Regulation of low shear flow-induced HAEC VCAM-1 expression and monocyte adhesion

Sumathy Mohan1, Natarajan Mohan2, Anthony J. Valente3, and Eugene A. Sprague1

1 Division of Cardiovascular and Special Intervention and 2 Division of Radiation Oncology, Department of Radiology and 3 Department of Medicine, University of Texas Health Science Center, San Antonio, Texas 78284-7800


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We recently reported that prolonged exposure of human aortic endothelial cells (HAEC) to low shear stress flow patterns is associated with a sustained increase in the activated form of the transcriptional regulator nuclear factor-kappa B (NF-kappa B). Here we investigate the hypothesis that low shear-induced activation of NF-kappa B is responsible for enhanced expression of vascular cell adhesion molecule (VCAM-1) resulting in augmented endothelial cell-monocyte (EC-Mn) adhesion and that this activation is dependent on intracellular oxidant activity. Before exposure to low shear (2 dyn/cm2) for 6 h, HAEC were preincubated with or without the antioxidants pyrrolidine dithiocarbamate (PDTC) or N-acetyl-L-cysteine (NAC). PDTC strongly inhibited low shear-induced activation of NF-kappa B, expression of VCAM-1, and EC-Mn adhesion. Paradoxically, NAC exerted a positive effect on low shear-induced VCAM-1 expression and EC-Mn adhesion and only slightly downregulated NF-kappa B activation. However, cytokine-induced NF-kappa B activation and VCAM-1 expression are blocked by both PDTC and NAC. These data suggest that NF-kappa B plays a key role in low shear-induced VCAM-1 expression and that pathways mediating low shear- and cytokine-induced EC-Mn adhesion may be differentially regulated.

transcription regulator; hemodynamics; atherosclerosis; human aortic endothelial cell; nuclear factor-kappa B; vascular cell adhesion molecule


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIAL CELLS LINING the vascular system are subjected to different hemodynamic blood flow patterns depending on arterial geometry. Early lesions of atherosclerosis tend to occur in regions of the vessel that experience low shear stress and reversing flow patterns such as arterial bifurcations and curved segments of the large elastic arteries (2, 11, 29). These lesion-prone areas are characterized by the enhanced recruitment, adhesion, and transendothelial migration of monocytes, leading to the development of foam cells in the subendothelial space (3, 9). Although there are several reports associating low shear stress areas and early atherosclerotic lesions (2, 11, 29), the molecular mechanisms involved in the cellular interactions at the lesion-prone areas have not been clearly defined. In vitro studies from our laboratory using human aortic endothelial cells (HAEC) (26) and in vivo studies by others using animal models (31) have shown that low shear stress enhances the expression of the vascular cell adhesion molecule (VCAM-1; CD106). VCAM-1 binds to those cells expressing the integrin very late antigen-4 (VLA-4), such as monocytes, T and B lymphocytes, basophils, and eosinophils (10). The increased expression of VCAM-1 in the arterial intima of human atherosclerotic plaques correlates with the specific enhanced recruitment of monocytes that subsequently leads to the progression of atherosclerotic lesions (21). A recent report from our laboratory demonstrated that exposing HAEC to prolonged low shear stress also induces a sustained activation of a key transcriptional regulator, nuclear factor-kappa B (NF-kappa B) (16). Studies of molecular structural analysis have shown that the 5' promoter regions of many adhesion molecules, including VCAM-1, contain one or more binding sites for NF-kappa B (5, 6, 13, 14). On induction by a variety of stimuli, the activated form of NF-kappa B is translocated to the nucleus and transactivates genes containing functional NF-kappa B binding sites. In the present study, we investigate 1) whether low shear stress-induced NF-kappa B in HAEC is responsible for the enhanced expression of VCAM-1 and 2) whether the blockade of NF-kappa B activation by the structurally different antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) inhibits low shear stress-induced VCAM-1 expression and associated endothelial monocyte adhesion. We demonstrate that the endothelial monocyte adhesion that is augmented by low shear stress, through activation of NF-kappa B and enhanced VCAM-1 expression, is efficiently inhibited by PDTC. Paradoxically, NAC, a general antioxidant, had a positive effect on low shear-induced VCAM-1 expression and monocyte adhesion. Monoclonal antibodies to VCAM-1 specifically blocked most of the low shear-induced endothelial cell-monocyte adhesion. On the other hand, in the presence of NAC, VCAM-1 antibodies only partially blocked the low shear-induced endothelial cell-monocyte adhesion, suggesting that low shear in the presence of NAC induces adhesion molecules other than VCAM-1.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell culture. HAEC (Clonetics/BioWhittaker, San Diego, CA) were cultured in MCDB-131 medium (Sigma, St. Louis, MO) containing 10% bovine calf serum (BCIS, HyClone, Kansas City, KS) enriched with 250 ng/ml fibroblast growth factor (Pepro Tech, Rocky Hill, NJ), 1 mg/ml epidermal growth factor (Pepro Tech), 1 mg/ml hydrocortisone (Sigma), 100 U/ml penicillin, and 100 mg/ml streptomycin (Mediatech, Herndon, VA). Cells from passages 4-7 were used for all the experiments.

