The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development.

Lymphedema is a debilitating disease with no effective cure and affects an estimated 250 million individuals worldwide. Prior studies have identified mutations in piezo-type mechanosensitive ion channel component 1 (PIEZO1), angiopoietin 2 (ANGPT2), and tyrosine kinase with Ig-like and EGF-like domains 1 (TIE1) in patients with primary lymphedema. Here, we identified crosstalk between these molecules and showed that activation of the mechanosensory channel PIEZO1 in lymphatic endothelial cells (LECs) caused rapid exocytosis of the TIE ligand ANGPT2, ectodomain shedding of TIE1 by disintegrin and metalloproteinase domain-containing protein 17 (ADAM17), and increased TIE/PI3K/AKT signaling, followed by nuclear export of the transcription factor FOXO1. These data establish a functional network between lymphedema-associated genes and provide what we believe to be the first molecular mechanism bridging channel function with vascular signaling and intracellular events culminating in transcriptional regulation of genes expressed in LECs. Our study provides insights into the regulation of lymphatic function and molecular pathways involved in human disease.

Identification Of A Higher Risk Lymphedema Phenotype And Associations With Cytokine Gene Polymorphisms.

CONTEXT: Breast cancer-related lymphedema (BCRL) is chronic condition that occurs in 5% to 75% of women following treatment for breast cancer. However, little is known about the risk factors and mechanisms associated with a worse BCRL profile. OBJECTIVES: Identify distinct BCRL profiles in women with the condition (i.e., lower vs. higher risk phenotype) and evaluate for associations with pro- and anti-inflammatory genes. METHODS: Latent class profile analysis (LCPA) was used to identify the BCRL profiles using phenotypic characteristics evaluated prior to surgery. Candidate gene analyses were done to identify cytokine genes associated with the two BCRL profiles. RESULTS: Of the 155 patients evaluated, 35.5% (n = 55) were in the Lower and 64.5% (n = 100) were in the Higher Risk classes. Risk factors for membership in the Higher class included: lower functional status, having sentinel lymph node biopsy, axillary lymph node dissection, mastectomy, higher number of positive lymph nodes, and receipt of chemotherapy. Polymorphisms for interleukin (IL)1-beta and IL6 were associated with membership in the Higher Risk class. CONCLUSION: The readily available and clinically relevant phenotypic characteristics associated with a worse BCRL profile can be used by clinicians to identify higher risk patients. If confirmed, these characteristics can be tested in predictive risk models. In addition, the candidate gene findings may guide the development of mechanistically-based interventions to decrease the risk of BCRL.

Perturbed collagen metabolism underlies lymphatic recanalization failure in Gata2 heterozygous deficient mice.

Lymphedema has become a global health issue following the growing number of cancer surgeries. Curative or supportive therapeutics have long been awaited for this refractory condition. Transcription factor GATA2 is crucial in lymphatic development and maintenance, as GATA2 haploinsufficient disease often manifests as lymphedema. We recently demonstrated that Gata2 heterozygous deficient mice displayed delayed lymphatic recanalization upon lymph node resection. However, whether GATA2 contributes to lymphatic regeneration by functioning in the damaged lymph vessels' microenvironment remains explored. In this study, our integrated analysis demonstrated that dermal collagen fibers were more densely accumulated in the Gata2 heterozygous deficient mice. The collagen metabolism-related transcriptome was perturbed, and collagen matrix contractile activity was aberrantly increased in Gata2 heterozygous embryonic fibroblasts. Notably, soluble collagen placement ameliorated delayed lymphatic recanalization, presumably by modulating the stiffness of the extracellular matrix around the resection site of Gata2 heterozygous deficient mice. Our results provide valuable insights into mechanisms underlying GATA2-haploinsufficiency-mediated lymphedema and shed light on potential therapeutic avenues for this intractable disease.

Apelin-VEGF-C mRNA delivery as therapeutic for the treatment of secondary lymphedema.

