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Role of Mitogen-activated Protein Kinases in 4-Hydroxy-2-nonenal-induced Actin Remodeling and Barrier Function in Endothelial Cells

2004; Elsevier BV; Volume: 279; Issue: 12 Linguagem: Inglês

10.1074/jbc.m311184200

1083-351X

Peter V. Usatyuk, Viswanathan Natarajan,

Nitric Oxide and Endothelin Effects

In vivo and in vitro studies indicate that 4-hydroxy-2-nonenal (4-HNE), generated by cellular lipid peroxidation or after oxidative stress, affects endothelial permeability and vascular tone. However, the mechanism(s) of 4-HNE-induced endothelial barrier function is not well defined. Here we provide evidence for the first time on the involvement of mitogen-activated protein kinases (MAPKs) in 4-HNE-mediated actin stress fiber formation and barrier function in lung endothelial cells. Treatment of bovine lung microvascular endothelial cells with hydrogen peroxide (H2O2), as a model oxidant, resulted in accumulation of 4-HNE as evidenced by the formation of 4-HNE-Michael protein adducts. Exposure of cells to 4-HNE, in a dose- and time-dependent manner, decreased endothelial cell permeability measured as transendothelial electrical resistance. The 4-HNE-induced permeability changes were not because of cytotoxicity or endothelial cell apoptosis, which occurred after prolonged treatment and at higher concentrations of 4-HNE. 4-HNE-induced changes in transendothelial electrical resistance were calcium independent, as 4-HNE did not alter intracellular free calcium levels as compared with H2O2 or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 μm) resulted in phosphorylation of ERK1/2, JNK, and p38 MAPKs, and actin cytoskeleton remodeling. Furthermore, pretreatment of bovine lung microvascular endothelial cells with PD 98059 (25 μm), an inhibitor of MEK1/2, or SP 600125 (25 μm), an inhibitor of JNK, or SB 202190 (25 μm), an inhibitor of p38 MAPK, partially attenuated 4-HNE-mediated barrier function and cytoskeletal remodeling. These results suggest that the activation of ERK, JNK, and p38 MAP kinases is involved in 4-HNE-mediated actin remodeling and endothelial barrier function. In vivo and in vitro studies indicate that 4-hydroxy-2-nonenal (4-HNE), generated by cellular lipid peroxidation or after oxidative stress, affects endothelial permeability and vascular tone. However, the mechanism(s) of 4-HNE-induced endothelial barrier function is not well defined. Here we provide evidence for the first time on the involvement of mitogen-activated protein kinases (MAPKs) in 4-HNE-mediated actin stress fiber formation and barrier function in lung endothelial cells. Treatment of bovine lung microvascular endothelial cells with hydrogen peroxide (H2O2), as a model oxidant, resulted in accumulation of 4-HNE as evidenced by the formation of 4-HNE-Michael protein adducts. Exposure of cells to 4-HNE, in a dose- and time-dependent manner, decreased endothelial cell permeability measured as transendothelial electrical resistance. The 4-HNE-induced permeability changes were not because of cytotoxicity or endothelial cell apoptosis, which occurred after prolonged treatment and at higher concentrations of 4-HNE. 4-HNE-induced changes in transendothelial electrical resistance were calcium independent, as 4-HNE did not alter intracellular free calcium levels as compared with H2O2 or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 μm) resulted in phosphorylation of ERK1/2, JNK, and p38 MAPKs, and actin cytoskeleton remodeling. Furthermore, pretreatment of bovine lung microvascular endothelial cells with PD 98059 (25 μm), an inhibitor of MEK1/2, or SP 600125 (25 μm), an inhibitor of JNK, or SB 202190 (25 μm), an inhibitor of p38 MAPK, partially attenuated 4-HNE-mediated barrier function and cytoskeletal remodeling. These results suggest that the activation of