver and interobserver reliability, the CM and LS3D method to measure the upper limb in a group of women with upper limb lymphedema. METHODS: There were 200 women with upper limb lymphedema (mean age, 64 +/- 9 years; body mass index, 24.72 +/- 2.94 kg/m(2)) involved in this study. Arm measurements were obtained with both the CM and LS3D method. Statistical analysis was conducted to compare the CM and LS3D method. RESULTS: Both the CM and LS3D method have a satisfactory level of agreement, but we found some statistically significant differences in terms of some measurements (both circumferential and volume measurements). CONCLUSIONS: The data obtained in this study indicate that the LS3D method could represent a reliable, valid method to measure arm circumferences and volumes in arms with lymphedema, suitable for daily clinical use. It combines precision, reproducibility, and ease of use with the possibility of measuring geometric parameters and shape information of scanned limbs.

BACKGROUND: Diffuse pulmonary lymphangiomatosis (DPL) mainly affects the lung and pleura. There are very few pathological reports of lung damage accompanied by diffuse involvement of the extrapulmonary lymph nodes and surrounding soft tissue. The clinicopathological significance of coexistence of pulmonary and extrapulmonary lesions is unknown. METHODS: Here, we report a 16-year-old male patient. The pathological specimens of the supraclavicular lymph node and soft tissue together with the lung biopsy were analyzed by pathological observation and immunohistochemical staining. Literatures were reviewed and clinical and imaging findings were discussed. RESULTS: The patient presented with coughing and expectoration for 1 year and intermittent hemoptysis for 4 months. Ultrasound revealed swollen lymph nodes in bilateral neck, left armpit, and pubic symphysis. Chest CT scan showed diffuse grid and linear shadows, bilateral pleural thickening, and nodule formation. Multiple enlarged lymph nodes were mainly investigated in bilateral hilar, mediastinal, para-aortic, lesser curvature, and retroperitoneal. Supraclavicular lymph node biopsy confirmed the lymphatic hyperplasia and expansion in the capsule and surrounding soft tissue. The thoracoscopic examination found bloody chylothorax on the left chest. And lung biopsy showed the lymphatic vessel hyperplasia and expansion on the pleura and adjacent lung tissue. Immunohistochemical stains showed that the lymphatic endothelial cells were positive for D2-40 and CD31. Lymphangiomatosis involving the pulmonary and extrapulmonary lymph nodes and surrounding soft tissue was diagnosed based on the aforementioned histological findings. CONCLUSION: Lymphangiomatosis of superficial lymph node mainly involves the capsule of lymph nodes and its surrounding soft tissue. The information obtained from the lymph node biopsy can prompt and assist the diagnosis of DPL.

Lymphedema is a chronic, progressive disease with no curative treatment. Breast cancer therapy is the most common cause of secondary lymphedema in the developed world. Treatment includes nonsurgical and surgical strategies. Conservative measures are reserved for subclinical lymphedema. Surgical options are divided into physiologic (to restore function) and reductive (to remove diseased tissue). Early stage disease is managed with physiologic procedures. Reductive treatment is reserved for moderate to severe staged disease owing to high morbidity. Surgical options effectively decrease edema and improve quality of life. However, further research is necessary to best establish management of lymphedema.

Lymphedema is a localized form of tissue swelling resulting from excessive retention of lymphatic fluid in the interstitial compartment. It is caused by impaired lymphatic drainage. Lymphedema is a chronic progressive disease with serious physical and psychosocial implications. It can be challenging to diagnose, especially in obese patients and in those with coexisting venous disease. We performed PubMed and Google Scholar searches of the English-language literature (1966–2017) using the terms lymphedema, lymphedema management, and lymphatic complications. Relevant publications were manually reviewed for additional resources. There are currently no standard guidelines for the diagnosis of lymphedema. There is no cure yet for lymphedema, and the objective for management is to limit disease progression and prevent complications.

