Update August 2019

The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-alpha/beta signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-kappaB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.

In this manuscript, the authors report their ambitious effort to define the transcriptional regulation of fetal pulmonary lymphatic endothelial cell (PLEC) development. By cell sorting and RNA microarray analysis of mouse E16.5 and E18.5 (late gestation) fetal lung tissue, CD45-/Epcam-/CD31+/Vegfr3+/Lyve1+/Pdpn+ primary PLECs were isolated, then analyzed for gene expression. IFN signaling, sphingosine kinase 1,Ccbe1 related genes were upregulated, as well as genes implicated the coagulation and complement cascades, cell/matrix adhesion related genes. Taken together, the molecular signature identified by these studies may lead to research further defining the roles/importance of these molecules in fetal lung development and the vulnerability of the lungs in premature infants whose lungs are not fully developed.

Combined germline and somatic second hit inactivating mutations of the RASA1 gene, which encodes a negative regulator of the Ras signaling pathway, cause blood and lymphatic vascular lesions in the human autosomal dominant vascular disorder capillary malformation-arteriovenous malformation (CM-AVM). How RASA1 mutations in endothelial cells (EC) result in vascular lesions in CM-AVM is unknown. Here, using different murine models of RASA1-deficiency, we found that RASA1 was essential for the survival of EC during developmental angiogenesis in which primitive vascular plexuses are remodeled into hierarchical vascular networks. RASA1 was required for EC survival during developmental angiogenesis because it was necessary for export of collagen IV from EC and deposition in vascular basement membranes. In the absence of RASA1, dysregulated Ras mitogen-activated protein kinase (MAPK) signal transduction in EC resulted in impaired folding of collagen IV and its retention in the endoplasmic reticulum (ER) leading to EC death. Remarkably, the chemical chaperone, 4-phenylbutyric acid, and small molecule inhibitors of MAPK and 2-oxoglutarate dependent collagen IV modifying enzymes rescued ER retention of collagen IV and EC apoptosis and resulted in normal developmental angiogenesis. These findings have important implications with regards an understanding of the molecular pathogenesis of CM-AVM and possible means of treatment.

The surface of bovine serum-derived exosomes (EXOs) are modified with alpha-d-mannose for facile interaction with mannose receptors on dendritic cells (DCs) and for efficient delivery of immune stimulators to the DCs. The surface of the EXOs is modified with polyethylene glycol (PEG) without particle aggregation (approximately 50 nm) via the incorporation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) into the lipid layer of the EXO, compared to chemical conjugation by N-hydroxysuccinimide activated PEG (NHS-PEG). PEG modification onto the exosomal surface significantly decreases the non-specific cellular uptake of the EXOs into the DCs. However, the EXOs with mannose-conjugated PEG-DSPE (EXO-PEG-man) exhibit excellent intracellular uptake into the DCs and boost the immune response by the incorporation of adjuvant, monophosphoryl lipid A (MPLA) within the EXO. After an intradermal injection, a higher retention of EXO-PEG-man is observed in the lymph nodes, which could be used for the efficient delivery of immune stimulators and antigens to the lymph nodes in vivo.

Hyaluronan (HA) is associated with innate immune response activation and may be a marker of allograft dysfunction in lung transplant recipients. This was a prospective, single center study comparing levels of bronchioalveolar lavage (BAL) and serum HA and the HA immobilizer LYVE-1 in lung transplant recipients with and without acute cellular rejection (ACR). Chronic lung allograft dysfunction (CLAD)-free survival was also evaluated based on HA and LYVE-1 levels. 78 recipients were enrolled with a total of 115 diagnostic biopsies and 1.5 years of median follow-up. Serum HA was correlated with BAL HA (r = 0.25, p = 0.01) and with serum LYVE-1 (r = 0.32, p = 0.002). There was significant variation in HA and LYVE-1 over time, regardless of ACR status. Levels of serum HA (median 74.7 vs 82.7, p = 0.69), BAL HA (median 149.4 vs 134.5, p = 0.39), and LYVE-1 (mean 190.2 vs 183.8, p = 0.72) were not associated with ACR. CLAD-free survival was not different in recipients with any episode of elevated serum HA (HR = 1.5, 95% CI = 0.3–7.7, p = 0.61) or BAL HA (HR = 0.94, 95% CI = 0.2–3.6, p = 0.93). These results did not differ when stratified by bilateral transplant status. In this small cohort, serum HA, BAL HA, and LYVE-1 levels are not associated with ACR or CLAD-free survival in lung transplant recipients.

Zebrafish are well-established as a model of vascular development. The genetic tractability, external development, permeability to small molecules and optical transparency of zebrafish larvae are all attributes that make this model attractive to the vascular biologist. There are an increasing number of lymphatic reporter lines that enable the visualization of zebrafish lymphatic vessel growth in vivo; these tools, coupled with either forward or reverse genetics, have provided new insights into the process of lymphatic specification and development. Zebrafish larvae have three main lymphatic networks: the trunk lymphatics, the intestinal lymphatics, and the facial lymphatics and it is therefore possible to use zebrafish to determine network-specific roles for molecules implicated in lymphatic development. This chapter provides protocols for visualization and analysis of facial lymphatic development in the zebrafish and may be applied in developmental or drug discovery studies.

Sensitive and specific monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues because PDPN is known as a lymphatic endothelial cell maker in pathology. Recently, we established anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, horse, goat, tiger, alpaca, and Tasmanian devil. However, anti-bear PDPN (bPDPN) has not been established yet. In this study, we immunized mice with bPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/bPDPN) cells, and screened mAbs against bPDPN using flow cytometry. One of the mAbs, PMab-247 (IgG1, kappa), specifically detected CHO/bPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-247 for the functional analyses of bPDPN.

The lymphatic system is an active player in the pathogenesis of several human diseases, including lymphedema and cancer. Relevant models are needed to advance our understanding of lymphatic biology in disease progression to improve therapy and patient outcomes. Currently, there are few 3D in vitro lymphatic models that can recapitulate the physiological structure, function, and interactions of lymphatic vessels in normal and diseased microenvironments. Here, we developed a 3D microscale lymphatic vessel (muLYMPH) system for generating human lymphatic vessels with physiological tubular structure and function. Consistent with characteristics of lymphatic vessels in vivo, the endothelium of cultured vessels was leaky with an average permeability of 1.38x10(−5) +/- 0.29x10(−5)cm/s as compared to 0.68x10(−5) +/- 0.13x10(−5)cm/s for blood vessels. This leakiness also resulted in higher uptake of solute by the lymphatic vessels under interstitial flow, demonstrating recapitulation of their natural draining function. The vessels secreted appropriate growth factors and inflammatory mediators. Our system identified the follistatin/activin axis as a novel pathway in lymphatic vessel maintenance and inflammation. Moreover, the muLYMPH system provided a platform for examining crosstalk between lymphatic vessels and tumor microenvironmental components, such as breast cancer-associated fibroblasts (CAFs). In co-culture with CAFs, vessel barrier function was significantly impaired by CAF-secreted IL-6, a possible pro-metastatic mechanism of lymphatic metastasis. Targeted blocking of the IL-6/IL-6R signaling pathway with an IL-6 neutralizing antibody fully rescued the vessels, demonstrating the potential of our system for screening therapeutic targets. These results collectively demonstrate the muLYMPH system as a powerful model for advancing lymphatic biology in health and disease.

Lymphatic vascular development involves specification of lymphatic endothelial progenitors that subsequently undergo sprouting, proliferation and tissue growth to form a complex second vasculature. The Hippo pathway and effectors Yap and Taz control organ growth and regulate morphogenesis and cellular proliferation. Yap and Taz control angiogenesis but a role in lymphangiogenesis remains to be fully elucidated. Here we show that YAP displays dynamic changes in lymphatic progenitors and Yap1 is essential for lymphatic vascular development in zebrafish. Maternal and Zygotic (MZ) yap1 mutants show normal specification of lymphatic progenitors, abnormal cellular sprouting and reduced numbers of lymphatic progenitors emerging from the cardinal vein during lymphangiogenesis. Furthermore, Yap1 is indispensable for Vegfc-induced proliferation in a transgenic model of Vegfc overexpression. Paracrine Vegfc-signalling ultimately increases nuclear YAP in lymphatic progenitors to control lymphatic development. We thus identify a role for Yap in lymphangiogenesis, acting downstream of Vegfc to promote expansion of this vascular lineage.

To elucidate the effect of simultaneously fed carbohydrate or protein on lipid absorption, lymphatic lipid transports in the rat intestine were observed with or without simultaneous feeding of glucose or amino acids. A lipid emulsion containing 40 mumol/h (35.4 mg/h) of triolein, 2.74 mumol/h (1.06 mg/h) of cholesterol, 7.8 mumol/h (6.08 mg/h) of egg phosphatidylcholine without any additive (the Control group), with 560 mmol/h (300 mg/h) of glucose (the Glucose group), or with 400 mmol/h (150 mg/h) of amino acids (the Amino Acids group), was infused intraduodenally at 3 mL/h for 8 h into mesenteric lymph-fistula rats. The amounts of triglyceride transported in lymph for 8 h were 185 +/- 12 (mean +/- SE) mg in the Amino Acids group (n = 4), 175 +/- 3 mg in the Glucose group (n = 5), and 147 +/- 7 mg in the Control group (n = 4), respectively, with a statistically significant difference (P < 0.05) among the groups. The amounts of cholesterol transported in lymph for 8 h of the Amino Acid group and the Glucose group seemed to be larger than that of the Control group. The amount of phosphatidylcholine transported in lymph for 8 h were 16.4 +/- 1.0 mg in the Amino Acids group, 15.7 +/- 0.4 mg in the Glucose group, and 12.4 +/- 0.3 mg in the Control group, respectively, with a statistically significant difference (P < 0.01) among the groups. Simultaneous glucose or amino acids feeding enhanced lymphatic lipid transport in the rat intestine during lipid feeding.

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.

Hippocratic Corpus, a collection of Greek medical literature, described the functional anatomy of the lymphatic system in the fifth century B.C. Subsequent studies in cadavers and surgical patients firmly established that lymphatic vessels drain extravasated interstitial fluid, also known as lymph, into the venous system at the bilateral lymphovenous junctions. Recent advances revealed that lymphovenous valves and platelet-mediated hemostasis at the lymphovenous junctions maintain life-long separation of the blood and lymphatic vascular systems. Here, we review murine models that exhibit failure of blood-lymph separation to highlight the novel mechanisms and molecular targets for the modulation of lymphatic disorders. Specifically, we focus on the transcription factors, cofactors, and signaling pathways that regulate lymphovenous valve development and platelet-mediated lymphovenous hemostasis, which cooperate to maintain blood-lymph separation.

Podoplanin (PDPN) has been utilized as a lymphatic endothelial cell marker especially in pathological diagnoses. Therefore, sensitive and specific monoclonal antibodies (mAbs) targeting PDPN are needed for immunohistochemical analyses using formalin-fixed paraffin-embedded tissues. Recently, anti-PDPN mAbs against many species, such as human, mouse, rat, rabbit, dog, cat, bovine, pig, and horse were established in our studies. However, anticetacean (whale) PDPN (wPDPN) has not been established yet. In this study, we immunized mice with wPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/wPDPN) cells, and screened mAbs against wPDPN using flow cytometry. One of the mAbs, PMab-237 (IgG1, kappa), specifically detected CHO/wPDPN cells by flow cytometry and immunohistochemistry. Our findings suggest the potential usefulness of PMab-237 for the functional analyses of wPDPN.

Recent studies of brain meningeal lymphatic endothelial cells in mouse and zebrafish have raised interest in their function. In this issue of Developmental Cell, Chen et al. (2019) reveal new developmental roles for these cells in facilitating tissue repair and guiding vascular re-growth in the central nervous system.

The origin of the mammalian lymphatic vasculature has been studied for more than a century; however, details regarding organ-specific lymphatic development remain unknown. A recent study reported that cardiac lymphatic endothelial cells (LECs) stem from venous and non-venous origins in mice. Here, we identified Isl1-expressing progenitors as a potential non-venous origin of cardiac LECs. Genetic lineage tracing with Isl1-Cre reporter mice suggested a possible contribution from the Isl1-expressing pharyngeal mesoderm constituting the second heart field to lymphatic vessels around the cardiac outflow tract as well as to those in the facial skin and the lymph sac. Isl1(+) lineage-specific deletion of Prox1 resulted in disrupted LYVE1(+) vessel structures, indicating a Prox1-dependent mechanism in this contribution. Tracing back to earlier embryonic stages revealed the presence of VEGFR3(+) and/or Prox1(+) cells that overlapped with the Isl1(+) pharyngeal core mesoderm. These data may provide insights into the developmental basis of heart diseases involving lymphatic vasculature and improve our understanding of organ-based lymphangiogenesis.

Lung lymphatics maintain fluid homoeostasis by providing a drainage system that returns fluid, cells and metabolites to the circulatory system. The 3D structure of the human pulmonary lymphatic network is essential to lung function, but it is poorly characterised. Image-based 3D mathematical modelling of pulmonary lymphatic microfluidics has been limited by the lack of accurate and representative image geometries. This is due to the microstructural similarity of the lymphatics to the blood vessel network, the lack of lymphatic-specific biomarkers, the technical limitations associated with image resolution in 3D, and sectioning artefacts present in 2D techniques. We present a method that combines lymphatic specific (D240 antibody) immunohistochemistry (IHC), optimised high-resolution X-ray microfocus computed tomography (muCT) and finite-element mathematical modelling to assess the function of human peripheral lung tissue. The initial results identify lymphatic heterogeneity within and between lung tissue. Lymphatic vessel volume fraction and fractal dimension significantly decreases away from the lung pleural surface (p < 0.001, n = 25 and p < 0.01, n = 20, respectively). Microfluidic modelling successfully shows that in lung tissue the fluid derived from the blood vessels drains through the interstitium into the lymphatic vessel network and this drainage is different in the subpleural space compared to the intralobular space. When comparing lung tissue from health and disease, human pulmonary lymphatics were significantly different across five morphometric measures used in this study (p </ = 0.0001). This proof of principle study establishes a new engineering technology and workflow for further studies of pulmonary lymphatics and demonstrates for the first time the combination of correlative muCT and IHC to enable 3D mathematical modelling of human lung microfluidics at micrometre resolution.

