Angiogenesis depends upon EPHB4-mediated export of collagen IV from vascular endothelial cells.

Capillary malformation-arteriovenous malformation (CM-AVM) is a blood vascular anomaly caused by inherited loss-of-function mutations in RASA1 or EPHB4 genes, which encode p120 Ras GTPase-activating protein (p120 RasGAP/RASA1) and Ephrin receptor B4 (EPHB4). However, whether RASA1 and EPHB4 function in the same molecular signaling pathway to regulate the blood vasculature is uncertain. Here, we show that induced endothelial cell-specific (EC-specific) disruption of Ephb4 in mice resulted in accumulation of collagen IV in the EC ER, leading to EC apoptotic death and defective developmental, neonatal, and pathological angiogenesis, as reported previously in induced EC-specific RASA1-deficient mice. Moreover, defects in angiogenic responses in EPHB4-deficient mice could be rescued by drugs that inhibit signaling through the Ras pathway and drugs that promote collagen IV export from the ER. However, EPHB4-mutant mice that expressed a form of EPHB4 that is unable to physically engage RASA1 but retains protein tyrosine kinase activity showed normal angiogenic responses. These findings provide strong evidence that RASA1 and EPHB4 function in the same signaling pathway to protect against the development of CM-AVM independent of physical interaction and have important implications for possible means of treatment of this disease.

5q14.3 deletion neurocutaneous syndrome: Contiguous gene syndrome caused by simultaneous deletion of RASA1 and MEF2C: A progressive disease.

We report the case of a young girl who was presented with complex clinical symptoms caused by the deletion of contiguous genes: RASA1 and MEF2C, located on chromosome 5q14.3. Specifically, the diagnosis of her skin disorder and vascular malformations involving central nervous system is consistent with a RASopathy. The child's neurological manifestations are observed in most patients suffering from 5q14.3 by deletion or mutation of the MEF2C gene. A review of the literature allowed us to conclude that the contiguous deletion of genes RASA1 and MEF2C fulfills the criteria for the diagnosis of a Neurocutaneous syndrome as proposed by Carr et al. [2011]. We also assessed the penetrance of RASA1 and clinical manifestations of MEF2C according to the type of deletion. This child described presents the complete symptomatology of both deleted genes. We would also like to highlight the progression of the disorder.

Cutting Edge: Codeletion of the Ras GTPase-Activating Proteins (RasGAPs) Neurofibromin 1 and p120 RasGAP in T Cells Results in the Development of T Cell Acute Lymphoblastic Leukemia.

Ras GTPase-activating proteins (RasGAPs) inhibit signal transduction initiated through the Ras small GTP-binding protein. However, which members of the RasGAP family act as negative regulators of T cell responses is not completely understood. In this study, we investigated potential roles for the RasGAPs RASA1 and neurofibromin 1 (NF1) in T cells through the generation and analysis of T cell-specific RASA1 and NF1 double-deficient mice. In contrast to mice lacking either RasGAP alone in T cells, double-deficient mice developed T cell acute lymphoblastic leukemia/lymphoma, which originated at an early point in T cell development and was dependent on activating mutations in the Notch1 gene. These findings highlight RASA1 and NF1 as cotumor suppressors in the T cell lineage.

RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity.

Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations.

Caspase-3 protects stressed organs against cell death.

The ability to generate appropriate defense responses is crucial for the survival of an organism exposed to pathogenesis-inducing insults. However, the mechanisms that allow tissues and organs to cope with such stresses are poorly understood. Here we show that caspase-3-knockout mice or caspase inhibitor-treated mice were defective in activating the antiapoptotic Akt kinase in response to various chemical and environmental stresses causing sunburns, cardiomyopathy, or colitis. Defective Akt activation in caspase-3-knockout mice was accompanied by increased cell death and impaired survival in some cases. Mice homozygous for a mutation in RasGAP that prevents its cleavage by caspase-3 exhibited a similar defect in Akt activation, leading to increased apoptosis in stressed organs, marked deterioration of their physiological functions, and stronger disease development. Our results provide evidence for the relevance of caspase-3 as a stress intensity sensor that controls cell fate by either initiating a RasGAP cleavage-dependent cell resistance program or a cell suicide response.

