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Simvastatin modulates TNFα-induced adhesion molecules expression in human endothelial cells
Life Sciences 75 (2004) 1287 – 1302 www.elsevier.com/locate/lifescie
Simvastatin modulates TNFa-induced adhesion molecules expression in human endothelial cells D. Zapolska-Downar a, A. Siennicka a, M. Kaczmarczyk a, B. Kolodziej b, M. Naruszewicz a,* a
Clinical Biochemistry and Laboratory Diagnostic, Regional Center for Atherosclerosis Research, Pomeranian Medical University, ul. Powstan´co´w Wlkp. 72, PL-70-111 Szczecin, Poland b Pathomorphology, Pomeranian Medical University, Szczecin, Poland Received 5 November 2003; accepted 3 February 2004
Abstract Adhesion and transendothelial migration of leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. We investigated the effect of simvastatin, an inhibitor of HMG-CoA reductase administered to reduce plasma levels of LDLcholesterol, on the expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor a (TNFa). We found the expression to be significantly inhibited by the drug in a time and concentration-dependent manner and to a greater extent in the case of VCAM-1 as compared with ICAM-1. In TNFa-stimulated HUVEC, simvastatin decreased VCAM-1 and ICAM-1 mRNA levels, inhibited TNFa-induced activation of nuclear factor nB (NF-nB) and enhanced expression of peroxisome proliferator-activated receptor a (PPARa). These effects were associated with reduction of adherence of monocytes and lymphocytes to HUVEC. The present findings suggest that the benefits of statins in vascular disease may include the inhibition of expression of VCAM-1 and ICAM-1 through effects on NF-nB. D 2004 Elsevier Inc. All rights reserved. Keywords: Simvastatin; Inflammation; Endothelial cells; Cell adhesion molecules; Transcription factors
* Corresponding author. Tel.: +48-91-826074; fax: +48-91-466-1490. E-mail address: [email protected] (M. Naruszewicz). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.005
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Introduction Mononuclear cell recruitment into the vascular wall is a crucial step in the initiation and progression of atherosclerosis (Ross, 1999; Li et al., 1993). A causative role in this process has been ascribed to endothelial cell dysfunction. Various stimuli acting on the endothelium induce the expression of several cell adhesion molecules (CAM) responsible for interactions with leukocytes (Price and Loscalzo, 1999; Springer, 1994) among them selectins (E and P) and the immunoglobulin superfamily members: intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1). In the early phase of leukocyte extravasation, selectins bind to their ligands on the leukocyte surface initiating transcient adhesion and rolling of these cells on the endothelium. Stable adhesion and transendothelial migration follows, mediated by ICAM-1/ LFA-1, VCAM-1/VLA-4 and PECAM-1/PECAM-1 interactions. The expression of most of these molecules can be induced by inflammatory cytokines (e.g. TNFa and IL1), oxidative stress, and oxidized LDL (oxyLDL) (Springer, 1994; Kume et al., 1992). Enhanced expression of endothelial adhesion molecules has been observed in atherosclerotic vessels, apparently contributing to further recruitment of mononuclear leukocytes into the lesions (Davies et al., 1993; Manka et al., 1999; van der Wal et al., 1992). Clinical importance of these findings is corroborated by elevated levels of soluble forms of ICAM-1 and VCAM-1 in patients with documented coronary artery disease (CAD) (Semaan et al., 2000) and in children with familial risk of atherosclerosis (Wojakowski and Gminski, 2001). Recent years have witnessed efforts to elucidate the molecular mechanisms responsible for induction of endothelial CAMs. It is now known that cytokines, oxidative stress, and oxyLDL stimulate transcription in several ways, one of them involving nuclear factor nB (NF-nB) (Collins and Cybulsky, 2001), a transcription factor present in the cytoplasm in an inactive complex with inhibitory proteins (InB). NF-nB release is preceded by activation of InB kinases (IKK) which phosphorylate InB, resulting in ubiquitination, dissociation and proteasomal degradation. NF-nB next migrates from the cytoplasm to the nucleus and stimulates transcription processes by targeting specific genes, some of them involved in the inflammatory response and regulation of CAM expression. Peroxisome proliferator-activated receptor a (PPARa) is another factor implicated in the regulation of the inflammatory response. It has been suggested that PPARa acts by inhibiting NF-nB (Fruchart et al., 1999). PPARa belongs to the superfamily of nuclear factor receptors that are ligand-activated transcription factors. Fatty acid derivatives and eicosanoids were identified as natural ligands for PPARs while fenofibrates, known activators of PPARa, were shown to suppress cytokine-induced VCAM-1 expression by inhibiting NF-nB (Marx et al., 1999). Statins have found widespread use in primary and secondary prevention of CAD. These drugs lower total and LDL cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (Faggiotto and Paoletti, 1999; Maron et al., 2000) and produce pleiotropic effects which may further contribute to their benefits in CAD. For example, statins were shown to interfere with processes taking place in the atherosclerotic lesion and directly inhibit the proliferation of endothelial cells, smooth muscle cells and macrophages (Negre-Aminou et al., 1997; Sakai et al., 1997). In human endothelial cells, statins upregulate the expression of nitric oxide (NO) synthase (Laufs and Liao, 1998), inhibit the expression of PAI-1 (Essig et al., 1998), and suppress leukocyte-endothelial interactions (Pruefer et al., 1999). Statins reduce the accumulation of cholesterol esters in macrophages exposed to oxyLDL
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(Kempen et al., 1991) and suppress tissue factor expression in cultured macrophages (Colli et al., 1997). Moreover, statins inhibit the secretion of matrix metalloproteinase 9 by macrophages, contributing to plaque stabilization (Bellosta et al., 1998). Considering the important role of mononuclear cell adhesion and transendothelial migration in atherogenesis and the fact that intravascular macrophages secrete TNFa which stimulates the expression of endothelial CAMs, we decided to study the effect of simvastatin on TNFa-induced expression of VCAM-1 and ICAM-1 by human endothelial cells. An attempt was also made to elucidate some aspects of the mechanism of action of the drug.
Materials and methods Materials Medium 199, fungizone, penicillin, streptomycin, gentamycin, heat-inactivated fetal bovine serum (FBS), collagenase, gelatin, glutamine, trypsin/EDTA solution and mevalonate were obtained from Sigma (USA). Recombinant TNFa (10 mg, 1 108 U/ml), random hexamers, reverse transcriptase, PCR buffer, Taq polymerase were from Boehringer Mannheim (Germany). Tissue culture plates were supplied by Costar (USA) and Ficoll-Paque by Pharmacia (Sweden). Monoclonal antibodies against VCAM-1 (CD106:FITC), ICAM-1 (CD54:FITC), CD14 (CD14:PE), CD45 (CD45:FITC), PECAM-1 (CD31:PE) and IgG1 (IgG1:FITC) were purchased from Becton Dickinson (USA). dATP, dTTP, dCTP, dGTP, and RNAsin were from Promega (USA). An ELISA-based kit for studying NF-nB activation was from Active Motif (Belgium). Simvastatin was kindly supplied by MSD (Poland). Endothelial cell isolation and culture Human umbilical vein endothelial cells (HUVEC) were obtained from umbilical cords by collagenase digestion as described by Jaffe et al. (1984). In brief, veins of umbilical cords were perfused with PBS to remove blood cells, filled with 0.1% collagenase (type Ia) and left for 8 min. at 37 jC. The resulting cellular suspension was supplemented with FBS and centrifuged (300 g for 10 min.). HUVEC were cultured in gelatin-coated 25 cm2 flasks, 6-well or 24-well tissue culture plates filled with Medium 199 supplemented with Earle’s salts, 20 mM HEPES, 100 U/ml penicillin, 100 Ag/ml streptomycin, 2.5 Ag/ ml fungisone, 2 mM glutamine and 20% FBS. Culture was at 37 jC under humidified 5% CO2 in room air and the medium was replaced every two days until confluence (3–5 days). HUVEC purity was ascertained by the cobblestone morphology typical for quiescent endothelial cells and staining for PECAM-1 (CD31). Isolation of peripheral blood mononuclear cells (PBMC) Immediately after collection, 25 ml of heparinized blood diluted with PBS was layered over 15 ml Ficoll-Paque and centrifuged (400g, 40 min., 22 jC). The mononuclear cell band was removed by aspiration, washed with PBS, centrifuged and suspended in the same medium as used for HUVEC culture, except that FBS concentration was reduced to 5%. Cells were next counted and used in the adhesion assay. PBMC consisted of approximately 10% monocytes and 90% lymphocytes.
