JS-K, a Novel Nitric Oxide (NO) Generator, Shows Potent Anti-Angiogenic Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3414-3414 ◽  
Author(s):  
Paul J. Shami ◽  
Gurmeet Kaur ◽  
Jagadambal Thillainathan ◽  
Lee Jia ◽  
Joseph E. Saavedra ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Cancer Therapeutics ◽  
Myelogenous Leukemia ◽  
Endothelial Cell Migration ◽  
In Vitro Assays ◽  
Endothelial Cell Proliferation

Abstract NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity in vitro and in vivo (Molecular Cancer Therapeutics 2:409-417,2003). The purpose of this study was to determine the effect of JS-K on angiogenesis. The anti-angiogenic properties of JS-K were tested in 3 different in vitro assays: proliferation, cord formation (reflecting new vessel formation) and migration using Human Umbilical Vein Endothelial Cells (HUVEC). JS-K inhibited the proliferation of HUVEC’s with a 50% inhibitory concentration (IC50) of 0.432, 0.466, and 0.505 μM at 24, 48, and 72 hours, respectively. At concentrations of 1 μM or above, HUVEC proliferation was totally inhibited. In the cord formation assay, treatment with JS-K lad to a decrease in both the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 μM, respectively. At a concentration of 1 μM, JS-K inhibited cord formation completely. JS-K inhibited cell migration at 5 hours using 10 ng/mL VEGF as a chemoattractant. At that time point, migration inhibition occurred at JS-K concentrations that did not affect cell growth with an IC50 of 0.493 μM. We conclude that JS-K is a potent inhibitor of 3 important elements of angiogenesis, namely endothelial cell proliferation, cord formation, and endothelial cell migration. These experiments identify a new mechanism by which JS-K and similar compounds may inhibit leukemia and solid tumor cell growth in vivo. Determining whether the anti-angiogenic effects of JS-K are NO-dependent will require further studies. (NO1-CO-12400).

Vascular endothelial growth factor (VEGF) in cartilage neovascularization and chondrocyte differentiation: auto-paracrine role during endochondral bone formation

2000 ◽  
Vol 113 (1) ◽  
pp. 59-69 ◽  
Author(s):  
M.F. Carlevaro ◽  
S. Cermelli ◽  
R. Cancedda ◽  
F. Descalzi Cancedda
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Hypertrophic Chondrocytes ◽  
Endothelial Cell Migration ◽  
Vegf Receptor ◽  
Hypertrophic Cartilage ◽  
Endothelial Growth Factor

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.

CYLD regulates angiogenesis by mediating vascular endothelial cell migration

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4130-4137 ◽  
Author(s):  
Jinmin Gao ◽  
Lei Sun ◽  
Lihong Huo ◽  
Min Liu ◽  
Dengwen Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial Cell ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
Vascular Endothelial

Cylindromatosis (CYLD) is a deubiquitinase that was initially identified as a tumor suppressor and has recently been implicated in diverse normal physiologic processes. In this study, we have investigated the involvement of CYLD in angiogenesis, the formation of new blood vessels from preexisting ones. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based 3-dimensional capillary sprouting assay. Disruption of CYLD also remarkably inhibits angiogenic response in vivo, as evidenced by diminished blood vessel growth into the angioreactors implanted in mice. Mechanistic studies show that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. Furthermore, we identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. Our findings thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Rac1 activation during endothelial cell migration and angiogenesis.

scholarly journals Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 349
Author(s):  
Devandir A. de Souza Junior ◽  
Carolina Santana ◽  
Gabriel V. Vieira ◽  
Constance Oliver ◽  
Maria Celia Jamur
Keyword(s):  
Endothelial Cells ◽  
Mast Cell ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Direct Action ◽  
Endothelial Cell Migration ◽  
Loop Formation ◽  
Mast Cell Protease

