Association of Lymphatic Abnormalities with Early Complications after Fontan Operation.

BACKGROUND: Increased central venous pressure is inherent in Fontan circulation but not strongly related to Fontan complication. Abnormalities of the lymphatic circulation may play a crucial role in early Fontan complications. METHODS: This was a retrospective, single-center study of patients undergoing Fontan operation from 2008 to 2015. The primary outcome was significant early Fontan complication defined as secondary in-hospital treatment due to peripheral edema, ascites, pleural effusions, protein-losing enteropathy, or plastic bronchitis. All patients received T2-weighted magnetic resonance images to assess abdominal and thoracic lymphatic perfusion pattern 6 months after Fontan completion with respect to localization, distribution, and extension of lymphatic perfusion pattern (type 1-4) and with application of an area score (0-12 points). RESULTS: Nine out of 42 patients developed early Fontan complication. Patients with complication had longer chest tube drainage (mean 28 [interquartile range [IQR]: 13-60] vs. 13 [IQR: 2-22] days, p = 0.01) and more often obstructions in the Fontan circuit 6 months after surgery (56 vs. 15%, p = 0.02). Twelve patients showed little or no abnormalities of lymphatic perfusion (lymphatic perfusion pattern type 1). Most frequently magnetic resonance imaging showed lymphatic congestion in the supraclavicular region (24/42 patients). Paramesenteric lymphatic congestion was observed in eight patients. Patients with early Fontan complications presented with higher lymphatic area score (6 [min-max: 2-10] vs. 2 [min-max: 0-8]), p = 0.001) and greater distribution and extension of thoracic lymphatic congestion (type 3-4: n = 5/9 vs. n = 1/33, p = 0.001). CONCLUSION: Early Fontan complication is related to hemodynamic factors such as circuit obstruction and to the occurrence and extent of lymphatic congestion.

Analysis of mTOR pathway expression in lymphatic malformation and related diseases.

The mammalian target of rapamycin (mTOR) inhibitor sirolimus is an effective treatment for difficult-to-treat lymphatic anomalies. However, little is known about the expression of mTOR pathway components in lymphatic anomalies. Here we investigated the expression pattern of mTOR pathway components and their phosphorylated forms (mTOR, p-mTOR, 4EBP1, p-4EBP1, S6K1 and p-S6K1) in normal lymphatic vessels and lymphatic anomalies using immunohistochemistry. We studied 18 patients of lymphatic anomalies, including lymphatic malformation (LM, n = 14), Kaposiform lymphangiomatosis (KLA, n = 2) and Kaposiform hemangioendothelioma (KHE, n = 2). Normal lymphatic vessels expressed 4EBP1, S6K1 and p-S6K1, but not p-4EBP1, mTOR or p-mTOR. The mTOR was detected in all lymphatic anomalies, whereas its activation form p-mTOR was detected in half cases of KLA and KHE but not in LM. All lymphatic anomalies expressed S6K1 and its activated form p-S6K1. The expression of 4EBP1 was also found in all lymphatic anomalies, but its activation was detected in approximately half of them. The activation of mTOR was seen in tumor (KLA and KHE) but not in malformation (LM), whereas the activation of S6K1 and 4EBP1 was seen in all and half of lymphatic anomalies, respectively.

Indications for tracheostomy in children with head and neck lymphatic malformation: analysis of a nationwide survey in Japan.

PURPOSE: Airway obstruction caused by lymphatic malformation (LM) in the head and neck may require a tracheostomy. We present the results of our analysis of a nationwide survey on the indications for tracheostomy in children with head and neck LM. METHODS: We analyzed data in relation to tracheostomy based on a questionnaire about 518 children with head and neck LM without mediastinal involvement. RESULTS: Tracheostomy was performed for 43 of the 518 children. Most (32/43) of these children were younger than 1 year of age and the tracheostomy was almost always performed for airway obstruction (40/43). The lesion was in contact with the airway in 32 (72%) of these children, but in only 58 (12%) of the 473 children who were managed without tracheostomy. When the maximum circumferential area of contact was compared, only 20 (27%) of 74 patients with maximum contact of less than a half-circle required tracheostomy, whereas 11 of 13 with maximum contact of more than a half-circle required tracheostomy (P = 0.0001). Six patients without airway contact required tracheostomy because of acute swelling caused by hemorrhage, infection, or both. CONCLUSIONS: Children with head and neck LM required tracheostomy to relieve airway obstruction. Tracheostomy should be considered if the lesion is in contact with the airway and surrounds more than a half-circle, and when it causes acute swelling.

Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

TNFΔARE Mice Display Abnormal Lymphatics and Develop Tertiary Lymphoid Organs in the Mesentery.

Chronic inflammatory diseases are associated with a persistent and enhanced response to environmental antigens. As an adaptive response to this exaggerated immune state, affected tissue typically develops tertiary lymphoid organs. Studies of Crohn disease (CD), a chronic inflammatory disease of the intestinal tract, report tertiary lymphoid organs present within the mucosal wall, along with other lymphatic diseases, such as lymphangiogenesis and obstructed lymphatic vessels. These observations suggest that downstream mesenteric lymphatic vessels and lymph drainage into mesenteric lymph nodes may be compromised. However, information is lacking on the morphologic features and functional status of mesenteric lymphatics in CD. Using confocal imaging, PCR, flow cytometry, and functional strategies, we addressed these questions in the established TNFΔARE mouse model of CD and found that this mouse model had many lymphatic abnormalities reminiscent of human CD. These abnormalities include intestinal lymphangiectasia, mesenteric lymph node lymphadenopathy, and lymphangiogenesis in both the mesentery and mucosa. Critically, TNFΔARE mice also present mesenteric tertiary lymphoid organs and have altered lymphatic transport of dendritic cells to mesenteric lymph nodes, two features likely to actively modulate immunity. Our findings provide key insights into lymphatic remodeling in the TNFΔARE mouse model. They shed light on the involvement of these lymphatic changes in immune dysfunctions observed in CD and suggest the lymphatic system as new target for therapeutic options.

Development of an international core outcome set for peripheral vascular malformations: the OVAMA project.

BACKGROUND: An important limitation in vascular malformation research is the heterogeneity in outcome measures used for the evaluation of treatment outcome. OBJECTIVES: To reach international consensus on a core outcome set (COS) for clinical research on peripheral vascular malformations: lymphatic (LM), venous (VM) and arteriovenous malformations (AVM). In this consensus study, we determined what domains should constitute the COS. METHODS: Thirty-six possibly relevant outcome domains were proposed to an international group of physicians, patients and the parents of patients. In a three-round e-Delphi process using online surveys, participants repeatedly rated the importance of these domains on a five-point Likert scale. Participants could also propose other relevant domains. This process was performed for LM, VM and AVM separately. Consensus was predefined as 80% agreement on the importance of a domain among both the physician group and the patient/parent group. Outcomes were then re-evaluated in an online consensus meeting. RESULTS: 167 physicians and 134 patients and parents of patients with LM (n = 50), VM (n = 71) and AVM (n = 29) participated in the study. After three rounds and a consensus meeting, consensus was reached for all three types of vascular malformations on the core domains of radiological assessment, physician-reported location-specific signs, patient-reported severity of symptoms, pain, quality of life, satisfaction and adverse events. Vascular malformation type-specific signs and symptoms were included for LM, VM and AVM, separately. CONCLUSIONS: Our recommendation is that therapeutic-efficacy studies on peripheral vascular malformations should measure at least these core outcome domains.

Angiopoietins as serum biomarkers for lymphatic anomalies.

Vascular anomalies can cause significant morbidity and mortality. Advances in diagnosis will be improved if noninvasive biomarkers can be identified, as obtaining a tissue biopsy can worsen the disease and precipitate complications. The goal of this study was to identify biomarkers for vascular anomaly patients to aid diagnosis and potentially give insights into pathogenesis. Blood was collected at baseline and then 6 and 12 months after treatment with the mTOR inhibitor sirolimus. Patients groups included generalized lymphatic anomaly (GLA), kaposiform lymphangiomatosis (KLA) and kaposiform hemangioendothelioma (KHE) with or without the Kasabach-Merritt phenomenon (KMP) coagulopathy. Serum was obtained from healthy controls selected to match the age and sex of the patients (21 days-28.5 years; 42% males; 58% females). Angiogenic and lymphangiogenic factors (VEGF-A, C, D, Ang-1 and Ang-2) were measured in serum using ELISA. In lymphatic anomaly patients, baseline levels of VEGF-A and VEGF-D were not different compared to controls. Angiopoietin-2 (Ang-2) levels were near controls levels in GLA patients but 10-fold greater in KLA patients and 14-fold greater in KHE patients when the KMP coagulopathy was present but not when it was absent. VEGF-C and angiopoietin-1 (Ang-1) levels were lower in KHE patients with KMP. Our analyses suggest that Ang-2 and Ang-1 can be used as biomarkers to help identify KLA and KHE patients with KMP coagulopathy with high sensitivity and specificity. After 12 months of sirolimus treatment, Ang-2 levels were lower in KLA and KHE with KMP patients compared to baseline levels and with most patients showing a clinical response. Hence, serum Ang-2 and Ang-1 levels may help in the diagnosis of patients with lymphatic anomalies and are concordant to sirolimus response.