Secondary lymphedema is a worldwide affliction that exacts a significant public health burden. This review examines the etiology, presentation, and management of secondary lymphedema. In addition, emerging adjunctive strategies are explored, specifically evidence from animal and pilot human studies regarding implantation of a collagen nanofibrillar scaffold (BioBridge; Fibralign Corporation, Union City, CA) in promoting lymphangiogenesis, preventing and treating lymphedema, and enhancing outcomes with lymphaticovenous anastomosis and vascularized lymph node transfer.

Until a few years ago, lymphatic vessels and lymphatic endothelial cells (LEC) were viewed as part of a passive conduit for lymph and immune cells to reach lymph nodes (LN). However, recent work has shown that LEC are active immunological players whose interaction with dendritic cells and T cells is of important immunomodulatory relevance. While the immunological interaction between LEC and other immune cells has taken a center stage, molecular analysis of LEC antigen processing and presentation machinery is still lagging. Herein we review the current knowledge of LEC MHC I and MHC II antigen processing and presentation pathways, Including the role of LEC in antigen phagocytosis, classical, and non-classical MHC II presentation, proteasome processing and MHC I presentation, and cross-presentation. The ultimate goal is to provide an overview of the LEC antigen processing and presentation machinery that constitutes the molecular basis for their role in MHC I and MHC II-restricted immune responses.

Since its introduction, the zebrafish has provided an important reference system to model and study cardiovascular development as well as lymphangiogenesis in vertebrates. A scientific workshop, held at the 2018 European Zebrafish Principal Investigators Meeting in Trento (Italy) and chaired by Massimo Santoro, focused on the most recent methods and studies on cardiac, vascular and lymphatic development. Daniela Panakova and Natascia Tiso described new molecular mechanisms and signaling pathways involved in cardiac differentiation and disease. Arndt Siekmann and Wiebke Herzog discussed novel roles for Wnt and VEGF signaling in brain angiogenesis. In addition, Brant Weinstein's lab presented data concerning the discovery of endothelium-derived macrophage-like perivascular cells in the zebrafish brain, while Monica Beltrame's studies refined the role of Sox transcription factors in vascular and lymphatic development. In this article, we will summarize the details of these recent discoveries in support of the overall value of the zebrafish model system not only to study normal development, but also associated disease states.

The interest in approaches to deliver therapeutics to the lymphatic system has increased in recent years as the lymphatics have been discovered to play an important role in a range of disease states such as cancer metastases, inflammatory and metabolic disease, and acute and critical illness. Therapeutic delivery to lymph has the potential to enhance treatment of these conditions. Currently much of the existing data explores therapeutic delivery to the lymphatic vessels and nodes that drain peripheral tissues and the intestine. Relatively little focus has been given to understanding the anatomy, function and therapeutic delivery to the peritoneal lymphatics. Gaining a better understanding of peritoneal lymphatic structure and function would contribute to the understanding of disease processes involving these lymphatics and facilitate the development of delivery systems to target therapeutics to the peritoneal lymphatics. This review explores the basic anatomy and ultrastructure of the peritoneal lymphatics system, the lymphatic drainage pathways from the peritoneum, and therapeutic and delivery system characteristics (size, lipophilicity and surface properties) that favour lymph uptake and retention after intraperitoneal delivery. Finally, techniques that can be used to quantify uptake into peritoneal lymph are outlined, providing a platform for future studies.

Endothelial cells (ECs), which line blood and lymphatic vessels, are generally described to come from the lateral plate mesoderm despite experimental evidence for a broader source of origin, including the paraxial mesoderm (PXM). Current dogma suggests that following specification from mesoderm, local environmental cues establish the distinct molecular and functional characteristics of ECs in different vascular beds. Here we present evidence to challenge this view, showing that lymphatic EC fate is imprinted during transition through the PXM lineage. We show that PXM-derived cells form the lymphatic endothelium of multiple organs and tissues, with a more restricted contribution to blood vessel endothelium. By deleting Prox1 specifically in PXM-derived cells, we show that this lineage is indispensable for lymphatic vessel development. Collectively, our data establish lineage history as a critical determinant of EC specialization, a finding with broad implications for our understanding of vascular development and heterogeneity.

Lymphangiogenesis is associated with some pathological conditions such as inflammation, tissue repair, and tumor growth. Recently, a paradigm shift occurred following the discovery of meningeal lymphatic structures in the human central nervous system (CNS); these structures may be a key drainage route for cerebrospinal fluid (CSF) into the peripheral blood and may also contribute to inflammatory reaction and immune surveillance of the CNS. Lymphatic vessels located along the dural sinuses absorb CSF from the adjacent subarachnoid space and brain interstitial fluid via the glymphatic system, which is composed of aquaporin-4 water channels expressed on perivascular astrocytic end-feet membranes. Magnetic resonance imaging (MRI) clearly visualized these lymphatic vessels in the human dura mater. The conception of some neurological disorders, such as multiple sclerosis and Alzheimer's disease, has been changed by this paradigm shift. Meningeal lymphatic vessels could be a promising therapeutic target for the prevention of neurological disorders. However, the involvement of meningeal lymphatic vessels in the pathophysiology has not been fully elucidated and is the subject of future investigations. In this article, to understand the involvement of meningeal lymphatic vessels in neurological disorders, we review the differences between lymphangiogenesis in the CNS and in other tissues during both developmental and adulthood stages, and pathological conditions that may be associated with meningeal lymphatic vessels in the CNS.

The lymphatic system extends its network of vessels throughout most of the body. Lymphatic vessels carry a fluid rich in proteins, immune cells, and long-chain fatty acids known as lymph. It results from an excess of interstitial tissue fluid collected from the periphery and transported centrally against hydrostatic pressure and protein concentration gradients. Thus, this one-way transport system is a key component in the maintenance of normal interstitial tissue fluid volume, protein concentration and fat metabolism, as well as the mounting of adequate immune responses as lymph passes through lymph nodes. In most cases, lymph is actively propelled via rhythmical phasic contractions through a succession of valve-bordered chambers constituting the lymphatic vessels. This contraction/relaxation cycle, or lymphatic pumping, is initiated in the smooth muscle cells present in the vessel wall by a pacemaker mechanism generating voltage-gated Ca(2+) channel-induced action potentials. The action potentials provide the depolarization and Ca(2+) influx essential for the engagement of the contractile machinery leading to the phasic constrictions of the lymphatic chambers and forward movement of lymph. The spontaneous lymphatic constrictions can be observed in isolated vessels in the absence of any external stimulation, while they are critically regulated by physical means, such as lymph-induced transmural pressure and flow rate, as well as diffusible molecules released from the lymphatic endothelium, perivascular nerve varicosities, blood and surrounding tissues/cells. In this chapter, we describe the latest findings which are improving our understanding of the mechanisms underlying spontaneous lymphatic pumping and discuss current theories about their physiological initiation.

Background: Radiation therapy has b een applied to prolong the duration of lymphedema. This study aimed to evaluate the effect of radiation on the development of lymphedema in a mouse hindlimb model. Methods and Results: A total of 24 Balb/c mice underwent the right popliteal lymph node excision and the afferent and efferent lymphatics blockage. The radiation group (n = 12) received a single 20 Gy radiation 1 day before surgery in the right hindlimb of each mouse, whereas the control group (n = 12) only received surgery without radiation. The right hindpaw thickness of each mouse was measured twice a week for 4 weeks. Fluorescence microscopy images using fluorescein isothiocyanate-dextran tracer were obtained once weekly. Immunohistochemical (IHC) staining images using anti-lymphatic vessel endothelial hyaluronan receptor-1 (anti-LYVE-1) were obtained at 4 weeks after surgery. The radiation group showed significant increase in the thickness of the right hind paws from 0.5 to 2 weeks compared with the control group. As for fluorescence lymphography, the radiation group showed a lower number of regenerated lymphatics and more congestion of tracers in the operated limb at the surgery sites at 1, 2, 3, and 4 weeks after surgery. For the IHC analysis, the radiation group showed a lower number of regenerated lymphatics per high-power field at the surgery site than the control group. Conclusion: Radiation therapy transiently aggravated the extent of lymphedema by inhibiting regenerated lymphatics in a mouse hindlimb model. However, it did not prolong the duration of lymphedema because the cutaneous interstitial flow contributes to the lymphatic fluid clearance.

BACKGROUND: Lymphatic endothelial cell (LEC) immunohistochemical markers have identified intestinal lymphatic vasculature abnormalities in humans with inflammatory bowel disease, but have not been used to evaluate intestinal lymphatic vasculature in a group of dogs with chronic inflammatory enteropathy (CIE). OBJECTIVES: To utilize LEC markers to identify and measure intestinal lymphatic vasculature in endoscopic biopsy samples of CIE dogs. To evaluate whether measured lymphatic vasculature variables correlate with serum albumin concentrations. ANIMALS: Twenty-four dogs with CIE; n = 13, serum albumin concentration <2.5 g/dL (CIE-protein-losing enteropathy [PLE]), n = 11, serum albumin concentration >/ = 2.5 g/dL (CIE-N). METHODS: Prospective study. Lymphatic endothelial cell immunolabeling with Prox-1 and LYVE-1 performed on endoscopic biopsy samples from 24 dogs with CIE. Duodenal and ileal villous lacteal width (VLW) and proprial mucosal lacteal width (MLW) were determined for each case and analyzed for correlation with serum albumin concentration. Lacteal dilatation scores using routine H&E histopathology were assessed for correlation with immunohistochemistry (IHC)-calculated VLW and MLW. RESULTS: Lower serum albumin concentrations were correlated with increased VLW (rho = -.4644; P = .02) and MLW (rho = -.6514; P < .001) in the ileum. Lymphatic endothelial cell IHC identified presumptive proprial mucosal lymphangiectasia in some dogs that was not recognized with routine H&E staining. Lacteal dilatation scores were correlated with VLW in duodenum (rho = .4634; P = .02) and ileum (rho = .5292; P = .008), but did not correlate with MLW. CONCLUSIONS AND CLINICAL IMPORTANCE: Lymphatic endothelial cell immunolabeling identified presumptive proprial mucosal lymphangiectasia in CIE dogs, particularly in the ileum of hypoalbuminemic dogs. Routine evaluation of villous lacteals likely underestimates abnormalities of the lymphatic vasculature in dogs with CIE.

Activated leukocyte cell adhesion molecule (ALCAM, CD166) is a cell adhesion molecule of the immunoglobulin superfamily and has been implicated in diverse pathophysiological processes including T cell activation, leukocyte trafficking, and (lymph)angiogenesis. However, exploring the therapeutic potential of ALCAM blockade in immune-mediated inflammatory disorders has been difficult due to the lack of antibodies with blocking activity toward murine ALCAM. In this study, we identified and characterized a monoclonal antibody with high affinity and specificity for murine ALCAM. This antibody reduced in vitro T cell activation induced by antigen-presenting dendritic cells (DCs) as well as (trans)migration of murine DCs across lymphatic endothelial monolayers. Moreover, it reduced emigration of DCs from in vitro-cultured human skin biopsies. Similarly, antibody-based blockade of ALCAM reduced (lymph)angiogenic processes in vitro and decreased developmental lymphangiogenesis in vivo to levels observed in ALCAM-deficient mice. Since corneal allograft rejection is an important medical condition that also involves (lymph)angiogenesis, DC migration and T cell activation, we investigated the therapeutic potential of ALCAM blockade in murine corneal disease. Blocking ALCAM lead to DC retention in corneas and effectively prevented corneal allograft rejection. Considering that we also detected ALCAM expression in human corneal DCs and lymphatics, our findings identify ALCAM as a potential novel therapeutic target in human corneal allograft rejection.

BACKGROUND: Lymphovenous anastomosis (LVA) is technically challenging and could be successfully performed with advanced operating microscope, super-microsurgical instruments, and indocyanine green (ICG) lymphography. This study was to compare the outcomes between side-to-end and end-to-end LVA configurations for unilateral extremity lymphedema. METHODS: Between April 2013 and June 2017, 58 patients who preoperatively had patent lymphatic ducts by ICG lymphography were indicated for LVA, including 20 upper limb lymphedema and 38 lower limb lymphedema. Either an end-to-end or a side-to-end LVA was used to anastomose the subdermal venule to lymphatic duct. The circumferential difference and episodes of cellulitis were used as outcome measurements. RESULTS: Twenty-three patients underwent an end-to-end LVA, and 35 patients had a side-to-end LVA. All cases had an immediate patency evaluated by ICG lymphography and patent blue assessments. All patients returned to their daily routine without the use of any compression garments. At an average follow-up of 16.5 (13.4–19.6) months, the improvement of circumferential difference 3.2 (1.8–4.6)% in side-to-end group was statistically greater than 2.2 (1–3.4)% in end-to-end group (p = 0.04). The overall episodes of cellulitis were significantly reduced from 1.7 (1.3–2.1) to 0.7 (0.3–1.1) times per year (p < 0.001), but no difference was observed between the two groups. CONCLUSIONS: Both side-to-end and end-to-end LVA configurations were effective surgical approaches for improving early-grade extremity lymphedema. Side-to-end LVA has the advantages of having a greater efficacy for lymph drainage while requiring only one anastomosis and eliminating the need to use compression garments.

BACKGROUND AND OBJECTIVES: Milroy disease is a form of congenital primary lymphedema affecting the lower limbs. When conservative management is ineffective, surgical treatment becomes necessary. The purpose of this study was to investigate the efficacy of vascularized lymph node transfer (VLNT) associated with extensive therapeutic lipectomy in the treatment of these patients. METHODS: In China Medical University Hospital, four patients have been diagnosed with Milroy disease and treated over an 8 year-period time. All patients presented with hereditary bilateral legs swelling since birth. All patients were treated with VLNT from the gastroepiploic region bilaterally associated with extensive therapeutic lipectomy. RESULTS: All procedures have been executed bilaterally and have been successful, without complications. The average follow-up of the patients was 20.2 +/- 2.8 months. The limbs treated presented an average circumference reduction of a 4.0 +/- 2.1 cm and patients did not experience cellulitis during follow-up. Patients expressed satisfaction with the procedure. CONCLUSIONS: VLNT together with therapeutic lipectomy proved to be a reliable technique in moderate cases of Milroy disease, providing an alternative path for lymph drainage, and reducing the lymph load and the excess of subcutaneous adipose tissues, thus improving patients' quality of life.

