Update FEBRUARY 2020

Renal allograft rejection can be prevented by immunological tolerance, which may be associated with de novo formed lymphatic vessels in the donor kidney after transplantation in man. A suitable mouse model of renal allograft rejection in which lymphangiogenesis can be deliberately induced in the graft is critical for elucidating the mechanisms responsible for the association between attenuated transplant rejection and abundance of lymphatic vessels. Here we describe the development of a novel mouse model of rapid renal transplant rejection in which transgenic induction of lymphangiogenesis in the immune-incompatible graft greatly extends its survival time. Thus, our novel approach may facilitate exploitation of lymphangiogenesis in the grafted organ.

Lymphatic function in yet another clinical setting is addressed in this manuscript which assesses the role of lymphangiogenesis in organ transplant rejection. There has been controversy regarding whether lymphatics contribute to or ameliorate acute organ rejection. In this concise study, the authors aim establish, via studies with a mouse model of acute renal transplant rejection, that lymphatic vessels are associated with decreased rather than increased renal transplant rejection. Donor kidneys from a mouse model with Doxycycline-induced VEGF-C expression, in turn stimulated lymphangiogenesis in the transplanted kidneys, revealed decreased organ rejection compared to controls. The function of lymphatic vessels in attenuating rejection may be related to increased clearance of inflammatory cells (associated with organ rejection) or other mechanisms. Interestingly, this notion was described in publications over 5 years ago,^1,2 however, the studies in the current manuscript provides a model to elucidate this more clearly. Of interest, another recent reference (see below) analyzes the of lymphatic anastomoses in organ (lung) transplantation in a mouse model. ³

BACKGROUND: Chronic progressive lymphoedema (CPL) is a disabling condition affecting various draft horse breeds, including Friesian horses. HYPOTHESIS: The high incidence of CPL in Friesian horses suggests a genetic component and a predisposing phenotype. ANIMALS: For the genomic study, 26 affected and 19 control horses were evaluated. Body measurements were taken from 28 affected and nine control Friesian horses. METHODS AND MATERIALS: Axiom(R) Equine Genotyping Array with a total of 307,474 single nucleotide polymorphism (SNPs) was used for the case/control genome-wide association study (GWAS). Height, weight and leg measurements (seven different body measurements) were obtained and compared between groups. RESULTS: No significant associations were identified with the GWAS. There were significant differences (P < 0.05) in gaskin length measurements between CPL-affected horses and controls. CONCLUSION: The current study suggests that the potential genetic contribution to CPL in Friesian horses is complex and may include other SNP-associated factors. Significantly increased gaskin length of affected horses suggests a phenotype prone to developing CPL.

BACKGROUND: Previously, we showed that lymphatic vessels (LVs) formed detours after lymphatic obstruction, contributing to preventing lymphedema. In this study, we developed detours using lymphatic ligation in mice and we identified the detours histologically. METHODS AND RESULTS: Under anesthesia, both hindlimbs in mice were subcutaneously injected with Evans blue dye to detect LVs. We tied the right collecting LV on the abdomen that passes through the inguinal lymph node (LN) at two points. The right and left sides comprised the operation and sham operation sides, respectively. Lymphography was performed to investigate the lymph flow after lymphatic ligation until day 30, using a near-infrared fluorescence imaging system. Anti-podoplanin antibody and 5-ethynyl-2′-deoxyuridine (EdU) were used to detect LVs and lymphangiogenesis. Within 30 days, detours had developed in 62.5% of the mice. Detours observed between two ligation sites were enlarged and irregular in shape. Podoplanin+ LVs, which were located in the subcutaneous tissue of the upper panniculus carnosus muscle, connected to collecting LVs at the upper portion from the cranial ligation site and at the lower portion from the caudal ligation site. EdU+ cells were not observed in these detours. The sham operation side showed normal lymph flow and did not show enlarged pre-collecting LVs until day 30. CONCLUSIONS: Detours after lymphatic ligation were formed not by lymphangiogenesis but through an enlargement of pre-collecting LVs that functioned as collecting LVs after lymphatic ligation. Further studies are required to explore the developmental mechanism of the lymphatic detour for treatment and effective care of lymphedema in humans.

OBJECTIVE: Hypertension is associated with renal immune cell accumulation and sodium retention. Lymphatic vessels provide a route for immune cell trafficking and fluid clearance. Whether specifically increasing renal lymphatic density can treat established hypertension, and whether renal lymphatics are involved in mechanisms of blood pressure regulation remain undetermined. Here, we tested the hypothesis that augmenting renal lymphatic density can attenuate blood pressure in established hypertension. METHODS: Transgenic mice with inducible kidney-specific overexpression of VEGF-D (‘KidVD+’ mice) and KidVD- controls were administered a nitric oxide synthase inhibitor, L-NAME, for 4 weeks, with doxycycline administration beginning at the end of week 1. To identify mechanisms by which renal lymphatics alter renal Na handling, Na excretion was examined in KidVD+ mice during acute and chronic salt loading conditions. RESULTS: Renal VEGF-D induction for 3 weeks enhanced lymphatic density and significantly attenuated blood pressure in KidVD+ mice whereas KidVD- mice remained hypertensive. No differences were identified in renal immune cells, however, the urinary Na excretion was increased significantly in KidVD+ mice. KidVD+ mice demonstrated normal basal sodium handling, but following chronic high salt loading, KidVD+ mice had a significantly lower blood pressure along with increased urinary fractional excretion of Na. Mechanistically, KidVD+ mice demonstrated decreased renal abundance of total NCC and cleaved ENaCalpha Na transporters, increased renal tissue fluid volume, and increased plasma ANP. CONCLUSION: Our findings demonstrate that therapeutically augmenting renal lymphatics increases natriuresis and reduces blood pressure under sodium retention conditions.

Lymphedema is a chronic accumulation of interstitial fluid due to inefficient lymph drainage. Major causes of lymphedema are malformations of lymphatic vessels, trauma, toxic damage and surgery. The swelling typically affects the limbs. Lymphedema may be primary, caused by genetic mutations and relatively rare, or secondary (acquired), due to external causes such as infections or surgery. Fluid accumulation induces pathological changes: activation of the inflammatory cascade, immune cell infiltration, tissue fibrosis, adipose accumulation. We focused on the inflammatory phenotype mediated by leukotriene B4, a lipid mediator of the inflammatory pathway, and the potential therapeutic effect of hydroxytyrosol. We conducted an electronic search in PubMed using “lymphedema”, “lymphedema pathway”, “hydroxytyrosol” as keywords. We found that lymphedema deregulates at least six molecular pathways and that hydroxytyrosol, a compound with antioxidant activity, can improve endothelial dysfunction, hemostatic and lipid profiles, and decrease oxidative stress and inflammation through inhibition of leukotriene B4 activity. This review is the first to highlight the possibility of using hydroxytyrosol to treat the secondary effects of lymphedema, especially inflammation. The possible effects of hydroxytyrosol on lymphedema should be tested in vitro and in vivo to find the best way to treat patients with lymphedema in order to improve their health status.

BACKGROUND: The evidence of pelvic lymph node metastases after radical prostatectomy (RP) with pelvic lymph node dissection (PLND) is one of the strongest prognostic factors for poor oncologic outcome. The extent of PLND, although representing a crucial step in RP, is still controversial. Currently, there is a critical drawback in clinical practice due to the lack of congruence between the known lymphatic drainage and cancer dissemination despite defined management by a surgical approach. We hypothesized the existence of alternative pathways for the lymphatic drainage of the prostate currently not considered in clinical daily practice. METHODS: We carried out a literature review of the anatomic description of nodal drainage of prostate reported by online databases (MEDLINE/PubMed, EBSCO, Web of Science, Ovid, and Scopus) and the original texts since the 18th century, with an additional anatomical dissection on a human cadaver to confirm theoretical data. RESULTS: The anatomical dissection study converged with the historical anatomical treatises in describing three groups of lymphatics devoted to carrying out prostatic nodal drainage. Apart from the ascending ducts from the cranial gland leading to the external iliac nodes; the lateral ducts leading to the hypogastric nodes; small lymphatic vessels from the posterior surface of the prostate, directed to the pararectal lymphatic plexus, in the direction of the lateral sacral lymph nodes and those at the sacral promontory (i.e., pararectal and presacral lymph nodes) were observed. CONCLUSIONS: Our preliminary findings demonstrate that lymphatic drainage of the prostate extends beyond standard nodal templates actually considered in surgical daily practice, despite the knowledge reported by historical anatomical treatises. Further anatomical and experimental evidence are needed to investigate anatomical variability in humans, as well as to add more topographical details.

Aim: To design lympho-targeted nanocarriers with the capacity to enhance the activity of associated drugs/antigens whose target is within the lymphatic system. Materials & methods: Inulin (INU)-based nanocapsules (NCs), negatively charged and positively charged chitosan NCs were prepared by the solvent displacement techniques. The NCs were produced in two sizes: small (70 nm) and medium (170–250 nm). Results: In vitro results indicated that small NCs interacted more efficiently with dendritic cells than the larger ones. The study of the NCs biodistribution in mice, using 3D reconstruction of the popliteal lymph node, showed that small INU NCs have the greatest access and uniform accumulation in different subsets of resident immune cells. Conclusion: Small and negatively charged INU NCs have a potential as lympho-targeted antigen/drug nanocarriers.

Head and neck lymphedema (HNL) is a disfiguring disease affecting over 90% of patients treated for head and neck cancer. Animal models of lymphedema are used to test pharmacologic and microsurgical therapies; however, no animal model for HNL is described in the literature to date. In this study we describe the first reproducible rat model for HNL. Animals were subjected to two surgical protocols: (1) lymphadenectomy plus irradiation; and (2) sham surgery and no irradiation. Head and neck expansion was measured on post-operative days 15, 30 and 60. Magnetic resonance imaging (MRI) was acquired at the same time points. Lymphatic drainage was measured at day 60 via indocyanine green (ICG) lymphography, after which animals were sacrificed for histological analysis. Postsurgical lymphedema was observed 100% of the time. Compared to sham-operated animals, lymphadenectomy animals experienced significantly more head and neck swelling at all timepoints (P < 0.01). Lymphadenectomy animals had significantly slower lymphatic drainage for 6 days post-ICG injection (P < 0.05). Histological analysis of lymphadenectomy animals revealed 83% greater subcutis thickness (P = 0.008), 22% greater collagen deposition (P = 0.001), 110% greater TGFbeta1(+) cell density (P = 0(.)04), 1.7-fold increase in TGFbeta1 mRNA expression (P = 0.03), and 114% greater T-cell infiltration (P = 0.005) compared to sham-operated animals. In conclusion, animals subjected to complete lymph node dissection and irradiation developed changes consistent with human clinical postsurgical HNL. This was evidenced by significant increase in all head and neck measurements, slower lymphatic drainage, subcutaneous tissue expansion, increased fibrosis, and increased inflammation compared to sham-operated animals.

In recent years, there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here, we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations.

The cardiac lymphatic vascular system and its potentially critical functions in heart patients have been largely underappreciated, in part due to a lack of experimentally accessible systems. We here demonstrate that cardiac lymphatic vessels develop in young adult zebrafish, using coronary arteries to guide their expansion down the ventricle. Mechanistically, we show that in cxcr4a mutants with defective coronary artery development, cardiac lymphatic vessels fail to expand onto the ventricle. In regenerating adult zebrafish hearts the lymphatic vasculature undergoes extensive lymphangiogenesis in response to a cryoinjury. A significant defect in reducing the scar size after cryoinjury is observed in zebrafish with impaired Vegfc/Vegfr3 signaling that fail to develop intact cardiac lymphatic vessels. These results suggest that the cardiac lymphatic system can influence the regenerative potential of the myocardium.

Recent studies have demonstrated that the brain is equipped with a lymphatic drainage system that is actively involved in parenchymal waste clearance, brain homeostasis and immune regulation. However, the exact anatomic drainage routes of brain lymph fluid (BLF) remain elusive, hampering the physiological study and clinical application of this system. In this study, we systematically dissected the anatomy of the BLF pathways in a rat model. Moreover, we developed a protocol to collect BLF from the afferent lymphatic vessels of deep cervical lymph nodes (dcLNs) and cerebrospinal fluid (CSF) from the fourth ventricle. Nuclear magnetic resonance spectroscopy showed that BLF contains more metabolites than CSF, suggesting that BLF might be a more sensitive indicator of brain dynamics under physiological and pathological conditions. Finally, we identified several metabolites as potential diagnostic biomarkers for glioma, Parkinson's disease and CNS infectious diseases. Together, these data may provide insight into the physiology of the lymphatic system in the brain and into the clinical diagnosis of CNS disorders.

The phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway has been implicated as a cancer target. Big pharma players and small companies have been developing small molecule inhibitors of PI3K and/or mTOR since the 1990s. Although four inhibitors have been approved, many open questions regarding tolerability, patient selection, sensitivity markers, development of resistances, and toxicological challenges still need to be addressed. Besides clear oncological indications, PI3K and mTOR inhibitors have been suggested for treating a plethora of different diseases. In particular, genetically induced PI3K/mTOR pathway activation causes rare disorders, known as overgrowth syndromes, like PTEN (phosphatase and tensin homolog) hamartomas, tuberous sclerosis complex (TSC), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth spectrum (PROS), and activated PI3-Kinase delta syndrome (PI3KCD, APDS). Some of those disorders likeTSC or hemimegalencephaly, which are one of the PROS disorders, also belong to a group of diseases called mTORopathies. This group of syndromes presents with additional neurological manifestations associated with epilepsy and other neuropsychiatric symptoms induced by neuronal mTOR pathway hyperactivation. While PI3K and mTOR inhibitors have been and still are intensively tested in oncology indications, their use in genetically defined syndromes and mTORopathies appear to be promising avenues for a pharmacological intervention.

Initial lymphatic vessels are made up of overlapped endothelial cells that act as unidirectional valves enabling one-way drainage of tissue fluid into the lumen of the initial lymphatics when there is a favourable pressure gradient. Initial lymphatics subsequently drain this fluid into the collecting lymphatics. This paper describes a computational model for a network of passive rat mesenteric lymphatic vessels with sparse secondary valves. The network was simulated with the secondary valves both operational and non-operational. The effects on the cycle-mean outflow-rate from the network of both inflammation and the resistance of the surrounding interstitium were considered. The cycle-mean outflow-rate is sensitive to vessel stiffness. If the influence of primary-valve resistance is reduced relative to that of interstitial resistance and intravascular resistance, there is no absolute advantage of extrinsic pumping, since maximum outflow-rate occurs when vessels are rigid. However, there is relative advantage, in that the outflow-rate at intermediate stiffness is higher with the secondary valves functioning than when they are deactivated. If primary-valve resistance dominates, then extrinsic pumping of non-rigid vessels provides absolute advantage. The nonlinear relation between pressure drop and flow-rate of the endothelial primary valves, combined with downstream compliance and pulsatile external pressure, constitutes a separate mechanism of pumping. By enabling the consideration of interactions between multiple phenomena (primary valves, secondary valves, a real network geometry with multiple branches, deformable vessel walls, interstitial resistance and external pressures), the model offers a perspective for delineating physiological phenomena that have not yet been fully linked to the biomechanics of fluid flow through initial lymphatic networks.

