“Large eaters” meet blood vessels: a new thematic series on macrophages and angiogenesis

Vascular Cell has launched a new series on macrophages and angiogenesis, a quickly evolving field critical to blood and lymphatic vessels during development, inflammation and tumorigenesis

Studies in Mice Reveal a Role for Anthrax Toxin Receptors in Matrix Metalloproteinase Function and Extracellular Matrix Homeostasis

The genes encoding Anthrax Toxin Receptors (ANTXRs) were originally identified based on expression in endothelial cells suggesting a role in angiogenesis. The focus of this review is to discuss what has been learned about the physiological roles of these receptors through evaluation of the Antxr knockout mouse phenotypes. Mice mutant in Antxr genes have defects in extracellular matrix homeostasis. We discuss how knowledge of physiological ANTXR function relates to what is already known about anthrax intoxication

Expression of HES and HEY genes in infantile hemangiomas

Background: Infantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs. Materials and Methods: HemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR. Results: HEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes. Conclusions: HES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells

The Effects of Genetic and Pharmacologic Loss of the Notch4 Protein on Angiogenesis

Angiogenesis is the process by which new blood vessels form, as cells undergoing hypoxia secrete Vascular Endothelial Growth Factor (VEGF), which prompts a tip cell phenotype in adjacent endothelial cells. The new tip cell expresses DLL4 and presents it on its surface, which binds to Notch receptors on neighboring cells and inhibits them from also becoming tip cells, forcing them to adopt the stalk cell phenotype. Notch signaling is critical to the tip/stalk cell fate decision, and both Notch1 and Notch4 are expressed in the vasculature, but not much is known about the different roles and parallel functions of Notch4 in contrast to the well-studied Notch1. In order to quantify the effects of removing Notch4, Notch4 mutants were generated via genetic mutations, or Notch4 function was inhibited by an anti-Notch4 neutralizing antibody. The vasculature of postnatal retina from control animals was compared to either knockout or anti-Notch4 treated mice on measures of radial outgrowth, tip cell count, vascular density, large vessels count, and branching. The experimental groups were then compared to each other to understand the degree to which pharmacologic inhibition recapitulates the genetic knockout phenotype. Our data shows that the Notch4 genetic knockout mice tend to exhibit decreased radial outgrowth and reduced branching. This supports the hypothesis that Notch1 and Notch4 have unique targets, as Notch1 knockouts show a stark increase in tip cell density, which is not seen in Notch4 genetic knockouts. In contrast, the pharmacologic Notch4 knockouts resembled the Notch4 genetic knockouts in radial outgrowth, but Notch1 mutants in tip cell density, though the reason why is not yet known. The mapping of angiogenic pathways is of consequence because it offers many avenues for studying human health. For example, new cancer research aims to reduce blood flow to tumors by decreasing angiogenesis, and published data suggests that knocking out Notch4 reduces tumor perfusion and growth. Therefore, a deeper understanding of the role of Notch4, and in turn, angiogenesis, could aid the development of new medicinal therapies for some of our most deadly diseases

Welcome to Journal of Angiogenesis Research

Angiogenesis is the growth of new blood vessels and is a key process which occurs during both physiological and pathological disease processes. Knowledge of the mechanisms through which this process is initiated and maintained will have a significant impact on the treatment of these diseases. Pathological angiogenesis occurs in major diseases such as cancer, diabetic retinopathies, age-related macular degeneration and atherosclerosis. In other diseases such as stroke and myocardial infarction, insufficient or improper angiogenesis results in tissue loss and ultimately higher morbidity and mortality

Lymphatics in health and disease: a new thematic series in vascular cell

Vascular Cell is launching new series on lymphatics, a vascular system required for physiological fluid balance and immunity, and whose damage leads to edema

Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity

In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis. We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression. Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression

Combined deficiency of Notch1 and Notch3 causes pericyte dysfunction, models CADASIL, and results in arteriovenous malformations

Pericytes regulate vessel stability and pericyte dysfunction contributes to retinopathies, stroke, and cancer. Here we define Notch as a key regulator of pericyte function during angiogenesis. In Notch1(+/-); Notch3(-/-) mice, combined deficiency of Notch1 and Notch3 altered pericyte interaction with the endothelium and reduced pericyte coverage of the retinal vasculature. Notch1 and Notch3 were shown to cooperate to promote proper vascular basement membrane formation and contribute to endothelial cell quiescence. Accordingly, loss of pericyte function due to Notch deficiency exacerbates endothelial cell activation caused by Notch1 haploinsufficiency. Mice mutant for Notch1 and Notch3 develop arteriovenous malformations and display hallmarks of the ischemic stroke disease CADASIL. Thus, Notch deficiency compromises pericyte function and contributes to vascular pathologies.Peer reviewe

Notch regulates the angiogenic response via induction of VEGFR-1

Notch is a critical regulator of angiogenesis and arterial specification. We show that ectopic expression of activated Notch1 induces endothelial morphogenesis in human umbilical vein endothelial cells (HUVEC) in a VEGFR-1-dependent manner. Notch1-mediated upregulation of VEGFR-1 in HUVEC increased their responsiveness to the VEGFR-1 specific ligand, Placental Growth Factor (PlGF). In mice and human endothelial cells, inhibition of Notch signaling resulted in decreased VEGFR-1 expression during VEGF-A-induced neovascularization. In summary, we show that Notch1 plays a role in endothelial cells by regulating VEGFR-1, a function that may be important for physiological and pathological angiogenesis

Inhibition of the Vascular Endothelial Cell (VE)-Specific Adhesion Molecule VE-Cadherin Blocks Gonadotropin-Dependent Folliculogenesis and Corpus Luteum Formation and Angiogenesis

Although it has been previously demonstrated that administration of anti-vascular endothelial growth factor (VEGF) receptor-2 antibodies to hypophysectomized (Hx) mice during gonadotropin-stimulated folliculogenesis and luteogenesis inhibits angiogenesis in the developing follicle and corpus luteum (CL), it is unclear which of the many components of VEGF inhibition are important for the inhibitory effects on ovarian angiogenesis. To examine whether ovarian angiogenesis can be more specifically targeted, we administered an antibody to VE-cadherin (VE-C), an interendothelial adhesion molecule, to Hx mice during gonadotropin stimulation. In tumor models and in vivo and in vitro assays, the anti-VE-C antibody E4G10 has been shown to specifically inhibit angiogenesis, but VE-C has yet to be inhibited in the context of ovarian angiogenesis. In addition to studying the effect on neovascularization in the follicular and luteal phases, we also examined the effect of E4G10 on established vessels of the CL of pregnancy. The results demonstrate that E4G10 specifically blocks neovascularization in the follicular and luteal phases, causing an inhibition of preovulatory follicle and CL development, a decrease in the vascular area, and an inhibition of function demonstrated by reduced hormone levels. However, when administered during pregnancy, unlike anti-VEGF receptor-2 antibody, E4G10 is unable to cause disruption of the established vessels of the mature CL. These data demonstrate that E4G10 causes a specific inhibition of neovascularization in the ovary without destabilizing preexisting vasculature