Shear stress experiments. HAEC were seeded on polyester film (10 × 19-cm Mylar sheets; Regal Plastics, San Antonio, TX) precoated with 2% gelatin and grown to near confluence, which was attained within 2-3 days. The cells on the slips were incubated in MCDB-131 medium containing 2% bovine calf serum in the absence of supplemented growth factors and hydrocortisone for 20 h before initiation of flow shear. Flow experiments were performed using the closed loop flow system described previously (22). In brief, HAEC-covered sheets were placed within rectangular parallel plate flow chambers, resulting in an available 90-cm2 cell surface area exposed to flow. In this flow system, the circulating medium flows by hydrostatic pressure from an upper reservoir through the flow chamber into a lower reservoir. The height difference between the two reservoirs determines the flow rate and the shear stress level in the cell chamber. Regardless of the shear regimen, flow rate was maintained at the same level through all chambers. Shear stress level was altered by adjusting the height of separation between the parallel plates. At any given flow rate, shear stress varies inversely with the square of chamber channel height. The medium was circulated back to the upper reservoir using a peristaltic roller pump (Master flex, Cole-Palmer Instrument, Chicago, IL). The temperature within the flow chamber was maintained at 37°C. The culture medium was buffered with 15 mM HEPES without bicarbonate to maintain the medium at a constant pH (pH 7.4). The flow chamber was designed so that it could be positioned on an inverted light microscope (Optiphot, Nikon, Japan) for continuous monitoring. Cells were subjected to 6 h of low shear stress (2 dyn/cm2) and harvested for nuclear protein extraction. As a control, slips with nearly confluent cells were incubated in the flow medium in a 37°C chamber under static conditions. Cells grown on 100-mm culture dishes and treated with 10 ng/ml interleukin-1beta (IL-1beta ; R&D Systems, Minneapolis, MN) for 4 h were used as positive controls.

Inhibition studies with antioxidants. The antioxidants PDTC and NAC were freshly prepared for every experiment. The stock solution of 1 M NAC or PDTC was prepared by dissolving NAC or PDTC in 1× PBS and was filter sterilized. For NAC, the pH was adjusted to pH 7.4 with 3 N NaOH before filter sterilization. Cells were preincubated with 100 µM PDTC for 1.5 h or with 30 mM NAC for 0.5 h and then subjected to low shear stress for 6 h. To study the dose-dependent regulation of VCAM-1 gene expression, PDTC concentrations in the range of 50, 75, and 100 µM or NAC concentrations in the range of 30, 40, and 50 mM were used. The cell viability at these concentrations of PDTC or NAC was determined by the trypan blue dye exclusion method. In addition, to ensure the reversible nature of the inhibitor, HAEC were treated with 100 µM PDTC or 30 mM NAC, and the cells were left in culture for 24 h. The cells were then challenged with a 10 ng/ml concentration of IL-1beta for 4 h and were harvested for nuclear protein extraction to study the NF-kappa B DNA binding activity.