Obesity, insulin resistance, dyslipidemia, and hypertension are fundamental clinical manifestations of the metabolic syndrome. Studies over the last few decades have implicated chronic inflammation and microvascular remodeling in the development of obesity and insulin resistance. Newer observations, however, suggest that dysregulation of the lymphatic system underlies the development of the metabolic syndrome. This review summarizes recent advances in the field, discussing how lymphatic abnormality promotes obesity and insulin resistance, and, conversely, how the metabolic syndrome impairs lymphatic function. We also discuss lymphatic biology in metabolically dysregulated diseases, including type 2 diabetes, atherosclerosis, and myocardial infarction.

OBJECTIVES: To investigate the cellular and molecular pathophysiology involved in the development of fibrotic skin of grade-3 lymphoedema patients with a focus on collagen types. METHODS: Fibrotic and normal skin biopsy samples obtained from grade-3 lymphoedema patients and normal individuals, respectively, were analysed by histopathology, quantitative real-time PCR and immunohistochemistry to examine collagen gene expression. RESULTS: Histopathologic analysis revealed epidermal changes such as orthokeratosis, hypergranulosis and irregular acanthosis in the skin biopsies. The thickened dermis contained nodules of haphazardly arranged thick collagen bundles. Real-time PCR data showed significant (P-value 0.0003) up-regulation of Collagen type I and type III gene transcripts in the fibrotic skin of patients resulting in 38.94-fold higher transcription of Collagen type III alpha-1 gene than of Collagen type I alpha-1 gene. Semi-quantification of the per cent of haematoxylin-DAB-stained area of immunohistochemistry images also showed significant (P < 0.0001) enhancement of both collagen proteins in the fibrotic skin of patients vs. normal human skin. CONCLUSIONS: Gene transcript analysis revealed significant up-regulation of Collagen type III vs. Collagen type I in fibrotic skin of limb nodules from patient biopsies. Histopathological and immunohistochemical analysis also revealed enhancement of Collagen types I and III in fibrotic vs. normal skin. The findings of this preliminary study indicate the potentially significant involvement of Collagen type III in the development of the fibrotic skin of grade-3 lymphoedema patients.

The mammalian heart contains multiple cell types that appear progressively during embryonic development. Advance in determining cardiac lineage diversification has often been limited by the unreliability of genetic tracers. Here we combine clonal analysis, genetic lineage tracing, tissue transplantation, and mutant characterization to investigate the lineage relationships between epicardium, arterial mesothelial cells (AMCs), and the coronary vasculature. We report a contribution of the second heart field (SHF) to a vasculogenic niche composed of AMCs and sub-mesothelial cells at the base of the pulmonary artery. Sub-mesothelial cells from this niche differentiate into lymphatic endothelial cells and, in close association with AMC-derived cells, contribute to and are essential for the development of ventral cardiac lymphatics. In addition, regionalized epicardial/mesothelial retinoic acid signaling regulates lymphangiogenesis, contributing to the niche properties. These results uncover a SHF vasculogenic contribution to coronary lymphatic development through a local niche at the base of the great arteries.

Contractile activity in the lymphatic vasculature is essential for maintaining fluid balance within organs and tissues. However, the mechanisms by which collecting lymphatics adapt to changes in fluid load and how these adaptations influence lymphatic contractile activity are unknown. Here we report a model of lymphatic injury based on the ligation of one of two parallel lymphatic vessels in the hind limb of sheep and the evaluation of structural and functional changes in the intact, remodelling lymphatic vessel over a 42-day period. We show that the remodelled lymphatic vessel displayed increasing intrinsic contractile frequency, force generation and vessel compliance, as well as decreasing flow-mediated contractile inhibition via the enzyme endothelial nitric oxide synthase. A computational model of a chain of lymphatic contractile segments incorporating these adaptations predicted increases in the flow-generation capacity of the remodelled vessel at the expense of normal mitochondrial function and elevated oxidative stress within the lymphatic muscle. Our findings may inform interventions for mitigating lymphatic muscle fatigue in patients with dysfunctional lymphatics.

Lymphangiogenesis plays important roles in normal fetal development and postnatal growth. However, its molecular regulation remains unclear. Here, we have examined the function of forkhead box protein O1 (FOXO1) transcription factor, a known angiogenic factor, in developmental dermal lymphangiogenesis using endothelial cell-specific FOXO1-deficient mice. FOXO1-deficient mice showed disconnected and dilated lymphatic vessels accompanied with increased proliferation and decreased apoptosis in the lymphatic capillaries. Comprehensive DNA microarray analysis of the causes of in vivo phenotypes in FOXO1-deficient mice revealed that the gene encoding C-X-C chemokine receptor 4 (CXCR4) was the most drastically downregulated in FOXO1-deficient primary lymphatic endothelial cells (LECs). CXCR4 was expressed in developing dermal lymphatic capillaries in wild-type mice but not in FOXO1-deficient dermal lymphatic capillaries. Furthermore, FOXO1 suppression impaired migration toward the exogenous CXCR4 ligand, C-X-C chemokine ligand 12 (CXCL12), and coordinated proliferation in LECs. These results suggest that FOXO1 serves an essential role in normal developmental lymphangiogenesis by promoting LEC migration toward CXCL12 and by regulating their proliferative activity. This study provides valuable insights into the molecular mechanisms underlying developmental lymphangiogenesis.

Lymphatic vessels play a critical role in mounting a proper immune response by trafficking peripheral immune cells to draining lymph nodes. Mast cells (MCs) are well known for their roles in type I hypersensitivity reactions but little is known about their secretory regulation in the lymphatic niche. MCs, as innate sensor and effector cells, reside close to mesenteric lymphatic vessels (MLVs) and their activation and ability to release histamine influences the lymphatic microenvironment in a histamine-NF-kB-dependent manner. Using an established experimental protocol involving surgical isolation of rat mesenteric tissue segments, including MLVs and surrounding perilymphatic tissues, we tested the hypothesis that perilymphatic mesenteric MCs possess histamine receptors (HRs) that bind and respond to histamine released from these same MCs. Under various experimental conditions, including inflammatory stimulation by LPS, we measured histamine in mesenteric perilymphatic tissues, evaluated expression of histidine decarboxylase in MCs along with the degree of MC degranulation, assessed the functional status of HRs in MCs, and evaluated the ability of histamine itself to induce MC activation. Finally, we evaluated the importance of MCs and HRs 1 and 2 for MLV-directed trafficking of CD11b/c-positive cells during acute tissue inflammation. Our data indicate the existence of a functionally potent MC-histamine autocrine regulatory loop, the elements of which are crucially important for acute inflammation-induced trafficking of the CD11b/c-positive cells towards MLVs. This MC-histamine loop serves as a first-line cellular servo control system, playing a key role in the innate and adaptive immune response as well as NF-kappaB-mediated maintenance of body homeostasis.

Lymphangioma of the tongue is a rare lymphatic malformation, and various authors have reported the successful use of sirolimus for its treatment. However, the safety of sirolimus in children needs further evaluation so that those who do not respond are not necessarily exposed to its potential adverse effects. We hypothesised that assessment of lymphangiogenesis can be used to predict whether the patient will respond to sirolimus, so we organised a prospective study after ethics committee approval had been given. After clinical and histological diagnoses of lymphangioma of the tongue had been confirmed, 16 patients were given sirolimus 0.8mg/day in three divided doses. Clinical response was assessed and compared with lymphatic microvessel density (LMVD), which was calculated immunohistochemically using the monoclonal antibody D2-40 as the lymphatic endothelial marker. Nine patients responded well, five partially, and two failed to respond. Mean (SD) LVD among the good responders was 21.00 (3.74), whereas among non-responders it was 8.00 (4.24). There was a significant difference in mean LVD between good responders, partial responders, and non-responders (p = 0.04). Sirolimus is effective in treating children with lymphangioma of the tongue, and lymphangiogenesis is a useful therapeutic predictive marker.

At steady state, the CNS parenchyma has few to no lymphocytes and less potent Ag-presentation capability compared with other organs. However, the meninges surrounding the CNS host diverse populations of immune cells that influence how CNS-related immune responses develop. Interstitial and cerebrospinal fluid produced in the CNS is continuously drained, and recent advances have emphasized that this process is largely taking place through the lymphatic system. To what extent this fluid process mobilizes CNS-derived Ags toward meningeal immune cells and subsequently the peripheral immune system through the lymphatic vessel network is a question of significant clinical importance for autoimmunity, tumor immunology, and infectious disease. Recent advances in understanding the role of meningeal lymphatics as a communicator between the brain and peripheral immunity are discussed in this review.

BACKGROUND: Establishing lung lymphatic drainage is thought to be important for successful lung transplantation. To date, there has been a complete absence of knowledge of how lymphatic connections are reestablished after lung transplant, despite evidence suggesting that this does indeed occur. The present study aimed to elucidate whether and how lymphatic anastomosis occurs after lung transplant. METHODS: An orthotopic murine model of lung transplant using lymphatic reporter mice and whole mount immunohistochemistry was used to evaluate the lymphatic vasculature and donor-host connections after lung transplantation. RESULTS: Immunohistochemistry of transplanted lungs demonstrated robust lymphatic vessels, and functional assays demonstrated lymphatic drainage in the transplanted lung that was comparable to that in native lungs. Lymphatic vessels in the donor lung exhibited active sprouting toward the host at the anastomosis within the first 3 days after lung transplantation, with more numerous and complex lymphatic sprouting developing thereafter. Donor lymphatic vessels were numerous at the site of anastomosis by day 14 after lung transplantation and formed physical connections with host lymphatic vessels, demonstrating a mechanism by which lymphatic drainage is reestablished in the transplanted lung. CONCLUSIONS: Lymphatic drainage after lung transplantation is established by active sprouting of donor lymphatic vessels towards the host and the formation of donor-host lymphatic connections at the level of the transplant anastomosis.

This seems to be the time to gain new knowledge about the meningeal lymphatic system and a deeper understanding of its anatomy and physiology. Although it is known that the meningeal lymphatics present in the layers of the brain, limited information is available about the role of this system in brain function. Here, for the first time we clearly demonstrate that the meningeal lymphatic pathway is involved in brain clearing from the blood after intracranial hemorrhage associated with hypoxia and forms a connective bridge between interstitial, cerebral spinal fluid and peripheral lymphatics. We also show that the development of methods to stimulate meningeal lymph flow after hemorrhagic evidence in the brain might be a neuroprotective strategy for effective recovery of the brain after a cerebrovascular catastrophe.

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-beta, the toxic driver of Alzheimer's disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.

Background Most lymphatic imaging examinations of the lower limb require intradermal or subcutaneous injection of tracer material into the foot to demonstrate the lymphatic vessels; however, no standard protocol exists, and single or multiple injections are applied at different sites. Purpose To determine the three-dimensional relationships between each lymphatic group of the lower limb and corresponding regional lymph nodes. Materials and Methods A total of 130 lower limbs (55 from men and 75 from women) from 83 fresh human cadavers were studied. Lymphatic vessels were first visualized by using indocyanine green fluorescent lymphography with 19 injection sites in the foot, classified into four distinct lymphatic groups (anteromedial, anterolateral, posteromedial, and posterolateral); dilute oil-based contrast material was then injected. Next, specimens were scanned with CT and three-dimensional images were analyzed. Results The anteromedial and anterolateral lymphatic groups of the lower-leg lymphatic vessels were independent of each other and connected to different regional lymph nodes in the inguinal region. The posteromedial group and the anteromedial group in the lower leg drained to the same inguinal lymph nodes. Only the posterolateral group of lymphatic vessels in the lower leg drained to the popliteal lymph nodes. Leg lymphatic drainage pathways were independent of genital pathways. Conclusion Standard injection sites at the web spaces between the toes did not help visualize some lymph nodes of the lower leg. Additional injection sites in the medial, lateral, and posterior aspect of the foot would be better for evaluating the whole lymphatic pathways and regional lymph nodes and for improving understanding of leg lymphedema. (c) RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Weiss and Liddel in this issue.

Podoplanin (PDPN)/T1alpha is utilized as a specific marker of lymphatic endothelial cells or type I alveolar cells of lung. Therefore, sensitive and specific monoclonal antibodies (mAbs) detecting PDPN are necessary for immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for immunohistochemical analyses to detect both lymphatic endothelial cells and type I alveolar cells of lung. However, it is difficult to distinguish lymphatic endothelial cells from type I alveolar cells in the bear lung. In this study, we showed that a novel anti-bPDPN mAb, PMab-241 stained only lymphatic endothelial cells, not type I alveolar cells of the lung in immunohistochemical analyses. These findings suggest that PMab-241 could be useful for staining lymphatic endothelial cells specifically in the bear lung tissues.

Calcitonin gene-related peptide (CGRP) regulates inflammation via signaling through receptor activity-modifying protein (RAMP) 1. Here, we investigated the role of RAMP1 signaling in growth of lymphatic vessels during inflammation. Lymphangiogenesis in the diaphragm of RAMP1-deficient ((−/−)) mice or their wild-type (WT) counterparts was induced by repeated intraperitoneal injection of lipopolysaccharide (LPS). Compared with WT mice, LPS-induced lymphangiogenesis in RAMP1(−/−) mice was suppressed. This was accompanied by the reduced expression of vascular endothelial growth factor (VEGF)-C and VEGF-D. The number of CD4(+) cells in diaphragm tissue from WT mice was greater than RAMP1(−/−) mice. Removing CD4(+) cells attenuated lymphangiogenesis and expression of VEGF-C and VEGF-D. CD4(+) cells isolated from RAMP1(−/−) mice exhibited reduced expression of VEGF-C and VEGF-D. The number of CD11b(+) cells from RAMP1(−/−) mice was higher than WT mice and was associated with the upregulated expression of genes related to pro-inflammatory macrophage phenotype and downregulation of reparative macrophage phenotype-related expression. When fluorescein isothiocyanate (FITC)-dextran was injected into the peritoneal cavity, the amount of residual FITC-dextran in WT mice was lower than that in RAMP1(−/−) mice. The present results suggest that RAMP1 signaling in immune cells plays a critical role in inflammation-related lymphangiogenesis; therefore, it represents a novel target for controlling lymphangiogenesis.

Animal models are of paramount importance in the research of lymphedema in order to understand the pathophysiology of the disease but also to explore potential treatment options. This mouse model allows researchers to induce significant lymphedema lasting at least 8 weeks. Lymphedema is induced using a combination of fractioned radiotherapy and surgical ablation of lymphatics. This model requires that the mice get a dose of 10 Gray (Gy) radiation before and after surgery. The surgical part of the model involves ligation of three lymph vessels and extraction of two lymph nodes from the mouse hindlimb. Having access to microsurgical tools and a microscope is essential, due to the small anatomical structures of mice. The advantage of this model is that it results in statistically significant lymphedema, which provides a good basis for evaluating different treatment options. It is also a great and easily available option for microsurgical training. The limitation of this model is that the procedure can be time consuming, especially if not practiced in advance. The model results in objectively quantifiable lymphedema in mice, without causing severe morbidity and has been tested in three separate projects.

