Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin remodeling and barrier function in endothelial cells.

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 (H(2)O(2)), 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 H(2)O(2) or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 microm) 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 microm), an inhibitor of MEK1/2, or SP 600125 (25 microm), an inhibitor of JNK, or SB 202190 (25 microm), 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.

Cell Culture-Bovine lung microvascular ECs (BLMVECs) cultured in MEM were maintained at 37°C in a humidified atmosphere of 5% CO 2 , 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 ϳ95% 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 (21,22). Resistance, in time course experiments, is expressed as normalized resistance.
Measurement of Intracellular Ca 2ϩ Concentration-BLMVECs were plated on glass coverslips (Hitachi Instruments) pretreated with 0.1% gelatin solution and grown to ϳ95% confluence in complete MEM. All procedures were carried out using as a basic media in mM: 116 NaCl, 5.37 KCl, 26.2 NaHCO 3 , 1.8 CaCl 2 , 0.81 MgSO 4 , 1.02 NaHPO 4 , 5.5 glucose, and 10 HEPES/HCl, pH 7.40. Cells were loaded with 5 M Fura-2 AM (23) 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% CO 2 , 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, [Ca 2ϩ ] 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 ϳ95% 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 Na 2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1 g/ml leupeptin and proteases inhibitor mixture (Roche Applied Science), scraped, sonicated on ice with a probe sonicator (15 s ϫ2), and centrifuged at 5000 ϫ 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 6ϫ 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 ϳ95% 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 (3ϫ 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 60ϫ 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 ϫ40). 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 Ͻ 0.05 unless otherwise stated. Data are expressed as mean Ϯ S.E.

RESULTS
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 H 2 O 2 (10 M 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, H 2 O 2 , 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 (9,24,25), and H 2 O 2 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 M) results in perivascular edema and endothelial cell disruption (12,18,19). However, the mechanism(s) of 4-HNEinduced 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 M), 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 (21,22). The decrease in TER peaked 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 (7,10,12,13). 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- (Fig. 4). Similar results were obtained with higher concentrations of 4-HNE (50 -100 M, data not shown). In contrast to 4-HNE, other oxidants, such as H 2 O 2 (27,28) and DPV (29 -31), which altered EC barrier function (Fig. 5), markedly stimulated [Ca 2ϩ ] i (Fig. 4). These results indicate that 4-HNE did not modulate intracellular calcium levels in BLM-VECs, but did induce ECs barrier dysfunction in a calciumindependent fashion. 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 Increases Michael Protein Adduct Formation-As
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 M) 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 M, and reached a plateau at 50 M (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 demonstrate that 4-HNE enhances phosphorylation of all three MAPKs in lung microvascular ECs.
Inhibition of MAPKs Blocks 4-HNE-induced EC Barrier Function-To further elucidate the role of MAP kinases in 4-HNE-induced EC permeability changes, we used selective and specific inhibitors of ERK, JNK, and p38 kinases (33)(34)(35). We chose to use 25 M 4-HNE, which was the lowest effective concentration to disrupt barrier function without cytotoxicity and apoptosis, to increase protein adduct formation and to enhance phosphorylation of MAP kinases. As shown in Fig. 9 Fig.  12, in control cells, the MAPK inhibitors had no effect on actin stress fibers, cell morphology, and cell-cell interactions. However, the exposure of cells to MAPK inhibitors, PD 98059 and SB 202190, partially prevented 4-HNE-mediated actin rearrangement, whereas the JNK inhibitor partially attenuated both actin rearrangement and gap junction. These results suggest that all three groups of mitogen-activated protein kinases, the ERK, JNK, and p38 MAPK, are involved in 4-HNE-mediated actin remodeling in BLMVECs. DISCUSSION Pulmonary endothelium serves as a semi-selective barrier between the plasma and interstitium to circulatory cells, macromolecules, and bioactive agents (26). Maintenance of this semi-selective barrier represents an important physiological process for vessel wall homeostasis and lung function. Injury to the endothelium results in barrier dysfunction with exudation of proteins and fluids within the interstitium and alveolar space (26). The onset and development of acute lung injury in acute respiratory distress syndrome and chronic obstructive pulmonary disease have been coupled to the activation and diapedesis of neutrophils in pulmonary microcirculation with subsequent release of proteases, inflammatory mediators, and ROS/reactive nitrogen intermediates (36). The ROS/reactive nitrogen intermediates react with cellular proteins, lipids, thiols, high-energy nucleotides, and DNA resulting in cell injury and dysfunction. 4-HNE, formed during inflammation and ox- idative stress, is one of the major biologically active aldehydes generated from peroxidation of membrane lipids (1). The toxic effects of 4-HNE are associated with several pathophysiological conditions, such as Parkinson and Alzheimer degenerative disorders, atherosclerosis, liver disorders, and lung diseases (1-3, 12, 37-40). Once formed, 4-HNE is relatively stable, can diffuse within or leak out of cells, and attack targets far from the site of origin (1). More recently, it has been observed that 4-HNE also modulates cellular DNA, RNA, protein synthesis, cell growth and differentiation (1-3), apoptosis (7,10,12,13), and microtubule remodeling (25). These effects of 4-HNE involve changes in intracellular calcium (41)(42)(43), modulation of signaling enzymes such as adenylate cyclase, protein kinase C, phospholipase D, phospholipase C, phosphatidylinositol 3-kinase (4 -6), and MAP kinases (7)(8)(9)(10)(11). Earlier studies on the infusion of 4-HNE (50 M) into rat lungs produced perivascular edema with vascular compression and early endothelial cell were subjected to 10% SDS-PAGE and probed with anti-phospho-ERK, anti-phospho-JNK, and pan antibodies as described in the legend to Fig. 6. For p38 MAP kinase, cell lysates were probed with pan-p38 MAP kinase, phospho-MAPKAPK-2, and pan-MAPKAPK-2 antibodies. Shown are representative blots from three independent experiments. The Western blots were analyzed by image analyzer and -fold change in phosphorylation of MAP kinases was normalized to total ERK, JNK, or p38 MAPK. *, significantly different from control (p Ͻ 0.05).

