5(S)-Hydroxyeicosatetraenoic Acid Stimulates DNA Synthesis in Human Microvascular Endothelial Cells via Activation of Jak/STAT and Phosphatidylinositol 3-Kinase/Akt Signaling, Leading to Induction of Expression of Basic Fibroblast Growth Factor 2*

To understand the role of eicosanoids in angiogenesis, we have studied the effect of lipoxygenase metabolites of arachidonic acid on human microvascular endothelial cell (HMVEC) DNA synthesis. Among the various lipoxygenase metabolites of arachidonic acid tested, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE) induced DNA synthesis in HMVEC. 5(S)-HETE also stimulated Jak-2, STAT-1, and STAT-3 tyrosine phosphorylation and STAT-3-DNA binding activity. Tyrphostin AG490, a specific inhibitor of Jak-2, significantly reduced tyrosine phosphorylation and DNA binding activity of STAT-3 and DNA synthesis induced by 5(S)-HETE. In addition, 5(S)-HETE stimulated phosphatidylinositol 3-kinase (PI3-kinase) activity and phosphorylation of its downstream targets Akt, p70S6K, and 4E-BP1 and their effector molecules ribosomal protein S6 and eIF4E. LY294002 and rapamycin, potent inhibitors of PI3-kinase and mTOR, respectively, also blocked the DNA synthesis induced by 5(S)-HETE. Interestingly, AG490 attenuated 5(S)-HETE-induced PI3-kinase activity and phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. 5(S)-HETE induced the expression of basic fibroblast growth factor 2 (bFGF-2) in a Jak-2- and PI3-kinase-dependent manner. In addition, a neutralizing anti-bFGF-2 antibody completely blocked 5(S)-HETE-induced DNA synthesis in HMVEC. Together these results suggest that 5(S)-HETE stimulates HMVEC growth via Jak-2- and PI3-kinase-dependent induction of expression of bFGF-2. These findings also reveal a cross-talk between Jak-2 and PI3-kinase in response to 5(S)-HETE in HMVEC.

Formation of new capillaries, a process known as angiogenesis, plays an important role in embryonic development and wound healing (21,22). Angiogenesis also plays a role in the progression of various proliferative diseases such as atherosclerosis, cancer, and diabetic retinopathy (21)(22)(23)(24)(25). Proliferation and motility of endothelial cells are essential events of angiogenesis (23). Factors such as fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) that influence endothelial cell proliferation and motility are, therefore, likely involved in embryonic development and disease processes (21)(22)(23)(24)(25). In recent years, some studies using pharmacological approaches have reported that eicosanoids play a role in angiogenesis, although the underlying mechanisms are yet to be investigated (26).
Janus kinases are a group of non-receptor tyrosine kinases that via phosphorylating modulate the activities of a group of transcriptional factors, namely signal transducers and activators of transcription (STATs) (27,28). STATs have been reported to be involved in the regulation of cell growth and differentiation (29 -31). Similarly, phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway has been reported to play a major role in agonist-induced cell survival and growth (32)(33)(34)(35). To understand the role of eicosanoids in angiogenesis, we have studied the effect of the LOX metabolites of arachidonic acid on HMVEC DNA synthesis. Here, we report for the first time that 5(S)-HETE, the 5-LOX metabolite of arachidonic acid, stimulates DNA synthesis in HMVEC, and this event is mediated via Jak-2-and PI3-kinase-dependent induction of expression of bFGF-2.
Cell Culture-HMVEC were bought from Clonetics (Walkersville, MD). Cells were grown in endothelial basal medium 2 (CC-3156) containing EGM-2 MV SingleQuots (CC-4147). Cultures were maintained at 37°C in a humidified 95% air, 5% CO 2 atmosphere. Cells were growth-arrested by incubating in endothelial basal medium 2 for 24 h and used to perform the experiments.
DNA Synthesis-HMVEC with and without appropriate treatments were pulse-labeled with 1 Ci/ml [ 3 H]thymidine for the indicated times. After labeling, cells were washed with cold phosphate-buffered saline, trypsinized, and collected by centrifugation. The cell pellet was suspended in cold 10% (w/v) trichloroacetic acid and vortexed vigorously to lyse cells. After standing on ice for 20 min, the cell lysis mixture was passed through a glass fiber filter (GF/C, Whatman). The filter was washed once with cold 5% trichloroacetic acid and once with cold 70% (v/v) ethanol. The filter was dried, placed in a liquid scintillation vial containing the scintillant fluid, and the radioactivity was measured in a liquid scintillation counter (LS 5000TA, Beckman Instruments).