Lymphedema of the lower limbs often contributes to the mobility impairment of morbidly obese patients. Defining novel costeffective protocols is important for reducing treatment costs. The study aimed to assess if Capacitive and Resistive Energy Transfer (TECAR) can reduce edema and the minimum number of sessions needed to observe volume reduction. Forty-eight severely obese subjects (age range: 46–78 years; BMI >40 kg/m2) with bilateral lower limb lymphedema were divided into three groups undergoing either manual lymphatic drainage, pressure therapy, or TECAR, in addition to a multidisciplinary rehabilitation program. They were compared to a control group composed by 12 women (age: 67.4 +/- 8.9 years, BMI: 44.6 +/- 4.1 Kg/m2) undergoing only the rehabilitation program. A handheld laser scanner 3D system was used for volume measurements. In addition, patients were evaluated with a Timed Up and Go (TUG) test and pain/heaviness of the lower limbs with a Visual Analog Scale (VAS). A significant volume reduction was observed after 6 sessions of TECAR: specifically, in the whole limb (PRE: 9.7 + 2.8 dm3; POST: 9.4 + 2.8 dm3; p < 0.05) and in the thigh (PRE: 3.5 + 1.3 dm3; POST: 3.3 + 1.2 dm3; p < 0.05). The TUG and VAS for pain showed a significant improvement in all groups. Our preliminary results suggest that TECAR can provide a relatively early reduction of lower limb edema with improvement of patients' function and pain.

BACKGROUND AND OBJECTIVES: Previously, we have shown that 9-cis retinoic acid (9-cis RA) stimulates lymphangiogenesis and limits postsurgical lymphedema in animal models when administered via daily intraperitoneal injections. In this study, we investigate whether a single-use depot 9-cis RA drug delivery system (DDS) implanted at the site of lymphatic injury can mitigate the development of lymphedema in a clinically relevant mouse limb model. METHODS: Hind limb lymphedema was induced via surgical lymphadenectomy and irradiation. Animals were divided into two treatment groups: (1) 9-cis RA DDS, (2) placebo DDS. Outcomes measured included paw thickness, lymphatic clearance and density, epidermal thickness, and collagen deposition. RESULTS: Compared with control animals, 9-cis RA-treated animals had significantly less paw swelling from postoperative week 3 (P = .04) until the final timepoint at week 6 (P = .0007). Moreover, 9-cis RA-treated animals had significantly faster lymphatic clearance (P < .05), increased lymphatic density (P = .04), reduced lymphatic vessel size (P = .02), reduced epidermal hyperplasia (P = .04), and reduced collagen staining (P = .10). CONCLUSIONS: Animals receiving 9-cis RA sustained-release implants at the time of surgery had improved lymphatic function and structure, indicating reduced lymphedema progression. Thus, we demonstrate that 9-cis RA contained within a single-use depot DDS has favorable properties in limiting pathologic responses to lymphatic injury and may be an effective strategy against secondary lymphedema.

Introduction Postoperative lymphatic drainage and lymphocele formation is a common seen complication after extended pelvic lymph node dissection (ePLND) in robot-assisted radical prostatectomy (RARP) operation. The aim of this study was to evaluate autologous fibrin glue as an additional treatment option to reduce the volume of lymphatic drainage and prevent lymphocele development. Materials and Method A total of 75 patients undergoing transperitoneal robot-assisted radical prostatectomy with extended pelvic lymph node dissection between January–July 2018 were enrolled in this study. Thirty-five patients who received autologous fibrin glue enrolled to study group, another 40 patients who did not receive to control group. Autologous fibrin glue was applied over the PLND areas. Age, body-mass index, pathological stages and number of removed lymph nodes were compared. The main endpoint was to compare postoperative lymphatic drainage volume and lymphocele formation rate between groups. Results There was not statistically significant difference between the groups respect to age, body-mass index, Gleason score, T-stage and number of removed lymph nodes. Autologous fibrin glue resulted in 50% (110ml vs. 210ml; P = 0.037) and 75% reduction of postoperative drainage volume (70ml vs. 270ml; P = <0.0001) in study group than control group at postoperative 2nd and 3th days, respectively. The total drainage volume was also 50% reduced in study group (277ml vs. 577ml; P = 0.004). The incidence of asymptomatic lymphocele was 20% (n = 7) and 37.5% (n = 15) in study and control groups, respectively (P = 0.112). One patient in control group developed symptomatic lymphocele. There were no immediate or late adverse effects in study group. Conclusion Autologous fibrin glue application reduced postoperative lymphatic drainage, and also lymphocele formation rate after extended PLND in robot-assisted radical prostatectomy operation.

BACKGROUND: To assess the efficacy of combined 5FU and Avastin injections in the treatment of primary pterygium METHODS: Sixteen eyes with primary pterygium received intralesional 5 fluorouracil and Avastin (2.5–5 mg) injections every 2 weeks for a maximum of five injections. Fourteen eyes of 14 patients received five injections, one eye received three injections and one eye received two injections. All eyes were followed at monthly intervals for 3 months after last injection. Tissue was obtained by surgical excision of primary pterygium from four eyes who received injections and three eyes with primary pterygium who did not receive injections (control) and subjected to immunohistological examination for beta fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), von-Willebrand factor (vWF), lymphatic vessel endothelial hyaluronan receptor (LYVE-1) and collagen-I. RESULTS: Pterygium progression was arrested in all patients. Sixty-two percent of patients had improvement of redness while 89% had reduced thickness of the lesion. VEGF, bFGF, EGF, vWF, LYVE-1 and collagen-I were all reduced in the injected samples. CONCLUSIONS: The injection of 5 fluorouracil and Avastin act synergistically to arrest progression and induce atrophy in primary pterygium. This is related to the effect of agents on fibroblasts, collagen, and vascular tissues. Such medical intervention is a safe and viable option in the management of primary pterygium though excision of residual tissue is still required in some cases. Longer follow up is needed to ascertain whether this will reduce the recurrence rate following excision.

INTRODUCTION: Genital lymphedema (GL) surgery can be either palliative or functional. Palliative procedures involve excision of the affected tissue and reconstruction by either local flaps or skin grafts. Reconstructive procedures aim to restore lymphatic flow through microsurgical lymphaticovenous anastomoses (LVAs). This systematic analysis of outcomes and complication rates aims to compare outcomes between these surgical treatment options for GL. METHODS: A systematic review of the PubMed database was performed with the following search algorithm: (lymphorrhea or lymphedema) and (genital or scrotal or vulvar) and (microsurgery or “surgical treatment”), evaluating outcomes, and complications after surgical treatment of GL. RESULTS: Twenty studies published between 1980 and 2016 met the inclusion criteria (total, 151 patients). Three main surgical treatments for GL were identified. Surgical resection and primary closure or skin graft was the most common procedure (46.4%) with a total complication rate of 10%. Surgical resection and flap reconstruction accounted for 39.1% of the procedures with an overall complication rate of 54.2%. Lympho venous shunt (LVA) procedures (14.5%) had a total complication rate of 9%. CONCLUSIONS: This review demonstrates a lack of consensus in both the preoperative assessment and surgical management of GL. Patients receiving excisional procedures tended to be later stage lymphedema. Patients in the excision and flap reconstruction group seemed to have the highest complication rates. Microsurgical LVAs may represent an alternative approach to GL, either alone or in combination with traditional procedures.

BACKGROUND: Detecting more number of functional lymphatic vessels is the essential point in lymphaticovenous anastomosis (LVA). The purpose of this prospective study was to elucidate the efficacy of multi-area injection in indocyanine green (ICG) lymphography in LVA. METHODS: We injected ICG into the first web spaces of the feet or the second web space of the hands, subcutaneously. In multi-area injection group, we injected additional ICG in other areas. We determined the incision design of LVA on the line at about 5 cm distal to dermal backflow point. In control group, we determined the incision site based on the linear pattern in ICG lymphography and lymphoscintigraphic findings. We performed LVA, and evaluated the circumference change and the intraoperative condition of the collecting lymphatic vessels based on Normal, Ectasis, Contraction, and Sclerosis Type (NECST) classification. RESULTS: Sixty patients (115 limbs) in multi-injection group and 49 patients (81 limbs) in control group were included. We injected ICG into an average of 1.9 sites in multi-injection group. The average number of anastomoses per limb was 3.3 in both groups. The average circumference change was −1.83% in multi-injection group and −0.34% in control group (P = .021). The percentage of the Ectasis type lymphatic vessels was 59.0% in multi-injection group and 40.2% in control group (P = 3.30 × 10(−5)). CONCLUSIONS: By injecting ICG into multiple sites in the affected limbs, we could detect a greater number of functional lymphatic vessels (Ectasis type) during LVA. This could lead to a better surgical result.

The mammalian circulatory system comprises both the cardiovascular system and the lymphatic system. In contrast to the closed, high-pressure and circular blood vascular circulation, the lymphatic system forms an open, low-pressure and unidirectional transit network from the extracellular space to the venous system. It plays a key role in regulating tissue fluid homeostasis, absorption of gastrointestinal lipids, and immune surveillance throughout the body. Despite the critical physiological functions of the lymphatic system, a complete understanding of the lymphatic vessels lags far behind that of the blood vasculatures due to the challenge of their visualization. During the last 20years, discoveries of underlying genes responsible for lymphatic vessel biology, combined with state-of-the-art lymphatic function imaging and quantification techniques, have established the importance of the lymphatic vasculature in the pathogenesis of cardiovascular diseases including lymphedema, obesity and metabolic diseases, dyslipidemia, hypertension, inflammation, atherosclerosis and myocardial infraction. In this review, we highlight the most recent advances in the field of lymphatic vessel biology, with an emphasis on the new identification techniques of lymphatic system, pathophysiological mechanisms of atherosclerosis and myocardial infarction, and new therapeutic perspectives of lymphangiogenesis.

BACKGROUND AND OBJECTIVES: Photoacoustic lymphangiography, which is based on photoacoustic technology, is an optical imaging that visualizes the distribution of light absorbing tissue components like hemoglobin or melanin, as well as optical absorption contrast imaging agents like indocyanine green (ICG) in the lymphatic channels, with high spatial resolution. In this report, we introduce the three-dimensional (3D) images of human lymphatic vessels obtained with photoacoustic lymphangiography. METHODS: We used the 3D photoacoustic visualization system (PAI-05). Some healthy subjects and lymphedema patients were recruited. To image the lymphatic structures of the limbs ICG was administered subcutaneously as in fluorescence lymphangiography. Photoacoustic images were acquired by irradiating the tissue using a laser at wavelengths of near-infrared region. On the same occasion, fluorescence images were also recorded. RESULTS: The lymphatic vessels up to the diameter of 0.2 mm could be observed three-dimensionally with the venules around them. In the patient-group, dermal backflow patterns were often observed as dense interconnecting 3D structures of lymphatic vessels. Collecting vessels passing below the dermis were also observed, which were not observed by fluorescence lymphography. CONCLUSIONS: Photoacoustic lymphangiography provided the detailed observation of each lymphatic vessel, leading to deeper understanding of 3D structures and physiological state of the vessel.

Secondary lymphedema is a common complication of cancer treatment resulting in progressive fibroadipose tissue deposition, increased risk of infections, and, in rare cases, secondary malignancies. Until recently, the pathophysiology of secondary lymphedema was thought to be related to impaired collateral lymphatic formation after surgical injury. However, more recent studies have shown that chronic inflammation-induced fibrosis plays a key role in the pathophysiology of this disease. In this review, we will discuss the evidence supporting this hypothesis and summarize recent publications demonstrating that lymphatic injury activates chronic immune responses that promote fibrosis and lymphatic leakiness, decrease collecting lymphatic pumping, and impair collateral lymphatic formation. We will review how chronic mixed T-helper cell inflammatory reactions regulate this process and how this response may be used to design novel therapies for lymphedema.

Tuberculosis used to be uncommon in the developed countries but seems to be still on rampant in developing countries. However, there seems to be an increasing occurrence in the developed countries too mainly due to low living conditions, increased migration, HIV immune-compromisation and inappropriate use of antitubercular drugs. Lymphatic tuberculosis is the second commonest extrapulmonary location of tuberculosis followed by genitourinary, bone and joint, miliary, meningeal and abdominal. Abdominal tuberculosis represents nearly 11%–16% of all extrapulmonary tuberculosis locations. Furthermore, abdominal tuberculosis co-exists with pulmonary tuberculosis in 10%–30% of patients. Abdominal tuberculosis remains difficult to diagnose due to non-specific symptoms, variable anatomical locations and lack of specific sensitive diagnostic tools. Diagnosis can be rarely suspected, especially in cases of isolated abdominal tuberculosis without clinical or radiological findings. We present a rare case of a patient with pulmonary tuberculosis combined with intra-abdominal lymphatic tuberculosis causing small intestine volvulus.

Lymphoscintigraphy represents the “gold standard” for diagnosis of lymphedema, but an important limitation is the lack of procedural standardization. The aim of this Italian expert panel was to provide a procedural standard for lymphoscintigraphy in the evaluation of lymphatic system disorders. Topic anaesthetic gels containing lidocaine should be avoided. Patients should remove compressive dressings. Total recommended activity for (99m)Tc-nanocolloid administration in adults is 74MBq, or 37MBq per limb and per investigated compartment, in single or multiple aliquots. 2–3 subcutaneous injections should be performed (II-III+/-I interdigital space of each hand/foot), avoiding intravascular injection. Deep lymphatic system of lower limbs should be evaluated in presence of dermal back-flow or lymphatic stasis (1–2 subfascial administrations in retro-malleolar or plantar region). Planar images should be acquired from injection site to liver with whole-body or serial static acquisitions 20’ and 90’ after subcutaneous administration. Additional information on lymphatic pathways is obtained after a quick and/or prolonged exercise protocol. SPECT/CT is recommended to study the thoracic, abdominal and pelvic territories. When required, deep lymphatic system of lower limbs should be evaluated with static acquisition 90’ after subfascial administration. The report should describe administration and imaging procedure, exercise protocol, qualitative and semi-quantitative analysis (wash-out rate, transport index), potential sources of error. Due to the essential role fulfilled by lymphoscintigraphy in clinical management of primary and secondary lymphedema, an effort for the standardization of this technique should be made to provide the clinicians with a homogeneous and reliable technical methodology.