FIG. 11. 4-HNE induces actin fiber rearrangement in ECs.
BLMVECs grown on glass coverslips to ϳ95% confluence were challenged with varying concentrations of 4-HNE (1-25 M) for 30 min. F-actin cytoskeletal organization was visualized following fixation of cells with 3.7% formaldehyde and staining with Alexa fluor phalloidin 568. Stained cells were examined by immunofluorescence microscope using an ϫ60 oil objective. Treatment of BLMVECs with 4-HNE resulted in the rearrangement of actin microfilament (vertical arrows) and formation of intercellular gaps (horizontal arrows). disruption (19). Treatment of human umbilical vein ECs with 4-HNE (1-10 M) for 24 h increased albumin transport through cell monolayers (13). Furthermore, 4-HNE affected blood brain barrier by altering capillary EC permeability (44). In the present study, we show for the first time a role for MAPKs in 4-HNE-induced actin remodeling and barrier function in lung microvascular ECs. Additionally, we detected the formation of protein adduct in the presence of 4-HNE by Western blotting with the 4-HNE-Michael adduct antibody. The novel finding of the present study is the role of ERK, JNK, and p38 MAPK regulating 4-HNE-induced barrier function, as measured by changes in TER in lung microvascular ECs. This conclusion is based on the ability of PD 98059, SP 600125, and SB 202190, specific pharmacological inhibitors of MEK, JNK, and p38, to prevent 4-HNE-dependent reduction in TER and actin stress fiber formation. We have earlier demonstrated that H 2 O 2 -mediated barrier function in lung microvascular ECs is regulated by p38 MAPK, but not by ERK or JNK (45). Although, both H 2 O 2 and 4-HNE activate ERK, JNK, and p38 MAPK, only 4-HNE-induced barrier function is regulated by all three MAPKs. At present the precise intracellular targets of three MAPKs are yet to be identified to establish the differential action of H 2 O 2 and 4-HNE on EC barrier dysfunction. It is conceivable that the specificity of different classes of MAPKs in altering EC barrier function is dictated by the intensity of the activation of different classes of MAPKs by various stimuli and duration of these signals. For example, activation of ERK by growth factors and phorbol esters is robust compared with p38 MAPK and JNK in NIH-3T3 cells (46). On the contrary, ROS and hyperosmolar stress are potent stimulators of JNK and p38 MAPK in fibroblasts and ECs (47). However, in rat vascular smooth muscle cells, ROS is a potent activator of big MAPK1 but not ERK (48).
Mechanism(s) of regulation of EC barrier function by MAPKs is unclear. Possible mechanism(s) include phosphorylation of targets like heat shock protein 27 (49), cytoskeletal proteins (50 -52), focal adhesion plaques, and adherens junction proteins including cadherins and catenins (28,52). Phosphorylation of heat shock protein 27 is mediated by the mitogenactivated kinase-activated protein kinase-2, a downstream target of activated p38 MAPK (49). Earlier studies have shown that agonist-or oxidant-mediated MAPK activation and actin stress fiber formation were attenuated by SB 203580, an inhibitor of p38 MAPK (49 -52). Our present study shows that 4-HNE is a potent modulator of actin microfilament in BLM-VECs leading to gap formation. The effect of 4-HNE on actin rearrangement was partially prevented by MAPK inhibitors suggesting an important role for ERK, p38 MAPK, and JNK in cytoskeletal regulation. We have earlier demonstrated that H 2 O 2 -mediated TER changes in lung microvascular ECs were dependent on the redox status of the cell as addition of Nacetylcysteine prior to oxidant treatment partially restored intracellular GSH level as well as barrier function (45). Furthermore, the oxidant-induced barrier dysfunction regulated by p38 MAPK was also redox sensitive in lung microvascular ECs (45). There is evidence in the literature that 4-HNE depletes antioxidants, in particular, GSH, promoting cellular damage (53). Thus depletion of intracellular GSH by 4-HNE may play a key role in the modulation of signaling enzymes involved in the regulation of barrier function.
