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Cancer Research 67, 4328, May 1, 2007. doi: 10.1158/0008-5472.CAN-06-3594
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

15(S)-Hydroxyeicosatetraenoic Acid–Induced Angiogenesis Requires STAT3-Dependent Expression of VEGF

Kalyan Srivastava, Venkatesh Kundumani-Sridharan, Baolin Zhang, Arun K. Bajpai and Gadiparthi N. Rao

Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Requests for reprints: Gadiparthi N. Rao, Department of Physiology, University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163. Phone: 901-448-7321; Fax: 901-448-7126; E-mail: grao{at}physio1.utmem.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
15(S)-Hydroxyeicosatetraenoic acid [15(S)-HETE] activated signal transducer and activator of transcription 3 (STAT3) as measured by its tyrosine phosphorylation, translocation from the cytoplasm to the nucleus, DNA binding, and reporter gene activity in human dermal microvascular endothelial cells (HDMVEC). Inhibition of STAT3 activation via adenovirus-mediated expression of its dominant-negative mutant suppressed 15(S)-HETE–induced HDMVEC migration and tube formation in vitro and aortic ring and Matrigel plug angiogenesis in vivo. 15(S)-HETE induced the expression of vascular endothelial growth factor (VEGF) in a time- and STAT3-dependent manner in HDMVEC. In addition, neutralizing anti-VEGF antibodies blocked 15(S)-HETE–induced HDMVEC migration and tube formation in vitro and aortic ring and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that 15(S)-HETE–induced angiogenesis requires STAT3-dependent expression of VEGF. In view of these findings, it is suggested that eicosanoids, particularly 15(S)-HETE, via its capacity to stimulate angiogenesis, may influence the progression of cancer and vascular disease. [Cancer Res 2007;67(9):4328–36]


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Angiogenesis, the formation of new blood vessels, is an adaptive phenomenon to meet the tissue/cellular requirement for oxygen/nutrients and thus plays an important role in development and wound healing (1, 2). Angiogenesis also plays a prominent role in the progression of cancer and vascular diseases such as atherosclerosis and restenosis (3–5). A large body of data indicates that lipoxygenases (LOX) are involved in the development of various types of cancers (6–9). It is known that oxidation of low-density lipoprotein (LDL) is a contributing factor in the pathogenesis of atherosclerosis (10, 11). In addition, many studies have shown that LOX, particularly 15-LOX, via its role in the oxidation of LDL, is also involved in the pathogenesis of atherosclerosis (12, 13). Furthermore, accumulated evidence suggests a role for LOX in restenosis as well (14). Towards understanding the mechanisms underlying the involvement of LOX in the pathogenesis of cancer and vascular diseases, it was reported that 12-hydroxyeicosatetraenoic acid [12(S)-HETE], the 12-LOX product of arachidonic acid, influences the growth and/or motility of cancer and vascular smooth muscle cells (15, 16). Similarly, 12(S)-HETE has been shown to stimulate angiogenesis (17). With regard to the role of other LOX and their products in the pathogenesis of the above mentioned diseases, it was shown that 15-LOX1, while inhibiting colorectal cancer cell growth (18, 19), stimulated prostate cancer cell growth (7, 8, 20). In contrast, 15-LOX2 inhibited prostate cancer growth (21). 15-LOX1, while preferentially converting linoleic acid to 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE], also metabolizes arachidonic acid to 15(S)-HETE and 12(S)-HETE (22). On the other hand, 15-LOX2 mainly converts arachidonic acid to 15(S)-HETE (22). However, only 13(S)-HODE mimicked the 15-LOX1 effects on the inhibition of colorectal cancer cell growth (18, 19). With regard to 15(S)-HETE, the arachidonic acid product of 15-LOX2, it exhibited proangiogenic activity in endothelial cells (23) and enhanced the growth of some cancer cell types such as erythroid cells (6). Thus, 15-LOX1/2 and their products 13(S)-HODE and 15(S)-HETE exhibited differential effects in different cancer cell types. Because 15(S)-HETE induced growth in some cancer cell types, and because its levels have been found increased in atherosclerosis (24), earlier, we attempted to understand the possible mechanisms by which this eicosanoid influences cancer and vessel wall diseases and showed that it stimulates angiogenesis (25). In addition, we have shown that 15(S)-HETE–induced angiogenesis depends on activation of phosphatidylinositol 3-kinase (PI3K)/Akt/S6K1 signaling.

The Janus-activated kinase/signal transducer and activator of transcription (Jak/STAT) signaling plays an important role in the regulation of cell growth, differentiation, and migration (26–29). Using pharmacologic approach, the involvement for the Jak/STAT pathway in the vessel wall diseases has also been reported (30). In addition, it was shown that STAT3 plays a role in angiogenesis in response to various stimulants (31, 32). To unravel the signaling mechanisms by which 15(S)-HETE influences angiogenesis, and thereby cancer and vascular diseases, here, we have extended our studies to examine the role of the Jak/STAT pathway. In the present communication, we report for the first time that 15(S)-HETE activates STAT3 in human dermal microvascular endothelial cells (HDMVEC). In addition, our results show that 15(S)-HETE–induced angiogenesis requires STAT3-dependent expression and release of vascular endothelial growth factor (VEGF).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Aprotinin, DTT, HEPES, leupeptin, phenylmethylsulfonyl fluoride (PMSF), sodium deoxycholate, and sodium orthovanadate were purchased from Sigma-Chemical Co. 15(S)-HETE was bought from Cayman Chemicals. Growth factor–reduced Matrigel was obtained from BD Biosciences. Phosphospecific anti-STAT3 and anti-STAT3 antibodies were bought from Cell Signaling Technology. Anti-VEGF antibodies and STAT3 consensus oligonucleotides (5'-GATCCTTCTGGGAATTCCTAGATC-3', 5'-GATCTAGGAATTCCCAGAAGGATC-3'; SC-2571) were purchased from Santa Cruz Biotechnology, Inc. Neutralizing anti-VEGF antibodies were bought from Chemicon International. Human VEGF ELISA kit was obtained from Pierce. T4 polynucleotide kinase was procured from Promega. FuGENE 6 transfection reagent was obtained from Roche Molecular Biochemicals. D-threo-[dichloroacetyl-1-14C] chloramphenicol (59 mCi/mmol) was from Amersham Biosciences. [{gamma} 32 P]ATP (3,000 Ci/mmol) was bought from NEN Life Science Products. All the primers were made by IDT.