A whole exome sequencing approach was recently used to detect a CELSR1 truncating variant associated with lymphedema in a large pedigree. Since this first report, no other similar associations have been reported in the literature. Here, we present the genetic results of 95 probands tested using a next generation sequencing panel that covered all known lymphedema-associated genes, including CELSR1. Five out of 95 probands (5.3%) were found to carry novel loss-of-function variants in CELSR1. Family segregation studies were possible in four out of five probands and showed possible sex-specific differences: CELSR1 variants showed almost complete penetrance in females and were associated with early-onset lymphedema, whereas in males they showed incomplete penetrance and were associated with late onset of the condition. Since the percentage of lymphedema patients carrying CELSR1 variants is not negligible, we do not hesitate to recommend including this gene in routine genetic testing.

Secondary upper limb lymphoedema is usually caused by lymphatic system dysfunction. Diagnosis is primarily based on clinical features. However, there are no distinct diagnostic criteria for lymphoedema. Although conventional lymphoscintigraphy is a useful technique to diagnose the severity of lymphoedema, the resultant data are two-dimensional. In this study, we examined the pathology of lymphoedema using single photon emission computed tomography-computed tomography lymphoscintigraphy (SPECT-CT LSG), a new technique that provides 3-dimensional information on lymph flow. We observed lymph flow pathways in the subcutaneous and muscle layers of the upper limbs. A significant positive correlation was found between the dermal back flow (DBF) type and the visualization of lymph nodes around the clavicle (p = 0.000266), the type of lymph flow pathways and the visualization of lymph nodes around the clavicle (p = 0.00963), and the DBF type and the lymph flow pathway (p = 0.00766). As the severity of lymphoedema increased, the DBF appeared more distally in the upper limb and the flow into the lymph nodes around the clavicle decreased, whereas the lymph flow pathways in the muscle layer became dominant. These findings demonstrate the features of lymphoedema pathology and the functional anatomy and physiology of the lymphatic system without the need for cadaver dissection.

OBJECTIVE: Intestinal lymphangiectasia (IL; primary or secondary) is an important cause of protein-losing enteropathy. We evaluated the clinicolaboratory profile, response to therapy, complications, and outcome of children with primary IL (PIL). METHODS: Consecutive children (</ = 18 years) diagnosed with PIL (clinical setting, typical small bowel histopathology, and exclusion of secondary causes) from 2007 to 2017 were evaluated. RESULTS: Twenty-eight children with PIL (16 boys, age at symptom onset-12 [1–192] months and at diagnosis 8 [1–18] years) were studied. Pedal edema (93%), chronic diarrhea (78.6%), and recurrent anasarca (64%) were the common presentations. Ascites, pleural, and pericardial effusion were seen in 64 (n-18; chylous-5, non-chylous-13), 18, and 18% cases, respectively. Hypoproteinemia, hypoalbuminemia, hypocalcemia, and lymphopenia were present in 82, 82, 75 and 39% cases, respectively. Duodenal biopsy established the diagnosis in 86% cases, while 14% required distal small bowel biopsies. Dietary therapy was given in all and 6 cases required additional therapy (octreotide-6, tranexamic acid-3, and total parenteral nutrition-1). Lymphedema (3/5 vs. 1/23), pleural effusion (4/5 vs. 1/23), and the need for additional therapy (4/5 vs. 2/23) were significantly more in patients with chylous ascites (n = 5) than those without chylous ascites (n = 23). Twenty-four cases in follow-up (39 [6–120] months) showed improvement; however, 8 required readmission (symptom recurrence-6 [25%], complication-2 [8.3%], Budd Chiari Syndrome-1, and abdominal B cell lymphoma-1). CONCLUSION: Presence of chylous ascites suggests severe disease in children with PIL. Majority of PIL children respond to dietary therapy; only 20% need additional therapy. Long-term follow-up is essential to monitor for symptoms relapse and complications.

Background: The skin's condition is altered in lymphedema patients, and evaluating this change is important. Some noninvasive methods for evaluating skin condition have been reported, especially in upper limb lymphedema. However, evaluating the skin in lower limb lymphedema remains challenging and is often limited to palpation. We aimed to develop a noninvasive skin evaluation method for lower limb lymphedema patients. Methods and Results: Twenty-five lower limb lymphedema patients were included. Skin induration and elasticity were measured using Indentometer((R)) IDM 400 and Cutometer((R)) MPA580. The relationship between the properties of skin from the healthy forearm and thigh, those of the affected thigh, and age was analyzed. Predicted skin induration age (IA) and elasticity age (EA) were calculated from the forearm, whereas actual values were calculated from the thigh, and the differences (DeltaIA and DeltaEA) were assessed. Patients were classified according to the International Society of Lymphology clinical staging system, and the differences in DeltaIA and DeltaEA were analyzed among the three groups (healthy, stage I/IIa, and stage IIb/III). Skin biopsy was performed in five unilateral lower limb lymphedema patients, and the dermal elastic fiber area was determined using microscopy with Elastica van Gieson staining. DeltaEA significantly increased with disease progression, but DeltaIA did not change significantly. Microscopy revealed elastic fiber filamentous changes, with decreased elastic fiber areas in lymphedema-affected skin. Conclusion: To our knowledge, this is the first report to evaluate lower limb skin elasticity in lymphedema quantitatively and noninvasively. DeltaEA is useful for evaluating skin condition progression in lymphedema patients.

Lymphatic filariasis (LF) is targeted for elimination by the year 2020. The Global Programme for Elimination of LF (GPELF) aims to achieve elimination by interrupting transmission through annual mass drug administration (MDA) of albendazole with ivermectin or diethylcarbamazine. The program has successfully eliminated the disease in 11 of the 72 endemic countries, putting in enormous efforts on systematic planning and implementation of the strategy. Mapping areas endemic for LF is a pre-requisite for implementing MDA, monitoring and evaluation are the components of programme implementation. This review was undertaken to assess how the mapping and impact monitoring activities have evolved to become more robust over the years and steered the LF elimination programme towards its goal. The findings showed that the WHO recommended mapping strategy aided 17 countries to delimit, plan and implement MDA in only those areas endemic for LF thereby saving resources. Availability of serological tools for detecting infection in humans (antigen/antibody assays) and molecular xenomonitoring (MX) in vectors greatly facilitated programme monitoring and evaluation in endemic countries. Results of this review are discussed on how these existing mapping and monitoring procedures can be used for re-mapping of unsurveyed and uncertain areas to ensure there is no resurgence during post-MDA surveillance. Further the appropriateness of the tests (Microfilaria (Mf)/antigenemia (Ag)/antibody(Ab) surveys in humans or MX of vectors for infection) used currently for post-MDA surveillance and their role in the development of a monitoring and evaluation strategy for the recently WHO recommended triple drug regimen in MDA for accelerated LF elimination are discussed.

INTRODUCTION AND OBJECTIVES: Chyluria, or the passage of chyle into the urine from anomalous lymphatic connections, results in a characteristic milky urine. In severe cases, it can cause significant morbidity from nutritional losses and immune suppression. Although predominantly associated with Wuchereria bancrofti infections, non-parasitic cases have also been described. Traditionally, surgical treatment has involved renal lymphatic disconnection using open or minimally invasive methods, occasionally aided by pre-operative imaging techniques like lymphangiography, or by identification of structures with laparoscopic magnification.

INTRODUCTION: Patients with pre-existing lymphedema who undergo total knee arthroplasty (TKA) for osteoarthritis (OA) are at high risk for periprosthetic joint infection. This complication usually requires removal of the implant. This study aimed to investigate whether surgical treatment of lymphedema reduces the rate of prosthesis removal in such patients. MATERIALS AND METHODS: We retrospectively reviewed our prospective database of patient information collected between January 2009 and December 2018. A total of 348 cases of lower extremity lymphedema were reviewed, and those who underwent total knee TKA for OA of the knee were included. Patient demographics, clinical data, lymphedema surgical history, and TKA surgical history including any episodes of removal were collected and analyzed. RESULTS: There were nine of 15 lymphedema patients with knee OA who subsequently underwent TKA. The mean patient age was 70.4 +/- 7.1 years. A total of 18 TKAs were performed in nine patients. The knee prosthesis removal rate was 66.7% (12/18). The prosthesis removal rate was 40% (2/5) in patients who underwent lymphedema microsurgery vs 76.9% (10/13) for those who did not (P = .03). CONCLUSIONS: Pre-existing lymphedema is associated with a high rate of knee prosthesis removal. Lymphedema microsurgery reduced the removal rate of knee prostheses. We recommend that lymphedema microsurgery be considered for patients who require TKA as a treatment for of the knee.

It has been suggested that the dynamics of the venous and lymphatic systems interact as a mutually dependent dual outflow system and that derangement of lymph flow could be reversed by surgical treatment of venous incompetence. In this report, we describe a patient in whom lymphatic function was restored after stripping of the great saphenous vein for varicosity. The patient was a 79-year-old woman who had varicose veins along the medial side of an edematous left leg. Lymphatic function was investigated using indocyanine green imaging to evaluate for the presence of lymphedema. Based on the findings, we made a diagnosis of bilateral varicosity of the great saphenous vein with left-sided lymphedema. The great saphenous vein was stripped between the groin and ankle on both sides. At 3 months after the stripping procedure, lymphatic flow was observed immediately after injection of indocyanine green in both legs along the medial side from the foot to the groin. We therefore determined that lymphatic flow had been restored after the stripping surgery. The functions of the venous and lymphatic systems are thought to be closely related, and that, if the function of one declines, the other will also be affected. Treatment of venous system, including stripping, may help to break the vicious cycle of lymphatic stasis and venous insufficiency.

Acute kidney injury (AKI) and chronic kidney disease (CKD) are interconnected syndromes with significant attributable morbidity and mortality. The disturbing trend of increasing incidence and prevalence of these clinical disorders highlights the urgent need for better understanding of the underlying mechanisms that are involved in pathogenesis of these conditions. Lymphangiogenesis and its involvement in various inflammatory conditions is increasingly recognized while its role in AKI and CKD remains to be fully elucidated. Here, we studied lymphangiogenesis in three models of kidney injury. Our results demonstrate that the main ligands for lymphangiogenesis, VEGF-C and VEGF-D, are abundantly present in tubules at baseline conditions and the expression pattern of these ligands is significantly altered following injury. In addition, we show that both of these ligands increase in serum and urine post-injury and suggest that such increment may serve as novel urinary biomarkers of AKI as well as in progression of kidney disease. We also provide evidence that irrespective of the nature of initial insult, lymphangiogenic pathways are rapidly and robustly induced as evidenced by higher expression of lymphatic markers within the kidney.

A 69-year-old woman was diagnosed as having non-small cell lung cancer (adenocarcinoma, T1aN3M1b). She had no history of surgery or abdominal trauma. She was treated with ramucirumab (10 mg/kg) plus docetaxel (60 mg/m(2)) intravenously (RAM + DTX) every 3 weeks. Although an enhanced CT examination showed a partial tumor response after eight courses of RAM + DTX, she gradually began to experience abdominal fullness with severe peripheral pitching edema. Her body weight increased by 18 kg in 2 months and RAM + DTX chemotherapy was discontinued. An enhanced CT examination showed a large amount of ascites and pleural effusion, with no obstructions of the central vein or lymphatic ducts. The ascites were white and milky in appearance and contained 527 mg/dL of triglyceride. In addition, her pleural effusion was also white and milky in appearance. No further increases in ascites and pleural effusion were observed thereafter. Four months after her last RAM + DTX chemotherapy, she continued to exhibit a partial response and no increases in ascites or pleural effusion were present. The chylous effusion might have been caused by the RAM + DTX chemotherapy.

Lymphatic vessels (LVs) are important in the regulation of tissue fluid homeostasis and the pathogenesis of tumor progression. We investigated the innervation of LVs and the response to agonists and antagonists of the autonomic nervous system in vivo. While skin-draining collecting LVs express muscarinic, alpha1- and beta2-adrenergic receptors on lymphatic endothelial cells and smooth muscle cells, intestinal lacteals express only beta-adrenergic receptors and muscarinic receptors on their smooth muscle cells. Quantitative in vivo near-infrared imaging of the exposed flank-collecting LV revealed that muscarinic and alpha1-adrenergic agonists increased LV contractility, whereas activation of beta2-adrenergic receptors inhibited contractility and initiated nitric oxide (NO)-dependent vasodilation. Tumor-draining LVs were expanded and showed a higher innervation density and contractility that was reduced by treatment with atropine, phentolamine, and, most potently, isoproterenol. These findings likely have clinical implications given the impact of lymphatic fluid drainage on intratumoral fluid pressure and thus drug delivery.

Bone marrow plasma cells of patients with myeloma are frequently aneuploid, have a high RNA content and typically show monoclonal immunoglobulin in their cytoplasm. Kappa-lambda co-expression in aneuploid tumor cells was restricted to patients with IgG lambda myeloma. Immunophenotype studies revealed early B cell expression often in association with mature B cell markers. Chromosomal aberrations were complex with a predominance of numeric but also structural lymphoma-type translocations; the latter were most prevalent in IgA myeloma. Specific translocations such as t(8;14) and t(11;14) were accompanied by aberrations of c-myc and bcl-l cellular genes. Effective salvage programs have been developed for melphalan-prednisone refractory myeloma. In comparing high-dose dexamethasone with vincristine-adriamycin + dexamethasone (VAD), VAD was superior particularly for relapsing myeloma (response rate of 60 vs. 23%). For VAD refractory myeloma, high-dose melphalan (HDM) programs were developed; total body irradiation (850 rad) followed by HDM 140 mg/m2 and supported by autologous bone marrow grafts was particularly effective and relatively well tolerated with all 4 patients still in remission from 3 to 15 months.

Background: Klippel-Trenaunay syndrome (KTS) is an overgrowth syndrome defined by capillary/venous/lymphatic malformations (CVLM) with soft tissue and/or bone hypertrophy. Whether KTS predisposes to cancer is not clear. Methods and Results: We surveyed members of the K-T Support Group (KTSG) and reviewed PubMed for “Klippel Trenaunay Syndrome” or “CVLM” and “cancer.” Individuals with cancer were reviewed for confirmation of KTS, tumor type, location, and age at presentation. Of 223 KTSG respondents, 24 (10.8%) reported 26 malignancies or benign brain tumors (diagnosed from 6 months to 68 years of age, median 41 years), including 3 who were younger than 18 years (2 with Wilms tumor). Nine of twenty-six cancers were basal cell carcinomas (4% of respondents). From 475 articles, we identified 11 cancers or brain tumors in 10 individuals with KTS. Four of these were in children (Wilms tumor n = 2; rhabdomyosarcoma n = 1; serous borderline tumor n = 1). Tumors in adults included basal cell carcinoma (n = 1), squamous cell carcinoma of skin (n = 2), and angiosarcoma, Hodgkin disease, glioblastoma, malignant hemangiopericytoma in one patient each. Ulceration or lymphedema associated with VLM or capillary malformations were associated with some basal cell or squamous cell carcinomas and angiosarcomas. Conclusions: The risk of embryonal cancer other than Wilms tumor in children with KTS does not appear to be higher than in the general population. Wilms tumor incidence is under 5%, and routine surveillance is not indicated. In adults, particular attention should be paid to skin in the area of malformations. These conclusions may not apply to all overgrowth syndromes with vascular malformations.