Electrophoretic Mobility Shift Assay-Nuclear extracts were prepared from treated or untreated HMVEC as described previously (36). The protein content of the nuclear extracts was determined using Micro BCA TM protein assay reagent kit (Pierce). Protein-DNA complexes were formed by incubating 5 g of nuclear protein in a total volume of 20 l consisting of 15 mM HEPES, pH 7.9, 3 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 4.5 g of bovine serum albumin, 2 g of poly(dI-dC), 15% glycerol, and 100,000 cpm of 32 P-labeled oligonucleotide probe for 30 min on ice. The protein-DNA complexes were resolved by electrophoresis on a 4% polyacrylamide gel using 1ϫ Tris-glycine-EDTA buffer (25 mM Tris-HCl, pH 8.5, 200 mM glycine, 0.1 mM EDTA). Double-stranded oligonucleotides were labeled with [␥-32 P]ATP using the T4 polynucleotide kinase kit (Invitrogen) following the supplier's protocol.
PI3-kinase Assay-PI3-kinase activity was measured according to a previously published protocol (37). After appropriate treatments, cells were lysed in 1 ml of lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM ␤-glycerophosphate, 1 mM dithiothreitol, 1 mM Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 2 M leupeptin, 10 units/ml aprotinin, and 400 M phenylmethylsulfonyl fluoride) for 20 min on ice. The cell lysates were cleared by centrifugation at 12,000 rpm for 15 min at 4°C. The protein content of the supernatants was determined as described above. Five hundred micrograms of protein from control and each treatment was immunoprecipitated with 5 l of anti-PI3-kinase antibodies for 2 h at 4°C followed by incubation with 40 l of 50% (w/v) protein A-Sepharose beads for an additional hour. The immunoprecipitates were washed 3 times with lysis buffer, 3 times with wash buffer, and 3 times with TNE buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 1 mM EDTA, and 10 M Na 3 VO 4 ). The kinase activity was measured by resuspending the immunoprecipitates in 30 l of TNE buffer and incubating with 10 l of 2 mg/ml phosphatidylinositol, 10 l of 100 mM MgCl 2 , 2 l of 100 mM ATP, and 20 Ci of [␥-32 P]ATP for 10 min at 22°C. The reaction was terminated by the addition of 20 l of 5 N HCl and 200 l of chloroform:methanol (1:1) mix. The aqueous and organic phases were separated by centrifugation at 2000 rpm for 10 min. The organic phase containing the phosphoinositide phosphates was spotted onto silica gel 60A TLC plate coated with 1% potassium oxalate and separated in a solvent system consisting of chloroform:methanol:water: ammonium hydroxide (90:70:14.6:5.4). The TLC plate was exposed to X-Omat AR x-ray film for 4 -6 h at Ϫ80°C and developed.
Western Blot Analysis-After appropriate treatments, HMVEC were rinsed with cold phosphate-buffered saline and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 l of lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxy-cholate, 0.1% SDS, 100 g/ml phenylmethylsulfonyl fluoride, 100 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM 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 amounts 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 Biosciences). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM 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 Biosciences).
Statistics-All the experiments were repeated three times with similar results. Data on DNA synthesis are presented as the mean Ϯ S.D. The treatment effects were analyzed by Student's t test. p values Ͻ 0.05 were considered to be statistically significant. In the case of PI3-kinase activity, electrophoretic mobility shift assay, and Western blot analysis, one representative set of data is shown.