ERK, JNK, and p38 MAP kinases is involved in 4-HNE-mediated actin remodeling and endothelial barrier function. Living systems constantly encounter free radicals, which are generated by either enzymatic or nonenzymatic mechanisms leading to oxidative stress. Polyunsaturated fatty acids of cellular membrane lipids are in particular vulnerable to free radical attack and undergo peroxidation, ultimately leading to the loss of both structural and function integrity of the cell. Membrane lipid peroxidation has been implicated in numerous pathological states such as atherosclerosis, diabetes, cancer, ischemia-reperfusion injury, and several vascular disorders (1Esterbauer H. Schaur R.J. Zollner H. Free Radical Biol. Med. 1999; 11: 81-128Crossref Scopus (5903) Google Scholar). Peroxidation of membrane lipids results in the generation of several highly reactive aldehydes, which react with proteins and nucleic acids and alter their functions. Among the aldehydes, 4-hydroxy-2-nonenal (4-HNE) 1The abbreviations used are: 4-HNE, 4-hydroxy-2-nonenal; BLMVECs, bovine lung microvascular endothelial cells; DPV, diperoxovanadate; ECs, endothelial cells; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; MEM, minimum essential media; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; TER, transendothelial electrical resistance; BAPTA, 1,2-bis(2-aminophenoyl)ethane-N,N,N′,N′-tetraacetic acid; PBS, phosphate-buffered saline; [Ca2+]i, intracellular Ca2+. has been identified as a potent cytotoxic agent that accumulates to concentrations of 10 μm to 5 mm both in vivo and in vitro (1Esterbauer H. Schaur R.J. Zollner H. Free Radical Biol. Med. 1999; 11: 81-128Crossref Scopus (5903) Google Scholar, 2Uchida K. Free Radical Biol. Med. 2000; 28: 1685-1696Crossref PubMed Scopus (537) Google Scholar, 3Dianzani M.U. Mol. Aspects Med. 2003; 24: 263-272Crossref PubMed Scopus (121) Google Scholar). Accumulation of 4-HNE invokes a wide range of biological activities including inhibition of protein and DNA synthesis (1Esterbauer H. Schaur R.J. Zollner H. Free Radical Biol. Med. 1999; 11: 81-128Crossref Scopus (5903) Google Scholar, 2Uchida K. Free Radical Biol. Med. 2000; 28: 1685-1696Crossref PubMed Scopus (537) Google Scholar, 3Dianzani M.U. Mol. Aspects Med. 2003; 24: 263-272Crossref PubMed Scopus (121) Google Scholar), stimulation of phospholipases C and D (4Natarajan V. Scribner W.M. Taher M.M. Free Radical Biol. Med. 1993; 15: 365-375Crossref PubMed Scopus (67) Google Scholar, 5Natarajan V. Scribner W.M. Vepa S. Am. J. Respir. Cell Mol. Biol. 1997; 17: 251-259Crossref PubMed Scopus (31) Google Scholar, 6Nitti M. Domenicotti C. d'Abramo C. Assereto S. Cottalasso D. Melloni E. Poli G. Biasi F. Marinari U.M. Pronzato M.A. Biochem. Biophys. Res. 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Med. 1999; 11: 81-128Crossref Scopus (5903) Google Scholar, 2Uchida K. Free Radical Biol. Med. 2000; 28: 1685-1696Crossref PubMed Scopus (537) Google Scholar, 12Hamilton Jr., R.F. Li L. Eschenbacher W.L. Szweda L. Holian A. Am. J. Physiol. 1998; 274: L8-L16PubMed Google Scholar, 18Eaton P. Li J.M. Hearse D.J. Shattock M.J. Am. J. Physiol. 1999; 276: H935-H943Crossref PubMed Google Scholar). Reactive oxygen species (ROS), and lipid peroxides, -mediated endothelial barrier function has been implicated in the pathogenesis of cardiovascular disorders including atherosclerosis and acute respiratory distress syndrome. 4-HNE generated by oxidative stress or exposure to ozone leads to apoptosis and pulmonary edema (10Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 12Hamilton Jr., R.F. Li L. Eschenbacher W.L. Szweda L. Holian A. Am. J. Physiol. 1998; 274: L8-L16PubMed Google Scholar, 13Herbst U. Toborek M. Kaiser S. Mattson M.P. Hennig B. J. Cell Physiol. 