Cell culture. HDMVEC were bought from Cascade Biologics. Cells were grown in medium 131 containing microvascular growth supplements (MVGS), 10 µg/mL gentamicin, and 0.25 µg/mL amphotericin B. Cultures were maintained at 37°C in a humidified 95% air and 5% CO2 atmosphere. Cells were quiesced by incubating in medium 131 for 24 h and used to perform the experiments unless otherwise indicated.

Cell migration assay Cell migration was done using a modified Boyden Chamber method as described by Nagata et al. (33). The cell culture inserts containing membranes with 10 mm in diameter and 8.0 µm pore size (Nalge Nunc International) were placed in a 24-well tissue culture plate (Costar, Corning, Inc.). The lower surface of the porous membrane was coated with 0.5% gelatin at 4°C overnight and then blocked with 0.1% heat-inactivated bovine serum albumin (BSA) at 37°C for 1 h. HDMVEC were quiesced for 24 h in medium 131, trypsinized, and neutralized with TNS. Cells were seeded into the upper chamber at 1 x 105 per well. Vehicle or 15(S)-HETE were added to the lower chamber at the indicated concentration. Both the upper and lower chambers contained medium 131. When the effect of dominant-negative STAT3 (dnSTAT3) mutant was tested on 15(S)-HETE–induced HDMVEC migration, cells were infected first with either Ad-GFP or Ad-dnSTAT3 at a multiplicity of infection (MOI) of 80 and quiesced before they were subjected to migration assay. Ser727 and Tyr705 were mutated to Ala and Phe, respectively, to construct the dnSTAT3 (28). In the case of testing the effect of neutralizing anti-VEGF antibodies on 15(S)-HETE–induced HDMVEC migration, cells were incubated with antibodies (3 µg/mL) for 30 min at 37°C and washed with medium 131. Cells were then seeded into each well, and wherever appropriate, the antibodies were added to both the upper and lower chambers before the addition of 15(S)-HETE. After 6 h of incubation at 37°C, non-migrated cells were removed from the upper side of the membrane with cotton swabs, and the cells on the lower surface of the membrane were fixed in methanol for 15 min. The membrane was then stained with 4',6-diamidino-2-phenylindole in Vectashield mounting medium (Vector Laboratories, Inc.) and observed under deconvolution fluorescence microscope. Cells were counted in five randomly selected squares per well and presented as number of migrated cells per field.

Tube formation assay. Tube formation assay was done as described by Nagata et al. (33). Twenty-four–well culture plates (Costar, Corning) were coated with growth factor–reduced Matrigel (BD Biosciences) in a total volume of 280 µl per well and allowed to solidify for 30 min at 37°C. HDMVEC were trypsinized, neutralized with TNS, and resuspended at 5 x 105/mL, and 200 µL of this cell suspension was added into each well. Vehicle or eicosanoid of interest, at the indicated concentration were added to the appropriate well, and the cells were incubated at 37°C for 6 h. When the effect of dnSTAT3 mutant was tested on 15(S)-HETE–induced HDMVEC tube formation, cells were infected first with either Ad-GFP or Ad-dnSTAT3 at a MOI of 80 and quiesced before they were subjected to tube formation. In the case of testing the effect of neutralizing anti-VEGF antibodies on 15(S)-HETE–induced HDMVEC tube formation, cells were incubated with antibodies (3 µg/mL) for 30 min at 37°C and washed with medium 131. Cells were then seeded into each well, and wherever appropriate, the antibodies were added to the well before the addition of 15(S)-HETE. Tube formation was observed under an inverted microscope (Model Eclipse TS100, Nikon). Images were captured with a CCD color camera (Model KP-D20AU, Hitachi) attached to the microscope, and tube length was measured using the NIH Image J 1.31v Program.

Western blot analysis. After appropriate treatments and rinsing with cold PBS, HDMVEC were lysed in 500 µL of lysis buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/mL PMSF, 100 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 mmol/L sodium orthovanadate) and scraped into 1.5-mL Eppendorf tubes. After standing on ice for 20 min, the cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell lysates containing equal amount of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond, Amersham Pharmacia Biotech). After blocking in 10 mmol/L Tris-HCl buffer (pH 8.0) containing 150 mmol/L sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidase–conjugated secondary antibodies. The antigen-antibody complexes were detected using chemiluminescence reagent kit (Amersham Pharmacia Biotech).

Electrophoretic mobility shift assay. After appropriate treatments, nuclear extracts were prepared from HDMVEC as described previously (27). The protein content of the nuclear extracts was determined using a Micro BCA Protein Assay Reagent kit (Pierce). Protein-DNA complexes were formed by incubating 5 µg of nuclear protein in a total volume of 20 µL consisting of 15 mmol/L HEPES (pH 7.9), 3 mmol/L Tris-HCl (pH 7.9), 60 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L DTT, 2.5 µg/mL of BSA, 1 µg/mL of poly(deoxyinosinic-deoxycytidylic acid), 15% glycerol, and 100,000 cpm of [32P]-labeled oligonucleotide probe for 30 min on ice. To find the presence of STAT3 in the protein-DNA complexes, the nuclear extracts were preincubated with anti-STAT3 antibodies (0.5 µg per reaction) for 30 min on ice before the addition of the labeled probe to it. The protein-DNA complexes were resolved by electrophoresis on a 4% polyacrylamide gel using 1x Tris-glycine-EDTA buffer [25 mmol/L Tris-HCl (pH 8.5), 200 mmol/L glycine, 0.1 mmol/L EDTA]. Double-stranded oligonucleotides were labeled with [{gamma} 32P]ATP using the T4 polynucleotide kinase kit (Promega) following the supplier's protocol.