Previous studies from our laboratory have shown that during angiogenesis in vitro, rmMCP-7 (recombinant mouse mast cell protease-7) stimulates endothelial cell spreading and induces their penetration into the matrix. The ability of rmMCP-7 to induce angiogenesis in vivo was assessed in the present study using a directed in vivo angiogenesis assay (DIVAA™). Vessel invasion of the angioreactor was observed in the presence of rmMCP-7 but was not seen in the control. Since integrins are involved in endothelial cell migration, the relationship between rmMCP-7 and integrins during angiogenesis was investigated. Incubation with rmMCP-7 resulted in a reduction in the levels of integrin subunits αv and β1 on SVEC4-10 endothelial cells during angiogenesis in vitro. Furthermore, the degradation of integrin subunits occurs both through the direct action of rmMCP-7 and indirectly via the ubiquitin/proteasome system. Even in the presence of a proteasome inhibitor, incubation of endothelial cells with rmMCP-7 induced cell migration and tube formation as well as the beginning of loop formation. These data indicate that the direct degradation of the integrin subunits by rmMCP-7 is sufficient to initiate angiogenesis. The results demonstrate, for the first time, that mMCP-7 acts in angiogenesis through integrin degradation.

Functionally defining the endothelial transcriptome, from Robo4 to ECSCR

2009 ◽  
Vol 37 (6) ◽  
pp. 1214-1217 ◽  
Author(s):  
Ana Raquel Verissimo ◽  
John M.J. Herbert ◽  
Victoria L. Heath ◽  
John A. Legg ◽  
Helen Sheldon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Critical Role ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
In Vitro Assays ◽  
Filamin A ◽  
Interfering Rna

We have applied search algorithms to expression databases to identify genes whose expression is restricted to the endothelial cell. Such genes frequently play a critical role in endothelial biology and angiogenesis. Two such genes are the roundabout receptor Robo4 and the ECSCR (endothelial-cell-specific chemotaxis regulator). Endothelial cells express both Robo1 and Robo4, which we have knocked down using siRNA (small interfering RNA) and then studied the effect in a variety of in vitro assays. Both Robo4 and Robo1 knockdown inhibited in vitro tube formation on Matrigel™. Transfection of Robo4 into endothelial cells increased the number of filopodial extensions from the cell, but failed to do so in Robo1-knockdown cells. Separate immunoprecipitation studies showed that Robo1 and Robo4 heterodimerize. We conclude from this and other work that a heteroduplex of Robo1 and Robo4 signals through WASP (Wiskott–Aldrich syndrome protein) and other actin nucleation-promoting factors to increase the number of filopodia and cell migration. Knockdown of the transmembrane ECSCR protein in endothelial cells also reduced chemotaxis and impaired tube formation on Matrigel™. Yeast two-hybrid analysis and immunoprecipitation studies showed that, in contrast with the roundabouts, ECSCR binds to the actin-modulatory filamin A. We conclude that all three of these genes are critical for effective endothelial cell migration and, in turn, angiogenesis.

scholarly journals CD112 Regulates Angiogenesis and T Cell Entry into the Spleen

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 169
Author(s):  
Erica Russo ◽  
Peter Runge ◽  
Neda Haghayegh Jahromi ◽  
Heidi Naboth ◽  
Angela Landtwing ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
T Cell ◽  
Cell Entry ◽  
Endothelial Cell Migration ◽  
Leukocyte Trafficking ◽  
Deficient Mice

Junctional adhesion proteins play important roles in controlling angiogenesis, vascular permeability and leukocyte trafficking. CD112 (nectin-2) belongs to the immunoglobulin superfamily and was shown to engage in homophilic and heterophilic interactions with a variety of binding partners expressed on endothelial cells and on leukocytes. Recent in vitro studies suggested that CD112 regulates human endothelial cell migration and proliferation as well as transendothelial migration of leukocytes. However, so far, the role of CD112 in endothelial cell biology and in leukocyte trafficking has not been elucidated in vivo. We found CD112 to be expressed by lymphatic and blood endothelial cells in different murine tissues. In CD112-deficient mice, the blood vessel coverage in the retina and spleen was significantly enhanced. In functional in vitro studies, a blockade of CD112 modulated endothelial cell migration and significantly enhanced endothelial tube formation. An antibody-based blockade of CD112 also significantly reduced T cell transmigration across endothelial monolayers in vitro. Moreover, T cell homing to the spleen was significantly reduced in CD112-deficient mice. Overall, our results identify CD112 as a regulator of angiogenic processes in vivo and demonstrate a novel role for CD112 in T cell entry into the spleen.