1999; 181: 295-303Crossref PubMed Scopus (107) Google Scholar); however, the involvement of 4-HNE in endothelial function is unclear. Therefore the present study was designed to examine the role of 4-HNE in lung endothelial permeability changes. It was hypothesized that 4-HNE mediates the activation of mitogen-activated protein kinases, cytoskeletal remodeling, and regulates endothelial barrier function. The present study shows for the first time that 4-HNE-mediated activation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK signaling pathways partly regulates actin rearrangement and changes in transendothelial cell electrical resistance (TER) in lung microvascular endothelial cells. Materials—Bovine lung microvascular endothelial cells (EC) (passage number 7) were purchased from Cell Systems (Kirkland, WA). Endothelial cell growth factor, minimum essential medium (MEM), sodium orthovanadate, trypsin/EDTA, EGTA, penicillin/streptomycin, fetal bovine serum, gelatin, trypan blue, albumin bovine (fraction V), and hydrogen peroxide, were obtained from Sigma. 4-HNE was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Poly(ADP-ribose) polymerase (PARP) antibody and cell lysis buffer were from Cell Signaling (Beverly, MA). Enhanced chemiluminescence (ECL) kit was from Amersham Biosciences. Non-essential amino acids and PBS were obtained from Biofluids Inc. (Rockville, MD). Fura-2 AM (cell permeable), 4-bromo-A23187, pluronic acid (F-127), Alexa fluor 488 and Alexa fluor phalloidin 568, Vybrant Apoptosis, and EnzChek Caspase-3 assay kits were obtained from Molecular Probes (Eugene, OR). Anti-phosphotyrosine antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-ERK1, anti-ERK2, and anti-phosphospecific ERK, anti-JNK, and anti-phosphospecific JNK, anti-p38, and anti-phosphospecific p38 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary goat anti-rabbit or anti-mouse IgG (H+L) horseradish peroxidase conjugates were obtained from Bio-Rad. Immunobilon-P, 0.45 mm, was from Millipore. Anti-HNE-Michael adduct antibody and MAPK inhibitors PD 98059, SP 6000125, and SB 202190 were obtained from Calbiochem (San Diego, CA). Crystallized diperoxovanadate (potassium salt), prepared by mixing equimolar amounts of hydrogen peroxide and sodium orthovanadate (20Shankar H.N. Ramasarma T. Mol. Cell. Biochem. 1993; 129: 19-29Crossref Scopus (29) Google Scholar) was kindly provided by Dr. T. Ramasarma, Indian Institute of Science, Bangalore, India. Cell Culture—Bovine lung microvascular ECs (BLMVECs) cultured in MEM were maintained at 37 °C in a humidified atmosphere of 5% CO2, 95% air and grown to contact-inhibited monolayer with typical cobblestone morphology. Cells from each flask were detached with 0.05% trypsin and resuspended in fresh medium and cultured on gold electrodes for electrical resistance determinations, or on glass coverslips for calcium and immunocytochemistry studies, or on 100-mm dishes for Western blotting. Measurement of Transendothelial Electrical Resistance—TER was measured in a electrical cell-substrate impedance sensing system, Applied Biophysics, Inc. (Troy, NY), with minor modifications. Briefly, endothelial cells were cultured on gold electrodes (8 electrodes per plate) until reaching ∼95% confluence. One hour before TER measurements, the cells were rinsed with MEM and incubated in serum-free media. Electrodes were placed into an electrical cell-substrate impedance incubator for 1 h to stabilize basal electrical resistance and pretreated with MAPK inhibitors as indicated. The total electrical resistance measured dynamically across the endothelial monolayer was determined by the combined resistance between the basal surface of the cell and the electrode, reflecting alterations in cell-cell adhesion and/or cell-matrix adhesion (21Giaever I. Keese C.R. Nature. 