Bovine Lung Microvascular Endothelial Cells Research Articles

Use of reflectance interference contrast microscopy to characterize the endothelial glycocalyx stiffness

Reflectance interference contrast microscopy (RICM) was used to study the mechanics of the endothelial glycocalyx. This technique tracks the vertical position of a glass microsphere probe that applies very light fluctuating loads to the outermost layer of the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx. Fluctuations in probe vertical position are used to estimate the effective stiffness of the underlying layer. Stiffness was measured before and after removal of specific glycocalyx components. The mean stiffness of BLMVEC glycocalyx was found to be ~7.5 kT/nm(2) (or ~31 pN/nm). Enzymatic digestion of the glycocalyx with pronase or hyaluronan with hyaluronidase increased the mean effective stiffness of the glycocalyx; however, the increase of the mean stiffness on digestion of heparan sulfate with heparinase III was not significant. The results imply that hyaluronan chains act as a cushioning layer to distribute applied forces to the glycocalyx structure. Effective stiffness was also measured for the glycocalyx exposed to 0.1%, 1.0%, and 4.0% BSA; glycocalyx compliance increased at two extreme BSA concentrations. The RICM images indicated that glycocalyx thickness increases with BSA concentrations. Results demonstrate that RICM is sensitive to detect the subtle changes of glycocalyx compliance at the fluid-fiber interface.

Stiffness and heterogeneity of the pulmonary endothelial glycocalyx measured by atomic force microscopy

The mechanical properties of endothelial glycocalyx were studied using atomic force microscopy with a silica bead (diameter ∼18 μm) serving as an indenter. Even at indentations of several hundred nanometers, the bead exerted very low compressive pressures on the bovine lung microvascular endothelial cell (BLMVEC) glycocalyx and allowed for an averaging of stiffness in the bead-cell contact area. The elastic modulus of BLMVEC glycocalyx was determined as a pointwise function of the indentation depth before and after enzymatic degradation of specific glycocalyx components. The modulus-indentation depth profiles showed the cells becoming progressively stiffer with increased indentation. Three different enzymes were used: heparinases III and I and hyaluronidase. The main effects of heparinase III and hyaluronidase enzymes were that the elastic modulus in the cell junction regions increased more rapidly with the indentation than in BLMVEC controls, and that the effective thickness of glycocalyx was reduced. Cytochalasin D abolished the modulus increase with the indentation. The confocal profiling of heparan sulfate and hyaluronan with atomic force microscopy indentation data demonstrated marked heterogeneity of the glycocalyx composition between cell junctions and nuclear regions.

Pulmonary Fibrosis Inducer, Bleomycin, Causes Redox-Sensitive Activation of Phospholipase D and Cytotoxicity Through Formation of Bioactive Lipid Signal Mediator, Phosphatidic Acid, in Lung Microvascular Endothelial Cells

The mechanisms of lung microvascular complications and pulmonary hypertension known to be associated with idiopathic pulmonary fibrosis (IPF), a debilitating lung disease, are not known. Therefore, we investigated whether bleomycin, the widely used experimental IPF inducer, would be capable of activating phospholipase D (PLD) and generating the bioactive lipid signal-mediator phosphatidic acid (PA) in our established bovine lung microvascular endothelial cell (BLMVEC) model. Our results revealed that bleomycin induced the activation of PLD and generation of PA in a dose-dependent (5, 10, and 100 µg) and time-dependent (2-12 hours) fashion that were significantly attenuated by the PLD-specific inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI). PLD activation and PA generation induced by bleomycin (5 µg) were significantly attenuated by the thiol protectant (N-acetyl-L-cysteine), antioxidants, and iron chelators suggesting the role of reactive oxygen species (ROS), lipid peroxidation, and iron therein. Furthermore, our study demonstrated the formation of ROS and loss of glutathione (GSH) in cells following bleomycin treatment, confirming oxidative stress as a key player in the bleomycin-induced PLD activation and PA generation in ECs. More noticeably, PLD activation and PA generation were observed to happen upstream of bleomycin-induced cytotoxicity in BLMVECs, which was protected by FIPI. This was also supported by our current findings that exposure of cells to exogenous PA led to internalization of PA and cytotoxicity in BLMVECs. For the first time, this study revealed novel mechanism of the bleomycin-induced redox-sensitive activation of PLD that led to the generation of PA, which was capable of inducing lung EC cytotoxicity, thus suggesting possible bioactive lipid-signaling mechanism/mechanisms of microvascular disorders encountered in IPF.

