Notch alters VEGF responsiveness in human and murine endothelial cells by direct regulation of VEGFR-3 expression

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Abstract

The Notch family of cell surface receptors and its ligands are highly conserved proteins that regulate cell fate determination, including those involved in mammalian vascular development. We report that Notch induces VEGFR-3 expression in vitro in human endothelial cells and in vivo in mice. In vitro, Notch in complex with the DNA-binding protein CBF-1/suppressor of hairless/Lag1 (CSL) bound the VEGFR-3 promoter and transactivated VEGFR-3 specifically in endothelial cells. Through induction of VEGFR-3, Notch increased endothelial cell responsiveness to VEGF-C, promoting endothelial cell survival and morphological changes. In vivo, VEGFR-3 was upregulated in endothelial cells with active Notch signaling. Mice heterozygous for null alleles of both Notch1 and VEGFR-3 had significantly reduced viability and displayed midgestational vascular patterning defects analogous to Notch1 nullizygous embryos. We found that Notch1 and Notch4 were expressed in normal and tumor lymphatic endothelial cells and that Notch1 was activated in lymphatic endothelium of invasive mammary micropapillary carcinomas. These results demonstrate that Notch1 and VEGFR-3 interact genetically, that Notch directly induces VEGFR-3 in blood endothelial cells to regulate vascular development, and that Notch may function in tumor lymphangiogenesis.

Authors

Carrie J. Shawber, Yasuhiro Funahashi, Esther Francisco, Marina Vorontchikhina, Yukari Kitamura, Stephanie A. Stowell, Valeriya Borisenko, Nikki Feirt, Simona Podgrabinska, Kazuko Shiraishi, Kallayanee Chawengsaksophak, Janet Rossant, Domenico Accili, Mihaela Skobe, Jan Kitajewski

PSGL-1–mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells

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Abstract

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2–Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2–Fc–stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Authors

Philippe Foubert, Jean-Sébastien Silvestre, Boussad Souttou, Véronique Barateau, Coralie Martin, Téni G. Ebrahimian, Carole Leré-Déan, Jean Olivier Contreres, Eric Sulpice, Bernard I. Levy, Jean Plouët, Gérard Tobelem, Sophie Le Ricousse-Roussanne

Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo

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Abstract

This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad–/–) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad–/– mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad–/– mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-α–induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with Nω-nitro-l-arginine methyl ester (l-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.

Authors

Raogo Ouedraogo, Yulan Gong, Brett Berzins, Xiandong Wu, Kalyankar Mahadev, Kelly Hough, Lawrence Chan, Barry J. Goldstein, Rosario Scalia

Antiinflammatory adaptation to hypoxia through adenosine-mediated cullin-1 deneddylation

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Abstract

A major adaptive pathway for hypoxia is hypoxic preconditioning (HPC), a form of endogenous protection that renders cells tolerant to severe challenges of hypoxia. We sought to define the antiinflammatory properties of HPC. cDNA microarray analysis of lung tissue from mice subjected to hypoxia or HPC identified a cluster of NF-κB–regulated genes whose expression is attenuated by HPC. Studies using an NF-κB luciferase reporter assay confirmed a significant suppression of NF-κB activation during HPC. HPC-elicited activity was conferrable, as a soluble supernatant from HPC-treated cells, and the active fraction was purified and identified as adenosine (Ado). Guided by recent studies demonstrating bacterial inhibition of NF-κB through cullin-1 (Cul-1) deneddylation, we found a dose-dependent deneddylation of Cul-1 by Ado receptor stimulation predominantly mediated by the Ado A2B receptor subtype. Further, siRNA-mediated repression of CSN5, a subunit of the COP9 signalosome responsible for deneddylation of Cul-1, partially reversed HPC-mediated inhibition of NF-κB. Cul-1 deneddylation was evident in a murine model of HPC and lost in animals lacking extracellular Ado (Cd73–/– mice). Taken together, these results demonstrate that HPC induces extracellular accumulation of Ado and suppresses NF-κB activity through deneddylation of Cul-1. These results define a molecular regulatory pathway by which Ado provides potent antiinflammatory properties.

