Nicola H. Dryden

Endothelial von Willebrand factor regulates angiogenesis

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin.

Tight regulation of the balance between apoptosis and survival is essential in angiogenesis. The ETS transcription factor Erg is required for endothelial tube formation in vitro. Inhibition of Erg expression in human umbilical vein endothelial cells (HUVECs), using antisense oligonucleotides, resulted in detachment of cell-cell contacts and increased cell death. Inhibition of Erg expression by antisense in HUVECs also lowered expression of the adhesion molecule vascular endothelial (VE)-cadherin, a key regulator of endothelial intercellular junctions and survival. Using chromatin immunoprecipitation, we showed that Erg binds to the VE-cadherin promoter. Furthermore, Erg was found to enhance VE-cadherin promoter activity in a transactivation assay. Apoptosis induced by inhibition of Erg was partly rescued by overexpression of VE-cadherin-GFP, suggesting that VE-cadherin is involved in the Erg-dependent survival signals. To show the role of Erg in angiogenesis in vivo, we used siRNA against Erg in a Matrigel plug model. Erg inhibition resulted in a significant decrease in vascularization, with increase in caspase-positive endothelial cells (ECs). These results identify a new pathway regulating angiogenesis and endothelial survival, via the transcription factor Erg and the adhesion molecule VE-cadherin.

Regulation of angiogenesis by ETS transcription factors.

Transcription factors of the ETS family are important regulators of endothelial gene expression. Here, we review the evidence that ETS factors regulate angiogenesis and briefly discuss the target genes and pathways involved. Finally, we discuss novel evidence that shows how these transcription factors act in a combinatorial fashion with others, through composite sites that may be crucial in determining endothelial specificity in gene transcription.

The transcription factor Erg regulates expression of histone deacetylase 6 and multiple pathways involved in endothelial cell migration and angiogenesis.

The endothelial ETS transcription factor Erg plays an important role in homeostasis and angiogenesis by regulating many endothelial functions including survival and junction stability. Here we show that Erg regulates endothelial cell (EC) migration. Transcriptome profiling of Erg-deficient ECs identified ∼ 80 genes involved in cell migration as candidate Erg targets, including many regulators of Rho- GTPases. Inhibition of Erg expression in HUVECs resulted in decreased migration in vitro, while Erg overexpression using adenovirus caused increased migration. Live-cell imaging of Erg-deficient HUVECs showed a reduction in lamellipodia, in line with decreased motility. Both actin and tubulin cytoskeletons were disrupted in Erg-deficient ECs, with a dramatic increase in tubulin acetylation. Among the most significant microarray hits was the cytosolic histone deacetylase 6 (HDAC6), a regulator of cell migration. Chromatin immunoprecipitation (ChIP) and transactivation studies demonstrated that Erg regulates HDAC6 expression. Rescue experiments confirmed that HDAC6 mediates the Erg-dependent regulation of tubulin acetylation and actin localization. In vivo, inhibition of Erg expression in angiogenic ECs resulted in decreased HDAC6 expression with increased tubulin acetylation. Thus, we have identified a novel function for the transcription factor Erg in regulating HDAC6 and multiple pathways essential for EC migration and angiogenesis.

The Transcription Factor Erg Inhibits Vascular Inflammation by Repressing NF-κB Activation and Proinflammatory Gene Expression in Endothelial Cells

Objective—To test whether ETS-related gene (Erg) inhibits tumor necrosis factor (TNF)-&agr;–dependent endothelial activation and inflammation. Methods and Results—Endothelial activation underlies many vascular diseases, including atherosclerosis. Endothelial activation by proinflammatory cytokines decreases expression of the ETS transcription factor Erg. By using human umbilical vein endothelial cells (HUVECs), we showed that Erg overexpression by adenovirus (AdErg) repressed basal and TNF-&agr;–induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM), and interleukin 8 (IL-8). Erg inhibited TNF-&agr;–dependent activation of the ICAM-1 promoter, nuclear factor (NF)-&kgr;B activity, and NF-&kgr;B p65 phosphorylation. Basal NF-&kgr;B activity was also inhibited by Erg overexpression. Chromatin immunoprecipitation showed that Erg binds to the ICAM-1 proximal promoter region, which contains 7 putative ETS binding sites. To test the anti-inflammatory role of Erg in vivo, we used a murine model of TNF-&agr;–dependent acute inflammation. The injection of AdErg into the paw decreased TNF-&agr;–induced inflammation compared with control. Finally, staining of human coronary plaques showed loss of Erg expression from the endothelium overlaying active plaque shoulders. Conclusion—We have identified a novel physiological anti-inflammatory pathway under the control of the transcription factor Erg; this pathway inhibits NF-&kgr;B–dependent transcription and TNF-&agr;–induced inflammation in vivo. These results suggest a novel approach to anti-inflammatory therapies.