A role for p120 RasGAP in thymocyte positive selection and survival of naive T cells.

Activation of the Ras small GTP-binding protein is necessary for normal T cell development and function. However, it is unknown which Ras GTPase-activating proteins (RasGAPs) inactivate Ras in T cells. We used a T cell-specific RASA1-deficient mouse model to investigate the role of the p120 RasGAP (RASA1) in T cells. Death of CD4(+)CD8(+) double-positive thymocytes was increased in RASA1-deficient mice. Despite this finding, on an MHC class II-restricted TCR transgenic background, evidence was obtained for increased positive selection of thymocytes associated with augmented activation of the Ras-MAPK pathway. In the periphery, RASA1 was found to be dispensable as a regulator of Ras-MAPK activation and T cell functional responses induced by full agonist peptides. However, numbers of naive T cells were substantially reduced in RASA1-deficient mice. Loss of naive T cells in the absence of RASA1 could be attributed in part to impaired responsiveness to the IL-7 prosurvival cytokine. These findings reveal an important role for RASA1 as a regulator of double-positive survival and positive selection in the thymus as well as naive T cell survival in the periphery.

The potential of capillary birthmarks as a significant marker for capillary malformation-arteriovenous malformation syndrome in children who had nontraumatic cerebral hemorrhage.

BACKGROUND/PURPOSE: Capillary malformation-arteriovenous malformation (CM-AVM) is a new autosomal dominant disorder with cutaneous capillary malformations (CM) and high-flow cerebral arteriovenous malformations (AVM). Patients may have Parkes-Weber syndrome. This study determined if cutaneous CM are a significant indicator of CM-AVM in children with cerebral bleeds. METHODS: Children with cerebral AVMs between 1991 and 2009 were reviewed. A family history of brain hemorrhage, AVMs, or cutaneous birthmarks was elicited. Patients and siblings were examined for CM and a family tree recorded. A brief questionnaire determined the family's opinion regarding screening for this syndrome. RESULTS: Of 30 families, 1 family had Parkes-Weber syndrome. In 3 families, both patient and relatives had CM. In 9 families, patients had no CM, but relatives had them. One family had hereditary hemorrhagic telangiectasia. From the survey, 80% of families would be concerned about vascular marks, and 87% of families would allow screening for cerebral AVMs. CONCLUSION: A family history of vascular marks may predict families at risk of having a cerebral AVM with hemorrhage. Most families would agree to screening. However, family history and physical examination alone do not confirm CM-AVM but form a useful screening tool to identify families needing further investigations with genetic testing and/or magnetic resonance imaging.

Role of p120 Ras-GAP in directed cell movement.

We have used cell lines deficient in p120 Ras GTPase activating protein (Ras-GAP) to investigate the roles of Ras-GAP and the associated p190 Rho-GAP (p190) in cell polarity and cell migration. Cell wounding assays showed that Ras-GAP-deficient cells were incapable of establishing complete cell polarity and migration into the wound. Stimulation of mutant cells with growth factor rescued defects in cell spreading, Golgi apparatus fragmentation, and polarized vesicular transport and partially rescued migration in a Ras-dependent manner. However, for directional movement, the turnover of stress fibers and focal adhesions to produce an elongate morphology was dependent on the constitutive association between Ras-GAP and p190, independent of Ras regulation. Disruption of the phosphotyrosine-mediated Ras-GAP/p190 complex by microinjecting synthetic peptides derived from p190 sequences in wild-type cells caused a suppression of actin filament reorientation and migration. From these observations we suggest that although Ras-GAP is not directly required for motility per se, it is important for cell polarization by regulating actin stress fiber and focal adhesion reorientation when complexed with 190. This observation suggests a specific function for Ras-GAP separate from Ras regulation in cell motility.