Angiotensin-Converting Enzyme Inhibition Prevents Arterial Nuclear Factor-κB Activation, Monocyte Chemoattractant Protein-1 Expression, and Macrophage Infiltration in a Rabbit Model of Early Accelerated Atherosclerosis
Background The migration of monocytes into the vessel wall is a critical event leading to the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is the main chemotactic factor involved in this phenomenon, and nuclear factor-κB (NF-κB) is one of the nuclear factors controlling its expression. ACE inhibitors have been useful in some experimental models of atherosclerosis. In this work, we addressed the hypothesis that angiotensin II (Ang II) may be implicated in the recruitment of monocytes into the vessel wall through the activation of NF-κB and the induction of MCP-1 expression.
Methods and Results Accelerated atherosclerosis was induced in the femoral arteries of rabbits by endothelial desiccation and atherogenic diet for 7 days. Atherosclerotic vessels exhibited an increase in NF-κB–like activity, and p50 and p65 NF-κB subunits were identified as components of this activity. MCP-1 (mRNA and protein) was also expressed in the injured vessels coincidently with the neointimal macrophage infiltration. ACE inhibition with quinapril reduced these three parameters. In cultured monocytic and vascular smooth muscle cells, Ang II elicited an increase in NF-κB activation and MCP-1 expression that was prevented by preincubation of cells with pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation.
Conclusions The present data support a role for Ang II in neointimal monocyte infiltration through NF-κB activation and MCP-1 expression in a model of accelerated atherosclerosis in rabbits. Our results suggest that ACE inhibitors may have a beneficial effect in early atherosclerosis.
The migration of monocytes into the subendothelial space of the vessel wall is an early event in the development of the atherosclerotic plaque.1 MCP-1, the main chemotactic factor involved in this process, is expressed in response to several stimuli both in vitro (LDL, IL-1, TNF-α, interferon-γ, macrophage colony–stimulating factor2 ) and in vivo (hypercholesterolemia3 and balloon injury4 ). During vascular injury, MCP-1 is expressed mainly by macrophages but also by endothelial cells and VSMCs.5
Within the human MCP-1 gene promoter region, two cis-acting elements have been identified: a remote κB region responsible for the induction of transcription after stimulation and a GC box (SP1 binding site) responsible for the maintenance of low levels of basal transcription.6 NF-κB is the main factor involved in the transcription of MCP-1 induced by LPS, IL-1β, TNF-α, and phorbol esters.6 The implication of NF-κB activation in the pathogenesis of atherosclerosis is an attractive emerging idea. The proliferation of VSMCs in response to thrombin is mediated by NF-κB.7 Moreover, vascular endothelial cells respond to oxidative stress with NF-κB activation and adhesion molecule generation.8
Recent data suggest that the renin-angiotensin system may play an important role in the pathogenesis of atherosclerosis. In humans, an upregulation of ACE was associated with an enhanced risk of myocardial infarction,9 and ACE inhibitors seem to reduce the incidence of reinfarction.10 Although several potential mechanisms of these effects, including plaque stabilization, have been proposed,11 there is not yet any clear explanation. In animal models, ACE inhibitors induced a reduction in neointima formation after vascular injury,12 and a beneficial effect in restenosis has been suggested.13 However, until now, clinical trials have failed to support this hypothesis.
A growing body of evidence indicates that Ang II affects circulating cells. Ang II receptors have been demonstrated on human monocytes.14 Moreover, Ang II stimulates chemotaxis of human mononuclear cells.15 Therefore, in this article we explore the hypothesis that Ang II could be responsible, at least in part, for the macrophage accumulation in the damaged artery. Overall, we have shown that during early accelerated atherosclerosis in rabbits, there is an increase in arterial NF-κB–like activity composed of at least p50 and p65. MCP-1 gene and protein expression was also increased in temporal correlation with macrophage infiltration of the neointima. Administration of the ACE inhibitor quinapril prevented the increase of NF-κB activity and MCP-1 expression and reduced the mononuclear cell infiltration of vessels. In cultured VSMCs and macrophages, Ang II elicited an upregulation of the MCP-1 gene and NF-κB activation. Our results clearly implicate Ang II as a new mediator of macrophage recruitment into vessel wall in a model of accelerated atherosclerosis.