Redox-active antioxidant modulation of lipid signaling in vascular endothelial cells: vitamin C induces activation of phospholipase D through phospholipase A2, lipoxygenase, and cyclooxygenase.

We have earlier reported that the redox-active antioxidant, vitamin C (ascorbic acid), activates the lipid signaling enzyme, phospholipase D (PLD), at pharmacological doses (mM) in the bovine lung microvascular endothelial cells (BLMVECs). However, the activation of phospholipase A(2) (PLA(2)), another signaling phospholipase, and the modulation of PLD activation by PLA(2) in the ECs treated with vitamin C at pharmacological doses have not been reported to date. Therefore, this study aimed at the regulation of PLD activation by PLA(2) in the cultured BLMVECs exposed to vitamin C at pharmacological concentrations. The results revealed that vitamin C (3-10 mM) significantly activated PLA(2) starting at 30 min; however, the activation of PLD resulted only at 120 min of treatment of cells under identical conditions. Further studies were conducted utilizing specific pharmacological agents to understand the mechanism(s) of activation of PLA(2) and PLD in BLMVECs treated with vitamin C (5 mM) for 120 min. Antioxidants, calcium chelators, iron chelators, and PLA(2) inhibitors offered attenuation of the vitamin C-induced activation of both PLA(2) and PLD in the cells. Vitamin C was also observed to significantly induce the formation and release of the cyclooxygenase (COX)- and lipoxygenase (LOX)-catalyzed arachidonic acid (AA) metabolites and to activate the AA LOX in BLMVECs. The inhibitors of PLA(2), COX, and LOX were observed to effectively and significantly attenuate the vitamin C-induced PLD activation in BLMVECs. For the first time, the results of the present study revealed that the vitamin C-induced activation of PLD in vascular ECs was regulated by the upstream activation of PLA(2), COX, and LOX through the formation of AA metabolites involving oxidative stress, calcium, and iron.

Effects of Lipopolysaccharide andMannheimia haemolyticaLeukotoxin on Bovine Lung Microvascular Endothelial Cells and Alveolar Epithelial Cells

Bovine respiratory disease resulting from infection with Mannheimia haemolytica commonly results in extensive vascular leakage into the alveoli. M. haemolytica produces two substances, lipopolysaccharide (LPS) and leukotoxin (LKT), that are known to be important in inducing some of the pathological changes. In the present study, we examined bovine pulmonary epithelial (BPE) cell and bovine lung microvascular endothelial cell monolayer permeability, as measured by trans-well endothelial and epithelial cell electrical resistance (TEER), after incubation with LPS, LKT, or LPS-activated neutrophils. Endothelial cell monolayers exposed to LPS exhibited significant decreases in TEER that corresponded with increased levels of proinflammatory cytokines, apoptosis, and morphological changes. In contrast, BPE cells exposed to LPS increased the levels of production of inflammatory cytokines but displayed no changes in TEER, apoptosis, or visible morphological changes. Both cell types appeared to express relatively equal levels of the LPS ligand Toll-like receptor 4. However, TEER in BPE cell monolayers was decreased when the cells were incubated with LPS-activated neutrophils. Although the incubation of BPE cells with LKT decreased TEER, this was not reduced by the incubation of LKT with a neutralizing antibody and was reversed when LKT was preincubated with the LPS-neutralizing compound polymyxin B. Because BPE cells did not express the LKT receptor CD11a/CD18, we infer that contaminating LPS was responsible for the decreased TEER. In conclusion, LPS triggered changes in endothelial cells that would be consistent with vascular leakage, but neither LPS nor LKT caused similar changes in epithelial cells, unless neutrophils were also present.

