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• W2045607632 abstract "15-Deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is a potent anti-angiogenic factor and induces endothelial cell apoptosis, although the mechanism remains unclear. In this study, 15d-PGJ2 was found to increase p53 levels of the human umbilical vein endothelial cells by stabilizing p53. Both 15d-PGJ2-induced apoptosis and the induction of p21Waf1 and Bax can be abolished by p53 small interfering RNA but not by peroxisome proliferator-activated receptor γ inhibitors. Moreover, 15d-PGJ2 activated JNK and p38 MAPK while inducing p53 phosphorylation at sites responsible for p53 activity. JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) pretreatment attenuated 15d-PGJ2-mediated apoptosis and suppressed the p21Waf1 and Bax expressions without affecting p53 protein accumulation. Pretreatment with SP600125 partially prevented the phosphorylation of p53 at serines 33 and 392 induced by 15d-PGJ2. 15d-PGJ2 was also found to induce reactive oxygen species generation and partially blocked nuclear factor-κB activity. Pretreatment with antioxidant N-acetylcysteine prevented the p53 accumulation, the phosphorylations of JNK and p38 MAPK, the inhibition of NF-κB activity, as well as the apoptosis induced by 15d-PGJ2. Using a mouse model of corneal neovascularization, it was demonstrated in vivo that 15d-PGJ2 induced reactive oxygen species generation, activated JNK and p38 MAPK, induced p53 accumulation/phosphorylation, and induced vascular endothelial cell apoptosis, which could be abolished by N-acetylcysteine, SP600125, SB203580, or a virus-derived amphipathic peptides-based p53 small interfering RNA. This is the first study that 15d-PGJ2 induces vascular endothelial cell apoptosis through the signaling of JNK and p38 MAPK-mediated p53 activation both in vitro and in vivo, further establishing the potential of 15d-PGJ2 as an anti-angiogenesis agent. 15-Deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2) is a potent anti-angiogenic factor and induces endothelial cell apoptosis, although the mechanism remains unclear. In this study, 15d-PGJ2 was found to increase p53 levels of the human umbilical vein endothelial cells by stabilizing p53. Both 15d-PGJ2-induced apoptosis and the induction of p21Waf1 and Bax can be abolished by p53 small interfering RNA but not by peroxisome proliferator-activated receptor γ inhibitors. Moreover, 15d-PGJ2 activated JNK and p38 MAPK while inducing p53 phosphorylation at sites responsible for p53 activity. JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) pretreatment attenuated 15d-PGJ2-mediated apoptosis and suppressed the p21Waf1 and Bax expressions without affecting p53 protein accumulation. Pretreatment with SP600125 partially prevented the phosphorylation of p53 at serines 33 and 392 induced by 15d-PGJ2. 15d-PGJ2 was also found to induce reactive oxygen species generation and partially blocked nuclear factor-κB activity. Pretreatment with antioxidant N-acetylcysteine prevented the p53 accumulation, the phosphorylations of JNK and p38 MAPK, the inhibition of NF-κB activity, as well as the apoptosis induced by 15d-PGJ2. Using a mouse model of corneal neovascularization, it was demonstrated in vivo that 15d-PGJ2 induced reactive oxygen species generation, activated JNK and p38 MAPK, induced p53 accumulation/phosphorylation, and induced vascular endothelial cell apoptosis, which could be abolished by N-acetylcysteine, SP600125, SB203580, or a virus-derived amphipathic peptides-based p53 small interfering RNA. This is the first study that 15d-PGJ2 induces vascular endothelial cell apoptosis