Assessment of angiogenesis-related parameters in juvenile idiopathic arthritis-associated uveitis.

PURPOSE: Juvenile idiopathic arthritis-associated uveitis (JIAU) may run a chronic and treatment-resistant course, and occasionally, alterations of the iris vasculature may be observed clinically. METHODS: Iris tissue (IT), aqueous humor (AH) and serum samples from patients with clinically inactive JIAU (n = 30), acute anterior uveitis (AAU; n = 18), and primary open angle glaucoma (POAG; n = 20) were obtained during trabeculectomy or cataract surgery. Samples were analyzed by RNA-Seq, qRT-PCR, LC-IMS, Western-Blot, and LEGENDplex™ analysis. Pattern of iris vasculature in JIAU patients was assessed qualitatively via fluorescein and indocyanine green angiography (FLA/ICGA). RESULTS: RNA-Seq of IT showed significantly differential expression (DE) of 136 genes between JIAU and POAG, of which 15 were associated with angiogenesis. qRT-PCR, performed to validate RNA-Seq results, showed upregulation of the angiogenesis-related genes Kdr, Angpt-1, Tie-1, Tie-2 and Mmrn2 in IT (JIAU vs POAG, p > 0.05). LC-IMS of IT revealed a total number of 56 DE proteins (JIAU vs POAG), of which Angiopoetin, Lumican and Decorin were associated with angiogenesis and showed increased (p > 0.05) expression on Western-Blot analysis. LEGENDplex™ analysis showed upregulation of ANGPT-2 in AH from JIAU compared to AAU and POAG, whereas VEGF was upregulated in AAU. Iris vascular leakage, hypoperfusion and neovascularization were observed by FLA/ICGA in JIA patients with treatment-refractory complicated course of uveitis. CONCLUSION: Angiogenesis-related factors could play a role in long-standing complicated JIAU, leading to clinically visible alterations in selected cases.

Standardization of diethylnitrosamine-induced hepatocellular carcinoma rat model with time based molecular assessment.

This study was intended (1) to develop a robust animal model for hepatocellular carcinoma (HCC) research, in which HCC tumors develop in a background of fibrosis or cirrhosis; and (2) to explore time-dependent regulatory changes in key molecular markers during disease advancement and HCC development. With the aim of establishing such HCC model, male Sprague-Dawley rats were injected with diethylnitrosamine (DEN) at a dose of 30 mg/kg twice a week for 10 weeks then once a week from 12th to 16th weeks. The rats were kept under observation until 18th week. At defined time intervals (2nd, 4th, 12th, and 18th week), serum biomarkers and microscopic components of tissue samples were used to investigate the chronic progression of liver disease, while gene and protein analysis was used to monitor expression patterns during HCC development. DEN-intoxicated rats manifested inflammation at week 4, fibrosis at week 12 and cirrhosis with early HCC tumors at week 18. Molecular analysis revealed that key markers of inflammation (Il-1ß, Il-6, and Tnf-α), fibrosis (Tgf-ß1, Col1α1, Col3α1, and Timp-1), and angiogenesis (Hif1-α and Vegf) were promptly (P ≤ 0.001) up-regulated at week 4, week 12 and week 18, respectively. Oxidative stress (iNos, Cyp2e1, and Sod1) and pro-apoptotic (Bax) markers showed significant upregulation from week 4 to week 12. However, Sod1 and Bax expressions dropped after week 12 and reached a minimum at 18th week. Strikingly, expressions of anti-apoptotic (Bcl-2) and cell proliferation (Pcna, Hgf, and Afp) markers were abruptly increased at week 18. Collectively, we describe an 18-week HCC model in DEN-intoxicated rats that exhibit chronic inflammation, oxidative imbalance, advance fibrosis/cirrhosis, halted apoptosis, and angiogenic sprouting, progressively.

Quantitative assessment and clinical relevance of VEGFRs-positive tumor cells in refractory brain tumors.

