Endothelial cell expression of mutant Map2k1 causes vascular malformations in mice.

Extracranial arteriovenous malformation (AVM) is a congenital vascular anomaly causing disfigurement, bleeding, ulceration, and pain. Most lesions are associated with somatic MAP2K1 activating mutations in endothelial cells (ECs). The purpose of this study was to determine if EC expression of mutant activated MAP2K1 is sufficient to produce vascular malformations in mice. We generated mice with a ROSA26 allele containing a lox-stop-lox gene trap (GT), Map2k1 cDNA with an activating p.K57N missense mutation, an internal ribosomal entry site, and green fluorescent protein cDNA (R26GT-Map2k1-GFP). We expressed mutant MAP2K1 and GFP in ECs of fetal and newborn mice using Tg-Cdh5Cre or Tg-Cdh5CreER alleles. Tg-Cdh5Cre+/-;R26GT-Map2k1-GFP/+ animals that express mutant MAP2K1 in ECs in utero developed diffuse vascular abnormalities and died by embryonic (E) day 16.5. Tg-Cdh5CreER+/-;R26GT-Map2k1-GFP/+ animals in which mutant MAP2K1 expression was induced in ECs by tamoxifen at postnatal (P) day 1 developed vascular malformations in the brain, ear, and intestines by P23. The lesions consisted of abnormal networks of blood vessels containing recombined and non-recombined ECs. In conclusion, expression of MAP2K1 p.K57N is sufficient to cause vascular malformations in mice. This model can be used to study the malformation process and for pre-clinical pharmacologic studies.

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels.

OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. Approach and Results: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) ß3, a downstream effector of Gαq. Activated PLCß3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCß3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCß3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

How we approach lymphedema in the pediatric population.

Lymphedema in children is rare; however, it is usually a progressive and chronic condition. Accurate diagnosis of lymphedema in the pediatric population often takes several months and sometimes is delayed for years. Lymphedema can be isolated or associated with genetic syndromes, thus it is very important to identify the correct diagnosis, to select carefully which patients to refer for genetic testing, and to initiate appropriate treatment in a timely fashion. In this article, we review key information about diagnosis of lymphedema, associated conditions and syndromes, and current treatment modalities.

How we approach lymphedema in the pediatric population.

Lymphedema in children is rare; however, it is usually a progressive and chronic condition. Accurate diagnosis of lymphedema in the pediatric population often takes several months and sometimes is delayed for years. Lymphedema can be isolated or associated with genetic syndromes, thus it is very important to identify the correct diagnosis, to select carefully which patients to refer for genetic testing, and to initiate appropriate treatment in a timely fashion. In this article, we review key information about diagnosis of lymphedema, associated conditions and syndromes, and current treatment modalities.

Human endothelial colony-forming cells provide trophic support for pluripotent stem cell-derived cardiomyocytes via distinctively high expression of neuregulin-1.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Lower extremity lymphatic function predicted by body mass index: a lymphoscintigraphic study of obesity and lipedema.

BACKGROUND/OBJECTIVES: Patients with obesity and lipedema commonly are misdiagnosed as having lymphedema. The conditions share phenotypic overlap and can influence each other. The purpose of this study was to delineate obesity-induced lymphedema, obesity without lymphedema, and lipedema in order to improve their diagnosis and treatment. SUBJECTS/METHODS: Our Lymphedema Center database of 700 patients was searched for patients with obesity-induced lymphedema (OIL), obesity without lymphedema (OWL), and lipedema. Patient age, sex, diagnosis, cellulitis history, body mass index (BMI), and treatment were recorded. Only subjects with lymphoscintigraphic documentation of their lymphatic function were included. RESULTS: Ninety-eight patients met inclusion criteria. Subjects with abnormal lymphatic function (n = 46) had a greater BMI (65 ± 12) and cellulitis history (n = 30, 65%) compared to individuals with normal lymphatic function [(BMI 42 ± 10); (cellulitis n = 8, 15%)] (p < 0.001). Seventeen patients had a history of lipedema and two exhibited abnormal lymphatic function (BMI 45, 54). The risk of having lower extremity lymphedema was predicted by BMI: BMI < 40 (0%), 40-49 (17%), 50-59 (63%), 60-69 (86%), 70-79 (91%), ≥80 (100%). Five patients with OIL (11%) underwent resection of massive localized lymphedema (MLL) or suction-assisted lipectomy. Three individuals (18%) with lipedema were treated with suction-assisted lipectomy. CONCLUSIONS: The risk of lymphedema in patients with obesity and lipedema can be predicted by BMI; confirmation requires lymphoscintigraphy. Individuals with OIL are at risk for cellulitis and MLL. Patients with a BMI > 40 are first managed with weight loss. Excisional procedures can further reduce extremity size once BMI has been lowered.