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Pretreatment of HUVEC Second-passage HUVEC were grown to confluence in 6- or 24-well plates, At confluence, FBS concentration in the medium was reduced to 5% and simvastatin was added. In an additional experiment, the medium containing simvastatin at 5 AM was supplemented with 100 AM mevalonate. Simvastatin and mevalonate were dissolved in ethanol. Control values were obtained by adding 0.05% ethanol only. Next, HUVEC were incubated with TNFa (100 U/ml) for defined periods in the absence or presence of simvastatin and mevalonate. Under all conditions cell viability was greater than 90% as judged by trypan blue exclusion. Measurement of VCAM-1 and ICAM-1 expression in HUVEC HUVEC were incubated for 30 min., 12 h or 24 h, without or with simvastatin (0.1, 1.0 or 5.0 AM) or with simvastatin (5 AM) + mevalonate (100 AM). TNFa (100 U/ml) was added and incubation continued for 8 h. Next, cells were washed with PBS and treated for 30 min. at 4 jC with saturating amounts of PE-conjugated anti-PECAM-1 (CD31) and FITC-conjugated anti-ICAM-1 (CD54) or antiVCAM-1 (CD106) monoclonal antibodies (Mab). For isotype control, cells were treated with FITCconjugated mouse anti-IgG1 Mab. Cells were washed with PBS, fixed in 1% paraformaldehyde, harvested by mild trypsinisation, washed again and centrifuged at 300 g for 10 min. prior to analysis by flow cytometry (FACS Calibur, Becton Dickinson). After correction for unspecific binding (isotype control), mean specific fluorescence intensity (MFI) was measured in each channel (10,000 cells per sample). Adhesion assay HUVEC grown to confluence in 24-well plates were pretreated with simvastatin (5 AM) or simvastatin (5AM) + mevalonate (100 AM) for 24 h and stimulated with TNFa (100 U/ml) for 8 h prior to the adhesion assay. Briefly, HUVEC were washed with PBS and coincubated for 30 min. with PBMC suspension (106 cells/ml). Next, HUVEC were washed twice with PBS to remove nonadherent cells and were treated for 30 min. at 4 jC with saturating amounts of mouse FITC-conjugated anti-CD45 and PE-conjugated anti-CD14 Mab. Cells were washed with PBS, fixed in 1% paraformaldehyde, washed again, harvested by mild trypsinisation, centrifuged and counted prior to analysis by flow cytometry (10,000 cells per sample). The proportion of monocytes and lymphocytes in the suspension was established by measuring fluorescence I (FLI–CD45) and fluorescence II (FL II–CD14). The absolute number of monocytes and lymphocytes adhering to endothelial cells was calculated in relation to the total number of cells obtained after trypsinisation. The results were expressed as percent of monocytes and lymphocytes added. Quantitation of VCAM-1, ICAM-1 and PPARa gene expression For quantitation of VCAM-1 and ICAM-1 gene expression, HUVEC were grown to confluence in 6-well plates, pretreated for 24 h with simvastatin (5 AM) or simvastatin (5 AM) + mevalonate (100 AM) and stimulated with TNFa (100 U/ml) for 4 h. For quantitation of PPARa gene expression, HUVEC were grown to confluence in 6-well plates and incubated with simvastatin (0.1, 1.0 or 5.0
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AM) for 24 h. Total RNA from 106 cells was isolated according to Chomczynski and Sacchi (1987) using TRIZOL. RNA concentrations were determined by spectrophotometry at 260 nm. RNA (500 ng) was dissolved in 20 Al of the reaction mixture containing dATP, dTTP, dCTP, and dGTP at 2.5 mM each, 20 U RNAsin, 100 pM random hexamers and 20 U MMLV reverse transcriptase. Incubation was carried out at 37 jC for 60 min. The temperature of the reaction was then raised to 94 jC for 5 min. to inactivate the enzyme and finally reduced to 4 jC. An aliquot of cDNA (5 Al of RT mixture) was dissolved in 25 Al of the reaction solution containing 10 PCR buffer (final Mg2 + concentration was 1.5 mM), dATP, dTTP, dCTP, and dGTP at 2.5 mM each, 10 pM up- and downstream primers (PPARa, VCAM-1, ICAM-1, GAPDH and h-actin) and 1 U Taq polymerase. For semiquantitative analyses, linearity of amplification in relation to the number of PCR cycles was established in preliminary experiments for PPARa, VCAM-1, ICAM-1, GAPDH and h-actin cDNAs. Optimal amplification to detect differences among the samples corresponded to 28 cycles for PPARa, 16 for VCAM-1, 18 for ICAM-1, 22 for GAPDH and 22 for h-actin. The following primer pairs were used: VCAM-1: sense 5V-CCCTTGACCGGCTGGAGATT-3V Antisense 5V-CTGGGGGCAACATTGACATAAAGTG-3V ICAM-1: sense: 5V-TGAAGGCCACCCCAGAGGACAAC-3V Antisense 5V-CCCATTATGACTGCGGCTGCTGCTACC-3V GAPDH: sense 5V-GAGTCAACGGATTTGGTCGT-3V Antisense 5V-GTTGTCATGGATGACCTTGG-3V PPARa: sense 5V-GCC CCT CCT CGG TGA CTT ATC-3V Antisense 5V-ATG ACC CGG GCT TTG ACC TT-3V h-actin: sense 5V-CCTCGCCTTTGCCGATCC-3V antisense 5V-GGATCTTCATGAGGTAGTCAGTC-3V Amplification products obtained with PCR (VCAM-1 241 bp, ICAM-1 409 bp, GAPDH 482 bp, PPARa 454 bp and h-actin 626 cDNA) were electrophoretically separated on 3 % agarose gel. Ethidium bromide stained bands of PPARa, VCAM-1, ICAM-1 and GAPDH were photographed with DS-34 Polaroid camera. Band intensity was densitometrically measured with the gel analysis macro attached supplied with the NIH Image (available free from: http://rsb.info.nih.gov/nih-image/). VCAM-1 and ICAM-1 signals were normalized to cDNA levels of the housekeeping gene GAPDH, PPARa to h-actin and expressed as a ratio. Measurement of NF-jB activation To measure NF-nB activation, confluent HUVEC were pretreated without or with simvastatin (5 AM) or with simvastatin (5 AM) + mevalonate (100 AM) for 24 h and exposed to TNFa (100 U/ml) for 2 h. NF-nB activation was measured in whole cell extract with an ELISA kit. Briefly, HUVEC were washed with ice-cold PBS, scraped into tubes and centrifuged. The pellet was lysed with Complete Lysis Buffer containing dithiothreitol and a protease inhibitor cocktail. After centrifugation at 14,000 rpm and 4 jC for 20 min., the protein concentration in the supernatant (whole cell extract) was determined with a Bradford-based assay. To determine NF-nB activation with ELISA, an oligonucleotide containing the NF-nB consensus binding site (5V-GGGACTTTCC-3V) specific for the active form of NF-nB was
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immobilized to a 96-well plate, whereupon the wells were filled for 1 h with 10 Ag of the whole cell extract. Three washings followed and incubation continued for 1 h with the primary antibody recognizing an epitope on p65 that is accessible only when NF-nB is activated and bound to its target DNA. Wells were next washed and the secondary antibody conjugated to horseradish peroxidase was added to achieve a sensitive readout by spectrophotometry at 450 nm. The experiments were performed in duplicate and the results were expressed as OD450. Statistical analysis The results were expressed as mean F SEM. Differences were analyzed by one-way ANOVA followed by Fisher’s protected least significant difference test. The level of significance was taken as p < 0.05.
