SVEP1 IS IMPORTANT FOR MORPHOGENESIS OF LYMPHATIC SYSTEM: POSSIBLE IMPLICATIONS IN LYMPHEDEMA

SVEP1, also known as Polydom, is alarge extracellular mosaic protein with functionsin protein interactions and adhesion.Since Svep1 knockout animals show severeedema and lymphatic system malformations, theaim of this study is to evaluate the presence ofSVEP1 variants in patients with lymphedema.We analyzed DNA from 246 lymphedemapatients for variants in known lymphedemagenes, 235 of whom tested negative and underwenta second testing for new candidate genes,including SVEP1, as reported here. We foundthree samples with rare heterozygous missensesingle-nucleotide variants in the SVEP1 gene.In one family, healthy members were found tocarry the same variants and reported somesubclinical edema. Based on our findings and areview of the literature, we propose SVEP1 as acandidate gene that should be sequenced inpatients with lymphatic malformations, with orwithout lymphedema, in order to investigateand add evidence on its possible involvement inthe development of lymphedema

ABNORMAL LYMPHATIC PHENOTYPE IN A CRISPR MOUSE MODEL OF THE HUMAN LYMPHEDEMA-CAUSING CONNEXIN47 R260C POINT MUTATION

Connexin proteins form gap junctionscontrolling exchange of ions and small moleculesbetween cells and play an important rolein movement of lymph within lymphatic vessels.Connexin47 (CX47) is highly expressed inlymphatic endothelial cells and CX47 missensemutations, i.e., R260C, cosegregate with primarylymphedema in humans. However, studiesutilizing CX47 knockout mice have failedto demonstrate any lymphatic anomalies. Tounravel the lymphatic consequences of expressinga mutant CX47 protein, we used CRISPRtechnology to create a mouse carrying a Cx47missense mutation (Cx47R259C) equivalent tothe human CX47R260C missense mutationassociated with human primary lymphedema.Intradermal Evans Blue dye injection identifieda 2-fold increase in regional lymph nodesin homozygous Cx47R259C mice compared towildtype, particularly in the jugular region (4.8± 0.4 and 2.0 ± 0.0, respectively, p0.01).Associated lymphatic channels were increasedin Cx47R259C mice and mesenteric lymphreflux occurred in homozygous Cx47R259Cmice but not in wildtype. Contractility ofsuperficial cervical lymphatics, assessed bypressure myography, was reduced in homozygousCx47R259C mice compared to wildtype.In conclusion, our data are the first to demonstratea role for the Cx47 protein in lymphaticanatomy and function. This phenotype is similarto that found with other valve deficientmouse mutants, e.g., in Foxc2. Of significance,this study is the first to use CRISPR technologyto develop a pre-clinical model of primarylymphedema and demonstrates the importanceof distinguishing between lack of and presenceof mutant protein when developing clinicallyrelevant animal models for translation ofpre-clinical findings

PHYSICAL THERAPY AFFECTS ENDOTHELIAL FUNCTION IN LYMPHEDEMA PATIENTS

Lymphedema arises due to a malfunction of the lymphatic system and can lead to massive tissue swelling. Complete decongestive therapy (CDT), consisting of manual lymphatic drainage (MLD) and compression bandaging, is aimed at mobilizing fluid and reducing volume in affected extremities. Lymphatic dysfunction has previously been associated with chronic inflammation processes. We investigated plasma ADMA as an indicator of endothelial function/inflammation before-, during- and after-CDT. Also assessed were vascular function parameters such as carotid-femoral pulse wave velocity (PWVcf), flow-mediated dilatation (FMD) and retinal microvasculature analysis. 13 patients (3 males and 10 females, 57 ± 8 years old (mean ± SD), 167.2 ± 8.3 cm height, 91.0 ± 23.5 kg weight), with lower limb lymphedema were included. Vascular function parameters were assessed on day 1, 2, 7, 14 and 21 of CDT, pre- and post-MLD. ADMA was significantly lower post-MLD (p=0.0064) and tended to reduce over three weeks of therapy (p=0.0506). PWVcf weakly correlated with FMD (r=0.361, p=0.010). PWVcf, FMD and retinal microvasculature analysis did not show changes due to physical therapy. The novel results from this study indicate that lymphedema does not affect endothelial function and lymphedema patients may therefore not have a higher risk of cardiovascular diseases. Our results further suggest that manual lymphatic drainage with or without full CDT could have potentially beneficial effects on endothelial function in lymphedema patients (by reducing ADMA levels), which has not been reported previously