Secondary lymphedema (LD) corresponds to a severe lymphatic dysfunction leading to the accumulation of fluid and fibrotic adipose tissue in a limb. Here, we identified apelin (APLN) as a powerful molecule for regenerating lymphatic function in LD. We identified the loss of APLN expression in the lymphedematous arm compared to the normal arm in patients. The role of APLN in LD was confirmed in APLN knockout mice, in which LD is increased and associated with fibrosis and dermal backflow. This was reversed by intradermal injection of APLN-lentivectors. Mechanistically, APLN stimulates lymphatic endothelial cell gene expression and induces the binding of E2F8 transcription factor to the promoter of CCBE1 that controls VEGF-C processing. In addition, APLN induces Akt and eNOS pathways to stimulate lymphatic collector pumping. Our results show that APLN represents a novel partner for VEGF-C to restore lymphatic function in both initial and collecting vessels. As LD appears after cancer treatment, we validated the APLN-VEGF-C combination using a novel class of nonintegrative RNA delivery LentiFlash® vector that will be evaluated for phase I/IIa clinical trial.

Thromboxane prostanoid signaling in macrophages attenuates lymphedema and facilitates lymphangiogenesis in mice : TP signaling and lymphangiogenesis.

BACKGROUND: Accumulating evidence suggests that prostaglandin E2, an arachidonic acid (AA) metabolite, enhances lymphangiogenesis in response to inflammation. However, thromboxane A2 (TXA2), another AA metabolite, is not well known. Thus, this study aimed to determine the role of thromboxane prostanoid (TP) signaling in lymphangiogenesis in secondary lymphedema. METHODS AND RESULTS: Lymphedema was induced by the ablation of lymphatic vessels in mouse tails. Compared with wild-type mice, tail lymphedema in Tp-deficient mice was enhanced, which was associated with suppressed lymphangiogenesis as indicated by decreased lymphatic vessel area and pro-lymphangiogenesis-stimulating factors. Numerous macrophages were found in the tail tissues of Tp-deficient mice. Furthermore, the deletion of TP in macrophages increased tail edema and decreased lymphangiogenesis and pro-lymphangiogenic cytokines, which was accompanied by increased numbers of macrophages and gene expression related to a pro-inflammatory macrophage phenotype in tail tissues. In vivo microscopic studies revealed fluorescent dye leakage in the lymphatic vessels in the wounded tissues. CONCLUSIONS: The results suggest that TP signaling in macrophages promotes lymphangiogenesis and prevents tail lymphedema. TP signaling may be a therapeutic target for improving lymphedema-related symptoms by enhancing lymphangiogenesis.

Recent Advances in Therapeutic Modalities Against Breast Cancer-Related Lymphedema: Future Epigenetic Landscape.

Background: Lymphedema is a significant postsurgical complication observed in the majority of breast cancer patients. These multifactorial etiopathogenesis have a significant role in the development of novel diagnostic/prognostic biomarkers and the development of novel therapies. This review aims to ascertain the epigenetic alterations that lead to breast cancer-related lymphedema (BCRL), multiple pathobiological events, and the underlying genetic predisposing factors, signaling cascades pertinent to the lapses in effective prognosis/diagnosis, and finally to develop a suitable therapeutic regimen. Methods and Results: We have performed a literature search in public databases such as PubMed, Medline, Google Scholar, National Library of Medicine and screened several published reports. Search words such as epigenetics to induce BCRL, prognosis/diagnosis, primary lymphedema, secondary lymphedema, genetic predisposing factors for BRCL, conventional therapies, and surgery were used in these databases. This review described several epigenetic-based predisposing factors and the pathophysiological consequences of BCRL, which affect the overall quality of life, and the interplay of these events could foster the progression of lymphedema in breast cancer survivors. Prognosis/diagnostic and therapy lapses for treating BCRL are highly challenging due to genetic and anatomical variations, alteration in the lymphatic vessel contractions, and variable expression of several factors such as vascular endothelial growth factor (VEGF)-E and vascular endothelial growth factor receptor (VEGFR) in breast cancer survivors. Conclusion: We compared the efficacy of various conventional therapies for treating BCRL as a multidisciplinary approach. Further substantial research is required to decipher underlying signaling epigenetic pathways to develop chromatin-modifying therapies pertinent to the multiple etiopathogenesis to explore the correlation between the disease pathophysiology and novel therapeutic modalities to treat BCRL.