1993; 366: 591-592Crossref PubMed Scopus (660) Google Scholar, 22Giaever I. Keese C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 88: 7896-7900Crossref Scopus (707) Google Scholar). Resistance, in time course experiments, is expressed as normalized resistance. Measurement of Intracellular Ca2+ Concentration—BLMVECs were plated on glass coverslips (Hitachi Instruments) pretreated with 0.1% gelatin solution and grown to ∼95% confluence in complete MEM. All procedures were carried out using as a basic media in mm: 116 NaCl, 5.37 KCl, 26.2 NaHCO3, 1.8 CaCl2, 0.81 MgSO4, 1.02 NaHPO4, 5.5 glucose, and 10 HEPES/HCl, pH 7.40. Cells were loaded with 5 μm Fura-2 AM (23Grynkiewicz G. Puenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) in 1 ml of the above media in the presence of 0.1% bovine serum albumin and 0.03% pluronic acid as recommended by the manufacturer's protocol at 37 °C in a cell culture incubator. Cells were loaded with Fura-2 AM for 15 min at 37 °C in 5% CO2, 95% air, rinsed twice, and inserted diagonally in the 1.0-cm acrylic cuvettes (Sarstedt, Newton, NC) filled with 3 ml of incubation media at 37 °C. Fura-2 fluorescence was measured with an Aminco-Bowman Series 2 luminescence spectrometer (SLM/Aminco, Urbana, IL) at excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. Intracellular free calcium, [Ca2+]i, in nanomolar was calculated from the 340/380 ratio using software and calibration curves. Preparation of Cell Lysates and Western Blotting—BLMVECs were grown on 100-mm culture dishes to ∼95% confluence. Before the experiment, cells were starved for 12-18 h by culturing in MEM containing only 2% of fetal bovine serum. Subsequent incubations were carried out in serum-free media. After treatment, the reaction was stopped by rinsing the dishes with ice-cold PBS containing 1 mm orthovanadate. ECs were lysed with 0.5-1 ml of nondenaturing cell lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm Na2EDTA, 1 mm EGTA, 1% Triton, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm Na3VO4, 1 μg/ml leupeptin and proteases inhibitor mixture (Roche Applied Science), scraped, sonicated on ice with a probe sonicator (15 s ×2), and centrifuged at 5000 × g in a microcentrifuge (4 °C for 5 min). Protein concentrations of the supernatants were determined using the Pierce protein assay kit. The supernatants, adjusted to 0.5-1 mg of protein/ml, were dissociated by boiling in 6× SDS sample buffer for 5 min, and samples were analyzed on 10% SDS-PAGE gels. Protein bands were transferred overnight (25 V, 4 °C) onto polyvinylidene difluoride (Millipore) membranes, probed with primary and secondary antibodies according to the manufacturer's protocol, and immunodetected by using enhanced chemiluminescence kit (Amersham Biosciences). The blots were scanned (UMAX Power Look II) and quantified by automated digitizing system UN-SCAN-IT GEL (Silk Scientific Corp.). Immunofluorescence Microscopy—BLMVECs grown on coverslips to ∼95% confluence were treated with 4-HNE or MAPK inhibitors as indicated in the figures, rinsed twice with PBS, and treated with 3.7% formaldehyde in PBS for 10 min at room temperature. Then cells were rinsed three times with PBS and permeabilized for 5 min with 0.25% Triton X-100 prepared in Tris-buffered saline containing 0.01% Tween 20 (TBST). After washing, cells were incubated for 30 min at room temperature in TBST blocking buffer containing 1% bovine serum albumin. 