Fig. 1. Simvastatin inhibits TNFa-induced cell surface expression of VCAM-1(A) and ICAM-1(B) in concentration dependent manner. Cells were pretreated without or with simvastatin (0.1, 1.0 or 5.0 AM) or with simvastatin (5 AM) + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 8 h, stained for VCAM-1 or ICAM-1 and analyzed by flow cytometry. Results are expressed as percent of TNFa-stimulated cells (100%) and are given as mean F SD from 7 experiments. **p < 0.01 and *p < 0.001, compared to cells stimulated with TNFa in the absence of simvastatin.
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Results Effect of simvastatin on VCAM-1 and ICAM-1 expression by TNFa-stimulated HUVEC VCAM-1 and ICAM-1 expression on the surface of endothelial cells was studied with flow cytometry. Expression was related to mean fluorescence intensity from FCS (forward scater) and PECAM-1 (CD31) gated cells, representing 80–90% of the cells analyzed. Expression of PECAM-1 was taken as a marker of endothelial viability, considering the fact that this CAM is always present on viable and disappears from dead cells (Weber et al., 1994). When compared with control cells, no significant changes were found in % of PECAM-1 positive cells in experiments with simvastatin or simvastatin and TNFa. As expected, HUVEC cultured for 8 h with TNFa demonstrated marked increase in VCAM-1 and ICAM-1 expression compared with resting cells (Figs. 1 and 2). Pretreatment of HUVEC with
Fig. 2. Simvastatin inhibits TNFa-induced cell surface expression of VCAM-1(A) and ICAM-1(B) in time dependent manner. Cells were pretreated without or with simvastatin (5.0 AM) for 30 min., 12 h or 24 h, stimulated with TNFa (100 U/ml) for 8 h, stained for VCAM-1 or ICAM-1 and analyzed by flow cytometry. Results are expressed as percent of TNFa-stimulated cells (100%) and are given as mean F SD from 7 experiments. ***p < 0.05, **p < 0.01 and *p < 0.001, compared to cells stimulated with TNFa in the absence of simvastatin.
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simvastatin (0.1–5.0 AM) for 24 h significantly reduced TNFa-induced VCAM-1 and ICAM-1 (Fig. 1) expression in a concentration-dependent manner, with maximal effect at 5 AM. At this concentration, expression of VCAM-1 was reduced by 28–44% (p < 0.001; n = 7) and ICAM-1 by 11– 25% (p < 0.001; n = 7). Mevalonate (100 AM) prevented the effect of simvastatin (Fig. 1B) on ICAM-1 but was only partly effective in the case of VCAM-1 (Fig. 1A), suggesting that inhibition of HMG-CoA reductase does not account for the whole effect of the drug on VCAM-1 expression. In addition, simvastatin inhibited VCAM-1 and ICAM-1 expression induced by TNFa in a timedependent manner with maximal reduction after 12 h of incubation for VCAM-1 and 24 h for ICAM-1 (Fig. 2). Effect of simvastatin on adhesiveness for monocytes and lymphocytes of TNFa-stimulated HUVEC In an attempt to determine the functional importance of inhibitory properties of simvastatin on TNFastimulated expression of VCAM-1 and ICAM-1, we studied the adhesion of freshly isolated monocytes and lymphocytes to HUVEC. Stimulation of HUVEC with TNFa increased the percentage of adherent monocytes from 19.0 F 2.9 to 26.5 F 3.3% (Fig. 3A) and the percentage of adherent lymphocytes
Fig. 3. Simvastatin inhibits adhesion of monocytes (A) and lymphocytes (B) to TNFa-stimulated HUVEC. Endothelial cells were pretreated without or with simvastatin (5.0 AM) or with simvastatin (5 AM) + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 8 h. Next, freshly isolated PBMC were coincubated for 30 min. Monocyte and lymphocyte adhesion was measured as described in the Material and Methods section. Results are expressed as percent of monocytes and lymphocytes added and are given as mean F SD from 9 experiments. *p < 0.001 compared to cells stimulated with TNFa in the absence of simvastatin.
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from 1.9 F 0.4 to 11.4 F 1.4% (Fig. 3B). Pretreatment for 24 h with 5 AM simvastatin significantly (p < 0.001; n = 9) reduced the adhesion of monocytes (Fig. 3A) to TNFa-stimulated HUVEC from 26.5 F 3.3 to 18.3 F 2.1% (reduction varied for individual populations but the mean percentage of adherent monocytes was typical for unstimulated cells). Adhesion of lymphocytes to TNFa-stimulated HUVEC (Fig. 3B) was reduced from 11.4 F 1.4 to 6.9 F 1.7 % (p < 0.001; n = 9). Mevalonate
Fig. 4. Simvastatin inhibits TNFa-induced VCAM-1 mRNA expression in HUVEC. Cells were pretreated without or with simvastatin (5.0 AM) or with simvastain + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 4 h. mRNA was extracted, reverse transcribed and amplified by PCR using specific primers for GAPDH and VCAM-1. Top: optical densities from 5 separate experiments after normalization to GAPDH (internal standard). Data are expressed as VCAM1/GAPDH ratio and are given as mean F SD from 5 experiments. *p < 0.001 compared to cells stimulated with TNFa in the absence of simvasatin. Photographs below show UV-illuminated gel electrophoregrams from a representative experiment.
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almost completely prevented the effect of simvastatin on monocytes and lymphocytes adhesion to endothelial cells. Effect of simvastatin on VCAM-1 and ICAM-1 mRNA levels in TNFa-stimulated HUVEC To explore mechanisms responsible for inhibition of VCAM-1 and ICAM-1 surface expression, we measured the respective mRNA levels using RT PCR. Stimulation of HUVEC with TNFa for 4
Fig. 5. Simvastatin inhibits TNFa-induced ICAM-1 mRNA expression in HUVEC. Cells were pretreated without or with simvastatin (5.0 AM) or with simvastain + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 4 h. mRNA was extracted, reverse transcribed and amplified by PCR using specific primers for GAPDH and ICAM-1. Top: optical densities from 5 separate experiments after normalization to GAPDH (internal standard). Data are expressed as ICAM-1/ GAPDH ratio and are given as mean F SD from 5 experiments. **p < 0.01 compared to cells stimulated with TNFa in the absence of simvasatin. Photographs below show UV-illuminated gel electrophoregrams from a representative experiment.
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h induced an approximately 13-fold increase in specific VCAM-1 PCR products (Fig. 4) and two-fold in specific ICAM-1 PCR products (Fig. 5). Pretreatment with simvastatin (5 AM) for 24 h significantly abolished induction of VCAM-1 and ICAM-1 transcripts by 20–48% (from 0.52 F 0.09 to 0.36 F 0.09; p < 0.001; n = 5) and 13–22% (from 0.97 F 0.07 to 0.81 F 0.1; p < 0.01; n = 5), respectively. These results closely reflect changes in surface expression of both CAMs described above. Mevalonate almost completely prevented the effect of simvastatin on VCAM-1 and ICAM-1 mRNA levels. Effect of simvastatin on TNFa-induced NF-jB p65 activation To determine whether NF-nB activation was involved in the effect of simvastatin on TNFa-stimulated CAM expression, we decided to measure the amount of p65 with DNA-binding activity in whole cell extracts of HUVEC. TNFa-stimulated HUVEC showed a marked increase (approx. 7-fold) in the levels of active form of NF-nB p65 measured with ELISA (Fig. 6). Pretreatment of HUVEC with simvastatin (5 AM) for 24 h significantly reduced NF-nB p65 activation by TNFa (from 0.44 F 0.061 to 0.25 F 0.041; p < 0.001; n = 9). Reduction varied in individual populations, ranging from 35 to 53%. Simvastatin retained its potency in the presence of mevalonate, reducing TNFa-stimulated levels of NF-nB from 0.44 F 0.066 to 0.377 F 0.07. Effect of simvastatin on PPARa expression Following suggestions that PPARa negatively interacts with the activated NF-nB signaling pathway, we decided to check whether simvastatin acts as PPARa activator. RT PCR analysis revealed that 24 h incubation of HUVEC with simvastatin at 1.0 and 5.0 AM significantly increased the level of specific
Fig. 6. Simvastatin inhibits TNFa-induced activation of NF-nB. Cells were pretreated without or with simvastatin (5.0 AM) or with simvastatin + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 2 h. A whole cell extract was obtained and NF-nB activation was measured with an ELISA kit. Results are expressed as OD450 and are given as mean F SD from 9 experiments. *p < 0.001 and ***p < 0.05, compared to cells stimulated with TNFa in the absence of simvastatin.