Synergistic Therapeutic Vascular Cytoprotection against Complement-Mediated Injury Induced via a PKCα-, AMPK-, and CREB-Dependent Pathway

Endothelial injury and dysfunction precede accelerated arterial disease in allograft vasculopathy and systemic autoimmune diseases and involve pathogenic Abs and complement. Recent reports suggest that switching to rapamycin from calcineurin antagonists reduces posttransplant vasculopathy and prolongs survival following cardiac transplantion. The majority of these patients also receive statin therapy. We examined potential mechanisms underlying this protective response in human endothelial cells and identified synergy between rapamycin and atorvastatin. Mechanistically, atorvastatin and rapamycin activated a protein kinase Cα, AMP-activated kinase, and CREB-dependent vasculoprotective pathway, which induced decay-accelerating factor (DAF) promoter activity via binding to the cAMP response element, mutation of which attenuated promoter activity. This response significantly increased endothelial cell surface DAF and enhanced protection against complement-mediated injury. Synergy with rapamycin was reproduced by simvastatin, whereas combining atorvastatin with cyclosporine or mycophenolate in place of rapamycin was ineffective. Importantly, synergy was reproduced in vivo, in which only atorvastatin and rapamycin therapy in combination was sufficient to induce DAF on murine aortic endothelium. We believe this pathway represents an important therapeutically inducible vasculoprotective mechanism for diseases mediated by pathogenic Abs and complement, including posttransplant vasculopathy and systemic lupus erythematosus. Although our study focuses on the vascular endothelium, the findings are likely to be broadly applicable, given the diverse cellular expression of DAF.

ICAM-2 regulates vascular permeability and N-cadherin localization through ezrin-radixin-moesin (ERM) proteins and Rac-1 signalling

BackgroundEndothelial junctions control functions such as permeability, angiogenesis and contact inhibition. VE-Cadherin (VECad) is essential for the maintenance of intercellular contacts. In confluent endothelial monolayers, N-Cadherin (NCad) is mostly expressed on the apical and basal membrane, but in the absence of VECad it localizes at junctions. Both cadherins are required for vascular development. The intercellular adhesion molecule (ICAM)-2, also localized at endothelial junctions, is involved in leukocyte recruitment and angiogenesis.ResultsIn human umbilical vein endothelial cells (HUVEC), both VECad and NCad were found at nascent cell contacts of sub-confluent monolayers, but only VECad localized at the mature junctions of confluent monolayers. Inhibition of ICAM-2 expression by siRNA caused the appearance of small gaps at the junctions and a decrease in NCad junctional staining in sub-confluent monolayers. Endothelioma lines derived from WT or ICAM-2-deficient mice (IC2neg) lacked VECad and failed to form junctions, with loss of contact inhibition. Re-expression of full-length ICAM-2 (IC2 FL) in IC2neg cells restored contact inhibition through recruitment of NCad at the junctions. Mutant ICAM-2 lacking the binding site for ERM proteins (IC2 ΔERM) or the cytoplasmic tail (IC2 ΔTAIL) failed to restore junctions. ICAM-2-dependent Rac-1 activation was also decreased in these mutant cell lines. Barrier function, measured in vitro via transendothelial electrical resistance, was decreased in IC2neg cells, both in resting conditions and after thrombin stimulation. This was dependent on ICAM-2 signalling to the small GTPase Rac-1, since transendothelial electrical resistance of IC2neg cells was restored by constitutively active Rac-1. In vivo, thrombin-induced extravasation of FITC-labeled albumin measured by intravital fluorescence microscopy in the mouse cremaster muscle showed that permeability was increased in ICAM-2-deficient mice compared to controls.ConclusionsThese results indicate that ICAM-2 regulates endothelial barrier function and permeability through a pathway involving N-Cadherin, ERMs and Rac-1.