Induction of Focal Atherosclerosis
Atherosclerosis was induced according to a previously described technique, with minimal modifications.16 Male New Zealand White rabbits were anesthetized by intramuscular injection of 5 mg/kg xylazine (Rompun, Bayer AG) and 35 mg/kg ketamine (Ketolar, Parke-Davis). Local relief of pain was achieved by subcutaneous injection of 2% lidocaine (Braun). A prophylactic intramuscular injection of 125 mg/kg cefazoline (Llorente Laboratories) was administered 30 minutes before the surgical procedure; all the techniques were done under sterile conditions. Blood (10 mL) was drawn from the ear vein for biochemical studies and serum ACE activity measurement. Arterial pressure was estimated by sphygmomanometer in the femoral artery. Proximal bilateral femoral arteriotomies were performed, and proximal and distal ligatures were placed to isolate a segment of ≈20 mm, which was cannulated with a 27-gauge needle. A vent was made by needle puncture, and blood was removed by a flush of saline. Endothelial damage was induced by the passage of industrial nitrogen at a rate of 80 mL/min for 8 minutes. The isolated segments were then flushed again with saline, and the ligatures were removed. Hemostasis was achieved by local pressure, and the wound was closed with a 4.0 vicryl subcuticular suture. Two days before the surgical procedure, the animals were randomized to quinapril (Parke-Davis) or no treatment. Quinapril was freshly dissolved every other day in the drinking water and given to the rabbits at 1 mg·kg−1·d−1. The consumption of water was measured every other day to confirm the appropriate administration of the drug. After the surgical procedure, the animals were given a 2% cholesterol/6% peanut oil diet (Letica) and maintained for 7 days in individual cages. We used 20 animals weighing 3.5 to 4 kg (mean, 3617±223 g) for mRNA expression and immunohistochemistry. Eight were untreated, 8 were treated with quinapril, and 4 were controls (fed standard chow and with no experimental intervention). Six more (3 treated and 3 untreated) were used for NF-κB determination. Five additional animals were killed after 2 days of quinapril consumption with no surgical intervention to quantify vascular ACE activity.
Harvesting of Vessels
At the time they were killed, animals were anesthetized with ketamine/xylazine, and 10 mL blood was drawn from the ear vein for biochemical determinations. Both femoral arteries were exposed, and ligatures were placed to isolate the damaged segments and flushed with saline. One of the arteries was removed, the adventitial layer was carefully peeled off, and the artery was immediately snap-frozen in liquid nitrogen. The aorta was ligated, flushed with saline, removed, and also snap-frozen in liquid nitrogen. The animals were euthanatized with an overdose of pentobarbital (Abbot), and the other femoral artery was cannulated and fixed in situ with 100 mL 4% buffered formaldehyde at 100 mm Hg pressure. Then it was removed and kept for 24 hours in the same buffer and after that in 70% ethanol until it was embedded in paraffin.
Measurement of ACE Activity and Serum Cholesterol
ACE activity determinations were done in serum and in samples of frozen uninjured artery (aorta) as previously described.17 Artery samples were homogenized in 0.05 mol/L HEPES, 0.1 mol/L NaCl, and 0.05% Triton X-100, pH 7.5, and centrifuged at 12 000g for 10 minutes at 4°C. The resulting supernatant was used for analysis of tissue ACE activity.
Serum cholesterol was measured by standard techniques and with an enzymatic kit (Biomerieux).
