Trametinib as a promising therapeutic option in alleviating vascular defects in an endothelial KRAS-induced mouse model.

Somatic activating Kirsten rat sarcoma viral oncogene homologue (KRAS) mutations have been reported in patients with arteriovenous malformations. By producing LSL-Kras (G12D); Cdh5 (PAC)-CreERT2 [iEC-Kras (G12D*)] mice, we hoped to activate KRAS within vascular endothelial cells (ECs) to generate an arteriovenous malformation mouse model. Neonatal mice were treated daily with tamoxifen from postnatal (PN) days 1-3. Mortality and phenotypes varied amongst iEC-Kras (G12D*) pups, with only 31.5% surviving at PN14. Phenotypes (focal lesions, vessel dilations) developed in a consistent manner, although with unpredictable severity within multiple soft tissues (such as the brain, liver, heart and brain). Overall, iEC-Kras (G12D*) pups developed significantly larger vascular lumen areas compared with control littermates, beginning at PN8. We subsequently tested whether the MEK inhibitor trametinib could effectively alleviate lesion progression. At PN16, iEC-Kras (G12D*) pup survival improved to 76.9%, and average vessel sizes were closer to controls than in untreated and vehicle-treated mutants. In addition, trametinib treatment helped normalize iEC-Kras (G12D*) vessel morphology in PN14 brains. Thus, trametinib could act as an effective therapy for KRAS-induced vascular malformations in patients.

Genetics of vascular anomalies.

Vascular anomalies are developmental defects of the vasculature and encompass a variety of disorders. The identification of genes mutated in the different malformations provides insight into the etiopathogenic mechanisms and the specific roles the associated proteins play in vascular development and maintenance. A few familial forms of vascular anomalies exist, but most cases occur sporadically. It is becoming evident that somatic mosaicism plays a major role in the formation of vascular lesions. The use of Next Generating Sequencing for high throughput and "deep" screening of both blood and lesional DNA and RNA has been instrumental in detecting such low frequency somatic changes. The number of novel causative mutations identified for many vascular anomalies has soared within a 10-year period. The discovery of such genes aided in unraveling a holistic overview of the pathogenic mechanisms, by which in vitro and in vivo models could be generated, and opening the doors to development of more effective treatments that do not address just symptoms. Moreover, as many mutations and the implicated signaling pathways are shared with cancers, current oncological therapies could potentially be repurposed for the treatment of vascular anomalies.

Trauma Can Induce Telangiectases in Hereditary Hemorrhagic Telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease of the fibrovascular tissue resulting in visceral vascular malformations and (muco-) cutaneous telangiectases with recurrent bleedings. The mechanism behind the disease is not fully understood; however, observations from HHT mouse models suggest that mechanical trauma may induce the formation of abnormal vessels. To assess the influence of environmental trauma (mechanical or light induced) on the number of telangiectases in patients with HHT, the number of telangiectases on the hands, face, and lips were counted on 103 HHT patients possessing at least three out of four Curaçao criteria. They were then surveyed for information concerning their dominant hand, exposure to sunlight, and types of regular manual work. Patients developed more telangiectases on their dominant hand and lower lip (Wilcoxon rank sum test: p < 0.001). Mechanical stress induced by manual work led to an increased number of telangiectases on patients' hands (Mann-Whitney U test: p < 0.001). There was also a positive correlation between sun exposure and the number of telangiectases on the lips (Mann-Whitney U test: 0.027). This study shows that mechanical and UV-induced trauma strongly influence the formation of telangiectases in HHT patients. This result has potential implications in preventive measures and on therapeutic approaches for HHT.

A Clinical Feasibility Study to Image Angiogenesis in Patients with Arteriovenous Malformations Using 68Ga-RGD PET/CT.

