6-Mercaptopurine Inhibits Atherosclerosis in Apolipoprotein E*3-Leiden Transgenic Mice Through Atheroprotective Actions on Monocytes and Macrophages
Objective— 6-Mercaptopurine (6-MP), the active metabolite of the immunosuppressive prodrug azathioprine, is commonly used in autoimmune diseases and transplant recipients, who are at high risk for cardiovascular disease. Here, we aimed to gain knowledge on the action of 6-MP in atherosclerosis, with a focus on monocytes and macrophages.
Methods and Results— We demonstrate that 6-MP induces apoptosis of THP-1 monocytes, involving decreased expression of the intrinsic antiapoptotic factors B-cell CLL/Lymphoma-2 (Bcl-2) and Bcl2-like 1 (Bcl-xL). In addition, we show that 6-MP decreases expression of the monocyte adhesion molecules platelet endothelial adhesion molecule-1 (PECAM-1) and very late antigen-4 (VLA-4) and inhibits monocyte adhesion. Screening of a panel of cytokines relevant to atherosclerosis revealed that 6-MP robustly inhibits monocyte chemoattractant chemokine-1 (MCP-1) expression in macrophages stimulated with lipopolysaccharide (LPS). Finally, local delivery of 6-MP to the vessel wall, using a drug-eluting cuff, attenuates atherosclerosis in hypercholesterolemic apolipoprotein E*3-Leiden transgenic mice (P<0.05). In line with our in vitro data, this inhibition of atherosclerosis by 6-MP was accompanied with decreased lesion monocyte chemoattractant chemokine-1 levels, enhanced vascular apoptosis, and reduced macrophage content.
Conclusion— We report novel, previously unrecognized atheroprotective actions of 6-MP in cultured monocytes/macrophages and in a mouse model of atherosclerosis, providing further insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.
Atherosclerosis is the major cause of cardiovascular disease; it usually progresses over decades in silence, until the vessel lumen is severely narrowed and compromises the required blood flow to limbs or organs. Fatal consequences of atherosclerosis are sudden myocardial infarction and stroke, resulting from ruptured lesions. Transplant recipients and patients experiencing chronic systemic inflammatory conditions, such as inflammatory bowel diseases, multiple sclerosis, and systemic lupus erythematosus,1–3 are at higher risk of developing cardiovascular disease.4,5 These patients are treated with immunosuppressive drugs to prevent transplant rejection or to treat their chronic inflammatory conditions. Among these drugs is azathioprine, and it has been proposed that azathioprine is among the more beneficial immunosuppressive drugs with regard to cardiovascular disease.6
See accompanying article on page 1494
The prodrug azathioprine is rapidly converted to 6-mercaptopurine (6-MP) and is subsequently further metabolized.5 The major mechanism of action of 6-MP is believed to be inhibition of purine synthesis. Other described mechanisms of action of 6-MP and its metabolites include incorporation into DNA and RNA and apoptosis of CD4+ T cells through inhibition of Rac1 activation.5,7 Recently, enhancement of the transcriptional activity of the Nur77-family of nuclear receptors has been proposed to contribute to biological effects of 6-MP.8,9 In agreement with the latter, we have shown that 6-MP inhibits smooth muscle cell (SMC) proliferation and attenuates SMC-rich lesion formation involving Nur77.10
Only very limited knowledge is available on the influence of 6-MP on atherosclerosis and its direct effect on monocytes and macrophages, key players in atherosclerosis development.4 Here, we report that 6-MP induces monocyte apoptosis, attenuates monocyte adhesion, and inhibits expression and secretion of monocyte chemoattractant chemokine-1 (MCP-1) by macrophages. We subsequently demonstrate that local delivery of 6-MP to the vessel wall inhibits early atherosclerosis development in hypercholesterolemic apolipoprotein E (ApoE)*3-Leiden transgenic mice. In line with our in vitro findings, this inhibition of atherosclerosis in response to 6-MP was accompanied with decreased lesion MCP-1, as well as with reduced macrophage content. We propose that our data unveil important novel insight into the effect of the immunosuppressive drug azathioprine in atherosclerosis.
For more detailed information on the materials and methods used in this study, please refer to the supplemental material, available online at http://atvb.ahajournals.org.
Cell Culture and Real-Time RT-PCR
Cells were cultured as described previously.11 Real-time RT-PCR was performed with the MyIQ system with SYBR-green mix (Biorad, Veenendaal, The Netherlands) using gene specific primers (Supplemental Table I).