Lymphedema is a localized form of tissue swelling resulting from excessive retention of lymphatic fluid in the interstitial compartment and caused by impaired lymphatic drainage. Lymphedema is classified as primary or secondary. Primary lymphedema is caused by developmental lymphatic vascular anomalies. Secondary lymphedema is acquired and arises as a result of an underlying systemic disease, trauma, or surgery. We performed PubMed and Google Scholar searches of the English-language literature (1966–2017) using the terms lymphedema, cancer-related lymphedema, and lymphatic complications. Relevant publications were manually reviewed for additional resources. This progressive chronic disease has serious implications on patients' quality of life. It is often misdiagnosed because it mimics other conditions of extremity swelling. There is no definitive cure for lymphedema. However, with proper diagnosis and management, its progression and potential complications may be limited.

BACKGROUND: The method of lymphatic venous anastomosis (LVA), including its indications or preoperative examinations, has not been established. The purpose of this study is to reveal the possible application of preoperative echography in surgical LVA outcome. METHODS: We performed a retrospective case-control study on patients with lower limb lymphedema who underwent LVA between August 15, 2013 and August 15, 2014. As a preoperative examination, we used venous echography to identify subcutaneous veins in the echo group, while we only used Accuvein visualizing system in the control group. The operation time, number of anastomoses, and limb circumference were compared between the two groups. RESULTS: Seventeen patients (34 limbs) were included in the echo group, and 21 patients (42 limbs) were included in the control group. The average follow-up period was 11.9 (6–16) and 12.4 (6–27) months, respectively. The average operation time in the echo group was 258.6 min, and that in the control group was 216.5 min. The average number of anastomoses was 9.8 and 7.0 in the echo and control group, respectively. The average time per anastomosis was 27.4 and 32.6 min, respectively. The diameter of the vein had a tendency to be larger in the echo group than in the control group. In 5.8% of the echo group, we observed a circumference increase, compared with 23.8% in the control group. CONCLUSIONS: Preoperative venous echography allowed surgeons to increase the number of anastomoses performed within the operating time, resulting in improvement of surgical outcomes.

BACKGROUND: Lymphoedema is a chronic, debilitating condition caused by a compromised lymphatic system. In recent years, the success of treating upper extremity lymphoedema with liposuction has been translated to patients with lower extremity lymphoedema (LEL), yet there remains a paucity of clinical evidence firmly supporting its use within this patient group. METHODS: 69 patients with LEL (72 legs) were consecutively treated with liposuction by a single surgeon. Compression garments were applied in theatre and continued postoperatively. RESULTS: Mean preoperative volume of oedema was 4372 mL (range 229–15,166 mL), and mean volume of aspirate was 4550 mL (range 575–12,150 mL). There were no major surgical complications. An average reduction in volume of leg oedema of 85% was found at 3 months (n = 72), 88% at 1 year (n = 60), 94% at 2 years (n = 41) and 90% at 5 years (n = 15). CONCLUSIONS: We have demonstrated that liposuction combined with continuous compression therapy (CCT) is a safe and effective technique for treatment of primary and secondary LEL, with a significant reduction of the original excess limb volume. Male patients with primary lymphoedema have the poorest outcomes. Limbs with secondary lymphoedema respond best to this treatment.

Cancer treatments with axillary or pelvic lymph nodes dissection and radiation place patients at lifelong risk for the development of secondary lymphedema. Our aim was to evaluate the role of stress lymphoscintigraphy for early detection and management of secondary lymphedema. METHODS: Stress lymphoscintigraphy was performed within 1 year after surgery and the completion of chemotherapy and radiation treatments. All patients were classified by the International Society of Lymphology clinical stages from 0 to 3. A dose of 50 MBq of 99mTc-HSA-nanocolloidal in 0.4mL was injected intradermally at the first and fourth intermetacarpal spaces on the hand, for the upper limb with edema, or at the first intermetatarsal space and at the lateral malleolus for lower extremities. Two planar static scans at rest were acquired immediately after tracer injection. Stress scans were acquired after weight lifting for upper extremity or stepping for 2 minutes for lower-extremity edema. After that, the patients underwent prolonged muscular exercise limited by symptoms, and later scans were acquired at 60 minutes to visualize regional lymph nodes and the effects of sustained muscular exercise. Transport Index was evaluated. RESULTS: Five patterns of lymphoscintigraphy were observed. In our experience, patients with types I to III pattern benefit from an exercise program as a first-line treatment. Patterns IVand V seem to be predictive of lymphedema. CONCLUSIONS: The abnormal patterns found may provide the basis for earlier complex physical therapy or microsurgical treatment of lymphatic disorders in patients resulting in improved outcomes.