Aagenaes syndrome/lymphedema cholestasis syndrome 1 is caused by a founder variant in the 5'-untranslated region of UNC45A.

BACKGROUND & AIMS: Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. METHODS: A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. RESULTS: One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). CONCLUSIONS: c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. IMPACT AND IMPLICATIONS: The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

In-silico assessment of high-risk non-synonymous SNPs in ADAMTS3 gene associated with Hennekam syndrome and their impact on protein stability and function.

Hennekam Lymphangiectasia-Lymphedema Syndrome 3 (HKLLS3) is a rare genetical disorder caused by mutations in a few genes including ADAMTS3. It is characterized by lymphatic dysplasia, intestinal lymphangiectasia, severe lymphedema and distinctive facial appearance. Up till now, no extensive studies have been conducted to elucidate the mechanism of the disease caused by various mutations. As a preliminary investigation of HKLLS3, we sorted out the most deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs) that might affect the structure and function of ADAMTS3 protein by using a variety of in silico tools. A total of 919 nsSNPs in the ADAMTS3 gene were identified. 50 nsSNPs were predicted to be deleterious by multiple computational tools. 5 nsSNPs (G298R, C567Y, A370T, C567R and G374S) were found to be the most dangerous and can be associated with the disease as predicted by different bioinformatics tools. Modelling of the protein shows it can be divided into segments 1, 2 and 3, which are connected by short loops. Segment 3 mainly consists of loops without substantial secondary structures. With prediction tools and molecular dynamics simulation, some SNPs were found to significantly destabilize the protein structure and disrupt the secondary structures, especially in segment 2. The deleterious effects of mutations in segment 1 are possibly not from destabilization but from other factors such as the change in phosphorylation as suggested by post-translational modification (PTM) studies. This is the first-ever study of ADAMTS3 gene polymorphism, and the predicted nsSNPs in ADAMST3, some of which have not been reported yet in patients, will serve for diagnostic purposes and further therapeutic implications in Hennekam syndrome, contributing to better diagnosis and treatment.

Possible biallelic inheritance in TIE1 in a family with congenital lymphedema, intestinal lymphangiectasia and cutis aplasia.

A short report of two male siblings born with cutis aplasia, lymphedema and intestinal lymphangiectasia, one found to carry bi-allelic variants in the TIE1 gene known to be associated with congenital lymphedema.

Prevalence of lymphedema among Anderson-Fabry disease patients: A report from the Fabry registry.

BACKGROUND: Anderson-Fabry disease (AFD) is a rare X-linked lysosomal storage disease due to a genetic variation in the α-galactosidase A (GLA) gene. As a result, the activity of the α-galactosidase A (AGAL-A) enzyme is reduced or absent, which causes sphingolipid deposition within different body parts. AFD typically manifests with cardiovascular, renal, cerebrovascular, and dermatologic involvement. Lymphedema is caused by sphingolipid deposition within lymphatics. Lymphedema can cause intolerable pain and limit daily activities. Very limited data exist on lymphedema in AFD patients. METHODS: Using data from the Fabry Registry (NCT00196742) with 7671 patients included (44% males and 56% females), we analyzed the prevalence of lymphedema among AFD patients who were ever assessed for lymphedema and studied the age of first reported lymphedema. Additionally, we assessed whether patients received AFD-specific treatment at some point during their clinical course. The data was stratified by gender and phenotype. RESULTS: Our study showed that lymphedema occurred in 16.5% of the Fabry Registry patients who were ever assessed for lymphedema (n = 5487). Male patients when compared to female patient have higher prevalence (21.7% vs 12.7%) and experienced lymphedema at a younger age (median age at first reported lymphedema of 43.7 vs 51.7 years). When compared to other phenotypes, classic phenotype has the highest prevalence of lymphedema with the earliest reported lymphedema. Among those who reported lymphedema, 84.5% received AFD-specific treatment during their clinical course. CONCLUSIONS: Lymphedema is a common manifestation of AFD in both genders, with a tendency to present later in female patients. Recognition of lymphedema can offer an important opportunity for intervention and potential impact on associated morbidity. Additional future studies are needed to characterize the clinical implications of lymphedema in AFD patients and identify additional treatment options for this growing population.