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Fig. 7. Simvastatin induces PPARa mRNA synthesis in HUVEC. Cells were incubated with simvastatin (5.0, 1.0 or 0.1 AM) for 24 h. mRNA was extracted, reverse transcribed and amplified by PCR using specific primers for h-actin and PPARa. Top: optical densities from 6 separate experiments after normalization to h-actin (internal standard). Data are expressed as PPARa/hactin ratio and are given as mean F SD from 6 experiments. *p < 0.001 compared to control cells. Photographs below show UV-illuminated gel electrophoregrams from a representative experiment.
PPARa PCR products from 0.7 F 0.11 to 1.39 F 0.38 and 1.44 F 0.87, respectively (p < 0.001; n = 6) (Fig. 7).
Discussion Recruitment of mononuclear cells by adhesion to the vascular wall and transendothelial migration plays an important role in atherogenesis (Ross, 1999). Monocytes transform into macrophages and macrophage-derived foam cells during early stages of atherosclerosis. Subsequently, macrophages enhance endothelial cell adhesiveness with cytokines like TNFa and IL-1, oxyLDL and oxygen free
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radicals and release metalloproteinases which digest components of connective tissue, contributing to plaque instability and its eventual rupture. This process appears be the cause of clinical manifestation of atherosclerosis in the form of CAD or myocardial infarction (Bellosta et al., 1998). Our in vitro study has demonstrated that simvastatin modulates TNFa-induced expression of VCAM1 and ICAM-1. The drug appears to reduce both surface protein and specific mRNA levels by inhibiting NF-nB. The effect was significantly greater for VCAM-1 than ICAM-1 and was time and concentrationdependent. Our results are in accordance with previous reports. It has been shown that fluvastatin reduces TNFa-stimulated levels of soluble selectin E and ICAM-1 released by HUVEC to the culture medium (Mueck et al., 2001). Atorvastatin and simvastatin were able to reduce oxyLDL-induced VCAM-1 and ICAM-1 expression in endothelial cells from human coronary arteries (Li et al., 2003). Meroni et al. (2001) demonstrated that fluvastatin at doses similar to those used by us inhibits the adhesion of monocytes to HUVEC and reduces expression of selectin E and ICAM-1 stimulated by cytokines or LPS. The pleiotropic effects of statins have already been documented (Faggiotto and Paoletti, 1999; Maron et al., 2000). We have now demonstrated that simvastatin may interfere with atherogenesis through direct action on endothelial cells leading to suppression of their pro-adhesive properties. The inhibitory effect of the drug was more pronounced in the case of VCAM-1 than ICAM-1. This finding is notable in view of the anti-atherosclerotic properties of simvastatin considering the fact that interactions of VCAM-1 with VLA-4 play an essential role in selective accumulation of monocytes and lymphocytes in the vascular wall during atherogenesis (Cybulsky et al., 2001). As in other in vitro studies, our experiments with simvastatin were performed at concentrations ranging from 0.1 to 5 AM. The reported peak serum concentrations of lovastatin after a single therapeutic dose of 40 mg are 0.1–0.3 AM (Pentikainen et al., 1992), matching the lowest concentration used by us. Although maximal inhibitory effect was seen at 5 AM, we believe that due to time constraints in vitro the reaction threshold can only be achieved with higher concentrations. Furthermore, chronic administration of lipophilic drugs may be associated with their accumulation in target tissues. The decreased expression of VCAM-1 and ICAM-1 prompted us to examine the effect of simvastatin on NF-nB, a key transcription factor implicated in the regulation of a variety of genes participating in immune and inflammatory responses, including genes encoding VCAM-1 and ICAM-1 (Collins, 1993). We have found elevated levels of p65 in TNFa-stimulated endothelial cells and demonstrated that simvastatin inhibits TNFa-stimulated activation of NF-nB. Our results are in accordance with previous reports. Atorvastatin reduced NF-nB activation in smooth muscle cells and monocytes stimulated with TNFa (Ortego et al., 1999). Similarly, simvastatin and atorvastatin inhibited the activation of NF-nB in response to oxyLDL in HUVEC (Li et al., 2003) and fluvastatin was able to prevent NF-nB translocation in response to TNFa in human endothelial cells (Meroni et al., 2001). In a rabbit model of atherosclerosis, atorvastatin reduced the activity of NF-nB in vascular macrophages and smooth muscle cells (Bustos et al., 1998). The effect of simvastatin was partly reversed by mevalonate, suggesting a link between inhibition of HMG-CoA reductase and activity of this drug. However, mevalonate was only partly effective in suppressing the effect of simvastatin on p65 levels and VCAM-1 expression, suggesting that inhibition of HMG-CoA reductase is not the sole factor involved. It is worth noting that TNFa-stimulated expression of VCAM-1 was suppressed by simvastatin after an incubation period as short as 30 min. Oxygen free radicals have been implicated in the release of NF-nB from its complex with inhibitory proteins (Collins, 1993). Moreover, a redox-sensitive mechanism has been linked with regulation of
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VCAM-1 gene expression (Offermann and Medford, 1994). Several studies have confirmed that antioxidants like N-acetylcysteine (NAC), pyrrolidine dicarbamate and probucol inhibit NF-nB activation and VCAM-1 expression without any effect on ICAM-1 (Marui et al., 1993; Chen et al., 2001). It has been shown that simvastatain decrease superoxide formation in human monocyte-derived macrophages in response to activation by phorbol mystrate acetate (Giroux et al., 1993). Additionally, we have found that PPARa mRNA levels in HUVEC were increased by simvastatin. Our results agree with the report of Inoue et al. (Inoue et al., 2000) on the effects of simvastatin in HUVEC. Increased PPARa protein and mRNA levels were found together with reduced levels of mRNA for IL-1, IL-6 and cycloxygenase-2. In our opinion, this finding may be related to the effect of simvastatin on VCAM-1 and NF-nB levels in view of reports that PPARa are inhibitors of NF-nB activation (Fruchart et al., 1999). Morever, fenofibrates, known activators of PPARa, were shown to suppress cytokine-induced VCAM-1 expression by inhibiting NF-nB. Since PPARa have potent antiinflammatory properties in vascular cells, our results indicate that simvastatin may suppress the function of stimulated endothelial cells via an anti-inflammatory system mediated at least by PPARa. In conclusion, this is the first study to show that simvastatin reduces expression of adhesion molecules in human endothelial cells and decreases mononuclear cell adhesion by modifying NF-nB. Since mononuclear cell adhesion followed by transendothelial migration plays an important role in atherogenesis, our results suggest that simvastatin may help reduce the risk of atherosclerosis not only by decreasing plasma LDL levels, but also by direct action on the vascular endothelium.
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Comparative pharmacokinetics of lovastatin, simvastatin and pravastatin in humans. Journal of Clinical Pharmacology 32 (2), 136 – 140. Price, D.T., Loscalzo, J., 1999. Cellular adhesion molecules and atherogenesis. The American Journal of Medicine 107 (1), 85 – 97. Pruefer, D., Scalia, R., Lefer, A.M., 1999. Simvastatin inhibits leukocyte-endothelial cell interactions and protects against inflammatory processes in normocholesterolemic rats. Arteriosclerosis Thrombosis Vascular Biolology 19 (12), 2894 – 2900.