Nitroaspirin (NCX-4016), an NO Donor, is Antiangiogenic Through Induction of Loss of Redox-Dependent Viability and Cytoskeletal Reorganization in Endothelial Cells

We recently reported that NCX-4016, a derivative of aspirin containing a nitro moiety that releases nitric oxide (NO) in a sustained fashion in biologic systems, is a potent cytotoxic agent inhibiting the proliferation of cisplatin-resistant human ovarian cancer cells. Therefore, we hypothesize that NCX-4016 possesses antiangiogenic properties. Our study with the bovine lung microvascular endothelial cells (BLMVECs) revealed that NCX-4016 significantly induced the loss of redox-dependent cell viability in a dose- and time-dependent manner, as assayed by the redox-sensitive Alamar blue cell viability assay. Fluorescence microscopy of cells labeled with NO-specific fluorophore (DAF-FM) confirmed that NCX-4016 generated significant levels of intracellular NO. NO donors, including S-nitroso-N-acetylpenicillamine, spermine NONOate, and isosorbide dinitrite, were less effective in causing loss of cell viability. Thiol-protectant, N-acetylcysteine, significantly attenuated the NCX-4016-induced loss of cell viability, suggesting the role of alteration of thiol-redox status therein. NCX-4016 also suppressed oxygen consumption, decreased transendothelial electrical resistance (EC barrier dysfunction), and induced actin cytoskeletal reorganization in BLMVECs. The in vitro assay with human umbilical vein ECs and BLMVECs revealed that NCX-4016, in a dose-dependent manner, significantly inhibited angiogenesis with almost complete inhibition at a 100-microM concentration, suggesting that NCX-4016 can act as an antiangiogenic drug.

Angiopoietin-2 is increased in severe sepsis: Correlation with inflammatory mediators

Angiopoietin (Ang)-2 is an endothelium-specific growth factor, regulated by proinflammatory stimuli, that destabilizes vascular endothelium and increases vascular leakage; consequently, Ang-2 may contribute to sepsis pathophysiology. We have studied 1) serum Ang-2 levels in critically-ill patients and investigated potential relationships with inflammatory mediators and indices of disease severity and 2) the effect of sepsis-related inflammatory mediators on Ang-2 production by lung endothelium in vitro. Prospective clinical study followed by cell culture studies. General intensive care unit and research laboratory of a university hospital. Human and bovine lung microvascular endothelial cells and 61 patients (32 men). Patients were grouped according to their septic stage as having: no systemic inflammatory response syndrome (n = 6), systemic inflammatory response syndrome (n = 8), sepsis (n = 16), severe sepsis (n = 18), and septic shock (n = 13). Cells were exposed to lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-6. Patients' serum Ang-2 levels were significantly increased in severe sepsis as compared with patients with no systemic inflammatory response syndrome or sepsis (p < .05 by analysis of variance). Positive linear relationships were observed with: serum tumor necrosis factor-alpha (rs = 0.654, p < .001), serum interleukin-6 (rs = 0.464, p < .001), Acute Physiology and Chronic Health Evaluation II score (rs = 0.387, p < .001), and Sequential Organ Failure Assessment score (rs = 0.428, p < .001). Multiple regression analysis revealed that serum Ang-2 is mostly related to serum tumor necrosis factor-alpha and severe sepsis. Treatment of human lung microvascular endothelial cells with all mediators resulted in a concentration-dependent Ang-2 reduction. Treatment of bovine lung microvascular endothelial cells with lipopolysaccharide and tumor necrosis factor-alpha increased Ang-2 release, and interleukin-6 reduced basal Ang-2 levels. First, patients' serum Ang-2 levels are increased during severe sepsis and associated with disease severity. The strong relationship of serum Ang-2 with serum tumor necrosis factor-alpha suggests that the latter may participate in the regulation of Ang-2 production in sepsis. Second, inflammatory mediators reduce Ang-2 release from human lung microvascular endothelial cells, implying that this vascular bed may not be the source of increased Ang-2 in human sepsis.

Redox Regulation of 4-Hydroxy-2-nonenal-mediated Endothelial Barrier Dysfunction by Focal Adhesion, Adherens, and Tight Junction Proteins

