Cellular adhesion and migration of inflammatory cells are early steps in the pathogenesis of atherosclerotic lesion formation.1 Members of the superfamilies of integrins, selectins, and immunoglobulines play an important role in the induction of cellular adhesion.2,3 Inhibition of cell migration showed protective effects on progression of atherosclerotic lesions.4 Recent in vivo studies demonstrated antiatherosclerotic effects of the adenosine analogue 3-Deazaadenosine (c3 Ado) and reduced expression of VCAM-1 and ICAM-1.5 c3 Ado is also known to inhibit synthesis of homocysteine, which is associated with vascular disease.6 Therefore, the antiatherosclerotic effects of c3 Ado might be partially explained by reduced homocysteine concentrations.7 Furthermore, c3 Ado has been shown to display other effects, such as antiviral activity on HIV-1,8 inhibition of platelet-derived growth factor (PDGF),9 and reduced chemotaxis of inflammatory cells in patients with rheumatoid arthritis.10
Since inflammatory mechanisms not only play a role in initiation of early atherosclerotic lesions but also in weakening of advanced plaques, we investigated the effects of c3 Ado in a model of already established vascular disease to evaluate potential plaque-stabilizing effects of the compound. Plaque stabilization as a potential therapeutic tool gains increasing recognition because patients with coronary heart disease predominantly become clinically apparent in a stage of rather advanced atherosclerosis.11
Animals and Drug Treatment
Female apolipoprotein E-deficient mice (strain name B6.129P2) on a C57BL/6J background (n = 35) were kept within the animal care facility of the University of Heidelberg. At the age of 35 weeks, 16 mice were started on a chow diet supplemented with 3-Deazaadenosine (0.06 mg/g, according to a dose of 10 mg/kg bodyweight/day) for 21 weeks; 15 mice received a regular chow diet. The dosage was chosen according to previously published animal studies5,7 and according to a pilot study conducted in our laboratory. The housing and care of animals and all the procedures done in this study were in accordance with the guidelines and regulations of the local Animal Care Committee. All animals remained healthy during the period on a 12-hour light/dark cycle in a temperature-controlled environment with free water and food supply. The average daily intake of food was 6.1 g per animal.
Animal Sacrifice and Preparation of Tissues
At the age of 56 weeks, mice were heavily sedated (Avertin; Aldrich, Milwaukee, WI), blood was collected from the inferior vena cava, and the animals were sacrificed by exsanguination. The mice were perfused with 10 mL of phosphate-buffered saline at physiological pressure via the left ventricle, and thoracic aortas were ligated distal of the brachiocephalic artery and removed for subsequent gel-shift analysis. Afterwards, brachiocephalic arteries were perfused with 4% buffered formalin, dissected out, embedded in paraffin, and serially sectioned (5 μM). Every fifth section was stained with a modified Movat's pentachrome stain.12 Van Kossa staining was used to identify vascular calcification.13
Determination of Plasma Lipid Concentration and Homocysteine Levels
Total serum cholesterol, phosphotungstic acid/magnesium chloride-precipitated high-density lipoprotein (HDL) cholesterol, and trigylcerides were determined enzymatically in heparinized plasma. Low-density lipoprotein (LDL) cholesterol was determined indirectly by Friedewald`s formula. Homocysteine levels were analyzed with a standard method by the laboratory department of our institution.
Evaluation of Plaque Composition and Lesion Size
Sections were evaluated by an investigator who was blinded to the study protocol. Each section was evaluated for characteristic features of plaque morphology and composition, such as frequencies of large necrotic cores (defined as occupying more than two thirds of the plaque volume), intraplaque hemorrhage (defined as the presence of red blood cells independent of microvessels), calcification (defined on the basis of positive van Kossa staining), and lateral xanthomas (defined as clusters of foam cells located in the lateral margin of the lesion). Size of the necrotic cores and thickness of the fibrous caps were determined by using computer-assisted morphometry (Image Pro; Media Cybernetics, Silver Spring, MD) and are reported as maximum area of the necrotic core, percentage of necrotic core relative to the total plaque area, and minimal thickness of the fibrous cap. The cross-sectional area and thickness of the lesion were also evaluated by computer-assisted morphometry and are reported as maximum lesions area, maximum lesion thickness, and maximum percentage of the stenosis per animal.