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Simvastatin modulates TNFa-induced adhesion molecules expression in human endothelial cells D. Zapolska-Downar a, A. Siennicka a, M. Kaczmarczyk a, B. Kolodziej b, M. Naruszewicz a,* a
Clinical Biochemistry and Laboratory Diagnostic, Regional Center for Atherosclerosis Research, Pomeranian Medical University, ul. Powstan´co´w Wlkp. 72, PL-70-111 Szczecin, Poland b Pathomorphology, Pomeranian Medical University, Szczecin, Poland Received 5 November 2003; accepted 3 February 2004
Abstract Adhesion and transendothelial migration of leukocytes into the vascular wall is a crucial step in atherogenesis. Expression of cell adhesion molecules by endothelial cells plays a leading role in this process. We investigated the effect of simvastatin, an inhibitor of HMG-CoA reductase administered to reduce plasma levels of LDLcholesterol, on the expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) by human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor a (TNFa). We found the expression to be significantly inhibited by the drug in a time and concentration-dependent manner and to a greater extent in the case of VCAM-1 as compared with ICAM-1. In TNFa-stimulated HUVEC, simvastatin decreased VCAM-1 and ICAM-1 mRNA levels, inhibited TNFa-induced activation of nuclear factor nB (NF-nB) and enhanced expression of peroxisome proliferator-activated receptor a (PPARa). These effects were associated with reduction of adherence of monocytes and lymphocytes to HUVEC. The present findings suggest that the benefits of statins in vascular disease may include the inhibition of expression of VCAM-1 and ICAM-1 through effects on NF-nB. D 2004 Elsevier Inc. All rights reserved. Keywords: Simvastatin; Inflammation; Endothelial cells; Cell adhesion molecules; Transcription factors
* Corresponding author. Tel.: +48-91-826074; fax: +48-91-466-1490. E-mail address: [email protected] (M. Naruszewicz). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.03.005
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Introduction Mononuclear cell recruitment into the vascular wall is a crucial step in the initiation and progression of atherosclerosis (Ross, 1999; Li et al., 1993). A causative role in this process has been ascribed to endothelial cell dysfunction. Various stimuli acting on the endothelium induce the expression of several cell adhesion molecules (CAM) responsible for interactions with leukocytes (Price and Loscalzo, 1999; Springer, 1994) among them selectins (E and P) and the immunoglobulin superfamily members: intracellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1). In the early phase of leukocyte extravasation, selectins bind to their ligands on the leukocyte surface initiating transcient adhesion and rolling of these cells on the endothelium. Stable adhesion and transendothelial migration follows, mediated by ICAM-1/ LFA-1, VCAM-1/VLA-4 and PECAM-1/PECAM-1 interactions. The expression of most of these molecules can be induced by inflammatory cytokines (e.g. TNFa and IL1), oxidative stress, and oxidized LDL (oxyLDL) (Springer, 1994; Kume et al., 1992). Enhanced expression of endothelial adhesion molecules has been observed in atherosclerotic vessels, apparently contributing to further recruitment of mononuclear leukocytes into the lesions (Davies et al., 1993; Manka et al., 1999; van der Wal et al., 1992). Clinical importance of these findings is corroborated by elevated levels of soluble forms of ICAM-1 and VCAM-1 in patients with documented coronary artery disease (CAD) (Semaan et al., 2000) and in children with familial risk of atherosclerosis (Wojakowski and Gminski, 2001). Recent years have witnessed efforts to elucidate the molecular mechanisms responsible for induction of endothelial CAMs. It is now known that cytokines, oxidative stress, and oxyLDL stimulate transcription in several ways, one of them involving nuclear factor nB (NF-nB) (Collins and Cybulsky, 2001), a transcription factor present in the cytoplasm in an inactive complex with inhibitory proteins (InB). NF-nB release is preceded by activation of InB kinases (IKK) which phosphorylate InB, resulting in ubiquitination, dissociation and proteasomal degradation. NF-nB next migrates from the cytoplasm to the nucleus and stimulates transcription processes by targeting specific genes, some of them involved in the inflammatory response and regulation of CAM expression. Peroxisome proliferator-activated receptor a (PPARa) is another factor implicated in the regulation of the inflammatory response. It has been suggested that PPARa acts by inhibiting NF-nB (Fruchart et al., 1999). PPARa belongs to the superfamily of nuclear factor receptors that are ligand-activated transcription factors. Fatty acid derivatives and eicosanoids were identified as natural ligands for PPARs while fenofibrates, known activators of PPARa, were shown to suppress cytokine-induced VCAM-1 expression by inhibiting NF-nB (Marx et al., 1999). Statins have found widespread use in primary and secondary prevention of CAD. These drugs lower total and LDL cholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (Faggiotto and Paoletti, 1999; Maron et al., 2000) and produce pleiotropic effects which may further contribute to their benefits in CAD. For example, statins were shown to interfere with processes taking place in the atherosclerotic lesion and directly inhibit the proliferation of endothelial cells, smooth muscle cells and macrophages (Negre-Aminou et al., 1997; Sakai et al., 1997). In human endothelial cells, statins upregulate the expression of nitric oxide (NO) synthase (Laufs and Liao, 1998), inhibit the expression of PAI-1 (Essig et al., 1998), and suppress leukocyte-endothelial interactions (Pruefer et al., 1999). Statins reduce the accumulation of cholesterol esters in macrophages exposed to oxyLDL
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(Kempen et al., 1991) and suppress tissue factor expression in cultured macrophages (Colli et al., 1997). Moreover, statins inhibit the secretion of matrix metalloproteinase 9 by macrophages, contributing to plaque stabilization (Bellosta et al., 1998). Considering the important role of mononuclear cell adhesion and transendothelial migration in atherogenesis and the fact that intravascular macrophages secrete TNFa which stimulates the expression of endothelial CAMs, we decided to study the effect of simvastatin on TNFa-induced expression of VCAM-1 and ICAM-1 by human endothelial cells. An attempt was also made to elucidate some aspects of the mechanism of action of the drug.
Materials and methods Materials Medium 199, fungizone, penicillin, streptomycin, gentamycin, heat-inactivated fetal bovine serum (FBS), collagenase, gelatin, glutamine, trypsin/EDTA solution and mevalonate were obtained from Sigma (USA). Recombinant TNFa (10 mg, 1 108 U/ml), random hexamers, reverse transcriptase, PCR buffer, Taq polymerase were from Boehringer Mannheim (Germany). Tissue culture plates were supplied by Costar (USA) and Ficoll-Paque by Pharmacia (Sweden). Monoclonal antibodies against VCAM-1 (CD106:FITC), ICAM-1 (CD54:FITC), CD14 (CD14:PE), CD45 (CD45:FITC), PECAM-1 (CD31:PE) and IgG1 (IgG1:FITC) were purchased from Becton Dickinson (USA). dATP, dTTP, dCTP, dGTP, and RNAsin were from Promega (USA). An ELISA-based kit for studying NF-nB activation was from Active Motif (Belgium). Simvastatin was kindly supplied by MSD (Poland). Endothelial cell isolation and culture Human umbilical vein endothelial cells (HUVEC) were obtained from umbilical cords by collagenase digestion as described by Jaffe et al. (1984). In brief, veins of umbilical cords were perfused with PBS to remove blood cells, filled with 0.1% collagenase (type Ia) and left for 8 min. at 37 jC. The resulting cellular suspension was supplemented with FBS and centrifuged (300 g for 10 min.). HUVEC were cultured in gelatin-coated 25 cm2 flasks, 6-well or 24-well tissue culture plates filled with Medium 199 supplemented with Earle’s salts, 20 mM HEPES, 100 U/ml penicillin, 100 Ag/ml streptomycin, 2.5 Ag/ ml fungisone, 2 mM glutamine and 20% FBS. Culture was at 37 jC under humidified 5% CO2 in room air and the medium was replaced every two days until confluence (3–5 days). HUVEC purity was ascertained by the cobblestone morphology typical for quiescent endothelial cells and staining for PECAM-1 (CD31). Isolation of peripheral blood mononuclear cells (PBMC) Immediately after collection, 25 ml of heparinized blood diluted with PBS was layered over 15 ml Ficoll-Paque and centrifuged (400g, 40 min., 22 jC). The mononuclear cell band was removed by aspiration, washed with PBS, centrifuged and suspended in the same medium as used for HUVEC culture, except that FBS concentration was reduced to 5%. Cells were next counted and used in the adhesion assay. PBMC consisted of approximately 10% monocytes and 90% lymphocytes.