4-Hydroxy-2-nonenal (4-HNE), one of the major biologically active aldehydes formed during inflammation and oxidative stress, has been implicated in a number of cardiovascular and pulmonary disorders. 4-HNE has been shown to increase vascular endothelial permeability; however, the underlying mechanisms are unclear. Hence, in the current study, we tested our hypothesis that 4-HNE-induced changes in cellular thiol redox status may contribute to modulation of cell signaling pathways that lead to endothelial barrier dysfunction. Exposure of bovine lung microvascular endothelial cells (BLMVECs) to 4-HNE induced reactive oxygen species generation, depleted intracellular glutathione, and altered cell-cell adhesion as measured by transendothelial electrical resistance. Pretreatment of BLM-VECs with thiol protectants, N-acetylcysteine and mercaptopropionyl glycine, attenuated 4-HNE-induced decrease in transendothelial electrical resistance, reactive oxygen species generation, Michael protein adduct formation, protein tyrosine phosphorylation, activation of ERK, JNK, and p38 MAPK, and actin cytoskeletal rearrangement. Treatment of BLMVECs with 4-HNE resulted in the redistribution of FAK, paxillin, VE-cadherin, beta-catenin, and ZO-1, and intercellular gap formation. Western blot analyses confirmed the formation of 4-HNE-derived Michael adducts with the focal adhesion and adherens junction proteins. Also, 4-HNE decreased tyrosine phosphorylation of FAK without affecting total cellular FAK contents, suggesting the modification of integrins, which are natural FAK receptors. 4-HNE caused a decrease in the surface integrin in a time-dependent manner without altering total alpha5 and beta3 integrins. These results, for the first time, revealed that 4-HNE in redox-dependent fashion affected endothelial cell permeability by modulating cell-cell adhesion through focal adhesion, adherens, and tight junction proteins as well as integrin signal transduction that may lead dramatic alteration in endothelial cell barrier dysfunction during heart infarction, brain stroke, and lung diseases.

Vitamin C-induced activation of phospholipase D in lung microvascular endothelial cells: Regulation by MAP kinases

Our earlier studies have shown that vitamin C at pharmacological doses (mM) induces loss of redox-dependent viability in bovine lung microvascular endothelial cells (BLMVECs) that is mediated by oxidative stress. Therefore, here, we investigated the vitamin C-induced activation of the lipid signaling enzyme, phospholipase D (PLD) in BLMVECs. Monolayer cultures of BLMVECs were treated with vitamin C (0–10 mM) for different time periods (0–2 h) and the activity of PLD was determined. Vitamin C induced activation of PLD in BLMVECs in a time- and dose-dependent fashion that was significantly attenuated by antioxidants, p38 mitogen-activated protein kinase (p38 MAPK)-specific inhibitor (SB203580), extracellular signal-regulated protein kinase (ERK)-specific inhibitor (PD98059), and transient transfection of cells with dominant-negative (DN)-p38 MAPK and DN-ERK1/ERK2. Vitamin C also induced phosphorylation and enhanced the activities of p38 MAPK and ERK in BLMVECs in a time-dependent fashion. It was also evident that vitamin C induced translocation of PLD 1 and PLD 2, association of p38 MAPK and ERK with PLD 1 and PLD 2, threonine phosphorylation of PLD 1 and PLD 2 and SB203580- and PD98059-inhibitable threonine phosphorylation of PLD 1 in BLMVECs. Transient transfection of BLMVECs with DN-p38 MAPK and DN-ERK1/ERK2 resulted in marked attenuation of vitamin C-induced phosphorylation of threonine in PLD 1 and PLD 2. We, for the first time, showed that vitamin C at pharmacological doses, activated PLD in the lung microvascular ECs through oxidative stress and MAPK activation.

Protein Kinase C-dependent Protein Kinase D Activation Modulates ERK Signal Pathway and Endothelial Cell Proliferation by Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.

BMK1/ERK5 Is a Novel Regulator of Angiogenesis by Destabilizing Hypoxia Inducible Factor 1α

Big MAP kinase 1 (BMK1 or ERK5) is a key mediator of endothelial cell (EC) function as shown by impaired embryonic angiogenesis and vascular collapse in BMK1 knockout mice. Hypoxia inducible factor 1alpha (HIF1alpha), a potent mediator of angiogenesis, is positively regulated by the MAP kinases, ERK1/2. Because BMK1 deficiency is associated with impaired angiogenesis we hypothesized that BMK1 might regulate HIF1alpha. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1alpha and BMK1 cDNAs, and stimulated by hypoxia. HIF1alpha activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1alpha activation. Hypoxia (1% O2, 24 hours) stimulated HIF1alpha activity by 5.1+/-0.6 fold. In the presence of dominant negative (DN)-BMK1, which inhibited BMK1 activity, hypoxia induced HIF1alpha activity was enhanced significantly to 6.4+/-0.4 fold. BMK1 activation by constitutively active (CA)-MEK5 inhibited HIF1alpha activity by 46+/-4%, suggesting BMK1 functions as a negative regulator of HIF1alpha activation. Activation of BMK1 reduced HIF1alpha protein levels. Ubiquitination inhibitors (30 micromol/L ALLN, 2 micromol/L lactacystin, or 100 nmol/L MG132) reduced the BMK1-mediated effect on HIF1alpha expression by >80%, suggesting that BMK1 stimulated HIF1alpha proteolysis. The negative effect of BMK1 on HIF1alpha was functionally important because transfection with CA-MEK5 significantly decreased EC migration by 68+/-10%, and inhibited angiogenesis (in vitro Matrigel assay) by 76+/-7%. In summary, BMK1 is a novel negative regulator of HIF1alpha and angiogenesis by increasing HIF1alpha ubiquitination and inhibiting HIF1alpha activity in endothelial cells.