Paraffin-embedded arteries were cross-sectioned into pieces 4 μm thick at 5-mm intervals from the proximal to the distal end, dewaxed, and rehydrated. Macrophages were identified with a monoclonal antibody for rabbit macrophages (anti–RAM-11, DAKO).18 MCP-1 was detected with a polyclonal goat anti-human MCP-1 antibody (Immunogenex Corp). Endogenous peroxidase activity in the sections was quenched by incubation in 3% hydrogen peroxide:methanol (1:1) for 30 minutes. Nonspecific antibody binding was blocked by incubation of the tissue section for 1 hour in suppressor serum consisting of 6% goat serum and 4% BSA in PBS (pH 7) for RAM-11 and 6% horse serum and 4% BSA in PBS (pH 7) for anti–MCP-1. RAM-11 (84 μg/mL in 1% goat serum and 4% BSA in PBS) was applied for 1 hour, and anti–MCP-1 (70 μg/mL in 1% horse serum and 4% BSA in PBS) was applied overnight. Secondary antibodies (goat anti-mouse IgG HRPO-conjugated [Seralab] for RAM-11 and donkey anti-goat IgG HRPO-conjugated [The Binding Site] for anti–MCP-1) diluted 1:100 in 4% BSA were applied for 30 minutes, then sections were stained for 10 minutes at room temperature with 0.05% 3,3′-diaminobenzidine tetrahydrocloride (DAKO) and 0.01% hydrogen peroxide in PBS. Finally, sections were counterstained with hematoxylin and mounted in Pertex (Medite). In each experiment, negative controls without the primary antibody or with an unrelated antibody were included to check for nonspecific staining.
For quantification, the sections with the maximal lesion in each animal were chosen. Computer-assisted morphometric analysis was performed with the Cue-2 semiautomatic image analysis system (Olympus). The arterial cross sections stained with the antibodies were digitized with an Olympus microscope (BH-2) connected to a CCD video camera and to the Cue-2 image analysis system. After image acquisition, a gray value ranging from 0 to 255 was assigned to each pixel. The labeled areas in the intima and the media were delimited, and after image enhancement (converting near-white pixels to white and near-black pixels to black and stretching the remaining pixels uniformly over the ranges of gray-level values) and segmentation (transformation of image into a binary image) to set a threshold value, automatic analysis was performed. Results were expressed as inmunostained area and fractional area of intima and media.
The morphometric analysis was performed with the NewSketch 1212 graphic tablet (Genius) linked to a microcomputer. Sections with the maximal lesion were chosen for quantification. The area was obtained by tracing the perimeters of the lumen and the internal and external elastic laminae.
Smooth muscle cells. Rat thoracic aortic VSMCs were isolated and cultured by a modification of the method of Owens et al.19 Briefly, adhering fat and connective tissue were removed by blunt dissection from the thoracic aorta. Vessels were opened longitudinally and preincubated in DMEM (Whitaker) containing 1 mg/mL collagenase (type II, 290 U/mg), penicillin (100 U/mL), streptomycin (100 μg/mL), and glutamine (2 mmol/L) (Sigma) for 15 to 20 minutes at 37°C in 95% air/5% CO2. Then aortas were minced into 1-mm pieces, incubated for an additional 1.5 to 2 hours, and rinsed twice with PBS to remove the cells, which were counted and seeded at a concentration of 104 cells/cm2 in plastic culture flasks (Costar). Cells were harvested for passaging at 2-week intervals and used between the 2nd and 10th passages.
U937 cell line. U937 cells (human monocytic cell line) were obtained from the American Type Culture Collection (1593-CRL) and were cultured in RPMI medium (Whitaker) with 10% FCS.