Lymphatic vasculature is crucial for metastasis in triple-negative breast cancer (TNBC); however, cellular and molecular drivers controlling lymphovascular metastasis are poorly understood. We define a macrophage-dependent signaling cascade that facilitates metastasis through lymphovascular remodeling. TNBC cells instigate mRNA changes in macrophages, resulting in beta4 integrin-dependent adhesion to the lymphovasculature. beta4 integrin retains macrophages proximal to lymphatic endothelial cells (LECs), where release of TGF-beta1 drives LEC contraction via RhoA activation. Macrophages promote gross architectural changes to lymphovasculature by increasing dilation, hyperpermeability, and disorganization. TGF-beta1 drives beta4 integrin clustering at the macrophage plasma membrane, further promoting macrophage adhesion and demonstrating the dual functionality of TGF-beta1 signaling in this context. beta4 integrin-expressing macrophages were identified in human breast tumors, and a combination of vascular-remodeling macrophage gene signature and TGF-beta signaling scores correlates with metastasis. We postulate that future clinical strategies for patients with TNBC should target crosstalk between beta4 integrin and TGF-beta1.

Secondary Angiosarcoma (Stewart-Treves Syndrome) is a rare malignant cutaneous lesion, which arises in chronic lymphedema of the extremity, often observed after breast cancer treatment. We reviewed the history and the oncological outcome of two patients with this disease. Multimodal therapy including hyperthermic isolated limb perfusion with TNF-alpha and Melphalan, combined with radical resection of the affected skin and subcutaneous tissue including the fascia, with large safety margins may probably lead to better survival.

PURPOSE: Regional nodal irradiation for women with breast cancer is known to be an important risk factor for the development of upper extremity lymphedema, but tools to accurately predict lymphedema risks for individual patients are lacking. This study sought to develop and validate a nomogram to predict lymphedema risk following axillary surgery and radiotherapy in women with breast cancer. METHODS: Data from 1,832 women accrued on the MA.20 trial between March 2000 and February 2007 were used to create a prognostic model with National Cancer Institute Common Toxicity Criteria Version 2.0 grade 2 or higher lymphedema as the primary end point. Multivariable logistic regression estimated model performance. External validation was performed on data from a single large academic cancer center (N = 785). RESULTS: In the MA.20 trial cohort, three risk factors were predictive of lymphedema risk: BMI (adjusted odds ratio 1.05 per unit BMI; 95% CI, 1.03 to 1.08, p < 0.001), extent of axillary surgery (adjusted odds radio for 8–11 lymph nodes removed, 3.28; 95% CI, 1.53 to 7.89, p = 0.004; 12–15 lymph nodes, 4.04; 95% CI, 1.76 to 10.26, p = 0.002, >/ = 16 nodes, 5.08; 95% CI, 2.26 to 12.70, p < 0.001), and extent of nodal irradiation (adjusted odds radio for limited, 1.66; 95% CI, 1.08 to 2.56, p = 0.02; for extensive, 2.31; 95% CI, 1.28 to 4.10, p = 0.004). A nomogram was created from these data that predicted lymphedema risk with reasonable accuracy confirmed by both internal (concordance index, 0.69; 95% CI 0.64 to 0.74) and external validation (concordance index, 0.71; 95% CI 0.66 to 0.76). CONCLUSIONS: The nomogram created from the MA.20 randomized trial data utilizing clinical information may be useful for lymphedema screening and risk stratification for therapeutic intervention trials.

Proliferation of aberrant, dysfunctional lymphatic vessels around solid tumors is a common histologic finding. Studies have shown that abnormalities in lymphatic function result in accumulation of inflammatory cells with an immunosuppressive profile. We tested the hypothesis that dysfunctional lymphatic vessels surrounding solid tumors regulate changes in the tumor microenvironment and tumor-specific immune responses. Using subcutaneously implanted mouse melanoma and breast cancer tumors in a lymphatic endothelial cell-specific diphtheria toxin receptor transgenic mouse, we found that local ablation of lymphatic vessels increased peritumoral edema, as compared with controls. Comparative analysis of the peritumoral fluid demonstrated increases in the number of macrophages, CD4(+) inflammatory cells, F4/80(+)/Gr-1(+) (myeloid-derived suppressor cells), CD4(+)/Foxp3(+) (Tregs) immunosuppressive cells, and expression of inflammatory cytokines such as TNFalpha, IFNgamma, and IL1beta following lymphatic ablation. Tumors grown in lymphatic ablated mice exhibited reduced intratumoral accumulation of cytotoxic T cells and increased tumor PD-L1 expression, causing rapid tumor growth, compared with tumors grown in nonlymphatic-ablated mice. Our study suggests that lymphatic dysfunction plays a role in regulating tumor microenvironments and may be therapeutically targeted in combination with immunotherapy to prevent tumor growth and progression.

This pilot, double-blind, randomized, placebo-controlled study is aimed at evaluating the effectiveness of low-level laser therapy (LLLT) as a complementary treatment to complete decongestive therapy (CDT) treating lymphedema among breast cancer patients for 12 months post-intervention. Study population was breast cancer patients who were diagnosed and referred to lymphedema clinic for CDT. Participants (n = 22) were randomized and assigned into either an active laser intervention group or an inactive laser placebo-control group. Active LLLT was administered to participants twice a week at the beginning of each CDT session. Outcome measures included lymphedema symptoms, symptom distress, and limb volume by an infrared perometer. Participants in the active and placebo laser groups were comparable in demographic and clinical predictors of lymphedema. In comparison with the placebo group (83.3%), significantly fewer participants in the active laser group (55.6%) reported more than one lymphedema symptom (p = 0.012) at 12 months post-intervention. Significantly, more patients in the active laser group (44.4%) reported less than two impaired limb mobility symptoms in comparison with the placebo group (33.3%) at 12 months post-intervention (p = 0.017). The active laser group had statistically significant improvements in symptom distress of sadness (p = 0.005) from 73 to 11% and self-perception (p = 0.030) from 36 to 0% over time from baseline to 12-months post-intervention. There was no significant reduction in limb volume. Findings of the trial demonstrated significant benefits of complementary LLLT for relieving symptoms and improvement of emotional distress in breast cancer patients with lymphedema.

Tumor-associated lymphatic vessels (LV) serve as a route of cancer dissemination through the prometastatic crosstalk between lymphatic endothelial cells (LECs) lining the LVs and cancer cells. Compared to blood endothelial cell-derived angiocrine factors, however, LEC-secreted factors in the tumor microenvironment and their roles in tumor metastasis are poorly understood. Here, we report that ELK3 expressed in LECs contributes to the dissemination of cancer cells during tumor growth by providing oncogenic miRNAs to tumor cells through exosomes. We found that conditioned medium from ELK3-suppressed LECs (LCM) lost its ability to promote the migration and invasion of breast cancer cells such as MDA-MB-231, Hs578T and BT20 in vitro. Suppression of ELK3 in LECs diminished the ability of LECs to promote tumor growth and metastasis of MDA-MB-231 in vivo. Exosomes derived from LECs significantly increased the migration and invasion of MDA-MB-231 in vitro, but ELK3 suppression significantly diminished the pro-oncogenic activity of exosomes from LECs. Based on the miRNA expression profiles of LECs and functional analysis, we identified miR-503-3p, miR-4269 and miR-30e-3p as downstream targets of ELK3 in LECs, which cause the above phenotype of cancer cells. These findings strongly suggest that ELK3 expressed in LECs is a major regulator that controls the communication between the tumor microenvironment and tumors to support cancer metastasis.

The origin of blood and lymphatic vessels in high-grade serous adenocarcinoma of ovary (HGSOC) is uncertain. We evaluated the potential of cancer stem cells (CSCs) in HGSOC to contribute to their formation. Using spheroids as an in vitro model for CSCs, we have evaluated their role in primary malignant cells (PMCs) in ascites from previously untreated patients with HGSOC and cell lines. Spheroids from PMCs grown under specific conditions showed significantly higher expression of endothelial, pericyte and lymphatic endothelial markers. These endothelial and lymphatic cells formed tube-like structures, showed uptake of Dil-ac-LDL and expressed endothelial nitric oxide synthase confirming their endothelial phenotype. Electron microscopy demonstrated classical Weibel-Palade bodies in differentiated cells. Genetically, CSCs and the differentiated cells had a similar identity. Lineage tracking using green fluorescent protein transfected cancer cells in nude mice confirmed that spheroids grown in stem cell conditions can give rise to all three cells. Bevacizumab, a monoclonal antibody that targets vascular endothelial growth factor inhibited the differentiation of spheroids to endothelial cells in vitro. These results suggest that CSCs contribute to angiogenesis and lymphangiogenesis in serous adenocarcinoma of the ovary, which can be inhibited.

Hyaluronan (HA) is a glycosaminoglycan with a simple structure but diverse and often opposing functions. The biological activities of this polysaccharide depend on its molecular weight and the identity of interacting receptors. HA is initially synthesized as high molecular-weight (HMW) polymers, which maintain homeostasis and restrain cell proliferation and migration in normal tissues. These HMW-HA functions are mediated by constitutively expressed receptors including CD44, LYVE-1, and STABILIN2. During normal processes such as tissue remodeling and wound healing, HMW-HA is fragmented into low molecular weight polymers (LMW-HA) by hyaluronidases and free radicals, which promote inflammation, immune cell recruitment and the epithelial cell migration. These functions are mediated by RHAMM and TLR2,4, which coordinate signaling with CD44 and other HA receptors. Tumor cells hijack the normally tightly regulated HA production/fragmentation associated with wound repair/remodeling, and these HA functions participate in driving and maintaining malignant progression. However, elevated HMW-HA production in the absence of fragmentation is linked to cancer resistance. The controlled production of HA polymer sizes and their functions are predicted to be key to dissecting the role of microenvironment in permitting or restraining the oncogenic potential of tissues. This review focuses on the dual nature of HA in cancer initiation vs. resistance, and the therapeutic potential of HA for chemo-prevention and as a target for cancer management.

Obesity is linked to the risk of breast cancer and treatment-related lymphedema (BCRL). Thus, knowledge of how obesity, or more specifically total body fat percentage (TBF) and body mass index (BMI), affect measurements that are used to detect or track lymphedema is clinically important. Tissue dielectric constant (TDC) is one measure used to help characterize lymphedema features, detect its presence, and assess treatment-related changes. The goal of this research was to determine the extent to which TDC values depend on TBF and BMI. TDC was measured on both forearms (2.5mm depth) in 250 women (18–72 years) along with TBF (impedance, 50KHz). TBF was 12.2%–54.4% (median = 29.3%) and BMI was 14.7Kg/m2–44.3 Kg/m2 (median = 22.6 Kg/m2). TDC values and interarm ratios were compared between subgroups that had TBF and BMI values in lower vs. upper quartiles. Subjects in the upper quartile had slightly lower TDC values (1.3 TDC units, p < 0.01) that was at most a 5% differential. Contrastingly, TDC interarm ratios were not dependent on TBF or BMI levels. These findings suggest that when tracking lymphedema changes using the TDC method, treatment-related or temporal changes in a woman's TBF or BMI are unlikely to significantly impact TDC values or their interarm ratios.

Background: Neoangiogenesis has proven to be a relevant pathogenetic mechanism in gastric cancer (GC) and lymphatic spread represents an important well-known prognostic factor. Vascular endothelial growth factor C (VEGF-C) plays a key role in lymphangiogenesis and its blood levels in GC patients are easily measurable. This analysis aimed to investigate the prognostic role of preoperative VEGF-C blood levels. Methods: VEGF-C serum levels were determined by enzyme-linked immunoadsorbent assay (ELISA) in 186 patients observed at our institution from January 2004 until December 2009 and 82 healthy subjects. Statistical analyses were performed using SPSS 21.0. Results: VEGF-C levels were significantly higher in GC patients (median: 287.4 pg/mL; range, 76.2–865.2 pg/mL) than in the control group (median VEGF-C: 31 pg/mL; range, 12–97 pg/mL). A significant correlation between VEGF-C levels, T, N and tumor stage has been described. The median overall survival (OS) was statistically significantly higher in pts with low serum VEGF-C levels [median: not reached (NR) vs. 26 months; P < 0.0001]. Higher preoperative VEGF-C levels correlated also with earlier disease relapse and poor disease-free survival (DFS) (median NR in each subgroup, P = 0.005). Furthermore, high VEGF-C levels [hazard ratio (HR) = 2.7; P = 0.018] and tumor grading (HR = 0.44; P = 0.007) were independent prognostic factors for OS at multivariate analysis. Conclusions: Our study showed that increased VEGF-C levels are significantly associated with advanced regional lymph node involvement and poor OS and DFS in pts with resected GC paving the way to a possible application as prognostic factor in the clinical practice.

Objectives: Melanoma induces lymphangiogenesis by secreting lymphangiogenic growth factors. The aim of this study was to examine the role of tumour lymphangiogenesis in survival of patients with cutaneous melanoma. Methods: Immunostaining of one hundred melanoma specimens was done with lymphatic-specific antibody D2–40. The quantification of tumour lymphangiogenesis—lymphatic vessel density (LVD) and lymphatic vessel area (LVA)—was calculated by computer-assisted morphometric analysis. Results: High intratumoural LVD, high peritumoural LVD, male gender, greater tumour thickness and Clark level IV/V were significantly associated with shorter disease-free survival (p = 0.001, p = 0.004, p = 0.004, p = 0.000 and p = 0.008, respectively) and melanoma-specific survival (p = 0.002, p = 0.002, p = 0.001, p = 0.000 and p = 0.017, respectively), while the trunk melanoma site was significantly associated only with shorter disease-free survival (p = 0.033). No significant association of LVA with survival was found. At multivariate analysis, peritumoural LVD [hazard ratio (HR) = 2.143, 95% confidence interval (CI) 1.097–4.189, p = 0.026)] and melanoma thickness (HR = 1.276, 95%CI 1.106–1.473, p = 0.001) were independent predictors of disease-free survival, while intratumoural LVD (HR = 3.446, 95%CI 1.465–8.109, p = 0.005), peritumoural LVD (HR = 2.742, 95%CI 1.313–5.725, p = 0.007) and gender (HR = 2.880, 95%CI 1.304–6.362, p = 0.009) were independent predictors of melanoma-specific survival. Conclusion: capital TE, Cyrillichis study shows that LVD enables better prediction of survival than melanoma thickness and other clinical-pathological parameters. Intratumoural LVD is the most significant predictor of melanoma-specific survival, while only peritumoural LVD has a significant impact on both, a disease-free survival and a melanoma-specific survival.