Electrophoretic mobility shift analysis. After flow shear or IL-1beta stimulation, cells were washed in ice-cold PBS and harvested at the indicated time periods. Nuclear proteins were extracted by methods reported by Mohan et al. (16). In brief, the cell pellet was resuspended in 400 µl of cold buffer A [10 mM HEPES-KOH, pH 7.9 at 4°C, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol (DTT), and a cocktail of protease inhibitors containing aprotinin, antipain, leupeptin, and bestain at final concentrations recommended by the manufacturer (Boehringer Mannheim, Indianapolis, IN)]. The cells were allowed to swell in buffer A on ice for 10 min, followed by centrifugation for 10 s at 14,000 rpm in a Microfuge centrifuge at 4°C. The pellet was resuspended in 50 µl of cold buffer B (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and a cocktail of protease inhibitors) and further incubated on ice for 20 min for high-salt extraction. The samples were centrifuged for 10 min at 14,000 rpm at 4°C, and the clear supernatants were transferred to prechilled tubes. The total protein concentrations were measured using the bicinchoninic acid method, following the manufacturer's protocol (Pierce, Rockford, IL). For electrophoretic mobility shift analysis (EMSA), a double-stranded oligonucleotide containing a tandem repeat of the consensus sequences of 5'-GGG-GAC-TTT-CC-3' was end labeled with T4 polynucleotide kinase (Promega, Madison, WI). Free unbound radioisotope was separated by a push column device (Stratagene, La Jolla, CA). The binding reaction was performed by mixing nuclear extract (8 µg of total protein), 0.1 µg of poly(dI-dC) (Pharmacia Fine Chemicals), and [gamma -32P]ATP-labeled NF-kappa B-specific oligonucleotide probe [0.5 ng DNA; ~50,000 counts/min (cpm); Amersham, Arlington Heights, IL] in binding buffer containing 10 mM Tris · Cl (pH 7.5), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, and 20% (vol/vol) glycerol. For competition assay, the nuclear extract was preincubated with unlabeled homologous NF-kappa B oligonucleotide for 5 min on ice. This procedure was followed by addition of labeled NF-kappa B probe. Incubations were performed at ambient temperature for 20 min. Subsequently, all samples were electrophoresed using 6% polyacrylamide gels in Tris-glycine buffer. The gels were then dried and autoradiographed. Estimation of NF-kappa B activation was performed by quantitative analysis using the National Institutes of Health (NIH) 1.58b19 image analysis software package with an integrated density program.

Northern analysis. Total cellular RNA was isolated from HAEC by using the Ultraspec reagent, following the manufacturer's protocol (Biotecx, Houston, TX). Total RNA (10-15 µg) was separated on 1% agarose-formaldehyde gels in the presence of ethidium bromide, transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), and immobilized by baking at 80°C for 2 h. Both prehybridization and hybridization were performed at 42°C for 18 h in 6× SSPE buffer (pH 7.4; 900 mM NaCl, 50 mM NaPO4, and 5 mM EDTA). After prehybridization, the membrane was hybridized with a cDNA probe encoding human VCAM-1. The VCAM-1 probe was a Xba I-Apa I fragment cloned in pRc/CMV vector and generously donated by Dr. Daniel K. Burns (Hoffman-LaRoche, Nutley, NJ). The probe was labeled with [alpha -32P]dATP (ICN Pharmaceuticals, Costa Mesa, CA) using a Megaprime primer labeling kit (Amersham), yielding a specific activity of ~1 × 109 cpm/µg DNA. Autoradiography was performed with an intensifying screen at -70°C. Quantitative analysis of VCAM-1 mRNA expression was performed by using the NIH 1.58b19 image analysis software package with an integrated density program.