EPHB4 mutation causes adult and adolescent-onset primary lymphedema.

Primary lymphedema results from the anomalous development of the lymphatic system and typically presents during infancy, childhood, or adolescence. Adult-onset primary lymphedema is rare and mutations associated with this condition have not been identified. The purpose of this investigation was to search for variants that cause adult-onset primary lymphedema. We discovered an autosomal dominant EPHB4 mutation in a patient who developed unilateral leg lymphedema at age 39 years; the same mutation affected his son who presented with the disease at 14 years of age.

Verrucous Venous Malformation-Subcutaneous Variant.

BACKGROUND: Verrucous venous malformation (VVM), previously called "verrucous hemangioma," typically involves the dermis and the subcutaneous fat. We have encountered patients with VVM confined to the hypodermis. MATERIALS AND METHODS: During a nearly 20-year period, 13 patients, aged 2-17 years, presented with a subcutaneous mass in the limb without clinically obvious epidermal alterations. Consequently, operative excisions did not include the skin. RESULTS: Histopathologically, the specimens were composed of blood-filled channels with morphologic characteristics of capillaries and veins that infiltrated adipose tissue. Aggregates often formed nodules with variable fibrosis and a component of large and radially oriented vessels. A diagnosis of VVM was supported by endothelial immunopositivity for GLUT-1 (25%-75% immunopositive channels in 16/16 specimens); D2-40 (1%-25% channels in 14/15 specimens); and Prox-1 (1%-50% of channels in 14/16 specimens). A MAP3K3 mutation was identified by droplet digital PCR in 3 of the 6 specimens. CONCLUSIONS: Diagnosis of VVM in this uncommon location is challenging because of absence of epidermal changes and lack of dermal involvement. Imaging is not pathognomonic, and mimickers are many. Appropriate immunohistochemical stains and molecular analysis contribute to the correct diagnosis.

Lipoblastoma phenotype contains a somatic PIK3CA mutation.

Lipoblastoma typically occurs in childhood and is associated with rearrangements of the PLAG1 gene. We present a patient with an isolated mass thought to be a lipoblastoma clinically, radiographically, and histologically. The lesion was diagnosed as a PIK3CA-adipose lesion after the tissue was negative for PLAG1 rearrangement and contained a somatic PIK3CA mutation (H1047R). Although PIK3CA variants are associated with PROS (PIK3CA-related overgrowth spectrum), this report illustrates a non-syndromic, lipoblastoma phenotype caused by a PIK3CA mutation.

Somatic MAP2K1 Mutations Are Associated with Extracranial Arteriovenous Malformation.

Arteriovenous malformation (AVM) is a fast-flow, congenital vascular anomaly that may arise anywhere in the body. AVMs typically progress, causing destruction of surrounding tissue and, sometimes, cardiac overload. AVMs are difficult to control; they often re-expand after embolization or resection, and pharmacologic therapy is unavailable. We studied extracranial AVMs in order to identify their biological basis. We performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) on AVM tissue from affected individuals. Endothelial cells were separated from non-endothelial cells by immune-affinity purification. We used droplet digital PCR (ddPCR) to confirm mutations found by WES and WGS, to determine whether mutant alleles were enriched in endothelial or non-endothelial cells, and to screen additional AVM specimens. In seven of ten specimens, WES and WGS detected and ddPCR confirmed somatic mutations in mitogen activated protein kinase kinase 1 (MAP2K1), the gene that encodes MAP-extracellular signal-regulated kinase 1 (MEK1). Mutant alleles were enriched in endothelial cells and were not present in blood or saliva. 9 of 15 additional AVM specimens contained mutant MAP2K1 alleles. Mutations were missense or small in-frame deletions that affect amino acid residues within or adjacent to the protein's negative regulatory domain. Several of these mutations have been found in cancers and shown to increase MEK1 activity. In summary, somatic mutations in MAP2K1 are a common cause of extracranial AVM. The likely mechanism is endothelial cell dysfunction due to increased MEK1 activity. MEK1 inhibitors, which are approved to treat several forms of cancer, are potential therapeutic agents for individuals with extracranial AVM.