Objective: To explore the relationships between expression of CD44v6, lymphatic vessel density (LVD) and the clinicopathological parameters of patients. Methods: One hundred early gastric cancer tissues, 55 high-grade gastric intraepithelial neoplasia (HGIN) tissues, 60 low-grade gastric intraepithelial neoplasia (LGIN) tissues and 60 chronic superficial gastritis tissues were collected and set as gastric cancer group, HGIN group, LGIN group and gastritis group respectively. The expression of CD44v6 and LVD of patients in all the groups were detected using two-step immunohistochemical method to analyze the relationships between the expression of CD44v6 and lymphatic vessel density in early gastric cancer tissues and their relationships with the clinicopathological parameters of patients. The values of LVD in predicting lymph node metastasis in early gastric cancer were evaluated using receiver operating characteristic (ROC) curve. Results: The positive expression of CD44v6 and LVD in the gastritis group, LGIN group, HGIN group and gastric cancer group gradually increased. The positive expression of CD44v6 and LVD in early gastric cancer tissues were in no correlation with the gender, age, tumor site, maximum diameter, differentiation degree and invasion depth (P > 0.05) and in a correlation with lymphatic metastasis and lymphatic vessel invasion (P < 0.06). The positive expression of CD44v6 in the early gastric cancer tissues was in a positive correlation with LVD (P < 0.05). The analysis of ROC curves suggested that the area under ROC curve of predicting lymphatic metastasis of early gastric cancer with LVD was 0.837 (95% CI: 0.756 ∼ 0.910), and the cut-off value was 14; the corresponding sensitivity and specificity were 63.6% and 90.2 respectively. Conclusion: The expression of CD44v6 and LVD in early gastric cancer tissues are in a close correlation with the clinicopathologic features, and joint detection of expression of CD44v6 and LVD can be taken as the indicator of gastric cancer metastasis.

Post-partum breast cancer patients, or breast cancer patients diagnosed within 10 years of last childbirth, are ∼3–5 times more likely to develop metastasis in comparison to non-post-partum, or nulliparous, patients. Additionally, post-partum patients have increased tumor-associated lymphatic vessels and LN involvement, including when controlled for size of the primary tumor. In pre-clinical, immune-competent, mouse mammary tumor models of post-partum breast cancer (PPBC), tumor growth and lymphogenous tumor cell spread occur more rapidly in post-partum hosts. Here we report on PD-L1 expression by lymphatic endothelial cells and CD11b+ cells in the microenvironment of post-partum tumors, which is accompanied by an increase in PD-1 expression by T cells. Additionally, we observed increases in PD-L1 and PD-1 in whole mammary tissues during post-partum mammary gland involution; a known driver of post-partum tumor growth, invasion, and metastasis in pre-clinical models. Importantly, implantation of murine mammary tumor cells during post-partum mammary gland involution elicits a CD8+ T cell population that expresses both the co-inhibitory receptors PD-1 and Lag-3. However, upon anti-PD-1 treatment, during post-partum mammary gland involution, the involution-initiated promotional effects on tumor growth are reversed and the PD-1, Lag-3 double positive population disappears. Consequently, we observed an expansion of poly-functional CD8+ T cells that produced both IFNgamma and TNFalpha. Finally, lymphatic vessel frequency decreased significantly following anti-PD-1 suggesting that anti-PD-1/PD-L1 targeted therapies may have efficacy in reducing tumor growth and dissemination in post-partum breast cancer patients.

CD147 (basigin; EMMPRIN), hyaluronan, and hyaluronan receptors (e.g., CD44) are intimately involved in several phenomena that underlie malignancy. A major avenue whereby they influence tumor progression is most likely their role in the characteristics of cancer stem cells (CSCs), subpopulations of tumor cells that exhibit chemoresistance, invasiveness, and potent tumorigenicity. Both CD147 and hyaluronan have been strongly implicated in chemoresistance and invasiveness, and may be drivers of CSC characteristics, since current evidence indicates that both are involved in epithelial-mesenchymal transition, a crucial process in the acquisition of CSC properties. Hyaluronan is a prominent constituent of the tumor microenvironment whose interactions with cell surface receptors influence several signaling pathways that lead to chemoresistance and invasiveness. CD147 is an integral plasma membrane glycoprotein of the Ig superfamily and cofactor in assembly and activity of monocarboxylate transporters (MCTs). CD147 stimulates hyaluronan synthesis and interaction of hyaluronan with its receptors, in particular CD44 and LYVE-1, which in turn result in activation of multiprotein complexes containing members of the membrane-type matrix metalloproteinase, receptor tyrosine kinase, ABC drug transporter, or MCT families within lipid raft domains. Multivalent hyaluronan-receptor interactions are essential for formation or stabilization of these lipid raft complexes and for downstream signaling pathways or transporter activities. We conclude that stimulation of hyaluronan-receptor interactions by CD147 and the consequent activities of these complexes may be critical to the properties of CSCs and their role in malignancy. Anat Rec, 2019. (c) 2019 Wiley Periodicals, Inc.,

Lymphatic metastasis plays an important role in cancer progression and prognosis. However, conventional small-molecule chemotherapy drugs inefficiently access the lymphatic system, making the effective eradication of lymphatic metastases difficult without dose-limiting toxicity. Various formulation and nanomedicine-based approaches can be used to significantly enhance the trafficking of small-molecule, peptide and protein drugs toward the lymphatic system to enhance drug exposure at sites of lymphatic cancer growth. However, a number of obstacles exist in translating improved lymphatic exposure into improved chemotherapeutic outcomes. This review highlights the opportunities and challenges inherent in employing formulation and nanomedicinal approaches to improve chemotherapeutic drug activity within the lymphatic system and, importantly, at sites of lymphatic cancer metastasis.

Purpose: Our previous study proved that FOXM1 regulates colorectal cancer (CRC) cell metastasis through epithelial-mesenchymal transition program. The aim of this study is to further explore the underlying mechanism of FOXM1 in CRC. Materials and methods: In this study, we detected the mRNA and protein expressions of FOXM1 and beta-catenin in CRC tissues and their corresponding normal-appearing tissues (NATs) by quantitative reverse transcription-PCR and western blot analysis, respectively. Then the potential link between FOXM1 and beta-catenin in CRC tissues was analyzed. Furthermore, we systematically analyzed the biological functions of FOXM1 in CRC cells after reconstitution of FOXM1 expression in vitro. Moreover, the mechanism of FOXM1-promoted CRC progression by improving beta-catenin nuclear translocation was also discussed. Results: Our data demonstrated that FOXM1 and beta-catenin were upregulated in CRC tissues compared with the corresponding NATs (P < 0.05). Clinicopathologic analysis revealed that increased FOXM1 (or beta-catenin) expression positively correlated with some clinicopathologic features, such as tumor size, TNM stage, lymphatic metastasis, and distant metastasis (P < 0.05). Meanwhile, the possible relationships between FOXM1 and beta-catenin in CRC samples were evaluated using SPSS software, and a significant positive correlation was found (P < 0.05). In vitro data demonstrate that elevated FOXM1 expression exerted oncogenic effects on CRC via activation of beta-catenin signaling pathway. The inhibition of beta-catenin by siRNAs significantly attenuates FOXM1-induced malignant activities. Conclusion: The data suggested that FOXM1/beta-catenin is critical for malignancy of CRC, which may constitute a potential therapeutic strategy for CRC.

BACKGROUND: Controversy in axillary reverse mapping in axillary lymph node dissection (ALND) possibly results from incomplete recognition of the arm lymphatic system (ALS) and its compromise to oncological safety. The iDEntification and Preservation of ARm lymphaTic system (DEPART) technique facilitates complete identification of ALS; therefore, its use may decrease the occurrence of arm lymphedema. This study aimed to examine the arm lymphedema rate, locoregional recurrence, and feasibility to perform DEPART in ALND. METHODS: Patients from February 2013 to October 2017 from two tertiary referral centers were randomly assigned to two groups. In the study group, indocyanine green and methylene blue (MB) were utilized to identify arm sentinel nodes, and 0.1 ml MB was injected into the arm sentinel nodes to reveal the subsequent-echelon nodes and lymphatics. Gross arm lymph nodes were examined by intraoperative partial frozen section and were removed if positive. Arm lymphedema, local recurrence, regional recurrence, and distant metastasis were recorded at different follow-up examinations. RESULTS: Arm sentinel nodes were identified in 573 (83.2%) patients. Subsequent-echelon nodes and lymphatics were visualized in 558 (97.4%) patients. Metastatic arm nodes were identified in 38 (6.8%) patients. The arm lymphedema rate was 3.3% (18/543) in the study group versus 15.3% (99/648) in the control group (p < 0.001) after 37-month median follow-up. Regional recurrence showed no difference between the two groups (1.4% and 1.2%, respectively) (p = 0.392). CONCLUSIONS: DEPART can benefit breast cancer patients who undergo ALND, reducing the arm lymphedema rate without adversely affecting the morbidity of regional recurrence.

Vascular anomaly is a general term that includes all vascular malformations, vascular tumors, and other congenital vascular defects. Vascular malformation is the most common term, and it describes blood vessels that are abnormally formed at birth. Vascular malformations can develop in any part of the body. The most common location is in the lower extremities. Vascular malformations involve arteries, veins, or lymphatic vessels, or a combination of these. Our patient was a 22-year-old man with an arteriovenous malformation in his left forearm. He was admitted due to increased pain and swelling on his left forearm over the previous 9 months. He had 1 arterial feeder derived from the ulnar artery and 2 venous drainage systems at the magnetic resonance angiography. We used indocyanine green fluorescence angiography to assess the arteriovenous malformation during surgery. We found that it was a very useful and unique technique for assessing the anomalies of the vascular anatomy and eradicating the nidus of the arteriovenous malformation. It could prove to be very helpful in avoiding significant blood loss during surgery.

BACKGROUND: Propranolol hydrochloride is the first-line agent recommended for the treatment of infantile hemangiomas (IH). Serious adverse effects of propranolol therapy for hemangiomas are infrequent. CASE PRESENTATION: We report a case presented in deep hypoglycemic coma during his treatment with propranolol for IH. Through our case report and the review of the literature, we aimed to underline the importance of recognizing adverse effects during propranolol therapy. Although propranolol has a long history of safe and effective use in infants and children, pediatricians should be aware that life-threatening adverse effects can happen during propranolol therapy for IH. CONCLUSION: Early identification of these adverse effects can be of great importance for patient management and prognosis. It must certainly be noted that not just early identification among doctors, but education for parents is crucial.

BACKGROUND: It has been 15 years since sirolimus, an mTOR inhibitor, received Food and Drug Administration approval to prevent acute rejection in kidney transplantation, and 8 years since its analog everolimus acquired the same status. Since then, these drugs have become more and more utilized and their immunosuppressive and antiproliferative properties have been tested in a great variety of clinical conditions, often achieving excellent results. Despite such positive evidence, the on-label indications for these rapalogs are still very restrictive, especially in children. AIMS: The aims of this study were to describe our center's experience with sirolimus and everolimus in managing rare pediatric conditions for which mTOR inhibitors have been reported as a therapeutic option, although without conclusive approval from regulatory agencies, and to evaluate safety and tolerability of the treatment at the prescribed doses. METHODS: All the subjects who received off-label sirolimus or everolimus at the Pediatric Department of the IRCCS Burlo Garofolo in the last 13 years were included. For each disease found in our case series, we reviewed the current scientific literature. RESULTS: Off-label treatment with rapalogs was prescribed in 16 children (11 males, 5 females, median age of 9.5 years, range 1–16 years). Seven had immunologic disorders: four autoimmune lymphoproliferative syndrome (ALPS), one multicentric Castleman disease (mCD), one activated PI3K delta kinase syndrome (APDS), and one immunodysregulation with polyendocrinopathy enteropathy X-linked (IPEX). Eight had proliferative disorders or vascular anomalies: one cystic lymphangioma, two Bannayan-Riley-Ruvalcaba syndrome (BRRS), one blue rubber bleb nevus syndrome (BRBNS), two tuberous sclerosis complex (TSC), and one low-flow mixed arterial and venous malformation. One case had congenital hyperinsulinism (CHI). The average dosage administered was 1 mg/m(2) for sirolimus and 7 mg/m(2) for everolimus. We experienced a good measurable clinical improvement in 14 patients. Nobody experienced serious adverse events (SAEs). The therapy was interrupted in two cases, for lack of efficacy and poor tolerance in one case and for occurrence of bacterial pneumonia in the other one. A review of the literature identified 101 published reports that met our inclusion criteria. CONCLUSIONS: Although use of mTOR inhibitors has been considered to be complicated, our experience shows that, using low dosages, it is possible to obtain relevant clinical improvements, with a good profile of safety and tolerability.

Congenital hemangiomas are benign vascular tumors, categorized by their postnatal behavior as rapidly involuting, non-involuting, or partially involuting. They are typically solitary, with a predilection for the head or limbs near a joint. We present two infants with small, multifocal congenital nonprogressive hemangiomas of the skin, one associated with hepatic and intracranial lesions, and another with an in utero intracranial hemorrhage and hydrocephalus. These cases further extend the differential diagnosis of congenital multifocal vascular lesions or “hemangiomatosis.”