through the signaling of JNK and p38 MAPK-mediated p53 activation both in vitro and in vivo, further establishing the potential of 15d-PGJ2 as an anti-angiogenesis agent. Neovascularization is involved in important pathological processes such as age-related macular degeneration, arthritis, and solid tumor growth. Hypoxia and inflammation-mediated vascular endothelial cell growth factor (VEGF) 2The abbreviations used are: VEGF, vascular endothelial cell growth factor; 15d-PGJ2, 15-deoxy-Δ(12,14)-prostaglandin J2; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; PPARγ, peroxisome proliferator-activated receptor γ; PEDF, pigment epithelium-derived factor; siRNA, small interfering RNA; NF-κB, nuclear factor-κB; ROS, reactive oxygen species; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; FITC, fluorescein isothiocyanate; EC, endothelial cell; CGZ, ciglitazone; TGZ, troglitazone; DCF, 2′,7′-dichlorofluorescein; RT, reverse transcriptase; PBS, phosphate-buffered saline; eNOS, endothelial nitric-oxide synthase. 2The abbreviations used are: VEGF, vascular endothelial cell growth factor; 15d-PGJ2, 15-deoxy-Δ(12,14)-prostaglandin J2; HUVEC, human umbilical vein endothelial cells; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; PPARγ, peroxisome proliferator-activated receptor γ; PEDF, pigment epithelium-derived factor; siRNA, small interfering RNA; NF-κB, nuclear factor-κB; ROS, reactive oxygen species; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; NAC, N-acetylcysteine; FITC, fluorescein isothiocyanate; EC, endothelial cell; CGZ, ciglitazone; TGZ, troglitazone; DCF, 2′,7′-dichlorofluorescein; RT, reverse transcriptase; PBS, phosphate-buffered saline; eNOS, endothelial nitric-oxide synthase. induction is generally accepted as the driving force of new vessel growth (1Zhang S.X. Ma J.X. Prog. Retin Eye Res. 2006; 26: 1-37Crossref PubMed Scopus (200) Google Scholar). Angiogenesis is thus a target of therapy, and there is an active search for agents capable of arresting both new vessel growth in vivo and the proliferation of vessel endothelial cells (EC) in vitro. Among these agents is 15-deoxy-Δ(12,14)-prostaglandin J2 (15d-PGJ2). 15d-PGJ2 has been reported to act as an anti-angiogenic factor by inducing EC apoptosis (2Bishop-Bailey D. Hla T. J. Biol. Chem. 1999; 274: 17042-17048Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 7Erl W. Weber C. Zernecke A. Neuzil J. Vosseler C.A. Kim H.J. Weber P.C. Eur. J. Immunol. 2004; 34: 241-250Crossref PubMed Scopus (18) Google Scholar) and suppressing angiogenic factor-induced EC proliferation, tube-like differentiation, and VEGF receptor expression (2Bishop-Bailey D. Hla T. J. Biol. Chem. 1999; 274: 17042-17048Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 3Xin X. Yang S. Kowalski J. Gerritsen M.E. J. Biol. Chem. 1999; 274: 9116-9121Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). An earlier study reported that the rat corneal neovascularization induced by VEGF can be significantly suppressed by co-implanted 15d-PGJ2 (3Xin X. Yang S. Kowalski J. Gerritsen M.E. J. Biol. Chem. 1999; 274: 9116-9121Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). Interestingly, it remains unclear whether 15d-PGJ2 can suppress the progression of existing neovessels. In this regard, the apoptotic-inducing capacity of 15d-PGJ2 in vivo is also unclear.15d-PGJ2 is a member of the cyclopentenone prostaglandins and is synthesized in many cell types in response to extrinsic stimuli (8Murakami M. Nakatani Y. Kuwata H. Kudo I. Biochim. Biophys. Acta. 2000; 1488: 159-166Crossref PubMed Scopus (83) Google Scholar). 