A core human primary tumor angiogenesis signature identifies the endothelial orphan receptor ELTD1 as a key regulator of angiogenesis.

Limited clinical benefits derived from anti-VEGF therapy have driven the identification of new targets involved in tumor angiogenesis. Here, we report an integrative meta-analysis to define the transcriptional program underlying angiogenesis in human cancer. This approach identified ELTD1, an orphan G-protein-coupled receptor whose expression is induced by VEGF/bFGF and repressed by DLL4 signaling. Extensive analysis of multiple cancer types demonstrates significant upregulation of ELTD1 in tumor-associated endothelial cells, with a higher expression correlating with favorable prognosis. Importantly, ELTD1 silencing impairs endothelial sprouting and vessel formation in vitro and in vivo, drastically reducing tumor growth and greatly improving survival. Collectively, these results provide insight into the regulation of tumor angiogenesis and highlight ELTD1 as key player in blood vessel formation

Combination of Reverse and Chemical Genetic Screens Reveals Angiogenesis Inhibitors and Targets

We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/ T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis

A Genome-Wide Scan of Ashkenazi Jewish Crohn's Disease Suggests Novel Susceptibility Loci

Crohn's disease (CD) is a complex disorder resulting from the interaction of intestinal microbiota with the host immune system in genetically susceptible individuals. The largest meta-analysis of genome-wide association to date identified 71 CD–susceptibility loci in individuals of European ancestry. An important epidemiological feature of CD is that it is 2–4 times more prevalent among individuals of Ashkenazi Jewish (AJ) descent compared to non-Jewish Europeans (NJ). To explore genetic variation associated with CD in AJs, we conducted a genome-wide association study (GWAS) by combining raw genotype data across 10 AJ cohorts consisting of 907 cases and 2,345 controls in the discovery stage, followed up by a replication study in 971 cases and 2,124 controls. We confirmed genome-wide significant associations of 9 known CD loci in AJs and replicated 3 additional loci with strong signal (p<5×10−6). Novel signals detected among AJs were mapped to chromosomes 5q21.1 (rs7705924, combined p = 2×10−8; combined odds ratio OR = 1.48), 2p15 (rs6545946, p = 7×10−9; OR = 1.16), 8q21.11 (rs12677663, p = 2×10−8; OR = 1.15), 10q26.3 (rs10734105, p = 3×10−8; OR = 1.27), and 11q12.1 (rs11229030, p = 8×10−9; OR = 1.15), implicating biologically plausible candidate genes, including RPL7, CPAMD8, PRG2, and PRG3. In all, the 16 replicated and newly discovered loci, in addition to the three coding NOD2 variants, accounted for 11.2% of the total genetic variance for CD risk in the AJ population. This study demonstrates the complementary value of genetic studies in the Ashkenazim

Correlating Global Gene Regulation to Angiogenesis in the Developing Chick Extra-Embryonic Vascular System

International audienceBACKGROUND: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis. METHOLOGY/PRINCIPAL FINDINGS: We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process. CONCLUSIONS/SIGNIFICANCE: Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis

DUAL EFFECTS OF FLAVONOID QUERCETIN ON RAT BASOPHILIC LEUKEMIA (RBL-2H3) CELLS : INHIBITS HISTAMINE RELEASE AND REDUCES CELL GROWTH

The dual effects of the flavonoid quercetin on rat basophilic leukemia cells (RBL-2H3), tumor analog mast cells, was studied. Quercetin is known as an anti-inflammatory drug that inhibits mast cell secretion of histamine. This study aims to investigate the consequences of varying incubation times with quercetin on 2H3 cells to histamine release inhibitory action and the effects of long time incubation to the morphology of the cells. The effect of quercetin on histamine synthesis was also observed. The histamine release from the cells was inhibited by quercetin as expected, but the ability of secretion was rapidly recovered when quercetin was removed before challenging. Incubation up to 6 hours decreased the inhibitory action, but longer than 6 hours increased the inhibitory activity. In long time incubation, the cells exhibited cell damage, decreased cell growth, morphological changes, and detachment from the underlying surface in proportion with the concentration of quercetin. The instant and reversible inhibitory effects of quercetin appear to represent a first phase of actions, while reduced cell growth, elevated cell damage and morphological changes seem to be connected to a second phase. Consequently, quercetin could be considered as a compound that acts dually on RBL-2H3 cells.Key words : quercetin, histamine, RBL-2H3 cell