4-HNE-protein adduct formation was measured after treatment of cells with primary 4-HNE-Michaels adducts antibody (1:100 dilution in blocking buffer for 1 h). Cells were thoroughly rinsed with TBST (3× 5 min) followed by staining with Alexa fluor 488 (1:200 dilution in blocking buffer for 1 h) as secondary antibody. Actin stress fibers were determined by staining of cells on coverslips with Alexa fluor phalloidin 568. Cells were examined by Nikon Eclipse TE 2000-S immunofluorescence microscopy with a Hamamatsu digital camera (Japan) using a 60× oil immersion objective and MetaVue software (Universal Imaging Corp.). Assessment of Cell Viability and Apoptosis of ECs—Attached and floating BLMVECs, after 4-HNE treatment, were harvested and analyzed for cell viability, necrosis, and apoptosis. Cells were stained with trypan blue, and counted for trypan blue-excluded (viable) and trypan blue-stained cells (necrotic). Apoptosis was determined with annexin V Alexa fluor 488/propidium iodide using the Vybrant apoptosis kit (Molecular Probes). After staining with Alexa fluor 488 annexin V and propidium iodide according to the manufacturer's protocol, apoptotic cells showed green fluorescence, dead cells showed red and green fluorescence, and live cells showed little or no fluorescence as measured by immunofluorescence microscopy (lens ×40). Additionally, programmed cell death was investigated by measurements of caspase-3 activity and cleavage of PARP. Briefly, BLMVECs were treated with 4-HNE, cells were lysed in lysis buffer containing 100 mm NaCl, 1 mm EDTA, 10 mm Tris-HCl, pH 7.5, and 0.01% Triton X-100. Caspase-3 activity in total cell lysates was determined by a fluorometric EnzChek caspase-3 assay kit (Molecular Probes) using 7-amino-4-methylcoumarin-derived substrate Z-DEVD-AMC by spectrofluorimeter using 342/441 nm excitation/emission wavelengths, respectively. 4-HNE-induced processing of caspase-3 was also characterized by cleavage of PARP and analyzed by Western blotting using anti-PARP antibody. Statistics—Analysis of variance with Student-Newman-Keel's test was used to compare clearance rates of two or more different treatment groups. The level of significance was taken to be p < 0.05 unless otherwise stated. Data are expressed as mean ± S.E. Oxidant Stress Increases 4-HNE Formation—As a first step in examining the role of 4-HNE in endothelial cell function, we examined 4-HNE formation under oxidative stress. BLMVECs were incubated with varying concentrations of H2O2 (10 μm to 1 mm) for 1 h, and cell lysates were subjected to SDS-PAGE and Western blotting with anti-4-HNE-Michael adduct antibody. As shown in Fig. 1A, H2O2, in a dose-dependent manner, induced production of 4-HNE as measured by 4-HNE-adduct formation with proteins of 40-203 kDa. In control cells, immunofluorescence microscopy (Fig. 1B) indicated the presence of 4-HNE-protein adducts predominantly in the perinuclear region as observed in earlier studies (9Parola M. Robino G. Marra F. Pinzani M. Bellomo G. Leonarduzzi G. Chiarugi P. Camandola S. Poli G. Waeg G. Gentilini P. Dianzani M.U. J. Clin. Invest. 1998; 102: 1942-1950Crossref PubMed Scopus (276) Google Scholar, 24Leonarduzzi G. Arkan M.C. Basaga H. Chiarpotto E. Sevanian A. Poli G. Free Radical Biol. Med. 2000; 28: 1370-1378Crossref PubMed Scopus (192) Google Scholar, 25Neely M.D. Amarnath V. Weitlauf C. Montine T.J. Chem. Res. Toxicol. 2002; 15: 40-47Crossref PubMed Scopus (13) Google Scholar), and H2O2 treatment increased the Michael adduct distribution throughout the cell. 4-HNE Causes EC Barrier Function—Generation of 4-HNE under oxidative stress or exposure of isolated lungs to 4-HNE (50 μm) results in perivascular edema and endothelial cell disruption (12Hamilton Jr., R.F. Li L. Eschenbacher W.L. Szweda L. Holian A. Am. J. Physiol. 1998; 274: L8-L16PubMed Google Scholar, 18Eaton P. Li J.M. Hearse D.J. Shattock M.J. Am. J. Physiol. 1999; 276: H935-H943Crossref PubMed Google Scholar, 19Compton C.N. Franko A.P. Murray M.T. Diebel L.N. Dulchavsky S.A. J. Trauma. 1998; 44: 783-788Crossref PubMed Scopus (30) Google Scholar). However, the mechanism(s) of 4-HNE-induced lung injury is not well understood. To assess the effect of 4-HNE on EC barrier function, BLMVECs were challenged with varying concentrations of 4-HNE (1-100 μm), and EC permeability changes were measured as TER generated across the monolayer. As shown in Fig. 2, 4-HNE in a time- and dose-dependent fashion decreased TER that reflects the increase in EC permeability (21Giaever I. Keese C.R. Nature. 