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Different effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the nuclear factor-κB mediated expression of ICAM-1 in human coronary endothelial and smooth muscle cells

Background Activation of nuclear factor-κB (NF-κB) is one of the key events in early atherosclerosis and restenosis. We hypothesized that tumor necrosis factor-α (TNF-α) induced and NF-κB mediated expression of intercellular adhesion molecule-1 (ICAM-1) can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides (RelA p65 and NF-κB1 p50). Results Smooth muscle cells (SMC) from human coronary plaque material (HCPSMC, plaque material of 52 patients), SMC from the human coronary media (HCMSMC), human endothelial cells (EC) from umbilical veins (HUVEC), and human coronary EC (HCAEC) were successfully isolated (HCPSMC, HUVEC), identified and cultured (HCPSMC, HCMSMC, HUVEC, HCAEC). 12 hrs prior to TNF-α stimulus (20 ng/mL, 6 hrs) RelA p65 and NF-κB1 p50 (1, 2, 4, 10, 20, and 30 μM) and controls were added for a period of 18 hrs. In HUVEC and HCAEC there was a dose dependent inhibition of ICAM-1 expression after adding of both RelA p65 and NF-κB1 p50. No inhibitory effect was seen after incubation of HCMSMC with RelA p65 and NF-κB1 p50. A moderate inhibition of ICAM-1 expression was found after simultaneous addition of RelA p65 and NF-κB1 p50 to HCPSMC, no inhibitory effect was detected after individual addition of RelA p65 and NF-κB1 p50. Conclusions The data point out that differences exist in the NF-κB mediated expression of ICAM-1 between EC and SMC. Experimental antisense strategies directed against RelA p65 and NF-κB1 p50 in early atherosclerosis and restenosis are promising in HCAEC but will be confronted with redundant pathways in HCMSMC and HCPSMC.

The NF-κB system might be a potential pharmacological target to interfere with chemotactic and adhesive mechanisms within the vessel wall. The prototypic NF-κB dimer, consisting of the subunits RelA p65 and NF-κB1 p50, is present in the cytosol in an inactive state, bound to the inhibitory protein that are collectively termed IκB. Activation of NF-κB by a multitude of stimuli, including inflammatory or lymphoproliferative cytokines, reactive oxygen intermediates and micro-organisms, requires the phosphorylation and proteolytic removal of IκB from the dimeric complex. This is followed by an intermediate translocation of activated NF-κB to the nucleus, where the dimer interacts with regulatory kb elements in promoters and enhancers, thereby controlling inducible gene transcription [8,14]. Recently first reports on inhibitory strategies of NF-κB mediated activities in human EC and SMC have been published [15][16][17][18][19].
ICAM-1 is one of various adhesion molecules that is activated via NF-κB pathway. The expression of ICAM-1 by SMC in human atheroma [20] and in hyperplastic lesions produced by experimental balloon injury [21] indicates that augmented ICAM-1 expression constitutes a marker of SMC activation of considerable in vivo relevance. It has been demonstrated that TNF-α increases the expression of ICAM-1 in human arterial SMC from peripheral [22] and coronary arteries [23,24] in a time and dose dependent manner. Recently our laboratory has reported [25] that high dose aspirin (5 mM) inhibits expression of ICAM-1 in human coronary vascular cells. In order to investigate a more specific inhibition of ICAM-1 expression we analysed the effect of antisense RelA p65 and NF-κB1 p50 oligonucleotides on the NF-κB-mediated expression of ICAM-1.

Intracellular uptake of FITC-labeled antisense
The intracellular uptake of fluorosense oligonucleotides in HUVEC, HCAEC, HCMSMC (Fig. 1), and HCPSMC was confirmed with fluorescence microscopy and flow cytometry. After fluorescence microscopical examination of HUVEC, HCAEC, HCMSMC, and HCPSMC the uptake of fluoresense started 1 hr after incubation. The maximal uptake was reached after 8 hrs, this level was kept for 24 and 48 hrs after incubation.
In flow cytometry examination baseline fluorescence was 2.77 in HUVEC, 4.18 in HCAEC, 1.88 in HCMSMC, and 4.68 in HCPSMC. 18 hrs after adding of fluorosense oligonucleotides the fluorescence intensity was increased to 82.03 and 89.11 in HUVEC and HCAEC ( Fig. 2A,B), respectively to 129.78 and 67.58 in HCMSMC and HCPSMC (Fig. 2C,D).