Clinical Efficacy of Herbal Medicine for Pediatric Lymphatic Malformations: A Pilot Study.

BACKGROUND: Lymphatic malformations (LMs) are congenital malformations of the lymphatic system that commonly affect the head and neck region and cause marked cosmetic and functional complications. In this pilot study, we present eight children with LMs treated using an herbal medicine for this indication. METHODS: Between January 2009 and May 2014, eight children (four boys, four girls) with LMs were treated using oral administration of an herbal medicine, Eppikajyutsuto (TJ-28; Tsumura, Tokyo, Japan), as monotherapy. RESULTS: Four of the cases were macrocystic and four were mixed micro- and macrocystic. The mean treatment duration was 7.2 ± 2.9 months (range 5-12 mos). The mean LM volume shrinkage on magnetic resonance imaging was 54.5 ± 38.3% (macrocystic 73.6 ± 27.0%; mixed micro- and macrocystic 35.4 ± 41.5%). One of four macrocystic lesions had a marked reduction, two had a moderate reduction, and one had no response. A marked reduction was observed in three of the four mixed micro- and macrocystic cases; the other mixed cystic case had no response. The treatment was well tolerated, without severe adverse events. CONCLUSIONS: This preliminary study demonstrates the beneficial effects of TJ-28. Further evaluations of this therapeutic modality are warranted.

Abdominal Lymphatic Malformation Presenting as Acute Abdominal Pain: A Common Pediatric Complaint, but an Unusual Diagnosis.

We present the clinical and radiological findings involving a mesenteric lymphatic malformation causing volvulus in a toddler presenting with acute abdominal pain, as well as its treatment options.

Essential role of the zinc finger transcription factor Casz1 for mammalian cardiac morphogenesis and development.

Chromosome 1p36 deletion syndrome is one of the most common terminal deletions observed in humans and is related to congenital heart disease (CHD). However, the 1p36 genes that contribute to heart disease have not been clearly delineated. Human CASZ1 gene localizes to 1p36 and encodes a zinc finger transcription factor. Casz1 is required for Xenopus heart ventral midline progenitor cell differentiation. Whether Casz1 plays a role during mammalian heart development is unknown. Our aim is to determine 1p36 gene CASZ1 function at regulating heart development in mammals. We generated a Casz1 knock-out mouse using Casz1-trapped embryonic stem cells. Casz1 deletion in mice resulted in abnormal heart development including hypoplasia of myocardium, ventricular septal defect, and disorganized morphology. Hypoplasia of myocardium was caused by decreased cardiomyocyte proliferation. Comparative genome-wide RNA transcriptome analysis of Casz1 depleted embryonic hearts identifies abnormal expression of genes that are critical for muscular system development and function, such as muscle contraction genes TNNI2, TNNT1, and CKM; contractile fiber gene ACTA1; and cardiac arrhythmia associated ion channel coding genes ABCC9 and CACNA1D. The transcriptional regulation of some of these genes by Casz1 was also found in cellular models. Our results showed that loss of Casz1 during mouse development led to heart defect including cardiac noncompaction and ventricular septal defect, which phenocopies 1p36 deletion syndrome related CHD. This suggests that CASZ1 is a novel 1p36 CHD gene and that the abnormal expression of cardiac morphogenesis and contraction genes induced by loss of Casz1 contributes to the heart defect.

Why increased nuchal translucency is associated with congenital heart disease: a systematic review on genetic mechanisms.