Tissue sections of the brachiocephalic artery were dewaxed and rehydrated. Detection of macrophages was performed using monoclonal rat anti-mouse antibody (anti-Mac-2, Accurat Chemie), detection of smooth muscle cells by using an anti-smooth muscle actin antibody (Dianova, Hamburg, Germany), of ICAM-1 and VCAM-1 using a polyclonal goat anti-mouse antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) and of the proinflammatory cytokine Egr-1 using a polyclonal rabbit anti-mouse antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), according to the manufacturers' protocols. Sections were then incubated with the biotinylated secondary antibody, rinsed 3x with PBS and incubated for 10 min with streptavidin at room temperature. AEC-chromogen substrate (Invitrogen, Karlsruhe, Germany) was used for visualisation. The extent of positive staining within the lesions was determined using computer-assisted morphometry and expressed in μM2 (Image Pro, Media Cybernetics, Silver Spring, USA).
Preparation of Nuclear Extracts
Aortas were seperated from connective tissue and homogenized in 400 μL of hypotonic buffer [10 mmol/L HEPES, pH 7.9; 10 mmol/L KCL; 0.1 mmol/L EDTA; 0.1 mmol/L EGTA; 2 mmol/L dithiothreitol (DTT)] supplemented with proteinase and phosphatase inhibitors (5 μg/mL E64; 1 mmol/L NaF; 0.2 mmol/L Na3VO4; 0.5 mg/L Pefabloc), incubated on ice for 15 mine, and afterwards supplemented with 25 μL of 10% NP-40. The nuclei were recovered by centrifugation (14,000 rpm, 1 min, 4°C). The nuclear pellets were resuspended in 50 μL of buffer (20 mmol/L HEPES, pH 7.9; 0.4 mol/L NaCl; 1 mmol/L EDTA; 1 mmol/L EGTA; 2 mmol/L DTT supplemented with 5 μg/mL E64; 1 mmol/L NaF; 0.2 mmol/L Na3VO4; 0.5 mg/mL Pefabloc). After centrifugation (14,000 rpm, 5 min, 4°C), the nuclear protein was collected and stored at -80°C until used.
Electrophoretic Mobility Shift Assay (EMSA)
Protein concentrations were measured by the Bradford method.14 Nuclear extracts (10 μg of protein in each assay) were incubated with labeled oligonucleotide probes. The sequences of the oligonucleotides used in the present study were as follows: NF-κB, 5′-AGTTGAGGGGACTTTCCCAGGC-3′, AP-1, 5′-CTGGGGTGAGTCATCCCTT-3′. The oligonucleotides (1.75 pmol/μL) were labeled with [γ32P]-ATP by using T4 polynucleotide kinase. Specific activities used in each assay were around 10,000 cpm. LPS-treated RAW cells were used as positive, untreated cells as negative controls. A 100-fold excess of unlabeled oligonucleotides was used for cold inhibition. Binding reactions were resolved on 4% native polyacrylamide gel and exposed to x-ray film for 12 to 24 h. Gels were analyzed using densitometric analysis (Bio-Rad Laboratories, Hercules, CA).
Evaluation of Soluble ICAM-1, VCAM-1, IL-10, and IL-1β
Serum samples from venous blood taken before sacrifice were stored at -80°C. Soluble ICAM-1 and VCAM-1 as well as IL-10 and IL-1β concentrations were analyzed with commercial enzyme immunoassays (Quantikine; R&D Systems, Inc., Minneapolis, MN) according to the instructions of the manufacturer.
All data are expressed as mean ± SD. Significant differences between means in plasma lipid profiles were determined with the 2-tailed unpaired Student t test. For analysis of plaque morphometry and areas of positive staining, groups were compared using the 2-tailed Mann-Whitney U test. For evaluation of plaque morphology, groups were compared using the chi-square test. P < 0.05 was considered statistically significant.
Body Weight, Plasma Lipid Levels, and Homocysteine Levels
At the time of sacrifice, animals showed no significant differences in body weights, total cholesterol, LDL-cholesterol, HDL-cholesterol, and total triglycerides (Table 1).