15d-PGJ2 is an end product of the cyclooxygenase pathways, in which 15d-PGJ2 is produced by dehydration of prostaglandin D2 (9Fukushima M. Fatty Acids. 1992; 47: 1-12Abstract Full Text PDF PubMed Scopus (186) Google Scholar). In contrast to other prostaglandins that have specific transmembrane receptors, no specific 15d-PGJ2 cell surface receptor has been identified to date. 15d-PGJ2 has been shown to act through direct interactions with its intracellular targets; for example, it is known to be a ligand of the nuclear transcriptional factor peroxisome proliferator-activated receptor γ (PPARγ) (10Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2713) Google Scholar, 11Kliewer S.A. Lenhard J.M. Willson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar). PPARγ binding to 15d-PGJ2 allows translocation from the cytoplasm into the nucleus to regulate a variety of genes involved in cell differentiation, lipid biosynthesis, glucose metabolism, immune response, and vasculature (12Rizzo G. Fiorucci S. Curr. Opin. Pharmacol. 2006; 6: 421-427Crossref PubMed Scopus (111) Google Scholar, 13Duan S.Z. Usher M.G. Mortensen R.M. Circ. Res. 2008; 102: 283-294Crossref PubMed Scopus (232) Google Scholar). Notably, the cyclopentenone moiety of 15d-PGJ2 contains an electrophilic carbon that can react covalently with nucleophiles such as the free sulfhydryls of GSH and cysteine residues in cellular proteins (14Uchida K. Shibata T. Chem. Res. Toxicol. 2007; 21: 138-144Crossref PubMed Scopus (126) Google Scholar). Most PPARγ ligands lack the electrophilic cyclopentenone. 15d-PGJ2 thus induces some PPARγ-independent biological actions through its electrophilic activity, such as inhibition of nuclear factor-κB (NF-κB) signaling through covalent modifications of critical cysteine residues in IκB kinase and the DNA-binding domains of NF-κB subunits (15Straus D.S. Pascual G. Li M. Welch J.S. Ricote M. Hsiang C.H. Sengchanthalangsy L.L. Ghosh G. Glass C.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4844-4849Crossref PubMed Scopus (940) Google Scholar).The induction of apoptosis in proliferating ECs is an available strategy in the treatment of diseases relative to neovascularization. The mechanism of 15d-PGJ2 induction of EC apoptosis has been suggested to be through the activation of PPARγ (2Bishop-Bailey D. Hla T. J. Biol. Chem. 1999; 274: 17042-17048Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 6Dong Y.G. Chen D.D. He J.G. Guan Y.Y. Acta Pharmacol. Sin. 2004; 25: 47-53PubMed Google Scholar). Interestingly, our recent study on pigment epithelium-derived factor (PEDF) identified the sequential activation of PPARγ and p53 as a signaling mechanism of EC apoptosis (16Ho T.C. Chen S.L. Yang Y.C. Liao C.L. Cheng H.C. Tsao Y.P. Cardiovasc. Res. 2007; 76: 213-223Crossref PubMed Scopus (115) Google Scholar). PPARγ is thus a potential mechanism for 15d-PGJ2-induced apoptosis. However, a recent study indicates that 15d-PGJ2-induced HUVEC apoptosis is PPARγ-independent (7Erl W. Weber C. Zernecke A. Neuzil J. Vosseler C.A. Kim H.J. Weber P.C. Eur. J. Immunol. 2004; 34: 241-250Crossref PubMed Scopus (18) Google Scholar). The PPARγ-independent effect is also supported by evidence that the cyclopentenone ring alone can dose-dependently induce HUVEC apoptosis (5Vosseler C.A. Erl W. Weber P.C. Biochem. Biophys. Res. Commun. 