RESULTS AND DISCUSSION
To understand the role of eicosanoids in angiogenesis, we have studied the effect of various LOX metabolites of arachidonic acid on HMVEC DNA synthesis. Quiescent HMVEC were treated with and without 1 M of indicated eicosanoids for 24 h, Equal amounts of protein (40 g) from control and each treatment were analyzed by Western blotting for pJak-2, pSTAT-1, and pSTAT-3 using their phospho-specific antibodies. As a loading control, the blots were reprobed with either anti-Jak-2 or anti-STAT-3 antibodies. and DNA synthesis was measured by pulse-labeling cells with 1 Ci/ml [ 3 H]thymidine for the last 2 h of the 24-h incubation period. Among the LOX metabolites of arachidonic acid tested, only 5(S)-HETE induced DNA synthesis by about 80% over control ( Fig. 1). To learn the signaling events of 5(S)-HETEinduced DNA synthesis, the role of the Jak/STAT pathway was studied. Quiescent HMVEC were treated with and without 5(S)-HETE (1 M) for the indicated times, and cell extracts were prepared. Equal amounts of protein from control and various times of 5(S)-HETE-treated HMVEC were analyzed for tyrosine phosphorylation of Jak-2, STAT-1, and STAT-3 using their phospho-specific antibodies. 5(S)-HETE stimulated tyrosine phosphorylation of Jak-2, STAT-1, and STAT-3 in a timedependent manner (Fig. 2). A maximum of a 2-3-fold increase in tyrosine phosphorylation of Jak-2 and STAT-3 was observed at 5 min of 5(S)-HETE treatment, and these levels were sustained for at least 1 h. In the case of STAT-1, only a slight increase in its tyrosine phosphorylation levels was observed at 5 min of 5(S)-HETE treatment compared with control, and these increases were also sustained for at least 1 h. To test the specificity of 5(S)-HETE on the phosphorylation of Jak-2 and STAT-3, quiescent HMVEC were treated with and without 1 M 5(S)-, 12(S)-or 15(S)-HETE for 30 min, and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for tyrosine phosphorylation of Jak-2 and STAT-3 using their phospho-specific antibodies. The effect of 5(S)-HETE on tyrosine phosphorylation of Jak-2 and STAT-3 was found significantly higher than that of 12(S)-or 15(S)-HETE (Fig. 3). This result suggests that the observed effects of 5(S)-HETE on stimulation of tyrosine phosphorylation of Jak-2 and STAT-3 are relatively specific. To find whether Jak-2 phosphorylates and activates STAT-3, we tested the effect of AG490, a potent and specific inhibitor of Jak-2 (38), on tyrosine phosphorylation of STAT-3. AG490 at 25 M concentration significantly inhibited 5(S)-HETE-stimulated tyrosine phosphorylation of STAT-3 (Fig. 4A). These results suggest a role for Jak-2 in tyrosine phosphorylation of STAT-3 by 5(S)-HETE. To investigate whether the increases in STAT-3 tyrosine phosphorylation lead to its activation, quiescent HMVEC were treated with and without 5(S)-HETE (1 M) in the presence and absence of AG490 (25 M) for 2 h, and nuclear extracts were prepared. Equal amounts of nuclear protein from control and each treatment were analyzed by electrophoretic mobility shift assay for DNA binding activity using 32 P-labeled STAT-3 consensus oligonucleotide as a probe. Consistent with its phosphorylation state, a 2-fold increase in STAT-3-DNA binding activity was observed at 2 h of 5(S)-HETE treatment (Fig. 4B). In addition, use of anti-STAT-3 antibodies caused a supershift of the STAT-3-DNA complex. This result suggests the presence of STAT-3 in the protein-DNA complex formed in response to 5(S)-HETE in HMVEC. Growth-arrested HMVEC were treated with and without 1 M of 5(S)-, 12(S)-, or 15(S)-HETE for 30 min, and cell extracts were prepared. Equal amounts of protein (40 g) from control and each treatment were analyzed by Western blotting for pJak-2 and pSTAT-3 using their phospho-specific antibodies. As a loading control, the same blot was reprobed with anti-Jak-2 antibodies.

FIG. 4. AG490, a potent inhibitor of Jak-2, reduces 5(S)-HETEinduced tyrosine phosphorylation and DNA binding activity of STAT-3 in HMVEC.
A, growth-arrested HMVEC were treated with and without 5(S)-HETE (1 M) in the presence and absence of AG490 (25 M) for 5 min, and cell extracts were prepared. Equal amounts of protein (40 g) from control and each treatment were analyzed by Western blotting for pSTAT-3 using its phospho-specific antibodies. As a loading control, the same blot was reprobed with anti-STAT-3 antibodies. B, growth-arrested HMVEC were treated with and without 5(S)-HETE (1 M) in the presence and absence of AG490 (25 M) for 2 h, and nuclear extracts were prepared. Five micrograms of nuclear protein from control and each treatment were incubated with 100,000 cpm of 32 P-labeled STAT-3 consensus oligonucleotide probe, and the DNAprotein complexes were separated by electrophoretic mobility shift assay and subjected to autoradiography. For supershift analysis, the DNA-protein complexes were incubated with 1 g of anti-STAT-3 antibodies for 2 h before subjecting the complexes to electrophoretic mobility shift assay. For competitive analysis, nuclear protein was first incubated with excessive cold oligos followed by incubation with labeled oligos. For clarity of the appearance of supershifts, longer exposure autoradiographic signals of the respective lanes in the left panel are shown in right panel. Ab, antibody.