Proper functioning of the lymphatic system is required for normal immune responses, fluid balance, and lipid reabsorption. Multiple regulatory mechanisms are employed to ensure the correct formation and function of lymphatic vessels; however, the epigenetic modulators and mechanisms involved in this process are poorly understood. Here, we assess the regulatory role of mouse Dot1l, a histone H3 lysine (K) 79 (H3K79) methyltransferase, in lymphatic formation. Genetic ablation of Dot1l in Tie2(+) endothelial cells (ECs), but not in Lyve1(+) or Prox1(+) lymphatic endothelial cells (LECs) or Vav1(+) definitive hematopoietic stem cells, leads to catastrophic lymphatic anomalies, including skin edema, blood-lymphatic mixing, and underdeveloped lymphatic valves and vessels in multiple organs. Remarkably, targeted Dot1l loss in Tie2(+) ECs leads to fully penetrant lymphatic aplasia, whereas Dot1l overexpression in the same cells results in partially hyperplastic lymphatics in the mesentery. Genetic studies reveal that Dot1l functions in c-Kit(+) hemogenic ECs during mesenteric lymphatic formation. Mechanistically, inactivation of Dot1l causes a reduction of both H3K79me2 levels and the expression of genes important for LEC development and function. Thus, our study establishes that Dot1l-mediated epigenetic priming and transcriptional regulation in LEC progenitors safeguard the proper lymphatic development and functioning of lymphatic vessels.

OBJECTIVE: Aging is a major risk factor for numerous neurological disorders and the mechanisms underlying brain aging remain elusive. Recent animal studies demonstrated tight relationship between impairment of glymphatic pathway, meningeal lymphatic vessels and aging. However, the relationship in human brain remains uncertain. METHODS: In this observational cohort study, patients underwent MRI before and at multiple time points after intrathecal administration of contrast agent. Head T1-Weighted Imaging was performed to assess function of glymphatic pathway and head high-resolution T2-Fluid Attenuated Inversion Recovery to visualize putative meningeal lymphatic vessels (pMLVs). We measured the signal unit ratio (SUR) of 6 locations in glymphatic pathway and pMLVs, defined percentage change in SUR from baseline to 39 hour as the clearance of glymphatic pathway and pMLVs, and then analyzed their relationships with aging. RESULTS: In all patients (n = 35), the SUR of glymphatic pathway and pMLVs changed significantly after intrathecal injection of contrast agent. The clearance of both glymphatic pathway and pMLVs were related to aging (all p < 0.05). The clearance of pMLVs was significantly related to the clearance of the glymphatic pathway (all p < 0.05), and the clearance of glymphatic pathway was significantly faster in patients with early filling of pMLVs than those with late filling (all p < 0.05). INTERPRETATION: We revealed that both glymphatic pathway and pMLVs might be impaired in aging human brain, through the novel clinically-available method to simultaneously visualize their clearance. Our findings also support that in humans, pMLVs are the downstream of the glymphatic pathway. This article is protected by copyright. All rights reserved.

Chikungunya fever (CHIKF) is an arbovirus characterized by acute fever, myalgia and polyarthralgia. Lymphedema in the lower limbs (LL) was observed in several patients during an outbreak of CHIKF in the state of Pernambuco (Brazil) in 2016. No reports on lymphatic vessels disease due to CHIKF have been described. The aim of the study was to follow lymphatic abnormalities in the LL of 16 patients with CHIKF, using lymphoscintigraphy.An observational, prospective study with patients in the acute phase of CHIKF (confirmed serological diagnosis) with LL edema submitted to clinical evaluation and lymphoscintigraphy at baseline and after 90 days.Sixteen patients (81% females) participated in this study. All patients presented with lower limb lymphedema, being 15 (94%) bilateral. Of the 31 limbs affected by lymphedema, 24 (77%) presented abnormalities in lymphatic drainage by lymphoscintigraphy. The delay to visualize pelvic lymph nodes was the most frequent lymphoscintigraphic abnormality, observed in 16 (51,6%) LL. Nine (56%) patients were clinically reevaluated after 90 days, and all 18 LL remained with lymphedema. A second lymphoscintigraphy showed persistent abnormalities in 13 (72%) of the 18 LL.CHIKF can lead to lymphedema, and lymphedema may persist or progress after 3 months of the acute phases of the disease.

Background: Lymphedema includes primary lymphedema (P-LE) and secondary lymphedema (S-LE), which is a chronic progressive disease. The former group is further classified as congenital and acquired P-LE (AP-LE); its etiology is unclear, and only a few studies on its pathophysiology exist. We hypothesized that an autoimmune disease or self-inflammatory mechanism occurs in lymphatic vessels, leading to obstruction. Methods and Results: We enrolled 46 patients with lymphedema who underwent lymphaticovenous anastomosis (LVA) from January to October 2015. Collecting lymph ducts were obtained during LVA. We performed hematoxylin/eosin staining and immunostaining for LYVE-1, IL-1beta, IL-6, and TNF-alpha. There were no substantial histological differences between the two types of lymphedema, whereas some differences in expression of inflammatory cytokines, including interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha, were observed. Only a few inflammatory cells could be seen around the vessels. Although no significant differences in expression of IL-1beta were found between AP-LE and S-LE, TNF-alpha was more highly expressed in the smooth muscle layer in AP-LE patients than in S-LE patients. There were no significant morphological differences in the collecting ducts of lymphatic vessels between S-LE and P-LE. Nevertheless, higher levels of TNF-alpha accumulation were found in the thick smooth muscle layer of P-LE patients than in that of S-LE patients. Conclusion: TNF-alpha-related inflammation in collecting ducts of lymphatic vessels is an important characteristic of the pathology of P-LE. TNF-alpha inhibitors might improve symptoms of AP-LE.

Background: Lymphedema is a disorder in which lymph accumulates in the interstitial spaces due to poor lymphatic flow resulting from hypoplasia or aplasia of the lymphatic vessels, or to morpho-functional alterations that impair lymphatic flow. Lymphedema is a debilitating condition associated initially with inflammation that then degenerates into hardening of affected tissues and the formation of ulcers on the skin of affected limbs. No definitive treatment is available. The only therapy for lymphedema consists of physiotherapy, surgery, and compression to reduce impairment, which only treats the symptoms, not the causes. A possible new therapy that could reinforce the treatment of lymphedema progression and complications is electrical stimulation (ES). Many studies underline the effects of electric currents on the different cell mechanisms associated with disease. Methods and Results: In this review, we summarize the effects of ES on the molecular and cellular processes involved in the pathophysiology of lymphedema, highlighting their therapeutic potential for edema reduction, ulcer repair, and restoration of lymphatic flow in vitro and in vivo. Conclusions: ES exerts its effect on the main stages that characterize lymphedema, from its onset to ulcer formation. There are few evidences on lymphatic models and more molecular studies are needed to understand the mechanism of action of this application in the treatment of lymphedema.

BACKGROUND: This study documents the development and evaluation of a comprehensive multidisciplinary model for the assessment and personalized care of patients with lymphoedema. METHODS: The Australian Lymphoedema Education Research and Treatment (ALERT) programme originated as an advanced clinic for patients considering surgery for lymphoedema. The programme commenced liposuction surgery in May 2012 and then introduced lymph node transfer in 2013 and lymphovenous anastomosis (LVA) in 2016. An outpatient conservative treatment clinic was established in 2016. ALERT commenced investigations with indocyanine green (ICG) lymphography in late 2015, leading to the creation of a diagnostic assessment clinic offering ICG in 2017. RESULTS: Since 2012, 1200 new patients have been referred to ALERT for assessment of lymphoedema for a total of 5043 episodes of care. The introduction of ICG lymphography in 2015 initially allowed better screening for LVA, but is now used not only to guide surgical options, but also as a diagnostic tool and to guide manual lymphatic drainage massage. The total number of new patients who attended the surgical assessment clinic to December 2018 was 477, with 162 patients (34.0 per cent) undergoing surgery. CONCLUSION: The ALERT programme has developed a multidisciplinary model of care for personalized lymphoedema treatment options based on clinical, imaging and ICG lymphography. Patients are selected for surgery based on several individual factors.

Liposuction is the treatment of choice for solid predominant extremity lymphedema. The classic lymphedema liposuction technique does not remove skin excess created following bulk removal. The skin excess is presumed to resolve with spontaneous skin contracture. We investigated the technique of simultaneously performing liposuction with immediate skin excision in patients with solid predominant lymphedema and compared the outcome with that from the classic technique. Methods: Modified liposuction with skin excision (mLIPO) and standard liposuction without skin excision (sLIPO) were offered to patients with solid predominant extremity lymphedema. Skin traction of 4 cm and undulating skin mobility constituted positive “flying squirrel” sign. Patients with negative “flying squirrel” sign were excluded. mLIPO patients underwent skin excision. Surgical outcomes and postoperative complications were compared. Results: The study enrolled 15 and 26 patients into the sLIPO and mLIPO groups, respectively. mLIPO patients demonstrated statistically significant decrease in seroma/hematoma, contour irregularity, and skin necrosis, while experiencing increased procedural satisfaction. Conclusions: Skin excision following liposuction for solid predominant lymphedema is safe. It decreases postoperative complication and improves surgical outcome.

Congenital chylothorax is an uncommon condition but represents the main cause of congenital pleural effusion during the neonatal period. It usually appears before birth, both as an isolated disorder or in association with hydrops fetalis, negatively affecting the subsequent neonatal outcome. Prenatal treatment is usually considered to ensure a satisfactory lung development in case of moderate to severe pleural effusion or in the presence of hydrops, although consensus on treatment timing and modalities has not been reached to date. Both medical and surgical therapeutic strategies are available to treat this condition and novel treatment options have been recently attempted with acceptable results in both prenatal and post-natal setting. The heterogeneous clinical presentation of congenital chylothorax together with its rarity, its numerous etiologies and the absence of a highly effective treatment renders the diagnostic and therapeutic approach difficult to standardize. In addition, adequate visualization of the lymphatic system is complex, especially in small neonates, although new promising techniques have been developed lately and may contribute to improved management of this serious but infrequent condition. This review focuses on the current evidence base for the diagnosis and treatment options for congenital chylothorax, suggesting a rational diagnostic and therapeutic approach both in the prenatal and in the neonatal period.

Purpose: The aim of the study was to evaluate the usefulness of thermography in the diagnosis of lymphedema. Methods: The study included 43 women after axillary dissection caused by breast cancer, whose mean age was 64.4 +/− 7.8 years. The subjects were divided into two groups, with the criterion for division being the appearance of secondary lymphedema. The size of lymphedema was verified by Limb Volumes Professional 5.0; body surface temperatures were registered using a thermal camera. Results: In the control group, the difference in the volume of the limb on the operated side and the contralateral limb was 7.5 +/− 77.7 mL (3.6%). The temperature on the nonoperated side tended to be higher than on the opposite side. In the test group, the difference in size between the limbs was 54.5 +/− 312.7 mL (29.3%). In the case of mild or moderate edema, a reverse tendency was observed. While measuring the difference in the mean surface temperatures of both limbs belonging to two groups, a significance of differences at the level of p = 0.04 was observed. Moreover, a negative Spearman correlation coefficient (−0.34) was observed between the size of secondary lymphedema and the surface temperature. Conclusion: Noninvasive thermography can provide a valuable supplementary tool in the diagnostics of secondary lymphedema.

BACKGROUND: Podoconiosis is a type of elephantiasis characterised by swelling of the lower legs. It is often confused with other causes of tropical lymphedema and its global distribution is uncertain. Here we synthesise the available information on the presence of podoconiosis to produce evidence consensus maps of its global geographical distribution. METHODS AND FINDINGS: We systematically searched available data on podoconiosis in SCOPUS and MEDLINE from inception, updated to 10 May, 2019, and identified observational and population-based studies reporting podoconiosis. To establish existence of podoconiosis, we used the number of cases reported in studies and prevalence data with geographical locations. We then developed an index to assess evidence quality and reliability, assigning each country an evidence consensus score. Using these summary scores, we then developed a contemporary global map of national-level podoconiosis status. There is evidence of podoconiosis in 17 countries (12 in Africa, three in Latin America, and two in Asia) and consensus on presence in six countries (all in Africa). We have identified countries where surveillance is required to further define the presence or absence of podoconiosis. We have highlighted areas where evidence is currently insufficient or conflicting, and from which more evidence is needed. CONCLUSION: The global distribution of podoconiosis is not clearly known; the disease extent and limits provided here inform the best contemporary map of the distribution of podoconiosis globally from available data. These results help identify surveillance needs, direct future mapping activities, and inform prevention plans and burden estimation of podoconiosis.

Single site Multiple Lymphatic-Venous Anastomoses (MLVA) provides optimal functional and cosmetic results with low complication rates in treatment of lower limb lymphedema. However, no evidence exists in literature concerning the use of this technique in treatment of peno-scrotal lymphedema. We report a case of a 44-year-old male patient who developed secondary peno-scrotal lymphedema with severe lymphorrhea, following a laser treatment for scrotal pustolosis, leading to recurrent infections and finally an established peno-scrotal lymphedema. Utilizing MLVA, a complete remission of scrotal lymphedema was achieved with significant volume reduction of the penile lymphedema. The post-operative course was uneventful with clear improvement in lymphatic flow demonstrable on lymphoscintigraphy (6 months) and no recurrence of scrotal lymphedema at 2 years follow-up. This article reports very promising results of a novel application of MLVA in the treatment of genital lymphedema and suggests that MLVA provides the possibility to shunt both superficial and deep lymphatics to improve the lymphatic drainage from the peno-scrotal area using a single surgical site.

Lymphoscintigraphy is a well-established radiologic examination to evaluate lymphatic function. We conducted a systematic review of the use of lymphoscintigraphy for evaluation of lymphedema treatment. We hypothesized that this radiologic examination could add relevant findings of treatment outcomes of lymphedema patients. We conducted a systematic review of articles in PubMed, without any time frame or language limitations, about the use of lymphoscintigraphy for the evaluation of lymphedema treatment. Articles were excluded if they investigated other uses of lymphoscintigraphy, such as diagnosis or prevention of lymphedema. Abstracts, presentations, reviews, and meta-analyses were also excluded. Of 101 potential articles found in the literature, 5 fulfilled our study eligibility criteria, and they were all case series. These articles included a total of 327 patients, most of whom had breast cancer-related lymphedema. Interventions included lymph node or vessel transfer (3 of 5 articles), complex decongestive therapy (1 of 5), and adipose-derived stem cell injection (1 of 5). The authors of these studies used lymphoscintigraphy to investigate the treatment functional outcomes, prognostic value, and complications. Lymphoscintigraphy detected lymphangiogenesis in transferred lymph nodes, and it was able to predict patient response to complex decongestive therapy. Studies that used lymphoscintigraphy to evaluate lymphedema treatment demonstrated its flexibility to provide various types of information. We hope this review will support future studies.

Although the sympathetic nerve system has been described as a modulator of lymphatic circulation, it has not been targeted in the treatment of lymphedema. We conducted a systematic review of publications assessing the use of sympathetic nerve block in lymphedema treatment. We hypothesized that sympathetic nerve block may be a promising treatment option for lymphedema patients. We conducted a comprehensive systematic review of the published literature on the use of sympathetic nerve block in lymphedema treatment using the PubMed database. Eligibility criteria excluded papers that reported other types of lymphedema treatment or any other anesthesiology procedure. Abstracts, presentations, reviews, and meta-analyses were also excluded. Extracted data included the year of study, country, author affiliation, type of study, patient characteristics, nerve block technique, and key findings. From 81 potential papers, eight studies fulfilled the eligibility criteria. All papers identified were clinical, reporting on a total of 187 patients. Sympathetic nerve block was proposed with local anesthetics, whether or not associated with triamcinolone. Treatment with a nerve block promoted lymphedema improvement expressed by decreased limb circumference and patient-reported outcomes. Large randomized clinical trials are still pending, but sympathetic nerve block seems to be a promising alternative for lymphedema patients who do not respond to conservative therapy.