Western Blotting, ELISA, and Fluorescence-Activated Cell Sorting Analysis
Western blotting was performed to detect both cleaved and uncleaved poly(ADP-ribose) polymerase 1 (PARP1) (BD Pharmingen, Breda, the Netherlands), and α-tubulin (Cedar Lane, Hornby, Ontario, Canada) served as loading control. Protein levels of MCP-1 were determined by an ELISA (DuoSet, R&D Systems), according to the manufacturer’s instructions. To measure apoptosis by fluorescence-activated cell sorting (FACS), approximately 5×105 cells were suspended in 50 μL of Annexin-V binding buffer (Molecular Probes) and stained with propidium iodine (PI; 100 μg/mL; Molecular Probes-Invitrogen, Breda, the Netherlands) and with allophycocyanin (APC) or fluorescein isothiocyanate (FITC)–labeled Annexin-V (Molecular Probes-Invitrogen) at dilutions of 1:50 and 1:100, respectively. Analysis was performed with a FACSCalibur instrument, using CellQuest software (BD Biosciences, Breda, the Netherlands).
Mouse Femoral Artery Cuff Model
Animal care and experimental procedures were approved by the animal experimental committee at our institute. Male ApoE*3-Leiden transgenic mice in a C57Bl/6 background aged 12 weeks (n=6/group) were fed a high-fat Western diet containing 15% cacao butter and 1% cholesterol (Arie Blok, Woerden, the Netherlands), which was initiated 1 week before cuff placement and continued until the end of the experiment. At time of cuff placement, mice were anesthetized with an intraperitoneal injection of 5 mg/kg midazolam (Roche, Basel, Switzerland), 0.5 mg/kg medetomidine (Orion, Helsinki, Finland), and 0.05 mg/kg fentanyl (Janssen, Geel, Belgium). Control or 6-MP-eluting cuffs (1% and 2.5% w/w) were placed loosely around the femoral artery and manufactured as described before.12 Plasma cholesterol levels were determined with a colorimetric assay (Biomerieux).
Immunohistochemistry and Quantification
Immunohistochemistry was performed using antibodies against Mac3 (M3/84, BD Pharmingen), MCP-1 (M-18, Santa Cruz Biotechnology, Heidelberg, Germany), or cleaved caspase 3 (Asp175, Cell Signaling Technology, Danvers, Mass) followed by horseradish peroxidase–conjugated donkey anti-rat antibodies (Jackson Immunoresearch Laboratories, West Grove, Penn) and 3,3′ diaminobenzidine (DAB) substrate color development (Immunologic, Duiven, the Netherlands). The positively stained area was determined using image analysis software (Leica Qwin). TUNEL assays were performed according to the manufactory and visualized by immunofluorescence (In Situ Cell Death Detection Kit, Roche Applied Science, Almere, the Netherlands), and nuclei were counterstained with Hoechst.
All data are presented as mean±SEM and were analyzed using the Student t test. Probability values <0.05 were considered statistically significant.
6-MP Decreases THP-1 Monocyte Numbers Through Apoptosis Induction
Recruitment and proliferation of monocytes have a central role in atherosclerosis development. To evaluate the effect of 6-MP on monocytes, we stimulated quiescent THP-1 monocytes with FCS in the presence or absence of 6-MP and observed that 6-MP treatment resulted in a decreased number of monocytes in time compared with control conditions (Figure 1A). To assess whether the decreased number of THP-1 monocytes by 6-MP involves apoptosis, we examined cleavage of poly ADP-ribose polymerase-1 (PARP1), which may be considered a readout for caspase activity in the cell. Clear PARP1 cleavage was observed, indicating that 6-MP indeed induces apoptosis of THP-1 monocytes (Figure 1B).
We next analyzed the percentage of apoptotic cells by fluorescence-activated cell sorting and observed that the percentage of late apoptotic (PI+/Annexin-V+) cells, as well as the percentage of early apoptotic (PI−/Annexin-V+) cells, was increased in response to 6-MP treatment (9.0- and 5.3-fold, respectively, P<0.05, Figure 1C). This effect on apoptosis was observed already at a concentration of 1 μmol/L 6-MP (Supplemental Figure I). 6-MP is well known to inhibit purine synthesis.5 In line with this, addition of the purine adenine fully rescued effects of 6-MP on monocyte apoptosis, which confirms inhibition of purine synthesis as the main mechanism of action by which 6-MP induces apoptosis of THP-1 monocytes (Figure 1D).