Arteriovenous malformations (AVMs) have an inherent capacity to form new blood vessels, resulting in excessive lesion growth, and this process is further triggered by the release of angiogenic factors. 68Ga-labeled arginine-glycine-aspartate tripeptide sequence (RGD) PET/CT imaging may provide insight into the angiogenic status and treatment response of AVMs. This clinical feasibility study was performed to demonstrate that 68Ga-RGD PET/CT imaging can be used to quantitatively assess angiogenesis in peripheral AVMs. Methods: Ten patients with a peripheral AVM (mean age, 40 y; 4 men and 6 women) and scheduled for endovascular embolization treatment were prospectively included. All patients underwent 68Ga-RGD PET/CT imaging 60 min after injection (mean dose, 207 ± 5 MBq). Uptake in the AVM, blood pool, and muscle was quantified as SUVmax and SUVpeak, and a descriptive analysis of the PET/CT images was performed. Furthermore, immunohistochemical analysis was performed on surgical biopsy sections of peripheral AVMs to investigate the expression pattern of integrin αvß3Results:68Ga-RGD PET/CT imaging showed enhanced uptake in all AVM lesions (mean SUVmax, 3.0 ± 1.1; mean SUVpeak, 2.2 ± 0.9). Lesion-to-blood and lesion-to-muscle ratios were 3.5 ± 2.2 and 4.6 ± 2.8, respectively. Uptake in blood and muscle was significantly higher in AVMs than in background tissue (P = 0.0006 and P = 0.0014, respectively). Initial observations included uptake in multifocal AVM lesions and enhanced uptake in intraosseous components in those AVM cases affecting bone integrity. Immunohistochemical analysis revealed cytoplasmatic and membranous integrin αvß3 expression in the endothelial cells of AVMs. Conclusion: This feasibility study showed increased uptake in AVMs with angiogenic activity, compared with surrounding tissue without angiogenic activity, suggesting that 68Ga-RGD PET/CT imaging can be used as a tool to quantitatively determine angiogenesis in AVMs. Further studies will be conducted to explore the potential of 68Ga-RGD PET/CT imaging for guiding current treatment decisions and for assessing response to antiangiogenic treatment.

Vascular Anomalies Caused by Abnormal Signaling within Endothelial Cells: Targets for Novel Therapies.

Vascular anomalies arise as a consequence of improper development and maintenance of the vasculature. Our knowledge on the pathophysiological bases of vascular anomalies has skyrocketed during the past 5 years. It is becoming clear that common intracellular signaling pathways are often activated by mutations, causing endothelial cell dysfunction. These mutations cause hyperactivation of two major intracellular signaling pathways that may be controlled by inhibitors developed for cancer treatment. Although we do not know yet all the downstream effects, it has become evident that normalization of the abnormal signaling is an interesting target for therapy. This is a major paradigm change, as developmental malformations were considered to be inert to any molecular treatment.

GATA2 null mutation associated with incomplete penetrance in a family with Emberger syndrome.

INTRODUCTION: GATA2 mutations are associated with several conditions, including Emberger syndrome which is the association of primary lymphedema with hematological anomalies and an increased risk for myelodysplasia and leukemia. OBJECTIVE: To describe a family with Emberger syndrome with incomplete penetrance. METHODS: A DNA sequencing of GATA2 gene was performed in the parents and offspring (five individuals in total). RESULTS: The family consisted of 5 individuals with a GATA2 null mutation (c.130G>T, p.Glu44*); three of them were affected (two of which were deceased) while two remained unaffected at the age of 40 and 13 years old. The three affected siblings (two boys and one girl) presented with lymphedema of the lower limbs, recurrent warts, epistaxis and recurrent infections. Two died due to hematological abnormalities (AML and pancytopenia). In contrast, the two other family members who carry the same mutation (the mother and one brother) have not presented any symptoms and their blood tests remain normal. DISCUSSION: Incomplete penetrance may indicate that GATA2 haploinsufficiency is not enough to produce the phenotype of Emberger syndrome. It could be useful to perform whole exome or genome sequencing, in cases where incomplete penetrance or high variable expressivity is described, in order to probably identify specific gene interactions that drastically modify the phenotype. In addition, skewed gene expression by an epigenetic mechanism of gene regulation should also be considered.

How to manage patients with hereditary haemorrhagic telangiectasia.