1993; 366: 591-592Crossref PubMed Scopus (660) Google Scholar, 22Giaever I. Keese C.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 88: 7896-7900Crossref Scopus (707) Google Scholar). The decrease in TER peaked at 2 h of 4-HNE (25 μm) treatment, and further increases in 4-HNE concentrations (50 and 100 μm) had no additional effect on TER (control, 1036 ± 72 ohms; 25 μm 4-HNE, 662 ± 87 ohms; 50 μm 4-HNE, 637 ± 83 ohms; 100 μm 4-HNE, 668 ± 64 ohms). These data indicate that 4-HNE modulates endothelial cell permeability in vitro. Effect of 4-HNE on Cell Viability and Apoptosis—Earlier studies have shown that exposure of mammalian cells to 4-HNE induces cell death, apoptosis, and necrosis (7Cheng J.Z. Singhal S.S. Sharma A. Saini M. Yang Y. Awasthi S. Zimniak P. Awasthi Y.C. Arch. Biochem. Biophys. 2001; 392: 197-207Crossref PubMed Scopus (101) Google Scholar, 10Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 12Hamilton Jr., R.F. Li L. Eschenbacher W.L. Szweda L. Holian A. Am. J. Physiol. 1998; 274: L8-L16PubMed Google Scholar, 13Herbst U. Toborek M. Kaiser S. Mattson M.P. Hennig B. J. Cell Physiol. 1999; 181: 295-303Crossref PubMed Scopus (107) Google Scholar). The ability of 4-HNE to induce cell death and apoptosis in BLMVECs was investigated by assessment of plasma membrane integrity with trypan blue and apoptosis with annexin V immunofluorescence microscopy, caspase-3 activity, and cleavage of PARP. Treatment of BLMVECs with 4-HNE (25-100 μm) for 2 h had no significant effect on cell viability as determined by trypan blue exclusion (Fig. 3A); however, exposure to 100 μm 4-HNE for 4 h resulted in ∼15% inclusion of trypan blue (Fig. 3A). Complimentary assays including annexin V immunofluorescence microscopy, caspase-3 activity, and PARP cleavage were carried out to measure 4-HNE-induced apoptosis in BLMVECs. At lower concentrations of 4-HNE (25-50 μm), there was no significant induction of apoptosis up to 4 h of treatment; however, 4-HNE at 100 μm induced an increase in the number of annexin V and propidium iodide-positive cells (∼4-8-fold compared with control cells) at 4 h of exposure to 4-HNE (Fig. 3B). To further establish the effect of higher concentrations of 4-HNE in inducing apoptosis, caspase-3 activity and PARP cleavage were measured. BLMVECs were exposed to varying concentrations of 4-HNE (1-100 μm) for 4 h and caspase-3 activity was measured using 7-amino-4-methylcoumarin-derived Z-DEVD substrate. As shown in Fig. 3C, 4-HNE treatment resulted in a 7-9-fold increase in caspase-3 activity at 50 and 100 μm concentrations, respectively. However, at lower doses of 4-HNE (1-25 μm) there was no significant change in caspase-3 activity (Fig. 3C) confirming the induction of apoptosis at higher, but not at lower doses of 4-HNE. Furthermore, PARP cleavage, as assessed by appearance of ∼89-kDa protein, was detected by 4 h after 4-HNE (50 and 100 μm) treatment of BLMVECs (Fig. 3D). No degradation of the ∼116-kDa PARP to the ∼89-kDa protein fragment was seen by 2 h after 4-HNE (25-100 μm) treatment or by 4 h after 25 μm 4-HNE (Fig. 3D). These results suggest that in BLMVECs, doses of 4-HNE from 50 μm induced appreciable cell death and apoptosis. Based on these data, all the experiments related to 4-HNE-mediated barrier dysfunction and cytoskeletal remodeling were carried out with 25 μm 4-HNE. Effect of 4-HNE on Changes in Intracellular Calcium—Based on earlier experiments that demonstrated the intracellular calcium dependence of ROS-induced EC permeability changes (26Dudek S.M. Garcia J.G. J. Appl. Physiol. 2001; 91: 1487-1500Crossref PubMed Scopus (837) Google Scholar, 27Lum H. Roebuck K.A. Am. J. Physiol. 