Flow cytometry. VCAM-1 protein expression was analyzed by fluorescence-activated cell sorting (FACS) analysis. HAEC subjected to low shear stress for 6 h with or without PDTC or NAC treatments were fixed with 1% formaldehyde for 10 min. Recombinant human tumor necrosis factor-alpha (TNF-alpha ; Promega) at a concentration of 10 ng/ml was used as a positive inducer. The cells were then detached from the plastic slips and probed with monoclonal anti-VCAM-1 antibody (Endogen, Woburn, MA) and FITC-labeled anti-mouse IgG antibody (Sigma) after blocking with 1% fetal bovine serum. In addition to no-shear test controls, cells were also incubated with isotype-matched (IgG1) immunoglobulin as a negative control. The labeled cells (~30,000 cells) were analyzed by EPICS ELITE flow cytometer (Coulter, Miami, FL). Statistical analysis was performed by the use of the Immuno-4 Analysis program (Coulter).

Monocyte adhesion assay. Whole blood, collected in vacutainer tubes containing EDTA from healthy volunteers by vein puncture was used within 4 h of collection. The monocytes were isolated by standard buoyant density centrifugation technique using NycoPrep 1.068 (Nycomed Pharma, Oslo, Norway). Cells obtained were further purified on Optiprep (Nycomed Pharma) to eliminate platelet contamination. Cell viability was determined by the trypan blue dye exclusion method. Purity of monocyte preparation was determined by labeling the cells with mouse monoclonal anti-macrophage antibodies (Enzo Diagnostics, Farmingdale, NY) followed by Texas red-conjugated goat anti-mouse antibodies (Calbiochem, San Diego, CA). Typically, >95% of the cells showed positive staining. For adhesion studies, the purified monocytes (1.67 × 104 cells/ml) were introduced into 150 ml of circulating medium and allowed to circulate at low shear for an additional 1 h after completion of the designated experimental flow regimen. The slips were then washed in PBS solution, fixed in methanol for 5 min, and stained with Giemsa stain (Sigma), which allows light microscopic identification of adherent monocytes. Under high-power light microscopy (×400), total adherent monocytes per high-power field were visualized and counted. No-shear control slips were subjected to 1 h of low shear with the monocytes in the circulation and used for comparison with PDTC- or NAC-incubated and low shear-subjected HAEC.

Antibody blocking assay. Endothelial cell-monocyte adhesion assay was also performed under low shear stress in the presence of 4 µg/ml anti-VCAM-1 antibody obtained from Endogen. HAEC incubated with or without NAC (30 mM) for 45 min and subjected to low shear stress for 5 h were further incubated with anti-VCAM-1 blocking antibodies at a concentration of 4 µg/ml for 45 min at 37°C. After incubation, the cells were further subjected to low shear stress in the presence of freshly purified monocytes for 1 h, and the number of monocytes adhering to the endothelial cells after a wash with PBS were counted as described above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Low fluid shear stress-induced NF-kappa B activation is inhibited by PDTC and NAC. To investigate whether the structurally different antioxidants PDTC and NAC can attenuate the low shear-induced as well as the cytokine-induced activation of NF-kappa B, HAEC were either subjected to 6 h of low shear stress (2 dyn/cm2) or treated with 10 ng/ml IL-1beta . Cells treated with IL-1beta were used as positive controls in EMSA experiments (Fig. 1C, lane 2). Quantitative analysis of the autoradio-gram, performed using the NIH 1.58b19 image analysis program, showed that nuclear extracts from cells exposed to 6 h of low shear (Fig. 1A, lane 2) contained a 5.2-fold higher NF-kappa B DNA binding activity than no-shear controls (P < 0.001; Fig. 1D). Preincubation with 100 µM PDTC strongly inhibited the low shear-induced activation of NF-kappa B in HAEC (P < 0.05; Fig. 1A, lane 3). In contrast, 30 mM NAC only slightly inhibited this activation (P < 0.001; Fig. 1A, lane 4). As an internal control, EMSA was performed with a probe for the ubiquitous factor octamer binding protein (OCT-1). No change in OCT-1 binding activity was observed, irrespective of the inhibitor treatment, indicating that the effect on NF-kappa B was specific (Fig. 1B). The final concentrations of PDTC (100 µM) and NAC (30 mM) chosen for inhibition studies were found to be nontoxic to the cells. Viability tests performed with various concentrations of PDTC and NAC by the trypan blue dye exclusion method indicated that cells were >90% viable after the incubations with either 100 µM PDTC or 30 mM NAC (Fig. 2, A and B). In a parallel experiment, cells treated with the inhibitors were subsequently cultured in inhibitor-free medium for a further 24 h and challenged with IL-1beta (10 ng/ml) for 4 h. EMSA results showed the induction of NF-kappa B DNA binding activity after IL-1beta treatment (Fig. 2C, lanes 2 and 4). This clearly indicated that the effects of the antioxidants are reversible and not cytotoxic. Therefore, the observed inhibition of NF-kappa B activation by the antioxidants is unlikely to have resulted from the effects of cell death.