To gain further insight into the mechanism underlying the enhanced apoptosis in response to 6-MP, we analyzed gene expression of B-cell CLL/Lymphoma (Bcl)-x, Bcl-xL, and Bcl-2, which are key regulatory proteins in the intrinsic apoptotic signaling pathway.13 Although expression of Bcl-x was not modulated by 6-MP, we observed that the antiapoptotic factors Bcl-xL and Bcl-2 (41.2% and 68.8% inhibition, respectively, P<0.05, Figure 1E through 1G) were potently inhibited by 6-MP, which is consistent with the observed apoptosis induction.
6-MP Inhibits Adhesion of THP-1 Monocytes
The influx of monocytes into the vessel wall is highly relevant in the initiation and progression of atherosclerosis. To investigate potential effects of 6-MP in this process, we analyzed expression levels of the critical monocyte adhesion molecules very late antigen-4 (VLA-4), integrin-β2, and platelet endothelial adhesion molecule-1 (PECAM-1) in response to 6-MP.4 Expression of integrin-β2 was not modulated in THP-1 monocytes by 6-MP, but we did observe that 6-MP reduced expression of the integrins PECAM-1 and, most potently, VLA-4 (73.7% and 50.0% inhibition, respectively, P<0.05, Figure 2A through 2C).
Downregulation of PECAM-1 and VLA-4 suggested that 6-MP may inhibit monocyte adhesion. We therefore assessed adhesion of THP-1 monocytes after 24 hours of treatment with 6-MP, a time point at which cell numbers are not yet affected by 6-MP. We demonstrate that 24 hours of 6-MP treatment did not modulate adhesion of THP-1 monocytes to gelatin-coated surfaces in response to phorbol 12-myristate 13-acetate treatment (Figure 2D), whereas adhesion of the cells to fibronectin-coated surfaces was disturbed (23.3% less adhesion at 30 minutes., P<0.05, Figure 2E). These data are in full agreement with the knowledge that VLA-4, most strongly suppressed by 6-MP, interacts with both vascular cell adhesion molecule-1 and fibronectin.14
6-MP Inhibits MCP-1 in Macrophages
Lesion macrophages are major players in atherosclerosis and locally secrete abundant amounts of cytokines. To investigate the influence of 6-MP on inflammatory gene expression in macrophages, THP-1 monocytes were matured into macrophages by phorbol 12-myristate 13-acetate. In phorbol 12-myristate 13-acetate–matured macrophages, even high concentrations of 6-MP only modestly reduced viability, as measured by counting cells, as well as by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay both in resting macrophages and in LPS-activated macrophages (Figure 3A and Supplemental Figure II). In addition, 6-MP did not noticeably change the cellular morphology of macrophages (Supplemental Figure IIIA through IIIC), nor did it modulate macrophage differentiation, as is shown by unchanged mRNA expression levels of CD11b and of the scavenger receptors CD36 and scavenger receptor-A (Supplemental Figure IIID through IIIF).
To examine a potential effect of 6-MP on inflammatory gene expression, we cultured macrophages and induced inflammatory gene expression by stimulating the cells with LPS, a relevant stimulus in atherosclerosis.15 Using real-time RT-PCR analysis, we assayed mRNA expression levels of tissue factor and of a panel of cytokines involved in atherosclerosis, including interleukin (IL)-1β; IL-6; IL-8; tumor necrosis factor-α; MCP-1 (CCL2); macrophage inflammatory protein-1α (CCL3); macrophage inflammatory protein-1β (CCL4); regulated on activation, normal T cell expressed and secreted (RANTES; CCL5); and pulmonary and activation-regulated chemokine (PARC; CCL18). This approach led us to the observation that 6-MP robustly suppressed mRNA expression of the potent chemokine MCP-1 (Table 1).
Quantification of MCP-1 protein levels in the culture medium of LPS-stimulated macrophages by ELISA revealed that MCP-1 protein secretion was also reduced in response to 6-MP (58.3% inhibition at 10 μmol/L 6-MP, P<0.05, Figure 3B). The inhibitory action of 6-MP on secreted MCP-1 protein levels was reversed by addition of adenine (Figure 3B). In line with our findings in THP-1-derived macrophages, we also observed a strong inhibition of MCP-1 mRNA expression by 6-MP in primary human macrophages, both in unstimulated macrophages and in LPS or tumor necrosis factor-α activated macrophages (P<0.05, Figure 3C).
