Nuclear translocation of cGAS orchestrates VEGF-A-mediated angiogenesis

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Regulation of endothelial cell prostaglandin synthesis by glutathione

Prostaglandin synthesis in in vitro systems is dependent on glutathione and peroxide concentrations. We tested the effects of glutathione depletion and H2O2 exposure on prostaglandin synthesis in cultured porcine aortic endothelial cells. Depletion of glutathione using buthionine sulfoximine (BSO), diethylmaleate, and 2,4-chlorodinitrobenzene increased prostaglandin synthetic capacity. Production of prostacyclin, but not prostaglandin E2, from exogenous arachidonic acid was significantly greater than in controls. Glutathione depletion also resulted in enhanced production of prostacyclin from exogenous prostaglandin H2. These responses were not due to direct effects of glutathione-depleting agents on prostaglandin synthetic enzymes. Exposure to H2O2 also altered prostaglandin synthetic capacity in endothelial cells. While 5 microM H2O2 stimulated prostaglandin production from exogenous arachidonate, 25 and 50 microM were found to be inhibitory. Prostaglandin synthetic capacity was greater in BSO-treated cells which were exposed to 5 and 10 microM H2O2 than in cells exposed to H2O2 alone. However, prostaglandin synthetic capacity was greatly reduced in BSO-treated cells exposed to 50 microM H2O2. Thus, normal levels of cellular glutathione exert an inhibitory influence on prostaglandin synthesis. However, glutathione depletion increases the sensitivity of prostaglandin synthesis to inhibition by 50 microM H2O2.     

Regulation of brain endothelial cells migration and angiogenesis by P-glycoprotein/caveolin-1 interaction

We have investigated the involvement of P-glycoprotein (P-gp)/caveolin-1 interaction in the regulation of brain endothelial cells (EC) migration and tubulogenesis. P-gp overexpression in MDCK-MDR cells was correlated with enhanced cell migration whereas treatment with P-gp inhibitors CsA or PSC833 reduced it. Transfection of RBE4 rat brain endothelial cells with mutated versions of MDR1, in the caveolin-1 interaction motif, decreased the interaction between P-gp and caveolin-1, enhanced P-gp transport activity and cell migration. Moreover, down-regulation of caveolin-1 in RBE4 cells by siRNA against caveolin-1 stimulated cell migration. Interestingly, the inhibition of P-gp/caveolin-1 interaction increased also EC tubulogenesis. Furthermore, decrease of P-gp expression by siRNA inhibited EC tubulogenesis. These data indicate that the level of P-gp/caveolin-1 interaction can modulate brain endothelial angiogenesis and P-gp dependent cell migration.     

Regulation of tumor angiogenesis by thrombospondin-1

Angiogenesis plays a critical role in the growth and metastasis of tumors. Thrombospondin-1 (TSP-1) is a potent angiogenesis inhibitor, and down-regulation of TSP-1 has been suggested to alter tumor growth by modulating angiogenesis in a variety of tumor types. Expression of TSP-1 is up-regulated by the tumor suppressor gene, p53, and down-regulated by oncogenes such as Myc and Ras. TSP-1 inhibits angiogenesis by inhibiting endothelial cell migration and proliferation and by inducing apoptosis. In addition, activation of transforming growth factor beta (TGF-beta) by TSP-1 plays a crucial role in the regulation of tumor progression. An understanding of the molecular basis of TSP-1-mediated inhibition of angiogenesis and tumor progression will aid in the development of novel therapeutics for the treatment of cancer.     

Fbxw7 controls angiogenesis by regulating endothelial notch activity

Notch signaling controls fundamental aspects of angiogenic blood vessel growth including the selection of sprouting tip cells, endothelial proliferation and arterial differentiation. The E3 ubiquitin ligase Fbxw7 is part of the SCF protein complex responsible for the polyubiquitination and thereby proteasomal degradation of substrates such as Notch, c-Myc and c-Jun. Here, we show that Fbxw7 is a critical regulator of angiogenesis in the mouse retina and the zebrafish embryonic trunk, which we attribute to its role in the degradation of active Notch. Growth of retinal blood vessel was impaired and the Notch ligand Dll4, which is also a Notch target, upregulated in inducible and endothelial cell-specific Fbxw7(iECKO) mutant mice. The stability of the cleaved and active Notch intracellular domain was increased after siRNA knockdown of the E3 ligase in cultured human endothelial cells. Injection of fbxw7 morpholinos interfered with the sprouting of zebrafish intersegmental vessels (ISVs). Arguing strongly that Notch and not other Fbxw7 substrates are primarily responsible for these phenotypes, the genetic inactivation of Notch pathway components reversed the impaired ISV growth in the zebrafish embryo as well as sprouting and proliferation in the mouse retina. Our findings establish that Fbxw7 is a potent positive regulator of angiogenesis that limits the activity of Notch in the endothelium of the growing vasculature.     