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Pretreatment of HUVEC Second-passage HUVEC were grown to confluence in 6- or 24-well plates, At confluence, FBS concentration in the medium was reduced to 5% and simvastatin was added. In an additional experiment, the medium containing simvastatin at 5 AM was supplemented with 100 AM mevalonate. Simvastatin and mevalonate were dissolved in ethanol. Control values were obtained by adding 0.05% ethanol only. Next, HUVEC were incubated with TNFa (100 U/ml) for defined periods in the absence or presence of simvastatin and mevalonate. Under all conditions cell viability was greater than 90% as judged by trypan blue exclusion. Measurement of VCAM-1 and ICAM-1 expression in HUVEC HUVEC were incubated for 30 min., 12 h or 24 h, without or with simvastatin (0.1, 1.0 or 5.0 AM) or with simvastatin (5 AM) + mevalonate (100 AM). TNFa (100 U/ml) was added and incubation continued for 8 h. Next, cells were washed with PBS and treated for 30 min. at 4 jC with saturating amounts of PE-conjugated anti-PECAM-1 (CD31) and FITC-conjugated anti-ICAM-1 (CD54) or antiVCAM-1 (CD106) monoclonal antibodies (Mab). For isotype control, cells were treated with FITCconjugated mouse anti-IgG1 Mab. Cells were washed with PBS, fixed in 1% paraformaldehyde, harvested by mild trypsinisation, washed again and centrifuged at 300 g for 10 min. prior to analysis by flow cytometry (FACS Calibur, Becton Dickinson). After correction for unspecific binding (isotype control), mean specific fluorescence intensity (MFI) was measured in each channel (10,000 cells per sample). Adhesion assay HUVEC grown to confluence in 24-well plates were pretreated with simvastatin (5 AM) or simvastatin (5AM) + mevalonate (100 AM) for 24 h and stimulated with TNFa (100 U/ml) for 8 h prior to the adhesion assay. Briefly, HUVEC were washed with PBS and coincubated for 30 min. with PBMC suspension (106 cells/ml). Next, HUVEC were washed twice with PBS to remove nonadherent cells and were treated for 30 min. at 4 jC with saturating amounts of mouse FITC-conjugated anti-CD45 and PE-conjugated anti-CD14 Mab. Cells were washed with PBS, fixed in 1% paraformaldehyde, washed again, harvested by mild trypsinisation, centrifuged and counted prior to analysis by flow cytometry (10,000 cells per sample). The proportion of monocytes and lymphocytes in the suspension was established by measuring fluorescence I (FLI–CD45) and fluorescence II (FL II–CD14). The absolute number of monocytes and lymphocytes adhering to endothelial cells was calculated in relation to the total number of cells obtained after trypsinisation. The results were expressed as percent of monocytes and lymphocytes added. Quantitation of VCAM-1, ICAM-1 and PPARa gene expression For quantitation of VCAM-1 and ICAM-1 gene expression, HUVEC were grown to confluence in 6-well plates, pretreated for 24 h with simvastatin (5 AM) or simvastatin (5 AM) + mevalonate (100 AM) and stimulated with TNFa (100 U/ml) for 4 h. For quantitation of PPARa gene expression, HUVEC were grown to confluence in 6-well plates and incubated with simvastatin (0.1, 1.0 or 5.0
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AM) for 24 h. Total RNA from 106 cells was isolated according to Chomczynski and Sacchi (1987) using TRIZOL. RNA concentrations were determined by spectrophotometry at 260 nm. RNA (500 ng) was dissolved in 20 Al of the reaction mixture containing dATP, dTTP, dCTP, and dGTP at 2.5 mM each, 20 U RNAsin, 100 pM random hexamers and 20 U MMLV reverse transcriptase. Incubation was carried out at 37 jC for 60 min. The temperature of the reaction was then raised to 94 jC for 5 min. to inactivate the enzyme and finally reduced to 4 jC. An aliquot of cDNA (5 Al of RT mixture) was dissolved in 25 Al of the reaction solution containing 10 PCR buffer (final Mg2 + concentration was 1.5 mM), dATP, dTTP, dCTP, and dGTP at 2.5 mM each, 10 pM up- and downstream primers (PPARa, VCAM-1, ICAM-1, GAPDH and h-actin) and 1 U Taq polymerase. For semiquantitative analyses, linearity of amplification in relation to the number of PCR cycles was established in preliminary experiments for PPARa, VCAM-1, ICAM-1, GAPDH and h-actin cDNAs. Optimal amplification to detect differences among the samples corresponded to 28 cycles for PPARa, 16 for VCAM-1, 18 for ICAM-1, 22 for GAPDH and 22 for h-actin. The following primer pairs were used: VCAM-1: sense 5V-CCCTTGACCGGCTGGAGATT-3V Antisense 5V-CTGGGGGCAACATTGACATAAAGTG-3V ICAM-1: sense: 5V-TGAAGGCCACCCCAGAGGACAAC-3V Antisense 5V-CCCATTATGACTGCGGCTGCTGCTACC-3V GAPDH: sense 5V-GAGTCAACGGATTTGGTCGT-3V Antisense 5V-GTTGTCATGGATGACCTTGG-3V PPARa: sense 5V-GCC CCT CCT CGG TGA CTT ATC-3V Antisense 5V-ATG ACC CGG GCT TTG ACC TT-3V h-actin: sense 5V-CCTCGCCTTTGCCGATCC-3V antisense 5V-GGATCTTCATGAGGTAGTCAGTC-3V Amplification products obtained with PCR (VCAM-1 241 bp, ICAM-1 409 bp, GAPDH 482 bp, PPARa 454 bp and h-actin 626 cDNA) were electrophoretically separated on 3 % agarose gel. Ethidium bromide stained bands of PPARa, VCAM-1, ICAM-1 and GAPDH were photographed with DS-34 Polaroid camera. Band intensity was densitometrically measured with the gel analysis macro attached supplied with the NIH Image (available free from: http://rsb.info.nih.gov/nih-image/). VCAM-1 and ICAM-1 signals were normalized to cDNA levels of the housekeeping gene GAPDH, PPARa to h-actin and expressed as a ratio. Measurement of NF-jB activation To measure NF-nB activation, confluent HUVEC were pretreated without or with simvastatin (5 AM) or with simvastatin (5 AM) + mevalonate (100 AM) for 24 h and exposed to TNFa (100 U/ml) for 2 h. NF-nB activation was measured in whole cell extract with an ELISA kit. Briefly, HUVEC were washed with ice-cold PBS, scraped into tubes and centrifuged. The pellet was lysed with Complete Lysis Buffer containing dithiothreitol and a protease inhibitor cocktail. After centrifugation at 14,000 rpm and 4 jC for 20 min., the protein concentration in the supernatant (whole cell extract) was determined with a Bradford-based assay. To determine NF-nB activation with ELISA, an oligonucleotide containing the NF-nB consensus binding site (5V-GGGACTTTCC-3V) specific for the active form of NF-nB was
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immobilized to a 96-well plate, whereupon the wells were filled for 1 h with 10 Ag of the whole cell extract. Three washings followed and incubation continued for 1 h with the primary antibody recognizing an epitope on p65 that is accessible only when NF-nB is activated and bound to its target DNA. Wells were next washed and the secondary antibody conjugated to horseradish peroxidase was added to achieve a sensitive readout by spectrophotometry at 450 nm. The experiments were performed in duplicate and the results were expressed as OD450. Statistical analysis The results were expressed as mean F SEM. Differences were analyzed by one-way ANOVA followed by Fisher’s protected least significant difference test. The level of significance was taken as p < 0.05.