RNA Extraction and Northern Blot Analysis
U937 and VSMCs were growth-arrested by incubation in 0.5% FCS medium for 24 hours and then incubated with the corresponding stimuli. Frozen femoral arteries were pulverized in a metallic chamber. RNA was obtained by the acid guanidinium thiocyanate–phenol-chloroform method20 and quantified by absorbance at 260 nm in duplicate. RNA (5 μg) from each animal of every group was pooled, and 20 μg of the pool and 30 μg of RNA from the cells were denatured and electrophoresed in a 1% agarose-formaldehyde gel, transferred to nylon membranes (Genescreen, New England Nuclear), and fixed by exposure to UV light for 3 minutes and baking at 80°C for 90 minutes. The membranes were prehybridized for at least 4 hours at 42°C in 50% formamide, 1% SDS, 5×SSC, 5×Denhardt’s solution, 0.1 mg/mL denatured salmon sperm DNA, and 50 mmol/L sodium phosphate buffer, pH 6.5. Hybridization was carried out at 42°C overnight with 20% dextran sulfate and [α-32P]-labeled denatured probe. The membranes were washed with 2×SSC, 0.1% SDS for 30 minutes at room temperature and then with 0.2× SSC, 0.1% SDS at 55°C for 15 minutes. Autoradiography was performed by standard methods. The cDNA probes for human MCP-1 (JE*/pGEM-hJE34) and 28S rRNA (HHCD07) were obtained from the American Type Culture Collection. Human MCP-1 probe was checked for hybridization with rat mRNA and used for hybridization both with human U937 cells and with rat VSMCs. The rabbit MCP-1 probe was obtained from the PCR product as explained below. Films were scanned by the Image Quant densitometer (Molecular Dynamics). 28S ribosomal RNA or ethidium bromide staining was used as internal control.
Reverse Transcription and Semiquantitative PCR Analysis
RNA (1 μg) from each animal was reverse transcribed to single-stranded cDNA by incubation with 20 μL reverse transcription mixture (5 mmol/L MgCl2, 10 mmol/L Tris-HCl [pH 8.8], 50 mmol/L KCl, 0.1% Triton X-100, 1 mmol/L dNTP mixture, 20 U rRNAsin [ribonuclease inhibitor], 15 U avian myeloblastosis virus reverse transcriptase, and 50 ng oligo dT) at 42°C for 30 minutes according to the manufacturer’s instructions (Promega). PCR was conducted in the presence of [α-32P]dCTP for 25, 30, 35, and 40 cycles in the same conditions for MCP-1 and GAPDH, used as internal control (1 minute at 54°C to allow annealing of the primers, 3 minutes at 72°C for primer extension, and 1 minute at 94°C to denature the double-stranded DNA). The following primers were used for rabbit MCP-121 : sense, 5′-TGTGCTTGCCCAGCCAGATG-3′ and antisense, 5′-GTGTCTGCATTTTCTTGTCC3′, which yielded a product of 230 bp. For GAPDH22 : sense, 5′-AATGCATCCTGCACCACCAA-3′ and antisense, 5′-ATACTGTTACTTATACCGATG-3′, which yielded a product of 515 bp. The DNA products from the PCR reactions were analyzed on a 4% polyacrylamide/urea gel in TBE buffer. The polyacrylamide gels were dried, exposed to x-ray films, and scanned with the IQ densitometer.
From tissue. For protein extraction from tissue samples, the method of Negoro et al23 was used, with modifications. Briefly, frozen arterial pieces were pulverized in a metallic chamber and resuspended in 1 mL cold extraction buffer containing 20 mmol/L HEPES-NaOH (pH 7.6), 20% (vol/vol) glycerol, 0.35 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, and 1 μg/mL pepstatin A. The homogenate was vigorously shaken for 30 minutes, insoluble materials were precipitated by centrifugation at 40 000g for 30 minutes at 4°C, and the supernatant was dialyzed overnight against a binding buffer containing 20 mmol/L HEPES-NaOH (pH 7.6), 20% (vol/vol) glycerol, 0.1 mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L DTT, and 0.5 mmol/L PMSF. The dialysate was cleared by centrifugation at 10 000g for 15 minutes at 4°C and frozen at −80°C in aliquots until use. Protein concentration was quantified by the BCA method.
