The angiogenic regulator CD13/APN is a transcriptional target of Ras signaling pathways in endothelial morphogenesis

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1818-1826 ◽  
Author(s):  
Shripad V. Bhagwat ◽  
Nenad Petrovic ◽  
Yasuhiro Okamoto ◽  
Linda H. Shapiro
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Blood Vessels ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Endothelial Cell Migration ◽  
Proximal Promoter ◽  
Limiting Factor ◽  
Ras Signaling

Angiogenesis, the formation of new blood vessels, is a critical step for tumor growth and metastasis and an integral component of the pathologic inflammatory response in arthritis and the proliferative retinopathies. The CD13/aminopeptidase N (CD13/APN) metalloprotease is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. Here, we show that cytokine induction of CD13/APN in endothelial cells is regulated by distinct Ras effector pathways involving Ras/mitogen-activated protein kinase (MAPK) or PI-3K. Signals transduced by activated Ras, Raf, and mitogen-induced extracellular kinase (MEK) stimulate transcription from theCD13/APN proximal promoter. Inhibition of these pathways and extracellular signal–regulated serine/threonine kinase (ERK-2) and PI-3K by expression of dominant-negative proteins or chemical inhibitors prevented induction of CD13/APNtranscription in response to basic fibroblast growth factor (bFGF). We show that Ras-induced signal transduction is required for growth factor–induced angiogenesis, because inhibition of downstream mediators of Ras signaling (MEK or PI-3K) abrogated endothelial cell migration, invasion, and morphogenesis in vitro. Reintroduction of CD13/APN, a shared downstream target of these pathways, overrode the suppressive effect of these inhibitors and restored the function of endothelial cells in migration/invasion and capillary morphogenesis assays. Similarly, inhibition of MEK abrogated cell invasion and the formation of endothelial-lined capillaries in vivo, which was effectively rescued by addition of exogenous CD13/APN protein. These studies provide strong evidence that CD13/APN is an important target of Ras signaling in angiogenesis and is a limiting factor in angiogenic progression.

Akt-1 Regulates Angiogenesis in Skin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 845-845
Author(s):  
Tatiana Byzova ◽  
Juhua Chen ◽  
Payaningal R. Somanath
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Vessels ◽  
Cell Function ◽  
Active Form ◽  
Endothelial Cell Migration ◽  
Matrix Composition ◽  
Adhesive Properties ◽  
Endothelial Cell Function

Abstract The major mechanism to adapt to ischemic conditions is the development of neovascularization, i.e. angiogenesis, a process driven by members of VEGF family of growth factors. Phosphoinositide 3-kinase/Akt pathway is a critical component of the signaling network that regulates endothelial cell function related to angiogenesis. VEGF treatment of endothelial cells results in rapid phosphorylation of Akt. Our studies demonstrated that Akt kinase activity is necessary for VEGF-induced and integrin-mediated endothelial cell adhesion and migration. Moreover, cell transfection with a constitutive active form of Akt (myr-Akt) leads to increased function of integrin receptors. Using Akt-1 null mice we found that Akt-1 controls VEGF-induced and integrin-dependent endothelial cell responses in vitro. Impaired endothelial cell migration and adhesion to extracellular matrix and a reduced rate of cell proliferation were observed in Akt-1 (−/−) endothelial cells compared to WT. There are three Akt isoforms with different tissue distribution, however, it appears that Akt-1 is a predominant isoform in skin and in skin microvasculature. This observation prompted us to perform series of in vivo experiments designed to assess the angiogenic response in skin in the absence of Akt-1. Angiogenesis assay using matrigel plugs revealed that the weight and hemoglobin content of matrigel plugs is about two fold higher in Akt (−/−) mice compared to WT mice. Tumor angiogenesis also appears to be enhanced in Akt(−/−) mice, resulting in the significantly lower degree of tumor necrosis. Blood vessels in Akt (−/−) mice appear to be smaller in diameter and have reduced laminin content. Our analysis revealed significant changes in blood vessel wall matrix composition of Akt (−/−) mice as compared to WT animals. These changes resulted in increased vascular permeability in skin of Akt (−/−) mice. Akt-1 is known to target multiple cellular processes including adhesive properties, cell survival, transcription and translation. It appears that the phenotype of Akt-1 (−/−) mice depends on the equilibrium between pro-angiogenic and anti-angiogenic roles of Akt-1 and reveals a central role for Akt-1 in the regulation of matrix production and maturation of blood vessels.

Platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its α receptor

Blood ◽  
2002 ◽  
Vol 99 (6) ◽  
pp. 2045-2053 ◽  
Author(s):  
Francesco De Marchis ◽  
Domenico Ribatti ◽  
Claudia Giampietri ◽  
Alessandro Lentini ◽  
Debora Faraone ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Cell Function ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Platelet Derived Growth Factor ◽  
Fibroblast Growth

Abstract Basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) modulate vascular wall cell function in vitro and angiogenesis in vivo. The aim of the current study was to determine how bovine aorta endothelial cells (BAECs) respond to the simultaneous exposure to PDGF-BB and bFGF. It was found that bFGF-dependent BAEC migration, proliferation, and differentiation into tubelike structures on reconstituted extracellular matrix (Matrigel) were inhibited by PDGF-BB. The role played by PDGF receptor α (PDGF-Rα) was investigated by selective stimulation with PDGF-AA, by blocking PDGF-BB-binding to PDGF-Rα with neomycin, or by transfecting cells with dominant-negative forms of the receptors to selectively impair either PDGF-Rα or PDGF-Rβ function. In all cases, PDGF-Rα impairment abolished the inhibitory effect of PDGF-BB on bFGF-directed BAEC migration. In addition, PDGF-Rα phosphorylation was increased in the presence of bFGF and PDGF, as compared to PDGF alone, whereas mitogen-activated protein kinase phosphorylation was decreased in the presence of PDGF-BB and bFGF compared with bFGF alone. In vivo experiments showed that PDGF-BB and PDGF-AA inhibited bFGF-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and that PDGF-BB inhibited bFGF-induced angiogenesis in Matrigel plugs injected subcutaneously in CD1 mice. Taken together these results show that PDGF inhibits the angiogenic properties of bFGF in vitro and in vivo, likely through PDGF-Rα stimulation.

Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function

1999 ◽  
Vol 112 (12) ◽  
pp. 2049-2057
Author(s):  
P. Gillis ◽  
U. Savla ◽  
O.V. Volpert ◽  
B. Jimenez ◽  
C.M. Waters ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Barrier Function ◽  
Keratinocyte Growth Factor ◽  
Mitogen Activated Protein Kinase ◽  
Microvascular Endothelial Cell ◽  
Vascular Permeability Factor ◽  
Small Vessels

Keratinocyte growth factor (KGF), also called fibroblast growth factor-7, is widely known as a paracrine growth and differentiation factor that is produced by mesenchymal cells and has been thought to act specifically on epithelial cells. Here it is shown to affect a new cell type, the microvascular endothelial cell. At subnanomolar concentrations KGF induced in vivo neovascularization in the rat cornea. In vitro it was not effective against endothelial cells cultured from large vessels, but did act directly on those cultured from small vessels, inducing chemotaxis with an ED50 of 0.02-0.05 ng/ml, stimulating proliferation and activating mitogen activated protein kinase (MAPK). KGF also helped to maintain the barrier function of monolayers of capillary but not aortic endothelial cells, protecting against hydrogen peroxide and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induced increases in permeability with an ED50 of 0.2-0.5 ng/ml. These newfound abilities of KGF to induce angiogenesis and to stabilize endothelial barriers suggest that it functions in microvascular tissue as it does in epithelial tissues to protect them against mild insults and to speed their repair after major damage.

Kallistatin is a new inhibitor of angiogenesis and tumor growth

Blood ◽  
2002 ◽  
Vol 100 (9) ◽  
pp. 3245-3252 ◽  
Author(s):  
Robert Q. Miao ◽  
Jun Agata ◽  
Lee Chao ◽  
Julie Chao
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Gene Delivery ◽  
Tumor Growth ◽  
Blood Vessels ◽  
Vascular Function ◽  
Hind Limb ◽  
Heparin Binding

Abstract Kallistatin is a unique serine proteinase inhibitor (serpin) and a heparin-binding protein. It has been localized in vascular smooth muscle cells and endothelial cells of human blood vessels, suggesting that kallistatin may be involved in the regulation of vascular function. Our previous study showed that kallistatin plays a role in neointima hyperplasia. In this study, we investigated the potential role of kallistatin in angiogenesis in vitro and in vivo. Purified human kallistatin significantly inhibited vascular endothelial growth factor (VEGF)– or basic fibroblast growth factor (bFGF)–induced proliferation, migration, and adhesion of cultured endothelial cells. Kallistatin attenuated VEGF- or bFGF-induced capillary density and hemoglobin content in subcutaneously implanted Matrigel plugs in mice. To further investigate the role of kallistatin in angiogenesis, we prepared adenovirus carrying the human kallistatin cDNA (Ad.HKBP) and evaluated the effect of kallistatin gene delivery on spontaneous angiogenesis in a rat model of hind-limb ischemia. Local kallistatin gene delivery significantly reduced capillary formation and regional blood perfusion recovery in the ischemic hind limb after removal of the femoral artery. Furthermore, a single intratumoral injection of Ad.HKBP into pre-established human breast tumor xenografts grown in athymic mice resulted in significant inhibition of tumor growth. CD31 immunostaining of tumor sections showed a decreased number of blood vessels in the kallistatin-treated group as compared to the control. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and tumor growth.

scholarly journals p38 MAP kinase negatively regulates endothelial cell survival, proliferation, and differentiation in FGF-2–stimulated angiogenesis

2002 ◽  
Vol 156 (1) ◽  
pp. 149-160 ◽  
Author(s):  
Taro Matsumoto ◽  
Ingela Turesson ◽  
Majlis Book ◽  
Pär Gerwins ◽  
Lena Claesson-Welsh
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial Cells ◽  
Chorioallantoic Membrane ◽  
Collagen Gel ◽  
P38 Map Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Chick Chorioallantoic Membrane

