Capillary malformations.

Capillary malformation (CM), or port wine birthmark, is a cutaneous congenital vascular anomaly that occurs in 0.1%-2% of newborns. Patients with a CM localized on the forehead have an increased risk of developing a neurocutaneous disorder called encephalotrigeminal angiomatosis or Sturge-Weber syndrome (SWS), with complications including seizure, developmental delay, glaucoma, and vision loss. In 2013, a groundbreaking study revealed causative activating somatic mutations in the gene (GNAQ) encoding guanine nucleotide-binding protein Q subunit α (Gαq) in CM and SWS patient tissues. In this Review, we discuss the disease phenotype, the causative GNAQ mutations, and their cellular origin. We also present the endothelial Gαq-related signaling pathways, the current animal models to study CM and its complications, and future options for therapeutic treatment. Further work remains to fully elucidate the cellular and molecular mechanisms underlying the formation and maintenance of the abnormal vessels.

Ablation of Venous Malformations by Photothermal Therapy with Intravenous Gold Nanoshells.

Venous malformations (VMs) consist of hugely enlarged and dysmorphic veins. These lesions cause significant disfigurement, pain, and complications such as bleeding and coagulopathy. Pharmacotherapy for the treatment of VMs has limited efficacy and potentially limiting toxicity. Current treatment for patients with VMs entails life-long pharmacotherapy or surgical procedures. Here we explored whether intravenously administered agents can be used to destroy VMs by photothermal therapy (PTT), using gold nanoshells (AuNSs) that generated heat following irradiation with near-infrared (NIR) light. In a murine model of VMs, intravenous AuNSs accumulated within the VMs. Irradiation of the VMs induced marked regression and even elimination. Nanoparticle-based photothermal therapy can provide effective therapy for VMs, which are otherwise relatively refractory to treatment.

MEK inhibition reduced vascular tumor growth and coagulopathy in a mouse model with hyperactive GNAQ.

Activating non-inherited mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) gene family have been identified in childhood vascular tumors. Patients experience extensive disfigurement, chronic pain and severe complications including a potentially lethal coagulopathy termed Kasabach-Merritt phenomenon. Animal models for this class of vascular tumors do not exist. This has severely hindered the discovery of the molecular consequences of GNAQ mutations in the vasculature and, in turn, the preclinical development of effective targeted therapies. Here we report a mouse model expressing hyperactive mutant GNAQ in endothelial cells. Mutant mice develop vascular and coagulopathy phenotypes similar to those seen in patients. Mechanistically, by transcriptomic analysis we demonstrate increased mitogen activated protein kinase signaling in the mutant endothelial cells. Targeting of this pathway with Trametinib suppresses the tumor growth by reducing vascular cell proliferation and permeability. Trametinib also prevents the development of coagulopathy and improves mouse survival.

NRASQ61R mutation in human endothelial cells causes vascular malformations.

Somatic mutations in NRAS drive the pathogenesis of melanoma and other cancers but their role in vascular anomalies and specifically human endothelial cells is unclear. The goals of this study were to determine whether the somatic-activating NRASQ61R mutation in human endothelial cells induces abnormal angiogenesis and to develop in vitro and in vivo models to identify disease-causing pathways and test inhibitors. Here, we used mutant NRASQ61R and wild-type NRAS (NRASWT) expressing human endothelial cells in in vitro and in vivo angiogenesis models. These studies demonstrated that expression of NRASQ61R in human endothelial cells caused a shift to an abnormal spindle-shaped morphology, increased proliferation, and migration. NRASQ61R endothelial cells had increased phosphorylation of ERK compared to NRASWT cells indicating hyperactivation of MAPK/ERK pathways. NRASQ61R mutant endothelial cells generated abnormal enlarged vascular channels in a 3D fibrin gel model and in vivo, in xenografts in nude mice. These studies demonstrate that NRASQ61R can drive abnormal angiogenesis in human endothelial cells. Treatment with MAP kinase inhibitor U0126 prevented the change to a spindle-shaped morphology in NRASQ61R endothelial cells, whereas mTOR inhibitor rapamycin did not.

A transcription factor is the target of propranolol treatment in infantile hemangioma.

Propranolol is a nonselective ß-adrenergic receptor (AR) blocker that has been the first-line therapy for problematic infantile hemangioma (IH), the most frequent childhood vascular tumor. Although IHs are benign and eventually regress spontaneously, at least 15% of patients require treatment. Despite the extensive use of propranolol for IH treatment, its mode of action remains unclear. In this issue of the JCI, Seebauer et al. investigated the cellular and molecular consequences of propranolol treatment on IH vascular tumor formation in a murine model of IH. The efficacy of propranolol was independent of its ß-AR blocker activity and was attributable to the direct targeting of the transcription factor SOX18, which, in turn, reduced hemangioma blood vessel formation. We believe these results will guide clinical translation for the use of more efficient and safer therapies for IH and possibly for other vascular anomalies in which SOX18 plays a role.