Serum homocysteine levels were analyzed in a subgroup of animals (n = 10 per group). Levels were significantly lower in c3Ado treated animals (4.4 ± 0.4 μmol/L) as compared with the control group (5.7 ± 0.3 μmol/L; P < 0.05).
Morphometric evaluation showed no significant effect of c3Ado treatment on maximum lesion area (268,025 ± 53,232 μM2 versus 277,778 ± 73,605 μM2), maximum lesion thickness (368.7 ± 57.6 μM versus 355.2 ± 92.1 μM), and maximum lesion stenosis (72.9 ± 7.7% versus 67.2 ± 6.9%) (Figure 1).
Evaluation of signs of plaque stability such as frequency of large necrotic cores, intraplaque hemorrhage, lateral xanthomas, and presence of cholesterol crystals showed no significant difference between c3Ado-treated and control mice (Table 2). Size of necrotic cores (142,546 ± 40,019 μM2 versus 166,652 ± 65,155 μM2), area of necrotic cores relative to maximum plaque area (53.7 ± 14.1% versus 60.3 ± 17.8%) and thickness of fibrous caps (12.8 ± 4.5 μM versus 10.4 ± 3.4 μM) were not significantly different in the c3Ado-treated, as compared with the control mice (Figure 2). Frequency of intraplaques calcification was significantly reduced in the treatment (10 of 16; 63%), as compared with the control group (15 of 15; 100%; P < 0.05) (Table 2; Figure 3, panel H).
Gel Shift Analysis
Electrophoretic mobility shift assays (EMSAs) of nuclear extracts and subsequent densitometric evaluation showed no significant differences of DNA binding activity of the transcription factors AP-1 and NF-kB in aortic tissue (data not shown).
Immunoassays of Adhesion Molecules and Cytokines
Immunoassays of sera did not show significantly different concentrations of soluble ICAM-1, soluble VCAM-1, IL-1β, and IL-10 between the groups (Table 3).
Immunohistochemistry and Special Stainings
Immunohistochemistry showed significantly reduced staining against α actin in the c3Ado group (9.5 ± 5.5%), as compared with control mice (18.3 ± 6.1%; P < 0.001; Table 4; Fig 3, panel F). Staining against the adhesion molecule ICAM-1 was also reduced in the c3Ado group (8.3 ± 6.7% versus 15.1 ± 10.3; P < 0.05; Table 3). Plaque calcification as evaluated by van Kossa staining was also significantly reduced in the treated mice (19.8 ± 17.6% versus 33.2 ± 14.1%; P < 0.05; Table 4; Figure 3, panel H). Staining against Mac-2 (Figure 3, panel E), the transcription factor early growth response 1 (Egr-1), and VCAM-1 showed no significant difference between the 2 groups (Table 4).
In the present study, long-term administration of the adenosine analogue c3Ado over a period of 21 weeks did not alter progression of advanced atherosclerotic lesions in brachiocephalic arteries of apolipoprotein E-deficient mice. This contrasts with previous observations in early stages of atherosclerosis, in which c3Ado was shown to be effective in preventing formation and progression of lesions in C57/BL6J mice5 as well as in young apo E−/− mice.7 There are several mechanisms by which c3Ado might be protective in early stages of atherosclerotic plaque formation. First, elevated serum homocysteine levels are an independent risk factor for cardiovascular disease, and c3Ado is one of the most potent inhibitors of homocysteine synthesis.15-17 In our study, we also observed a significant reduction of homocysteine levels in the group of c3Ado-treated animals. It is possible that elevated homocysteine levels, mainly through their deteriorating effects on endothelial function, play a prominent role in early lesion formation rather than in progression and destabilization of already established atherosclerotic plaques. It is possible that impaired blood vessel reactivity, as observed in hyperlipidemic mice, can be improved by treatment with c3Ado. Second, the expression of adhesion molecules, such as ICAM-1 and VCAM-1, and concomitant monocytes infiltration were shown to be reduced in c3Ado-treated animals.18,19 In the present study, we did also observe a significant reduction of ICAM-1, but not of VCAM-1 expression. Again, it is possible that expression of adhesion molecules plays an important role only in an initial vascular response to cholesterol accumulation in the intima3,20 and that their inhibition comes too late in advanced atherosclerotic disease to alter plaque morphology. The hypothesis that the effect of c3Ado on expression of adhesion molecules becomes less significant as lesions progress is supported by observations in c3Ado-treated mice, where only aortic intimal cells without fatty lesions showed reduced ICAM-1 mRNA levels.7 Some studies demonstrated a potential role of elevated serum levels of soluble ICAM-1 and VCAM-1 in progression of atherosclerosis.21,22 If concentrations of circulating adhesion molecules are as important as their expression in tissue, this could also explain the lack of effect on preventing lesion progression in our study.