The p38 mitogen–activated protein kinase (p38) is activated in response to environmental stress and inflammatory cytokines. Although several growth factors, including fibroblast growth factor (FGF)-2, mediate activation of p38, the consequences for growth factor–dependent cellular functions have not been well defined. We investigated the role of p38 activation in FGF-2–induced angiogenesis. In collagen gel cultures, bovine capillary endothelial cells formed tubular growth-arrested structures in response to FGF-2. In these collagen gel cultures, p38 activation was induced more potently by FGF-2 treatment compared with that in proliferating cultures. Treatment with the p38 inhibitor SB202190 enhanced FGF-2–induced tubular morphogenesis by decreasing apoptosis, increasing DNA synthesis and cell proliferation, and enhancing the kinetics of cell differentiation including increased expression of the Notch ligand Jagged1. Overexpression of dominant negative mutants of the p38-activating kinases MKK3 and MKK6 also supported FGF-2–induced tubular morphogenesis. Sustained activation of p38 by FGF-2 was identified in vascular endothelial cells in vivo in the chick chorioallantoic membrane (CAM). SB202190 treatment enhanced FGF-2–induced neovascularization in the CAM, but the vessels displayed abnormal features indicative of hyperplasia of endothelial cells. These results implicate p38 in organization of new vessels and suggest that p38 is an essential regulator of FGF-2–driven angiogenesis.

scholarly journals Nogo-B receptor is essential for angiogenesis in zebrafish via Akt pathway

Blood ◽  
2010 ◽  
Vol 116 (24) ◽  
pp. 5423-5433 ◽  
Author(s):  
Baofeng Zhao ◽  
Changzoon Chun ◽  
Zhong Liu ◽  
Mark A. Horswill ◽  
Kallal Pramanik ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Umbilical Vein ◽  
Endothelial Cell Migration ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor

Abstract Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro. To investigate NogoB-NgBR function in vivo, we cloned the zebrafish ortholog of both genes and studied loss of function in vivo using morpholino antisense technology. Zebrafish ortholog of Nogo-B is expressed in somite while expression of zebrafish NgBR is localized in intersomitic vessel (ISV) and axial dorsal aorta during embryonic development. NgBR or Nogo-B knockdown embryos show defects in ISV sprouting in the zebrafish trunk. Mechanistically, we found that NgBR knockdown not only abolished its ligand Nogo-B–stimulated endothelial cell migration but also reduced the vascular endothelial growth factor (VEGF)–stimulated phosphorylation of Akt and vascular endothelial growth factor–induced chemotaxis and morphogenesis of human umbilical vein endothelial cells. Further, constitutively activated Akt (myristoylated [myr]Akt) or human NgBR can rescue the NgBR knockdown umbilical vein endothelial cell migration defects in vitro or NgBR morpholino-caused ISV defects in vivo. These data place Akt at the downstream of NgBR in both Nogo-B– and VEGF-coordinated sprouting of ISVs. In summary, this study identifies the in vivo functional role for Nogo-B and its receptor (NgBR) in angiogenesis in zebrafish.

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 471-478 ◽  
Author(s):  
W. Risau ◽  
H. Sariola ◽  
H.G. Zerwes ◽  
J. Sasse ◽  
P. Ekblom ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Blood Vessels ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Activity Increase ◽  
Vasculogenesis And Angiogenesis

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.

scholarly journals Integrin αvβ3 Requirement for Sustained Mitogen-activated Protein Kinase Activity during Angiogenesis

1998 ◽  
Vol 140 (5) ◽  
pp. 1255-1263 ◽  
Author(s):  
Brian P. Eliceiri ◽  
Richard Klemke ◽  
Staffan Strömblad ◽  
David A. Cheresh
Keyword(s):  
Endothelial Cells ◽  
Growth Factors ◽  
Growth Factor ◽  
Map Kinase ◽  
Blood Vessels ◽  
Integrin Αvβ3 ◽  
Protein Kinase Activity ◽  
Mitogen Activated Protein ◽  
Erk Activity

Angiogenesis depends on growth factors and vascular cell adhesion events. Integrins and growth factors are capable of activating the ras/MAP kinase pathway in vitro, yet how these signals influence endothelial cells during angiogenesis is unknown. Upon initiation of angiogenesis with basic fibroblast growth factor (bFGF) on the chick chorioallantoic membrane (CAM), endothelial cell mitogen-activated protein (MAP) kinase (ERK) activity was detected as early as 5 min yet was sustained for at least 20 h. The initial wave of ERK activity (5–120 min) was refractory to integrin antagonists, whereas the sustained activity (4–20 h) depended on integrin αvβ3, but not β1 integrins. Inhibition of MAP kinase kinase (MEK) during this sustained αvβ3-dependent ERK signal blocked the formation of new blood vessels while not influencing preexisting blood vessels on the CAM. Inhibition of MEK also blocked growth factor induced migration but not adhesion of endothelial cells in vitro. Therefore, angiogenesis depends on sustained ERK activity regulated by the ligation state of both a growth factor receptor and integrin αvβ3.

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