It is estimated that one in every six patients undergoing solid cancer treatment will develop secondary lymphedema. We conducted a systematic review of publications assessing the potential use of pharmacotherapy agents in lymphedema treatment. The search was conducted on PubMed and eligibility criteria excluded papers that investigated other therapies or focused on primary lymphedema. From 285 potential papers found in the literature, seven studies fulfilled the eligibility criteria. Different types of therapies were proposed, but all of them interfered with inflammation in the lymphedema tissue. Interestingly, the majority of publications were clinical, and three authors conducted randomized, placebo-controlled, double-blinded clinical studies. Promising results were observed for the oral administration of ketoprofen or selenium and topical tacrolimus. Pharmacotherapy agents were successfully described in lymphedema treatment in clinical and experimental studies. The benefits of delivering ketoprofen, selenium, or tacrolimus in lymphedema were noticed, and these therapies were easily delivered and well-tolerated.

Different delivery mechanisms have been proposed in the literature for targeted therapies in the treatment of lymphedema. They vary from simple and direct injection to sophisticated induction of gene expression in a targeted tissue. We conducted a systematic review of publications assessing the use of viral vectors for gene transfer in lymphedema treatment. We hypothesized that viral vectors are an effective way to deliver targeted therapy in lymphedema treatment. We conducted a comprehensive systematic review of the published literature on targeted therapies associated with lymphedema surgery using the PubMed database. Eligibility criteria excluded papers that reported use of viral vectors for other medical conditions. Abstracts, presentations, reviews, meta-analyses, and non-English language articles were also excluded. From 21 potential articles found in the literature, fourteen fulfilled study eligibility criteria. Positive outcomes in terms of lymphangiogenesis were seen. The viral vectors used included adenovirus and recombinant adeno-associated virus. Most of the genes expressed were growth factors, but expression of dominant-negative transforming growth factor-beta1 receptor-II or Prox1 was also proposed. Five studies targeted genetic expression on lymphedema tissue, five on transplanted lymph nodes, two on skeletal muscle, and one on adipose-derived stem cells. Publications assessing use of viral vectors for gene transfer in lymphedema treatment demonstrated that it is an effective mechanism of delivering targeted therapies. However, to date, all studies were experimental and further studies must be performed before translating these therapies into clinical practice.

It is estimated that 140 to 200 million people are affected by lymphedema worldwide. Many studies have proposed targeted therapies that can be delivered systemically or locally to treat lymphedema. Since lymphedema primarily affects the skin and subcutaneous tissues, topical approaches to therapy should be considered as an attractive proposition as they can avoid systemic complications. In light of this, we conducted a systematic review of publications that analyzed the use of topical approaches to delivering targeted therapies in the treatment of lymphedema. We hypothesized that topical approaches resulted in the satisfactory treatment of lymphedema. We conducted a systematic review of publications on PubMed. The main eligibility criterion was that the articles should primarily investigate the use of topical approaches to delivering targeted therapies in the treatment of lymphedema. Consequently, we excluded papers that investigated any other delivery approaches or medical conditions. Of the 174 potential studies found in the literature, six were found to fulfill our eligibility criteria. All these studies were experimental ones on small animals (mice). The authors generally proposed different types of therapies, which could be clustered into two main groups: 1) induction of lymphangiogenesis [vascular endothelial growth factor C (VEGF-C) hydrogel or fibroblast growth factor]; and 2) modulation of inflammation (tacrolimus or topical collagen gel or troxerutin-phosphatidylcholine). All studies presented positive outcomes, demonstrating that topical therapy is a promising route for delivering growth factors and anti-inflammatory agents in the treatment of lymphedema. However, studies were conducted under heterogeneous protocols, and the safe application of these therapies in humans has not been assessed. Further studies are necessary to confirm the benefits and safety of targeted topical therapy on patients with lymphedema.

The Global Programme to Eliminate Lymphatic Filariasis was launched in 2000 to eliminate lymphatic filariasis (LF) as a public health problem by 1) interrupting transmission through mass drug administration (MDA) and 2) offering basic care to those suffering from lymphoedema or hydrocele due to the infection. Although impressive progress has been made, the initial target year of 2020 will not be met everywhere. The World Health Organization recently proposed 2030 as the new target year for elimination of lymphatic filariasis (LF) as a public health problem. In this letter, LF modelers of the Neglected Tropical Diseases (NTDs) Modelling Consortium reflect on the proposed targets for 2030 from a quantitative perspective. While elimination as a public health problem seems technically and operationally feasible, it is uncertain whether this will eventually also lead to complete elimination of transmission. The risk of resurgence needs to be mitigated by strong surveillance after stopping interventions and sometimes perhaps additional interventions.

BACKGROUND: The objective of this study was to compare the economic impact of complete decongestive therapy and lymphovenous bypass in the management of upper extremity lymphedema. METHODS: Economics were modeled for a patient with breast cancer-related lymphedema undergoing three different clinical pathways: (1) complete decongestive therapy alone; (2) lymphovenous bypass no longer requiring ongoing complete decongestive therapy; or (3) lymphovenous bypass requiring ongoing complete decongestive therapy. Activity-based cost analysis identified costs incurred with complete decongestive therapy and lymphovenous bypass. Costs were retrieved from supplier price lists, physician fee schedules, lymphedema therapists, and literature reviews. The net present value of all costs incurred for each clinical pathway were calculated. RESULTS: The estimated net present value of all costs for a patient with breast cancer-related lymphedema undergoing treatment were as follows: (1) complete decongestive therapy alone ($30,400); (2) lymphovenous bypass no longer requiring ongoing complete decongestive therapy ($15,000); or (3) lymphovenous bypass requiring ongoing complete decongestive therapy ($42,100). The expected net present value of all costs for lymphovenous bypass was $26,800, which was comparable to that of complete decongestive therapy alone. Sensitivity analysis demonstrated that the expected net present value of lymphovenous bypass was dependent on the patient's life expectancy, number of bypass anastomoses, and likelihood of discontinuing complete decongestive therapy. CONCLUSIONS: Lymphedema has substantial ongoing costs irrespective of the treatment modality. The cost of lymphovenous bypass appears comparable to that of complete decongestive therapy alone-the surgical costs of lymphovenous bypass are offset by the savings from discontinued ongoing therapy. Despite its limitations as a theoretical economic model, this study provides insight into the potential economic impact of lymphovenous bypass.

BACKGROUND: Lymphedema is a chronic condition that carries a significant physical, psychosocial, and economic burden. The authors' program was established in 2017 with the aims of providing immediate lymphatic reconstruction in high-risk patients undergoing lymphadenectomy and performing delayed lymphatic reconstruction in patients with chronic lymphedema. The purpose of this study was to describe the authors' clinical experience in the first year. METHODS: A retrospective review of our clinical database was performed on all individuals presenting to the authors' institution for lymphatic surgery consideration. Patient demographics, clinical characteristics, and surgical management were reviewed. RESULTS: A total of 142 patients presented for lymphatic surgery evaluation. Patients had a mean age of 54.8 years and an average body mass index of 30.4 kg/m. Patients with lymphedema were more likely to be referred from an outside facility compared to patients seeking immediate lymphatic reconstruction (p < 0.001). For patients with lymphedema, the most common cause was breast cancer related. Thirty-two percent of all patients evaluated underwent a lymphatic procedure. Of these, 32 were immediate lymphatic reconstructions and 13 were delayed lymphatic reconstructions. In the authors' first year, 94 percent of eligible patients presenting for immediate lymphatic reconstruction underwent an intervention versus only 38 percent of eligible lymphedema patients presenting for delayed lymphatic reconstruction (p < 0.001). CONCLUSIONS: First-year review of our lymphatic surgery experience has demonstrated clinical need evidenced by the number of patients and high percentage of outside referrals. As a program develops, lymphatic surgeons should expect to perform more time-sensitive immediate lymphatic reconstructions, as evaluation of chronic lymphedema requires development of a robust team for workup and review.

Less-invasive surgeries, such as lymphaticovenular anastomosis (LVA), are the widely accepted intervention for lymphedema. This study aimed to assess the outcomes of flow-oriented LVA modification on lymphatic malformation (LM). Methods: We included 19 patients diagnosed with LM mixed type or microcystic type, who came to our clinic from June 2015 to December 2017. Under general anesthesia, all patients were administered an indocyanine green lymphography injection subcutaneously. In the case of a strong inflow, the patient underwent afferent lymph vessel of LM to venous anastomosis (LMVA). Otherwise, the side wall of LMVA was performed to the cysts. Outcomes were classified into the following groups based on the size changes: treatment effect (TE) 4 = >80% reduction rate; TE 3 = 50%–80% reduction rate; TE 2 = 20%–50% reduction rate; and TE 1 = 0%–20% reduction rate. Results: All cases underwent surgery, with no case having an increased size. The results were as follows: TE 4 = 4 (21%) patients; TE 3 = 6 (32%) patients; TE 2 = 5 (26%) patients; and TE 1 = 4 (21%) patients. No case required study termination due to disease progression. Minor complication occurred in 3 cases. One vesicle increased at the labial mucosa and one wound dehiscence that epithelized within 1 month. Conclusion: LMVA could be a novel, minimally invasive lymph flow-oriented surgical method for intractable LM.

With improving survival of children with complex congenital heart disease (CCHD), postoperative complications, like protein-losing enteropathy (PLE) are increasingly encountered. A 3-year-old girl with surgically corrected CCHD (ventricular inversion/L-transposition of the great arteries, ventricular septal defect, pulmonary atresia, post-double switch procedure [Rastelli and Glenn]) developed chylothoraces. She was treated with pleurodesis, thoracic duct ligation and subsequently developed chylous ascites and PLE (serum albumin </ = 0.9 g/dL) and was malnourished, despite nutritional rehabilitation. Lymphangioscintigraphy/single-photon emission computed tomography showed lymphatic obstruction at the cisterna chyli level. A segmental chyle leak and chylous lymphangiectasia were confirmed by gastrointestinal endoscopy, magnetic resonance (MR) enterography, and MR lymphangiography. Selective glue embolization of leaking intestinal lymphatic trunks led to prompt reversal of PLE. Serum albumin level and weight gain markedly improved and have been maintained for over 3 years. Selective interventional embolization reversed this devastating lymphatic complication of surgically corrected CCHD.

Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by uncontrolled growth of smooth muscle -like cells in the lungs that can spread via the lymphatic system to other parts of the body. The current treatment for LAM, oral rapamycin, is limited by its low oral bioavailability and side effects. This study aims to develop an inhaled formulation of rapamycin solid lipid nanoparticles (Rapa-SLNs) to avoid first-pass metabolism, increase invivo half-life and facilitate entry into the lymphatic system through the lungs. Rapa-SLNs were manufactured using a hot evaporation technique and freeze-dried overnight with 5% (w/v) mannitol and before being assessed further for particle characteristics and in vitro aerosol performance and release. The formulation's ability to penetrate through bronchial epithelial layer was evaluated using a Calu-3 cell model, while its ability to interfere with the LAM intracellular cascade was evaluated using Mouse Embryonic fibroblast (MEF) cells deficient for the tuberous sclerosis complex 2 (TSC2) and compared with rapamycin solution. Results showed that the Rapa- SLNs had the appropriate size (237.5+/−1.8nm), charge (−11.2), in vitro aerosol performance (MMAD = 5.4+/−0.4mum) and sustained release profile suitable for entry into the lymphatic system via the pulmonary route. Additionally, the nanoparticles were transported at a faster rate across the bronchial epithelial layer compared to free rapamycin solution. The formulation also showed similar mTOR (mammalian target of Rapamycin) inhibition properties compared to free rapamycin, and was able to significantly decrease the amount of proliferation in TSC2 negative MEF cells. This formulation is therefore a promising alternative treatment for LAM patients, as it could potentially reduce problems associated with low bioavailability and side effects of current oral treatment.

BACKGROUND: Chylous leak is an uncommon complication after head and neck surgery and typically results from a lesion of the thoracic duct (TD). Beside conservative treatment, different minimally invasive and surgical procedures exist, of which almost all lead to a total closure of the TD. METHODS: We report on a rare case of microsurgical lymphovenous anastomosis to treat a TD lesion. An additional systematic review on surgical procedures to treat TD lesions with special attention to lymphovenous anastomoses was performed according to the PRISMA guidelines. RESULTS: A 52-year-old patient with a chylous fistula after modified radical neck dissection was successfully treated by a lymphovenous anastomosis of the TD and external jugular vein with additional coverage by sternocleidomastoid muscle flap. The patient showed a complete resolution of chylous leak with an uneventful postoperative course.The systematic search of literature yielded 684 articles with 4 case reports on lymphovenous anastomosis in chylous leak with a high success rate. Other surgical techniques include transcervical, thoracoscopic, or video-assisted thoracoscopic TD ligation, either alone or combined with a local muscle flap. CONCLUSIONS: Lymphovenous anastomosis of the TD is a feasible and safe technique allowing for treatment of cervical TD lesions, especially if minimally invasive procedures fail. Compared with other techniques, lymphatic circulation can successfully be maintained.

Background: There are many techniques of monitoring leg lymphedema during physical therapy. Taking volumetric measurements with a tape measure is among the most common clinically, and changes in volume are typically used to measure therapy efficacy. This study shows how the Kinect infrared (IR) sensor with custom algorithms can assess leg circumferences and volumes comparable with tape measurements taken by a trained therapist while exploring regional leg changes to determine uniformity of change. Methods and Results: Leg volumes were measured in 38 lymphedema patients using the tape measure circumference method and the Kinect IR system. Changes in circumferences in various leg regions over the course of therapy were analyzed in 23 patients. The leg circumferences (R(2) = 0.9522) and volumes (R(2) = 0.9847) strongly correlated between the two methods. The Bland-Altman analysis indicated a circumference percent different bias of 1.6 (6.2%), requiring a minor correction factor between the two methods. Over the course of therapy, patients with a reduction in leg volume, defined as a change >6.5% have greater reduction most distal to the body. Conclusion: The Kinect IR system explored can be used clinically for leg volume measurements to monitor leg lymphedema patients over the course of their therapy. Implementing analysis of regional leg changes can better inform physical therapy to improve efficacy of treatment.

Manual Lymphatic Drainage (MLD) appears to stimulate lymphatic contraction, aid in the development of secondary derivation pathways, and stimulate the appearance of collateral pathways that could function as the main drainage routes of the limb in case of lymphedema. Through stretching, call up maneuvers are used to stimulate lymphangion reflex contraction and, therefore, lymphatic function. The aim was to describe the presence of areas and pathways of collateral lymphatic drainage under basal conditions and to determine, using Indocyanine Green (ICG) lymphography, whether an increase in these pathways occurs after 30 min of manual lymphatic stimulation with only call up maneuvers according to the Leduc Method((R)(R)). In this prospective analytical study (pretest-posttest), the frequency of presentation of areas and collateral lymphatic pathways was analyzed in 19 patients with secondary lymphedema of the upper limb after breast cancer using an infrared camera. Analyses were completed at three time points: after ICG injection, at baseline (pretest), and after the application of MLD (post-test). The Leduc Method maneuvers were applied to the supraclavicular and axillary nodes, chest, back, Mascagni, and Caplan pathways. The areas visualized in the pretest continued to be visible in the posttest. Additional pathways and fluorescent areas were observed after the maneuvers. The McNemar test showed statistical significance (p = 0.008), the odds ratio was infinite, and the Cohen's g value was equal to 0.5. Manual stimulation by call up maneuvers increased the observation frequency of areas and collateral lymphatic pathways. Therefore, ICG lymphography appears to be a useful tool for bringing out the routes of collateral bypass in secondary lymphoedema after cancer treatment.