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)1 and 2 signaling is a potent activator of tumor angiogenesis. Although the expressions of VEGFR1 and VEGFR2 were initially thought to be limited to the endothelial cells, it is now known that both the receptors are expressed in tumor cells. This is the first study wherein VEGFRs-positive tumor cells are quantitatively evaluated for brain tumors with upregulated VEGF/VEGFR signaling. The percentage of VEGFRs-positive tumor cells was quantitatively evaluated in various brain tumors (10 glioblastomas, 22 neurofibromatosis type 2 [NF2]-related schwannomas, 21 sporadic schwannomas, 27 chordomas, 36 meningiomas, 29 hemangioblastomas, 11 hemangiopericytoma, and 13 ependymomas) using immunohistochemistry. VEGF-A expression was also analyzed using quantitative real-time polymerase chain reaction. Double immunofluorescence staining using anti-PDGFR-ß and anti-CD34 antibody, microvessel density, and vessel diameter were analyzed to evaluate the vascular characteristics. Chordomas demonstrated an extremely higher percentage of VEGFR1 and VEGFR2-positive tumor cells than other tumors. In contrast, meningiomas and hemangiopericytomas showed few VEGFRs-positive tumor cells. The percentage of positive tumor cells in chordomas, hemangioblastomas, and NF2 schwannomas was associated with clinical courses, such as shorter progression free survival, and growth speed. Glioblastomas and NF2 schwannomas showed larger tumor vessels without pericyte coverage. The present study is the first to quantitatively analyze VEGFR1- and VEGFR2- positive tumor cells in various types of refractory brain tumors. This novel parameter significantly correlated with the progressive clinical courses.

Assessment of Expression of Homeobox A5 in Endometrial Cancer on the mRNA and Protein Level.

BACKGROUND: Endometrial cancer is one of the most common gynecological cancer in the developed countries and occurs mainly in postmenopausal women. Angiogenesis is important for cancer formation as it provides nutrients for growing tumor mass. Most tumors do not show detectable Homeobox A5 (HOXA5 level), suggesting its potential role as a cancer suppressor. It was demonstrated that HOXA5 is involved in the progression of various types of cancer and the loss of its expression correlates with higher pathological grade and poorer outcome. OBJECTIVE: The aim of the study was to evaluate HOXA5 expression at transcriptome and protein levels. MATERIALS AND METHODS: The study enrolled 45 women diagnosed with endometrial cancer and 15 without neoplastic changes. The histopathological examination allowed us to divide cancer tissue samples according to the degree of histological differentiation: G1, 17; G2, 15; G3, 13. The expression of the HOXA5 protein was determined by immunohistochemistry. Microarray and RT-qPCR techniques were used to assess HOXA5 expression at the mRNA level. RESULTS: The reaction to the HOXA5 protein was only visible in glandular cells in G1 endometrial cancer and was lower compared to the control. In grades 2 and 3, reactions were noted at the limit of the method's sensitivity. In addition, reduced HOXA5 expression was observed at the transcriptome level. CONCLUSION: HOXA5 may become a potential complementary molecular marker, allowing early detection of neoplastic changes in the endometrium. It also seems that detection of HOXA5 at the mRNA and protein levels may be helpful in improving the accuracy of diagnosis and planning effective oncological therapy.

Assessment of Stabilization and Activity of the HIFs Important for Hypoxia-Induced Signalling in Cancer Cells.

Blood vessels in tumors contain chaotic branching structures and leaky vessel lumens, resulting in uneven supply of oxygen in the tumor microenvironment. High metabolic and proliferation rate of tumor cells further depletes the local oxygen supply. Therefore, hypoxia is a common phenomenon in multiple solid malignancies. Hypoxia-inducible factors (HIFs) regulate the transcription of a spectrum of genes, which are vitally important for tumor cell adaption under hypoxia, and shape the tumor microenvironment to become more favorable for progression. HIFs are involved in almost every step of cancer development through inducing angiogenesis, metabolic reprogramming, metastasis, cancer stemness maintenance, chemoresistance, and immune evasion. Here, we describe methods for the assessment of HIF activity, as well as identification of novel transcriptional targets of HIFs in vitro and in vivo.

Assessment of the direct effects of DDAH I on tumour angiogenesis in vivo.