Fig. 1. Simvastatin inhibits TNFa-induced cell surface expression of VCAM-1(A) and ICAM-1(B) in concentration dependent manner. Cells were pretreated without or with simvastatin (0.1, 1.0 or 5.0 AM) or with simvastatin (5 AM) + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 8 h, stained for VCAM-1 or ICAM-1 and analyzed by flow cytometry. Results are expressed as percent of TNFa-stimulated cells (100%) and are given as mean F SD from 7 experiments. **p < 0.01 and *p < 0.001, compared to cells stimulated with TNFa in the absence of simvastatin.
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Results Effect of simvastatin on VCAM-1 and ICAM-1 expression by TNFa-stimulated HUVEC VCAM-1 and ICAM-1 expression on the surface of endothelial cells was studied with flow cytometry. Expression was related to mean fluorescence intensity from FCS (forward scater) and PECAM-1 (CD31) gated cells, representing 80–90% of the cells analyzed. Expression of PECAM-1 was taken as a marker of endothelial viability, considering the fact that this CAM is always present on viable and disappears from dead cells (Weber et al., 1994). When compared with control cells, no significant changes were found in % of PECAM-1 positive cells in experiments with simvastatin or simvastatin and TNFa. As expected, HUVEC cultured for 8 h with TNFa demonstrated marked increase in VCAM-1 and ICAM-1 expression compared with resting cells (Figs. 1 and 2). Pretreatment of HUVEC with
Fig. 2. Simvastatin inhibits TNFa-induced cell surface expression of VCAM-1(A) and ICAM-1(B) in time dependent manner. Cells were pretreated without or with simvastatin (5.0 AM) for 30 min., 12 h or 24 h, stimulated with TNFa (100 U/ml) for 8 h, stained for VCAM-1 or ICAM-1 and analyzed by flow cytometry. Results are expressed as percent of TNFa-stimulated cells (100%) and are given as mean F SD from 7 experiments. ***p < 0.05, **p < 0.01 and *p < 0.001, compared to cells stimulated with TNFa in the absence of simvastatin.
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simvastatin (0.1–5.0 AM) for 24 h significantly reduced TNFa-induced VCAM-1 and ICAM-1 (Fig. 1) expression in a concentration-dependent manner, with maximal effect at 5 AM. At this concentration, expression of VCAM-1 was reduced by 28–44% (p < 0.001; n = 7) and ICAM-1 by 11– 25% (p < 0.001; n = 7). Mevalonate (100 AM) prevented the effect of simvastatin (Fig. 1B) on ICAM-1 but was only partly effective in the case of VCAM-1 (Fig. 1A), suggesting that inhibition of HMG-CoA reductase does not account for the whole effect of the drug on VCAM-1 expression. In addition, simvastatin inhibited VCAM-1 and ICAM-1 expression induced by TNFa in a timedependent manner with maximal reduction after 12 h of incubation for VCAM-1 and 24 h for ICAM-1 (Fig. 2). Effect of simvastatin on adhesiveness for monocytes and lymphocytes of TNFa-stimulated HUVEC In an attempt to determine the functional importance of inhibitory properties of simvastatin on TNFastimulated expression of VCAM-1 and ICAM-1, we studied the adhesion of freshly isolated monocytes and lymphocytes to HUVEC. Stimulation of HUVEC with TNFa increased the percentage of adherent monocytes from 19.0 F 2.9 to 26.5 F 3.3% (Fig. 3A) and the percentage of adherent lymphocytes
Fig. 3. Simvastatin inhibits adhesion of monocytes (A) and lymphocytes (B) to TNFa-stimulated HUVEC. Endothelial cells were pretreated without or with simvastatin (5.0 AM) or with simvastatin (5 AM) + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 8 h. Next, freshly isolated PBMC were coincubated for 30 min. Monocyte and lymphocyte adhesion was measured as described in the Material and Methods section. Results are expressed as percent of monocytes and lymphocytes added and are given as mean F SD from 9 experiments. *p < 0.001 compared to cells stimulated with TNFa in the absence of simvastatin.
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from 1.9 F 0.4 to 11.4 F 1.4% (Fig. 3B). Pretreatment for 24 h with 5 AM simvastatin significantly (p < 0.001; n = 9) reduced the adhesion of monocytes (Fig. 3A) to TNFa-stimulated HUVEC from 26.5 F 3.3 to 18.3 F 2.1% (reduction varied for individual populations but the mean percentage of adherent monocytes was typical for unstimulated cells). Adhesion of lymphocytes to TNFa-stimulated HUVEC (Fig. 3B) was reduced from 11.4 F 1.4 to 6.9 F 1.7 % (p < 0.001; n = 9). Mevalonate
Fig. 4. Simvastatin inhibits TNFa-induced VCAM-1 mRNA expression in HUVEC. Cells were pretreated without or with simvastatin (5.0 AM) or with simvastain + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 4 h. mRNA was extracted, reverse transcribed and amplified by PCR using specific primers for GAPDH and VCAM-1. Top: optical densities from 5 separate experiments after normalization to GAPDH (internal standard). Data are expressed as VCAM1/GAPDH ratio and are given as mean F SD from 5 experiments. *p < 0.001 compared to cells stimulated with TNFa in the absence of simvasatin. Photographs below show UV-illuminated gel electrophoregrams from a representative experiment.
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almost completely prevented the effect of simvastatin on monocytes and lymphocytes adhesion to endothelial cells. Effect of simvastatin on VCAM-1 and ICAM-1 mRNA levels in TNFa-stimulated HUVEC To explore mechanisms responsible for inhibition of VCAM-1 and ICAM-1 surface expression, we measured the respective mRNA levels using RT PCR. Stimulation of HUVEC with TNFa for 4
Fig. 5. Simvastatin inhibits TNFa-induced ICAM-1 mRNA expression in HUVEC. Cells were pretreated without or with simvastatin (5.0 AM) or with simvastain + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 4 h. mRNA was extracted, reverse transcribed and amplified by PCR using specific primers for GAPDH and ICAM-1. Top: optical densities from 5 separate experiments after normalization to GAPDH (internal standard). Data are expressed as ICAM-1/ GAPDH ratio and are given as mean F SD from 5 experiments. **p < 0.01 compared to cells stimulated with TNFa in the absence of simvasatin. Photographs below show UV-illuminated gel electrophoregrams from a representative experiment.