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a rare lymphatic anomaly with significant morbidity and mortality. KLA is characterized by diffuse multifocal lesions comprised of focal areas of “kaposiform” spindled cells accompanying malformed lymphatic channels. The goal of this study was to identify activated signaling pathways in cells isolated from three KLA patients for the purpose of testing new therapies. PROCEDURE: Cells were obtained from the lungs of one patient isolated at autopsy and the spleen of two patients removed in surgery due to disease complications. A protein kinase array was performed on the KLA cell lysates and normal lymphatic endothelial cells. RESULTS: Higher activation of key signaling pathways in the KLA cells, including PRAS40, AKT1/2/3, and ERK-1/2, was identified by protein kinase array and confirmed by Western blot analysis. This indicated a role for highly activated PI3K-AKT and MAPK-ERK-1/2 signaling pathways in KLA cells. Cell proliferation studies assessed PI3K inhibitors (LY294002; BYL719), AKT inhibitor ARQ092, mTOR inhibitor rapamycin, and MAPK inhibitor U0126. These studies demonstrated that PI3K-AKT-mTOR and MAPK signaling are important mediators of KLA cell proliferation. BYL719 and rapamycin were more effective at inhibiting KLA cell proliferation than U0126. CONCLUSIONS: Our studies using cells from KLA patient lesions demonstrate that these cells are highly proliferative and the PI3K-AKT-mTOR and MAPK pathways are promising therapeutic targets. Development and clinical trials of PI3K, AKT, and MAPK inhibitors for cancer treatment and the data in this study lend support for early clinical trials assessing the efficacy of these inhibitors in KLA patients.

Pseudomyogenic hemangioendothelioma (PMH) is a rare neoplasm with vascular and sarcomatous elements, unpredictable course, and uncommon metastatic or fatal potential. Although systemic chemotherapy has been reported with variable success, generally accepted treatment is aggressive surgery with wide margins. Evidence-based treatment options are lacking, and lack of clear prognostic features poses a risk of undertreatment or overtreatment with associated morbidity and mortality. We report the use of initial systemic therapy with oral sirolimus (SIR) and IV zoledronic acid (ZA) to induce a sustained clinical response and avoidance of amputation in a 6-year-old boy. At 37 months after diagnosis, our patient remains in sustained clinical remission as documented by x-ray, MRI, and PET-CT with return of normal mobility/activity and resolution of swelling and pain. Literature review identified 20 cases of pediatric and young adult patients with PMH, of which 7 received some form of systemic therapy. To the best of our knowledge, our patient represents the youngest reported case of PMH and the first successful and limb-sparing utilization of systemic chemotherapy as primary treatment for PMH.

Intussusception in lymphatic vessels has received less attention than in blood vessels. In tumors and pseudotumors of blood vessels with intravascular papillary structures, including sinusoidal hemangioma and intravascular papillary endothelial hyperplasia, we observed exuberant intussusceptive angiogenesis, as well as the similarity between papillae (term used by pathologists) and pillars/folds (hallmarks of intussusceptive angiogenesis). A similar response could be expected in lymphangiomas (lymphatic malformations and reactive processes rather than tumors) with papillae. The aim of this work is to assess whether papillae/pillars/folds and associated structures (vessel loops and septa) are present in lymphangiomas, and to establish the characteristics and formation of these structures. For this purpose, we selected lymphangiomas with intraluminal papillae (n: 18), including cystic, cavernous, circumscriptum, and progressive types, of which two cases of each type with a greater number of papillae were used for serial histologic sections and immunohistochemistry. The studies showed a) dilated lymphatic spaces giving rise to lymphatic-lymphatic vascular loops, which dissected and encircled perilymphatic structures (interstitial tissue structures/ITSs and pillars/posts), b) ITSs and pillars, surrounded by anti-podoplanin-positive endothelial cells, protruding into the lymphatic spaces (papillary aspect), and c) splitting, remodeling, linear arrangement, and fusion of papillae/pillars/folds, forming papillary networks and septa. In conclusion, as occurs in blood vessel diseases, the development of lymphatic vessel loops, papillae/pillars/folds, and septa (segmentation) supports intussusceptive lymphangiogenesis and suggests a piecemeal form of intussusception. This intussusceptive lymphangiogenesis in lymphatic diseases can provide a basis for further studies of lymphatic intussusception in other conditions, with clinical and therapeutic implications. This article is protected by copyright. All rights reserved.

Generalized lymphatic anomaly (GLA) is characterized by diffuse or multicentric proliferation of dilated lymphatic vessels resembling common lymphatic malformation, and thoracic lesions can be related to a poor prognosis. Sirolimus, an inhibitor of the mammalian target of rapamycin, is effective against vascular anomalies with few severe adverse drug reactions. Here, we report the case of a patient with intractable hemothorax pleural effusion due to GLA who was treated with sirolimus and experienced disseminated intravascular coagulation. Although a standard treatment for GLA has not been established, pleural fluid might be reduced using the Kampo medicine Eppikajyutsuto.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by multi-systemic vascular dysplasia affecting 1 in 5000 people worldwide. Individuals with HHT suffer from many complications including nose and gastrointestinal bleeding, anemia, iron deficiency, stroke, abscess, and high-output heart failure. Identification of the causative gene mutations and the generation of animal models have revealed that decreased transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP) signaling and increased vascular endothelial growth factor (VEGF) signaling activity in endothelial cells are responsible for the development of the vascular malformations in HHT. Perturbations in these key pathways are thought to lead to endothelial cell activation resulting in mural cell disengagement from the endothelium. This initial instability state causes the blood vessels to response inadequately when they are exposed to angiogenic triggers resulting in excessive blood vessel growth and the formation of vascular abnormalities that are prone to bleeding. Drugs promoting blood vessel stability have been reported as effective in preclinical models and in clinical trials indicating possible interventional targets based on a normalization approach for treating HHT. Here, we will review how disturbed TGF-beta and VEGF signaling relates to blood vessel destabilization and HHT development and will discuss therapeutic opportunities based on the concept of vessel normalization to treat HHT.

Kaposiform lymphangiomatosis (KLA) is a vascular anomaly featuring lymphatic expansion. It has no known cause, no effective treatment, and is associated with high morbidity. Proliferative cells from 3 KLA patient lesions were characterized relative to adiopose-derived mesenchyme stem cells (ADSCs) and cells derived from a patient with the related disease kaposiform hemangioendothelioma (KHE). KLA cells variably expressed markers of mesenchyme stem cells (CD73, CD90, CD105, CD146) and lacked endothelial cell markers (CD31, CD34) as determined by flow cytometry. They expressed markers of vascular pericytes (neural/glial antigen 2, alpha-smooth muscle actin, platelet-derived growth factor-beta receptor, and CXCL12) as determined by quantitative reverse transcription polymerase chain reaction. Lesion cells transcribed vascular markers VEGFC and VEGFD, as well as VCAM-1, the latter of which was confirmed by flow cytometry, consistent with angiogenic MSC-like pericytes. Furthermore, conditioned medium from each was shown to promote the proliferation of growth factor-starved lymphatic endothelial cells. Unlike kaposiform hemangioendothelioma-derived MSC-like pericytes and ADSCs, KLA isolates were defective in support of vascular network formation in co-cultures with either vascular or lymphatic endothelial cells. Genetic analysis by whole exome sequencing revealed novel variant alleles in 2 populations of KLA cells (BAD, TSC1) that may bear on aberrant pericyte growth and function.

Infantile hemangiomas are the most common tumors of infancy and are often managed with oral beta-blockers to address or prevent associated complications. However, treatment with propranolol can occasionally be associated with sleep disturbances, which in some cases are severe enough to warrant discontinuation or replacement with another agent. We herein report four cases in which treatment with propranolol resulted in significant sleep disturbances prompting substitution with atenolol, which in some cases resolved these issues.

The discoveries of new genes underlying genetic skin diseases have occurred at a rapid pace, supported by advances in DNA sequencing technologies. These discoveries have translated to an improved understanding of disease mechanisms at a molecular level and identified new therapeutic options based on molecular targets. This article highlights just a few of these recent discoveries for a diverse group of skin diseases, including tuberous sclerosis complex, ichthyoses, overgrowth syndromes, interferonopathies, and basal cell nevus syndrome, and how this has translated into novel targeted therapies and improved patient care.

RATIONALE AND OBJECTIVES: Lymphangiomatosis is a rare disease characterized by the widespread presence of lymphangiomas in any part of the body. In previous studies, lymphatic vessel abnormalities in lymphangiomatosis have only rarely been mentioned. The objective of this paper is to discuss the imaging features of lymphangiomatosis, including cystic lesions and lymphatic abnormalities, on computed tomography lymphangiography (CTL). MATERIALS AND METHODS: All 34 patients who were diagnosed with lymphangiomatosis underwent direct lymphangiography followed by CTL. The CTL images were independently analyzed by two experienced radiologists. The CTL image analyses included assessment of the features of cystic lesions and lymphatic vessel abnormalities. RESULTS: (i) CTL revealed several cystic lesions ranging in size from 3 mm to 14 cm; the lesions were located in the neck and shoulders (61.8%), mediastinum (52.9%), retroperitoneum (70.6%), and pelvis and perineum (64.7%). (ii) Approximately 29.4% of patients showed abnormal contrast medium accumulation in cystic masses. (iii) Approximately 67.6% of patients showed lymphatic reflux. (iv) Dilated lymphatic vessels were distributed in the mediastinum (38.2%), retroperitoneum (50.0%), pelvis and lower limbs (47.1%). (v) Finally, 8.8% of patients had perineal lymphatic fistulae, and 2.9% of patients had chyluria. CONCLUSION: Lymphangiomatosis is a type of systemic lymphatic abnormality that is accompanied by multiple cystic lesions. The therapeutic measures for lymphangiomatosis are determined by the size of the cystic lesions. Furthermore, the prognosis of lymphangiomatosis is affected by lymphatic abnormalities.

Lymphatic malformations (LMs) are disfiguring congenital anomalies characterized by aberrant growth of lymphatic vessels. They are broadly categorized histopathologically as macrocystic and microcystic. Although sclerotherapy has shown some success in the treatment of macrocystic malformations there has been less progress with developing treatment strategies for microcystic malformations. In this study we characterized lymphatic endothelial cells isolated from lymphatic and lymphaticovenous malformations. When compared to cells from normal lymphatic vessels, we found that the primary cultured malformed cells are morphologically different and also exhibited differences in binding, proliferation, migration, and tube formation. Transcriptome analysis identified several genes whose expression was substantially higher in malformed compared to normal lymphatic endothelium, including DIRAS3 and FOXF1. Further analysis of LM tissue samples revealed distinguishing gene expression patterns that could pave the way to understanding the molecular pathogenesis of LMs. Based on gene expression signatures we propose a new hypothesis that the subtype of localized LMs could be formed because of disruptions in lymph node development. This article is protected by copyright. All rights reserved.

BACKGROUND: Somatic overgrowth conditions, including Proteus syndrome, Sturge-Weber syndrome, and PIK3CA-related overgrowth spectrum, are caused by post-zygotic pathogenic variants, result in segmental mosaicism, and give rise to neural, cutaneous and/or lipomatous overgrowth. These variants occur in growth-promoting pathways leading to cellular proliferation and expansion of tissues that arise from the affected cellular lineage. METHODS: We report on 80 serial patients evaluated for somatic overgrowth conditions in a diagnostic laboratory setting, including three prenatal patients. In total, 166 tissues from these 80 patients were subjected to targeted sequencing of an 8-gene panel capturing 10.2 kb of sequence containing known pathogenic variants associated with somatic overgrowth conditions. Deep next-generation sequencing was performed with the IonTorrent PGM platform at an average depth typically >5,000x. RESULTS: Likely pathogenic or pathogenic variants were identified in 36 individuals and variants of unknown significance in four. The overall molecular diagnostic yield was 45% but was highly influenced by both submitted tissue type and phenotype. In the prenatal setting, two patients had pathogenic variants identified in cultured amniocytes but in a third patient, the pathogenic variant was only present in post-natal tissues. Finally, expanding the test to include full gene sequencing of PIK3CA in contrast to targeted sequencing identified likely pathogenic variants in 3 of 7 patients that tested negative on the original panel. CONCLUSION: Next-generation sequencing has enabled sensitive detection of somatic pathogenic variants associated with overgrowth conditions. However, as the pathogenic variant allele frequency varies by tissue type within an individual, submission of affected tissue(s) greatly increases the chances of a molecular diagnosis.

Proteus syndrome (PS) is an ultra-rare disease characterized by progressive, disproportionate, segmental overgrowth caused by a somatic gain-of-function mutation p.Glu17Lys in the oncogene AKT1. The disease has high morbidity and mortality rates due to the increased risk for patients to develop cancer and progressive overgrowth. A teenage patient with severe PS phenotype developed a pelvic recurrence of low-grade serous ovarian carcinoma (LGSOC). Taking into consideration, recent results of the use of AKT inhibitors both in PS and AKT-mutant cancers, we treated the patient on a compassionate basis, with miransertib (ARQ 092), a potent, selective, allosteric AKT inhibitor. Targeted deep sequencing assay of PI3K/AKT pathway genes of the affected overgrowth lesion (cerebriform connective tissue nevus) and the tumor tissues detected the same activating AKT1 mutation in both. Treatment with miransertib led to a complete remission of the cancer and a significant improvement in the patients' everyday life. The treatment is still ongoing at 22 months. This is the first report showing the therapeutic effects of an AKT inhibitor on both benign and malignant tissues that harbor the same pathogenic AKT1 mutation. The present article showed that personalized medicine is feasible in ultra-rare diseases.

Proteus syndrome is a mosaic, progressive overgrowth disorder caused by a somatic activating variant c.49G>A p.(E17K) in AKT1. The presentation in affected individuals is variable, with a diversity of tissues demonstrating abnormalities. Common manifestations include skin and bony overgrowth, vascular malformations, cysts, and benign tumors. We used two methods to create mouse models that had endogenously-regulated mosaic expression of the Proteus syndrome variant. Variant allele fractions (VAFs) ranged from 0–50% across numerous tissues in 44 Proteus syndrome mice. Mice were phenotypically heterogeneous with lesions rarely observed before 12 months of age. Vascular malformations were the most frequent finding with a total of 69 found in 29 of 44 Proteus syndrome mice. Twenty-eight cysts and ectasia, frequently biliary, were seen in 22 of 44 Proteus syndrome mice. Varying levels of mammary hyperplasia were seen in 10 of 16 female Proteus syndrome mice with other localized regions of hyperplasia and stromal expansion noted in several additional animals. Interestingly, 27 of 31 Proteus syndrome animals had non-zero blood VAF which is in contrast to the human disorder where it is rarely seen in peripheral blood. Identification of variant-positive cells by green fluorescent protein staining in chimeric Proteus syndrome mice showed that in some lesions, hyperplastic cells were predominantly GFP/Akt1E17K-positive and showed increased pAKT signal compared to GFP-negative cells. However, hyperplastic mammary epithelium was a mixture of GFP/Akt1E17K-positive and negative cells with some GFP/Akt1E17K-negative cells also having increased pAKT signal suggesting that the variant-positive cells can induce lesion formation in a non-cell autonomous manner.