2003; 307: 322-326Crossref PubMed Scopus (17) Google Scholar). In addition, several pro-apoptotic signals induced by 15d-PGJ2 have been shown to be independent of PPARγ in cell types other than ECs. These include accumulation of the p53 tumor suppressor protein in SH-SY5Y human neuroblastoma cells (17Kondo M. Shibata T. Kumagai T. Osawa T. Shibata N. Kobayashi M. Sasaki S. Iwata M. Noguchi N. Uchida K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7367-7372Crossref PubMed Scopus (163) Google Scholar) and the activation of p38 mitogen-activated protein kinase (MAPK) in human articular chondrocytes (18Shan Z.Z. Masuko-Hongo K. Dai S.M. Nakamura H. Kato T. Nishioka K. J. Biol. Chem. 2004; 279: 37939-53790Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and in a human pancreatic cancer cell line (19Hashimoto K. Farrow B.J. Evers B.M. Pancreas. 2004; 28: 153-159Crossref PubMed Scopus (31) Google Scholar). Based on this conflicting information, the involvement of PPARγ remains to be clarified. Unlike PPARγ, the involvement of p53 in EC apoptosis induced by 15d-PGJ2 is more plausible. p53 is a well established pro-apoptotic protein. p53 is involved in the apoptosis or cell cycle arrest of ECs induced by PEDF (16Ho T.C. Chen S.L. Yang Y.C. Liao C.L. Cheng H.C. Tsao Y.P. Cardiovasc. Res. 2007; 76: 213-223Crossref PubMed Scopus (115) Google Scholar), adenovirus-mediated p53 gene transfer (20Teodoro J.G. Parker A.E. Zhu X. Green M.R. Science. 2006; 313: 968-971Crossref PubMed Scopus (152) Google Scholar), and paclitaxel (Taxol) (21Pasquier E. Carré M. Pourroy B. Camoin L. Rebaï O. Briand C. Braguer D. Mol. Cancer Ther. 2004; 3: 1301-1310PubMed Google Scholar). Moreover, p53 protein expression is induced by 15d-PGJ2 (6Dong Y.G. Chen D.D. He J.G. Guan Y.Y. Acta Pharmacol. Sin. 2004; 25: 47-53PubMed Google Scholar, 17Kondo M. Shibata T. Kumagai T. Osawa T. Shibata N. Kobayashi M. Sasaki S. Iwata M. Noguchi N. Uchida K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7367-7372Crossref PubMed Scopus (163) Google Scholar). However, the necessity of p53 in 15d-PGJ2-induced EC apoptosis has never been established.MAPKs, including stress-activated c-Jun NH2-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK), have been found to respond to a variety of extracellular stimuli and to determine cell fate under stress (22Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-3131Crossref PubMed Scopus (5026) Google Scholar, 23Chang L. Karin M. Nature. 2001; 410: 37-40Crossref PubMed Scopus (4332) Google Scholar). Emerging evidence indicates that 15d-PGJ2 can activate MAPKs in ECs. For example, 15d-PGJ2 can enhance DNA binding of AP-1 by inducing c-Jun phosphorylation via JNK activation (4Zernecke A. Erl W. Fraemohs L. Lietz M. Weber C. FASEB J. 2003; 17: 1099-1101Crossref PubMed Scopus (27) Google Scholar, 24Chen N.G. Han X. Biochem. Biophys. Res. Commun. 2001; 282: 717-722Crossref PubMed Scopus (44) Google Scholar). 15d-PGJ2 has also been shown to activate p38 MAPK in ECV304 cells (6Dong Y.G. Chen D.D. He J.G. Guan Y.Y. Acta Pharmacol. Sin. 2004; 25: 47-53PubMed Google Scholar). However, the potential involvement of these kinases in the EC apoptosis induced by 15d-PGJ2 has not been established. Here we demonstrate that 15d-PGJ2 induces apoptosis of HUVECs and ECs in chemical burn-induced vessels on mouse cornea through the signaling of p53 and that p53 activation is achieved by JNK and p38 MAPK-mediated modulation of p53 phosphorylation.EXPERIMENTAL PROCEDURESCell Culture and Treatment—HUVECs (Cascade Biologics, Inc., Portland, OR) were grown in Medium 200 with Low Serum Growth Supplement (LSGS kit, supplement contains 1.9% fetal bovine serum, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, 1 μg/ml hydrocortisone, and 10 ng/ml epidermal growth factor). Culture plates were coated with 2% gelatin. Cells (passages 4-8) were cultured at 37 °C in a humidified atmosphere of 5% CO2. To prepare 15d-PGJ2 (Cayman Chemical, Ann Arbor, MI), the original solvent methyl acetate was evaporated