CAT assay. HDMVEC were plated evenly onto 100-mm dishes and grown in medium 131 supplemented with MVGS and G/A. At 50% to 80% confluence, medium was replaced with medium 131, and cells were transfected with pSIE-CAT plasmid using FuGENE 6 reagent according to the manufacturer's instructions (Roche Diagnostics). The pSIE-CAT plasmid contains three copies of sis-inducible elements (equivalent to GAS) from the IRF-1 gene (kindly provided by Dr. H. Young, National Cancer Institute-Frederick Research Cancer and Development Center, Frederick, MD). Twelve hours after transfection in medium 131, cells were quiesced for 24 h and treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated time periods, and cell extracts were prepared. Wherever the effect of dnSTAT3 mutant was tested on 15(S)-HETE–induced CAT activity, cells were first infected with either Ad-GFP or Ad-dnSTAT3 at a MOI of 80 followed by transfection with pSIE-CAT plasmid DNA. HDMVEC extracts were normalized for protein and assayed for CAT activity using [14C]chloramphenicol and acetyl-CoA as substrates. The substrate and products were extracted with ethyl acetate, separated by TLC, and subjected to autoradiography.

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation assay was done on HDMVEC by using chromatin immunoprecipitation assay kit following supplier's protocol (Upstate Biotechnology). STAT3-DNA complexes were immunoprecipitated using anti-STAT3 antibody (SC-482, Santa Cruz Biotechnology). Preimmune serum (SC-2338) was used as a negative control. Precipitated DNA was extracted using phenol-chloroform, and the DNA fragments from the aqueous phase were purified using QIAquick columns (Qiagen). The purified DNA was used as a template for PCR amplification using primers (forward, 5'-TTGGTGCCAAATTCTTCTCC-3'; reverse, 5'-CATACGTCCTCACTCTCGAA-3') flanking the putative STAT-binding site located at –848 in human VEGF promoter region (34). The PCR products were resolved on 2% agarose gel and stained with ethidium bromide.

Rat aortic ring assay. Rat aortic ring assay was done as described previously by Masson et al. (35). Briefly, male Fischer-344 rats (age 8–12 weeks) were sacrificed with cervical dislocation. The thoracic aorta was immediately removed and placed in cold DMEM. The periaortic fibroadipose tissue was carefully removed with fine microdissecting forceps and iridectomy scissors without damaging the aortic wall. One-millimeter-long aortic rings were sectioned and extensively rinsed in DMEM. To study the role of STAT3 on microvessel formation, the aortic rings were first incubated with adenovirus [5 x 109 plaque-forming units (pfu)/mL] expressing dnSTAT3 in incomplete medium 131 for 6 h at 37°C in 95% air/5% CO2 incubator. Rings were then embedded in rat tail interstitial collagen gel (1.5 mg/mL) prepared by mixing 7.5 volumes of 2 mg/mL collagen, 1 volume of 10x MEM, and 1.5 volume of NaHCO3 (15.6 mg/mL, pH 7.4). Three milliliters of medium 131 with and without containing 0.1 µmol/L 15(S)-HETE was then added into each well. To test the effect of neutralizing anti-VEGF antibodies, the rings were incubated with and without 15(S)-HETE (0.25 µmol/L) in the presence and absence of 3 µg/mL of antibodies. The microvessel formation was observed 7 days after incubation under the Nikon Eclipse 50i microscope at x4 magnification equipped with a camera. The length and number of microvessels formed were evaluated as a degree of angiogenesis using ImageJ software (NIH).

Matrigel plug angiogenesis assay. Matrigel plug assay was done essentially as described by Medhora et al. (36). C57BL/6 mice (8 weeks old) were lightly anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and injected s.c. with 0.5 mL of Matrigel that was premixed with vehicle or 50 µmol/L 15(S)-HETE along the dorsal midline (due to its highly labile characteristic feature, one higher bolus dose of 15(S)-HETE was added to the Matrigel to facilitate its continuous availability over a period of several days). The injection was made rapidly with a B-D 26G1/2 needle to ensure the entire content was delivered as a single plug. Wherever the effect of Ad-GFP, Ad-dnSTAT3 (5 x 109 pfu/mL), or neutralizing anti-VEGF antibodies (3 mg/mL) were tested on 15(S)-HETE–induced angiogenesis, they were added to the Matrigel before injecting into mice. The mice were allowed to recover, and 7 days later, unless otherwise stated, the animals were sacrificed by inhalation of CO2, and the Matrigel plugs were harvested from underneath the skin. The plugs were homogenized in 1 mL of deionized water on ice and cleared by centrifugation at 10,000 rpm for 6 min at 4°C. The supernatant was collected and used in duplicate to measure hemoglobin content with Drabkin's reagent along with hemoglobin standard essentially according to the manufacturer's protocol (Sigma-Chemical). The absorbance was read at 540 nm in an ELISA plate reader (Spectra Max 190, Molecular Devices). These experiments were repeated at least thrice with four mice for each group, and the values are expressed as g/dL of hemoglobin/mg plug.

VEGF ELISA. VEGF released into the culture media was measured using an ELISA kit following the manufacturer's instructions (Pierce).