BACKGROUND: Breast cancer-related lymphedema affects more than 400,000 survivors in the United States. In 2009, lymphatic microsurgical preventive healing approach (LYMPHA) was first described as a surgical technique to prevent lymphedema by bypassing divided arm lymphatics into adjacent veins at the time of an axillary lymph node dissection. We describe the first animal model of LYMPHA. METHODS: In Yorkshire pigs, each distal hind limb lymphatic system was cannulated and injected with a different fluorophore (human serum albumin-conjugated indocyanine green or Evans Blue). Fluorescence-assisted resection and exploration imaging system was used to map the respective lymphangiosomes to the groin. Baseline lymphatic clearance of each hind limb lymphangiosome was obtained by measuring the fluorescence of each dye from centrally obtained blood samples. A lymphadenectomy versus lymphadenectomy with LYMPHA was then performed. The injections were then repeated to obtain clearance rates that were compared against baseline values. RESULTS: Human serum albumin-conjugated indocyanine green and Evans Blue allowed for precise lymphatic mapping of each respective hind limb using fluorescence-assisted resection and exploration imaging. Lymphatic clearance from the distal hind limb dropped 68% when comparing baseline clearance versus after a groin lymphadenectomy. In comparison, lymphatic clearance dropped only 21% when comparing baseline clearance versus a lymphadenectomy with LYMPHA. CONCLUSIONS: We describe the first animal model for LYMPHA, which will enable future studies to further evaluate the efficacy and potential limitations of this technique. Of equal importance, we demonstrate the power of optical imaging to provide real-time lymphatic clearance rates for each hind limb.

The transcription factor SOX18 has been shown to play a role in the development of hair, blood and lymphatic vessels. Mutations in SOX18 result in hereditary lymphedema, with the unique clinical association of hypotrichosis and telangiectasia (HLTS). Some patients present with additional disease features which may be explained by the location of SOX18 mutation. We report a patient with hypotrichosis-lymphedema-telangiectasia syndrome (HLTS) confirmed by detection of a novel mutation in the SOX18 gene. Few cases of HTLS have been reported in the literature. We reviewed all cases reported to date to delineate the clinical manifestations that allow us to prompt diagnosis of this syndrome for appropriate management and genetic counseling.

The World Health Organization's Global Program to Eliminate Lymphatic Filariasis (LF) has reduced LF transmission worldwide, but millions remain affected by filarial lymphedema. Tools for clinically monitoring lymphedema in developing nations are limited. We tested a novel, portable, infrared three-dimensional imaging system (3DIS) against water displacement (WD) and tape measurement of limb circumference (TMLC) among patients with filarial leg lymphedema in Galle, Sri Lanka. Outcomes were accuracy and reproducibility of imaging system measurements. In parallel, we also tested the reproducibility of skin thickness ultrasound (STU) measurements. We examined 52 patients (104 limbs) with lymphedema of stages 0–6 (N = 28, 19, 20, 21, 2, 4, and 10, respectively). 3DIS measurements correlated nearly perfectly with WD (r(2) = 0.9945) and TMLC values (r(2) > 0.9801). The median time required to acquire imaging system measurements for both legs was 2.1 minutes, compared with 17, 7, and 29 minutes, respectively, for WD, TMLC, and STU. Median interexaminer coefficients of variation (CVs) for volume measurements were 1.1% (interquartile range [IQR] 0.5–2.1%) for WD and 1.7% (IQR 1.2–2.4%) for the 3DIS. CVs for circumference measurements were 1.4% (IQR 0.8–2.4%) by TMLC and 1.3% (0.8–1.9%) by 3DIS. Median interexaminer CV for STU was 13.7% (IQR 8.5–21.3%). The portable imaging system noninvasively provided accurate and reproducible limb volume and circumference measurements in approximately 2 minutes per patient. This portable technology has the potential to greatly improve assessment and monitoring of lymphedema in the clinic and in the field.