Impact of antisense on TNF-α mediated expression of ICAM-1 in HUVEC
Addition of antisense NF-κB1 p50 oligonucleotide to HUVEC caused only a very small inhibition of the TNF-α induced expression of ICAM-1 (Fig. 4A). In HUVEC the expression of ICAM-1 was 100, 93, and 91% after incubation with antisense NF-κB1 p50 oligonucleotide in a concentration of 1, 2, and 4 µM/mL and 86, 91, and 93% after incubation with antisense NF-κB1 p50 oligonucleotide in a concentration of 10, 20, 30 µM/mL. Incubation of HUVEC with antisense RelA p65 oligonucleotide caused a moderate reduction of the expression of ICAM-1 (Fig. 4A). Expression of ICAM-1 was 66, 71, and 75% after incubation with antisense RelA p65 oligonucleotide in a concentration of 1, 2, and 4 µM/mL and 75, 89, 77% after incubation with antisense RelA p65 oligonucleotide in a concentration of 10, 20, and 30 µM/mL. Simultaneous adding of antisense RelA p65 and NF-κB1 p50 oligonucleotides to HUVEC caused a dose dependent inhibition of the expression of ICAM-1. Expression of ICAM-1 was 90, 80, 63, and 49% after incubation with antisense RelA p65 and NF-κB1 p50 oligonucleotides in a concentration of 1, 2, 4 and 10 µM/mL. The further increase of the concentration to 20 and 30 µM/mL did not further increase the inhibitory effect. The expression of ICAM-1 was 61% after incubation with 20 µM/mL and 53% after incubation with 30 µM/mL (Fig. 4A, Fig.  3A,B). Mismatched antisense RelA p65 and NF-κB1 p50 oligonucleotides did not affect the expression of ICAM-1 in HUVEC, no matter whether added individually or simultaneously.

Impact of antisense on TNF-α mediated expression of ICAM-1 in HCMSMC
In HCMSMC adding of antisense NF-κB1 p50 oligonucleotides caused a moderate increase of the expression of TNF-α induced expression of ICAM-1 (Fig. 4C). The expression of ICAM-1 was 117, 122, and 130% after incubation with antisense NF-κB1 p50 oligonucleotides in a concentration of 1, 2, and 4 µM/mL and 117, 113, and 135% after incubation with antisense NF-κB1 p50 oligonucleotides in a concentration of 10, 20, 30 µM/mL. Antisense RelA p65 oligonucleotides had a small inhibitory effect on the expression of ICAM-1 in HCMSMC. The only exception was the maximal concentration of antisense RelA p65 oligonucleotides (30 µM/mL) with a neutral effect. The expression of ICAM-1 was 81, 91, and 86% after incubation with antisense RelA p65 oligonucleotides in a concentration of 1, 2, and 4 µM/mL. After addition of antisense RelA p65 oligonucleotides in a concentration of 10 and 20 µM/mL the expression of ICAM-1 was 86 and 81% (Fig. 4C). After adding of antisense RelA p65 oligonucleotides in the maximal concentration of 30 µM/mL no inhibitory effect on the surface expression of ICAM-1 was detected. Simultaneous incubation of HCMSMC with antisense RelA p65 and NF-κB1 p50 oligonucleotides caused a small stimulatory effect on the expression of ICAM-1 in HCMSMC. The percentage of HCMSMC with positive expression of ICAM-1 was 103, 100, and 110% after incubation with antisense RelA p65 and NF-κB1 p50 oligonucleotides in a concentration of 1, 2, and 4 µM/mL and 110, 103, and 107% after incubation with antisense RelA p65 and NF-κB1 p50 oligonucleotides in a concentration of 10, 20, and 30 µM/mL (Fig.  4C). Mismatched antisense RelA p65 and NF-κB1 p50 oligonucleotides did not affect the expression of ICAM-1 in HCMSMC, no matter whether added individually or simultaneously.

Discussion
In this study we investigated the role of RelA p65 and NF-κB1 p50 for the TNF-α mediated expression of ICAM-1 in HUVEC and human coronary vascular cells. The major findings of the study are: 1) intracellular uptake of antisense RelA p65 and NF-κB1 p50 oligonucleotides was confirmed by the detection of FITC-labeled antisense RelA p65 and NF-κB1 p50 oligonucleotides in the intracellular space. 2) In HUVEC and HCAEC simultaneous incubation with antisense RelA p65 and NF-κB1 p50 oligonucleotides caused a dose dependent inhibition of ICAM-1 expression. The inhibitory effect was almost doubled if compared to the effect after selective addition of antisense RelA p65 oligonucleotides, selective incubation with antisense NF-κB1 p50 oligonucleotides had no effect. 3) Simultaneous incubation with antisense RelA p65 and NF-κB1 p50 oligonucleotides exhibited a moderate inhibitory effect on expression of ICAM-1 in HCPSMC but no effect in HCMSMC. No inhibitory effect was seen after selective addition of antisense RelA p65 and NF-κB1 p50 oligonucleotides to HCMSMC and HCPSMC.