Adenosine A2B receptor stimulates angiogenesis by inducing VEGF and eNOS in human microvascular endothelial cells

Angiogenesis is critical to wound repair due to its role in providing oxygen and nutrients that are required to support the growth and function of reparative cells in damaged tissues. Adenosine receptors are claimed to be of paramount importance in driving wound angiogenesis by inducing VEGF. However, the underlying mechanisms for the regulation of adenosine receptors in VEGF as well as eNOS remain poorly understood. In the present study, we found that adenosine and the non-selective adenosine receptor agonists (NECA) induced tube formation in HMEC-1 in a dose-dependent manner. Adenosine or NECA (10 µmol/L) significantly augmented the number and length of the segments in comparison with the control. Simultaneously, VEGF and eNOS were significantly upregulated following the administration of 10 µmol/L NECA, while they were suppressed after A_2B AR genetic silencing and pharmacological inhibition by MRS1754. In addition, VEGF expression and eNOS bioavailability elimination significantly reduced the formation of capillary-like structures. Furthermore, the activation of A_2B AR by NECA significantly increased the intracellular cAMP levels and concomitant CREB phosphorylation, eventually leading to the production of VEGF in HMEC-1. However, the activated PKA-CREB pathway seemed to be invalidated in the induction of eNOS. Moreover, we found that the elicited PI3K/AKT signaling in response to the induction of NECA assisted in regulating eNOS but failed to impact on VEGF generation. In conclusion, the A_2B AR activation-driven angiogenesis via cAMP-PKA-CREB mediated VEGF production and PI3K/AKT-dependent upregulation of eNOS in HMEC-1.     

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.     

Regulation of lymphocyte clustering by CD30-mediated ICAM-1 up-regulation

CD30 is expressed transiently on activated B and T lymphocytes and constitutively on several B- and T cell lymphomas. CD30 functions include participation in negative selection of thymocytes, costimulation of activated T cells, isotype switching of B cells, and regulation of the effector activity of cytotoxic lymphocytes. Although CD30 is not a marker for T helper 2 (TH2) cells, it may participate in the polarization of TH1 and TH2 cells. The pleiotropic functions of CD30 are initiated by interaction of CD30-expressing cells with other immune competent cells expressing CD30-L and providing the signals for modulation of effector cell activity. Here, we report that CD30 signals generated by anti-CD30 on activated, normal murine T cells strongly up-regulate the expression of intercellular adhesion molecule 1 (ICAM-1, CD54), and to a lesser extent, ICAM-2 (CD102). CD30 signals moreover delay the subsequent decline of ICAM expression. CD30 cross-linking did not alter the expression of CD11a/CD18 (LFA-1), the counter receptor for ICAM abundant on T cells. CD30-mediated ICAM-1 up-regulation is independent of cytokine secretion and appears to be transmitted directly through NF-kappaB activation. CD30-mediated up-regulation of ICAM-1 expression led to a significant increase in cluster formation of lymph node cells. Increased lymphocyte self-aggregation mediated by CD30 may set the stage for fraternal signaling to modulate lymphocyte function.     

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser¹¹⁷⁷ under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr⁴⁹⁵ in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG.In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.     

Human lactoferrin upregulates expression of KDR/Flk-1 and stimulates VEGF-A-mediated endothelial cell proliferation and migration

Lactoferrin (LF) is a multifunctional iron-binding glycoprotein, which plays a variety of biological processes including immunity. In this study, we demonstrate that human LF upregulates KDR/Flk-1 mRNA and protein levels in HUVECs at an optimal concentration of 5 microg/ml, which subsequently promotes the VEGF-induced proliferation and migration of the endothelial cells. Exposure of HUVECs to LF significantly increased VEGF-induced ERK MAP kinase phosphorylation. The maximal stimulation of KDR/Flk-1 expression by LF was correlated with LF-induced increase in cell proliferation and migration. These findings suggest that LF may stimulate in vivo angiogenesis via upregulation of KDR/Flk-1 expression in endothelial cells.     

