VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation.

Vascular endothelial growth factor (VEGF) guides the path of new vessel sprouts by inducing VEGF receptor-2 activity in the sprout tip. In the stalk cells of the sprout, VEGF receptor-2 activity is downregulated. Here, we show that VEGF receptor-2 in stalk cells is dephosphorylated by the endothelium-specific vascular endothelial-phosphotyrosine phosphatase (VE-PTP). VE-PTP acts on VEGF receptor-2 located in endothelial junctions indirectly, via the Angiopoietin-1 receptor Tie2. VE-PTP inactivation in mouse embryoid bodies leads to excess VEGF receptor-2 activity in stalk cells, increased tyrosine phosphorylation of VE-cadherin and loss of cell polarity and lumen formation. Vessels in ve-ptp(-/-) teratomas also show increased VEGF receptor-2 activity and loss of endothelial polarization. Moreover, the zebrafish VE-PTP orthologue ptp-rb is essential for polarization and lumen formation in intersomitic vessels. We conclude that the role of Tie2 in maintenance of vascular quiescence involves VE-PTP-dependent dephosphorylation of VEGF receptor-2, and that VEGF receptor-2 activity regulates VE-cadherin tyrosine phosphorylation, endothelial cell polarity and lumen formation.

Transcriptional profiling of human glioblastoma vessels indicates a key role of VEGF-A and TGFß2 in vascular abnormalization.

Glioblastoma are aggressive astrocytic brain tumours characterized by microvascular proliferation and an abnormal vasculature, giving rise to brain oedema and increased patient morbidity. Here, we have characterized the transcriptome of tumour-associated blood vessels and describe a gene signature clearly associated with pleomorphic, pathologically altered vessels in human glioblastoma (grade IV glioma). We identified 95 genes differentially expressed in glioblastoma vessels, while no significant differences in gene expression were detected between vessels in non-malignant brain and grade II glioma. Differential vascular expression of ANGPT2, CD93, ESM1, ELTD1, FILIP1L and TENC1 in human glioblastoma was validated by immunohistochemistry, using a tissue microarray. Through qPCR analysis of gene induction in primary endothelial cells, we provide evidence that increased VEGF-A and TGFß2 signalling in the tumour microenvironment is sufficient to invoke many of the changes in gene expression noted in glioblastoma vessels. Notably, we found an enrichment of Smad target genes within the distinct gene signature of glioblastoma vessels and a significant increase of Smad signalling complexes in the vasculature of human glioblastoma in situ. This indicates a key role of TGFß signalling in regulating vascular phenotype and suggests that, in addition to VEGF-A, TGFß2 may represent a new target for vascular normalization therapy.

Analysis of VEGF--a regulated gene expression in endothelial cells to identify genes linked to angiogenesis.

Angiogenesis is important for many physiological processes, diseases, and also regenerative medicine. Therapies that inhibit the vascular endothelial growth factor (VEGF) pathway have been used in the clinic for cancer and macular degeneration. In cancer applications, these treatments suffer from a "tumor escape phenomenon" where alternative pathways are upregulated and angiogenesis continues. The redundancy of angiogenesis regulation indicates the need for additional studies and new drug targets. We aimed to (i) identify novel and missing angiogenesis annotations and (ii) verify their significance to angiogenesis. To achieve these goals, we integrated the human interactome with known angiogenesis-annotated proteins to identify a set of 202 angiogenesis-associated proteins. Across endothelial cell lines, we found that a significant fraction of these proteins had highly perturbed gene expression during angiogenesis. After treatment with VEGF-A, we found increasing expression of HIF-1α, APP, HIV-1 tat interactive protein 2, and MEF2C, while endoglin, liprin ß1 and HIF-2α had decreasing expression across three endothelial cell lines. The analysis showed differential regulation of HIF-1α and HIF-2α. The data also provided additional evidence for the role of endothelial cells in Alzheimer's disease.

Transcriptional profiling reveals a critical role for tyrosine phosphatase VE-PTP in regulation of VEGFR2 activity and endothelial cell morphogenesis.