From cells. Cells were made quiescent for 24 hours in 0.5% FCS medium, and then 5×106 cells were incubated with the stimuli for different periods of time. Cells were collected, washed with cold PBS, and resuspended in 5 cell-pellet vol buffer A (in mmol/L: HEPES 10 [pH 7.8], KCl 15, MgCl2 2, EDTA 0.1, DTT 1, and PMSF 1). After 10 minutes on ice, the cells were pelleted, resuspended in 2 vol buffer A, and homogenized. Nuclei were centrifuged at 1000g for 10 minutes, washed twice in buffer A, and resuspended in 2 vol buffer A. Then 3 mol/L KCl was added drop by drop to reach 0.39 mol/L KCl. Nuclei were extracted for 1 hour at 4°C and centrifuged at 100 000g for 30 minutes. Supernatant was dialyzed in buffer C (mmol/L: HEPES 50 [pH 7.8], KCl 50, PMSF 1, EDTA 0.1, and DTT 1, and 10% glycerol) and then cleared by centrifugation and stored at −80°C. Protein concentration was determined by the BCA method.
Electrophoretic Mobility Shift Assays
Gel shift assays were performed with a commercial kit according to the instructions of the manufacturer (Promega). Briefly, NF-κB consensus oligonucleotide (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was [32P]-end-labeled by incubation for 10 minutes at 37°C with 10 U T4 polynucleotide kinase (Promega) in a reaction containing 10 μCi [γ-32P]ATP (3000 Ci/mmol) (Amersham), 70 mmol/L Tris-HCl, 10 mmol/L MgCl2, and 5 mmol/L DTT. The reaction was stopped by the addition of EDTA to a final concentration of 0.05 mol/L. Nuclear or cellular protein (10 μg) was equilibrated for 10 minutes in a binding buffer containing 4% glycerol, 1 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L DTT, 50 mmol/L NaCl, 10 mmol/L Tris-HCl (pH 7.5), and 50 μg/mL poly(dI-dC) (Pharmacia LKB). When competition assays were performed, the cold probe was added to this buffer 10 minutes before the addition of the labeled probe. Labeled probe (0.35 pmol) was added to the reaction and incubated for 20 minutes at room temperature. For supershift assays, 1 μg anti-p65 (Santa Cruz Biotechnology Inc) or anti-p50 (Chemikon) antibody was added and incubated for 1 hour. The reaction was stopped by addition of gel loading buffer (250 mmol/L Tris-HCl, 0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) and run on a nondenaturing, 4% acrylamide gel at 100 V at room temperature in TBE. The gel was dried and exposed to x-ray film.
Results are expressed as mean±SD (unless specified). Significance was established with GraphPAD InStat (GraphPAD Software). Student’s t test and Wilcoxon’s nonparametric test were used to compare the data. Differences were considered significant when P<.05.
Total serum cholesterol of rabbits on a high-fat diet for 7 days increased 10-fold over the basal values of the same animals before they started the diet (643±343 versus 60±24 mg/dL).
Measurement of ACE Activity
Quinapril (1 mg·kg−1·d−1) was administered to the animals from 2 days before the induction of the focal atherosclerosis (day −2) until they were killed. At the moment of injury (day 0), there was a significant diminution in serum ACE activity (2.5±0.9×10−2 versus 8.5±3×10−2 U/mL, P<.008) and in arterial blood pressure (61±16 versus 83±12 mm Hg, P<.0001). However, at this time there was no significant change between vascular ACE activity (determined in the aorta) in quinapril-treated and control rabbits (1.2±0.2×10−2 U/mg, n=5, versus 1.1±0.4×10−2 U/mg, n=4). At death (day 7), serum ACE activity could not be quantified because of the turbidity caused by the hyperlipidemia. Vascular ACE activity, however, diminished ≈40% in quinapril-treated versus nontreated rabbits.
Immunohistochemistry and Morphological Analysis
Injured arteries were paraffin-embedded, and serial sections were stained with the RAM-11 or the anti–MCP-1 antibody. As shown in Fig 1⇓ (bottom photomicrographs), no control (healthy) animal showed any stain with the antibodies. Untreated animals presented a marked stain with the anti–MCP-1 antibody coinciding with a certain number of macrophages in the incipient neointima (Fig 1⇓, top). By contrast, quinapril treatment (Fig 1⇓, middle) was associated with a significant diminution of macrophage infiltration (17±16 versus 4897±2998 μm2, P<.03) and MCP-1 staining (3378±693 versus 8809±2585 μm2, P<.02), Fig 1⇓, graph. No significative changes were noted in the mild neointima formation, although a certain trend toward normalization was found in the quinapril-treated group (intima/media ratio: 0.125±0.024 versus 0.165±0.03, P=.3; maximal lesion: 28 770±4746 versus 45 859±9372 μm2, P=.15). No staining was observed in the negative controls included in each experiment (not shown).