survival (32)(33)(34)(35). In recent years, it was reported that the LOX and cytochrome P450 monooxygenase metabolites of arachidonic acid, particularly, 12(S)-HETE and 14, 15epoxyeicosatrienoic acid, play a role in cell survival (39 -41). Because cell survival activity is required for cell growth, we wanted to study the effect of 5(S)-HETE on activation of PI3-kinase/Akt signaling. Quiescent HMVEC were treated with and without 5(S)-HETE in the presence and absence of LY294002 (25 M), a potent and specific inhibitor of PI3kinase, and cell extracts were prepared. Equal amounts of protein from control and each treatment were assayed for PI3-kinase activity. 5(S)-HETE stimulated PI3-kinase activity 2-fold, and this response was completely inhibited by LY294002 (Fig. 6). We next tested the effect of 5(S)-HETE on the phosphorylation of PI3-kinase downstream targets, Akt  (44,45), and eIF4E (46). 5(S)-HETE stimulated serine phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E (Fig. 7). LY294002 completely inhibited 5(S)-HETE-stimulated phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. To test the role of PI3kinase/Akt in 5(S)-HETE-induced HMVEC growth, we next studied the effect of LY294002 on 5(S)-HETE-induced DNA synthesis. LY294002 significantly inhibited 5(S)-HETEinduced DNA synthesis in HMVEC (Fig. 8). Consistent with this finding, rapamycin (50 ng/ml), a potent inhibitor of mTOR, also significantly blocked 5(S)-HETE-induced DNA synthesis in HMVEC (Fig. 8). To validate the inhibitory effects of LY294002 and rapamycin and, therefore, the role of the PI3-kinase/Akt pathway in 5(S)-HETE-induced growth, we tested the effect of cyclosporin A (1 M), a potent inhibitor of calcineurin (47) that supposedly does not affect the PI3kinase/Akt pathway, on 5(S)-HETE-stimulated DNA synthesis. Cyclosporin A had no effect on 5(S)-HETE-stimulated DNA synthesis in HMVEC (data not shown). Previous studies from other laboratories report that constitutively active STAT-5 stimulates hematopoietic cell survival and growth, and it involves activation of PI3-kinase (48). Because 5(S)-HETE induced DNA synthesis in HMVEC and this phenomenon required activation of both Jak-2 and PI3-kinase, we were intrigued to learn whether there is any cross-talk between these two events. To address this, we tested the effect of AG490 on 5(S)-HETE-induced PI3-kinase activity and phosphorylation of its downstream molecules. Interestingly, 5(S)-HETE-induced increases in PI3-kinase activity and Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E phosphorylation were inhibited about 50% by AG490 (Fig. 9). These results suggest a requirement for Jaks, particularly Jak-2, in FIG. 9. 5(S)-HETE-stimulated PI3-kinase activity and phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E exhibit a requirement for Jak-2 in HMVEC. Growth-arrested HMVEC were treated with and without 5(S)-HETE (1 M) in the presence and absence of AG490 (25 M) for the indicated times, and cell extracts were prepared. A, equal amounts of protein (400 g) from control and each treatment were immunoprecipitated with 3 g of anti-PI3-kinase antibodies, and the kinase activity was measured in the immunocomplexes as described in legend to Fig. 6. PIP3, inositol 1,4,5trisphosphate. B, equal amounts of protein (40 g) from control and each treatment were analyzed by Western blotting for Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E phosphorylation using their phospho-specific antibodies. For loading control, the blot was reprobed only with anti-Akt antibodies because the same blot was used for probing with all the indicated phospho-specific antibodies. the activation of PI3-kinase and its downstream targets by 5(S)-HETE in HMVEC.
