Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jun 15;111(12):5581-91.
doi: 10.1182/blood-2007-11-126680. Epub 2008 Apr 11.

An essential role for SRC-activated STAT-3 in 14,15-EET-induced VEGF expression and angiogenesis

Sergey Y Cheranov  1 , Manjula Karpurapu, Dong Wang, Baolin Zhang, Richard C Venema, Gadiparthi N Rao
Affiliations

An essential role for SRC-activated STAT-3 in 14,15-EET-induced VEGF expression and angiogenesis

Sergey Y Cheranov et al. Blood. .

Abstract

To understand the molecular mechanisms underlying 14,15-epoxyeicosatrienoic acid (14,15-EET)-induced angiogenesis, here we have studied the role of signal transducer and activator of transcription-3 (STAT-3). 14,15-EET stimulated the tyrosine phosphorylation of STAT-3 and its translocation from the cytoplasm to the nucleus in human dermal microvascular endothelial cells (HDMVECs). Adenovirus-mediated delivery of dominant negative STAT-3 substantially inhibited 14,15-EET-induced HDMVEC migration, and tube formation and Matrigel plug angiogenesis. 14,15-EET activated Src, as measured by its tyrosine phosphorylation and blockade of its activation by adenovirus-mediated expression of its dominant negative mutant, significantly attenuated 14,15-EET-induced STAT-3 phosphorylation in HDMVECs and the migration and tube formation of these cells and Matrigel plug angiogenesis. 14,15-EET induced the expression of vascular endothelial cell growth factor (VEGF) in a time- and Src-STAT-3-dependent manner in HDMVECs. Transfac analysis of VEGF promoter revealed the presence of STAT-binding elements and 14,15-EET induced STAT-3 binding to this promoter in vivo, and this interaction was inhibited by suppression of Src-STAT-3 signaling. Neutralizing anti-VEGF antibodies completely blocked 14,15-EET-induced HDMVEC migration and tube formation and Matrigel plug angiogenesis. These results reveal that Src-dependent STAT-3-mediated VEGF expression is a major mechanism of 14,15-EET-induced angiogenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
14,15-EET activates STAT-3 in HDMVECs. Quiescent HDMVECs were treated with and without 14,15-EET (0.1 μM) for the indicated times and either cell extracts (A) or the cytoplasmic and nuclear factions (C) were prepared and analyzed by Western blotting for pSTAT-3 using its phosphospecific antibodies. (B) The bar graph represents the quantitative analysis of the time course effect of 14,15-EET on tyrosine phosphorylation of STAT-3 in triplicate. Error bars represent SD. The blot in panel A was reprobed with anti–STAT-3 antibodies for normalization. For testing the purity of the cytoplasmic and nuclear preparations, the blot in panel C was reprobed sequentially with anti–STAT-3 and anti-p53 antibodies. *P < .01 versus control.
Figure 2
Figure 2
Adenovirus-mediated expression of dnSTAT-3 suppresses 14,15-EET–induced HDMVEC migration and tube formation in vitro and Matrigel plug angiogenesis in vivo. (A,B) HDMVECs were transduced with Ad-GFP or Ad-dnSTAT-3 at an MOI of 80, quiesced, and subjected to 14,15-EET–induced migration (A,B) or tube formation (C,D). (E,F) C57BL/6 mice were injected subcutaneously with 0.5 mL Matrigel premixed with vehicle or 50 μM 14,15-EET with and without Ad-GFP or Ad-dnSTAT-3 (5 × 109 pfu/mL). One week later, the animals were killed and the Matrigel plugs were harvested from underneath the skin and either analyzed for hemoglobin content using Drabkin reagent or immunostained for CD31 expression using anti-CD31 anti-bodies. The values in the bar graphs in panels B, D, and E are the means plus or minus SD of 3 independent experiments or 4 animals. *P < .01 vs Ad-GFP; **P < .01 vs AD-GFP + 14,15-EET.
Figure 3
Figure 3
14,15-EET activates Src in HDMVECs. (A) Quiescent HDMVECs were treated with and without 14,15-EET (0.1 μM) for the indicated times, and cell extracts were prepared and analyzed by Western blotting for pSrc using its phosphospecific antibodies. The blot was reprobed with anti-Src antibodies for normalization. (B) The bar graph represents the quantitative analysis of the time course effect of 14,15-EET on tyrosine phosphorylation of Src in triplicate. *P < .01 vs control. Error bars represent SD.
Figure 4
Figure 4
Adenovirus-mediated expression of dnSrc inhibits 14,15-EET–induced STAT-3 phosphorylation in HDMVECs. HDMVECs that were transduced with Ad-GFP or Ad-dnSrc at an MOI of 80 were treated with and without 14,15-EET (0.1 μM) for the indicated times, and cell extracts were prepared and analyzed by Western blotting for pSTAT-3 using its phosphospecific antibodies. The blot was reprobed sequentially with anti–STAT-3 antibodies for normalization and anti-Src antibodies for overexpression of Src.
Figure 5
Figure 5