This overview provides insight into the underlying genetic mechanism of the high incidence of cardiac defects in fetuses with increased nuchal translucency (NT). Nuchal edema, the morphological equivalent of increased NT, is likely to result from abnormal lymphatic development and is strongly related to cardiac defects. The underlying genetic pathways are, however, unknown. This study aims to present a systematic overview of genes involved in both cardiac and lymphatic development in mouse embryos. A search of PubMed and the Mammalian Phenotype Browser was performed. Fifteen candidate genes involved in both cardiac and lymphatic development were identified: Adrenomedullin; Chicken ovalbumin upstream promoter-transcription factor 2 (COUP-TFII); Cyp51; Ephrin-B2; Forkhead box protein C2 (Foxc2); Nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1); Neurofibromatosis type 1 (Nf1); Phosphoinositide 3-kinase encoding isoform p110α (Pik3ca); Podoplanin; Prospero-related homeobox 1 (Prox1); T-box 1 (Tbx1); Tyrosine kinase with immunoglobulin-like and endothelial growth factor-like domains 1 (Tie1); vascular endothelial growth factor (Vegf)-A; Vegf receptor-3 (Vegfr-3); and Vascular endothelial zinc finger 1 (Vezf1). Mutations in all but one gene (Pik3ca) resulted in both a cardiac defect and nuchal edema. Candidate genes - mainly encoding for endothelium - are involved in both cardiac and lymphatic development. Alterations in candidate genes are associated with the strong relation between increased NT and cardiac defects.

Lymphoscintigraphy patterns in newborns and children with congenital lymphatic dysplasia.

We performed lymphoscintigraphy on 31 patients (newborns and children) affected by congenital lymphatic dysplasia according to our previously published protocol. Congenital lymphatic dysplasia may present with various degrees of clinical severity, ranging from nonimmune hydrops fetalis with visceral effusions to lymphedema alone. We recommend that lymphoscintigraphy should be strongly considered in all patients with signs of lymphatic dysplasia, including those with minimal and initial signs of lymphatic impairment, in order to obtain a very early diagnosis and to start treatment. Lymphoscintigraphy is safe and useful in the diagnosis of lymphatic dysplasia in the newborn and children. Moreover, it is well tolerated by patients and well accepted by their parents.

[Lymphedema: anatomy, physiology and pathophysiology of lymphedema, definition and classification of lymphedema and lymphatic vascular malformations]. / Das Lymphödem - Anatomie, Physiologie und Pathophysiologie des Lymphödems, Definition und Klassifizierung des Lymphödems und lymphatischer Gefäßmalformationen.

Lymphedema is a chronic disease associated with a congenital or acquired disorder of the lymphatic vessels or lymph nodes. Untreated lymphedema can lead to complications and disability. Clinical Lymphology deals not only with lymphedema of the extremities but also of the head, the genitals and the internal organs (lymphostatic enteropathy, chylaskos, chylothorax, chylopericard etc). Symptoms of this disorder are often misdiagnosed or not recognized. Ignorance and trivialization of lymphedema causes insufficient treatment, which then is not carried out to the extent as it is possible today by scientific findings. Even today delayed or not optimal treatment causes a long ordeal for many patients.The fact that lymphedema for those affected is a major psychological and social burden, which is limiting the quality of life, has also often been unregarded. The knowledge of anatomy, physiology and pathophysiology as well as the knowledge of causes are necessary for diagnosis, so that early treatment can be initiated.

Aberrant lymphatic development in euploid fetuses with increased nuchal translucency including Noonan syndrome.

OBJECTIVE: Increased nuchal translucency in the human fetus is associated with aneuploidy, structural malformations and several syndromes such as Noonan syndrome. In 60­70% of the Noonan syndrome cases, a gene mutation can be demonstrated. Previous research showed that aneuploid fetuses with increased nuchal translucency (NT) demonstrate an aberrant lymphatic endothelial differentiation. METHOD: Fetuses with increased NT and normal karyotype (n = 7) were compared with euploid controls having normal NT (n = 5). A Noonan syndrome gene mutation was found in three out of seven fetuses with increased NT. Endothelial differentiation was evaluated by immunohistochemistry using lymphatic markers (PROX-1, Podoplanin, LYVE-1) and blood vessel markers vascular endothelial growth factor-A (VEGF-A), Neuropilin-1 (NP-1), Sonic hedgehog, von Willebrand factor, and the smooth muscle cell marker, smooth muscle actin. RESULTS: Nuchal edema and enlarged jugular lymphatic sacs (JLSs) were observed in fetuses with increased NT, together with abnormal lymphatic endothelial differentiation i.e. the presence of blood vessel characteristics, including high levels of VEGF-A and NP-1 expression. The enlarged JLSs contained erythrocytes and were surrounded by smooth muscle cells. CONCLUSION: This study shows an aberrant lymphatic endothelial differentiation in fetuses with increased NT and a normal karyotype (including Noonan syndrome fetuses), as was previously reported before in aneuploid fetuses.