Functional genomics of vascular endothelial cells

Angiogenesis, the formation of new blood vessels from preexisting ones, is a process involved in normal development as well as in several pathological conditions, such as cancer, ischemic heart disease, wound healing and certain retinal complications. Antiangiogenic targeting is therefore a promising new therapeutic principle. However, few blood vessel-specific drug targets have been identified, and information is still limited about endothelial cell (EC)-specific molecular processes. Here we aimed at identifying novel key players and signaling pathways during angiogenesis, and to determine the EC-specific core transcriptome in vivo. During angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A. We found that Delta-like 4 (DLL4)/Notch1 signaling regulated the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the developing postnatal retina. Inhibition of Notch-signaling led to excessive tip cell formation, and increased vascular density. Conversely, activation of Notch-signaling led to fewer tip cells and reduced vessel density. DLL4/Notch1-signaling between ECs therefore restricts tip cell formation in response to VEGF, leading to correct sprouting and branching patterns. We also found that blocking VEGF receptor 3 (VEGFR-3) signaling with antibodies resulted in decreased sprouting, vascular density, vessel branching, and EC proliferation. Antibodies against VEGFR-3 and VEGFR-2 in combination had additive effects. Notch inhibition led to endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3. These findings suggest that Notch and VEGFR-3 signaling may constitute new targets for anti-angiogenic therapy. In order to identify additional candidate vascular drug targets, we combined publicly available gene expression data with own transcriptional profiles of mouse microvasculature. In this way we identified 58 gene transcripts with broad and specific expression in microvascular endothelium, of which 32 presently lack known functions in vascular biology. 7 of the 32 genes showed considerably enriched expression in the microvasculature, namely: Eltd1, Gpr116, Ramp2, Slc9a3r2, Slc43a3, and NM_023516. The 32 gene products were all predicted to be cell surface expressed, or implicated in cell signaling processes, and are therefore interesting as putative microvascular drug targets. We also identified yet another set of new candidate vascular targets by combining reverse- and chemical genetics. In the reverse genetics screen, 50 genes were knocked down in zebrafish and 16 of these were found to be necessary for developmental angiogenesis. In the chemical genetics screen, 28 compounds targeting 69 proteins selectively inhibited endothelial sprouting. The reverse- and chemical genetics screens identified an overlap of three members of a superfamily of serine/threonine (S/T) protein phosphatases, Ppp1ca, Ppp1cc and Ppp4c, and one compound, Endothall, targeting that family. Treatment of zebrafish with Endothall led to a dose-dependent effect on lumen formation, similar to that seen in zebrafish knockdowns of the identified S/T protein phosphatases. The discoveries made in this study span from detailed insights into specific endothelial signaling pathways to global effects on endothelial gene expression, representing different angles of angiogenesis and vascular biology research. Overall, the results in this study contribute to the understanding of the vasculature and its transcriptome

Notch3 Is Necessary for Blood Vessel Integrity in the Central Nervous System

Objective— Vascular smooth muscle cells (VSMC) are important for contraction, blood flow distribution, and regulation of blood vessel diameter, but to what extent they contribute to the integrity of blood vessels and blood–brain barrier function is less well understood. In this report, we explored the impact of the loss of VSMC in the Notch3 −/− mouse on blood vessel integrity in the central nervous system. Approach and Results— Notch3 −/− mice showed focal disruptions of the blood–brain barrier demonstrated by extravasation of tracers accompanied by fibrin deposition in the retinal vasculature. This blood–brain barrier leakage was accompanied by a regionalized and patchy loss of VSMC, with VSMC gaps predominantly in arterial resistance vessels of larger caliber. The loss of VSMC appeared to be caused by progressive degeneration of VSMC resulting in a gradual loss of VSMC marker expression and a progressive acquisition of an aberrant VSMC phenotype closer to the gaps, followed by enhanced apoptosis and cellular disintegration in the gaps. Arterial VSMC were the only mural cell type that was morphologically affected, despite Notch3 also being expressed in pericytes. Transcriptome analysis of isolated brain microvessels revealed gene expression changes in Notch3 −/− mice consistent with loss of arterial VSMC and presumably secondary transcriptional changes were observed in endothelial genes, which may explain the compromised vascular integrity. Conclusions— We demonstrate that Notch3 is important for survival of VSMC, and reveal a critical role for Notch3 and VSMC in blood vessel integrity and blood–brain barrier function in the mammalian vasculature. </jats:sec