Statistics. All the experiments were repeated thrice, with a similar pattern of results. Data are presented as mean ± SD. The treatment effects were analyzed by Student's t test, and P < 0.05 was considered statistically significant. In the case of CAT assay, electrophoretic mobility shift assay (EMSA), and Western blotting, one representative set of data is shown.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
15(S)-HETE activates STAT3 in HDMVEC. To elucidate the mechanisms of 15(S)-HETE–induced angiogenesis, we have studied the role of STAT3. Quiescent HDMVEC were treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated times, and an equal amount of protein from each treatment was analyzed for phosphorylated STAT3 using its phosphospecific antibodies. 15(S)-HETE stimulated tyrosine (Tyr705) phosphorylation of STAT3 in a time-dependent manner with about 2-fold increase at 1 min (Fig. 1A ). 15(S)-HETE–induced STAT3 phosphorylation sustained for 1 h. Upon phosphorylation, STATs form homodimers or heterodimers with other members of their family and translocate from the cytoplasm to the nucleus where they bind to promoter regions and influence the gene transcription (37). To study STAT3 translocation, cells were treated with and without 15(S)-HETE (0.1 µmol/L) for various time periods, and the cytoplasmic and nuclear extracts were prepared and analyzed for phosphorylated STAT3 levels as described above. Phosphorylated STAT3 was detected only in the nuclear fractions of both control and treated cells, and its levels were found 2-fold higher in 15(S)-HETE–treated cells compared with untreated cells (Fig. 1B). To confirm the nuclear translocation of STAT3, the blot was reprobed with anti-p53 antibodies. As expected, p53 was found only in the nuclear fractions, a result that confirms the purity of the cytoplasmic and nuclear preparations. Furthermore, sequential reprobing of the membrane with anti-STAT3 antibodies indicated that the majority of STAT3 was present in the cytoplasm, and only a small fraction of it was phosphorylated in response to 15(S)-HETE (Fig. 1B).


Figure 1
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  		Figure 1. 15(S)-HETE activates STAT3 in HDMVEC. Quiescent HDMVEC were treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated times, and either cell extracts (A) or cytoplasmic and nuclear factions (B) were prepared and analyzed by Western blotting for phosphorylated STAT3 (pSTAT3) using its phosphospecific antibodies. The blot in (A) was reprobed with anti-STAT3 antibodies for normalization. For testing the purity of cytoplasmic and nuclear preparations, the blot in (B) was reprobed sequentially with anti-STAT3 and anti-p53 antibodies.

 
To test whether the increased translocation of STAT3 also leads to its increased DNA binding activity, an equal amount of nuclear protein from control and 0.1 µmol/L 15(S)-HETE–treated HDMVEC was analyzed for DNA binding activity using [32P]-labeled STAT3 consensus oligonucleotides as a probe. A time-dependent increase in STAT3 DNA binding activity was observed in response to 15(S)-HETE treatment (Fig. 2A ). Preincubation of nuclear extract with anti-STAT3 antibodies (0.5 µg) reduced STAT3 DNA binding activity by 80% (Fig. 2A). In addition to the major prominent upper band, depending upon the exposure time of the X-ray film to the gel, a faint lower band was observed in some EMSA, which indicates the possible heterodimers of STAT3 with other members of the STAT family of transcriptional factors. These results confirm the presence of STAT3 in 15(S)-HETE–induced protein-DNA complexes. To find whether the increased DNA binding activity correlates with its ability of transcriptional transactivation, cells were transfected with pSIE-CAT reporter gene plasmid DNA in which the transcription of the CAT gene is regulated by three copies of sis-inducing elements, quiesced, and treated with and without 15(S)-HETE for various time periods. Cell extracts were prepared and analyzed for CAT activity using [14C]chloramphenicol and acetyl-CoA as substrates. A time-dependent increase in STAT3 reporter gene activity was observed in response to 15(S)-HETE (Fig. 2B). To confirm these results, we also studied the effect of dnSTAT3. HDMVEC were infected with Ad-GFP or Ad-dnSTAT3 with a MOI of 80, quiesced, and treated with and without 15(S)-HETE (0.1 µmol/L) for 1 h. Nuclear extracts were prepared and analyzed for STAT3 DNA binding activity as described above. Adenovirus-mediated expression of dnSTAT3 significantly inhibited 15(S)-HETE–induced STAT3 DNA binding activity (Fig. 3A ). In case of testing the effect of dnSTAT3 on 15(S)-HETE–induced STAT3 reporter gene activity, after infection with either Ad-GFP or Ad-dnSTAT3, cells were transfected with pSIE-CAT plasmid DNA, quiesced, and treated with and without 15(S)-HETE (0.1 µmol/L) for 6 h. Cell extracts were prepared and analyzed for CAT activity. Adenovirus-mediated expression of dnSTAT3 also inhibited 15(S)-HETE–induced STAT3 reporter gene activity by 60% (Fig. 3B and C).


Figure 2
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  		Figure 2. 15(S)-HETE stimulates STAT3-dependent transcriptional transactivation in HDMVEC. A, quiescent HDMVEC were treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated times, and nuclear extracts were prepared. The nuclear extracts containing an equal amount of protein from control and each treatment were assayed for STAT3 DNA binding activity using its [32P]-labeled consensus double-stranded oligonucleotides as a probe. To detect the presence of STAT3 in the protein-DNA complex, the nuclear proteins of control and 15(S)-HETE–treated HDMVEC were preincubated with anti-STAT3 antibodies (1 µg) and subjected to STAT3 DNA binding reaction. B, HDMVEC were transfected with pSIE-CAT plasmid DNA (3 µg per dish), quiesced, and treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated times, and cell extracts were prepared and assayed for CAT activity using [14C]chloramphenicol and acetyl-CoA as substrates.