Reduced TIMP-2 in hypoxia enhances angiogenesis

2011 ◽  
Vol 300 (3) ◽  
pp. C557-C566 ◽  
Author(s):  
Nitza Lahat ◽  
Haim Bitterman ◽  
Miri Engelmayer-Goren ◽  
Doron Rosenzweig ◽  
Lea Weiss-Cerem ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Protein Secretion ◽  
Endothelial Cell Migration ◽  
Human Endothelial Cells ◽  
Monocyte Migration

Hypoxia, which characterizes ischemia, trauma, inflammation, and solid tumors, recruits monocytes, immobilizes them, and alters their function, leading to an anti-inflammatory and proangiogenic phenotype. Monocyte extravasation from the circulation and their migration in tissues are partially mediated by the balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). The mechanisms evoked by hypoxia that regulate monocyte migration and activation are not entirely clear. Specifically, the effect of hypoxia on TIMPs in these cells has hardly been investigated. We show that hypoxia reduces TIMP-2 secretion from human primary monocytes and from the monocyte-like cell lines U937 and THP-1 by three- to fourfold ( P < 0.01), by inhibiting TIMP-2 transcription through mechanisms that involve the transcription factor SP-1. Hypoxia also lowers TIMP-2 protein secretion from human endothelial cells (by 2-fold, P < 0.05). TIMP-2 levels do not influence the reduced migration of THP-1 cells in hypoxia; however, low TIMP-2 levels enhance endothelial cell migration/proliferation, their ability to form tubelike structures in vitro, and the appearance of mature blood vessels in a Matrigel plug assay in vivo. Thus we conclude that reduced TIMP-2 levels secreted from both hypoxic monocytes and endothelial cells are proangiogenic.

Orally administered lenalidomide (CC-5013) is anti-angiogenic in vivo and inhibits endothelial cell migration and Akt phosphorylation in vitro

2005 ◽  
Vol 69 (1-2) ◽  
pp. 56-63 ◽  
Author(s):  
Keith Dredge ◽  
Rebecca Horsfall ◽  
Simon P. Robinson ◽  
Ling-Hua Zhang ◽  
Ling Lu ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Endothelial Cell Migration ◽  
Akt Phosphorylation

scholarly journals Urokinase-type plasminogen activator is induced in migrating capillary endothelial cells.

1987 ◽  
Vol 105 (6) ◽  
pp. 2535-2541 ◽  
Author(s):  
M S Pepper ◽  
J D Vassalli ◽  
R Montesano ◽  
L Orci
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Plasminogen Activator ◽  
Cellular Migration ◽  
Endothelial Cell Migration ◽  
Vascular Lesions ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Cellular migration is an essential component of invasive biological processes, many of which have been correlated with an increase in plasminogen activator production. Endothelial cell migration occurs in vivo during repair of vascular lesions and angiogenesis, and can be induced in vitro by wounding a confluent monolayer of cells. By combining the wounded monolayer model with a substrate overlay technique, we show that cells migrating from the edges of an experimental wound display an increase in urokinase-type plasminogen activator (uPA) activity, and that this activity reverts to background levels upon cessation of movement, when the wound has closed. Our results demonstrate a direct temporal relationship between endothelial cell migration and uPA activity, and suggest that induction of uPA activity is a component of the migratory process.

scholarly journals Expression of Connexin43 Stimulates Endothelial Angiogenesis Independently of Gap Junctional Communication In Vitro

2021 ◽  
Vol 22 (14) ◽  
pp. 7400
Author(s):  
Christoph Koepple ◽  
Zizi Zhou ◽  
Lena Huber ◽  
Matthias Schulte ◽  
Kjestine Schmidt ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Intercellular Communication ◽  
Endothelial Cell Migration ◽  
Dye Transfer ◽  
Cx43 Expression ◽  
Angiogenic Potential ◽  
Junctional Communication

Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy.

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