Background: Primary lymphedema is genetically heterogeneous. Two of the most common forms of primary lymphedema are Milroy disease (MD) and lymphedema-distichiasis syndrome (LDS). This study aims to look further into the pathogenesis of the two conditions by analyzing the lymphoscintigram images from affected individuals to ascertain if it is a useful diagnostic tool. Methods and Results: The lymphoscintigrams of patients with MD and LDS were analyzed, comparing the images and transport parameters of the two genotypes against a control population. Lymphoscintigrams were available for 12 MD and 16 LDS patients (all genetically proven diagnoses). Eight of the 12 (67%) lymph scans performed on patients with MD demonstrated little or no uptake from the initial lymphatics and poor visualization of the inguinal lymph nodes. These changes were consistent with a “functional aplasia,” that is, the lymphatic vessels were present but appeared to be ineffective in absorbing the interstitial fluid into the lymphatic system. In patients with LDS the lymphoscintigraphic appearances were different. In 12 of the 16 scans (75%), the lymph scans were highly suggestive of lymphatic collector reflux. Quantification revealed a significantly reduced uptake of tracer within the inguinal lymph nodes and a higher residual activity in the feet at 2 hours in MD compared with LDS and compared with controls. Conclusion: Lymphoscintigraphic imaging and quantification can be characteristic in specific genetic forms of primary lymphedema and may be useful as an additional tool for in-depth phenotyping, leading to a more accurate diagnosis and providing insight into the underlying mechanism of disease.

Breast tumor progression is a complex process involving intricate crosstalk between the primary tumor and its microenvironment. In the context of breast tumor-lymphatic interactions, it is unclear how breast cancer cells alter the gene expression of lymphatic endothelial cells and how these transcriptional changes potentiate lymphatic dysfunction. Thus, there is a need for in vitro lymphatic vessel models to study these interactions. In this work, a tumor-lymphatic microfluidic model is developed to study the differential conditioning of lymphatic vessels by estrogen receptor-positive (i.e., MCF7) and triple-negative (i.e., MDA-MB-231) breast cancer cells. The model consists of a lymphatic endothelial vessel cultured adjacently to either MCF7 or MDA-MB-231 cells. Quantitative transcriptional analysis reveals expression changes in genes related to vessel growth, permeability, metabolism, hypoxia, and apoptosis in lymphatic endothelial cells cocultured with breast cancer cells. Interestingly, these changes are different in the MCF7-lymphatic cocultures as compared to the 231-lymphatic cocultures. Importantly, these changes in gene expression correlate to functional responses, such as endothelial barrier dysfunction. These results collectively demonstrate the utility of this model for studying breast tumor-lymphatic crosstalk for multiple breast cancer subtypes.

BACKGROUND: Extremity lymphedema is a dreaded complication of ilioinguinal or axillary lymphadenectomy. In conventional lymph node dissection, no effort is performed to maintain or reestablish extremity lymphatic circulation. We hypothesized that immediate lymphatic reconstruction (ILR) could be a reproducible procedure to maintain functional lymphatic flow after ilioinguinal and axillary lymphadenectomy in patients with malignant melanoma. This is the first report describing prophylactic ILR in patients with melanoma who underwent complete lymph node dissection for gross nodal disease. METHODS: We report a case series of 22 malignant melanoma patients who had axillary or ilioinguinal lymph node dissection for bulky locoregional invasion with immediate lymphatic reconstruction. A novel method to identify and select lymphatics with high flow using fluorescent lymphangiogram with indocyanine green dye gradient software is described. Surgical details, common difficulties, as well as indications are discussed. Instructional videos are also provided. RESULTS: Our technique is reproducible, since we have successfully completed immediate lymphatic reconstruction in 22 cases consecutively. Intradermal indocyanine green injections allowed for visualization of 1 to 3 transected lymphatics after lymphadenectomy. An average of 1.8 lymphaticovenous bypass (range 1–3) was performed per patient. CONCLUSION: Reestablishment of lymphatic circulation after ilioinguinal or axillary lymphadenectomy in patients with melanoma characterizes a novel method that may reduce the problem of upper and lower extremity iatrogenic lymphedema. This is particularly important given the emergence of new adjuvant treatment modalities that considerably improve patients' survival after lymphadenectomy.

Introduction: The phenomenon of lymph node metastasis has been known for a long time. However, the underlying mechanism by which malignant tumor cells are able to break loose from the primary tumor site remains unclear. In particular, two competing fluid sensitive migration mechanisms have been reported in the experimental literature: (i) autologous chemotaxis (Shields et al. in Cancer Cell 11:526–538, 2007) which gives rise to downstream migration; (ii) an integrin-mediated and strain-induced upstream mechanism (Polacheck et al. in PNAS 108:11115–11120, 2011). How can these two competing mechanisms be used as a means for metastatic behavior in a realistic tumor setting? Excessive fluid flow is typically produced from leaky intratumoral blood vessels and collected by lymphatics in the peritumoral region giving rise to a heterogeneous fluid velocity field and a corresponding heterogeneous cell migration behavior, quite different from the experimental setup. Method: In order to shed light on this issue there is a need for tools which allow one to extrapolate the observed single cell behavior in a homogeneous microfluidic environment to a more realistic, higher-dimensional tumor setting. Here we explore this issue by using a computational multiphase model. The model has been trained with data from the experimental results mentioned above which essentially reflect one-dimensional behavior. We extend the model to an envisioned idealized two-dimensional tumor setting. Result: A main observation from the simulation is that the autologous chemotaxis migration mechanism, which triggers tumor cells to go with the flow in the direction of lymphatics, becomes much more aggressive and effective as a means for metastasis in the presence of realistic IF flow. This is because the outwardly directed IF flow generates upstream cell migration that possibly empowers small clusters of tumor cells to break loose from the primary tumor periphery. Without this upstream stress-mediated migration, autologous chemotaxis is inclined to move cells at the rim of the tumor in a homogeneous and collective, but space-demanding style. In contrast, inclusion of realistic IF flow generates upstream migration that allows two different aspects to be synthesized: maintain the coherency and solidity of the the primary tumor and at the same time cleave the outgoing waves of tumor cells into small clusters at the front that can move collectively in a more specific direction.

PURPOSE: We aimed to evaluate the potential role of quantitative methods associated with lymphoscintigraphy for the assessment of severity of lymphedema post-operatively in patients with breast cancer who did not show definite dermal backflow activity on the lymphoscintigraphy. METHODS: We evaluated 47 lymphoscintigraphies without dermal backflow in patients with lymphedema who received a mastectomy and axillary dissection or sentinel lymph node dissection for invasive ductal carcinoma of the breast. The quantitative asymmetry indices (QAIs) of both arms were calculated for each axilla, upper arm, forearm, and the whole arm. The QAI was defined as the radiopharmaceutical uptake ratio of the affected side to the unaffected side. Arm circumference was measured at four locations per arm to identify the maximal circumference difference (MCD) between affected and unaffected sides. RESULTS: The total and forearm QAIs of each side arm were significantly higher in the group with above moderate stage lymphedema compared with the mild stage group. Previous radiotherapy also had a significant effect on radiotracer retention expressed as QAI. The MCD was significantly correlated with QAI values of the forearm and the whole arm. The QAI of axillary areas was not significantly correlated with circumferential measurements of the arm. CONCLUSIONS: The QAIs have significant value for the diagnosis and severity of lymphedema and may therefore potentially be used as an objective tool for the assessment of lymphedema.

BACKGROUND: Most studies on lower limb lymphedema have been conducted in gynecologic cancer patients who underwent surgery for gynecologic malignancy. This study aimed to evaluate the risk factors for lower limb lymphedema development in gynecologic cancer patients who underwent initial treatment. METHODS: A retrospective cohort design was used to follow 903 gynecologic cancer patients who underwent treatment at Kurume University Hospital between January 1, 2013 and December 31, 2015. Data analyses were performed in 356 patients, and the patients were followed up until December 31, 2017. The model comprised two components to facilitate statistical model construction. Specifically, a discrete survival time model was constructed, and a complementary log-log link model was fitted to estimate the hazard ratio. Associations between risk factors were estimated using generalized structural models. RESULTS: The median follow-up period was 1083 (range 3–1819) days, and 54 patients (15.2%) developed lower limb lymphedema, with a median onset period of 240 (range 3–1415) days. Furthermore, 38.9% of these 54 patients developed lower limb lymphedema within 6 months and 85.2% within 2 years. International Federation of Gynecology and Obstetrics stage, radiotherapy, and number of lymph node dissections (>/ = 28) were significant risk factors. CONCLUSION: Simultaneous examination of the relationship between lower limb lymphedema and risk factors, and analysis among the risk factors using generalized structural models, enabled us to construct a clinical model of lower limb lymphedema for use in clinical settings to alleviate this condition and improve quality of life.

BACKGROUND: This study was to investigate the lymphoscintigraphy findings for the diagnosis and severity in unilateral gynecological cancer-related lymphedema (GCRL) and to correlate lymphoscintigraphy stages with the clinical findings. METHODS: Patients with unilateral GCRL who underwent lymphoscintigraphy were staged using the presence of ileo-inguinal lymph nodes, distal-lymphatic ducts, and dermal backflow findings. Taiwan Lymphoscintigraphy Staging (TLS) was divided into three patterns and seven stages: normal drainage (L-0); partial obstruction (P-1, P-2, and P-3); and total obstruction (T4, T-5, and T-6). Correlations between clinical lymphedema severity and TLS were evaluated using analysis of variance and multivariable linear regression analyses. RESULTS: A total of 141 patients with unilateral GCRL were divided as follows: 6 (4.3%) in normal drainage, 56 (39.7%) in partial-obstruction, and 79 (56%) in total obstruction. Cellulitis episodes, circumferential difference, and computed tomography (CT) volumetric difference were shown to be statistically different between TLS stages (P < .001 for all). Total obstruction stages were the most significant factors associated with the severity of circumferential difference (beta = 19.72, 25.54, 32.42, respectively; P < .05) and CT volumetric difference (beta = 36.04, 45.12, 52.78, respectively; P < .01). CONCLUSIONS: Total lymphatic obstruction was present in 56% of unilateral GCGL. Lymphoscintigraphy stages were statistically correlated with episodes of cellulitis, circumferential difference and CT volumetric difference in unilateral GCRL.

Immune surveillance against pathogens and tumours in the central nervous system is thought to be limited owing to the lack of lymphatic drainage. However, the characterization of the meningeal lymphatic network has shed light on previously unappreciated ways that an immune response can be elicited to antigens that are expressed in the brain (1–3). Despite progress in our understanding of the development and structure of the meningeal lymphatic system, the contribution of this network in evoking a protective antigen-specific immune response in the brain remains unclear. Here, using a mouse model of glioblastoma, we show that the meningeal lymphatic vasculature can be manipulated to mount better immune responses against brain tumours. The immunity that is mediated by CD8 T cells to the glioblastoma antigen is very limited when the tumour is confined to the central nervous system, resulting in uncontrolled tumour growth. However, ectopic expression of vascular endothelial growth factor C (VEGF-C) promotes enhanced priming of CD8 T cells in the draining deep cervical lymph nodes, migration of CD8 T cells into the tumour, rapid clearance of the glioblastoma and a long-lasting antitumour memory response. Furthermore, transfection of an mRNA construct that expresses VEGF-C works synergistically with checkpoint blockade therapy to eradicate existing glioblastoma. These results reveal the capacity of VEGF-C to promote immune surveillance of tumours, and suggest a new therapeutic approach to treat brain tumours.

Angiosarcoma is a rare malignant tumour, which accounts for 1–2% of all malignant soft-tissue tumours. Most cases of angiosarcoma arise spontaneously, and malignant transformation of vascular malformation to angiosarcoma is extremely rare. We describe the case of a 70-year-old woman with a massive arteriovenous malformation in her shoulder, which gradually enlarged, despite repeated surgeries and radiation therapy over 53 years. She also presented with rapidly growing haemorrhagic masses in her oral cavity. Excision biopsy was performed, and the pathohistological diagnosis was angiosarcoma. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose accumulation in the oral cavity and right shoulder, the latter of which was consistent with the location of the arteriovenous malformation. The masses in the oral cavity were diagnosed as metastatic angiosarcoma from the right shoulder, where the massive arteriovenous malformation was suspected to have malignantly transformed. This report describes a possible case of malignant transformation of arteriovenous malformation to angiosarcoma.

Lymphatic infiltration (LI) is a key factor affecting the treatment of patients with colorectal cancer (CRC). Thus, the aim of this study was to develop and validate a nomogram for individual preoperative prediction of LI in patients with CRC.We conducted a retrospective analysis of 664 patients who received their initial diagnosis of CRC at our center. Those patients were allocated to a training dataset (n = 468) and a validation dataset (n = 196). The least absolute shrinkage and selection operator regression model was used for data dimension reduction and feature selection. The nomogram was constructed from the training dataset and internally verified using the concordance index (C-index), calibration, area under the receiver operating characteristic curve and decision curve analysis (DCA).The enhancement computed tomography reported N1/N2 classification, preoperative tumor differentiation, elevated carcinoembryonic antigen, and carbohydrate antigen19–9 level were selected as variables for the prediction nomogram. Encouragingly, the nomogram showed favorable calibration with C-index 0.757 in the training cohort and 0.725 in validation cohort. The DCA signified that the nomogram was clinically useful. The Kaplan-Meier survival curve showed that patients with LI had a worse prognosis and could benefit from postoperative adjuvant chemotherapy. Use common clinicopathologic factors, a non-invasive scale for individualized preoperative forecasting of LI was established conveniently. LI prediction has great significance for risk stratification of prognosis and treatment of resectable CRC.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been widely used to treat non-small cell lung cancer (NSCLC) because they inhibit tumour growth and metastasis. However, the underlying mechanisms are not fully understood. Here, we investigate whether anti-lymphangiogenesis mechanisms contribute to the anti-tumour effects of EGFR-TKIs. Three different EGFR-TKIs (Gefitinib, Afatinib, and AZD9291) were used to determine the possible biological effects of EGFR-TKIs on lymphangiogenesis in vitro and in vivo. EGFR-TKIs inhibited human lymphatic endothelial cells (HLEC) proliferation, migration and tube formation at the indicated concentrations. Conditioned medium from human lung adenocarcinoma HCC827 cells treated with EGFR-TKIs also inhibited HLEC migration and tube formation. EGFR-TKIs inhibited VEGFC secretion, which further influenced HLEC behaviour in vitro. Afatinib inhibited tumour growth and lymphangiogenesis in the HCC827 xenograft mouse model. The densities and tube diameters of the lymphatic vessels were decreased in a dose-dependent manner, as shown by lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) staining. EGFR-TKIs also inhibited the expression of important lymphangiogenesis regulatory factors vascular endothelial growth factor 2/3 (VEGF2/3), VEGFC, and chemokine receptor 7 (CCR7) as shown by immunocytochemistry (IHC) staining. Additional assays confirmed that the JAK/STAT3 signalling pathways play important roles in the anti-lymphangiogenesis process induced by EGFR-TKIs. Inhibition of lymphangiogenesis is another important role that the three EGFR-TKIs play in the treatment of lung cancer and the Janus kinase/signal transducers and activators of transcription 3 (JAK/STAT3) maybe an important signalling pathway regulating lymphangiogenesis, which provides a new idea for clinical therapy of lung cancer.