A Xenograft Model for Venous Malformation.

Xenograft models allow for an in vivo approach to monitor cellular functions within the context of a host microenvironment. Here we describe a protocol to generate a xenograft model of venous malformation (VM) based on the use of human umbilical vein endothelial cells (HUVEC) expressing a constitutive active form of the endothelial tyrosine kinase receptor TEK (TIE2 p.L914F) or patient-derived EC containing TIE2 and/or PIK3CA gene mutations. Hyperactive somatic TIE2 and PIK3CA mutations are a common hallmark of VM in patient lesions. The EC are injected subcutaneously on the back of athymic nude mice to generate ectatic vascular channels and recapitulate histopathological features of VM patient tissue histology. Lesion plugs with TIE2/PIK3CA-mutant EC are visibly vascularized within 7-9 days of subcutaneous injection, making this a great tool to study venous malformation.

Kaposiform lymphangiomatosis treated with multimodal therapy improves coagulopathy and reduces blood angiopoietin-2 levels.

Kaposiform lymphangiomatosis (KLA) is a rare, life-threatening congenital lymphatic malformation. Diagnosis is often delayed due to complex indistinct symptoms. Blood angiopoietin-2 (ANG2) levels are elevated in KLA and may be useful as a biomarker to monitor disease status. We report a 7-year-old male child with easy bruising, inguinal swelling, and consumptive coagulopathy, diagnosed with KLA. A multimodal treatment regimen of prednisone, sirolimus, vincristine, and adjunctive zoledronate was used. Plasma ANG2 levels were highly elevated at diagnosis but decreased during treatment. The patient showed significant clinical improvement over a 38-month period and normalization of ANG2 levels correlated with resolution of the coagulopathy.

A Patient-Derived Xenograft Model for Venous Malformation.

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7‒9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation.

Capillary lymphatic venous malformations (CLVM) are complex vascular anomalies characterized by aberrant and enlarged lymphatic and blood vessels. CLVM appear during fetal development and enlarge after birth, causing life-long complications such as coagulopathy, pulmonary embolism, chronic pain, and disfigurement. Treatment includes surgical debulking, amputation, and recurrent sclerotherapy. Somatic, mosaic mutations in the 110-kD catalytic α-subunit of phosphoinositide-3-kinase (PIK3CA) gene have been previously identified in affected tissues from CLVM patients; however, the cell population harboring the mutation is still unknown. In this study, we hypothesized that endothelial cells (EC) carry the PIK3CA mutations and play a major role in the cellular origin of CLVM. We isolated EC from the lesions of seven patients with CLVM and identified PIK3CA hotspot mutations. The CLVM EC exhibited constitutive phosphorylation of the PI3K effector AKT as well as hyperproliferation and increased resistance to cell death compared to normal EC. Inhibitors of PIK3CA (BYL719) and AKT (ARQ092) attenuated the proliferation of CLVM EC in a dose-dependent manner. A xenograft model of CLVM was developed by injecting patient-derived EC into the flanks of immunocompromised mice. CLVM EC formed lesions with enlarged lymphatic and vascular channels, recapitulating the patient histology. EC subpopulations were further obtained by both immunomagnetic separation into lymphatic EC (LEC) and vascular EC (VEC) and generation of clonal populations. By sequencing these subpopulations, we determined that both LEC and VEC from the same patient express the PIK3CA mutation, exhibit increased AKT activation and can form lymphatic or vascular lesions in mouse.

Constitutive Active Mutant TIE2 Induces Enlarged Vascular Lumen Formation with Loss of Apico-basal Polarity and Pericyte Recruitment.

Abnormalities in controlling key aspects of angiogenesis including vascular cell migration, lumen formation and vessel maturation are hallmarks of vascular anomalies including venous malformation (VM). Gain-of-function mutations in the tyrosine kinase receptor TIE2 can cause VM and induce a ligand-independent hyperactivation of TIE2. Despite these important findings, the TIE2-dependent mechanisms triggering enlarged vascular lesions are not well understood. Herein we studied TIE2 p.L914F, the most frequent mutation identified in VM patients. We report that endothelial cells harboring a TIE2-L914F mutation display abnormal cell migration due to a loss of front-rear polarity as demonstrated by a non-polarized Golgi apparatus. Utilizing a three-dimensional fibrin-matrix based model we show that TIE2-L914F mutant cells form enlarged lumens mimicking vascular lesions present in VM patients, independently of exogenous growth factors. Moreover, these abnormal vascular channels demonstrate a dysregulated expression pattern of apico-basal polarity markers Podocalyxin and Collagen IV. Furthermore, in this system we recapitulated another pathological feature of VM, the paucity of pericytes around ectatic veins. The presented data emphasize the value of this in vitro model as a powerful tool for the discovery of cellular and molecular signals contributing to abnormal vascular development and subsequent identification of novel therapeutic approaches.