2001; 280: C719-C741Crossref PubMed Google Scholar, 28Vepa S. Scribner W.M. Parinandi N.L. English D. Garcia J.G. Natarajan V. Am. J. Physiol. 1999; 277: L150-L158PubMed Google Scholar), we investigated the effect of 4-HNE on [Ca2+]i. In Fura-2-loaded BLMVECs, 4-HNE (25 μm) did not elevate [Ca2+]i (Fig. 4). Similar results were obtained with higher concentrations of 4-HNE (50-100 μm, data not shown). In contrast to 4-HNE, other oxidants, such as H2O2 (27Lum H. Roebuck K.A. Am. J. Physiol. 2001; 280: C719-C741Crossref PubMed Google Scholar, 28Vepa S. Scribner W.M. Parinandi N.L. English D. Garcia J.G. Natarajan V. Am. J. Physiol. 1999; 277: L150-L158PubMed Google Scholar) and DPV (29Usatyuk P.V. Fomin V.P. Shi S. Garcia J.G. Schaphorst K. Natarajan V. Am. J. Physiol. 2003; 285: L1006-L1017Google Scholar, 30Mikalsen S.O. Kaalhus O. J. Biol. Chem. 1998; 273: 10036-10045Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 31Shi S. Garcia J.G. Roy S. Parinandi N.L. Natarajan V. Am. J. Physiol. 2000; 279: L441-L451Crossref PubMed Google Scholar), which altered EC barrier function (Fig. 5), markedly stimulated [Ca2+]i (Fig. 4). These results indicate that 4-HNE did not modulate intracellular calcium levels in BLMVECs, but did induce ECs barrier dysfunction in a calcium-independent fashion.Fig. 5Effect of 4-HNE and ROS on transendothelial electrical resistance. A, BLMVECs grown on gold microelectrodes to ∼95% confluence were challenged with 4-HNE (25 μm), DPV (5 μm), or H2O2 (100 μm) and followed by measurement of TER as described under "Experimental Procedures." Shown is a representative tracing from four independent experiments. B, changes in TER (ohms) of 4-HNE, DPV, or H2O2 addition were calculated from A. *, significantly different from control (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) 4-HNE Increases Michael Protein Adduct Formation—As the aldehyde group in 4-HNE forms Schiff's base with -NH2 residues in proteins (1Esterbauer H. Schaur R.J. Zollner H. Free Radical Biol. Med. 1999; 11: 81-128Crossref Scopus (5903) Google Scholar, 17Doorn J.A. Petersen D.R. Chem. Res. Toxicol. 2002; 11: 1445-1450Crossref Scopus (270) Google Scholar, 32Minekura H. Kumagai T. Kawamoto Y. Nara F. Uchida K. Biochem. Biophys. Res. Commun. 2001; 282: 557-561Crossref PubMed Scopus (34) Google Scholar), we examined the formation of 4-HNE-mediated protein adducts in ECs. Treatment of BLMVECs with 4-HNE, in a dose- and time-dependent fashion, increased protein adduct formation as determined by Western blotting with anti-4-HNE-Michael adduct antibody (Fig. 6A). Modification of EC proteins of 40-45, 60-80, and 100-130 kDa by 4-HNE was detected with 5 μm 4-HNE, which was dramatically increased at higher concentrations. Interestingly, the formation of 4-HNE adduct was observed as early as 15 min after treatment and peaked at 45 min of 4-HNE treatment (Fig. 6B). These results show that 4-HNE induces the formation of protein adduct in ECs. 4-HNE Activates ERK1/2, JNK, and p38 MAPK—To investigate the role of MAPKs in 4-HNE-induced barrier dysfunction, BLMVECs were treated with different 4-HNE concentrations or with 4-HNE (25 μm) for varying times, and cell lysates were analyzed for enhanced phosphorylation of ERK1/2, JNK, and p38 MAPK by Western blotting with phosphospecific antibodies. As shown in Fig. 7, 4-HNE stimulated ERK1/2, JNK, and p38 MAPK as evidenced by enhanced phosphorylation of threonine/tyrosine residues. The activation of ERK1/2, JNK, and p38 MAPK by 4-HNE was dose-dependent with increased phosphorylation detected at concentrations as low as 25 μm, and reached a plateau at 50 μm (Fig. 7). As shown in Fig. 8, the phosphorylation of ERK1/2 peaked at 15 min and decreased thereafter; however, activation of JNK and p38 MAPK increased from 15 to 60 min after 4-HNE treatment. These data dem