RARE PECAM1 VARIANTS IN THREE FAMILIES WITH LYMPHEDEMA

PECAM1 is a member of the immunoglobulin superfamily and is expressed in monocytes, neutrophils, macrophages and other types of immune cells as well as in endothelial cells. PECAM1 function is crucial for the development and maturation of B lymphocytes. The aim of this study was to link rare PECAM1 variants found in lymphedema patients with the development of lymphatic system malformations. Using NGS, we previously tested 246 Italian lymphedema patients for variants in 29 lymphedema-associated genes and obtained 235 negative results. We then tested these patients for variants in the PECAM1 gene. We found three probands with rare variants in PECAM1. All variants were heterozygous missense variants. In Family 1, the unaffected mother and brother of the proband were found to carry the same variant as the proband. Lymphoscintigraphy was performed to determine possible lymphatic malformations and showed that in both cases a bilateral slight reduction in the speed and lymphatic clearance of the lower limbs. PECAM1 function is important for lymphatic vasculature formation. We found variants in PECAM1 that may be associated with susceptibility to lymphedema

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2021 ISL NEW

ASSESSMENT OF LYMPHEDEMA WITH LYMPHOSCINTIGRAPHY: CAN NODAL QUANTIFICATION HELP?

Lymphoscintigraphy with combinedqualitative and quantitative analysis is reportedto be a more sensitive approach to diagnoselymphedema in comparison with the conventionalclinical analysis. Our study seeks toevaluate the diagnostic performance of lowerlimb lymphoscintigraphy with amalgamation ofqualitative and quantitative analysis by measuringthe ilio-inguinal nodal uptake. Thisprospective observational study was comprisedof 86 patients (172 limbs) diagnosed with lowerlimb lymphedema. After a thorough clinicalgrading of edema, radionuclide lymphoscintigraphywas performed as per a dedicatedinstitutional protocol. Ilio-inguinal nodalquantification of tracer uptake was computedalong with the visual study of the scans.Additionally, the corresponding mean nodaluptake percentage for each grade of lymphedemawas assessed and a cut off nodal uptakepercentage to differentiate between normal andabnormal limbs was defined. Although quantitativeanalysis with nodal uptake percentageprovides objective criteria to diagnose lymphedema,it can only act as an adjunct to qualitativemethod without replacing it. Finally,standardization of procedure for quantitativelymphoscintigraphy is needed including thepotential for combining both rate of clearanceof tracer from injection site and nodal uptakefor quantification

BONE DEVELOPMENT AND FRACTURE HEALING IS NORMAL IN MICE THAT HAVE A DEFECT IN THE DEVELOPMENT OF THE LYMPHATIC SYSTEM

Ectopic lymphatics form in bone andpromote bone destruction in diseases such asGorham-Stout disease, generalized lymphaticanomaly, and kaposiform lymphangiomatosis.However, the role lymphatics serve in normalbone development and repair is poorly understood.The objective of this study was tocharacterize bone development and fracturehealing in mice that have a defect in thedevelopment of the lymphatic vasculature. Wefound that bones in wild-type adult mice andmouse embryos did not have lymphatics. Wealso found that bone development was normalin Vegfr3(Chy/Chy) embryos. These mice do nothave lymphatics and die shortly after birth. Todetermine whether lymphatics serve a role inpostnatal bone development and fracturehealing, we analyzed bones from Vegfr3(wt/Chy)mice. These mice are viable and have fewerlymphatics than wild-type mice. We found thatpostnatal bone development and fracturehealing was normal in Vegfr3(wt/Chy) mice. Takentogether, our results suggest that lymphatics donot play a major role in normal bonedevelopment or repair