Regulation of vascular endothelial growth factor receptor-2 activity by caveolin-1 and plasma membrane cholesterol

The stimulation of vascular endothelial growth factor receptor-2 (VEGFR-2) by tumor-derived VEGF represents a key event in the initiation of angiogenesis. In this work, we report that VEGFR-2 is localized in endothelial caveolae, associated with caveolin-1, and that this complex is rapidly dissociated upon stimulation with VEGF. The kinetics of caveolin-1 dissociation correlated with those of VEGF-dependent VEGFR-2 tyrosine phosphorylation, suggesting that caveolin-1 acts as a negative regulator of VEGF R-2 activity. Interestingly, we observed that in an overexpression system in which VEGFR-2 is constitutively active, caveolin-1 overexpression inhibits VEGFR-2 activity but allows VEGFR-2 to undergo VEGF-dependent activation, suggesting that caveolin-1 can confer ligand dependency to a receptor system. Removal of caveolin and VEGFR-2 from caveolae by cholesterol depletion resulted in an increase in both basal and VEGF-induced phosphorylation of VEGFR-2, but led to the inhibition of VEGF-induced ERK activation and endothelial cell migration, suggesting that localization of VEGFR-2 to these domains is crucial for VEGF-mediated signaling. Dissociation of the VEGFR-2/caveolin-1 complex by VEGF or cyclodextrin led to a PP2-sensitive phosphorylation of caveolin-1 on tyrosine 14, suggesting the participation of Src family kinases in this process. Overall, these results suggest that caveolin-1 plays multiple roles in the VEGF-induced signaling cascade.     

Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase

Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.     

Naftidrofuryl-driven regulation of endothelial ICAM-1 involves nitric oxide

Naftidrofuryl is a selective inhibitor of the 5-HT2 receptor expressed on human endothelial cells. This drug has been used over the years to cope with cerebral or peripheral ischemic accidents; however, no clear mechanism of action of this molecule has been highlighted to explain its vascular effects. In the present work, we demonstrate that the involvement of nitric oxide can account for the effects of naftidrofuryl. Indeed, naftidrofuryl potently inhibited the TNF-alpha-triggered increase of intercellular adhesion molecule-1 (ICAM-1) expression as well as stress fiber formation in human umbilical vein endothelial cells (HUVEC). Moreover, naftidrofuryl induced the expression of type II nitric oxide synthase (NOS II) messenger and protein, leading to a noticeable increase in nitric oxide synthesis. Furthermore, using the specific NOS II inhibitor 1400W, we verified that the observed effects of naftidrofuryl were NOS II-dependent. The biology of nitric oxide accounts for the reduction of the vasospasm associated with stroke and the strong inhibition of platelet aggregation. In conclusion, our work provides evidence for the inhibition of leukocyte recruitment by downregulation of CD54/ICAM-1, an additional key factor to be dealt with during thrombotic accidents. Importantly, it also highlights a novel NOS II-dependent mechanism of action for naftidrofuryl.     

Regulation of locomotion and cell-cell contact area by the LFA-1 and ICAM-1 adhesion receptors.

We demonstrate complementary differences in the behavior of B lymphoblastoid cells adhering to LFA-1 or its counter-receptor ICAM-1. The interaction of B lymphoblastoid cells with glass-supported planar bilayers bearing LFA-1 or ICAM-1 was observed by time-lapse video microscopy, and the distribution of adhesion receptors on cells interacting with the planar bilayers was studied by immunofluorescence microscopy. B lymphoblasts formed a large contact area and crawled rapidly (up to 25 microns/min) on planar bilayers bearing ICAM-1. In contrast, these cells attached to planar bilayers bearing LFA-1 through a fixed point about which the cells actively pivoted, using a single stalk-like projection. Phorbol ester-stimulated lymphoblasts, which adhere more strongly to ICAM-1-bearing substrates than unstimulated lymphoblasts, were still capable of locomotion on ICAM-1. Phorbol ester stimulation of B lymphoblasts on planar bilayers bearing LFA-1 promoted a rapid conversion from "stalk" attachment to symmetrical spreading of the cell on the substrate. Cellular LFA-1 remained uniformly distributed on the cell surface during interaction with bilayers bearing purified ICAM-1 as determined by immunofluorescence. In contrast, ICAM-1 was concentrated in the stalk-like structure through which the unstimulated B lymphoblasts adhered to LFA-1 in planar bilayers, but ICAM-1 immunofluorescence became more uniformly distributed over the cell surface within minutes of phorbol ester addition. Neither LFA-1 or ICAM-1 colocalized with the prominent staining of filamentous actin in the ruffling membrane regions. Interaction through cell surface LFA-1 and ICAM-1, 2, or 3 promotes different cellular morphologies and behaviors, the correlation of which with previously observed patterns of lymphocyte interaction with different cell types is discussed.