Vitamin C-Induced Loss of Redox-Dependent Viability in Lung Microvascular Endothelial Cells

Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction.

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.

Simultaneous measurement of oxygenation in intracellular and extracellular compartments of lung microvascular endothelial cells.

A new technique is described for simultaneous determination of intra- and extracellular oxygen concentrations [partial pressure of O(2) (pO(2))] in bovine lung microvascular endothelial cells (BLMVECs) using electron paramagnetic resonance (EPR) oximetry. The measurements were performed in BLMVEC suspensions of a 20- micro l volume containing 4,000 cells. The extracellular pO(2) was measured using a trityl EPR probe [triarylmethyl (TAM), 10 micro M], a tricarboxylate anion radical, that stays exclusively in the extracellular space. The intracellular oxygen was measured using a pre-internalized particulate spin probe, lithium 5,9,14,18,23,27,32,36-octa-n-butoxy-naphthalocyanine (LiNc-BuO). Because there is a wide discrepancy in the reported values of cellular oxygenation by and large due to differences in the methods employed, we utilized the dual EPR probe technique to measure the oxygen gradient that apparently exists across the cell membrane. The intra- and extracellular pO(2) values were 139 +/- 2.5 and 157 +/- 3.6 mm Hg, respectively, for cells exposed to room air. A fairly smaller gradient of oxygen was observed in cells exposed to 7.5% oxygen (pO(2) = 57 mm Hg). In summary, this study confirms the feasibility of simultaneous and accurate measurements of intra- and extracellular pO(2) using LiNc-BuO and TAM EPR oximetry probes.

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.

Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells.

Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.

Lung endothelial heparan sulfates mediate cationic peptide-induced barrier dysfunction: a new role for the glycocalyx.

The endothelial glycocalyx is believed to play a major role in microvascular permeability. We tested the hypothesis that specific components of the glycocalyx, via cytoskeletal-mediated signaling, actively participate in barrier regulation. With the use of polymers of arginine and lysine as a model of neutrophil-derived inflammatory cationic proteins, we determined size- and dose-dependent responses of cultured bovine lung microvascular endothelial cell permeability as assessed by transendothelial electrical resistance (TER). Polymers of arginine and lysine >11 kDa produced maximal barrier dysfunction as demonstrated by a 70% decrease in TER. Monomers of l-arginine and l-lysine did not alter barrier function, suggesting a cross-linking requirement of cell surface "receptors". To test the hypothesis that glycosaminoglycans (GAGs) are candidate receptors for this response, we used highly selective enzymes to remove specific GAGs before polyarginine (PA) treatment and examined the effect on TER. Heparinase III attenuated PA-induced barrier dysfunction by 50%, whereas heparinase I had no effect. To link changes in barrier function with structural alterations, we examined actin organization and syndecan localization after PA. PA induced actin stress fiber formation and clustering of syndecan-1 and syndecan-4, which were significantly attenuated by heparinase III. PA-induced cytoskeletal rearrangement and barrier function did not involve myosin light chain kinase (MLCK) or p38 MAPK, as ML-7, a specific MLCK inhibitor, or SB-20358, a p38 MAPK inhibitor, did not alter PA-induced barrier dysfunction. In summary, lung endothelial cell heparan sulfate proteoglycans are key participants in inflammatory cationic peptide-induced signaling that links cytoskeletal reorganization with subsequent barrier dysfunction.

Hyperoxia alters phorbol ester-induced phospholipase D activation in bovine lung microvascular endothelial cells.