Hereditary haemorrhagic telangiectasia is a rare systemic autosomal dominantly inherited disorder of the fibrovascular tissue with a wide variety of clinical manifestations. Diagnosis is based on the clinical Curaçao criteria or molecular genetic testing. Dilated vessels can develop into telangiectases or larger vascular malformations in various organs, calling for an interdisciplinary approach. Epistaxis and gastrointestinal bleeding can result from these vascular defects. Various conservative and interventional treatments have been described for these conditions. However, no optimal therapy exists. Treatment can become especially difficult due to progressive anaemia or when anticoagulant or anti-thrombotic therapy becomes necessary. Screening for pulmonary arteriovenous malformations (PAVM) should be performed in all confirmed and suspected patients. Treatment by percutaneous transcatheter embolotherapy and antibiotic prophylaxis is normally effective for PAVM. Cerebral or hepatic vascular malformations and rare manifestations need to be evaluated on a case-by-case basis to determine the best course of action for treatment.

Genetics of vascular malformations.

Vascular anomalies are developmental defects of the vasculature and encompass a variety of disorders. The majority of these occur sporadically, yet a few are reported to be familial. The identification of genes mutated in the different malformations provides insight into their etiopathogenic mechanisms and the specific roles the associated proteins play in vascular development and maintenance. It is becoming evident that somatic mosaicism plays a major role in the formation of vascular lesions. The importance of utilizing Next-Generating Sequencing (NGS) for high-throughput and "deep" screening of both blood and lesional DNA and RNA is thus emphasized, as the somatic changes are present in low quantities. There are several examples where NGS has already accomplished discovering these changes. The identification of all the causative genes and unraveling of a holistic overview of the pathogenic mechanisms should enable generation of in vitro and in vivo models and lead to development of more effective treatments, not only targeted on symptoms.

Common and distinctive pathogenetic features of arteriovenous malformations in hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 animal models--brief report.

OBJECTIVE: Hereditary hemorrhagic telangiectasia is a genetic disorder characterized by visceral and mucocutaneous arteriovenous malformations (AVMs). Clinically indistinguishable hereditary hemorrhagic telangiectasia 1 and hereditary hemorrhagic telangiectasia 2 are caused by mutations in ENG and ALK1, respectively. In this study, we have compared the development of visceral and mucocutaneous AVMs in adult stages between Eng- and Alk1-inducible knockout (iKO) models. APPROACH AND RESULTS: Eng or Alk1 were deleted from either vascular endothelial cells (ECs) or smooth muscle cells in adult stages using Scl-CreER and Myh11-CreER lines, respectively. Latex perfusion and intravital spectral imaging in a dorsal skinfold window chamber system were used to visualize remodeling vasculature during AVM formation. Global Eng deletion resulted in lethality with visceral AVMs and wound-induced skin AVMs. Deletion of Alk1 or Eng in ECs, but not in smooth muscle cells, resulted in wound-induced skin AVMs. Visceral AVMs were observed in EC-specific Alk1-iKO but not in Eng-iKO. Intravital spectral imaging revealed that Eng-iKO model exhibited more dynamic processes for AVM development when compared with Alk1-iKO model. CONCLUSIONS: Both Alk1- and Eng-deficient models require a secondary insult, such as wounding, and ECs are the primary cell type responsible for the pathogenesis. However, Alk1 but not Eng deletion in ECs results in visceral AVMs.

Venous malformation-causative TIE2 mutations mediate an AKT-dependent decrease in PDGFB.

Mutations in the endothelial cell (EC) tyrosine kinase receptor TIE2 cause inherited and sporadic forms of venous malformation. The recurrent somatic mutation L914F and common germline mutation R849W differ in terms of phosphorylation level, as well as sub-cellular localization and trafficking of the receptor. Previous studies have shed light on certain pathogenic properties of R849W, but the mechanisms of action of L914F are unknown. We used global gene expression profiling to study the effects of L914F on ECs. We found that L914F strongly dysregulates genes involved in vascular development, cell migration and extracellular matrix processing, while R849W has weak effects. We also demonstrate, for the first time, that TIE2-mutant ECs are deficient in the production of PDGFB, both in vitro and ex vivo in patient tissues. This defect is mediated by the chronic, ligand-independent activation of AKT by the mutant receptors. Inadequate secretion of the major mural cell attractant likely plays an important role in the development of abnormal vascular channels, contributing to the characteristic paucity of surrounding vascular smooth muscle cells.

Physiological and genomic consequences of adrenergic deficiency during embryonic/fetal development in mice: impact on retinoic acid metabolism.