Endothelial MAP2K1 mutations in arteriovenous malformation activate the RAS/MAPK pathway.

Arteriovenous malformation (AVM) is a locally destructive congenital vascular anomaly caused by somatic mutations in MAP2K1. The mutation is isolated to endothelial cells (ECs). The purpose of this study was to determine the effects of mutant MAP2K1 on EC signaling and vascular network formation. Pathway effects were studied using both mutant MAP2K1 (K57N) human AVM tissue and human umbilical vein endothelial cells (HUVECs) engineered to overexpress the MAP2K1 (K57N) mutation. Western blot was used to determine cell signaling along the RAS/MAPK pathway. Geltrex tube formation assays were performed to assess EC vascular network formation. Cells were treated with a MAP2K1 inhibitor (Trametinib) to determine its effect on signaling and vascular tube formation. Human mutant MAP2K1-AVM ECs had similar baseline MEK1 and ERK1/2 expression with controls; however, mutant MAP2K1-AVM ECs produced significantly more phosphorylated ERK1/2 than wild-type ECs. Mutant MAP2K1 HUVECs demonstrated significantly more phosphorylated ERK1/2 than control HUVECs. Trametinib reduced the phosphorylation of ERK1/2 in mutant cells and prevented the ability of ECs to form vascular networks. AVM MAP2K1 mutations activate RAS/MAPK signaling in ECs. ERK activation and vascular network formation are reduced with Trametinib. Pharmacotherapy using MAP2K1 inhibitors may prevent the formation or progression of AVMs.

Congenital Disseminated Pyogenic Granuloma: Characterization of an Aggressive Multisystemic Disorder.

OBJECTIVE: To describe the clinical, radiologic, and histopathologic features of "congenital disseminated pyogenic granuloma" involving various organs with high morbidity related to cerebral hemorrhagic involvement. STUDY DESIGN: We searched the database of the Vascular Anomalies Center at Boston Children's Hospital from 1999 to 2019 for patients diagnosed as having multiple vascular lesions, visceral vascular tumors, congenital hemangiomatosis, multiple pyogenic granulomas, or multiple vascular lesions without a definite diagnosis. A retrospective review of the medical records, photographs, histopathologic, and imaging studies was performed. Only patients with imaging studies and histopathologic diagnosis of pyogenic granuloma were included. RESULTS: Eight children (5 male, 3 female) had congenital multifocal cutaneous vascular tumors. Lesions also were found in the brain (n = 7), liver (n = 4), spleen (n = 3), muscles (n = 4), bone (n = 3), retroperitoneum (n = 3), and intestine/mesentery (n = 2). Less commonly affected were the spinal cord, lungs, kidneys, pancreas, and adrenal gland (n = 1 each). The mean follow-up period was 21.8 months. The cerebral and visceral lesions were hemorrhagic with severe neurologic sequelae. The histopathologic diagnosis was pyogenic granuloma with prominent areas of hemorrhage and necrosis. The endothelial cells had enlarged nuclei, pale cytoplasm and were immunopositive for CD31 and negative for D2-40 and glucose transporter 1. CONCLUSIONS: Congenital disseminated pyogenic granuloma is a distinct multisystemic aggressive disorder that primarily affects the skin, brain, visceral organs, and musculoskeletal system. Differentiation of this entity from other multiple cutaneous vascular lesions is critical because of possible cerebral hemorrhagic involvement.

Arteriovenous malformation phenotype resembling congenital hemangioma contains KRAS mutations.

Extracranial arteriovenous malformation (AVM) is most commonly caused by a somatic mutation in MAP2K1. We report two patients with vascular anomalies that had an unclear clinical diagnosis most consistent with either an AVM or congenital hemangioma. Lesions were cutaneous, reddish-purple with telangiectasias, present at birth, and had defined borders. Histopathology indicated AVM and both lesions contained somatic KRAS mutations. A rare AVM phenotype exists that shares clinical features with congenital hemangioma.