OBJECTIVES: At our institution, in vivo facial nerve mapping (FNM) is used during vascular anomaly (VAN) surgeries involving the facial nerve (FN) to create an FN map and prevent injury. During mapping, FN anatomy seemed to vary with VAN type. This study aimed to characterize FN branching patterns compared to published FN anatomy and VAN type. STUDY DESIGN: Retrospective study of surgically relevant facial nerve anatomy. METHODS: VAN patients (n = 67) with FN mapping between 2005 and 2018 were identified. Results included VAN type, FN relationship to VAN, FNM image with branch pattern, and surgical approach. A Fisher exact test compared FN relationships and surgical approach between VAN pathology, and FN branching types to published anatomical studies. MATLAB quantified FN branching with Euclidean distances and angles. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) analyzed quantitative FN patterns amongst VAN types. RESULTS: VANs included were hemangioma, venous malformation, lymphatic malformation, and arteriovenous malformation (n = 17, 13, 25, and 3, respectively). VAN FN patterns differed from described FN anatomy (P < .001). PCA and HCA in MATLAB-quantified FN branching demonstrated no patterns associated with VAN pathology (P = .80 and P = .91, one-way analysis of variance for principle component 1 (PC1) and priniciple component 2 (PC2), respectively). FN branches were usually adherent to hemangioma or venous malformation as compared to coursing through lymphatic malformation (both P = .01, Fisher exact). CONCLUSIONS: FN branching patterns identified through electrical stimulation differ from cadaveric dissection determined FN anatomy. This reflects the high sensitivity of neurophysiologic testing in detecting small distal FN branches. Elongated FN branches traveling through lymphatic malformation may be related to abnormal nerve patterning in these malformations. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

BACKGROUND: Vascular anomalies (VA), characterized by the abnormal development or growth of blood and/or lymphatic vessels, encompasses a spectrum of conditions with a range of symptoms and complications. VA are frequently associated with cutaneous complications that can cause significant morbidity. Systemic sirolimus has previously been shown to be effective in the treatment of complicated VA. There are limited studies to date on the use of topical sirolimus for the treatment of cutaneous manifestations of VA. METHODS: Retrospective review of medical records of pediatric patients with VA treated with topical sirolimus at a single quaternary pediatric institution. Response was determined by clinical subjective and objective measures of improvement. RESULTS: Twenty-three patients with cutaneous VA manifestations were treated with topical sirolimus. Median age was 14 (range 4–27 years). The main indication for treatment was complication of lymphatic blebbing (82%, n = 19) including lymphatic fluid leakage, bleeding, pain, pruritus, swelling, or recurrent infection. Treatment course ranged from 109 to 1424 days with median of 622 days. No major side effects were reported. Eighty-six percent of patients (n = 20) had subjective or objective improvement of cutaneous lesions. Lymphatic blebbing complications improved in 90% (n = 17) of individuals. Eighty-two percent (n = 14) of patients not receiving concurrent systemic sirolimus demonstrated improvement with topical therapy. One patient electively stopped treatment due to pruritus and burning sensation. CONCLUSION: Topical sirolimus appears to be a beneficial therapy for lymphatic blebbing associated with lymphatic malformations or mixed malformations with a lymphatic component, although benefit in other VA remains unclear. Topical sirolimus was well-tolerated with minimal side effects.

Background: The initial recommendation propranolol usage in managing infantile hemangioma was in 2008 followed by various researches assessing the dosage, efficacy and other parameters. Itraconazole is a world-wide tolerated antifungal but only a few studies have focused on its assessment in the treatment of IH. Objective: This study aim was to investigate the newly proposed antifungal drug ICZ and characterize different aspects of its usage as antiangiogenic drug. Methods: This is an interventional clinical trial to assess the efficacy of ICZ versus propranolol in the treatment of infantile hemangioma with studying the change in serum Ang2. A total of 36 pediatric patients were divided into two equal groups first treated with oral itraconazole, second treated by oral propranolol. Results: Response to treatment was observed using modified IH score. In itraconazole treated infants, good response was observed in 44.4% of the patients. This was slightly higher than the propranolol group which showed 22.2% with good response. We observed a decrease in serum ang2 level after usage of ICZ & propranolol and the change in serum angiopoietin2 level before and after treatment in each group was statistically significant (p < 0.001). Conclusion: Oral itraconazole can be an equivalent option for oral propranolol while safer and shorter treatment periods.

We present an interesting and unusual case of a 57-year-old woman presenting with symptoms concerning for a bowel obstruction, and diagnostic imaging concerning for an internal hernia. The patient underwent an emergency laparotomy and was found to have chylous ascites throughout the abdomen, and the extravasation of chyle into the mesentery giving an appearance of a white mesentery. During this case, we will present all of the findings and discuss the unusual underlying pathology.

BACKGROUND: Lymphatic malformations remain a challenge for patients and plastic surgeons. Promising results have been reported using intralesional laser photocoagulation (ILP) for treating vascular anomalies. BACKGROUND DATA: The objective of our study is to review the long-term results of a large series of lymphatic malformations in patients treated with ILP. MATERIALS AND METHODS: A retrospective review of 190 head and neck lymphatic malformations in patients were treated by ILP with an neodymium-yttrium-aluminum-garnet (Nd:YAG) laser (1064 nm) over a period of 20 years (January 1997-January 2016). Patients' ages ranged from 10 months to 12 years and 6 months (mean, 1 year and 11 months). The patient group consisted of 98 female and 92 male patients. RESULTS: Patients were treated with an Nd:YAG laser (Sharplan, Inc., Allendale, New Jersey) delivered through a 600-mum optical fiber. Laser power was set at 7 to 15 W and delivered with a pulse duration of 7 to 15 seconds. All patients demonstrated improvement as judged by clinical assessment of the reduction in lesion size (range, 65%–100%; mean reduction, 85%). One hundred fifty-two (80%) patients had a more than 65% reduction of the volume in lymphatic malformations at 3 months after 1 treatment, and 171 (90%) patients had a more than 85% reduction of the volume after 2 treatments. Excellent results were seen in 19 (10%) patients after 3 treatments. Postoperative complications were related to photocoagulation that was delivered too extensively or superficially, with resultant ulceration, infection, induration, and scarring. CONCLUSIONS: Throughout the course of our long-term study, ILP using an Nd:YAG laser is an effective treatment modality for lymphatic malformations. Complications can be avoided if the potential for harm is kept in mind.

Complex lymphatic anomalies include a variety of disorders with overlapping clinical, histological and imaging features. The often-confusing nomenclature used for lymphatic anomalies limits timely diagnosis and treatment. The updated 2018 classification of the International Society for the Study of Vascular Anomalies divides lymphatic anomalies into several subsets.(1) Newer imaging techniques including intranodal and magnetic resonance lymphangiography have improved our understanding of anatomy and function of the lymphatic system. Advances in medical, interventional, and surgical treatments have opened a realm of new therapeutic options for patients with complex lymphatic disorders.

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.

In recent years, numerous overgrowth syndromes have been found to be caused by pathogenic DNA sequence variants in “epigenes,” genes that encode proteins that function in epigenetic regulation. Epigenetic marks, including DNA methylation (DNAm), histone modifications and chromatin conformation, have emerged as a vital genome-wide regulatory mechanism that modulate the transcriptome temporally and spatially to drive normal developmental and cellular processes. Evidence suggests that epigenetic marks are layered and engage in crosstalk, in that disruptions of any one component of the epigenetic machinery impact the others. This interdependence of epigenetic marks underpins the recent identification of gene-specific DNAm signatures for a variety of disorders caused by pathogenic variants in epigenes. Here, we discuss the power of DNAm signatures with respect to furthering our understanding of disease pathophysiology, enhancing the efficacy of molecular diagnostics and identifying new targets for therapeutics of overgrowth syndromes. These findings highlight the promise of the field of epigenomics to provide unprecedented insights into disease mechanisms generating a host of opportunities to advance precision medicine.

Megalencephaly-capillary malformation syndrome (MCAP) is a brain overgrowth disorder characterized by cortical malformations (specifically polymicrogyria), vascular anomalies, and segmental overgrowth secondary to somatic activating mutations in the PI3K-AKT-MTOR pathway (PIK3CA). Cases of growth failure and hypoglycemia have been reported in patients with MCAP, raising the suspicion for unappreciated growth hormone (GH) deficiency. Here we report an observational multicenter study of children with MCAP and GH deficiency. Eleven participants were confirmed to have GH deficiency, all with very low or undetectable circulating concentrations of insulin-like growth factor-1 and insulin-like growth factor binding protein-3. Seven underwent GH stimulation testing and all had insufficient responses with a median GH peak of 3.7 ng/ml (range 1.1–8.6). Growth patterns revealed a drastic decline in length z-scores within the first year of life but then stabilized afterward. Five were treated with GH; one discontinued due to inconsolability. The other four participants continued on GH with improvement in linear growth velocity. Other endocrinopathies were identified in 7 of the 11 participants in this cohort. This study indicates that GH deficiency is associated with MCAP and that children with MCAP and hypoglycemia and/or postnatal growth failure should be evaluated for GH deficiency and other endocrinopathies.

BACKGROUND: Infantile hemangioma (IH) is one of the most common tumors in infants. Its pathogenesis is complex and poorly understood. The risk factors of IH have been extensively studied from clinical and epidemiological perspectives in recent years, but the conclusions in the literature reports are inconsistent. To provide a reference for the prevention of hemangioma, we conducted a meta-analysis of the published studies of potential risk factors for IH. METHODS: The Cochrane Library, Ovid, PubMed, and Web of Science databases were searched systematically. Log odds ratios (log ORs), logistic regression standard errors and 95% confidence intervals (CIs) were used to compare the correlation between IH and potential risk factors. Review Manager 5.3.3 was used for the statistical analysis. RESULTS: Six studies were included and 17 potential risk factors were eventually evaluated. P values <0.05 were found for female gender (P < 0.01, OR 2.04, 95% CI 1.65–2.51), low birth weight (P < 0.01, OR 4.39, 95% CI 3.05–6.31), multiple gestation (P = 0.01, OR 2.39, 95% CI 1.21–4.71), preterm birth (P = 0.03, OR 2.37, 95% CI 1.07–5.23), progesterone therapy (P < 0.01, OR 2.73, 95% CI 2.12–3.51), and family history (P = 0.01, OR 1.98, 95% CI 1.16–3.38). CONCLUSIONS: This meta-analysis revealed that risk factors, including female gender, low birth weight, multiple gestation, preterm birth, progesterone therapy, and family history may affect the occurrence of IH.

Mutations in genes involved in chromatin remodelling have been implicated in broad phenotypes of congenital abnormalities and neurodevelopment. However, limited genotype-phenotype correlations are available for some of the rarest genetic disorders that affect chromatin regulation. We hereby describe a 12-year-old girl presented at birth with severe hypotonia, developmental delay, a mid-line capillary malformation and distinctive craniofacial features. During the natural history of her disease, the girl developed severe spasticity and drug-resistant seizures, leading to a diagnosis of Bohring-Opitz syndrome (BOS). We performed whole-exome sequencing (WES) and identified a de novo mutation in ASXL1 (c.2033dupG) which results in the introduction of a premature stop codon (p.R678fs*6). ASXL1 encodes a polycomb repressive complex protein implicated in chromatin regulation and de novo mutations are a known cause of BOS. Phenotypes with segmental craniofacial overgrowth associated to midline capillary malformations enlarge the clinical spectrum of BOS at onset and further expand the differential diagnosis in ASXL1 mutation carriers.

Vascular malformations of the extremities represent a wide spectrum of lesions, broadly divided into high-flow and low-flow categories. High-flow lesions include arteriovenous malformations and arteriovenous fistulas, while the more common low-flow lesions consist of venous and lymphatic malformations. The clinical presentation of vascular malformations is variable and can include extremity pain, discoloration, focal mass, or diffuse extremity enlargement. A vascular murmur can also be present and is more typical of high-flow lesions. While vascular malformations can often be diagnosed or strongly suspected by clinical features alone, imaging is often used to confirm the diagnosis, determine lesion characteristics and extent, and/or plan for treatment. Among the imaging options available, those usually appropriate for initial imaging of suspected vascular malformation are MR angiography without and with intravenous contrast, MRI without and with intravenous contrast, CT angiography with intravenous contrast, or US duplex Doppler. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed annually by a multidisciplinary expert panel. The guideline development and revision include an extensive analysis of current medical literature from peer reviewed journals and the application of well-established methodologies (RAND/UCLA Appropriateness Method and Grading of Recommendations Assessment, Development, and Evaluation or GRADE) to rate the appropriateness of imaging and treatment procedures for specific clinical scenarios. In those instances where evidence is lacking or equivocal, expert opinion may supplement the available evidence to recommend imaging or treatment.

Subjects with the rare autosomal dominant disease Hereditary Hemorrhagic Telangiectasia (HHT) may develop medical conditions that require antithrombotic therapy (AT). However, safety of AT is uncertain in these patients and the only data currently available derive from retrospective analyses of registries and/or databases. At the HHT Centre of the ‘Fondazione Policlinico Universitario A. Gemelli IRCCS’ (Rome, Italy), a prospective study is currently ongoing to evaluate the safety of AT in subjects affected by HHT. The study is enrolling subjects with a definite diagnosis of HHT who receive an AT prescription by one of the physicians of the HHT Centre. The primary outcome is the number of hemorrhagic events, distinguished in major, clinically relevant non-major (CRNM), and minor bleedings, according to the criteria of the International Society on Thrombosis and Hemostasis (ISTH). Another primary outcome is worsening of epistaxis upon initiation of AT, assessed using the internationally accepted Epistaxis Severity Score (ESS). Additional outcomes are changes in hemoglobin levels and changes in the need of blood transfusion after initiation of AT. Here, we present the results of an interim analysis, conducted on the 12 HHT subjects that have been enrolled so far. After a mean follow-up of 6.5 +/− 0.8 months, no major bleedings, no CRNM bleedings, and no minor bleedings different from epistaxis were recorded. Worsening of epistaxis upon initiation of AT was documented only in one patient, but did not require discontinuation of AT. There were no significant changes in the mean ESS measured before and after initiation of AT. There were no significant changes in hemoglobin levels and need for blood transfusion after initiation of AT. Although preliminary, these are the first prospective data on the safety of AT in HHT patients. Our interim analysis suggests that, when prescribed by experienced physicians in a multidisciplinary setting, AT is well tolerated by HHT patients. More patients and a longer follow-up are needed to confirm these findings.