To define molecular events accompanying formation of the 3-dimensional (3D) vascular tube, we have characterized gene expression during vascular endothelial growth factor (VEGF)-induced tubular morphogenesis of endothelial cells. Microarray analyses were performed comparing gene induction in growth-arrested, tube-forming endothelial cells harvested from 3D collagen cultures to that in proliferating endothelial cells cultured on fibronectin. Differentially expressed genes were clustered and analyzed for specific endothelial expression through publicly available datasets. We validated the contribution of one of the identified genes, vascular endothelial protein tyrosine phosphatase (VE-PTP), to endothelial morphogenesis. Silencing of VE-PTP expression was accompanied by increased VEGF receptor-2 (VEGFR2) tyrosine phosphorylation and activation of downstream signaling pathways. The increased VEGFR2 activity promoted endothelial cell cycle progression, overcoming the G(0)/G(1) arrest associated with organization into tubular structures in the 3D cultures. Proximity ligation showed close association between VEGFR2 and VE-PTP in resting cells. Activation of VEGFR2 by VEGF led to rapid loss of association, which was resumed with time in parallel with decreased receptor activity. In conclusion, we have identified genes, which may serve critical functions in formation of the vascular tube. One of these, VE-PTP, regulates VEGFR2 activity thereby modulating the VEGF-response during angiogenesis.

FGFR-1 regulates angiogenesis through cytokines interleukin-4 and pleiotrophin.

The role of fibroblast growth factors (FGFs) in blood vessel formation has remained unclear. We used differentiating stem-cell cultures (embryoid bodies) and teratomas to show that FGF receptor-1 (FGFR-1) exerts a negative regulatory effect on endothelial cell function in these models. Embryoid bodies lacking expression of FGFR-1 as a result of gene targeting (Fgfr-1(-/-)) displayed increased vascularization and a distinct, elongated vessel morphology. Teratomas derived from FGFR-1-deficient stem cells were characterized by an increased growth rate and abundant, morphologically distinct vessels. Transmission electron microscopy of the Fgfr-1(-/-) teratomas showed a compact and voluminous but functional endothelium, which anastomosed with the host circulation. The increased vascularization and altered endothelial cell morphology was dependent on secreted factor(s), based on the transfer of the Fgfr-1(-/-) vascular phenotype by conditioned medium to Fgfr-1(+/-) embryoid bodies. Antibody and transcript arrays showed down-regulation of interleukin-4 (IL-4) and up-regulation of pleiotrophin in Fgfr-1(-/-) embryoid bodies, compared with the heterozygous cultures. We used neutralizing antibodies to show that IL-4 and pleiotrophin act as negative and positive angiogenic regulators, respectively. We conclude that FGFR-1 negatively regulates endothelial cell function by altering the balance of modulatory cytokines.

Endocan is a VEGF-A and PI3K regulated gene with increased expression in human renal cancer.

An in vitro model of VEGF-A-induced angiogenesis was used to generate transcription profiles of human microvascular endothelial cells. Microarray analysis showed increased transcription of genes known to regulate angiogenesis, but also genes that previously have not been firmly associated with angiogenesis such as endocan, pinin, plakophilin, phosphodiesterase 4B and gelsolin. Increased endocan mRNA levels in response to VEGF-A in endothelial cells and in human renal cancer have previously been reported. We now show increased endocan protein levels in VEGF-A treated endothelial cells and in human renal clear cell carcinoma. Increased protein expression was observed both in tumor cells and in a subset of tumor vessels, while expression in normal kidney tissue was low. VEGF-A seemed to be a specific inducer of endocan transcription since FGF-2, PDGF-BB, HGF/SF and EGF did not alter expression levels. Inhibition of PI3K with LY294002 caused a 12-fold increase in endocan transcription suggesting a repressive function of PI3K. In contrast inhibition of Src or MEK, which are signaling pathways activated by VEGF-A, did not influence basal or VEGF-A-induced endocan levels. In conclusion our study shows that, among angiogenic growth factors, VEGF-A is a specific inducer of endocan transcription which is translated into increased protein levels in VEGF-A treated endothelial cells. Increased endocan protein expression in human renal cancer suggests a role in tumor growth.