Adenovirus-mediated expression of dnSrc suppresses 14,15-EET–induced HDMVEC migration and tube formation in vitro and Matrigel plug angiogenesis in vivo. (A,B) HDMVECs were transduced with Ad-GFP or Ad-dnSrc at an MOI of 80, quiesced, and subjected to 15(S)-HETE–induced migration (A,B) or tube formation (C,D). (E,F) C57BL/6 mice were injected subcutaneously with 0.5 mL Matrigel premixed with vehicle or 50 μM 14,15-EET with and without Ad-GFP or Ad-dnSrc (5 × 109 pfu/mL). One week later, the animals were killed and the Matrigel plugs were harvested from underneath the skin and analyzed for either hemoglobin content with Drabkin reagent or immunostained for CD31 expression using anti-CD31 antibodies. The values in the bar graphs in panels B, D, and E are the means plus or minus SD of 3 independent experiments or 4 animals. *P < .01 vs Ad-GFP; **P < .01 vs AD-GFP + 14,15-EET.
Figure 6
Figure 6
14,15-EET induces VEGF expression in a time-dependent manner in HDMVECs. Quiescent HDMVECs were treated with and without 14,15-EET (0.1 μM) for the indicated time periods and either RNA was isolated and analyzed for VEGF mRNA levels by RT-PCR (A), cell extracts were prepared and analyzed for VEGF levels by Western blotting (B), or the culture medium was collected and assayed for released VEGF by ELISA (C). The values in the bar graph in panel C are the means plus or minus SD of 3 independent experiments. *P < .01 vs control.
Figure 7
Figure 7
14,15-EET–induced VEGF expression is mediated by Src-STAT-3 signaling in HDMVECs. (A,B) HDMVECs were transduced with Ad-GFP, Ad-dnSrc, or Ad-dnSTAT-3 with an MOI of 80, quiesced, and treated with and without 14,15-EET (0.1 μM) for 2 hours, and RNA was isolated and analyzed for VEGF mRNA levels by RT-PCR (A), or for 6 hours, and the VEGF release into the culture medium was measured by ELISA (B). (C,E) Quiescent HDMVECs were treated with and without 14,15-EET (0.1 μM) for the indicated time periods and nuclear extracts were either prepared and analyzed by EMSA for STAT binding using [32P]-labeled STAT consensus sequence of VEGF promoter as a probe in vitro (C) or processed for ChIP analysis of STAT-3 binding to VEGF promoter in vivo (E). (D,F) HDMVECs that were transduced with Ad-GFP, Ad-dnSrc, or Ad-dnSTAT-3 with an MOI of 80 and quiesced were treated with and without 14,15-EET (0.1 μM) either for 30 minutes and nuclear extracts were prepared and analyzed by EMSA for STAT binding as described in panel C (D) or for 1 hour and processed for ChIP analysis of STAT-3 binding to VEGF promoter in vivo (F). The values in the bar graph in panel B are the means plus or minus SD of 3 independent experiments. *P < .01 vs Ad-GFP; **P < .01 vs AD-GFP + 14,15-EET.
Figure 8
Figure 8
Neutralizing anti-VEGF antibodies suppress 14,15-EET–induced HDMVEC migration and tube formation in vitro and Matrigel plug angiogenesis in vivo. Quiescent HDMVECs were treated with neutralizing anti-VEGF antibodies (3 μg/mL) for 30 minutes at 37°C followed by washing with medium 131. The cells were then subjected to 14,15-EET (0.1 μM)–induced migration (A,B) or tube formation (C,D) in the presence and absence of 3 μg/mL neutralizing anti-VEGF antibodies. (E,F) C57BL/6 mice were injected subcutaneously with 0.5 mL Matrigel premixed with vehicle or 50 μM 14,15-EET with and without 3 μg/mL neutralizing anti-VEGF antibodies. One week later, the animals were killed and the Matrigel plugs were harvested from underneath the skin and analyzed for either hemoglobin content with Drabkin reagent or immunostained for CD31 expression using anti-CD31 antibodies. Preimmune serum was added to Ad-GFP– and Ad-GFP + 14,15-EET–treated cells or mice. The values in the bar graphs in panels B, D, and E are the means plus or minus SD of 3 independent experiments or 4 animals. *P < .01 vs control; **P < .01 vs 14,15-EET.
Figure 9
Figure 9
The lack of effect of 14,15-EET on VEGF expression in HASMCs. Quiescent HASMCs were treated with and without 14,15-EET (0.1 μM) for the indicated time periods and either RNA was isolated and analyzed for VEGF mRNA levels by RT-PCR (A), cell extracts were prepared and analyzed for VEGF protein levels by Western blotting (B), or the culture medium was collected and assayed for released VEGF by ELISA (C). Error bars represent SD.
Figure 10
Figure 10
Schematic diagram showing how 14,15-EET induces angiogenesis in HDMVECs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Spector AA, Norris AW. Action of epoxyeicosatrienoic acids on cellular function. Am J Physiol. 2007;289:C996–C1012. - PubMed
    1. Fisslthaler B, Popp R, Kiss L, et al. Cytochrome P4502C is an EDHF synthase in coronary arteries. Nature. 1999;401:493–497. - PubMed
    1. Campbell WB, Harder DR. Endothelium-derived hyperpolarizing factors and vascular cytochrome P450 metabolites of arachidonic acid in the regulation of tone. Circ Res. 1999;84:484–488. - PubMed
    1. Michaelis UR, Fisslthaler B, Barbosa-Sicard E, Falck JR, Fleming I, Busse R. Cytochrome P450 epoxygenases 2C8 and 2C9 are implicated in hypoxia-induced endothelial cell migration and angiogenesis. J Cell Sci. 2005;118:5489–5498. - PubMed
    1. Chen JK, Capdevila J, Harris RC. Over expression of C-terminal Src kinase blocks 14,15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis. J Biol Chem. 2000;275:13789–13792. - PubMed

Publication types

MeSH terms

-