NOTCH1: Review of its role in lymphatic development and study of seven families with rare pathogenic variants.

BACKGROUND: We developed a Next-Generation-Sequencing (NGS) protocol to screen the most frequent genetic variants related to lymphedema and a group of candidate genes. The aim of the study was to find the genetic cause of lymphedema in the analyzed patients. METHODS: We sequenced a cohort of 246 Italian patients with lymphatic malformations. In the first step, we analyzed genes known to be linked to lymphedema: 235 out of 246 patients tested negative for the most frequent variants and underwent testing for variants in a group of candidate genes, including the NOTCH1 gene, selected from the database of mouse models. We also performed in silico analysis to observe molecular interactions between the wild-type and the variant amino acids and other protein residues. RESULTS: Seven out of 235 probands, five with sporadic and two with familial lymphedema, were found to carry rare missense variants in the NOTCH1 gene. CONCLUSIONS: Our results propose that NOTCH1 could be a novel candidate for genetic predisposition to lymphedema.

Dysfunction of dermal initial lymphatics of the arm and upper body quadrant causes congenital arm lymphedema.

OBJECTIVE: The objective of this study was to explore the pathologic process underlying primary lymphedema. METHODS: Twenty-seven patients with unilateral congenital arm lymphedema who visited our clinic from January 1, 2014, to May 30, 2019, were enrolled. The patients' clinical signs and the findings of indocyanine green (ICG) lymphography, skin tissue immunohistochemical staining, and whole exome sequencing of tissue and blood were evaluated. RESULTS: Among the 27 patients, 11 were diagnosed with stage II and 16 were diagnosed with stage III lymphedema. No lymphatic vessels were visualized in the affected arm in 25 of 27 (93%) patients who underwent ICG lymphography; likewise, no lymphatics were found in the territories of axillary lymph node drainage in the trunk, irrespective of any anomalies of the axillary lymph nodes. In only two (7%) patients, an unclear lymphatic trunk gradually appeared in the dorsum of the affected hand. The number of initial lymphatics was increased in the skin specimens of all nine patients in whom lymphatics were not demonstrated by ICG lymphography. Among 14 tested patients, we found compound heterozygote variants in the PIEZO1 gene in only one (7%) patient. Two missense variants, c.4072C>T; p.Arg1358Cys and c.5033C>T; p. Ala1678Val, were identified and found to have been inherited from the father and mother, respectively. No other pathogenic or likely pathogenic variants of currently known lymphedema-related genes were identified in the remaining 13 patients. No genetic difference was found between the lymphedematous and nonedematous healthy skin tissue of the same person. CONCLUSIONS: Segmental or regional dysfunction of the dermal initial lymphatics causes congenital arm lymphedema and may have implications for clinical treatment.

Clinical Factors in Trunk Capillary Malformations in the Neonate: When to Suspect Other Associated Malformations? A Case-Control Study.

INTRODUCTION: Capillary malformations (CMs) can be sporadic or syndromic, in association with other underlying venous malformation (VM) or lymphatic malformation (LM). The objective of this study is to describe the clinical patterns in the neonate that allow us to differentiate sporadic CMs from those associated with other vascular malformations. MATERIALS AND METHODS: A case-control study was performed in neonates with CM located in the trunk, followed at our institution between 2008 and 2018. The patients were divided into two groups: group A (cases: CM associated with VM or LM) and group B (controls: sporadic CM without associated malformations). Demographic and clinical variables collected in the clinical history were evaluated (color, location, multifocality, bilaterality, position regarding the vascular axis, and involvement of the midline). RESULTS: Thirty-eight patients were included (18 cases and 20 controls) without differences in gender and age. In group A, the totality of patients presented CM with uniform color and lateral location (p < 0.001). In this group, bilateral and multifocal involvements were lower than in group B, without significant differences between both groups. The distribution of CMs in group A was always parallel to the vascular axis and the midline was always respected, without observing these characteristics in the group B (p < 0.001). CONCLUSION: The presence of a CM in the trunk of a neonate with uniform color, lateral location, parallel position to the vascular axis, and absence of involvement of the midline should make us suspect other underlying vascular malformations, which should be studied with complementary tests.