Effects of antisense p65 and p50 in HUVEC and HCAEC
Recently it has been reported that a decoy doublestranded oligonucleotides containing a high affinity kb sequence can specifically inhibit NF-κB-dependent transcription of gene products [29,30]. Lockyer et al. [15] and Gawaz et al. [16] demonstrated that in HUVEC the expression of ICAM-1 can be inhibited via the adding of the inhibitory protein IκB. The effect of antisense RelA p65 and NF-κB1 p50 oligonucleotides to the expression of adhesion molecules in cultures of HUVEC and HCAEC has not yet been described. After adding simultaneously antisense RelA p65 and NF-κB1 p50 oligonucleotides the expression of ICAM-1 in HUVEC and HCAEC was inhibited in a dose dependent manner. The inhibitory effect was almost 50% in HUVEC and even more than 40% in HCAEC. The stronger effect in HCAEC can be partially explained by the fact that the stimulatory effect of TNFα is stronger in HCAEC in comparison to HUVEC [23]. Selective addition of mere antisense RelA p65 had a moderate inhibitory effect, no effect was documented after selective adding of antisense NF-κB1 p50. A predominance of RelA p65 in the dimers of the NF-κB complex has already been suggested by Brand et al. [11]. The inhibition of ICAM-1 expression in HUVEC and HCAEC after simultaneous addition of antisense RelA p65 and NF-κB1 p50 was almost twice as high as the effect after addition of mere antisense RelA p65. Therefore it can be speculated that a simultaneous administration of antisense RelA p65 and NF-κB1 p50 is necessary if a sufficient inhibition of NF-κB mediated activities shall be achieved.

Effects of antisense p65 and p50 in HCMSMC and HCPSMC
Although many authors describe the activation of NF-κB in endothelial cells, relatively few data exist concerning the activation of NF-κB in SMC. Lawrence et al. [31] demonstrated that bovine SMC exhibit basal, constitutive NF-κB activity in vitro, Cercek et al. [32] found an increased activity of NF-κB after balloon injury in rat iliofemoral arteries. First investigations of NF-κB activation in human arterial SMC were reported by Bourcier et al. [33], Marayama [34], and Hishikawa et al. [17]. In an earlier report we have demonstrated that the expression of ICAM-1 in HCMSMC and HCPSMC is increased in a time and dose dependent manner after treatment with TNF-α in a concentration of 2.5 -20 ng/mL [23].
Recently Katsuyama et al. [35] reported that in rat vascular smooth muscle cells a strong inhibition of NF-κB mediated effects can be achieved by dexamethasone or aspirin. Only few groups investigated strategies to inhibit activation of NF-κB in human vascular SMC. Staels et al. [18] reported that the activation of NF-κB in human aortic SMC can be inhibited by peroxisome proliferator-ac-tivated alpha receptors (PPARalpha), Speir et al. [19] demonstrated in HCMSMC that aspirin inhibits the cytomegalo-virus-induced activation of NF-κB.
The group of Hishikawa et al. [17] recently demonstrated with electrophoretic mobility shift assays that in HCMS-MC the TNF-α induced activation of NF-κB can be inhibited by antisense RelA p65 and NF-κB1 p50 oligonucleotides. If the NF-κB system is sufficiently inhibited it should be expected that NF-κB mediated effects are inhibited as well. In the study presented we added antisense RelA p65 and NF-κB1 p50 oligonucleotides to HCMSMC and HCPSMC and investigated the expression of ICAM-1. However TNF-α induced and NF-κB mediated expression of ICAM-1 was not inhibited in HCMSMC, only a moderate inhibitory effect was seen in HCPSMC. Different inhibitory effects in HCMSMC and HCPSMC however are difficult to explain because the stimulatory effect of TNF-a on the expression of ICAM-1 is almost equal in HCPSMC and HCMSMC [23]. Furthermore in HCPSMC no inhibitory effect was found after selective addition of antisense RelA p65 or NF-κB1 p50 oligonucleotides. Therefore it is likely that the moderate inhibitory effect found after addition of antisense RelA p65 and NF-κB1 p50 oligonucleotides to HCPSMC is a nonspecific effect.
Keeping in mind the negative effects in HCMSMC and the non specific effects in HCPSMC it is allowed to speculate that in HCPSMC and HCMSMC activation of ICAM-1 is triggered via different pathways, probably via different members of the NF-κB family [36]. This hypothesis is supported by Lindner et al. [37] investigating the role of the NF-κB system after balloon catheter injury in the rat carotid artery. Lindner and colleagues [37] reported that in rat vascular SMC not only high levels of NF-κB1 p50 and RelA p65 but although high levels of NF-κB2 p52, c-Rel, and RelB were expressed.