under a gentle stream of nitrogen, and then it was redissolved in PBS before adding to the medium. Treatments with 15d-PGJ2 (10 μm, unless specified), recombinant PEDF derived from Escherichia coli (25Tsao Y.P. Ho T.C. Chen S.L. Cheng H.C. Life Sci. 2006; 79: 545-550Crossref PubMed Scopus (85) Google Scholar), MAPKs inhibitors, PPARγ antagonists, NF-κB inhibitors (Calbiochem), or NAC (Sigma) were performed 4 h after seeding (5 × 105 cells/well of 6-well plate) in LSGS medium.Animals and Treatment—BALB/c mice, weighing 25-35 g, were anesthetized with injections of ketamine. An alkaline burn was created by touching the cornea for 20 s with a 3-mm-diameter disk containing 1 n NaOH. The ocular surface was then irrigated with 20 ml of physiological saline. At 3 days after the injury, the mice were randomly divided into the 15d-PGJ2 treatment group and control group, with 10 mice per group. For treatment, 20 μl of 20 μm 15d-PGJ2 or 15d-PGJ2 in combination with 80 μm MAPKs inhibitors was dropped onto the cornea, two times at an interval of 4 h. For NAC treatment, corneas were covered with 20 μl of 10 mm NAC for 30 min, before treatment with 15d-PGJ2. The control group received 20 μl of saline eye drops. For evaluation of apoptosis, the corneas were treated twice at an interval of 4 h, and corneas were harvested 24 h after the second treatment. At the end of the treatment, corneas with iris intact were dissected from the eyes for evaluation of ROS generation or apoptosis without fixation. Corneas designated for immunofluorescent assay were first fixed with 4% paraformaldehyde for 2 h. All animal experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.Evaluation of Apoptosis—The percentage of HUVEC apoptosis was calculated using TACS annexin V-FITC kit (R & D Systems, Minneapolis, MN). Stained cells were analyzed by flow cytometry (FACSCalibur; Beckman) as described previously (16Ho T.C. Chen S.L. Yang Y.C. Liao C.L. Cheng H.C. Tsao Y.P. Cardiovasc. Res. 2007; 76: 213-223Crossref PubMed Scopus (115) Google Scholar). The percentage of annexin V-positive cells was also confirmed by in situ staining according to the manufacturer's instruction. The cell number was monitored by counterstaining with Hoechst 33342. The nuclei were calculated in 10 randomly selected fields of the three different chambers (∼7200 cells). Specimens were examined and photographed on a Zeiss epifluorescence microscope (×40, 10 fields/sample). Pictures were recorded on Zeiss software.To determine whether 15d-PGJ2 has any apoptotic effect on vascular ECs, the corneas were incubated with 10% goat serum and 1% bovine serum albumin for 30 min at 4 °C and then double-labeled with annexin V-FITC (R & D Systems) and PECAM-1 (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. Following labeling, the cornea was washed twice in PBS and fixed with 4% paraformaldehyde for 20 min. The cornea was incubated with rhodamine-conjugated goat anti-mouse IgG antibody (1:500 dilution; Santa Cruz Biotechnology) for 1 h at room temperature, and cell nuclei were monitored by counterstaining with Hoechst 33342 for 2 min. After final washes and mounting, apoptotic cells were counted in randomly selected fields using a Leica confocal microscope (×40, 10 fields/cornea).Western Blot Analysis—Cells were scraped into lysis buffer (150 μl/35-mm well) containing 20 mm HEPES (pH 7.4), 1% SDS, 150 mm NaCl, 1 mm EGTA, 5 mm β-glycerophosphate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Samples containing 20 μg of protein were analyzed by SDS-PAGE and then were electrotransferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) and processed for immunoblot analysis. Antibodies