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.

Towards a biofunctionalized vascular prosthesis: immune cell trapping via a growth factor receptor.

To improve the clinical performance of vascular prostheses, which is inacceptably low for implants with small diameters (< 6 mm), biofunctionalization of synthetic implants by endothelialization has become a major, although still unreached, aim. In order to be able to recruit native endothelial progenitor cells (EPCs) to luminal implant surfaces from the blood stream, we generated monoclonal antibodies against the EPC-specific vascular endothelial growth factor receptor 2 (VEGFR-2). Employing the very efficient genetic immunization strategy, > 10 000 hybridoma clones were generated. Screening with various deletion mutants of VEGFR-2, 49 highly-specific monoclonal antibodies (mAbs) covering all seven Ig domains of VEGFR-2 were selected. mAb 9H10 was characterized in detail. Once immobilized on synthetic surfaces, mAb 9H10 allowed, within min, nearly 100-fold enrichment of VEGFR-2-expressing cells under continuous flow conditions. Cell trapping was cell-type specific and essentially not affected by competing VEGFR-2-negative cells. To exclude that the antibody would adversely modify receptor responses, four different in vitro assays were employed. Cell proliferation, angiogenic tube formation, acetylated low-density lipoprotein uptake and VEGFR-2 phosphorylation remained unaffected, suggesting that the antibody did not interfere with the receptor functioning of human umbilical vascular endothelial cells. The molecular and cellular characteristics make the selected monoclonal antibody a very promising tool for the biofunctionalization of vascular implants. Copyright © 2016 John Wiley & Sons, Ltd.

Assessing heterogeneity of peroxisomes: isolation of two subpopulations from rat liver.

Peroxisomes exhibit a heterogeneous morphological appearance in rat liver tissue. In this respect, the isolation and subsequent biochemical characterization of peroxisome species from different subcellular prefractions should help to solve the question of whether peroxisomes indeed diverge into functionally specialized subgroups in one tissue. As a means to address this question, we provide a detailed separation protocol for the isolation of peroxisomes from both the light (LM-Po) and the heavy (HM-Po) mitochondrial prefraction for their subsequent comparative analysis. Both isolation strategies rely on centrifugation in individually adapted Optiprep gradients. In case of the heavy mitochondrial fraction, free flow electrophoresis is appended as an additional separation step to yield peroxisomes of sufficient purity. In view of their morphology, peroxisomes isolated from both fractions are surrounded by a continuous single membrane and contain a gray-opaque inner matrix. However, beyond this overall similar appearance, HM-Po exhibit a smaller average diameter, float at lower density, and show a more negative average membrane charge when compared to LM-Po.

No evidence for a functional role of bi-directional Notch signaling during angiogenesis.

The Delta-Notch pathway is a signal exchanger between adjacent cells to regulate numerous differentiation steps during embryonic development. Blood vessel formation by sprouting angiogenesis requires high expression of the Notch ligand DLL4 in the leading tip cell, while Notch receptors in the trailing stalk cells are activated by DLL4 to achieve strong Notch signaling activity. Upon ligand binding, Notch receptors are cleaved by ADAM proteases and gamma-secretase. This releases the intracellular Notch domain that acts as a transcription factor. There is evidence that also Notch ligands (DLL1, DLL4, JAG1, JAG2) are processed upon receptor binding to influence transcription in the ligand-expressing cell. Thus, the existence of bi-directional Delta-Notch signaling has been proposed. We report here that the Notch ligands DLL1 and JAG1 are processed in endothelial cells in a gamma-secretase-dependent manner and that the intracellular ligand domains accumulate in the cell nucleus. Overexpression of JAG1 intracellular domain (ICD) as well as DLL1-ICD, DLL4-ICD and NOTCH1-ICD inhibited endothelial proliferation. Whereas NOTCH1-ICD strongly repressed endothelial migration and sprouting angiogenesis, JAG1-ICD, DLL1-ICD and DLL4-ICD had no significant effects. Consistently, global gene expression patterns were only marginally affected by the processed Notch ligands. In addition to its effects as a transcription factor, NOTCH1-ICD promotes cell adhesion to the extracellular matrix in a transcription-independent manner. However, JAG1-ICD, DLL1-ICD and DLL4-ICD did not influence endothelial cell adhesion. In summary, reverse signaling of Notch ligands appears to be dispensable for angiogenesis in cellular systems.

Cerebral cavernous malformation protein CCM1 inhibits sprouting angiogenesis by activating DELTA-NOTCH signaling.

Cerebral cavernous malformations (CCM) are frequent vascular abnormalities caused by mutations in one of the CCM genes. CCM1 (also known as KRIT1) stabilizes endothelial junctions and is essential for vascular morphogenesis in mouse embryos. However, cellular functions of CCM1 during the early steps of the CCM pathogenesis remain unknown. We show here that CCM1 represents an antiangiogenic protein to keep the human endothelium quiescent. CCM1 inhibits endothelial proliferation, apoptosis, migration, lumen formation, and sprouting angiogenesis in primary human endothelial cells. CCM1 strongly induces DLL4-NOTCH signaling, which promotes AKT phosphorylation but reduces phosphorylation of the mitogen-activated protein kinase ERK. Consistently, blocking of NOTCH activity alleviates CCM1 effects. ERK phosphorylation is increased in human CCM lesions. Transplantation of CCM1-silenced human endothelial cells into SCID mice recapitulates hallmarks of the CCM pathology and serves as a unique CCM model system. In this setting, the multikinase inhibitor Sorafenib can ameliorate loss of CCM1-induced excessive microvascular growth, reducing the microvessel density to levels of normal wild-type endothelial cells. Collectively, our data suggest that the origin of CCM lesions is caused by perturbed Notch signaling-induced excessive capillary sprouting, which can be therapeutically targeted.

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.

Peroxisomes from the heavy mitochondrial fraction: isolation by zonal free flow electrophoresis and quantitative mass spectrometrical characterization.

Peroxisomes are a heterogeneous group of organelles fulfilling reactions in a variety of metabolic pathways. To investigate if functionally different subpopulations can be found within a single tissue, peroxisomes from the heavy mitochondrial fraction (HM-Po) of the rat liver were isolated and compared to "classic" peroxisomes from the light mitochondrial fraction (LM-Po) using iTRAQ tandem mass spectrometry. Peroxisomes represent only a minor although significant proportion of the heavy mitochondrial fraction (2700g(max)) precluding a straightforward isolation by standard protocols. Thus, a new fractionation scheme suitable for a subsequent mass spectrometrical analysis was developed using a combination of centrifugation techniques and zonal free flow electrophoresis. On the basis of the iTRAQ-measurement, a variation of the peroxisomal protein pattern between both fractions could be determined and further confirmed by immunoblotting and enzyme activity assays for selected proteins: whereas peroxisomes from the light mitochondrial fraction contain high amounts of beta-oxidation enzymes, peroxisomes from the heavy mitochondrial fraction were dominated by enzymes fulfilling other functions. Among other findings, HM-Po was characterized by a high abundance of D-amino acid oxidase. This observation can be mirrored at the ultrastructural level, where tissue sections of liver peroxisomes show a heterogeneous staining for the enzymes activity, when visualized by the cerium technique.