Barden Chan

Endostatin Causes G1 Arrest of Endothelial Cells through Inhibition of Cyclin D1

Endostatin, a type XVIII collagen fragment, is a potent antiangiogenic molecule that inhibits endothelial cell migration, promotes apoptosis, and induces cell cycle arrest in vitro. We have investigated the mechanism by which endostatin causes G1 arrest in endothelial cells. Endostatin decreased the hyperphosphorylated retinoblastoma gene product and down-regulated cyclin D1 mRNA and protein. Importantly, endostatin was unable to arrest cyclin D1 overexpressing endothelial cells, suggesting that cyclin D1 is a critical target for endostatin action. Next, we analyzed cyclin D1 promoter activity in endothelial cells and found that endostatin down-regulated the cyclin D1 promoter. Using a series of deletion and mutant promoter constructs, we identified the LEF1 site in the cyclin D1promoter as essential for the inhibitory effect of endostatin. Finally, we showed that endostatin can repress cyclin D1 promoter activity in cells over-expressing β-catenin but not in cells over-expressing a transcriptional activator that functions through the LEF1 site and is insensitive to β-catenin. Collectively, our data pointed to a role for cyclin D1, and in particular, transcription through the LEF1 site as critical for endostatin actionin vitro and suggest that β-catenin is a target for endostatin.

Endostatin is a potential inhibitor of Wnt signaling

Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. To gain insight into ES-mediated signaling, we studied the effects of ES RNA on Xenopus embryogenesis and observed developmental abnormalities consistent with impaired Wnt signaling. ES RNA blocked the axis duplication induced by β-catenin, partially suppressed Wnt-dependent transcription, and stimulated degradation of both wild-type and “stabilized” forms of β-catenin, the latter suggesting that ES signaling does not involve glycogen synthase kinase 3. Moreover, ES uses a pathway independent of the Siah1 protein in targeting β-catenin for proteasome-mediated degradation. ES failed to suppress the effects of T cell–specific factor (TCF)-VP16 (TVP), a constitutive downstream transcriptional activator that acts independently of β-catenin. Importantly, these data were replicated in endothelial cells and also in the DLD-1 colon carcinoma cells with the mutated adenomatous polyposis coli protein. Finally, suppression of endothelial cell migration and inhibition of cell cycle by ES were reversed by TVP. Though high levels of ES were used in both the Xenopus and endothelial cell studies and the effects on β-catenin signaling were modest, these data argue that at pharmacological concentrations ES may impinge on Wnt signaling and promote β-catenin degradation.

Angiopoietin-1 requires p190 RhoGAP to protect against vascular leakage in vivo.

Angiopoietin-1 (Ang-1), a ligand of the endothelium-specific receptor Tie-2, inhibits permeability in the mature vasculature, but the mechanism remains unknown. Here we show that Ang-1 signals Rho family GTPases to organize the cytoskeleton into a junction-fortifying arrangement that enhances the permeability barrier function of the endothelium. Ang-1 phosphorylates Tie-2 and its downstream effector phosphatidylinositol 3-kinase. This induces activation of one endogenous GTPase, Rac1, and inhibition of another, RhoA. Loss of either part of this dual effect abrogates the cytoskeletal and anti-permeability actions of Ang-1, suggesting that coordinated GTPase regulation is necessary for the vessel-sealing effects of Ang-1. p190 RhoGAP, a GTPase regulatory protein, provides this coordinating function as it is phosphorylated by Ang-1 treatment, requires Rac1 activation, and is necessary for RhoA inhibition. Ang-1 prevents the cytoskeletal and pro-permeability effects of endotoxin but requires p190 RhoGAP to do so. Treatment with p190 RhoGAP small interfering RNA completely abolishes the ability of Ang-1 to rescue endotoxemia-induced pulmonary vascular leak and inflammation in mice. We conclude that Ang-1 prevents vascular permeability by regulating the endothelial cytoskeleton through coordinated and opposite effects on the Rho GTPases Rac1 and RhoA. By linking Rac1 activation and RhoA inhibition, p190 RhoGAP is critical to the protective effects of Ang-1 against endotoxin. These results provide mechanistic evidence that targeting the endothelium through Tie-2 may offer specific therapeutic strategies in life-threatening endotoxemic conditions such as sepsis and acute respiratory distress syndrome.

Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tiel, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tiel. We report here that Angl induces Tiel phosphorylation in endothelial cells. Tiel phosphorylation was, however, Tie2 dependent because 1) Angl failed to induce Tiel phosphorylation when Tie2 was down‐regulated in endothelial cells;2) Tiel phosphorylation was induced in the absence of Angl by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Angl phosphorylated a form of Tiel without kinase activity when coexpressed with Tie2, and Angl failed to phos‐phorylate Tiel when coexpressed with kinase‐defective Tie2. Angl‐mediated AKT and 42/44MAPK phosphor‐ylation is predominantly Tie2 mediated, and Tiel down‐regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tiel is to modulate blood vessel morphogenesis by virtue of its ability to down‐regulate Tie2‐driven signaling and endothelial survival. Our new observations help to explain why Tiel null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine‐tuned by the interplay between closely related receptors by a single growth factor.—Yuan, H. T., Venkatesha, S., Chan, B., Deutsch, U., Mam‐moto, T., Sukhatme, V. P., Woolf, A. S., Karumanchi, S. A. Activation of the orphan endothelial receptor Tiel modifies Tie2‐mediated intracellular signaling and cell survival. FASEB J. 2l, 3l7l–3l83 (2007)

Microarray analysis of in vitro pericyte differentiation reveals an angiogenic program of gene expression

The vasculature consists of endothelial cells (ECs) lined by pericyte/vascular smooth muscle cells (vSMCs). Pericyte/vSMCs provide support to the mature vasculature but are also essential for normal blood vessel development. To determine how pericyte‐EC communication influences vascular development, we used the well‐established in vitro model of TGFβ‐stimulated differentiation of 10T1/2 cells into pericyte/vSMCs. Microarray analysis was performed to identify genes that were differentially expressed by induced vs. uninduced 10T1/2 cells. We discovered that these cells show an angiogenic program of gene expression, with up‐regulation of several genes previously implicated in angiogenesis, including VEGF, IL‐6, VEGF‐C, HB‐EGF, CTGF, tenascin C, integrin α5, and Eph receptor A2. Up‐regulation of some genes was validated by Western blots and immunocytochemistry. We also examined the functional significance of these gene expression changes. VEGF and IL‐6 alone and in combination were important in 10T1/2 cell differentiation. Furthermore, we used a coculture system of 10T1/2 and human umbilical vein ECs (HUVECs), resulting in the formation of cordlike structures by the HUVECs. This cordlike structure formation was disrupted when neutralizing antibodies to VEGF or IL‐6 were added to the coculture system. The results of these studies show that factors produced by pericytes may be responsible for recruiting ECs and promoting angiogenesis. Therefore, a further understanding of the genes involved in pericyte differentiation could provide a novel approach for developing anti‐angiogenic therapies.

Critical roles of CD146 in zebrafish vascular development

In this report, we use zebrafish as a model system to understand the importance of CD146 in vascular development. Endothelial‐specific expression of CD146 was verified by whole‐mount in situ hybridization. Suppression of CD146 protein expression by antisense morpholino oligonucleotides (MO) resulted in poorly developed intersomitic vessels (ISVs). In CD146 morphants, we observed a lack of blood flow through the ISV region, despite that fluorescence microangiography showed that the ISVs were present. This finding suggests that the lumens of the developing ISVs may be too narrow for proper circulation. Additionally, remodeling of the caudal vein plexus into functional vascular tubes appeared to be affected. Suppression of CD146 protein expression resulted in a circulation shunt that caused the circulation to by‐pass part of the caudal artery/vein system. The same vascular defects were recapitulated by using a second morpholino oligonucleotide. This morphant expressed a truncated CD146 protein with amino acids V32 to T57 at the N terminus deleted in an in‐frame manner. This region, therefore, is likely to contain elements critical for CD146 function. This study provides the first in vivo functional assessment of CD146 in embryonic development by showing that knockdown of CD146 protein expression severely hinders vascular development in zebrafish. Developmental Dynamics 232:232–244, 2005.