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Fig. 1.   Inhibition of nuclear factor-kappa B (NF-kappa B) DNA binding activity by pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC) in low shear-subjected human aortic endothelial cells (HAEC). Cells, incubated for 20 h in complete medium containing 2% serum without growth factors, were treated with PDTC (100 µM) for 90 min or NAC (30 mM) for 30 min. Cells were then subjected to low shear for 6 h. Nuclear proteins were extracted, and electrophoretic mobility shift assay analyses were performed as described in MATERIALS AND METHODS. A: electrophoretic mobility shift analysis (EMSA) using 32P-labeled NF-kappa B-specific oligonucleotide probe showing NF-kappa B DNA binding activity. Lane 1, no shear (control); lane 2, low shear (2 dyn/cm2); lane 3, PDTC-pretreated cells subjected to low shear; lane 4, NAC-pretreated cells subjected to low shear. Autoradiogram is representative of at least 3 independent experiments. B: EMSA using 32P-labeled octamer binding protein (OCT-1)-specific oligonucleotide probe showing OCT-1 DNA binding activity. Lane 1, no shear (control); lane 2, low shear (2 dyn/cm2); lane 3, PDTC-pretreated cells subjected to low shear; lane 4, NAC-pretreated cells subjected to low shear. C: HAEC treated with 10 ng/ml interleukin-1beta (IL-1beta ) for 4 h used as positive control. Lane 1, untreated control; lane 2, IL-1beta -treated cells; lane 3, PDTC-pretreated cells subjected to IL-1beta induction. Arrows, specific bands of NF-kappa B DNA binding. D: total amount of NF-kappa B activation in nuclei from at least 3 independent experiments (means ± SE) was determined by quantitative analysis using NIH 1.58b19 image analysis software package with integrated density program. Background density on an autoradiogram was subtracted from densitometric data of each band.



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Fig. 2.   Viability of HAEC treated with PDTC or NAC at selected concentrations for 24 h. A and B: viability of HAEC in presence or absence of various concentrations of PDTC (A) and NAC (B). No. of viable cells was determined after 24 h by trypan blue dye exclusion method. Each point is arithmetic mean ± SD of 2 cell counts from 3 independent experiments and is expressed as a percentage of viability of untreated controls. C: NF-kappa B DNA binding activity in PDTC- or NAC-pretreated HAEC after IL-1beta induction. HAEC were pretreated with 100 µM PDTC or 30 mM NAC, left in culture for 24 h, and further induced with 10 ng/ml IL-1beta for 4 h. Cells were subjected to nuclear protein extraction, and EMSA was performed with 32P-labeled NF-kappa B probe. Autoradiogram shows untreated controls (lanes 1 and 3), cells pretreated with PDTC (lane 2), and cells pretreated with NAC (lane 4).