Interestingly, c3Ado treatment resulted in a significant reduction of α-smooth muscle actin (α-SMA) expression in atherosclerotic lesions. Previous studies demonstrate an effect of homocysteine on enhanced myofibroblast differentiation via upregulation of transforming growth factor-β1. Furthermore, homocysteine induces collagen type-1 and α-SMA expression in mouse aortic endothelial cells.23,24 c3Ado with its inhibitory effects on homocysteine synthesis could therefore affect organization of cellular microfilaments. The clinical relevance of reduced expression of α actin within the plaques is not entirely clear. The presence of smooth muscle cells within the protective fibrous cap is considered to enhance stability of the lesion, whereas enhanced proliferation of that cell type contributes to restenosis after angioplasty. Clinical trials investigating the effect of homocysteine-lowering therapy after coronary interventions shows controversial results.25,26 Extent of lesion calcification was significantly inhibited by c3Ado, possibly through inhibitory effects on homocysteine synthesis. Homocysteine was shown to induce calcification in cultured rat aortic smooth muscle cells27 and elevated levels also correlate with coronary artery calcification in humans.28 Alternatively, c3Ado may be affecting microfilaments by more indirect means such as altering intracellular calcium levels or the subcellular distribution of calcium, independently on its inhibition of S-adenosylhomocysteine hydrolase.29 Despite the effects of c3 Ado treatment on homocysteine levels and potentially related subsequent effects on lesion calcification and expression of ICAM-1 and α actin, the present study provides another important piece of evidence that homocysteine-lowering therapy fails to significantly improve relevant endpoints such as progression and composition of established lesions.
In previous studies, using the same animal model of advanced atherosclerotic disease, we were able to demonstrate reduced lesion progression and enhanced plaque stability after long-term administration of statins,30 modulators of the renin-angiotensin system (Blessing, unpublished data), and a direct thrombin inhibitor,31 at least in part, through their antiinflammatory effects, as evaluated by DNA-binding activity to NF-κB and AP-1. In the present study, c3Ado treatment did not reduce DNA-binding activity to NF-κB and AP-1. Previous studies demonstrated that c3Ado inhibits the transcriptional activity of NF-κB through the hindrance of p65 phosphorylation without reduction of its nuclear translocation and DNA-binding activity.6 It seems possible that attenuation of DNA binding activity of proinflammatory transcription factors, rather than their transcriptional activity, is a crucial mechanistic step in promoting plaque stability in that model.
The majority of experimental studies using pharmacological interventions have focused their efforts on the initiation of atherosclerotic lesions in part due to the lack of suitable animal models of advanced disease. This is despite the fact that in cardiovascular disease, the rupture of an advanced atherosclerotic plaque rather than continued narrowing of the blood vessel due to plaque progression leads to acute coronary events. Using the older apolipoprotein E-deficient mice, our laboratory was able to demonstrate that these mice develop advanced atherosclerotic lesions in the brachiocephalic arteries that have many of the morphologic features of advanced atherosclerotic lesions in humans.30-32
The present study could not demonstrate a significant effect of long-term administration of 3-Deazaadenosine on progression and stability of advanced atherosclerotic lesions. A potential antiatherosclerotic effect of c3Ado (eg, mediated through inhibition of adhesion molecules) might therefore be limited to clinically less important prevention of early lesion formation and does not seem to play a relevant role in modifying advanced atherosclerotic disease.
We thank Annette Buttler for excellent technical assistance.
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