MCP-1 Gene Expression in Injured Femoral Arteries
Because of the small amount of RNA obtained from each arterial sample, MCP-1 mRNA expression was studied in each individual artery by a semiquantitative PCR method by amplifying a fragment of MCP-1 in the same conditions as a fragment of the housekeeping gene GAPDH, as described in “Methods.” Densitometry of the bands obtained after the electrophoresis of the PCR products in polyacrylamide/urea gels showed that the amplification was linear up to 35 cycles both for MCP-1 and for GAPDH, and data for cycle 35 were used for calculations. Control animals showed no detectable expression of MCP-1, whereas this was significantly increased in untreated animals (6.3±1.2 AU; n=6). Quinapril-treated rabbits showed a significant diminution in arterial MCP-1 expression (3.4±1.4 AU; n=7; P<.04 versus untreated animals). A representative PCR obtained after pooling of 1 μg RNA from each animal in every study group is shown in Fig 2⇓.
Similar results (≈50% reduction in MCP-1 mRNA expression in the quinapril-treated group) were obtained when a pool of RNA from every experimental group was subjected to Northern blot analysis (Fig 2⇑).
NF-κB Activity in Arteries of Rabbits
Both injured femoral arteries of three different animals from each group (controls, untreated, and quinapril-treated) were removed, and cellular extracts were pooled. NF-κB activity was quantified in 10 μg total protein from each pool, and experiments were done in duplicate. Negative control experiments without cellular extracts and competition assays with a 50-fold excess of cold NF-κB and cold AP-1 oligonucleotides were performed to establish the specificity of the reaction. No signal was found in the reactions without cellular extracts, and the reaction was proved to be specific because cold NF-κB but not cold AP-1 decreased the signal of the retarded bands (not shown). In relation to controls, injured arteries from untreated animals showed a 7-fold increase in NF-κB activity that decreased to a 4-fold increase in quinapril-treated animals (Fig 3⇓).
Supershift assays with the cellular extracts obtained from the untreated animals were performed to characterize the NF-κB activity found in the arteries. Both the anti-p65 and anti-p50 antibodies reduced the intensity of the major band. In addition, the anti-p50 antibody induced the appearance of a supershifted complex. Therefore, p65 and p50 seem to be components of the NF-κB activity found in the artery wall.
MCP-1 Gene Expression in Monocytes and in VSMCs
Macrophages are primarily responsible for the increase in MCP-1 in atherosclerotic arteries,5 although VSMCs can also synthesize this chemokine. To show the effect of Ang II on MCP-1 expression, some experiments with cultured U937 cells and VSMCs were done. In preliminary studies, maximal MCP-1 expression was found at 6 hours of incubation in both mononuclear and vascular cells (data not shown). In subsequent experiments, growth-arrested cells were incubated for 6 hours in 0.5% FCS medium with Ang II (10−7, 10−9, and 10−10 mol/L). In U937 cells, Ang II (10−9 mol/L) elicited a 3-fold and TNF-α (positive control) a 6-fold increase in MCP-1 mRNA expression (Fig 4A⇓). MCP-1 gene expression in VSMCs was also triggered by Ang II, with a similar dose-response curve (Fig 4B⇓).
To analyze whether NF-κB mobilization could be involved in MCP-1 activation induced by Ang II, cells were preincubated for 1.5 hours with 200 μmol/L PDTC (Sigma), a substance that has been described as inhibiting NF-κB activation in several cell types.24 In all experiments, MCP-1 expression was blocked by preincubation with PDTC, suggesting that the increased MCP-1 mRNA expression elicited by Ang II was mediated by NF-κB activation in mononuclear cells and VSMCs (Fig 4⇑).