Nitric oxide (NO) has been strongly implicated in glioma progression and angiogenesis. The endogenous inhibitors of NO synthesis, asymmetric dimethylarginine (ADMA) and N-monomethyl-L-arginine (L-NMMA), are metabolized by dimethylarginine dimethylaminohydrolase (DDAH), and hence, DDAH is an intracellular factor that regulates NO. However, DDAH may also have an NO-independent action. We aimed to investigate whether DDAH I has any direct role in tumour vascular development and growth independent of its NO-mediated effects, in order to establish the future potential of DDAH inhibition as an anti-angiogenic treatment strategy. A clone of rat C6 glioma cells deficient in NO production expressing a pTet Off regulatable element was identified and engineered to overexpress DDAH I in the absence of doxycycline. Xenografts derived from these cells were propagated in the presence or absence of doxycycline and susceptibility magnetic resonance imaging used to assess functional vasculature in vivo. Pathological correlates of tumour vascular density, maturation and function were also sought. In the absence of doxycycline, tumours exhibited high DDAH I expression and activity, which was suppressed in its presence. However, overexpression of DDAH I had no measurable effect on tumour growth, vessel density, function or maturation. These data suggest that in C6 gliomas DDAH has no NO-independent effects on tumour growth and angiogenesis, and that the therapeutic potential of targeting DDAH in gliomas should only be considered in the context of NO regulation.

Hepatocellular carcinoma: Where there is unmet need.

Hepatocellular carcinoma (HCC) is a complex and heterogeneous tumor most commonly associated with underlying chronic liver disease, especially hepatitis. It is a growing problem in the United States and worldwide. There are two potential ways to prevent HCC. Primary prevention which is based on vaccination or secondary prevention involving agents that slow down carcinogenesis. Several pathways have been thought to play a role in the development of HCC; specifically, those involving vascular endothelial growth factor (VEGF)-mediated angiogenesis, WNT, phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and c-MET. Currently, there are only a limited number of drugs which have been proven as effective treatment options for HCC and several clinical trials are testing drugs which target aberrations in the pathways mentioned above. In this review, we discuss currently approved therapies, monotherapies and combination therapy for the treatment of HCC.

DCE-MRI assessment of the effect of Epstein-Barr virus-encoded latent membrane protein-1 targeted DNAzyme on tumor vasculature in patients with nasopharyngeal carcinomas.

BACKGROUND: EBV-encoded latent membrane protein 1 (EBV-LMP1) is an important oncogenic protein for nasopharyngeal carcinoma (NPC) and has been shown to engage a plethora of signaling pathways. Correspondingly, an LMP1-targeted DNAzyme was found to inhibit the growth of NPC cells both in vivo and in vitro by suppressing cell proliferation and inducing apoptosis. However, it remains unknown whether an LMP1-targeted DNAzyme would affect the vasculature of NPC. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been applied in the clinical trials of anti-angiogenic drugs for more than ten years, and Ktrans has been recommended as a primary endpoint. Therefore, the objective of the current study was to use DCE-MRI to longitudinally study the effect of an EBV-LMP1-targeted DNAzyme on the vasculature of patients with NPC. METHODS: Twenty-four patients were randomly divided into two groups: a combined treatment group (radiotherapy + LMP1-targeted DNAzyme) and a radiotherapy alone group (radiotherapy + normal saline). DCE-MRI scans were conducted 1 ~ 2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), upon completion of radiotherapy (RT 70 Gy), and three months after radiotherapy (3 months post-RT). Parameters of vascular permeability and intra- and extravascular volumes were subsequently obtained (e.g., Ktrans, kep, ve) using nordicICE software. RESULTS: Both Ktrans and kep values for NPC tumor tissues decreased for both groups after treatment. Moreover, a statistically significant difference in Ktrans values at the pre-therapy and post-therapy timepoints emerged earlier for the combined treatment group (RT 50 Gy, P =0.045) compared to the radiotherapy alone group (3 months post-RT, P = 0.032). For the kep values, the downward trend observed for both the combined treatment group and the radiotherapy alone group were similar. In contrast, ve values for all of the tumor tissues increased following therapy. CONCLUSIONS: The EBV-LMP1-targeted DNAzyme that was tested was found to accelerate the decline of Ktrans values for patients with NPC. Correspondingly, the LMP1-targeted DNAzyme treatments were found to affect the angiogenesis and microvascular permeability of NPC. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01449942. Registered 6 October 2011.