Authors

Joseph Khoury, Juan C. Ibla, Andrew S. Neish, Sean P. Colgan

Thrombospondins deployed by thrombopoietic cells determine angiogenic switch and extent of revascularization

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Abstract

Thrombopoietic cells may differentially promote or inhibit tissue vascularization by releasing both pro- and antiangiogenic factors. However, the molecular determinants controlling the angiogenic phenotype of thrombopoietic cells remain unknown. Here, we show that expression and release of thrombospondins (TSPs) by megakaryocytes and platelets function as a major antiangiogenic switch. TSPs inhibited thrombopoiesis, diminished bone marrow microvascular reconstruction following myelosuppression, and limited the extent of revascularization in a model of hind limb ischemia. We demonstrate that thrombopoietic recovery following myelosuppression was significantly enhanced in mice deficient in both TSP1 and TSP2 (TSP-DKO mice) in comparison with WT mice. Megakaryocyte and platelet levels in TSP-DKO mice were rapidly restored, thereby accelerating revascularization of myelosuppressed bone marrow and ischemic hind limbs. In addition, thrombopoietic cells derived from TSP-DKO mice were more effective in supporting neoangiogenesis in Matrigel plugs. The proangiogenic activity of TSP-DKO thrombopoietic cells was mediated through activation of MMP-9 and enhanced release of stromal cell–derived factor 1. Thus, TSP-deficient thrombopoietic cells function as proangiogenic agents, accelerating hemangiogenesis within the marrow and revascularization of ischemic hind limbs. As such, interference with the release of cellular stores of TSPs may be clinically effective in augmenting neoangiogenesis.

Authors

Hans-Georg Kopp, Andrea T. Hooper, M. Johan Broekman, Scott T. Avecilla, Isabelle Petit, Min Luo, Till Milde, Carlos A. Ramos, Fan Zhang, Tabitha Kopp, Paul Bornstein, David K. Jin, Aaron J. Marcus, Shahin Rafii

NF-κB regulation of endothelial cell function during LPS-induced toxemia and cancer

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Abstract

The transcription factor NF-κB is an important regulator of homeostatic growth and inflammation. Although gene-targeting studies have revealed important roles for NF-κB, they have been complicated by component redundancy and lethal phenotypes. To examine the role of NF-κB in endothelial tissues, Tie2 promoter/enhancer–IκBαS32A/S36A transgenic mice were generated. These mice grew normally but exhibited enhanced sensitivity to LPS-induced toxemia, notable for an increase in vascular permeability and apoptosis. Moreover, B16-BL6 tumors grew significantly more aggressively in transgenic mice, underscoring a new role for NF-κB in the homeostatic response to cancer. Tumor vasculature in transgenic mice was extensive and disorganized. This correlated with a marked loss in tight junction formation and suggests that NF-κB plays an important role in the maintenance of vascular integrity and response to stress.

Authors

Tatiana Kisseleva, Li Song, Marina Vorontchikhina, Nikki Feirt, Jan Kitajewski, Christian Schindler

Endothelial cell–restricted disruption of FoxM1 impairs endothelial repair following LPS-induced vascular injury

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Abstract

Recovery of endothelial integrity after vascular injury is vital for endothelial barrier function and vascular homeostasis. However, little is known about the molecular mechanisms of endothelial barrier repair following injury. To investigate the functional role of forkhead box M1 (FoxM1) in the mechanism of endothelial repair, we generated endothelial cell–restricted FoxM1-deficient mice (FoxM1 CKO mice). These mutant mice were viable and exhibited no overt phenotype. However, in response to the inflammatory mediator LPS, FoxM1 CKO mice displayed significantly protracted increase in lung vascular permeability and markedly increased mortality. Following LPS-induced vascular injury, FoxM1 CKO lungs demonstrated impaired cell proliferation in association with sustained expression of p27Kip1 and decreased expression of cyclin B1 and Cdc25C. Endothelial cells isolated from FoxM1 CKO lungs failed to proliferate, and siRNA-mediated suppression of FoxM1 expression in human endothelial cells resulted in defective cell cycle progression. Deletion of FoxM1 in endothelial cells induced decreased expression of cyclins, Cdc2, and Cdc25C, increased p27Kip1 expression, and decreased Cdk activities. Thus, FoxM1 plays a critical role in the mechanism of the restoration of endothelial barrier function following vascular injury. These data suggest that impairment in FoxM1 activation may be an important determinant of the persistent vascular barrier leakiness and edema formation associated with inflammatory diseases.