Cerebral arteriovenous malformation (AVM) presents a great health threat due to its high probability of rupture that can cause severe brain damage. Image segmentation alone is not sufficient to support AVM embolization procedure. In order to successfully navigate the catheter and perform embolization, the segmented blood vessels need to be classified into feeding arteries, draining veins and the AVM nidus. For this reason we address here the AVM localization and vessel decomposition problem. We propose in this paper a novel AVM localization and vessel delineation method using ordered thinning-based skeletonization. The main focus of vessel delineation is the delineation of draining veins since it is essential for the embolization procedure. The main contribution is a graph-based method for exact extraction of draining veins which, in combination with our earlier work on AVM detection, allows the AVM decomposition into veins, arteries and the nidus (with an emphasis on the draining veins). We validate the proposed approach on blood vessel phantoms representing the veins and the AVM structure, as well as on cerebral 3D digital rotational angiography (3DRA) images before and after embolization, paired with digital subtraction angiography (DSA) images. Results on AVM delineation show high correspondence to the ground truth structures and indicate potentials for use in surgical planning.

PurposeWe set out to facilitate the molecular diagnosis of patients with PIK3CA-related overgrowth spectrum (PROS), a heterogeneous somatic disorder characterized by variable presentations of segmental overgrowth, vascular malformations, skin lesions, and nephroblastomatosis, rare precursor lesions to Wilms tumor. Molecular diagnosis of PROS is challenging due to its mosaic nature, often requiring invasive biopsies. MethodsDigital droplet polymerase chain reaction (ddPCR) was used to analyze tissues including urine, saliva, buccal cells, and blood, from eight patients with PROS. Further analyses were performed on plasma and urine cell-free DNA (cfDNA).ResultsPIK3CA variants were detected in plasma cfDNA at levels up to 0.5% in 50% of tested samples. In addition, high levels of PIK3CA variants in urine cfDNA correlated with a history of nephroblastomatosis compared with patients without renal involvement (P<0.05).ConclusionDigital droplet PCR is a sensitive molecular tool that enables low-level variant detection of PIK3CA in various tissue types, providing an alternative diagnostic method. Furthermore, urine cfDNA is a candidate biomarker for nephroblastomatosis in PROS, which may be useful to refine screening guidelines for tumor risk in these patients.GENETICS in MEDICINE advance online publication, 4 January 2018; doi:10.1038/gim.2017.228.

With ever increasing demands to manage finite resources for health care utilization, we performed an investigation to identify inpatient clinical characteristics and trends in children with lymphatic malformations using the Kids' Inpatient Database, years 2000 to 2009, to help identify populations best suited for resource deployment. Subjects included children 18 years and below with International Classification of Diseases (ICD), ninth revision code: 228.1-lymphangioma, any site. In the United States, between 2000 and 2009, inpatient pediatric patients with lymphatic malformations most commonly affected children aged 3 years and younger, urban hospital locations, and the South and West regions. There was no significant change in age of children with lymphatic malformations or the distribution of their age from year to year, P = 0.948 and 0.4223, respectively. No significant evidence for seasonal variation or effect on inpatient admission was identified, P = 0.7071. A great majority of admissions (>96%) were in urban locations across each year. There was also no significant change in breakdown of admissions by geographic location, P = 0.7133. Further investigation may help to elucidate how to improve access to multidisciplinary vascular anomalies teams to optimize care for these children with unique and complex lymphatic malformations.