Discovery and functional assessment of gene variants in the vascular endothelial growth factor pathway.

Angiogenesis is a host-mediated mechanism in disease pathophysiology. The vascular endothelial growth factor (VEGF) pathway is a major determinant of angiogenesis, and a comprehensive annotation of the functional variation in this pathway is essential to understand the genetic basis of angiogenesis-related diseases. We assessed the allelic heterogeneity of gene expression, population specificity of cis expression quantitative trait loci (eQTLs), and eQTL function in luciferase assays in CEU and Yoruba people of Ibadan, Nigeria (YRI) HapMap lymphoblastoid cell lines in 23 resequenced genes. Among 356 cis-eQTLs, 155 and 174 were unique to CEU and YRI, respectively, and 27 were shared between CEU and YRI. Two cis-eQTLs provided mechanistic evidence for two genome-wide association study findings. Five eQTLs were tested for function in luciferase assays and the effect of two KRAS variants was concordant with the eQTL effect. Two eQTLs found in each of PRKCE, PIK3C2A, and MAP2K6 could predict 44%, 37%, and 45% of the variance in gene expression, respectively. This is the first analysis focusing on the pattern of functional genetic variation of the VEGF pathway genes in CEU and YRI populations and providing mechanistic evidence for genetic association studies of diseases for which angiogenesis plays a pathophysiologic role.

A metabolic shift favoring sphingosine 1-phosphate at the expense of ceramide controls glioblastoma angiogenesis.

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite sphingosine 1-phosphate (S1P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S1P over ceramide, which increases with increasing cancer grade. S1P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C18 ceramide, was on average 5-fold lower. Increased S1P content in the tumors was significantly correlated with increased sphingosine kinase 1 (SPHK1) and decreased sphingosine phosphate phosphatase 2 (SGPP2) expression. Inhibition of S1P production by cultured glioblastoma cells, using a highly potent and selective SPHK1 inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S1P balance is an important feature of human cancers and support the development of SPHK1 inhibitors as antiangiogenic agents for cancer therapy.

Molecular biology in breast cancer: intrinsic subtypes and signaling pathways.

The last decade has brought a breakthrough in the knowledge of the biology of breast cancer. The technological development, and in particular the high throughput technologies, have allowed researchers to inquire more deeply into the nature of the disease through the comparative study of large numbers of samples. The classification of breast cancer by traditional parameters has been joined by rankings based on gene expression. Among the most popular platforms are MammaPrint®, Oncotype DX® the wound-response model, the rate of two genes model, the genomic grade index and the intrinsic subtype model. The latter one provides the amplest biological information and allows for the classification of breast cancer into six intrinsic subtypes: luminal A, luminal B, HER2-enriched, basal-like, normal breast and claudin-low. These new classifications are not yet fully applicable to clinical practice not only because they have not been standardized, but also because they entail a substantial economic outlay. Nevertheless, they have provided valuable information on tumor biology that has led to a better understanding of the signaling pathways governing the processes of formation, maintenance and expansion of the tumors. Researchers now know more about the HER2, estrogen receptor, IGF1R, PI3K/AKT, mTOR, AMPK and angiogenesis pathways which has allowed for the development of new targeted therapeutics now being tested in ongoing clinical trials. In general, one can say that the last decade has changed the way researchers understand, classify and study breast cancer, and it has reshaped the way doctors diagnose and treat this disease. In addition, it has undoubtedly changed the search for alternative therapies by integrating molecular studies and the selection of study populations based on their molecular markers into clinical trials. The present review summarizes the advances that have allowed researchers to both better classify the disease, as well as explore some of the most important signaling pathways.

Adipose tissue-specific inhibition of hypoxia-inducible factor 1{alpha} induces obesity and glucose intolerance by impeding energy expenditure in mice.