NF-κB Activation in Monocytes and in VSMCs
Growth-arrested U937 cells were incubated with Ang II (10−7, 10−9, and 10−10 mol/L) for 30, 60, and 120 minutes, nuclear extracts were obtained, and the amount of active NF-κB was estimated by gel-shift assay. Optimal induction was found after 1 hour of stimulation, returning almost to basal levels at 2 hours. All subsequent experiments were done at 1 hour. The specificity of the reaction was established according to the same controls as described above. Ang II augmented NF-κB activity, being maximal at 10−9 mol/L (3-fold over basal). LPS, a strong inducer of NF-κB activation in macrophages, was used as positive control (Fig 5⇓). Preincubation of the cells with PDTC abolished NF-κB activation (not shown). Preincubation of the nuclear extracts for 1 hour with 1 μg anti-p50 or anti-p65 antibodies reduced the intensity of the bands, and anti-p50 antibody induced the appearance of a supershifted band.
The ability of Ang II to induce NF-κB activation in VSMCs was also assessed. As can be seen in Fig 6⇓, Ang II increased NF-κB activation with a time course and dose response similar to that in monocytic cells: 10−9 mol/L Ang II induced a 7-fold increase. NF-κB activation was abolished by preincubation of cells with 200 μmol/L PDTC for 1.5 hours. Preincubation of the cells with anti-p50 for 1 hour reduced the intensity of the main band, and anti-p65 almost abolished it (Fig 6⇓).
In this article, we show that in rabbits with early accelerated atherosclerosis, the administration of low doses of the ACE inhibitor quinapril decreased NF-κB activity, MCP-1 expression, and macrophage accumulation in the endothelium-injured arteries. In cultured mononuclear cells and VSMCs, Ang II elicited an upregulation of MCP-1 gene expression, probably through the activation of NF-κB. Since in human atheromatous lesions, the concentration of macrophages is great in fissured and ulcerated plaques,25 26 this effect of quinapril could contribute to the stabilization of the atherosclerotic plaque.
Ang II Participates in the Macrophage Recruitment Into the Neointima
In intact vessels, ACE activity is generated mainly by endothelial cells, whereas during the atherosclerotic process, the VSMCs from the neointima became the agent primarily responsible for the observed increase in ACE activity.13 The fact that both ACE inhibitors and type 1 Ang II receptor antagonists reduce neointimal proliferation suggests that Ang II may be directly implicated in this phenomenon.27 According to the present results, Ang II triggers the generation of MCP-1 by both mononuclear cells and VSMCs and could therefore participate in monocyte recruitment into the atherosclerotic lesion. The chemotactic activity of Ang II and its degradation products on mononuclear cells is well established.15 Ang II can also induce the generation of neutrophil chemoattractants by endothelial cells.28 We have observed that the administration of quinapril to animals with endothelial damage and atherogenic diet decreases MCP-1 expression and the number of macrophages invading the neointima. This effect could be due to the reduction in Ang II induced by quinapril and is supported by the in vitro studies in which Ang II directly elicited the gene expression of MCP-1.
However, ACE inhibitors also modify the generation of NO, and this could be another pathway by which these drugs could have a beneficial effect in reducing mononuclear cell accumulation. In endothelial cells, a diminution in the basal production of NO can induce an increase of MCP-1 production and NF-κB activity.29 Ang II can downregulate the expression of the inducible NO synthase triggered by cytokines.30 In a situation of vascular damage with increased cytokine levels, the blockade of Ang II generation could contribute to abolishing this negative effect of Ang II on NO generation.