Diffuse capillary malformation with overgrowth contains somatic PIK3CA variants.

Diffuse capillary malformation with overgrowth (DCMO) is a clinical diagnosis describing patients with multiple, extensive capillary malformations (CMs) associated with overgrowth and foot anomalies. The purpose of the study was to identify somatic variants in DCMO. Skin containing CM and overgrown subcutaneous adipose tissue was collected from patients with DCMO. Exons from 447 cancer-related genes were sequenced using OncoPanel. Variant-specific droplet digital PCR (ddPCR) independently confirmed the variants and determined variant allele frequencies (VAF). One subject contained a somatic PIK3CA p.G106V variant. A second patient had a PIK3CA p.D350G variant. VAF was 27% to 29% in skin and 16% to 28% in subcutaneous adipose. Variants were enriched in endothelial cells (VAF 50%-51%) compared to nonendothelial cells (1%-8%). DCMO is associated with somatic PIK3CA variants and should be considered on the PIK3CA-related overgrowth spectrum (PROS). Variants are present in both skin and subcutaneous adipose and are enriched in endothelial cells.

Fibroadipose Vascular Anomaly in the Upper Extremity: A Distinct Entity With Characteristic Clinical, Radiological, and Histopathological Findings.

PURPOSE: Fibroadipose vascular anomaly (FAVA) is an intramuscular vascular malformation that has been recently described as a distinct clinical entity. The clinical, radiological, and histopathological characteristics of FAVA in the upper extremity are reviewed. METHODS: This was a retrospective case series of upper-extremity FAVA lesions. RESULTS: We reviewed 19 patients with FAVA of the upper limb. Pain, stiffness, swelling, and flexion contractures were the most common presentations. Except for one lesion confined to the hand, all lesions either presented with or developed a contracture within 10 years. Ten patients underwent surgical debulking. Six required tendon transfer reconstruction and 3 necessitated a free functional muscle transfer. CONCLUSIONS: Fibroadipose vascular anomaly in the upper extremity requires an accurate diagnosis and may benefit from early referral to a multidisciplinary vascular anomaly center with experienced hand surgeons. Compression garments, propranolol, and sclerotherapy seem to be ineffective. Surgical resection focused on symptomatic regions with appropriate reconstruction may have benefit in salvage of limbs with compromised function. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Surgical Management of Fibroadipose Vascular Anomaly of the Lower Extremities.

BACKGROUND: Fibroadipose vascular anomaly (FAVA) is a recently-defined vascular malformation often involving the extremities and presenting in childhood. Patients may present to orthopaedic surgeons with pain, swelling, joint contractures, and leg length discrepancy. There is no established therapy or treatment paradigm. We report on outcomes following surgical excision for patients with this condition. METHODS: Between 2007 and 2016, all 35 patients that underwent excision of lower-extremity FAVA were retrospectively reviewed using a combination of medical records, radiologic findings, and telemedicine reviews. RESULTS: Mean age at initial presentation was 12.3±6.8 years. Mean follow-up from time of definitive diagnosis at our institution was 66 months (range: 12 to 161 mo). Mean follow-up after surgery was 35 months (range: 6 to 138 mo). Females were affected more than males (71% vs. 29%). The most common location of FAVA was in the calf (49%), followed by the thigh (40%). The most commonly involved muscle was gastrocnemius (29%), followed by the quadriceps (26%). At latest follow-up after surgery, there was an improvement in the proportion of patients with pain at rest (63% vs. 29%), pain with activity (100% vs. 60%), as well as analgesia use (94% vs. 37%). Fourteen patients (40%) had symptomatic residual disease or recurrence of FAVA requiring further treatment. Six patients (17%) required further surgery and 6 (17%) required further interventional radiologic procedures. Three patients (9%) required eventual amputation for intractable pain and loss of function. Lesions with direct nerve involvement were associated with persistent neuropathic symptoms at latest follow-up (P=0.002) as well as symptomatic residual disease and/or recurrence requiring further treatment (P=0.01). Seventeen patients (49%) had 19 preoperative joint contractures. Eighteen of the 19 contractures (95%) had sustained improvement at latest follow-up. CONCLUSIONS: In carefully selected patients, surgical excision of FAVA results in improvement of symptoms. However, symptomatic residual disease and/or recurrence are not uncommon. Direct nerve involvement is associated with a worse outcome. LEVEL OF EVIDENCE: Level IV-case series.