Two novel variations p.(Ser1275Thr) and p.(Ser1275Arg) in FLT4 causing prenatal hereditary lymphedema type 1.

BACKGROUND: Hereditary lymphedema 1 is a rare congenital condition, characterized by the development of chronic swelling in body parts. It is highly variable in expression and age of onset with different presentations: from feet edema to hydrops fetalis. This affection is genetically heterogeneous with autosomal dominant inheritance and incomplete penetrance due to a mutation in the FLT4 gene in most cases. CASES: In our study, we report on two fetuses harboring congenital lymphedema with FLT4 variation and review the prenatal confirmed ones of the literatures. Our cases were selected within fetuses explored by exome sequencing in a diagnosis setting. Prenatal ultrasonography showed hydrops fetalis in one case and an increased nuchal translucency with hydrothorax in the other. Comparative genomic hybridization array on amniocentesis was normal in both cases. Exome sequencing identified a variation p.(Ser1275Thr) and p.(Ser1275Arg) in fetus 1 and fetus 2 in the FLT4 gene, respectively. A de novo mutation at the same codon was reported in prenatal literature suggesting possible genotype phenotype correlation. CONCLUSION: Cystic hygroma/hydrops fetalis are possible manifestations of several disorders. This study illustrates how the integration of exome sequencing in prenatal clinical practice can facilitate the diagnosis and genetic counseling of heterogeneous developmental affections.

Redefining WILD syndrome: a primary lymphatic dysplasia with congenital multisegmental lymphoedema, cutaneous lymphovascular malformation, CD4 lymphopaenia and warts.

BACKGROUND: Primary lymphoedema (PL) syndromes are increasingly recognised as presentations of complex genetic disease, with at least 20 identified causative genes. Recognition of clinical patterns is key to diagnosis, research and therapeutics. The defining criteria for one such clinical syndrome, 'WILD syndrome' (Warts, Immunodeficiency, Lymphoedema and anogenital Dysplasia), have previously depended on a single case report. METHODS AND RESULTS: We present 21 patients (including the first described case) with similar clinical and immunological phenotypes. All had PL affecting multiple segments, with systemic involvement (intestinal lymphangiectasia/pleural or pericardial effusions) in 70% (n=14/20). Most (n=20, 95%) had a distinctive cutaneous lymphovascular malformation on the upper anterior chest wall. Some (n=10, 48%) also had hyperpigmented lesions resembling epidermal naevi (but probably lymphatic in origin). Warts were common (n=17, 81%) and often refractory. In contrast to the previous case report, anogenital dysplasia was uncommon-only found in two further cases (total n=3, 14%). Low CD4 counts and CD4:CD8 ratios typified the syndrome (17 of 19, 89%), but monocyte counts were universally normal, unlike GATA2 deficiency. CONCLUSION: WILD syndrome is a previously unrecognised, underdiagnosed generalised PL syndrome. Based on this case series, we redefine WILD as 'Warts, Immunodeficiency, andLymphatic Dysplasia' and suggest specific diagnostic criteria. The essential criterion is congenital multisegmental PL in a 'mosaic' distribution. The major diagnostic features are recurrent warts, cutaneous lymphovascular malformations, systemic involvement (lymphatic dysplasia), genital swelling and CD4 lymphopaenia with normal monocyte counts. The absence of family history suggests a sporadic condition, and the random distribution of swelling implicates mosaic postzygotic mutation as the cause.