Adrenergic hormones are essential for early heart development. To gain insight into understanding how these hormones influence heart development, we evaluated genomic expression changes in embryonic hearts from adrenergic-deficient and wild-type control mice. To perform this study, we used a mouse model with targeted disruption of the Dopamine ß-hydroxylase (Dbh) gene, whose product is responsible for enzymatic conversion of dopamine into norepinephrine. Embryos homozygous for the null allele (Dbh(-/-)) die from heart failure beginning as early as embryonic day 10.5 (E10.5). To assess underlying causes of heart failure, we isolated hearts from Dbh(-/-) and Dbh(+/+) embryos prior to manifestation of the phenotype and examined gene expression changes using genomic Affymetrix 430A 2.0 arrays, which enabled simultaneous evaluation of >22,000 genes. We found that only 22 expressed genes showed a significant twofold or greater change, representing ~0.1% of the total genes analyzed. More than half of these genes are associated with either metabolism (31%) or signal transduction (22%). Remarkably, several of the altered genes encode for proteins that are directly involved in retinoic acid (RA) biosynthesis and transport. Subsequent evaluation showed that RA concentrations were significantly elevated by an average of ~3-fold in adrenergic-deficient (Dbh(-/-)) embryos compared with controls, thereby suggesting that RA may be an important downstream mediator of adrenergic action during embryonic heart development.

TGF-ß signaling in endothelial cells, but not neuroepithelial cells, is essential for cerebral vascular development.

The various organs of the body harbor blood vessel networks that display unique structural and functional features; however, the mechanisms that control organ-specific vascular development and physiology remain mostly unknown. In the developing mouse brain, αvß8 integrin-mediated TGF-ß activation and signaling is essential for normal blood vessel growth and sprouting. Whether integrins activate TGF-ß signaling pathways in vascular endothelial cells (ECs), neural cells, or both, has yet to be determined. Here, we have generated and characterized mice in which TGF-ß receptors are specifically deleted in neuroepithelial cells via Nestin-Cre, or in ECs via a novel Cre transgenic strain (Alk1(GFPCre)) in which Cre is expressed under control of the endogenous activin receptor-like kinase 1 (Alk1) promoter. We report that deletion of Tgfbr2 in the neuroepithelium does not impact brain vascular development. In contrast, selective deletion of the Tgfbr2 or Alk5 genes in ECs result in embryonic lethality because of brain-specific vascular pathologies, including blood vessel morphogenesis and intracerebral hemorrhage. These data reveal for the first time that αvß8 integrin-activated TGF-ßs regulate angiogenesis in the developing brain via paracrine signaling to ECs.

Noninvasive tracking of cardiac embryonic stem cells in vivo using magnetic resonance imaging techniques.

Despite rapid advances in the stem cell field, the ability to identify and track transplanted or migrating stem cells in vivo is limited. To overcome this limitation, we used magnetic resonance imaging (MRI) to detect and follow transplanted stem cells over a period of 28 days in mice using an established myocardial infarction model. Pluripotent mouse embryonic stem (mES) cells were expanded and induced to differentiate into beating cardiomyocytes in vitro. The cardiac-differentiated mES cells were then loaded with superparamagnetic fluorescent microspheres (1.63 microm in diameter) and transplanted into ischemic myocardium immediately following ligation and subsequent reperfusion of the left anterior descending coronary artery. To identify the transplanted stem cells in vivo, MRI was performed using a Varian Inova 4.7 Tesla scanner. Our results show that (a) the cardiac-differentiated mES were effectively loaded with superparamagnetic microspheres in vitro, (b) the microsphere-loaded mES cells continued to beat in culture prior to transplantation, (c) the transplanted mES cells were readily detected in the heart in vivo using noninvasive MRI techniques, (d) the transplanted stem cells were detected in ischemic myocardium for the entire 28-day duration of the study as confirmed by MRI and post-mortem histological analyses, and (e) concurrent functional MRI indicated typical loss of cardiac function, although significant amelioration of remodeling was noted after 28 days in hearts that received transplanted stem cells. These results demonstrate that it is feasible to simultaneously track transplanted stem cells and monitor cardiac function in vivo over an extended period using noninvasive MRI techniques.