Background: Large involuted infantile hemangioma (IH) can cause facial disfigurement and functional impairment and constitute a major concern to children and parents. Liposuction for large involuted IH has been reported in some case reports. However, systematic study is seldom and the patient outcome is uneven.Methods: Thirteen eligible patients who underwent liposuction treatment at Shanghai Ninth People's Hospital between August 2016 and August 2018 were reviewed. IH lesions were evaluated according to their timing, components, involvement, and location. Liposuction was performed to address the volumetric residue. Results: Between August 2016 and August 2018, 13 patients, four boys and nine girls, with an average age of 8 +/− 4.4 years underwent the liposuction for large facial involuted IH. Clinical follow-up ranges from 3 to 18 months. In two cases, lipectomy and facelift were undertaken in conjunction with liposuction. One patient needs a secondary revision due to the residual facial asymmetry. All patients were satisfied with the outcomes. Conclusions: Liposuction for large involuted IH is reliable and underreported in the literature. With proper patient evaluation and good counseling on realistic expectations, liposuction for large facial involuted IH can be expected to achieve excellent results with a low complication rate in pediatric population.

Brain arteriovenous malformations (AVMs) are a significant cause of intracerebral hemorrhage in children and young adults. Currently, one third of patients have no viable treatment options. Vascular targeting agents (VTAs) are being designed to deliver pro-thrombotic molecules to the abnormal AVM vessels for rapid occlusion and cure. This study assessed the efficacy of a pro-thrombotic VTA targeting phosphatidylserine (PS) in a radiation-primed AVM animal model. The model AVM was surgically created in rats by anastomosis of the left external jugular vein to the adjacent common carotid artery. After 6 weeks, the AVM was irradiated (20 Gy) using gamma knife surgery (GKS). A PS-targeting VTA was created by conjugation of annexin V with human thrombin and administered intravenously 3 weeks post-GKS or sham. Unconjugated thrombin was used as a non-targeting control. AVM thrombosis and occlusion was monitored 3 weeks later by angiography and histology. Preliminary experiments established a safe dose of active thrombin for systemic administration. Subsequently, a single dose of annexin V-thrombin conjugate (0.77 mg/kg) resulted in angiographic AVM occlusion in sham (75%) and irradiated (63%) animals, while non-targeted thrombin did not. Lowering the conjugate dose (0.38 mg/kg) decreased angiographic AVM occlusion in sham (13%) relative to irradiated (80%) animals (p = 0.03) as did delivery of two consecutive doses of 0.38 mg/kg, 2 days apart (sham (0%); irradiated (78%); p = 0.003). These findings demonstrate efficacy of the PS-targeting VTA and the feasibility of a vascular targeting approach for occlusion of high-flow AVMs. Targeting specificity can be enhanced by radiation-sensitization and VTA dose modification.

Cerebral cavernous malformations are focal vascular lesions of the brain, occurring sporadically or as an autosomal dominant familial form. The genetic background influences not only the clinical course but also patients' consultation and the indication to treat. We here present the rare case of monozygotic male twins of a polyzygotic triplet birth, carrying a CCM1 mutation, inherited from the mother. Both twins showed an identical site and size of a large frontobasal lesion. The genetic segregation and the clinical course in affected family members are presented and discussed.

Capillary malformation-arteriovenous malformation syndrome (CM-AVM) is an autosomal dominant disorder caused by heterozygous mutations in RASA1 and EPHB4. Capillary stains in CM-AVM are compatible with Schobinger's phase I AVMs. Vascular laser has been classically contraindicated for the treatment of AVMs, as there is a fear of accelerating their progression. In this study, we have treated capillary stains in five CM-AVM patients with pulsed dye laser, with improvement and without worsening or recurrence of the lesions after 1 year of clinical and ultrasound follow-up.

BACKGROUND: Intracranial vascular abnormalities in Sturge-Weber syndrome, including leptomeningeal angiomatosis, anomalous cortical venous structures, and transmedullary developmental venous anomalies, are well recognized. Prominent vascular flow voids on T2-weighted magnetic resonance imaging (MRI) are occasionally identified in patients with Sturge-Weber syndrome, raising concern of arteriovenous malformations, a congenital high-flow vascular malformation with a risk of bleeding. METHODS: We report four patients with prominent flow voids on conventional MRI that suggested high-flow lesions. RESULTS: Diagnostic evaluation was performed with cerebral angiography in one patient and with a combination of magnetic resonance angiography and magnetic resonance venography in three patients. In all four patients, the conventional MRI-identified lesions represented prominent developmental venous anomalies and not arteriovenous malformations. CONCLUSIONS: This series highlights that developmental venous anomalies may appear in individuals with Sturge-Weber syndrome as unusually large and seemingly high-flow lesions on MRI. Noninvasive imaging with magnetic resonance angiography and magnetic resonance venography can be used in the management of such patients for further characterization of these vascular structures.

BACKGROUND: Cutaneous microcystic lymphatic malformations (CMLMs) are rare conditions in children and adults. They present as clusters of vesicles full of lymph and blood to various extents, inducing maceration, esthetic impairment, pain, and impaired quality of life. The treatment is challenging. Sirolimus is an inhibitor of mammalian target of rapamycin (mTOR) involved in angio-lymphangiogenesis. Topical sirolimus has recently been reported as effective in a few reports of patients with CMLMs. The objective is to compare the efficacy and safety of a 12-week application of 0.1% topical sirolimus versus topical vehicle in CMLMs in children and adults. METHODS: This French blinded multicenter within-person randomized controlled phase 2 trial aims to include 55 patients aged >/ = 6 years who have a primary CMLM. The CMLM will be divided into two equal areas that will be randomly allocated to 0.1% topical sirolimus or topical vehicle applied for 12 weeks. At the end of the 12-week period, the patient/parent will treat the whole area of CMLM with 0.1% topical sirolimus on remaining lesions, for eight more weeks. Patients will be seen at week 20 (treatment will be stopped) and at month 12 to evaluate long-term efficacy. The primary outcome will be improvement of the CMLM in the area treated with topical sirolimus compared to the area treated with topical vehicle by the investigator physician (blinded to the treatment) with the Physician Global Assessment score at week 12. Secondary outcomes will include: assessment of efficacy by independent experts on the basis of standardized photographs; impact on quality of life; efficacy for oozing, bleeding, erythema, and thickness evaluated by the investigators; and global efficacy as well as efficacy for functional and aesthetic impairment evaluated by the patient. Systemic passage of sirolimus will be measured at weeks 6, 12, and 20, and at week 16 for CMLMs >/ = 900 cm(2). DISCUSSION: For patients with CMLMs, topical sirolimus could be a non-invasive and well-tolerated therapeutic option. If the trial demonstrates efficacy and safety of this treatment, this result will lead to a real change in the management of this condition, and 0.1% sirolimus cream would become the first-line treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03972592. Registered on 3 June 2019. EU Clinical Trials Register EudraCT, 2018-001359-11.

AIMS: To evaluate the effectiveness and safety of topical beta-receptor blocker in treating superficial infantile haemangiomas (SIH) and compare the effectiveness and safety of topical beta-receptor blocker against other therapies. METHODS: A search of the literature using PubMed, Embase, Cochrane Review database, China National Knowledge Infrastructure and Wanfang were performed to identify the studies that estimated the effectiveness and safety of topical beta-receptor blocker in treating SIH, the fixed-effect or random-effects meta-analytical techniques were applied to assess the outcomes. RESULTS: Twenty studies, involving 2098 patients, were included to conduct this analysis. Topical propranolol and topical timolol were discovered to be as effective as oral propranolol in treating SIH (propranolol, odds ratio [OR] = 0.486, 95% confidence interval [CI] 0.165, 1.426, P = .189; timolol, OR = 0.955; 95%CI 0.700, 1.302; P = .769), and topical timolol was more effective than topical imiquimod (OR = 2.561; 95%CI 1.182, 5.550; P = .017), observation (OR = 18.458; 95%CI 5.660, 60.191; P < .001) and topical saline solutions (OR = 19.193; 95%CI 8.837, 41.683; P < .001) in treating SIH. The comparison between topical propranolol and oral propranolol led to no discovery of significant difference in the incidence of adverse effects (OR = 1.258; 95%CI 0.471, 3.358; P = .647). Compared with oral propranolol, topical timolol was associated with fewer incidences of adverse effects (OR = 0.191; 95%CI 0.043, 0.858; P = .031). No significant difference in the incidence of adverse effects was found when topical timolol and topical imiquimod were compared (OR = 0.077; 95%CI 0.005, 1.206; P = .068). CONCLUSION: This meta-analysis provided evidence that topical beta-receptor blockers (propranolol and timolol), especially timolol, may replace oral propranolol as a first-line treatment for SIH.

The purpose of this study was to develop a new solid paediatric formulation for propranolol hydrochloride (PR). This drug is used to treat various paediatric diseases, and recently received clearance to treat haemangioma. However, PR has a bitter salty taste that does not facilitate high rates of compliance among children, especially in liquid formulations. In addition, the solid formulations are designed for adults and often their dosage is not suitable for children that require a flexible dose based on their weight. Therefore, matrix microbeads of EUDRAGIT(R) E PO containing PR were manufactured to overcome these limitations. Nine different samples were prepared using the prilling-congealing technique with high yield. Using 2 nozzles, 300 and 450 mum (code n), the diameters obtained of microbeads (from 333 to 699 mum) were homogenous and appropriate to be swallowed by children. In this study, the ratio drug:matrix for the microbeads was also examined in detail: 1:25 (F1), 1:15 (F2) and 1:10 (F3) in aqueous and tert-butyl alcohol/aqueous (code t) media. Most of the examined microbeads were characterized by high percentage of encapsulation efficiency (22–100%) and drug loading (22–77 mg of drug per g of matrix) effective for the administration of low and high doses of PR. SEM analysis revealed a matrix with a radial or a spongy structure, with numerous pores that generated soft floating microbeads in aqueous solution. Release studies confirmed a low release and dissolution of the drug in artificial saliva, mainly F1n > F1 > F2nt, and a prompt dissolution in simulated gastric media. Finally, electronic tongue measurements revealed the ability of these formulations to mask the bitter drug taste, especially for the sample with a ratio 1:25 (F1n and F1). These samples were chemically and physically stable for six months. In conclusion, the projected microbeads F1, and F1n reached the goal of the study, and could be proposed as new solid oral formulations dedicated to use by children.

Specificity in signal transduction is determined by the ability of cells to “encode” and subsequently “decode” different environmental signals. Akin to computer software, this “signaling code” governs context-dependent execution of cellular programs through modulation of signaling dynamics and can be corrupted by disease-causing mutations. Class IA phosphoinositide 3-kinase (PI3K) signaling is critical for normal growth and development and is dysregulated in human disorders such as benign overgrowth syndromes, cancer, primary immune deficiency, and metabolic syndrome. Despite decades of PI3K research, understanding of context-dependent regulation of the PI3K pathway and of the underlying signaling code remains rudimentary. Here, we review current knowledge on context-specific PI3K signaling and how technological advances now make it possible to move from a qualitative to quantitative understanding of this pathway. Insight into how cellular PI3K signaling is encoded or decoded may open new avenues for rational pharmacological targeting of PI3K-associated diseases. The principles of PI3K context-dependent signal encoding and decoding described here are likely applicable to most, if not all, major cell signaling pathways.

Nadolol is a beta-adrenergic antagonist that has been shown to be efficacious in the treatment of infantile hemangioma. It has been suggested that this drug may have fewer side effects compared with the gold standard therapy, propranolol, because it does not exhibit membrane-stabilizing effects and has little ability to cross the blood-brain barrier. However, the pharmacokinetics and safety of nadolol in infants are not well understood, potentially making this therapy dangerous. beta-adrenergic antagonist toxicity causes bradycardia, hypotension, hypoglycemia, and even death. We report a case of a 10-week-old girl who was started on nadolol for infantile hemangioma, died 7 weeks later, and was found to have an elevated postmortem cardiac blood nadolol level of 0.94 mg/L. The infant had no bowel movements for 10 days before her death, which we hypothesize contributed to nadolol toxicity. Pharmacokinetics studies show a large fraction of oral nadolol either remains in the feces unchanged or is excreted into feces via the biliary system, allowing continued absorption over time in infants who stool infrequently. Propranolol may be a safer therapy overall. Not only does it have a shorter half-life, but propranolol is hepatically metabolized and renally eliminated, allowing for less drug accumulation in healthy infants with variable stooling patterns. We suggest that if nadolol is selected for therapy, pediatricians should instruct parents to monitor their infants' bowel movements closely and encourage early intervention in the event of decreased stooling. This intervention may greatly improve the safety of nadolol in this vulnerable patient population.

OBJECTIVES: Proteus syndrome (PS) is an extremely rare disorder with asymmetric and disproportionate bone overgrowth. Craniofacial abnormalities in PS are less frequent than skeletal abnormalities. Although there are recognized oral and maxillofacial manifestations of PS, few case reports describing these manifestations are available. Thus, the objective of this systematic review and case report is to describe oral and maxillofacial manifestations of PS and to report a PS case. METHODS: A 31-year-old male presented with restricted mouth opening and pain during mastication. A panoramic radiograph and an occlusal radiograph were obtained. Reports with relevant keywords were assessed. Data were summarized and demonstrated using a critical appraisal checklist for case reports. RESULTS: The panoramic radiograph demonstrated unilateral overgrowth of the mandible, impacted teeth, and deciduous prolonged retention. Thirteen PS case reports were identified. CONCLUSIONS: Proteus syndrome oral and maxillofacial manifestations may include dental agenesis, impacted teeth, malocclusion, asymmetric dental growth and maturation, frontal line displacement, asymmetric tongue enlargement, mandibular hemihypertrophy and asymmetry, presence of exostoses/hyperostosis, degenerative changes in the temporomandibular joint, alterations of maxillary and mandibular vertical and/or horizontal growth, and enlargement of mandibular canal and foramen. The PROSPERO systematic review registration number is CRD42019140942.

Importance: Oral propranolol is widely considered to be first-line therapy for complicated infantile hemangioma, but its use in patients with PHACE (posterior fossa malformations, hemangioma, arterial anomalies, cardiac defects, eye anomalies) syndrome has been debated owing to concerns that the cardiovascular effects of the drug may increase the risk for arterial ischemic stroke. Objective: To assess the incidence of adverse events among patients with PHACE syndrome receiving oral propranolol for infantile hemangioma. Design, Setting, and Participants: This multicenter retrospective cohort study assessed the incidence of adverse events among 76 patients with PHACE syndrome receiving oral propranolol for infantile hemangioma at 11 tertiary care, academic pediatric dermatology practices. Medical records from January 1, 2010, through April 25, 2017, were reviewed. Exposures: Patients received oral propranolol, 0.3 mg/kg/dose or more. Main Outcomes and Measures: The main outcome was the rate and severity of adverse events occurring throughout the course of treatment with oral propranolol, as documented in the medical records. Adverse events were graded from 1 to 5 using a scale derived from the Common Terminology Criteria for Adverse Events and were considered to be serious if they were grade 3 or higher. Results: A total of 76 patients (59 girls and 17 boys; median age at propranolol initiation, 56 days [range, 0–396 days]) met the inclusion criteria. There were no reports of serious adverse events (i.e., stroke, transient ischemic attack, or cardiovascular events) during treatment with oral propranolol. A total of 46 nonserious adverse events were reported among 29 patients (38.2%); the most commonly reported nonserious adverse events were sleep disturbances and minor gastrointestinal tract and respiratory tract symptoms. In a comparison with 726 infants who received oral propranolol for hemangioma but did not meet criteria for PHACE syndrome, there was no significant difference in the rate of serious adverse events experienced during treatment (0 of 76 patients with PHACE syndrome and 3 of 726 patients without PHACE syndrome [0.4%]). Conclusions and Relevance: This study found that oral propranolol was used to treat infantile hemangioma in 76 patients with PHACE syndrome and that no serious adverse events were experienced. These data provide support for the safety of oral propranolol in this patient population.

