Inhibition of Protein Kinase Cα Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo

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.

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Isoprostanes Inhibit Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration, Tube Formation, and Cardiac Vessel Sprouting In Vitro, As Well As Angiogenesis In Vivo via Activation of the Thromboxane A 2 Receptor

Circulation Research ◽

10.1161/circresaha.108.184036 ◽

2008 ◽

Vol 103 (9) ◽

pp. 1037-1046 ◽

Cited By ~ 66

Author(s):

Ralf A. Benndorf ◽

Edzard Schwedhelm ◽

Anke Gnann ◽

Raihana Taheri ◽

Ghainsom Kom ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Cell Migration ◽

Endothelial Cell ◽

Growth Factor ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Vascular Endothelial ◽

Endothelial Growth Factor

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

Journal of Cell Science ◽

10.1242/jcs.113.1.59 ◽

2000 ◽

Vol 113 (1) ◽

pp. 59-69 ◽

Cited By ~ 8

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.

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DARC Regulates Angiogenesis by Mediating Vascular Endothelial Cell Migration

10.21203/rs.3.rs-115340/v1 ◽

2020 ◽

Author(s):

Xiaolin Wang ◽

Yongqian Bian ◽

Yuejun Li ◽

Jing Li ◽

Congying Zhao ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Vascular Endothelial Cell ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Immunofluorescent Staining ◽

Control Group ◽

Rhoa Activation ◽

And Control

Abstract Background: DARC (The Duffy antigen receptor for chemokines) is a kind of glycosylated membrane protein that binds to members of the CXC chemokine family associated with angiogenesis and has recently been reported to be implicated in diverse normal physiologic processes. This study aimed to investigate the involvement of DARC in angiogenesis, which is known to generate new capillary blood vessels from preexisting ones. Methods: HDMECs (Human dermal microvascular endothelial cells) were divided into two groups (DARC overexpression group, and control group). We used Brdu staining to detect cell proliferation, and wound healing assay to detect cell migration. Then tube formation assay were observed. Also, western blot and immunofluorescent staining were used to estimate the relationship between DARC and RhoA (Ras homolog gene family, member A). Results: HDMECs proliferation, migration, and tube formation were inhibited significantly when DARC was overexpressed intracellular. DARC impaired microfilament dynamics and intercellular connection in migrating cells, and RhoA activation underlay the effect of DARC on endothelial cell. Furthermore, DARC inhibited the formation of new capillaries in vitro. Conclusion: Our ﬁndings revealed the role of DARC in the angiogenic process and provided a novel mechanism for RhoA activation during endothelial cell migration and angiogenesis.

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Inactivation of Foxo3a and Subsequent Downregulation of PGC-1α Mediate Nitric Oxide-Induced Endothelial Cell Migration

Molecular and Cellular Biology ◽

10.1128/mcb.00175-10 ◽

2010 ◽

Vol 30 (16) ◽

pp. 4035-4044 ◽

Cited By ~ 52

Author(s):

Sara Borniquel ◽

Nieves García-Quintáns ◽

Inmaculada Valle ◽

Yolanda Olmos ◽

Brigitte Wild ◽

...

Keyword(s):

Nitric Oxide ◽

Cell Migration ◽

Endothelial Cell ◽

Protein Kinase ◽

Endothelial Cell Migration ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Endothelial Migration ◽

Endothelial Nitric Oxide

ABSTRACT In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1α downregulation and investigated the regulatory pathway involved. PGC-1α negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1α. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1α downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1α, and we found that a functional FoxO binding site in the PGC-1α promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1α is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1α and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.

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Mast Cell Protease 7 Promotes Angiogenesis by Degradation of Integrin Subunits

Cells ◽

10.3390/cells8040349 ◽

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.

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

Blood ◽

10.1182/blood.v104.11.3414.3414 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3414-3414 ◽

Cited By ~ 1

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).

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Involvement of Akt1/protein kinase Bα in tumor conditioned medium-induced endothelial cell migration and survival in vitro

Journal of Cancer Research and Clinical Oncology ◽

10.1007/s00432-009-0601-9 ◽

2009 ◽

Vol 135 (11) ◽

pp. 1543-1550 ◽

Cited By ~ 9

Author(s):

Ming Li Tu ◽

Han Qin Wang ◽

Long Ju Chen ◽

Jin Chang Lu ◽

Fei Jiang ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Protein Kinase ◽

Conditioned Medium ◽

Endothelial Cell Migration

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Angiomotin

The Journal of Cell Biology ◽

10.1083/jcb.152.6.1247 ◽

2001 ◽

Vol 152 (6) ◽

pp. 1247-1254 ◽

Cited By ~ 254

Author(s):

Boris Troyanovsky ◽

Tetyana Levchenko ◽

Göran Månsson ◽

Olga Matvijenko ◽

Lars Holmgren

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Leading Edge ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Kringle Domains ◽

A Cell ◽

Cell Type Specific

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type–specific manner. We have used a construct encoding the kringle domains 1–4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

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