Lack of embryonic homozygous or adult heterozygous lymphatic phenotypes for a Sos1 mutation and lack of lymphatic embryonic phenotypes for a homozygous Cx47 mutation in mice.

We have studied the lymphatic phenotypes of 2 mutations, known to cause abnormalities of lymphatics in humans, in mice. The Cx47 R260C mutation (variably penetrant in humans heterozygous for it and causing limb lymphedema) had an adult mouse phenotype of hyperplasia and increased lymph nodes only in homozygous condition but we did not find any anatomical phenotype in day 16.5 homozygous embryos. Mice harboring the Sos1 mutation E846K (causing Noonan's in man which occasionally shows lymphatic dysplasia) had no adult heterozygous phenotype in lymphatic vessel appearance and drainage (homozygotes are early embryonic lethals) while day 16.5 heterozygous embryos also had no detectable anatomical phenotype.

Evaluation of Circulating MicroRNAs and Adipokines in Breast Cancer Survivors with Arm Lymphedema.

Breast cancer-related lymphedema (BCRL) is a form of secondary lymphedema that is characterized by abnormal swelling of one or both arms due to the accumulation of lymph fluid in the interstitial tissue spaces, resulting from obstruction of the lymphatic vessels due to surgery insults, radiotherapy, or chemotherapy. Due to the multifactorial nature of this condition, the pathogenesis of secondary lymphedema remains unclear and the search for molecular factors associated with the condition is ongoing. This study aimed to identify serum microRNAs and adipokines associated with BCRL. Blood was collected from 113 breast cancer survivors and processed to obtain serum for small RNA-sequencing (BCRL vs. non-BCRL, n = 7 per group). MicroRNAs that were differentially expressed (fold change >1.5, p < 0.05) between lymphedema cases and those without lymphedema were further quantified in a validation cohort through quantitative reverse transcription PCR (BCRL n = 16, non-BCRL, n = 83). Leptin and adiponectin levels were measured in a combined cohort (BCRL n = 23, non-BCRL n = 90) using enzyme-linked immunosorbent assays. Two of the most significantly upregulated microRNAs, miR-199a-3p and miR-151a-3p, were strongly correlated with the onset of lymphedema and diabetes mellitus in the BCRL group. Leptin levels were higher in the BCRL cohort compared to the non-BCRL cohort (p < 0.05). A metabolic syndrome biomarker, the adiponectin/leptin ratio, was found to be lower in the BCRL group than in the non-BCRL group (median: 0.28 vs. 0.41, p < 0.05). Extensive studies on the mechanisms of the identified microRNAs and association of leptin with arm lymphedema may provide new insights on the potential biomarkers for lymphedema that should be followed up in a prospective cohort study.

Clinical staging and genetic profiling of Korean patients with primary lymphedema using targeted gene sequencing.

Lymphedema is a progressive disease caused by lymphatic flow blockage in the lymphatic pathway. Primary (hereditary) lymphedema is caused by genetic mutations without secondary causes. We performed clinical profiling on Korean primary lymphedema patients based on their phenotypes using lymphoscintigraphy and made genetic diagnoses using a next-generation sequencing panel consisting of 60 genes known to be related to primary lymphedema and vascular anomalies. Of 27 patients included in this study, 14.8% of the patients had lymphedema of the upper extremities, 77.8% had lymphedema of the lower extremities and 7.4% had 4-limbs lymphedema. Based on the International Society of Lymphology staging, 14, 10, and 3 patients had stage 3, 2, and 1 lymphedema, respectively. Only one family was genetically confirmed to harbor likely pathogenic variants in CELSR1. The proband was carrying two likely pathogenic variants in CELSR1, while her symptomatic mother was confirmed to carry only one of the variants. Furthermore, two other variants of uncertain significance in CELSR1 were detected in other patients, making CELSR1 the most commonly altered gene in our study. The clinical and genetic profile of hereditary lymphedema reported here is the first such data series reported for South Korea.