Inhibition of VCAM-1 mRNA expression. The effect of the antioxidants on the low shear-induced expression of VCAM-1 was investigated by Northern blot analysis (Fig. 3, A and B). HAEC subjected to 6 h of low shear exhibited expression of VCAM-1 mRNA (P < 0.01; Fig. 3A, lane 2) in contrast to the no-shear (static) controls in which the expression of VCAM-1 mRNA was undetectable (Fig. 3A, lane 1). Quantitative analyses of the autoradiograms from two independent experiments were performed using the NIH 1.58b19 image analysis program (Fig. 3B). PDTC at a concentration of 100 µM (Fig. 3A, lane 3) clearly inhibited the low shear-induced expression of VCAM-1 mRNA expression, as did lower concentrations of PDTC (50 and 75 µM; Fig. 3C, lanes 6 and 7). On the other hand, 30 mM NAC had minimal effect on the low shear-induced VCAM-1 mRNA gene expression (P > 0.01; Fig. 3A, lane 4). Furthermore, increased concentrations (40 and 50 mM) of NAC not only failed to inhibit VCAM-1 gene expression (Fig. 3C, lanes 2 and 3) but, in contrast, resulted in an increase in the level of expression of VCAM-1 mRNA. On the basis of these results, all subsequent studies were performed using 100 µM PDTC and 30 mM NAC.




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Fig. 3.   Northern blot analysis showing vascular cell adhesion molecule (VCAM-1) mRNA expression in HAEC exposed to low shear alone or pretreated with different concentrations of either PDTC or NAC before low shear exposure. A: control (no shear; lane 1), 6 h of low shear (lane 2), 100 µM PDTC pretreatment followed by 6 h of low shear (lane 3), 30 mM NAC pretreatment followed by 6 h of low shear (lane 4). B: densitometric scanning of VCAM-1 mRNA expression. Autoradiographs were scanned, and changes in VCAM-1 mRNA levels were quantified using an integrated density program (NIH 1.58b19 image analysis software). Background density on an autoradiogram was subtracted from densitometric data for each band. Results of 2 independent experiments were summarized and expressed as means ± SD of stimulation in multiples over no-shear control. C: controls (no shear; lanes 1 and 5), 40 and 50 mM NAC pretreatment followed by 6 h of low shear (lanes 2 and 3, respectively), 6 h of low shear (lane 4), and 50 and 75 µM PDTC pretreatment followed by 6 h of low shear (lanes 6 and 7, respectively). Arrow, mRNA fraction of VCAM-1. Equal amount of RNA loading was confirmed by ethidium bromide staining.

Inhibition of VCAM-1 protein expression. FACS analysis was performed to determine low shear stress-induced VCAM-1 protein expression in HAEC. As shown in Table 1, 100 µM PDTC eliminated induced VCAM-1 protein expression. However, HAEC incubated with 30 mM NAC and then subjected to 6 h of low shear stress exhibited increased expression of VCAM-1 protein expression. As shown in Table 1, 5.3 ± 0.7% of cells stained positive, significantly higher than in the controls (P < 0.01). HAEC incubated with NAC without shear showed only background staining. TNF-alpha (10 ng/ml)-induced HAEC were used as positive controls (50.6 ± 6.8%). Both NAC and PDTC efficiently inhibited TNF-alpha -induced VCAM-1 expression.

                              
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Table 1.   Percent of FITC-positive cells expressing VCAM-1 adhesion molecule

Effect of PDTC and NAC on endothelial monocyte adhesion. The influence of the antioxidants on low shear-induced enhancement of endothelial monocyte adhesion was also studied. In concordance with results obtained in NF-kappa B activation and VCAM-1 mRNA and protein expression, PDTC at a final concentration of 100 µM inhibited the adhesion of monocytes to HAEC close to the levels observed with controls, as shown in Fig. 4. Again, similar to the effect on VCAM-1 mRNA and protein expression, treatment of low shear-subjected cells with 30 mM NAC enhanced the adhesion of monocytes significantly (P < 0.05) compared with the controls and also compared with cells subjected to low shear alone (P < 0.1).