Capillary malformation-arteriovenous malformation syndrome (CM-AVM) is a rare condition associated with mutations in the genes RASA1 and EPHB4. We present a challenging case of CM-AVM in a 17-month-old boy with permanent diplegia from an undiagnosed arteriovenous malformation underlying a large atypical capillary malformation over the lower thoracic spine. This case demonstrates that clinicians should have a low threshold for neuroimaging in the context of new neurologic symptoms in patients with atypical capillary malformations.

BACKGROUND: Generalized lymphatic anomaly (GLA) and Gorham-Stout disease (GSD) are rare complicated lymphatic malformations that occur in multiple body sites and are associated with significant morbidity and mortality. Treatment options have been limited, and conventional medical therapies have been generally ineffective. Emerging data suggest a role for sirolimus as a treatment option for complex lymphatic anomalies. PROCEDURE: Disease response was evaluated by radiologic imaging, quality of life (QOL), and clinical status assessments in children and young adults with GLA and GSD from a multicenter systematic retrospective review of patients treated with oral sirolimus and the prospective phase 2 clinical trial assessing the efficacy and safety of sirolimus in complicated vascular anomalies (NCT00975819). Sirolimus dosing regimens and toxicities were also assessed. RESULTS: Eighteen children and young adults with GLA (n = 13) or GSD (n = 5) received oral sirolimus. Fifteen patients (83%) had improvement in one or more aspects of their disease (QOL 78%, clinical status 72%, imaging 28%). No patients with bone involvement had progression of bone disease, and the majority had symptom or functional improvement on sirolimus. Improvement of pleural and pericardial effusion(s) occurred in 72% and 50% of affected patients; no effusions worsened on treatment. CONCLUSIONS: Sirolimus appears effective at stabilizing or reducing signs/symptoms of disease in patients with GLA and GSD. Functional impairment and/or QOL improved in the majority of individuals with GLA and GSD with sirolimus treatment.

Hereditary hemorrhagic telangiectasia (HHT), a genetic bleeding disorder leading to systemic arteriovenous malformations (AVMs), is caused by loss-of-function mutations in the ALK1-ENG-Smad1/5/8 pathway. Evidence suggests that HHT pathogenesis strongly relies on overactivated PI3K-Akt-mTOR and VEGFR2 pathways in endothelial cells (ECs). In the BMP9/10-immunoblocked (BMP9/10ib) neonatal mouse model of HHT, we report here that the mTOR inhibitor, sirolimus, and the receptor tyrosine-kinase inhibitor, nintedanib, could synergistically fully block, but also reversed, retinal AVMs to avert retinal bleeding and anemia. Sirolimus plus nintedanib prevented vascular pathology in the oral mucosa, lungs, and liver of the BMP9/10ib mice, as well as significantly reduced gastrointestinal bleeding and anemia in inducible ALK1-deficient adult mice. Mechanistically, in vivo in BMP9/10ib mouse ECs, sirolimus and nintedanib blocked the overactivation of mTOR and VEGFR2, respectively. Furthermore, we found that sirolimus activated ALK2-mediated Smad1/5/8 signaling in primary ECs-including in HHT patient blood outgrowth ECs-and partially rescued Smad1/5/8 activity in vivo in BMP9/10ib mouse ECs. These data demonstrate that the combined correction of endothelial Smad1/5/8, mTOR, and VEGFR2 pathways opposes HHT pathogenesis. Repurposing of sirolimus plus nintedanib might provide therapeutic benefit in HHT patients.

Phenotype-based diagnostic criteria were developed for Proteus syndrome in 1999 and updated in 2006. Subsequently, the causative mosaic gene alteration was discovered, the c.49G>A p.E17K variant in AKT1. As well, a number of overlapping overgrowth disorders attributable to mosaic PIK3CA variants have now been characterized, leading to the designation of PIK3CA-related overgrowth spectrum (PROS). Finally, ongoing work to better characterize Proteus syndrome has led to identification of additional features of that disorder that could be useful in diagnostic criteria. We have taken the opportunity of these discoveries to re-evaluate the Proteus syndrome diagnostic criteria. Here we propose a new set of diagnostic criteria that establishes a weighted, point-based system for the phenotypic attributes and then integrates that with the potential molecular test results to result in one of two designations: AKT1-related Proteus syndrome or AKT1-related overgrowth spectrum. A patient whose only manifestation is an AKT1 c.49G>A-positive tumor would receive neither of these designations. Here we review the rational basis of diagnostic criteria and argue that a unitary diagnostic entity is a distinct gene-phenotype dyad and that this should be the model for all mendelian disorders. The gene-alone or phenotype-alone approach is inadequate to rigorously delineate a unitary diagnostic entity.

Propranolol is a widely used beta blocker that consists of a racemic mixture of R and S stereoisomers. Only the S stereoisomer has significant activity against the beta-adrenergic receptor. A fortuitous clinical observation was made in an infant who received propranolol for cardiac disease, and regression of a hemangioma of infancy was noted. This has led to the widespread use of propranolol for the treatment of large and life-threatening hemangiomas of infancy. Infants receiving propranolol require monitoring to ensure that they do not suffer from side effects related to beta blockade. The exact mechanism of activity of propranolol in hemangioma of infancy is unknown. In this study, we treated hemangioma stem cells with both beta blockade active S- and inactive R-propranolol and looked for genes that were coordinately regulated by this treatment. Among the genes commonly downregulated, Angiopoietin-like 4 (ANGPTL4) was among the most regulated. We confirmed that propranolol isomers downregulated ANGPTL4 in endothelial cells, with greater downregulation of ANGPTL4 using the beta blockade inactive R-propranolol. ANGPTL4 is present in human hemangiomas of infancy. Finally, R-propranolol inhibited the growth of bEnd.3 hemangioma cells in vivo. The implication of this is that hemangioma growth can be blocked without the side effects of beta blockade. Given that humans have been exposed to racemic propranolol for decades and thus to R-propranolol, clinical development of R-propranolol for hemangiomas of infancy and other angiogenic diseases is warranted.

Hemangioma is one of the commonest benign vascular tumors among children. Propranolol is the first-line therapeutic drug for hemangioma. However, the effects and mechanisms of propranolol in hemangioma have not been thoroughly elaborated. In this study, the effects and mechanisms of propranolol were explored using hemangioma-derived endothelial cells (HemECs). The expression of GLUT1 were determined by immunofluorescence staining. qRT-PCR assay was conducted to detect the mRNA expressions of angiopoietin-2 (Ang-2) and Tie-2. Western blot assay was carried out to measure the protein levels of Ang-2, Tie-2, protein kinase-B (Akt) and phospholyrated-Akt (p-Akt). Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay and Western blot of Ki67 protein level. Cell apoptosis was measured by flow cytometry analysis and Western blot of Bax and Bcl-2 levels. We found that propranolol inhibited proliferation and induced apoptosis in human umbilical vein endothelial cells (HUVECs) and HemECs. Moreover, propranolol inhibited the expressions of Ang-2 and Tie-2 in HUVECs and HemECs. Functional analysis revealed that Ang-2 attenuated the effects of propranolol on HemEC proliferation and apoptosis. Mechanistical analysis showed that propranolol inhibited the Akt pathway by regulating Ang-2 expression in HemECs. Futhermore, inhibition of the Akt pathway attenuated the effects of Ang-2 on proliferation and apoptosis in HemECs. In conclusion, propranolol inhibited proliferation and induced apoptosis of HemECs via Akt pathway by down-regulating Ang-2 expression, which contributes to our understanding on the pathogenesis of hemangioma and promotes the development of therapeutic approaches for hemangioma.

Kaposiform hemangioendothelioma (KHE) is a locally aggressive vascular condition of childhood and is clinicopathologically related to tufted angioma (TA), a benign skin lesion. Due to their rarity molecular data are scarce. We investigated 7 KHE and 3 TA by comprehensive mutational analysis and genome-wide methylation profiling and compared the clustering, also with vascular malformations. Lesions were from 7 females and 3 males. The age range was 2 months to 9 years with a median of 10 months. KHEs arose in the soft tissue of the thigh (n = 2), retroperitoneum (n = 1), thoracal/abdominal (n = 1), supraclavicular (n = 1) and neck (n = 1). One patient presented with multiple lesions without further information. Two patients developed a Kasabach-Merritt phenomenon. TAs originated in the skin of the shoulder (n = 2) and nose/forehead (n = 1). Of the 5 KHEs and 2 TAs investigated by DNA sequencing, one TA showed a hot spot mutation in NRAS, and one KHE a mutation in RAD50. Unsupervised hierarchical clustering analysis indicated a common methylation pattern of KHEs and TAs, which separated from the homogeneous methylation pattern of vascular malformations. In conclusion, methylation profiling provides further evidence for KHEs and TAs potentially forming a spectrum of one entity. Using next generation sequencing, heterogeneous mutations were found in a subset of cases (2/7) without the presence of GNA14 mutations, previously reported in KHE and TA.

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors have been shown to have excellent effects in the management of kaposiform hemangioendothelioma (KHE); however, the mechanism of action is unclear. This study identified the expressions of mTOR pathway-related proteins in different vascular tumors to provide insight into the pathogenesis of KHE. METHODS: We retrospectively reviewed the pathologic specimens of 30 patients (KHE, 15; tufted angioma [TA], 5; infantile hemangioma [IH], 5; and lymphatic malformation [LM], 5). The immunohistochemical expression of mTOR-related proteins tuberous sclerosis complex 2 (TSC2), phosphatase and tensin homologue (PTEN), phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4EBP1), phosphorylated mTOR (p-mTOR), and phosphorylated ribosomal protein S6 kinase B1 (p-P70S6K) were analyzed using Image-Pro Plus software. KHE had the following pattern of expression in the spindle vascular endothelial cells: TSC2 (−); PTEN (−); p-4EBP1 (+); p-mTOR (+); and p-P70S6K (+). RESULTS: All 3 patients treated with sirolimus had good responses. The TA results were similar to KHE with no significant differences (p-4EBP1: p = 0.0687; p-mTOR: p = 0.0832). The expressions of TSC2, PTEN, p-4EBP1, p-mTOR, and p-P70S6K were negative or weakly positive in IH with a statistically significant difference compared to KHE (p-4EBP1: p < 0.001; p-mTOR: p < 0.001; p-P70S6K: p < 0.001). LM had no significant differences when compared to KHE. CONCLUSIONS: The absence of TSC2 and PTEN caused abnormal activation of the mTOR signaling pathway and may be involved in the pathogenesis of KHE. The expression of mTOR-related proteins in TA and LM was similar to KHE, unlike IH. The KHE pattern of expression [PTEN (−), TSC2 (−), p-mTOR (+), p-P70S6K (+), and p-4EBP1 (+)] suggested that sirolimus may be a good therapeutic choice.

Backgrounds and Objective: Blue rubber bleb nevus syndrome (BRBN) or Bean syndrome is a rare venous malformation (VM)-associated disorder, which mostly affects the skin and gastrointestinal tract in early childhood. Somatic mutations in TEK have been identified from BRBN patients; however, the etiology of TEK mutation-negative patients of BRBN need further investigation. METHOD: Two unrelated sporadic BRBNs and one sporadic VM were firstly screened for any rare non-silent mutation in TEK by Sanger sequencing and subsequently applied to whole exome sequencing to identify underlying disease causative variants. Overexpression assay and immunoblotting were used to evaluate the functional effect of the candidate disease causative variants. RESULTS: In the VM case, we identified the known causative somatic mutation in the TEK gene c.2740C>T (p.Leu914Phe). In the BRBN patients, we identified two rare germline variants in GLMN gene c.761C>G (p.Pro254Arg) and c.1630G>T(p.Glu544*). The GLMN-P254R-expressing and GLMN-E544X-expressing HUVECs exhibited increased phosphorylation of mTOR-Ser-2448 in comparison with GLMN-WT-expressing HUVECs, in vitro. CONCLUSION: Our results demonstrated that rare germline variants in GLMN might contribute to the pathogenesis of BRBN. Moreover, abnormal mTOR signaling might be the pathogenesis mechanism underlying the dysfunction of GLMN protein.

Objective: No active treatment is required in the majority of cases of infantile hemangioma (IH), while they proliferate and involute without sequelae. However, ulceration, bleeding, or destruction/obstruction of important structures may occur in 10% of cases during the proliferating phase. These lesions lead to a disfigured appearance with redundant skin, fibrofatty residuum, protruding surface, drooping, and scarring. This study focused on prevention and management of disfiguring scars in involuted IH. Approach: A retrospective photography and chart review were performed for patients with IH who visited our hospital (Shinshu University Hospital). Results: The study population consisted of 107 patients with IH. The lesions were located on the head and neck (59.8%), trunk (27.1%), upper limb (7.5%), or lower limb (5.6%). Twenty-four patients (22.4%) underwent surgical excision of the disfigured lesion after involution. The percentage of surgical interventions was highest for lesions in the head and neck area (28.9%) compared with other regions. The fibrofatty tissue and redundant skin after involution of each lesion were partly resected and sutured. All suture lines were finally set on the wrinkle line or the anatomical borderline. Innovation: Although total excision of the lesion was impossible in some cases, a natural surface contour was obtained. The operative scar was not visible in the residual damaged skin after involution. Conclusion: Effective preventive therapies during the proliferating phase are required to avoid tissue damage due to hyperexpansion of the surrounding tissue and surface breakdown to present excellent cosmetic results in patients with IH.

Background: Capillary malformation-arteriovenous malformation (CM-AVM) is an autosomal dominant disorder characterized by CMs, often in association with fast-flow vascular malformations. Alagille syndrome is an autosomal dominant multisystem disorder, usually involving hepatic, cardiac, ophthalmic, skeletal, or renal dysplasia. The combination of CM-AVM and Alagille syndrome in a patient presenting serious vascular malformations in the liver and heart has never been reported. Here, we report the case of a 20-month-old infant presenting these two diseases. Case presentation: The patient manifested port-wine stains, congenital heart disease, cholestasis with abnormal morphology, and vascular anomalies. Color Doppler (B-mode) ultrasonography, and radiological imaging including computed tomography (CT) with enhanced three-dimensional (3D) reconstruction and angiography, revealed a type II Abernethy malformation in the hepatic portal vein. The left hepatic lobe was enlarged showing dilation of the portal vein and the left artery. Whole exome sequencing (WES) identified a paternally inherited RASA1 heterozygous pathogenic variant p.(Ser219Ter) causing CM-AVM and a de novo NOTCH2 heterozygous variant p.(Met2042Thr) associated with Alagille syndrome. Conclusion: This is the first case of combined CM-AVM and Alagille syndrome presenting serious liver and heart abnormalities diagnosed using imaging technology and WES. The patient harbored variants in two genes: RASA1 and NOTCH2, which rarely contribute to aberrant vascular development. This report highlights the value of accurately diagnosing similar diseases and guiding therapy using genetic testing combined with careful clinical examinations.