Conclusions
The results point out that in HUVEC and HCAEC simultaneous antisense therapy with NF-κB1 p50 and RelA p65 oligonucleotids strongly inhibits activation NF-κB mediated expression of ICAM-1. After individual addition of antisense NF-κB1 p50 and RelA p65 oligonucleotides to HUVEC and HCAEC only RelA p65 had an inhibitory effect on TNF-α mediated expression of ICAM-1. However the superiority of the combined therapy was demonstrated by the fact that the inhibitory potential was almost doubled after the simultaneous therapy with RelA p65 and NF-κB1 p50.
In HCMSMC and HCPSMC the negative or nonspecific effects of antisense RelA p65 and NF-κB1 p50 oligonucleotides on TNF-α mediated expression of ICAM-1 indi-cate the existence of redundant pathways. In further studies it should be elucidated whether HCMSMC and HCPSMC express other members of the NF-κB family as well, such as NF-κB2 p52, c-Rel, and RelB. If these molecules are expressed in HCMSMC and HCPSMC, all of these single pathways have to be blocked for a sufficient inhibition of NF-κB-related effects.
All cell incubations were done at 37°C in a humidified atmosphere of air containing 5% C02. Tissues were collected with the patients' informed consent and approval by the Ethical Committee of the University of Ulm.
For flow cytometry investigations 2 × 105 HUVEC, HCAEC, HCMSMC, and HCPSMC were seeded in petri dishes (28 cm2) and the stock solution was added for a period of 18 hrs to the cultures. After 18 hrs cells were washed twice with PBS, detached by trypsin treatment, and suspended in phosphate-buffered saline, pH 7.2, containing 1% of bovine serum albumin (BSA) with 0.2% of sodium azide at 4°C. This buffer was used in all subsequent steps. Cells (100% gated) were analyzed immediately with a fluorescence-activated cell sorter (FACScan, Becton Dickinson, Heidelberg, Germany, Macintosh System Software 7.1.0).
Antisense RelA p65 and NF-κB1 p50 oligonucleotides is the revise complement of a target sequence as described by Ruben et al. [28]. The form is a phosphothioate DNA-Na-salt, lyophilized and diluted with oligo-buffer (Biognostik). Antisense RelA p65 and NF-κB1 p50 oligonucleotides are added for a period of 18 hrs in a concentration of, 1, 2, 4, 10, 20, and 30 µM. As controls mismatched antisense RelA p65 and NF-κB1 p50 oligonucleotides were used. Form and concentration is identical as described for antisense RelA p65 and NF-κB1 p50 oligonucleotides.

TNF-α induced expression of ICAM-1
Surface expression of ICAM-1 was investigated with indirect immunofluorescence microscopy. HUVEC, HCAEC, HCMSMC, and HCPSMC were seeded on round coverslips into 6-well plates (Falcon/Becton-Dickinson, Heidelberg, Germany). 18 hrs before fixation in 4% paraformaldehyde antisense RelA p65 and NF-κB1 p50 oligonucleotides and mismatched controls were added, 6 hrs before fixation cultures were stimulated with recombinant human TNF-α (20 ng/mL; Sigma, Deisenhofen, Germany). Human ICAM-1 was detected using monoclonal anti-ICAM-1 antibodies (clone 84H10, 2 µg/mL, Dianova Immunotech, Hamburg, Germany). Tetrarhodamine-isothiocyanate (TRITC)-labeled secondary antibodies were purchased from Dianova Immunotech. In 100 cells the expression of ICAM-1 was analysed in duplicate by two independent observers according the cri-teria negative, low and strong. Since both low and negative staining were found as basic expression of ICAM-1 in untreated controls, only a strong positive staining was rated as positive expression of ICAM-1. The inhibitory effect of antisense RelA p65 and NF-κB1 p50 oligonucleotides was calculated in relation to the relative stimulatory effect of TNF-α. For the investigations an immunofluorescence microscope was used (100 cell per concentration, magnification × 100).