Hypoxia in adipose tissue has been postulated as a possible contributor to obesity-related chronic inflammation, insulin resistance, and metabolic dysfunction. HIF1α (hypoxia-inducible factor 1α), a master signal mediator of hypoxia response, is elevated in obese adipose tissue. However, the role of HIF1α in obesity-related pathologies remains to be determined. Here we show that transgenic mice with adipose tissue-selective expression of a dominant negative version of HIF1α developed more severe obesity and were more susceptible to high fat diet-induced glucose intolerance and insulin resistance compared with their wild type littermates. Obesity in the transgenic mice was attributed to impaired energy expenditure and reduced thermogenesis. Histological examination of interscapular brown adipose tissue (BAT) in the transgenic mice demonstrated a markedly increased size of lipid droplets and decreased mitochondrial density in adipocytes, a phenotype similar to that in white adipose tissue. These changes in BAT of the transgenic mice were accompanied by decreased mitochondrial biogenesis and reduced expression of key thermogenic genes. In the transgenic mice, angiogenesis in BAT was decreased but was little affected in white adipose tissue. These findings support an indispensable role of HIF1α in maintaining the thermogenic functions of BAT, possibly through promoting angiogenesis and mitochondrial biogenesis in this tissue.

Assessment of angiogenesis by CD105 antigen in epithelial salivary gland neoplasms with diverse metastatic behavior.

BACKGROUND: Information on the biology of metastasis development in salivary gland tumors is scarce. Since angiogenesis seems associated with this phenomenon in other tumors, we sought to compare salivary gland tumors with diverse metastatic behavior in order to improve the knowledge and management of these lesions. METHODS: Samples from the most important salivary gland tumors were segregated according to its metastatic behavior and submitted to routine immunohistochemistry to identify vessels positive for CD105 expression. Frequency of positive cases and intratumoral microvessel density (IMD) was compared among the group of lesions. RESULTS: CD105 positive vessels were absent in normal salivary gland tissue, were rare in pleomorphic adenomas and adenoid cystic carcinomas (ACC), more common in polymorphous low-grade adenocarcinomas and highest in mucoepidermoid carcinomas. Only ACC with such feature were metastatic. IMD was higher in malignant rather than benign tumors. CONCLUSION: Immunostaining of CD105 in salivary gland tumors implies participation of angiogenesis in the development of malignant lesions, as well as some role for myoepithelial cells in the control of new vessel formation. In addition, suggest that ACC with positive CD105 vessels are at higher risk for metastasis.

Assessment of 193 candidate genes for retinopathy in African Americans with type 1 diabetes.

OBJECTIVE: To report in African Americans with type 1 diabetes the association of single-nucleotide polymorphisms in 193 candidate genes with diabetic retinopathy (DR) and/or its progression. METHODS: A custom panel of 1536 single-nucleotide polymorphisms located on 193 candidate genes for DR was genotyped in 437 African Americans with type 1 diabetes who participated in the New Jersey 725 study. Clinical evaluations at baseline and follow-up examinations included structured clinical interview, ocular examination, 7-field stereoscopic fundus photographs, and blood pressure measurements. Severity of DR was determined via masked grading of fundus photographs. Biological evaluations included blood and urine assays. RESULTS: Single-nucleotide polymorphisms in 13 candidate genes for DR involved in glucose metabolism, angiogenesis, inflammation, neurotransmission, hypertension, and retinal development were significantly associated with the prevalence of severe DR. Three of these genes were also significantly associated with progression of DR. Adjusting for sex, duration of diabetes, glycosylated hemoglobin, systemic hypertension, and total cholesterol did not alter the results. CONCLUSIONS: Our data support the role of genetic factors to account for severity and/or progression of DR in African Americans with type 1 diabetes and to identify several prime genes that likely contribute to the risk of DR.

MRI assessment of hemodynamic effects of angiopoietin-2 overexpression in a brain tumor model.