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h induced an approximately 13-fold increase in specific VCAM-1 PCR products (Fig. 4) and two-fold in specific ICAM-1 PCR products (Fig. 5). Pretreatment with simvastatin (5 AM) for 24 h significantly abolished induction of VCAM-1 and ICAM-1 transcripts by 20–48% (from 0.52 F 0.09 to 0.36 F 0.09; p < 0.001; n = 5) and 13–22% (from 0.97 F 0.07 to 0.81 F 0.1; p < 0.01; n = 5), respectively. These results closely reflect changes in surface expression of both CAMs described above. Mevalonate almost completely prevented the effect of simvastatin on VCAM-1 and ICAM-1 mRNA levels. Effect of simvastatin on TNFa-induced NF-jB p65 activation To determine whether NF-nB activation was involved in the effect of simvastatin on TNFa-stimulated CAM expression, we decided to measure the amount of p65 with DNA-binding activity in whole cell extracts of HUVEC. TNFa-stimulated HUVEC showed a marked increase (approx. 7-fold) in the levels of active form of NF-nB p65 measured with ELISA (Fig. 6). Pretreatment of HUVEC with simvastatin (5 AM) for 24 h significantly reduced NF-nB p65 activation by TNFa (from 0.44 F 0.061 to 0.25 F 0.041; p < 0.001; n = 9). Reduction varied in individual populations, ranging from 35 to 53%. Simvastatin retained its potency in the presence of mevalonate, reducing TNFa-stimulated levels of NF-nB from 0.44 F 0.066 to 0.377 F 0.07. Effect of simvastatin on PPARa expression Following suggestions that PPARa negatively interacts with the activated NF-nB signaling pathway, we decided to check whether simvastatin acts as PPARa activator. RT PCR analysis revealed that 24 h incubation of HUVEC with simvastatin at 1.0 and 5.0 AM significantly increased the level of specific
Fig. 6. Simvastatin inhibits TNFa-induced activation of NF-nB. Cells were pretreated without or with simvastatin (5.0 AM) or with simvastatin + mevalonate (100 AM) for 24 h prior to incubation without or with TNFa (100 U/ml) for 2 h. A whole cell extract was obtained and NF-nB activation was measured with an ELISA kit. Results are expressed as OD450 and are given as mean F SD from 9 experiments. *p < 0.001 and ***p < 0.05, compared to cells stimulated with TNFa in the absence of simvastatin.
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Fig. 7. Simvastatin induces PPARa mRNA synthesis in HUVEC. Cells were incubated with simvastatin (5.0, 1.0 or 0.1 AM) for 24 h. mRNA was extracted, reverse transcribed and amplified by PCR using specific primers for h-actin and PPARa. Top: optical densities from 6 separate experiments after normalization to h-actin (internal standard). Data are expressed as PPARa/hactin ratio and are given as mean F SD from 6 experiments. *p < 0.001 compared to control cells. Photographs below show UV-illuminated gel electrophoregrams from a representative experiment.
PPARa PCR products from 0.7 F 0.11 to 1.39 F 0.38 and 1.44 F 0.87, respectively (p < 0.001; n = 6) (Fig. 7).
Discussion Recruitment of mononuclear cells by adhesion to the vascular wall and transendothelial migration plays an important role in atherogenesis (Ross, 1999). Monocytes transform into macrophages and macrophage-derived foam cells during early stages of atherosclerosis. Subsequently, macrophages enhance endothelial cell adhesiveness with cytokines like TNFa and IL-1, oxyLDL and oxygen free
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radicals and release metalloproteinases which digest components of connective tissue, contributing to plaque instability and its eventual rupture. This process appears be the cause of clinical manifestation of atherosclerosis in the form of CAD or myocardial infarction (Bellosta et al., 1998). Our in vitro study has demonstrated that simvastatin modulates TNFa-induced expression of VCAM1 and ICAM-1. The drug appears to reduce both surface protein and specific mRNA levels by inhibiting NF-nB. The effect was significantly greater for VCAM-1 than ICAM-1 and was time and concentrationdependent. Our results are in accordance with previous reports. It has been shown that fluvastatin reduces TNFa-stimulated levels of soluble selectin E and ICAM-1 released by HUVEC to the culture medium (Mueck et al., 2001). Atorvastatin and simvastatin were able to reduce oxyLDL-induced VCAM-1 and ICAM-1 expression in endothelial cells from human coronary arteries (Li et al., 2003). Meroni et al. (2001) demonstrated that fluvastatin at doses similar to those used by us inhibits the adhesion of monocytes to HUVEC and reduces expression of selectin E and ICAM-1 stimulated by cytokines or LPS. The pleiotropic effects of statins have already been documented (Faggiotto and Paoletti, 1999; Maron et al., 2000). We have now demonstrated that simvastatin may interfere with atherogenesis through direct action on endothelial cells leading to suppression of their pro-adhesive properties. The inhibitory effect of the drug was more pronounced in the case of VCAM-1 than ICAM-1. This finding is notable in view of the anti-atherosclerotic properties of simvastatin considering the fact that interactions of VCAM-1 with VLA-4 play an essential role in selective accumulation of monocytes and lymphocytes in the vascular wall during atherogenesis (Cybulsky et al., 2001). As in other in vitro studies, our experiments with simvastatin were performed at concentrations ranging from 0.1 to 5 AM. The reported peak serum concentrations of lovastatin after a single therapeutic dose of 40 mg are 0.1–0.3 AM (Pentikainen et al., 1992), matching the lowest concentration used by us. Although maximal inhibitory effect was seen at 5 AM, we believe that due to time constraints in vitro the reaction threshold can only be achieved with higher concentrations. Furthermore, chronic administration of lipophilic drugs may be associated with their accumulation in target tissues. The decreased expression of VCAM-1 and ICAM-1 prompted us to examine the effect of simvastatin on NF-nB, a key transcription factor implicated in the regulation of a variety of genes participating in immune and inflammatory responses, including genes encoding VCAM-1 and ICAM-1 (Collins, 1993). We have found elevated levels of p65 in TNFa-stimulated endothelial cells and demonstrated that simvastatin inhibits TNFa-stimulated activation of NF-nB. Our results are in accordance with previous reports. Atorvastatin reduced NF-nB activation in smooth muscle cells and monocytes stimulated with TNFa (Ortego et al., 1999). Similarly, simvastatin and atorvastatin inhibited the activation of NF-nB in response to oxyLDL in HUVEC (Li et al., 2003) and fluvastatin was able to prevent NF-nB translocation in response to TNFa in human endothelial cells (Meroni et al., 2001). In a rabbit model of atherosclerosis, atorvastatin reduced the activity of NF-nB in vascular macrophages and smooth muscle cells (Bustos et al., 1998). The effect of simvastatin was partly reversed by mevalonate, suggesting a link between inhibition of HMG-CoA reductase and activity of this drug. However, mevalonate was only partly effective in suppressing the effect of simvastatin on p65 levels and VCAM-1 expression, suggesting that inhibition of HMG-CoA reductase is not the sole factor involved. It is worth noting that TNFa-stimulated expression of VCAM-1 was suppressed by simvastatin after an incubation period as short as 30 min. Oxygen free radicals have been implicated in the release of NF-nB from its complex with inhibitory proteins (Collins, 1993). Moreover, a redox-sensitive mechanism has been linked with regulation of
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VCAM-1 gene expression (Offermann and Medford, 1994). Several studies have confirmed that antioxidants like N-acetylcysteine (NAC), pyrrolidine dicarbamate and probucol inhibit NF-nB activation and VCAM-1 expression without any effect on ICAM-1 (Marui et al., 1993; Chen et al., 2001). It has been shown that simvastatain decrease superoxide formation in human monocyte-derived macrophages in response to activation by phorbol mystrate acetate (Giroux et al., 1993). Additionally, we have found that PPARa mRNA levels in HUVEC were increased by simvastatin. Our results agree with the report of Inoue et al. (Inoue et al., 2000) on the effects of simvastatin in HUVEC. Increased PPARa protein and mRNA levels were found together with reduced levels of mRNA for IL-1, IL-6 and cycloxygenase-2. In our opinion, this finding may be related to the effect of simvastatin on VCAM-1 and NF-nB levels in view of reports that PPARa are inhibitors of NF-nB activation (Fruchart et al., 1999). Morever, fenofibrates, known activators of PPARa, were shown to suppress cytokine-induced VCAM-1 expression by inhibiting NF-nB. Since PPARa have potent antiinflammatory properties in vascular cells, our results indicate that simvastatin may suppress the function of stimulated endothelial cells via an anti-inflammatory system mediated at least by PPARa. In conclusion, this is the first study to show that simvastatin reduces expression of adhesion molecules in human endothelial cells and decreases mononuclear cell adhesion by modifying NF-nB. Since mononuclear cell adhesion followed by transendothelial migration plays an important role in atherogenesis, our results suggest that simvastatin may help reduce the risk of atherosclerosis not only by decreasing plasma LDL levels, but also by direct action on the vascular endothelium.
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