This study aimed to verify the differentially expressed miRNAs (microRNAs) in hemangioma, and explore their roles in the pathogenesis of hemangioma in vivo and ex vivo. Real-time polymerase chain reaction (PCR) and western blot were used to measure reported differentially expressed miRNAs and their potential targets. In-silicon analysis and luciferase assay were conducted to find the target of miR-15a and miR-205. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flowcytometry were performed to examine the effect of dysregulation of miR-15a and miR-205 on the proliferation and apoptosis of endothelial cells. Among all candidate miRNAs, only miR-205 level was significantly downregulated whereas miR-15a was evidently upregulated in the hemangioma group. Accordingly, AKT3 was validated to be the direct target of miR-15a and miR-205. Using real-time PCR, the level of AKT1 was much higher in hemangioma group, whereas level of AKT3 was much lower in the hemangioma group, and in general expression level of ATK was upregulated in the hemangioma group. Furthermore, the ATK1 level of cells transfected with miR-205 mimics and ATK1 siRNA was substantially downregulated, and anti-miR-205 mimic significantly improved the level of AKT1, and meanwhile the level of ATK3 and PTEN were remarkably suppressed after transfection with miR-15a mimics and ATK3 siRNA, whereas notably overexpressed after introduction of anti-miR-15a. And miR-15a, AKT3 siRNA and anti-miR-205 evidently induced viability, and miR-205, AKT1 siRNA, and anti-miR-15a obviously promoted apoptosis of cells. CONCLUSION: miR-15a and miR-205 had different expression in hemangioma, may be novel therapeutic targets in the treatment of hemangioma by targeting AKT3 and AKT1.

Vascular anomalies can be correctly diagnosed in the majority of instances using the combination of clinical history, physical examination and imaging. In certain cases, the clinical work-up may be inconclusive or unavailable to the radiologist, and the imaging findings can be nonspecific, yielding more than one possible diagnosis. In this pictorial essay, we discuss diagnoses that can mimic vascular anomalies and highlight key differentiating imaging features.

General lymphatic anomaly (GLA) is a very rare disorder, characterised by multifocal lymphatic malformations into various tissues that is due to congenital abnormalities of lymphatic development. No treatment has ever proved its efficiency. We report a 22-year-old man with recurrent bronchial casts due to thoracic involvement of GLA. After a 6-month treatment with sildenafil (20 mg three times a day), a phosphodiesterase 5 inhibitor, chest CT scan showed a complete regression of ground-glass opacities and lung function test results improved substantially and remained stable for 1 year. The treatment was well tolerated.This observation suggests that sildenafil may be a therapeutic approach to be tested in thoracic involvement of GLA.

Capillary malformation-arteriovenous malformation (CM-AVM, MIM#608354) is a rare autosomal dominant disorder characterized by multiple cutaneous capillary malformations co-occurring with fast-flow vascular anomalies, such as arteriovenous malformation or fistula. Despite the identification of RASA1 as the first causative gene in Western patients with CM-AVM, there have been no literature reports of Japanese patients with this gene mutation. We herein report two Japanese pedigrees harboring multiple affected members with CM-AVM. Whole-exome sequencing in the two probands identified novel heterozygous mutations in RASA1, which were co-segregated with the disease in each family and were not reported in large-scale sequencing databases. One was a frameshift mutation and the other a splice-site mutation causing aberrant splicing, confirmed by a minigene assay. There were no other genes commonly disrupted among these probands. RASA1 was a major causative gene even in Japanese patients with CM-AVM, although obvious locus heterogeneity was known for this disease.

Port wine stain (PWS) is a congenital vascular malformation involving human skin. Approximately 15–20% of children a facial PWS involving the ophthalmic (V1) trigeminal dermatome are at risk for Sturge Weber syndrome (SWS), a neurocutaneous disorder with vascular malformations in the cerebral cortex on the same side of the facial PWS lesions. Recently, evidence has surfaced that advanced our understanding of the pathogenesis of PWS/SWS, including discoveries of somatic genetic mutations (GNAQ, PI3K), MAPK and PI3K aberrant activations, and molecular phenotypes of PWS endothelial cells. In this review, we summarize current knowledge on the etiology and pathology of PWS/SWS based on evidence that the activation of MAPK and/or PI3K contributes to the malformations, as well as potential futuristic treatment approaches targeting these aberrantly dysregulated signaling pathways. Current data support that: (1) PWS is a multifactorial malformation involving the entire physiological structure of human skin; (2) PWS should be pathoanatomically re-defined as “a malformation resulting from differentiation-impaired endothelial cells with a progressive dilatation of immature venule-like vasculatures”; (3) dysregulation of vascular MAPK and/or PI3K signaling during human embryonic development plays a part in the pathogenesis and progression of PWS/SWS; and (4) sporadic low frequency somatic mutations, such as GNAQ, PI3K, work as team players but not as a lone wolf, contributing to the development of vascular phenotypes. We also address many crucial questions yet to be answered in the future research investigations.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) has recently been distinguished as a novel subtype of generalized lymphatic anomaly (GLA), and is characterized by foci of spindle endothelial cells amid a background of malformed lymphatic channels. The etiology of these diseases remains unknown and diagnosis is confounded by their similar clinical findings. This study aimed to clarify differences in the clinical findings and plasma cytokine profiles of GLA and KLA patients. PROCEDURE: Clinical features data of GLA and KLA patients were obtained from a national survey. Differences in clinical findings, plasma levels of cytokines, and survival were analyzed. Plasma was obtained from healthy controls and GLA and KLA patients. Thirty-six angiogenic and lymphangiogenic factors were evaluated for cytokine concentration. RESULTS: Twenty-one patients with GLA and 11 with KLA were recruited. Mediastinal masses, hemorrhagic pericardial and pleural effusion, coagulation disorders, and thrombocytopenia were more frequent in KLA than in GLA. KLA had a significantly poorer outcome than GLA (P = 0.044). Soluble VEGFR3, angiopoietin 2, HGF, soluble HER2, tenascin C, and soluble HGFR levels were higher in KLA. Notably, soluble VEGFR3 and angiopoietin 2 levels were approximately 10-fold higher than those of other molecules measured. However, soluble VEGFR1 and soluble TIE2 were lower in KLA than in GLA and the controls. CONCLUSIONS: Patients with KLA have an unfavorable prognosis and serious symptoms (hemorrhagic pleural effusion and coagulation disorders). Our data indicate that eight angiogenic cytokines might be potential biomarkers of KLA.

BACKGROUND: Lymphatic anomalies (LAs) include several disorders in which abnormal lymphatic tissue invades the neck, chest, and various organs. Progressive cases may result in lethal outcomes and have proven difficult to treat. Sirolimus is showing promising results in the management of vascular anomalies. We examined the efficacy and safety of sirolimus treatment in patients with progressive LAs. METHODS: All patients with LAs treated with sirolimus from May 2015 to September 2018 were included. They received oral sirolimus once a day and the dose was adjusted so that the trough concentration remained within 5–15 ng/mL. We prospectively reviewed the response to drugs (the response rate of radiological volumetric change of the target lesion), severity scores, reported quality of life (QOL), and adverse effects at 6 months after administration. RESULTS: Twenty patients (five with cystic lymphatic malformation (LM), three with kaposiform lymphangiomatosis, three with generalized lymphatic anomaly, six with Gorham-Stout disease, and three with central conducting lymphatic anomaly) were treated with sirolimus at our institution. Fifty percent of patients (10/20) demonstrated a partial response by a radiological examination and a significant improvement in disease severity and QOL scores (P = 0.0020 and P = 0.0117, respectively). Ten patients who had no reduction in lesion size (stable disease group) showed no significant improvement in disease severity and QOL scores. Eighty percent of patients (16/20) had side effects, such as stomatitis, infection, and hyperlipidemia. CONCLUSIONS: Sirolimus impacts the reduction of the lymphatic tissue volume of LMs and could lead to improvement in clinical symptoms and QOL. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN000016580. Registered 19 February 2015.

Sturge-Weber syndrome (SWS) includes facial, leptomeningeal and choroidal hemangioma. The retinal vasculature is essentially normal. Rare cases of retinal vascular tortuosity and arterio-venous malformations have been reported. We report two cases with rare concomitant retinal vascular abnormalities along with SWS. Both the patients had nevus flammeus, hemifacial hypertrophy, and choroidal hemangioma. In one case, retinal cavernous hemangioma was seen in the affected eye. The other case revealed retinal neovascularization secondary to proliferative diabetic retinopathy in the eye with choroidal hemangioma.

The etiology of infantile haemangiomas is still not fully understood. The hypothesis of tissue hypoxia was corroborated by children presenting with large segmental facial haemangiomas (SFH) in association with ipsilateral cerebro- and/or cervicovascular anomalies (CVA). PHACE syndrome (posterior fossa malformations, haemangioma, arterial, cardiac, eye and/or sternal anomalies, OMIM 606519) can be diagnosed in about 31% of all children with large facial haemangiomas. Most children have only one extracutaneous manifestation(1). CVA were reported in up to 91% of all PHACE patients. Blood-flow limiting CVA confer a risk of arterial ischaemic stroke(2). CVA are thus the most prevalent and relevant extracutaneous manifestations of PHACE syndrome This article is protected by copyright. All rights reserved.

Lymphatic malformations (LM) are rare congenital low flow vascular malformations.(1) The goal of LM management is to maintain functionality, control associated symptoms, and preserve aesthetic integrity. It requires a multidisciplinary approach, considering surgery, sclerotherapy, observation and new, promising medical treatments.(2,3) Overtime, except in case of small well circumscribed lesions, it is obvious that complete surgical removal of the LM is almost always impossible because of infiltrative lesions.(4,5) Moreover, partial excision is classically considered to be associated with the risk of local recurrence.(6) Worsening of LM after surgery, especially occurrence of superficial lymphangiectasia (SL), is sometimes discussed. SL are bothersome for patients because of intermittent swelling, haemorrhage and leakage of lymph from superficial vesicles. The aim with this retrospective study was to assess whether surgery might be a triggering factor for SL occurrence. This article is protected by copyright. All rights reserved.

BACKGROUND: This study evaluates 6 years of prenatal rasopathy testing in the Netherlands, updates on previous data and gives recommendations for prenatal rasopathy testing. METHODS: 424 fetal samples, sent in for prenatal rasopathy testing in 2011–2016, were collected. Cohort 1 included 231 samples that were sequenced for 1–5 rasopathy genes. Cohort 2 included 193 samples that were analysed with a 14-gene next generation sequencing (NGS) panel. For all mutation-positive samples in both cohorts, the referring physician provided detailed ultrasound findings and postnatal follow-up. For 168 mutation-negative samples in cohort 2, solely clinical information on the requisition form was collected. RESULTS: In total, 40 (likely) pathogenic variants were detected (9.4%). All fetuses showed a variable degree of involvement of prenatal findings: increased nuchal translucency (NT)/cystic hygroma, distended jugular lymph sacs (JLS), hydrops fetalis, polyhydramnios, pleural effusion, ascites, cardiac defects and renal anomalies. An increased NT was the most common finding. Eight fetuses showed solely an increased NT/cystic hygroma, which were all larger than 5.5 mm. Ascites and renal anomalies appeared to be poor predictors of pathogenic outcome. CONCLUSION: Fetuses with a rasopathy show in general multiple ultrasound findings. The larger the NT and the longer it persists, the more likely it is to find a pathogenic variant. Rasopathy testing is recommended when the fetus shows an isolated increased NT >/ = 5.0 mm or when NT of >/ = 3.5 mm and at least one of the following ultrasound anomalies is present: distended JLS, hydrops fetalis, polyhydramnios, pleural effusion, ascites, cardiac defects and renal anomalies.

Long before the classification of vascular anomalies from the International Society for the Study of Vascular Anomalies (ISSVA) provided a framework to differentiate vascular anomalies, otherwise known as vascular birthmarks, it was recognized that patients with such lesions can present with acute life-threatening hemostatic and/or thrombotic complications, as well as chronic long-standing bleeding or thrombotic issues. Scenarios such as a rapidly growing vascular lesion with severe acute thrombocytopenia, a visceral hemorrhagic lesion, a lesion associated with repetitive and painful superficial thrombosis, and cases of unprovoked or post-procedural fatal pulmonary embolism highlight the wide spectrum of manifestations of abnormal coagulation in patients with vascular anomalies. The separation of vascular anomalies into two distinct groups, vascular tumors and vascular malformations, was followed by the characterization that their respective coagulopathies were due to either a derangement of platelets or to a disequilibrium of the patient's coagulation/fibrinolytic process. This configuration of coagulopathies will be the foundation for this two-chapter review series. In the initial review, coagulopathies where thrombocytopenia is the main feature will be characterized, whereas the second review will focus on vascular malformations that have a coagulation disorder secondary to some degree of coagulation consumption and/or fibrinolytic pathway derangement.

PURPOSE OF REVIEW: Vascular malformations (VaMs) are a consequence of disrupted morphogenesis that may involve arterial, capillary, venous, or lymphatic endothelium alone or in a combination. VaMs can have serious health impacts, leading to life-threatening conditions sometimes. Genetic mutations affecting proliferation, migration, adhesion, differentiation, and survival of endothelial cells, as well as integrity of extracellular matrix are believed to be the pathogenesis of these disorders. Here, we present an updated review of genetic mutations and potential therapeutic targets for VaMs. RECENT FINDINGS: Increased number of genetic mutations have been discovered in vascular anomalies via targeted deep sequencing. When a genetic defect is identified, it often presents in only a small percentage of cells within the malformation. In addition, mutations within the same gene may result in different clinical phenotypes. Management of VaMs can be challenging depending on the severity and functional impairment associated. There are no standard treatment algorithms available to date for VaMs, therefore the disorder has significant unmet clinical needs. Currently, the focus of therapeutic development is to target constitutively activated intracellular signaling pathways resulted from genetic mutations. SUMMARY: Knowledge about the genetic mutations and altered signaling pathways related to VaMs have improved our understanding about the pathogenesis of vascular anomalies and provided insights to the development of new targeted therapies.