 

Figure 3
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  		Figure 3. Adenovirus-mediated expression of dnSTAT3 inhibits 15(S)-HETE–induced STAT3 DNA binding and reporter gene activity. HDMVEC were transduced first with Ad-GFP or Ad-dnSTAT3 at a MOI of 80, quiesced, and treated with and without 15(S)-HETE (0.1 µmol/L) for 1 h, and nuclear extracts were prepared and analyzed for STAT3 DNA binding activity as described in Fig. 2 legend. B, after transduction with Ad-GFP or Ad-dnSTAT3 (80 MOI), cells were transfected with pSIE-CAT plasmid DNA (3 µg per dish), quiesced, and treated with and without 15(S)-HETE (0.1 µmol/L) for 6 h, and cell extracts were prepared and assayed for CAT activity as described in Fig. 2B legend. C, quantitative analysis of three independent experiments on CAT-reporter gene activity. *, P < 0.01, versus Ad-GFP; **, P < 0.01, versus AD-GFP + 15(S)-HETE.

 
STAT3 mediates 15(S)-HETE–induced HDMVEC migration and tube formation as well as aortic ring and Matrigel plug angiogenesis. Next, we tested the role of STAT3 in 15(S)-HETE–induced HDMVEC migration and tube formation. 15(S)-HETE stimulated HDMVEC cell migration by about 2-fold as measured by the modified Boyden chamber method, and adenovirus-mediated expression of dnSTAT3 attenuated this effect (Fig. 4A ). Similarly, 15(S)-HETE induced HDMVEC tube formation by about 2-fold, and this effect was also found to be STAT3 dependent as adenovirus-mediated expression of its dominant-negative mutant inhibited this activity (Fig. 4B). To obtain additional evidence for the role of STAT3 in 15(S)-HETE–induced angiogenesis, we used the aortic ring and Matrigel plug angiogenesis models. As shown in Fig. 4C, 15(S)-HETE at 0.1 µmol/L concentration stimulated the sprouting and elongation of microvessels from the aortic ring explants. In addition, preincubation of aortic rings with Ad-dnSTAT3 (5 x 109 pfu/mL) substantially attenuated 15(S)-HETE–induced aortic ring angiogenesis (Fig. 4C). Similarly, 15(S)-HETE (50 µmol/L) induced Matrigel plug angiogenesis, and dnSTAT3 significantly inhibited this effect (Fig. 4D).


Figure 4
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  		Figure 4. Adenovirus-mediated expression of dnSTAT3 suppresses 15(S)-HETE–induced HDMVEC migration and tube formation in vitro as well as aortic ring and Matrigel plug angiogenesis in vivo. A and B, HDMVEC were transduced with Ad-GFP or Ad-dnSTAT3 at a MOI of 80, quiesced, and subjected to 15(S)-HETE–induced migration (A) or tube formation (B). C, aortic rings were transduced first with Ad-GFP or Ad-dnSTAT3 at 5 x 109 pfu/mL and then subjected to 15(S)-HETE–induced angiogenesis. D, C57BL/6 mice were injected s.c. with 0.5 mL of Matrigel premixed with vehicle or 50 µmol/L 15(S)-HETE with and without Ad-GFP or Ad-dnSTAT3 (5 x 109 pfu/mL). One week later, the animals were sacrificed, and the Matrigel plugs were harvested from underneath the skin and analyzed for hemoglobin with Drabkin's reagent. Columns, means of four animals; bars, SD. *, P < 0.01, versus Ad-GFP; **, P < 0.01, versus AD-GFP + 15(S)-HETE.

 
15(S)-HETE–induced angiogenic activities are dependent on STAT3-mediated VEGF expression. It was reported that STAT3 mediates VEGF expression in response to some angiogenic stimulants (38, 39). To find whether 15(S)-HETE induces the expression of VEGF, and if so, the role of STAT3, quiescent HDMVEC were treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated times, and cell extracts were prepared. An equal amount of protein from control and treatment was then analyzed by Western blotting for VEGF using its specific antibodies. As shown in Fig. 5A , 15(S)-HETE induced the expression of VEGF in a time-dependent manner with a maximum 3-fold increase at 6 h, and these levels were sustained for at least 24 h. Similarly, 15(S)-HETE induced the release of VEGF into the culture medium in a time-dependent manner with a maximum of 3-fold effect at 6 h (Fig. 5B). Blockade of STAT3 by adenovirus-mediated expression of its dominant-negative mutant significantly blunted the 15(S)-HETE–induced VEGF expression and its release (Fig. 5C and D). Transfac analysis of human VEGF promoter revealed the presence of a putative STAT binding motif spanning from –848 to –841 (34). To obtain additional evidence for the role of STAT3 in 15(S)-HETE–induced VEGF expression, chromatin immunoprecipitation analysis was done using human VEGF promoter–specific primers flanking the putative STAT-binding motif. As shown in Fig. 6 , STAT3 binds to VEGF promoter in vivo in a time-dependent manner in response to 15(S)-HETE treatment. This result provides the direct evidence for the role of STAT3 in 15(S)-HETE–induced VEGF expression. To test the role of VEGF in 15(S)-HETE–induced angiogenesis, we next studied the effect of neutralizing anti-VEGF antibodies on 15(S)-HETE–induced HDMVEC migration and tube formation. Addition of neutralizing anti-VEGF antibodies (3 µg/mL) completely suppressed 15(S)-HETE–induced HDMVEC migration and tube formation (Fig. 7A and B ). Neutralizing anti-VEGF antibodies also inhibited 15(S)-HETE–induced aortic ring and Matrigel plug angiogenesis (Fig. 7C and D).