Among a variety of factors, bFGF-2 and VEGF are potent mitogens for endothelial cells and play an important role in angiogenesis (22,23). To test whether 5(S)-HETE stimulates HMVEC growth via induction of expression of angiogenic factors, we studied its effect on the expression of bFGF-2 and VEGF. Although having a modest effect on VEGF expression (1.7-fold), 5(S)-HETE induced the expression of bFGF-2 by 3-fold compared with control (Fig. 10). In addition, 5(S)-HETE-induced expression of bFGF-2 but not VEGF was attenuated by AG490 and LY294002, the inhibitors of Jak-2 and PI3-kinase, respectively (Fig. 10). To test the role of bFGF-2 in 5(S)-HETE-induced HMVEC DNA synthesis, quiescent cells were treated with and without 5(S)-HETE (1 M) in the presence and absence of neutralizing anti-bFGF-2 (3 g/ml) or anti-VEGF (3 g/ml) antibodies for 24 h, and DNA synthesis was measured by [ 3 H]thymidine incorporation. 5(S)-HETE-induced DNA synthesis was completely inhibited by neutralizing anti-bFGF-2 but not anti-VEGF antibodies (Fig. 11).
The important findings of the present study are as follows. 1) Among the LOX products of arachidonic acid examined, 5(S)-HETE significantly induced DNA synthesis in HMVEC; 2) 5(S)-HETE stimulated the tyrosine phosphorylation of Jak-2, STAT-1, and STAT-3 in a time-dependent manner; 3) AG490, a potent and specific inhibitor of Jak-2, substantially reduced tyrosine phosphorylation and DNA binding activity of STAT-3 and DNA synthesis induced by 5(S)-HETE; 4) 5(S)-HETE stimulated PI3-kinase activity and phosphorylation of its downstream molecules, Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E; 5) LY294002 and rapamycin, specific inhibitors of PI3-kinase and mTOR, respectively, blocked 5(S)-HETEinduced DNA synthesis; 6) PI3-kinase activity and the phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by 5(S)-HETE were sensitive to inhibition by AG490; 7) 5(S)-HETE induced expression of bFGF-2 in a Jak-2-and PI3-kinase-dependent mechanism; 8) a neutralizing anti-bFGF-2 but not anti-VEGF antibody blocked 5(S)-HETE-induced DNA synthesis in HMVEC. These results suggest that 5(S)-HETE stimulates HMVEC growth via Jak-2and PI3-kinase-dependent induction of expression of bFGF-2. A number of studies have reported that the LOX, cytochrome P450 monooxygenase, and cyclooxygenase metabolites of arachidonic acid stimulate growth in a variety of cell types, including cancer cells (13)(14)(15)(16)(17)(18)(19)(20). In recent years, it was also reported that the LOX and cytochrome P450 monooxygenase metabolites of arachidonic acid such as 12(S)-HETE and 14, 15-epoxyeicosatrienoic acid modulate survival activity in some cell types (39,41). Inhibition of 5-LOX caused apoptosis in malignant pleural mesothelial cells, and it was reversed by 5(S)-HETE (40). Furthermore, the protective effect of 5(S)-HETE was dependent on the production of VEGF. 5-LOX products of arachidonic acid such as leukotriene C 4 have also been shown to mediate epidermal growth factor-induced cytoskeletal rearrangement (9). The findings that eicosanoids, particularly the LOX products of arachidonic acid, mediate mitogen-induced signaling events via inhibition of GTPase activating proteins offer additional support for their role in cell growth (11,12,49). In addition to their role in the mitogenic signaling events cited above, some eicosanoids such as prostaglandin F 2␣ and 12(R)-hydroxy-5,8,14eicosatrienoic acid induce the expression of angiogenic factors bFGF-2 and VEGF in rat aortic smooth muscle cells and rabbit limbal microvessel endothelial cells, respectively (50,51). In the present study we show for the first time that 5(S)-HETE induces the expression of bFGF-2 and, thereby, stimulates an autocrine growth in HMVEC, and this phenomenon is dependent on activation of Jak-2 and PI3-kinase. Considering these novel observations, it is likely that 5(S)-HETE plays an important role in angiogenesis. A large body of pharmacological data indicates that the LOX metabolites of arachidonic acid are involved in tumorigenesis (14,15,17). In this aspect, the present study identifies 5(S)-HETE as one of the LOX products of arachidonic acid that is a potent mitogen for microvessel endothelial cells. Future studies are required to understand whether 5(S)-HETE also regulates the levels and/or activities of other molecules such as matrix metalloproteinases that are involved in angiogenesis.