INTRODUCTION: Soft tissue occlusion of the external auditory canal (EAC) can cause intense pruritis, recurrent foul smelling otorrhea, recurrent otitis externa, and conductive hearing loss. Occlusion of the EAC can be challenging to treat as the area is prone to circumferential scarring. METHODS: We describe the novel use of serial bipolar radiofrequency ablation (coblation) to treat 3 children with complete EAC occlusion from congenital and acquired conditions including lymphedema (1), microcystic lymphatic malformation (1), and venolymphatic malformation (1). RESULTS: Patients underwent a mean of 3 procedures with postoperative EAC stenting (7 days) and antibiotic and steroid aural preparations (10 days). Otologic symptoms resolved in all patients, and their EACs remained patent 14 months after last procedure (range, 4–32 months). One patient experienced a pinpoint tympanic membrane perforation that healed spontaneously 2 weeks later. CONCLUSIONS: Coblation of soft tissue stenosis of the EAC can be an effective treatment for this problematic condition.

Kaposiform hemangioendotheliomas are pediatric vascular tumors that do not metastasize. We present a patient with a thigh kaposiform hemangioendothelioma successfully treated using a systemic corticosteroid during infancy who was diagnosed with lymphedema in the extremity 9 years later. The observation that extremity kaposiform hemangioendothelioma could possibly be associated with lymphedema has implications for the care of patients with kaposiform hemangioendothelioma.

Congenital Lipomatous Overgrowth with Vascular, Epidermal, and Skeletal anomalies (CLOVES) and Klippel-Trenaunay (KTS) syndromes are caused by somatic gain-of-function mutations in PIK3CA, encoding a catalytic subunit of phosphoinositide 3-kinase. Affected tissue is needed to find mutations, since mutant alleles are not detectable in blood. Because some patients with CLOVES develop Wilms tumor, we tested urine as a source of DNA for mutation detection. We extracted DNA from the urine of 17 and 24 individuals with CLOVES and KTS, respectively, and screened 5 common PIK3CA mutation hotspots using droplet digital PCR. Six of 17 CLOVES participants (35%) had mutant PIK3CA alleles in urine. Among 8 individuals in whom a mutation had been previously identified in affected tissue, 4 had the same mutant allele in the urine. One study participant with CLOVES had been treated for Wilms tumor. We detected the same PIK3CA mutation in her affected tissue, urine, and tumor, indicating Wilms tumors likely arise from PIK3CA mutant cells in patients with CLOVES. No urine sample from a participant with KTS had detectable PIK3CA mutations. We suggest that urine, which has the advantage of being collected non-invasively, is useful when searching for mutations in individuals with CLOVES syndrome.

BACKGROUND We used indocyanine green (ICG) fluorescence imaging to completely resect lymphatic malformations (LMs). This is the first report of navigation surgery utilizing ICG fluorescence imaging for resection of LMs. CASE REPORT A 15-year-old boy was diagnosed with LMs in the abdominal wall. The extent of the tumor was determined by an ultrasound, and ICG (Diagnogreen(R), Daiichi-Sankyo Pharma, Tokyo, Japan) was injected subcutaneously and intradermally into the core and 2 marginal regions of the tumor (3 injections in total), respectively. During surgery, the extent of the tumor was confirmed with a photodynamic eye, and the tumor was completely resected. A fluorescent portion macroscopically estimated as normal was additionally resected and no residual fluorescence or tumor were confirmed in the remaining tissue. Abnormal lymphatic vessels were histopathologically observed in the additionally resected tissue, indicating the invasion of LMs. The surgery had a good outcome with no evidence of recurrence. CONCLUSIONS We performed near-infrared fluorescence-guided imaging surgery for the resection of LMs in the abdominal wall. This is a single case study; therefore, assessment of more cases is warranted for further validation. This procedure could provide significant benefit to patients requiring resection of LMs.

This series and literature review aimed to prenatally characterize the nature of axillary lymphangioma. A total of 30 cases, including our 5 cases, were analyzed. Insights gained from this review are as follows: Septate and nonseptate cysts seem to be different entities. The nonseptate type tends to be small and transient but more highly associated with aneuploidies. Septate cysts are very rarely associated with other abnormalities and hydrops fetalis, unlike cystic hygroma colli, but are more progressive with gestational age and associated with adverse outcomes. The cases with high flow have a higher risk of intralesional hemorrhage. Prenatal diagnosis is important for the route of timely delivery and possibly prenatal interventions. Shoulder dystocia is common and should always be taken into account for decisions on the route of delivery.