used in this study were for active p38 MAPK and active JNK (Promega, Madison, WI), p38 MAPK/SAPK2, JNK (Upstate Biotechnology, Lake Placid, NY), p53 (Chemicon, Temecula, CA), phospho-p53, phospho-NF-κB, p65 (Ser-536), c-Jun, ATF2, NF-κB, p65, phospho-IκB-α (Ser-32/36), IκB-α (Cell Signaling Technology, Beverley, MA), cleaved caspase-3 (Abcam Ltd., Cambridge, UK), and β-actin (Sigma). Proteins of interest were detected using the appropriate IgG-horseradish peroxidase secondary antibody (Santa Cruz Biotechnology) and ECL reagent (Amersham Biosciences). X-ray films were scanned on the model GS-700 Imaging Densitometer (Bio-Rad) and analyzed using Labworks 4.0 software. For quantification, blots of at least three independent experiments were used.p53 Small Interfering RNA Treatment—Human and mouse p53 and control siRNAs were purchased from Santa Cruz Biotechnology. For the transfection procedure, HUVECs were grown to 70% confluence, and siRNA was transfected using INTERFERin siRNA transfection reagent (PolyPlus-Transfection, San Marcos, CA) according to the manufacturer's instructions. The final concentration of siRNA was 10 nm. By 8 h after siRNA transfection, HUVECs were resuspended in new culture media with recovery for 36 h and then treated with 15d-PGJ2.Transfection of p53 siRNA into endothelial cell on mouse cornea was achieved by using the DeliverX Plus siRNA transfection kit (Panomics, Fremont, CA), a virus-derived amphipathic peptides-based kit following the manufacturer's instruction. Briefly, 5 μmol/liter working stock of mouse p53 siRNA or control siRNA was mixed with sonicated transfection reagent and incubated at 37 °C for 20 min to generate the working siRNA transfection complex. The final concentration of siRNA was 100 nm. Next, each mouse cornea was eye-dropped with 20 μl of sonicated siRNA transfection reagent alone (mock control) or siRNA transfection complex twice at an interval of 4 h, and recovery was for a further 24 h before 15d-PGJ2 treatment. The transfection efficiency was determined, in separated experiments, using a FITC-labeled nonsilencing control siRNA (Santa Cruz Biotechnology). Results revealed that almost all of vascular ECs and epithelial cells of cornea can be transfected and still maintain normal morphologies. No green fluorescence can be detected without using the transfection reagent.Semi-quantitative Reverse Transcriptase (RT)-PCR—Total RNA was extracted from HUVECs with TRIzol reagent (Invitrogen). Synthesis of cDNA was performed with 1 μg of total RNA at 50 °C for 50 min, using oligo(dT) primers and reverse transcriptase (Superscript III, Invitrogen). The amplification mixture (final volume, 20 μl) contained 1× Taq polymerase buffer, 0.2 mm dNTPs, 1.5 mm MgCl2, 1 μm primer pair, and 0.5 unit of TaqDNA polymerase (Invitrogen). cDNA was equalized in an 18-26 cycle amplification reaction with PPARγ primers 5′-CAGGAGCAGAGCAAAGAGGTG-3′ (forward)/5′-CAAACTCAAACTTGGGCTCCA-3′ (reverse) or p53 primers 5′-GCGCACAGAGGAAGAGAATC-3′ (forward)/5′-TGAGTCAGGCCCTTCTGTCT-3′ (reverse) yielding 300- and 330-bp products, respectively. The number of cycles for the primer sets (denaturation, 30 s, 94 °C; annealing, 30 s, 61 °C; and polymerization, 30 s, 72 °C) was chosen to be in the linear range of amplification. The PCR products were subjected to electrophoresis on 2% agarose gel, and the DNA was visualized by staining with ethidium bromide under ultraviolet irradiation. The intensities of the PCR products were quantified by densitometrically using a FUJI LAS-3000 system and multigauge version 1.01 software (FUJIFILM, Tokyo, Japan).Immunofluorescent—Mouse corneas were treated at 4 °C with