Authors

You-Yang Zhao, Xiao-Pei Gao, Yidan D. Zhao, Muhammad K. Mirza, Randall S. Frey, Vladimir V. Kalinichenko, I-Ching Wang, Robert H. Costa, Asrar B. Malik

Carcinoembryonic antigen–related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo

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Abstract

Carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1–/– mice, the Ceacam1 gene was deleted systemically, and in CEACAM1endo+ mice, CEACAM1 was overexpressed under the control of the endothelial cell–specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1–/– mice failed to establish new capillaries whereas in CEACAM1endo+ mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1–/– mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1endo+ mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.

Authors

Andrea Kristina Horst, Wulf D. Ito, Joachim Dabelstein, Udo Schumacher, Heike Sander, Claire Turbide, Jens Brümmer, Thomas Meinertz, Nicole Beauchemin, Christoph Wagener

Spare guanylyl cyclase NO receptors ensure high NO sensitivity in the vascular system

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Abstract

In the vascular system, the receptor for the signaling molecule NO, guanylyl cyclase (GC), mediates smooth muscle relaxation and inhibition of platelet aggregation by increasing intracellular cyclic GMP (cGMP) concentration. The heterodimeric GC exists in 2 isoforms (α1-GC, α2-GC) with indistinguishable regulatory properties. Here, we used mice deficient in either α1- or α2-GC to dissect their biological functions. In platelets, α1-GC, the only isoform present, was responsible for NO-induced inhibition of aggregation. In aortic tissue, α1-GC, as the major isoform (94%), mediated vasodilation. Unexpectedly, α2-GC, representing only 6% of the total GC content in WT, also completely relaxed α1-deficient vessels albeit higher NO concentrations were needed. The functional impact of the low cGMP levels produced by α2-GC in vivo was underlined by pronounced blood pressure increases upon NO synthase inhibition. As a fractional amount of GC was sufficient to mediate vasorelaxation at higher NO concentrations, we conclude that the majority of NO-sensitive GC is not required for cGMP-forming activity but as NO receptor reserve to increase sensitivity toward the labile messenger NO in vivo.

Authors

Evanthia Mergia, Andreas Friebe, Oliver Dangel, Michael Russwurm, Doris Koesling

Decreased vascular lesion formation in mice with inducible endothelial-specific expression of protein kinase Akt

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Abstract

To determine whether endothelial Akt could affect vascular lesion formation, mutant mice with a constitutively active Akt transgene, which could be inducibly targeted to the vascular endothelium using the tet-off system (EC-Akt Tg mice), were generated. After withdrawal of doxycycline, EC-Akt Tg mice demonstrated increased endothelial-specific Akt activity and NO production. After blood flow cessation caused by carotid artery ligation, neointimal formation was attenuated in induced EC-Akt Tg mice compared with noninduced EC-Akt Tg mice and control littermates. To determine the role of eNOS in mediating these effects, mice were treated with Nω-nitro-L-arginine methyl ester (L-NAME). Neointimal formation was attenuated to a lesser extent in induced EC-Akt Tg mice treated with L-NAME, suggesting that some of the vascular protective effects were NO independent. Indeed, endothelial activation of Akt resulted in less EC apoptosis in ligated arteries. Immunostaining demonstrated decreased inflammatory and proliferative changes in induced EC-Akt Tg mice after vascular injury. These findings indicate that endothelial activation of Akt suppresses lesion formation via increased NO production, preservation of functional endothelial layer, and suppression of inflammatory and proliferative changes in the vascular wall. These results suggest that enhancing endothelial Akt activity alone could have therapeutic benefits after vascular injury.

Authors

Yasushi Mukai, Yoshiyuki Rikitake, Ichiro Shiojima, Sebastian Wolfrum, Minoru Satoh, Kyosuke Takeshita, Yukio Hiroi, Salvatore Salomone, Hyung-Hwan Kim, Laura E. Benjamin, Kenneth Walsh, James K. Liao