Changes in [Ca 2ϩ ] i in response to agonists or ROS have been implicated in EC barrier function. Exposure of ECs to oxidants increased [Ca 2ϩ ] i , whereas chelation of extracellular calcium partly prevented the oxidant response suggesting that the influx of extracellular calcium and mobilization of intracellular calcium were involved in the regulation of barrier function (28). One possible mechanism of the increase in [Ca 2ϩ ] i by oxidants is through the hydrolysis of polyphosphoinositides by phospholipase C resulting in the generation of diacylglycerol and inositol 1,4,5-trisphosphate. Whereas diacylglycerol is an endogenous activator of classical and novel protein kinase C isoforms, inositol 1,4,5-trisphosphate binds to specific receptors on the endoplasmic reticulum releasing calcium (54). Recently, we reported that chelation of intracellular calcium with BAPTA attenuated the diperoxovanadate-mediated increase in [Ca 2ϩ ] i and changes in TER in ECs (29). However, chelation of extracellular calcium with EGTA only partially blocked DPV-induced intracellular calcium release and permeability changes (29). The present study shows that 4-HNE, compared with H 2 O 2 or DPV, did not increase intracellular calcium, although it decreased TER suggesting a calcium-independent mechanism of 4-HNE-mediated barrier function in lung microvascular ECs. In contrast to our data, earlier studies have demonstrated that 4-HNE modulated [Ca 2ϩ ] i in hepatocytes (41) and neurons (42,43). Furthermore, the data reported here do not exclude the possible inhibition of ion pumps in plasma membrane and/or in the endoplasmic reticulum by 4-HNE, as modification of the sulfhydryl groups in Ca 2ϩ -ATPase by 4-HNE causes changes in [Ca 2ϩ ] i (55). However, in BLMVECs, exposure to 4-HNE (1-100 M) for Ն1 h did not increased [Ca 2ϩ ] i (data not shown). Thus, the ability of 4-HNE to induce [Ca 2ϩ ] i seems to be dependent on the cell type studied.
There is considerable evidence for the role of cytoskeletal proteins including actin and actin-binding proteins, focal adhesion, and adherens junction proteins in regulation of EC barrier function (26,27). Recent studies have demonstrated that agonists such as thrombin and ROS potently and rapidly polymerized G-actin to F-actin and decreased TER in lung ECs, suggesting participation of actin reorganization in barrier function (26). In contrast to these edemic agonists, sphingosine 1-phosphate, a platelet-derived bioactive lipid mediator, enhanced TER and stabilized the reorganization of filamentous actin into prominently thickened cortical actin band (56). These data point out that agonist-dependent destabilization or stabilization of actin microfilaments with other cytoskeletal proteins regulate EC barrier function. In this context, it is relevant to point out that cortactin, an actin-binding protein, plays an important role in modifying cortical actin assembly and organization. Consistent with its ability to bind F-actin, cortactin localizes within the peripheral cell structures such as lamellipodia, pseudopodia, and membrane ruffles. Furthermore, agonist-induced tyrosine phosphorylation of cortactin by Src kinase is associated with cytoskeletal rearrangement (26). In the present study, we observed that 4-HNE induced actin stress fibers as well as intercellular gap formation suggesting a link between F-actin cytoskeletal rearrangement and permeability changes. However, it is unclear if 4-HNE enhances tyrosine phosphorylation of cortactin in lung microvascular ECs or if there is a co-localization of cortactin with actin in these stress fibers.
4-HNE acts as a non-oxidative, a highly reactive aldehyde, and a signaling molecule. It forms stable adducts with cysteine, lysine, and histidine by a Michael type of reaction that further undergo cyclization between the aldehyde and C-4 position of 4-HNE to form a hemiacetal structure (1,17). Our present results in BLMVECs exposed to physiological concentrations of 4-HNE are consistent with the earlier reports on formation of Michael adduct-type proteins. Although the current study has not characterized any of the Michael adducts formed in lung microvascular ECs, it has been demonstrated that HNE-protein adduct accumulation reflects cellular toxicity compromising tissue survival during heart ischemia, ischemia/reperfusion injury, or pulmonary diseases (18, 19, 38 -40). Exogenously added or endogenously generated in cells, 4-HNE modulates protein function; examples include Na-K-ATPase (57), glucose transporter (58), MAPKs (7)(8)(9)(10)(11)24), phospholipases (1,4,5,59,60), protein kinase C (6), IK␤ kinase (61), and gene expression of ␥-glutamylcysteine synthetase (62). Thus, 4-HNE generated during lipid peroxidation can serve as an extracellular and intracellular signaling molecule altering cellular responses to stress and toxicity.
In conclusion, we have shown that the lipid peroxidation product, 4-HNE, induces EC barrier function involving activation of ERK, JNK, and p38 MAPKs. We have also demonstrated that 4-HNE-mediated cytoskeletal rearrangement is also regulated by the MAPKs. The ability of 4-HNE to regulate EC barrier function has important implications for the role of this lipid peroxidation product in the physiology of vessel wall disorders and lung diseases.