Despite treatment efforts, the median survival in patients with glioblastoma multiforme, the most aggressive form of glioma, does not extend beyond 12-15 months. One of the major pathophysiological characteristics of these tumors is their ability to induce active angiogenesis. Thus, based on the lack of efficient therapies, agents that inhibit angiogenesis are particularly attractive as a therapeutic option. However, it has been recently proposed that although specifically targeting vascular endothelial growth factor, the main angiogenic factor, certainly leads to significant tumor regression, it could also be followed by tumor relapses. In this case, angiogenesis is driven by alternate pathways that include other angiogenic factors. One possible strategy to overcome this therapeutic obstacle is to overexpress antivascular factors such as angiopoietin-2 (Ang2). Here, by using MRI and histological analysis, we studied the vascular events involved in glioma growth impairment induced by Ang2 overexpression. Our results show that an increase in Ang2 expression, during the tumor growth, leads to a significant decrease in tumor growth ( approximately 86%) along with an increase in the survival median ( approximately 70%) but does not modify the tumor vascular area or cerebral blood volume. However, tumor Ang2-derived blood vessels display an abnormal, enlarged morphology. We show that the presence of Ang2 leads to an enhancement of tumor perfusion and a decrease in tumor vessel permeability. Based on our MR image evaluations of hemodynamic tumor vessel changes, we propose that Ang2-derived tumor vessels lead to an inadequate oxygenation of the tumor tissue, leading to impairment in tumor growth.

Developing and applying a gene functional association network for anti-angiogenic kinase inhibitor activity assessment in an angiogenesis co-culture model.

BACKGROUND: Tumor angiogenesis is a highly regulated process involving intercellular communication as well as the interactions of multiple downstream signal transduction pathways. Disrupting one or even a few angiogenesis pathways is often insufficient to achieve sustained therapeutic benefits due to the complexity of angiogenesis. Targeting multiple angiogenic pathways has been increasingly recognized as a viable strategy. However, translation of the polypharmacology of a given compound to its antiangiogenic efficacy remains a major technical challenge. Developing a global functional association network among angiogenesis-related genes is much needed to facilitate holistic understanding of angiogenesis and to aid the development of more effective anti-angiogenesis therapeutics. RESULTS: We constructed a comprehensive gene functional association network or interactome by transcript profiling an in vitro angiogenesis model, in which human umbilical vein endothelial cells (HUVECs) formed capillary structures when co-cultured with normal human dermal fibroblasts (NHDFs). HUVEC competence and NHDF supportiveness of cord formation were found to be highly cell-passage dependent. An enrichment test of Biological Processes (BP) of differentially expressed genes (DEG) revealed that angiogenesis related BP categories significantly changed with cell passages. Built upon 2012 DEGs identified from two microarray studies, the resulting interactome captured 17226 functional gene associations and displayed characteristics of a scale-free network. The interactome includes the involvement of oncogenes and tumor suppressor genes in angiogenesis. We developed a network walking algorithm to extract connectivity information from the interactome and applied it to simulate the level of network perturbation by three multi-targeted anti-angiogenic kinase inhibitors. Simulated network perturbation correlated with observed anti-angiogenesis activity in a cord formation bioassay. CONCLUSION: We established a comprehensive gene functional association network to model in vitro angiogenesis regulation. The present study provided a proof-of-concept pilot of applying network perturbation analysis to drug phenotypic activity assessment.

In vivo assessment of RAS-dependent maintenance of tumor angiogenesis by real-time magnetic resonance imaging.

New blood vessel formation is a prominent feature of human cancers and tumor progression and is frequently accompanied by the acquisition of an angiogenic phenotype associated with a switch in the balance of proangiogenic and antiangiogenic molecules. This study was designed to investigate the role of activated H-RAS on the angiogenic phenotype of melanoma that arises in the inducible Tyr/Tet-RAS Ink4a/Arf(-/-) model using in vivo imaging with histopathologic correlation. We show that loss of RAS activity in fully established melanomas led to a reduction in tumor volume, which was preceded by impairment of vascular function as determined by in vivo magnetic resonance imaging. This correlated with activation of apoptosis in host-derived endothelial cells as well as in tumor cells. Thus, real-time in vivo imaging provided evidence that maintenance of tumor angiogenesis requires activated RAS in this model system, and that loss of vascular integrity upon inactivation of RAS is an active process rather than a consequence of loss of tumor cell viability.