Elevation of Costal Cartilage Ear Construct for Microtia Using a V-Y Scalp Flap.

Autologous ear construction for microtia creates an auricle using a costal cartilage framework. To separate the construct from the mastoid, the most common methods incorporate the use of fascial flaps or skin grafting. The authors describe a V-Y skin and subcutaneous scalp advancement flap for ear elevation as part of autologous ear construction for microtia. The method is simple and reliable and offers advantages compared to other techniques.

Intramuscular fast-flow vascular anomaly contains somatic MAP2K1 and KRAS mutations.

BACKGROUND: The term "intramuscular hemangioma capillary type" (IHCT) refers to a fast-flow vascular lesion that is classified as a tumor, although its phenotype overlaps with arteriovenous malformation (AVM). The purpose of this study was to identify somatic mutations in IHCT. METHODS: Affected tissue specimens were obtained during a clinically indicated procedure. The diagnosis of IHCT was based on history, physical examination, imaging and histopathology. Because somatic mutations in cancer-associated genes can cause vascular malformations, we sequenced exons from 446 cancer-related genes in DNA from 7 IHCT specimens. We then performed mutation-specific droplet digital PCR (ddPCR) to independently test for the presence of a somatic mutation found by sequencing and to screen one additional IHCT sample. RESULTS: We detected somatic mutations in 6 of 8 IHCT specimens. Four specimens had a mutation in MAP2K1 (p.Q58_E62del, p.P105_I107delinsL, p.Q56P) and 2 specimens had mutations in KRAS (p.K5E and p.G12D, p.G12D and p.Q22R). Mutant allele frequencies detected by sequencing and confirmed by ddPCR ranged from 2 to 15%. CONCLUSIONS: IHCT lesions are phenotypically similar to AVMs and contain the same somatic MAP2K1 or KRAS mutations, suggesting that IHCT is on the AVM spectrum. We propose calling this lesion "intramuscular fast-flow vascular anomaly."

Arteriovenous malformation associated with a HRAS mutation.

The majority of extracranial arteriovenous malformations (AVMs) are caused by somatic mutations in MAP2K1. We report a somatic HRAS mutation in a patient who has a facial AVM associated with subcutaneous adipose overgrowth. We performed whole exome sequencing on DNA from the affected tissue and found a HRAS mutation (p.Thr58_Ala59delinsValLeuAspVal). Mutant allelic frequency was 5% in whole tissue and 31% in isolated endothelial cells (ECs); the mutation was not present in blood DNA or non-ECs. Somatic mutations in HRAS can cause AVM.

Somatic Activating Mutations in GNAQ and GNA11 Are Associated with Congenital Hemangioma.

Congenital hemangioma is a rare vascular tumor that forms in utero. Postnatally, the tumor either involutes quickly (i.e., rapidly involuting congenital hemangioma [RICH]) or partially regresses and stabilizes (i.e., non-involuting congenital hemangioma [NICH]). We hypothesized that congenital hemangiomas arise due to somatic mutation and performed massively parallel mRNA sequencing on affected tissue from eight participants. We identified mutually exclusive, mosaic missense mutations that alter glutamine at amino acid 209 (Glu209) in GNAQ or GNA11 in all tested samples, at variant allele frequencies (VAF) ranging from 3% to 33%. We verified the presence of the mutations in genomic DNA using a combination of molecular inversion probe sequencing (MIP-seq) and digital droplet PCR (ddPCR). The Glu209 GNAQ and GNA11 missense variants we identified are common in uveal melanoma and have been shown to constitutively activate MAPK and/or YAP signaling. When we screened additional archival formalin-fixed paraffin-embedded (FFPE) congenital cutaneous and hepatic hemangiomas, 4/8 had GNAQ or GNA11 Glu209 variants. The same GNAQ or GNA11 mutation is found in both NICH and RICH, so other factors must account for these tumors' different postnatal behaviors.