Figure 5
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  		Figure 5. 15(S)-HETE induces VEGF expression in STAT3-dependent manner in HDMVEC. A, quiescent HDMVEC were treated with and without 15(S)-HETE (0.1 µmol/L) for the indicated times, and cell extracts were prepared. An equal amount of protein from control and each treatment was analyzed by Western blotting for VEGF using its specific antibodies. B, the VEGF released into the culture medium of control and 0.1 µmol/L 15(S)-HETE–treated cells was measured by ELISA. C, conditions were the same as in (A) except that cells were transduced first with Ad-GFP or Ad-dnSTAT3 with a MOI of 80 and quiesced before they were subjected to treatment with and without 15(S)-HETE for 6 h and analyzed for VEGF levels. D, conditions were the same as in (B) except that the VEGF released into the culture medium was analyzed by ELISA. *, P < 0.01, versus control or Ad-GFP; **, P < 0.01 versus AD-GFP + 15(S)-HETE.

 

Figure 6
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  		Figure 6. STAT3 binds to VEGF promoter in vivo in response to 15(S)-HETE in HDMVEC. Chromatin immunoprecipitation was done with control and various time periods of 0.1 µmol/L 15(S)-HETE–treated HDMVEC using anti-STAT3 antibodies, and the resulting DNA fragments were subjected to PCR amplification using primers spanning –890 to –503 region of the human VEGF promoter.

 

Figure 7
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  		Figure 7. Neutralizing anti-VEGF antibodies suppress 15(S)-HETE–induced HDMVEC migration and tube formation in vitro as well as aortic ring and Matrigel plug angiogenesis in vivo. Quiescent HDMVEC were treated with neutralizing anti-VEGF antibodies (3 µg/mL) for 30 min at 37°C followed by washing with medium 131. The cells were then subjected to 0.1 µmol/L 15(S)-HETE–induced migration (A) and tube formation (B) in the presence and absence of 3 µg/mL neutralizing anti-VEGF antibodies. C, the aortic rings were incubated with and without 15(S)-HETE (0.25 µmol/L) in the presence and absence of 3 µg/mL neutralizing anti-VEGF antibodies for 1 wk, and microvessel formation was observed. D, C57BL/6 mice were injected s.c. with 0.5 mL Matrigel premixed with vehicle or 50 µmol/L 15(S)-HETE with and without 3 µg/mL neutralizing anti-VEGF antibodies. One week later, the animals were sacrificed, and the Matrigel plugs were harvested from underneath the skin and analyzed for hemoglobin with Drabkin's reagent. Columns, means of four animals; bars, SD.*, P < 0.01, versus control; **, P < 0.01, versus 15(S)-HETE alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of the present study are as follows: (a) 15(S)-HETE, the 15-LOX product of arachidonic acid, stimulates STAT3 in a time-dependent manner in HDMVEC. (b) 15(S)-HETE induced the expression of VEGF in STAT3-dependent manner. (c) 15(S)-HETE–induced angiogenesis requires STAT3-mediated expression and release of VEGF. Two 15-LOX (i.e., 15-LOX1 and 15-LOX2) have been reported in humans and both produce 15(S)-HETE from arachidonic acid as a major product (22, 38). However, both 15-LOX1 and 15-LOX2 have been reported to exert different effects in different cell types. It was shown that 15-LOX1, while inhibiting the growth of colorectal cancer cells (18, 19), stimulated prostate cancer cell growth (7, 8, 20). On the other hand, 15-LOX2 inhibited the growth of prostate cancer cells (21). Despite these differential effects of 15-LOX1 and 15-LOX2, their arachidonic acid product 15(S)-HETE influences growth in many other cell types including endothelial cells (6, 39). Towards understanding the possible mechanisms by which 15-LOX plays a role in cancer and vascular diseases, previously, we have shown that 15(S)-HETE induces angiogenesis (25). In the present study, we investigated the role of STAT3 in 15(S)-HETE–induced angiogenesis. STAT3 has been shown to be involved in the regulation of growth in various cell types in response to different growth factors and cytokines (26, 27, 40–44). In addition, numerous studies have shown that STAT3 mediates stimulant-induced angiogenesis (31, 32). In this aspect, the present results show that 15(S)-HETE also activates STAT3 in inducing angiogenesis.

VEGF plays a crucial role in endothelial cell growth and motility, thereby in angiogenesis (1, 2). In addition, VEGF seems to be a major mediator of angiogenesis in response to several stimulants (31, 38, 39, 45). Interestingly, 15(S)-HETE–induced angiogenesis also requires VEGF expression, and this response is dependent on activation of STAT3. This conclusion is supported by the observations that STAT3 binds to VEGF promoter in vivo in response to 15(S)-HETE treatment, and blockade of STAT3 activation inhibits 15(S)-HETE–induced VEGF expression in HDMVEC as well as the migration and tube formation of these cells. Similarly, neutralizing anti-VEGF antibodies significantly inhibited 15(S)-HETE–induced HDMVEC migration and tube formation in vitro as well as aortic ring and Matrigel plug angiogenesis in vivo. In lieu of these findings, it seems that 15(S)-HETE mimics other angiogenic stimulants, such as hypoxia and interleukin-8, in activating STAT3-VEGF signaling in endothelial cells (38, 39). Earlier, we have also reported that activation of PI3/Akt/mammalian target of rapamycin (mTOR)/S6K1 is required for 15(S)-HETE–induced angiogenesis (25). Because the inhibition of either the PI3/Akt/mTOR/S6K1 or STAT3 signaling resulted in almost complete suppression of 15(S)-HETE–induced angiogenesis, it is possible that these two signaling pathways play stage-specific events of angiogenesis. It was reported that PI3K/AKT signaling plays an important role in the sprouting of endothelial cells (46, 47). On the other hand, VEGF is needed for the survival and maintenance of the plasticity of the vessel (48, 49). Therefore, activation of both PI3K/Akt/S6K1 and STAT3 signaling may be necessary to perform different aspects of 15(S)-HETE–induced angiogenesis. If this was the case, then one would expect an equally important role for both signaling events in 15(S)-HETE–induced angiogenesis and inhibition of either one would result in the disruption of this process.

