Inhibition of Endothelial Notch Signaling Impairs Fatty Acid Transport and Leads to Metabolic and Vascular Remodeling of the Adult Heart.

BACKGROUND: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. METHODS: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. RESULTS: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice. CONCLUSIONS: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.

Synaptojanin-2 binding protein stabilizes the Notch ligands DLL1 and DLL4 and inhibits sprouting angiogenesis.

RATIONALE: The formation of novel blood vessels is initiated by vascular endothelial growth factor. Subsequently, DLL4-Notch signaling controls the selection of tip cells, which guide new sprouts, and trailing stalk cells. Notch signaling in stalk cells is induced by DLL4 on the tip cells. Moreover, DLL4 and DLL1 are expressed in the stalk cell plexus to maintain Notch signaling. Notch loss-of-function causes formation of a hyperdense vascular network with disturbed blood flow. OBJECTIVE: This study was aimed at identifying novel modifiers of Notch signaling that interact with the intracellular domains of DLL1 and DLL4. METHODS AND RESULTS: Synaptojanin-2 binding protein (SYNJ2BP, also known as ARIP2) interacted with the PDZ binding motif of DLL1 and DLL4, but not with the Notch ligand Jagged-1. SYNJ2BP was preferentially expressed in stalk cells, enhanced DLL1 and DLL4 protein stability, and promoted Notch signaling in endothelial cells. SYNJ2BP induced expression of the Notch target genes HEY1, lunatic fringe (LFNG), and ephrin-B2, reduced phosphorylation of ERK1/2, and decreased expression of the angiogenic factor vascular endothelial growth factor (VEGF)-C. It inhibited the expression of genes enriched in tip cells, such as angiopoietin-2, ESM1, and Apelin, and impaired tip cell formation. SYNJ2BP inhibited endothelial cell migration, proliferation, and VEGF-induced angiogenesis. This could be rescued by blockade of Notch signaling or application of angiopoietin-2. SYNJ2BP-silenced human endothelial cells formed a functional vascular network in immunocompromised mice with significantly increased vascular density. CONCLUSIONS: These data identify SYNJ2BP as a novel inhibitor of tip cell formation, executing its functions predominately by promoting Delta-Notch signaling.

Integrin cytoplasmic domain-associated protein-1 attenuates sprouting angiogenesis.

RATIONALE: The ICAP1 (integrin cytoplasmic domain-associated protein-1) is a specific intracellular binding protein of beta1-integrins and the cerebral cavernous malformation (CCM) protein CCM1. ICAP1 recruits CCM1 to the cell membrane and activates CCM1 by changing its conformation. Because CCM1 plays a critical role for cardiovascular development, we hypothesized that its activator ICAP1 is involved in vascular differentiation. OBJECTIVE: The objective of this study was to define the role of ICAP1 in endothelial cells. METHODS AND RESULTS: Loss of ICAP1 in primary human endothelial cells causes excessive angiogenic branching and network formation in vitro (3D sprouting angiogenesis) and in vivo (xenotransplantation of ICAP1-silenced human endothelial cells). ICAP1 increases cell motility and the initial formation of capillary sprouts but prevents vessel outgrowth. ICAP1 inhibits Rho kinase activity and ERK (extracellular signal-regulated kinase) phosphorylation and induces expression of the cell cycle inhibitors p21 and p27, leading to less endothelial proliferation. However, ICAP1 promotes endothelial survival and AKT phosphorylation. Global gene expression analyses revealed that the ICAP1 effects are mediated by strong activation of DELTA-NOTCH signaling. Active NOTCH1 or silencing of the NOTCH ligand DLL4 phenocopy the ICAP1 effects and blockade of NOTCH cleavage rescues the ICAP1-mediated defects in endothelial cells. Both ICAP1 and NOTCH1 reduce the expression of ESM1 (endothelial cell-specific molecule-1), and silencing of ESM1 disturbs vascular endothelial growth factor- or fibroblast growth factor 2-induced sprouting angiogenesis. CONCLUSIONS: In this study, we identified ICAP1 as a novel regulator to prevent excessive sprouting angiogenesis.

Endothelial Notch1 Activity Facilitates Metastasis.

Endothelial cells (ECs) provide angiocrine factors orchestrating tumor progression. Here, we show that activated Notch1 receptors (N1ICD) are frequently observed in ECs of human carcinomas and melanoma, and in ECs of the pre-metastatic niche in mice. EC N1ICD expression in melanoma correlated with shorter progression-free survival. Sustained N1ICD activity induced EC senescence, expression of chemokines and the adhesion molecule VCAM1. This promoted neutrophil infiltration, tumor cell (TC) adhesion to the endothelium, intravasation, lung colonization, and postsurgical metastasis. Thus, sustained vascular Notch signaling facilitates metastasis by generating a senescent, pro-inflammatory endothelium. Consequently, treatment with Notch1 or VCAM1-blocking antibodies prevented Notch-driven metastasis, and genetic ablation of EC Notch signaling inhibited peritoneal neutrophil infiltration in an ovarian carcinoma mouse model.

ANKS1B Interacts with the Cerebral Cavernous Malformation Protein-1 and Controls Endothelial Permeability but Not Sprouting Angiogenesis.

Cerebral cavernous malformations are fragile blood vessel conglomerates in the central nervous system that are caused by mutations in the CCM1/KRIT1, CCM2 or CCM3 genes. The gene products form a protein complex at adherens junctions and loss of either CCM protein disrupts endothelial cell quiescence leading to increased permeability and excessive angiogenesis. We performed a yeast 2-hybrid screen to identify novel proteins directly interacting with KRIT1. The ankyrin repeat and sterile alpha motif domain-containing protein 1B (ANKS1B) was identified as a novel binding partner of KRIT1. Silencing of ANKS1B or the related gene ANKS1A in primary human endothelial cells had no significant effects on cellular proliferation, migration and sprouting angiogenesis. However, silencing of ANKS1B expression disturbed endothelial cell barrier functions leading to increased permeability. Forced ANKS1B expression reduced permeability. This was independent of Rho kinase activity and the presence of KRIT1. Taken together, ANKS1B was identified as a novel KRIT1-interacting protein that selectively controls endothelial permeability but not angiogenesis.