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Fig. 4.   Monocyte adhesion to HAEC pretreated with PDTC or NAC followed by exposure to 6 h of low shear (LS; 2 dyn/cm2). Confluent HAEC were preconditioned to 100 µM PDTC or 30 mM NAC for 90 and 30 min, respectively, followed by exposure to 6 h of low shear. Monocytes (1.67 × 104 cells/ml) purified from peripheral blood were then added to circulation, and low shear flow regimen was continued for 1 h. At end of incubation, after thorough rinsing, cells were fixed and stained by Giemsa stain and number of monocytes adhering was counted under high-power (×400 magnification) microscopy. No-shear controls were also subjected to circulating monocytes for 1 h under low shear. Anti-VCAM-1 antibody (Ab) blocking studies were performed by incubating HAEC subjected to 5 h of low shear stress (with or without NAC) with 4 µg/ml monoclonal anti-VCAM-1 Ab for 45 min at 37°C. HAEC exposed to Ab were further subjected to low shear stress for 1 h with monocytes present in circulation. Cells were fixed and stained, and number of monocytes was counted. Adhesion assays were performed independently 4 times. Data are expressed as means ± SD in multiples of increase over no-shear controls (Cont).

Blocking of monocyte adhesion by anti-VCAM-1 antibodies. Antibodies were used to investigate the role of VCAM-1 antigen in low shear stress-induced monocyte adhesion to HAEC. Anti-VCAM-1 antibodies inhibited 89.7% of the monocyte adhesion to HAEC by blocking VCAM-1 expression induced by low shear stress. On the other hand, they inhibited only 43% of the monocyte adhesion when HAEC were incubated with NAC and further subjected to low shear stress.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Areas of the human vascular tree that experience low shear stress are known to be predisposed to atherosclerosis (25). The results of our studies reported here indicate that treatment of HAEC with low shear (2 dyn/cm2) induces the activation of the transcription factor complex NF-kappa B, the enhanced expression of VCAM-1 mRNA and protein, and the increased adhesion of peripheral blood monocytes mediated by VCAM-1. The differential effects of the antioxidants PDTC and NAC on this low shear-induced response suggest that the mechanism of low shear-induced VCAM-1 differs in some way from cytokine-induced VCAM-1 expression.

Several studies using cytokines or mitogens as stimuli have shown VCAM-1 induction to be mediated by the activation of transcription factor NF-kappa B (6, 13, 14, 27). NF-kappa B, a multisubunit transcription factor activated in response to proinflammatory stimuli, plays a pivotal role in the development of the cellular immune and inflammatory response (4). Two functional NF-kappa B binding sites have been identified in the promoter regions of the VCAM-1 gene, and these together with two GATA sites in the core promoter of the gene appear to be important in cytokine-induced VCAM-1 expression (20). Although previous studies in our laboratory had demonstrated that exposure of HAEC to low shear was associated with a sustained elevation in both NF-kappa B activation and VCAM-1 expression, it was not known whether the low shear-induced activation of NF-kappa B was involved in the upregulation of VCAM-1 expression and the enhanced monocyte adhesion. Because several studies have demonstrated that NF-kappa B can be induced primarily by oxidant-sensitive mechanisms and that antioxidants can selectively inhibit NF-kappa B activation in many cell lines (12, 17, 23, 24, 30), the antioxidants PDTC and NAC were used in this current study to define the role of NF-kappa B in low shear-induced VCAM-1 expression. The antioxidant PDTC has been shown to efficiently and specifically block NF-kappa B (8, 24, 32). PDTC may exert its effects by scavenging superoxide anions and thus prevent the generation of H2O2. PDTC may also chelate Fe2+ and thereby inhibit the Fenton reaction that generates hydroxyl radicals from H2O2 (8). Furthermore, PDTC may directly block NF-kappa B activation by reversibly inhibiting the release of the inhibitory subunit Ikappa -Balpha from the active NF-kappa B complex. NAC has antioxidant activity comparable to PDTC and acts on · HOCl and OH · radicals, but it has been reported to have no effect on superoxide radical (1).