15-LOX has been shown to play a role in atherosclerosis via oxidation of LDL (12, 13). In addition, increased levels of 15(S)-HETE, the 15-LOX product of arachidonic acid, were reported in atherosclerosis (24). Furthermore, angiogenesis has been shown to influence the plaque progression (4, 5). Therefore, the production of 15(S)-HETE and its capacity to induce angiogenesis may also exert a positive effect on the progression of atherosclerosis.

In brief, the present results provide new insights on the possible mechanisms by which 15-LOX/15(S)-HETE could influence the progression of cancer and vascular diseases.


   Acknowledgments
 
Grant support: NIH grant HL74860.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/27/06. Revised 2/ 4/07. Accepted 2/21/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:10931–4.[Free Full Text]
  2. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27–31.[CrossRef][Medline]
  3. Duda DG. Antiangiogenesis and drug delivery to tumors: bench to bedside and back. Cancer Res 2006;66:3967–70.[Abstract/Free Full Text]
  4. Khurana R, Simons M, Martin JF, et al. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation 2005;112:1813–24.[Abstract/Free Full Text]
  5. Winter PM, Morawski AM, Caruthers SD, et al. Molecular imaging of angiogenesis in early-stage atherosclerosis with alpha(v) beta3-integrin-targeted nanoparticles. Circulation 2003;108:2270–4.[Abstract/Free Full Text]
  6. Postoak D, Nystuen L, King L, et al. 15-Lipoxygenase products of arachidonate play a role in proliferation of transformed eythroid cells. Am J Physiol 1990;259:C849–53.[Medline]
  7. Kelavkar UP, Nixon JB, Cohen C, et al. Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis. Carcinogenesis 2001;22:1765–73.[Abstract/Free Full Text]
  8. Kelavkar UP, Glasgow W, Olson SJ, et al. Overexpression of 12/15-lipoxygense, an artholog of human 15-lipoxygenase-1, in the prostate tumors of TRAMP mice. Neoplasia 2004;6:821–30.[Medline]
  9. Sun Z, Sood S, Li N, et al. Involvement of the 5-lipoxygenae/leukotriene A4 hydrolase pathway in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamster cheek pouch, and inhibition of carcinogenesis by its inhibitors. Carcinogenesis 2006;27:1902–8.[Abstract/Free Full Text]
  10. Carew TE, Schwenke DC, Steinberg D. Anti-atherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks showing the progression of atherosclerosis in the WHHL rabbit. Proc Natl Acad Sci U S A 1987;84:7725–9.[Abstract/Free Full Text]
  11. Berliner JA, Territo MC, Sevanian A, et al. Minimally modified low-density lipoprotein stimulates monocytes endothelial interactions. J Clin Invest 1990;85:1260–6.[Medline]
  12. Yla-Herttuala S, Rosenfeld ME, Parthasarthy S, et al. Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low-density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Asci U S A 1990;87:6959–63.[CrossRef]
  13. Zhu H, Takahashi Y, Xu W, et al. Low density lipoprotein receptor-related protein-mediated membrane translocation of 12/15- lipoxygenase is required for oxidation of low density lipoprotein by macrophages. J Biol Chem 2003;278:13350–5.[Abstract/Free Full Text]
  14. Gu JL, Pei H, Thomas L, et al. Ribozyme-mediated inhibition of rat leukocyte-type 12- lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries. Circulation 2001;103:1446–52.[Abstract/Free Full Text]
  15. Liu B, Maher RJ, DeJonckheere JP, et al. 12(S)-HETE increases the motility of prostate tumor cells through selective activation of PKC alpha. Adv Exp Med Biol 1997;400B:707–18.
  16. Preston IR, Hill NS, Warburton RR, et al. Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation. Am J Physiol 2006;290:L367–74.
  17. Tang DG, Renaud C, Stojakovic S, et al. 12(S)-HETE is a mitogenic factor for microvascular endothelial cells: its potential role in angiogenesis. Biochem Biophys Res Commun 1995;211:462–8.[CrossRef][Medline]
  18. Hsi LC, Xi X, Lotan R, et al. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells. Cancer Res 2004;64:8778–81.[Abstract/Free Full Text]
  19. Shureiqi I, Jiang W, Zuo X, et al. The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells. Proc Natl Acad Sci U S A 2003;100:9968–73.[Abstract/Free Full Text]
  20. Hsi LC, Wison LC, Eling TE. Opposing effects of 15-lipoxygenase-1 and -2 metabolites on MAPK signaling in prostate. Alteration in peroxisome proliferators-activated receptor gamma. J Biol Chem 2002;277:40549–56.[Abstract/Free Full Text]
  21. Bhatia B, Maldonado CJ, Tang S, et al. Subcellular localization and tumor-suppressive functions of 15-lipoxygenase 2 (15-LOX2) and its splice variants. J Biol Chem 2003;278:25091–100.[Abstract/Free Full Text]
  22. Brash AR, Boeglin WE, Chang MS. Discovery of a second 15(S)-lipoxygenase in humans. Proc Natl Acad Sci U S A 1997;94:6148–52.[Abstract/Free Full Text]
  23. Graeber JE, Glaser BM, Setty BN, et al. 15-Hydroxyeicosatetraenoic acid stimulates migration of human retinal microvessel endothelium in vitro and neovascularization in vivo. Prostaglandins 1990;39:665–73.[CrossRef][Medline]