Glycolytic cancer cells lacking 6-phosphogluconate dehydrogenase metabolize glucose to induce senescence

We show that knockdown of 6‐phosphogluconate dehydrogenase (6PGD) of the pentose phosphate pathway (PPP) inhibits growth of lung cancer cells by senescence induction. This inhibition is not due to a defect in the oxidative PPP per se. NADPH and ribose phosphate production are normal in 6PGD knockdown cells and shutdown of PPP by knockdown of glucose‐6‐phosphate dehydrogenase (G6PD) has little effect on cell growth. Moreover, 6PGD knockdown cells can proliferate when the PPP is bypassed by using fructose instead of glucose in medium. Significantly, G6PD knockdown rescues proliferation of cells lacking 6PGD, suggesting an accumulation of growth inhibitory glucose metabolics in cells lacking 6PGD. Therefore, 6PGD inhibition may provide a novel strategy to treat glycolyic tumors such as lung cancer.

Receptor tyrosine kinase EphA2 mediates thrombin-induced upregulation of ICAM-1 in endothelial cells in vitro

Thrombin potently induces endothelial inflammation. One of the responses is upregulation of adhesion molecules such as ICAM-1, resulting in enhanced leukocyte attachment to the endothelium. In this report, we examine the contribution of EphA2 in thrombin-induced expression of ICAM-1 in human umbilical vein endothelial cells (HUVECs). We showed that thrombin transiently induced tyrosine- phosphorylation of EphA2 in a Src-kinase dependent manner. This transactivation was mediated through PAR-1, because a PAR-1 specific agonistic peptide also transactivated EphA2. Expression knockdown of endogenous EphA2 by siRNAs blocked ICAM-1 upregulation and leukocyte/endothelium attachment induced by thrombin. Overexpression of exogenous mouse EphA2 rescued both ICAM-1 expression and leukocyte attachment induced by thrombin in endogenous EphA2-knockdown HUVECs. Mechanistically, we showed EphA2 knockdown suppressed thrombin-induced serine 536 phosphorylation of NFkappaB, an event critical of ICAM-1 transcriptional upregulation. Collectively, our results strongly suggest EphA2 is a necessary component for thrombin-induced ICAM-1 upregulation.

Receptor tyrosine kinase Tie-1 overexpression in endothelial cells upregulates adhesion molecules.

Tie-1 is an endothelial specific cell surface protein whose biology remains poorly understood. Using an overexpression system in vitro, we examined whether Tie-1 activity in endothelial cells in vitro would elicit a proinflammatory response. We found that when overexpressed in endothelial cells in vitro, Tie-1 is tyrosine-phosphorylated. We also showed that Tie-1 upregulates VCAM-1, E-selectin, and ICAM-1, partly through a p38-dependent mechanism. Interestingly, upregulation of VCAM-1 and E-selectin by Tie-1 is significantly higher in human aortic endothelial cells than in human umbilical vein endothelial cells. Additionally, attachment of cells of monocytic lineage to endothelial cells is also enhanced by Tie-1 expression. Collectively, our data show that Tie-1 has a proinflammatory property and may play a role in the endothelial inflammatory diseases such as atherosclerosis.

Antiangiogenic property of human thrombin.

Using protein chromatography, we purified and identified human prothrombin from human plasma as antiangiogenic. Prothrombin significantly inhibited endothelial cell tube formation in vitro at 10 microg/ml. Importantly, it also inhibited bFGF-induced angiogenesis in Matrigel-plug assays performed in mice. The proteolytic activity of thrombin appeared to be critical for the antiangiogenic activity of prothrombin. For example, thrombin exhibited inhibitory effects on endothelial cell tube formation in vitro at 10 U/ml. Addition of lepirudin, a specific inhibitor of thrombin, completely blocked prothrombins and thrombins antiangiogenic effects in vitro. We also assessed the importance of thrombin receptors in angiogenesis. Using small peptides that activate different protease-activated receptors (PARs), we showed that activation of PAR-1 led to inhibition of endothelial cell tube formation in vitro and bFGF-induced angiogenesis in vivo. Collectively, our data suggest that thrombins proteolytic activity can be antiangiogenic.