methanol for 2 min and blocked with 10% goat serum and 5% bovine serum albumin for 1 h at room temperature. Corneas were stained with antibodies to monoclonal anti-mouse eNOS antibody (1:200; Pharmingen), active p38 MAPK or active JNK (1:500; Promega), or phospho-p53 (1:500; Cell Signaling Technology), or p53 (1:500, ab4060; Abcam Ltd.), or NF-κB p65 (1:500; Cell Signaling Technology) at 4 °C overnight, followed by incubation with both rhodamine-conjugated goat anti-mouse IgG antibody and FITC-conjugated goat anti-rabbit IgG antibody (1:500; Santa Cruz Biotechnology) for 1 h at room temperature. Nuclei were located by counterstaining with Hoechst 33342 for 20 min. After final washes and mounting, corneas were examined using a Leica confocal microscope (×40). p-p53 (Ser-392)-positive cells were counted in randomly selected fields (×40, 10 fields/cornea).Detection of ROS by H2DCFDA—The intracellular ROS generation was assayed using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and when oxidized by ROS it releases the green fluorescent compound 2′,7′-dichlorofluorescein (DCF).To detect ROS by spectrofluorometric assay, 1.2 × 104 cells were seeded in a 2% gelatin-coated 96-well plate and incubated for a further 4 h. After 15d-PGJ2 treatment, cells were washed with PBS (pH 7.4) and then incubated with fresh medium containing 5 μm H2DCFDA (Molecular Probes, Eugene, OR) in the dark for 15 min at 37 °C. Fluorescence (excitation, 488 nm; emission, 520 nm) was measured with a SpectraMAX GEMINI Reader (Molecular Devices, Sunnyvale, CA). The background fluorescence from control wells without the addition of H2DCFDA was subtracted from experimental readings.For monitoring the DCF fluorescence in HUVECs, cells were plated on 2% gelatin-coated plate in LSGS medium. After 15d-PGJ2 treatment and H2DCFDA exposure as described above, cells were washed two times with LSGS medium and fixed with 4% paraformaldehyde for 15 min. Nuclei were stained with Hoechst 33342. Fluorescence images were taken with an inverted fluorescence microscope (Olympus Optical Co., Ltd., Melville, NY).For detection of ROS in vascular ECs, after 15d-PGJ2 treatment for 1 h, corneas were dissected from animal eyes and transferred into a 12-well plate. Individual cornea was then incubated with 1 ml of LSGS medium containing 5 μm H2DCFDA for 20 min at 37 °C and then washed two times with LSGS medium. Corneas were then fixed with 4% paraformaldehyde for 15 min and stained with Hoechst 33342. Fluorescence images were taken with a Leica confocal microscope (10 fields/cornea).Determination of NF-κB Activation—NF-κB p50/p65 transcription factor colorimetric assay (SGT510, Chemicon International, Temecula, CA) was used to measure the active NF-κB in nuclear extracts by following the manufacturer's instructions. Briefly, nuclear extracts from 5 × 105 HUVECs were prepared using the NE-PER nuclear and cytoplasmic extraction kit (Pierce). Double-stranded biotinylated oligonucleotides containing the consensus sequence for NF-κB binding (5′-GGGACTTTCC-3′) were mixed with nuclear extract and assay buffer. After incubation, the mixture was transferred to the streptavidin-coated ELISA kit processed following the manufacturer's instruction and read at 450 nm using a SpectraMAX GEMINI Reader (Molecular Devices). For each experiment, triplicate samples were measured for statistical significance. The specificity of binding was confirmed by competition with unlabeled oligonucleotides.Statistical Analyses—Data are expressed as means ± S.D. of three to five independent experiments. The Mann-Whitney U test was used to determine statistically significant differences. p values <0.05 