Pediatric Lymphedema: Study of 180 Patients Referred to a Tertiary Lymphedema Clinic.

BACKGROUND: Lymphedema is due to dysfunction of the lymphatic system. It can be primary or secondary. Pediatric lymphedema is more often primary and is a chronic disease with a heavy burden on quality of life. METHODS: Medical records of patients under 18 years of age referred between 1996 and 2021 to the specialized lymphedema clinic at the Sainte-Justine University Hospital Center were reviewed. Demographic data, sex, age at presentation, location of the lymphedema, clinical features, genetic testing, symptoms, complications, investigations, and treatment were collected. RESULTS: Of 180 referred patients, lymphedema was confirmed in 151, and 137 were primary lymphedema. Median age of apparition of primary lymphedema was 7.00 years and was significantly lower in boys than in girls. Primary congenital lymphedema was more frequent in boys (51.0%, 27.3% in girls, P = .007), and onset of primary lymphedema during adolescence was more frequent in girls (53.4%, 25.0% in boys, P = .001). Lower limbs were the most impacted (88.3%). Sixty patients had genetic testing, and 38 (63.3%) of them were discovered to have a pertinent genetic mutation. The most common mutated gene was the FLT4 gene (in 9 patients). Seven patients (5.1%) had associated extensive/central lymphatic malformation and 24 (17.6%) had a polymalformative syndrome/syndromic lymphedema. CONCLUSIONS: Pediatric lymphedema is more frequent in girls, usually involves lower limb, and is most often sporadic, but often associated with a genetic mutation, and genetic testing should be performed.

Low Efficacy of Genetic Tests for the Diagnosis of Primary Lymphedema Prompts Novel Insights into the Underlying Molecular Pathways.

Lymphedema is a chronic inflammatory disorder caused by ineffective fluid uptake by the lymphatic system, with effects mainly on the lower limbs. Lymphedema is either primary, when caused by genetic mutations, or secondary, when it follows injury, infection, or surgery. In this study, we aim to assess to what extent the current genetic tests detect genetic variants of lymphedema, and to identify the major molecular pathways that underlie this rather unknown disease. We recruited 147 individuals with a clinical diagnosis of primary lymphedema and used established genetic tests on their blood or saliva specimens. Only 11 of these were positive, while other probands were either negative (63) or inconclusive (73). The low efficacy of such tests calls for greater insight into the underlying mechanisms to increase accuracy. For this purpose, we built a molecular pathways diagram based on a literature analysis (OMIM, Kegg, PubMed, Scopus) of candidate and diagnostic genes. The PI3K/AKT and the RAS/MAPK pathways emerged as primary candidates responsible for lymphedema diagnosis, while the Rho/ROCK pathway appeared less critical. The results of this study suggest the most important pathways involved in the pathogenesis of lymphedema, and outline the most promising diagnostic and candidate genes to diagnose this disease.

Impaired Fanconi anemia pathway causes DNA hypomethylation in human angiosarcomas.

Angiosarcomas (AS) is a rare soft tissue sarcomas with poor treatment options and a dismal prognosis. The abnormal DNA methylation pattern has been determined as the certain clinical relevance with different angiosarcoma subtypes. However, the profound mechanism is not clear. In present study, we studied thirty-six AS with or without chronic lymphedema, and reported that DNA damage was an important factor causing DNA methylation abnormality. Furthermore, we determined that the impaired Fanconi anemia (FA) pathway contributed to severe DNA damage in AS with chronic lymphedema. We also observed that the activated FANCD2 could facilitate DNMT1 recruitment on genomic DNA. Our study uncovers a novel regulatory mechanism of FA pathway on DNA methylation, and is a benefit to advanced understanding the pathogenesis of AS, as well as providing the potential therapeutic targets for AS treatment.