Signaling pathways and inhibitors of cells from patients with kaposiform lymphangiomatosis.

BACKGROUND: Kaposiform lymphangiomatosis (KLA) is a rare lymphatic anomaly with significant morbidity and mortality. KLA is characterized by diffuse multifocal lesions comprised of focal areas of "kaposiform" spindled cells accompanying malformed lymphatic channels. The goal of this study was to identify activated signaling pathways in cells isolated from three KLA patients for the purpose of testing new therapies. PROCEDURE: Cells were obtained from the lungs of one patient isolated at autopsy and the spleen of two patients removed in surgery due to disease complications. A protein kinase array was performed on the KLA cell lysates and normal lymphatic endothelial cells. RESULTS: Higher activation of key signaling pathways in the KLA cells, including PRAS40, AKT1/2/3, and ERK-1/2, was identified by protein kinase array and confirmed by Western blot analysis. This indicated a role for highly activated PI3K-AKT and MAPK-ERK-1/2 signaling pathways in KLA cells. Cell proliferation studies assessed PI3K inhibitors (LY294002; BYL719), AKT inhibitor ARQ092, mTOR inhibitor rapamycin, and MAPK inhibitor U0126. These studies demonstrated that PI3K-AKT-mTOR and MAPK signaling are important mediators of KLA cell proliferation. BYL719 and rapamycin were more effective at inhibiting KLA cell proliferation than U0126. CONCLUSIONS: Our studies using cells from KLA patient lesions demonstrate that these cells are highly proliferative and the PI3K-AKT-mTOR and MAPK pathways are promising therapeutic targets. Development and clinical trials of PI3K, AKT, and MAPK inhibitors for cancer treatment and the data in this study lend support for early clinical trials assessing the efficacy of these inhibitors in KLA patients.

RUNX represses Pmp22 to drive neurofibromagenesis.

Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor ß (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation.

Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VMs grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ (phospholipase C) and ERK (extracellular signal-regulated kinase) activity.

Cellular and molecular mechanisms of PIK3CA-related vascular anomalies.

The phosphoinositide 3-kinase (PI3K) pathway is a major mediator of growth factor signaling, cell proliferation and metabolism. Somatic gain-of-function mutations in PIK3CA, the catalytic subunit of PI3K, have recently been discovered in a number of vascular anomalies. The timing and origin of these mutations remain unclear although they are believed to occur during embryogenesis. The cellular origin of these lesions likely involves endothelial cells or an early endothelial cell lineage. This review will cover the diseases and syndromes associated with PIK3CA mutations and discuss the cellular origin, pathways and mechanisms. Activating PIK3CA 'hot spot' mutations have long been associated with a multitude of cancers allowing the development of targeted pharmacological inhibitors that are FDA-approved or in clinical trials. Current and future therapeutic approaches for PIK3CA-related vascular anomalies are discussed.

Combined mTOR and MEK inhibition is an effective therapy in a novel mouse model for angiosarcoma.

Angiosarcoma is an aggressive malignancy of vascular origin that occurs de novo or in the context of previous cancer therapy. Despite multi-modal aggressive treatment including surgical resection, chemotherapy, and radiation, five-year overall survival remains poor at 35%. Due to its rarity, little is known about its molecular pathology and clinical trials have been extremely difficult to conduct. Development of animal models for rare diseases like angiosarcoma is critical to improve our understanding of tumorigenesis and to test novel treatment regimens. A genetically engineered mouse model for angiosarcoma was generated by conditional deletion of Trp53, Pten, and Ptpn12 in endothelial cells. Tumors arising from these mice recapitulate the histology and molecular pathology of the human disease including hyperactivation of the PI3K/mTOR and MAPK signaling pathways. Treatment of tumor-bearing mice with mTOR or MEK inhibitors effectively inactivated signaling and resulted in reduced proliferation and elevated apoptosis leading to tumor regression. The effect of treatment on tumor growth was transient and proliferation was restored after a period of dormancy. However, combined inhibition of mTOR and MEK resulted in profound tumor regression which was sustained for the duration of treatment. These results suggest that angiosarcoma may be effectively treated by this drug combination. .

A xenograft model for venous malformation.