We investigated the effect of hyperoxia on phospholipase D (PLD) activation in bovine lung microvascular endothelial cells (BLMVECs). Generation of intracellular reactive oxygen species in BLMVECs exposed to hyperoxia for 2 or 24 h was three-fold higher compared with normoxic cells as measured by dichlorodihydrofluorescein di(acetoxymethyl ester) fluorescence. Exposure of BLMVECs to hyperoxia for 2 or 24 h attenuated 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated PLD activation compared with normoxic cells, however, hyperoxia did not alter basal PLD activity. Antioxidants, such as propyl gallate and pyrrolidine dithiocarbamate, reversed the effect of hyperoxia on TPA-induced PLD activity. Furthermore, the TPA-induced PLD activation was inhibited not only by the protein kinase C inhibitor, Go6976, but also by the tyrosine kinase inhibitor, genistein, and by the Src kinase specific inhibitor, PP-2, suggesting the involvement of protein kinase C and also tyrosine kinases in TPA-induced PLD activation. Western blot analysis of cell lysates from the hyperoxic (2 or 24 h) BLMVECs stimulated with TPA with anti-phosphotyrosine antibody showed an attenuation in overall tyrosine phosphorylation of proteins. In conclusion, we have demonstrated that hyperoxia enhanced the generation of reactive oxygen species in lung microvascular endothelial cells and attenuated TPA-induced protein tyrosine phosphorylation and PLD activation. As protein tyrosine phosphorylation and PLD play important roles in inflammatory responses, this could provide a mechanism for the regulation of endothelial barrier function during hyperoxic lung injury.

Fluid shear stress attenuates hydrogen peroxide-induced c-Jun NH2-terminal kinase activation via a glutathione reductase-mediated mechanism.

c-Jun NH2-terminal kinase (JNK) is activated by a number of cellular stimuli including reactive oxygen species (ROS). Previous studies have demonstrated that fluid shear stress (flow) inhibits cytokine-induced JNK activation in endothelial cells (ECs). In the present study, we show JNK activation by ROS in ECs and hypothesized that flow inhibits ROS-induced JNK activation in ECs via modulation of cellular protection systems against ROS. JNK was activated by 300 micro mol/L hydrogen peroxide (H2O2) in bovine lung microvascular ECs (BLMVECs) with a peak at 60 minutes after stimulation (6.3+/-1.2-fold increase). Preexposure of BLMVECs to physiological steady laminar flow (shear stress=12 dyne/cm2) for 10 minutes significantly decreased H2O2-induced JNK activation. Thioredoxin and glutathione are cellular antioxidants that protect cells against ROS. Flow induced a significant increase in the ratio of reduced glutathione to oxidized glutathione consistent with a 1.6-fold increase in glutathione reductase (GR) activity. Preincubation of BLMVECs with the GR inhibitor, 1,3 bis-(2 chloroethyl)-1-nitrosourea, abolished the inhibitory effect of flow. In contrast, preincubation of BLMVECs with azelaic acid, a specific inhibitor for thioredoxin reductase, did not alter the effect of flow on H2O2-induced JNK activation. Overexpression of GR mimicked the effect of flow to inhibit JNK activation. These results suggest that flow activates GR, an important regulator of the intracellular redox state of glutathione, and exerts a protective mechanism against oxidative stress in endothelial cells.

Cloning and Characterization of the Human Lung Endothelial-Cell-Specific Molecule-1 Promoter

Endothelial-cell-specific molecule-1 (ESM-1) is a cysteine-rich protein that is expressed primarily in endothelial cells of the lung, kidney and gut. In the present study, we have cloned and sequenced 3,888 bp of the 5′ flanking region of the human ESM-1 gene. The full-length promoter directed high-level expression of the luciferase reporter gene in bovine lung microvascular endothelial cells and bovine aortic endothelial cells, but not in nonendothelial cell types. In 5′ deletion analyses, a region spanning –81 to +58 was shown to contain information for endothelial-cell-specific expression. Mutational analysis in transient transfection assays uncovered an important role for an Ets-binding motif located between –77 and –74 and a cAMP-response-element (CRE)-like motif located between –68 and –62 in mediating high-level expression in endothelial cells. A second Ets site (–63 to –60) as well as a novel 6-bp palindromic sequence (–58 to –53) were found to inhibit expression. In DNase footprint analyses, both the Ets-binding motifs were protected specifically in endothelial cells, while the CRE-like element and palindrome were protected in endothelial and nonendothelial cells alike. Taken together, these results provide an important foundation for studying endothelial-cell-subtype-specific gene regulation.