  24. Henriksson P, Hamberg M, Diczfalusy U. Fornation of 15-HETE as a major hydroxyeicosatetranoic acid in the atherosclerotic vessel wall. Biochem Biophys Acta 1985;834:272–4.[Medline]
  25. Zhang B, Cao H, Rao GN. 15(S)-Hydroxyeicosatetraenoic acid induces angiogenesis via activation of PI3-Akt-mTOR-S6K1 signaling. Cancer Res 2005;65:7283–91.[Abstract/Free Full Text]
  26. Bromberg JF, Wrzeszczynska MH, Devgan G, et al. Stat3 as an oncogene. Cell 1999;98:295–303.[CrossRef][Medline]
  27. Yellaturu CR, Rao GN. Cytosolic phospholipase A2 is an effector of janus kinase/signal transducers and activators of transcription signaling and is involved in platelet-derived growth factor BB-induced growth in vascular smooth muscle cells. J Biol Chem 2003;278:9986–92.[Abstract/Free Full Text]
  28. Wu YY, Bradshaw RA. Activation of the STAT3 signaling pathway is required for differentiation by interleukin-6 in PC12-2 cells. J Biol Chem 2000;275:2147–56.[Abstract/Free Full Text]
  29. Neeli I, Liu Z, Dronadula N, et al. An essential role of Jak-2/STAT-3/cPLA2 axis in platelet-derived growth factor BB-induced vascular smooth muscle cell motility. J Biol Chem 2004;279:46122–8.[Abstract/Free Full Text]
  30. Seki Y, Kai H, Shibata R, et al. Role of the Jak-STAT pathway in rat carotid artery remodeling after vascular injury. Circ Res 2000;87:12–8.[Abstract/Free Full Text]
  31. Wei D, Le X, Zheng L, et al. Stat3 activation regulates the expression of vascular endothelial growth factor and human pancreatic cancer angiogenesis and metastasis. Oncogene 2003;22:319–29.[CrossRef][Medline]
  32. Burger M, Hartmann T, Burger JA, et al. KSHV-GPCR and CXCR2 transforming capacity and angiogenic response mediated through a JAK2–3-dependent pathway. Oncogene 2005;24:2067–75.[CrossRef][Medline]
  33. Nagata D, Mogi M, Walsh K. AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress. J Biol Chem 2003;278:31000–6.[Abstract/Free Full Text]
  34. Brogan IJ, Khan N, Isaac K, et al. Novel polymorphisms in the promoter and 5' UTR regions of the human vascular endothelial growth factor gene. Hum Immunol 1999;60:1245–9.[CrossRef][Medline]
  35. Masson V, Devy L, Grignet-Debrus C, et al. Mouse aortic ring assay: a new approach of the genetics of angiogenesis. Biol Proced Online 2002;4:24–31.[CrossRef][Medline]
  36. Medhora M, Daniels J, Mundey K, et al. Epoxygenase-driven angiogenesis in human lung microvascular endothelial cells. Am J Physiol Heart Circ Physiol 2003;284:H215–24.[Abstract/Free Full Text]
  37. Bromberg J, Darnell JE, Jr. The role of STATs in transcriptional control and their impact on cellular function. Oncogene 2000;19:2468–73.[CrossRef][Medline]
  38. Xu Q, Briggs J, Parks S, et al. Targeting Stat3 blocks both HIF-1 and VEGF expression induced by multiple oncogenic growth signaling pathways. Oncogene 2005;24:5552–60.[CrossRef][Medline]
  39. Repovic P, Fears CY, Gladson CL, et al. Oncostatin-M induction of vascular endothelial growth factor expression in astroglioma cells. Oncogene 2003;22:8117–24.[CrossRef][Medline]
  40. Schewe T, Halangk W, Heibsch C, et al. A lipoxygenase in rabbit reticulocytes which attacks phospholipids and intact mitochondria. FEBS Lett 1975;60:149–52.[CrossRef][Medline]
  41. Setty BN, Graeber JE, Stuart MJ. The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition. J Biol Chem 1987;262:17613–22.[Abstract/Free Full Text]
  42. Jenkins BJ, Grail D, Nheu T, et al. Hyperactivation of STAT-3 in gp130 mutant mice promotes gastric hyperproliferation and desensitizes TGF-ß signaling. Nat Med 2005;11:845–52.[CrossRef][Medline]
  43. Selander KS, Li L, Watson L, et al. Inhibition of gp130 signaling in breast cancer blocks constitutive activation of STAT-3 and inhibits in vivo malignancy. Cancer Res 2004;64:6924–33.[Abstract/Free Full Text]
  44. Hilfiker-Kleiner D, Hilfiker A, Fuchs M, et al. Signal transducer and activator of transcription 3 is required for myocardial capillary growth, control of interstitial matrix deposition, and heart protection from ischemic injury. Circ Res 2004;95:187–95.[Abstract/Free Full Text]
  45. Giavazzi R, Giuliani R, Coltrini D, et al. Modulation of tumor angiogenesis by conditional expression of fibroblast growth factor-2 affects early but not established tumors. Cancer Res 2001;61:309–17.[Abstract/Free Full Text]
  46. Adini I, Rabinovitz I, Sun JF, et al. RhoB controls Akt trafficking and stage-specific survival of endothelial cells during vascular development. Genes Dev 2003;17:2721–32.[Abstract/Free Full Text]
  47. Zhu WH, MacIntyre A, Nicosia RF. Regulation of angiogenesis by vascular endothelial growth factor and angiopoietin-1 in the rat aorta model. Am J Pathol 2002;161:823–30.[Abstract/Free Full Text]
  48. Baffert F, Thurston G, Rochon-Duck M, et al. Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasticity of adult blood vessels. Circ Res 2004;94:984–92.[Abstract/Free Full Text]
  49. Zacchigna S, Papa G, Antonini A, et al. Improved survival of ischemia cutaneous and musculocutaneous flaps after vascular endothelial growth factor gene transfer using adeno-associated virus vectors. Am J Pathol 2005;167:981–91.[Abstract/Free Full Text]



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