were considered significant.RESULTS15d-PGJ2 Dose-dependently Induces Apoptosis and Increases Protein Levels of p53 and PPARγ—Exposure of HUVECs to 10 μm or greater 15d-PGJ2 for 16 h increased the percentage of annexin V-positive apoptotic cells to 40% (Fig. 1A). Using 10 μm or greater 15d-PGJ2 markedly induced PPARγ and p53 protein accumulation at all time periods studied (Fig. 1B). RT-PCR showed that the levels of p53 mRNA were similar to untreated cells at all time periods studied, suggesting that transcription of p53 is not activated (Fig. 1C). Because the half-life of p53 protein is short in most primary cells, further experiments were performed to investigate whether 15d-PGJ2 can enhance the protein stability of p53 and showed that 15d-PGJ2 prolonged the half-life of p53 in HUVECs (Fig. 1D). On the other hand, the level of PPARγ mRNA was increased at 4-8 h, as compared with control (Fig. 1C). Pretreatment with actinomycin D for 3 h prior to 15d-PGJ2 exposure suppressed the PPARγ mRNA level, suggesting that the increase of PPARγ mRNA is transcription-dependent.p53 Is Critical for 15d-PGJ2-mediated Apoptosis of HUVECs and Vascular ECs on Mouse Cornea—To investigate whether p53 protein is required for 15d-PGJ2-mediated apoptosis, HUVECs were transfected with p53-specific siRNA before 15d-PGJ2 treatment. Western blotting verified that the p53 induction was specifically and significantly reduced by the cognate p53 siRNA (Fig. 2, A and B). Importantly, compared with either mock or control siRNA transfections, p53 siRNA significantly reduced 15d-PGJ2-induced apoptosis (Fig. 2C) as well as Bax and p21Waf1 expressions and procaspase-3 cleavages (Fig. 2B).FIGURE 2p53 siRNA suppresses the 15d-PGJ2-induced Bax and p21Waf1 expression and procaspase-3 cleavage and reduces the 15d-PGJ2-induced HUVEC apoptosis. A, HUVECs were transfected with p53 siRNA or control siRNA for 8 h and allowed to recover for a further 36 h. Cells were treated with 15d-PGJ2 for an additional 10 h, and cell lysate was then isolated for Western blot analysis of p53, Bax, and p21Waf1. Parts of the 15d-PGJ2-treated cells were harvested after treatment for 16 h for detecting the cleaved caspase-3 (17 kDa). “Mock (M)” indicates that cells were treated with transfection reagent. B, densitometric analysis of A.*, p < 0.02 versus mock-treated cells. #, p < 0.05 versus control siRNA-pretreated cells. C, apoptosis was quantified by using the annexin V-FITC apoptosis detection kit. *, p < 0.05 versus control siRNA-pretreated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether 15d-PGJ2 can also increase p53 protein in vivo, we investigated the ECs in abnormal vessels induced by alkali burn on mouse corneas. As shown in Fig. 3, immunofluorescent assay showed that there is a base-line p53 (green) in both vascular ECs and epithelial cells of cornea. The identity of ECs was confirmed by an endothelial cell antigen (eNOS; Fig. 3, red). Treatment of cornea with 15d-PGJ2 for 8 h specifically enhanced p53 protein in the vascular ECs but not in the corneal epithelium (Fig. 3, panel b). The induced p53 was localized at both of the cytoplasms as the distribution of eNOS (Fig. 3, yellow) and the nucleus (pale blue).FIGURE 315d-PGJ2 induces p53 protein expression in vascular ECs. Mouse corneas were subjected to alkali trauma and at 3 days later treated with either mock transfection reagent (virus-derived amphipathic peptide alone), control siRNA, or p53 siRNA transfection mixture. After recovery for a further 24 h, the corneas were treated with PBS or 20 μm 15d-PGJ2 for a further 8 h. The corneas wer" @default.
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