Several reports have shown that cytokine induction of VCAM-1 gene expression is specifically blocked by preincubation of cultured cells with 100 µM PDTC and 30 mM NAC (8, 32, 15). From our data presented here, it is clear that low shear stress-induced NF-kappa B activation, VCAM-1 mRNA and protein expression, and monocyte adhesion are effectively inhibited by PDTC at a concentration of 100 µM. NAC (30 mM), on the other hand, partially inhibited shear-induced NF-kappa B activation and, remarkably, did not suppress either mRNA or protein expression of VCAM-1. In fact, the data obtained from flow cytometer analysis clearly showed increased expression of VCAM-1 after the cells had been treated with the combination of 30 mM NAC and low shear stress of 2 dyn/cm2. In contrast, the TNF-alpha -induced VCAM-1 expression was efficiently downregulated by 30 mM NAC, as observed by others (15, 32). These results clearly indicate that NAC acts as a costimulator of low shear-induced VCAM-1 expression.

This effect of NAC is supported by several studies (18, 19, 28) reporting that low-molecular-weight thiols can act as prooxidants as well as antioxidants. This prooxidant effect of NAC has been reported in in vivo animal models: animals receiving low concentrations of NAC survived lipopolysaccharide-induced septic shock, whereas the mortality was very high among animals receiving high doses of NAC (28). The enhanced expression of VCAM-1 seen with low shear stress and NAC treatment correlated well with the augmented endothelial monocyte adhesion. The partial involvement of VCAM-1 in this increased adhesion was confirmed by blocking with monoclonal anti-VCAM-1 antibodies that inhibited the induced monocyte adhesion by 43%. The observed uninhibited portion of adhesion in these NAC-pretreated and low shear-subjected cells may have been due to the induction of adhesion molecules other than VCAM-1.

One possible explanation for the enhanced monocyte adhesion observed in the presence of low shear and 30 mM NAC may be that VCAM-1 expression is induced in HAEC by reactive oxygen species, especially superoxide radicals, which are not scavenged by NAC. It is also possible that PDTC may have inhibited the low shear-induced NF-kappa B activation by a mechanism other than its antioxidant attributes, such as its ability to prevent the uncoupling of Ikappa Balpha from the NF-kappa B complex. The prolonged time frame in which the cells are continuously exposed to low fluid shear stress and are also generating the superoxide radicals may induce the cells to express adhesion factors including VCAM-1 and soluble chemokine-like molecules that favor the binding of the monocytes. De Keulenaer et al. (7) recently showed in human endothelial cells exposed to oscillatory flow patterns that there is a sustained activation of prooxidant processes resulting in redox-sensitive gene expression, although the maximum levels of shear stress (±5 dyn/cm2) employed in this study exceeded the levels used in the present work.

In conclusion, our results indicate that effective blocking of NF-kappa B activation, as observed with PDTC pretreatment of HAEC, also blocks subsequent VCAM-1 and enhanced monocyte adhesion associated with low shear stress exposure. This further indicates that NF-kappa B activation plays a key role in low shear stress-induced VCAM-1 expression and monocyte adhesion. The paradoxical activation of VCAM-1 expression and monocyte adhesion by the antioxidant NAC provides a basis for further investigation into the low shear-induced endothelial cell-monocyte adhesion and signaling mechanisms relevant to NF-kappa B pathway and suggests that low shear-induced responses may be differentially mediated by oxidant-sensitive mechanisms compared with cytokine responses.


    ACKNOWLEDGEMENTS

We thank Jian Luo and Julie Meier for technical assistance in cell culture and shear experiments and Richard A. Salinas (Flow Cytometry Laboratory, Cancer Therapy and Research Center, San Antonio, TX) for performing FACS analysis. We also appreciate the photographic services of Cono Farias (Dept. of Radiology, University of Texas Health Science Center at San Antonio).


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants F32-HL-09694-01A1 (National Research Service Award to S. Mohan) and HL-52218.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: E. A. Sprague, Dept. of Radiology, Div. of Cardiovascular Interventions, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284-7800 (E-mail: sprague{at}uthscsa.edu).

Received 16 October 1998; accepted in final form 29 January 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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