Ang II Triggers MCP-1 mRNA Expression Via NF-κB Activation in Cultured Macrophages and VSMCs
Our results also show that the increase in MCP-1 expression induced by Ang II is mediated by the activation of NF-κB. The mechanisms by which Ang II triggers NF-κB activation are not completely elucidated. Although the pathway of activation of NF-κB is multifactorial, a serine-threonine protein kinase seems to be involved.31 In different cells such as VSMCs, Ang II increases protein tyrosine phosphorylation and activates several protein kinases, including protein kinase C.32 33 However, there is no direct evidence that this kinase plays any role in NF-κB mobilization. Ang II also phosphorylates the STAT family of transcription factors through the activation of type 1 Ang II receptor.34 Stat3-related protein is elicited by the Src oncogene tyrosine kinase,35 which is activated in VSMCs after stimulation with Ang II.36 Several lines of evidence indicate that reactive oxygen intermediates, in particular H2O2, serve as messengers in the activation pathway of NF-κB.37 In fact, radical scavengers such as PDTC decrease NF-κB activation and the gene expression of MCP-1 and other inflammatory genes.38 Recent data have shown that reactive oxygen intermediates also seem to be involved in the intracellular transduction of Ang II signal.39 Our data showing PDTC abrogation of Ang II effects are in agreement with this mechanism.
ACE Inhibition Decreases NF-κB Activation in Injured Vessels
Although previous studies had shown that in hyperlipidemic hamsters, the ACE inhibitor captopril inhibited macrophage–foam cell accumulation independently of blood pressure and plasma lipids, no possible explanations were approached.40 In this article, we demonstrate that the diminution of monocytes in the neointima of hyperlipidemic rabbits treated with quinapril was clearly linked to the reduction both in NF-κB activation and in MCP-1 mRNA and protein expression in the arterial wall. These findings are in agreement with the idea that activation of NF-κB and the subsequent expression of inflammatory genes are leading events in the development of atherosclerosis. NF-κB activity can be induced in a wide variety of cell types in response to treatment with agents such as IL-1, TNF-α, adhesion, oxidative stress, and oxidized LDL.41 In this study, we also show that Ang II is another factor that could be added to this list. Interestingly, constitutive NF-κB activity is essential for proliferation of cultured bovine VSMCs.42 Therefore, ACE inhibitors could modify vascular injury, modulating both the mononuclear cell infiltration and the proliferation of VSMCs.
Recent data have shown that the instability of the atherosclerotic plaque is closely related to its macrophage content.25 One could speculate that during early atherosclerosis in rabbits, the diminution in MCP-1 production and macrophage content in the neointima induced by quinapril could favor the stabilization of the lesion. In this sense, a diminution in the risk of acute ischemic events in patients receiving ACE inhibitors has been pointed out previously.10
Overall, the present data show that during early accelerated atherosclerosis in rabbits, there was an increase in NF-κB activation, MCP-1 expression (gene and protein), and macrophage accumulation in the artery wall that was prevented by quinapril treatment. Ang II increased MCP-1 mRNA expression and NF-κB activation in cultured mononuclear cells and VSMCs. The present experiments and those of previous authors suggest that Ang II is emerging as an important regulator of two important phenomena of atherosclerotic plaque formation, such as macrophage accumulation and VSMC proliferation. Our results support the idea that ACE inhibitors might have a beneficial effect in the earliest phases of atherosclerosis.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|MCP-1||=||monocyte chemoattractant protein-1|
|PCR||=||polymerase chain reaction|
|TBE||=||Tris-borate EDTA buffer (mmol/L: Tris-HCl 45, boric acid 45, EDTA 1)|
|TNF-α||=||tumor necrosis factor-α|
|VSMC||=||vascular smooth muscle cell|
This work was supported by grants from FISS (93/5389, 94/0370), Ministerio de Educación y Ciencia (MEC) (PB 94/0211), and Fundación Iñigo Alvarez de Toledo. M. Hernández-Presa is a fellow of MEC. The authors want to thank Dr C. Gómez-Guerrero and Dr C. Guijarro for helpful revision and comments on the manuscript and Dr S.L. Sarasa for technical help with the photographs.
- Received August 26, 1996.
- Accepted October 20, 1996.
- Copyright © 1997 by American Heart Association
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