Vascular malformations are defects caused by the abnormal growth of the vasculature. Among them, venous malformation (VM) is an anomaly characterized by slow-flow vascular lesions with abnormally shaped veins, typically in sponge-like configuration. VMs can expand over years causing disfigurement, obstruction of vital structures, thrombosis, bleeding, and pain. Treatments have been very limited and primarily based on supportive care, compression garments, sclerotherapy, and/or surgical resection. Sirolimus treatment has recently shown efficacy in some patients with complicated vascular anomalies, including VMs. Activating somatic TIE2 gene mutations have been identified in up to 60% of VMs and PIK3CA mutations have been found in another 25%. Here, we report a xenograft model of VM that reflects the patients' mutation heterogeneity. First, we established a protocol to isolate and expand in culture endothelial cells (VM-EC) from VM tissue or VM blood of nine patients. In these cells, we identified somatic mutations of TIE2, PIK3CA, or a combination of both. Both TIE2 and PIK3CA mutations induced constitutive AKT activation, while TIE2 mutations also showed high MAPK-ERK signaling. Finally, VM-EC implanted into immune-deficient mice generated lesions with ectatic blood-filled channels with scarce smooth muscle cell coverage, similar to patients' VM. This VM xenograft model could be instrumental to test the therapeutic efficacy of Sirolimus in the presence of the different TIE2 or PIK3CA mutations or to test for efficacy of additional compounds in targeting the specific mutated protein(s), thus enabling development of personalized treatment options for VM patients.

EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells.

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH+ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH+ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH+ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH+ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.

Endoglin regulates mural cell adhesion in the circulatory system.

The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for ß1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or ß1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5ß1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng (+/-) mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia.

Rapamycin improves TIE2-mutated venous malformation in murine model and human subjects.

Venous malformations (VMs) are composed of ectatic veins with scarce smooth muscle cell coverage. Activating mutations in the endothelial cell tyrosine kinase receptor TIE2 are a common cause of these lesions. VMs cause deformity, pain, and local intravascular coagulopathy, and they expand with time. Targeted pharmacological therapies are not available for this condition. Here, we generated a model of VMs by injecting HUVECs expressing the most frequent VM-causing TIE2 mutation, TIE2-L914F, into immune-deficient mice. TIE2-L914F-expressing HUVECs formed VMs with ectatic blood-filled channels that enlarged over time. We tested both rapamycin and a TIE2 tyrosine kinase inhibitor (TIE2-TKI) for their effects on murine VM expansion and for their ability to inhibit mutant TIE2 signaling. Rapamycin prevented VM growth, while TIE2-TKI had no effect. In cultured TIE2-L914F-expressing HUVECs, rapamycin effectively reduced mutant TIE2-induced AKT signaling and, though TIE2-TKI did target the WT receptor, it only weakly suppressed mutant-induced AKT signaling. In a prospective clinical pilot study, we analyzed the effects of rapamycin in 6 patients with difficult-to-treat venous anomalies. Rapamycin reduced pain, bleeding, lesion size, functional and esthetic impairment, and intravascular coagulopathy. This study provides a VM model that allows evaluation of potential therapeutic strategies and demonstrates that rapamycin provides clinical improvement in patients with venous malformation.

AKT hyper-phosphorylation associated with PI3K mutations in lymphatic endothelial cells from a patient with lymphatic malformation.

Lymphatic malformations (LM) are characterized by abnormal formation of lymphatic vessels and tissue overgrowth. The lymphatic vessels present in LM lesions may become blocked and enlarged as lymphatic fluid collects, forming a mass or cyst. Lesions are typically diagnosed during childhood and are often disfiguring and life threatening. Available treatments consist of sclerotherapy, surgical removal and therapies to diminish complications. We isolated lymphatic endothelial cells (LM-LEC) from a surgically removed microcystic LM lesion. LM-LEC and normal human dermal-LEC (HD-LEC) expressed endothelial (CD31, VE-Cadherin) as well as lymphatic endothelial (Podoplanin, PROX1, LYVE1)-specific markers. Targeted gene sequencing analysis in patient-derived LM-LEC revealed the presence of two mutations in class I phosphoinositide 3-kinases (PI3K) genes. One is an inherited, premature stop codon in the PI3K regulatory subunit PIK3R3. The second is a somatic missense mutation in the PI3K catalytic subunit PIK3CA; this mutation has been found in association with overgrowth syndromes and cancer growth. LM-LEC exhibited angiogenic properties: both cellular proliferation and sprouting in collagen were significantly increased compared with HD-LEC. AKT-Thr308 was constitutively hyper-phosphorylated in LM-LEC. Treatment of LM-LEC with PI3-Kinase inhibitors Wortmannin and LY294 decreased cellular proliferation and prevented the phosphorylation of AKT-Thr308 in both HD-LEC and LM-LEC. Treatment with the mTOR inhibitor rapamycin also diminished cellular proliferation, sprouting and AKT phosphorylation, but only in LM-LEC. Our results implicate disrupted PI3K-AKT signaling in LEC isolated from a human lymphatic malformation lesion.