6-MP Inhibits Atherosclerosis in Hypercholesterolemic ApoE*3-Leiden Transgenic Mice
Our data in cultured monocytes and macrophages suggested that 6-MP influences atherosclerosis development through multiple pathways. We therefore examined the effect of locally applied 6-MP in atherosclerosis by placing 6-MP eluting cuffs (or control cuffs) loosely around the femoral arteries of ApoE*3-Leiden transgenic mice fed a high-fat Western-type diet.16 Release of 6-MP from the cuffs was determined as described before.12 Briefly, 6-MP showed a sustained and dose-dependent release from the 6-MP eluting cuffs, which was 32.6±6.0 μg total release in 12 days for 1% 6-MP eluting cuffs and 59.4±2.8 μg total release in 12 days for 2.5% 6-MP eluting cuffs (Supplemental Figure IV). Two weeks after cuff placement, plasma cholesterol levels were elevated in all mice (≈9 mmol/L, Supplemental Figure V). Consecutive cross-sections of the cuffed arteries stained with HE-Lawson revealed the induction of atherosclerotic lesion formation (Figure 4A through 4C). Quantification of the intimal lesion area demonstrated that 6-MP decreased lesion size compared with control conditions. A 74.6% reduction was observed with cuffs containing 1% 6-MP and a 85.9% reduction was observed with cuffs containing 2.5% 6-MP compared with lesions induced by control cuffs (2423±541 and 1409±466 μm2, respectively, versus 9962±1644 μm2, P<0.05, Figure 4D and Table 2). Furthermore, the intima/media ratio and the percentage of lumen stenosis were considerably reduced by 6-MP, whereas media and total vessel surface areas were not influenced by 6-MP (Table 2). We next performed an immunohistochemical staining and quantified MCP-1 levels in the lesions by image analysis software. MCP-1 levels corrected for lesion surface area revealed that 6-MP inhibits MCP-1 levels in the atherosclerotic lesion (Figure 4E), which is in agreement with our data in cultured cells. To demonstrate that 6-MP similarly as in the cultured cells induces apoptosis in the vessel wall on local application, we performed TUNEL assays (Figure 4F and 4G) and analyzed cleaved caspase 3 protein expression (Figure 4H-I). In 6-MP-treated vessels, strong TUNEL staining was observed, as was enhanced expression of active caspase 3 (Figure 4F through 4H) in comparison with vessels exposed to a control cuff (Figure 4G through 4I).
To investigate whether the enhanced monocyte/macrophage cell death, as well as the reduced secretion of the chemokine MCP-1, resulted in reduced lesion macrophages, we stained macrophages in the atherosclerotic lesions by immunohistochemistry (Figure 4J through 4L). On quantification of the contribution of macrophages to the lesion surface, we unmasked decreased macrophage content in the intima of arteries treated with 1% or 2.5% 6-MP cuffs compared with lesions induced by control cuffs, as well as in the media of arteries (Figure 4M and 4N). The percentage of macrophage content in the media was also reduced in 1% and 2.5% 6-MP-treated vessels, compared with control-treated vessels (Table 2). Although the absolute macrophage content in the intima was reduced, the relative macrophage content in the intima was not modulated by 6-MP treatment (Table 2). The latter may be explained by our previous observations that 6-MP also reduces SMC proliferation, which raises the possibility that in these more complex lesions also, SMC recruitment to the intima is reduced.12,17
To study the effect of systemic 6-MP treatment on monocyte blood counts, we treated hyperlipidemic mice systemically with azathioprine. We applied doses of 2.5 to 25 μg of azathioprine per mouse per day (which is equivalent to 1.5 to 15 μg of 6-MP per mouse per day) in the food for 4 weeks, and we subsequently performed monocyte counts. We did not find significant changes in the number of monocytes by 6-MP (data not shown). These data indicate that normal development of monocytes is not affected by systemic 6-MP treatment.
Together, the data obtained in this mouse model of atherosclerosis are consistent with our in vitro observations and demonstrate that 6-MP modulates atherosclerosis development at least partially through antiatherogenic effects of this drug on monocytes and macrophages.
6-MP is derived from the prodrug azathioprine, which is in clinical practice a commonly used immunosuppressive drug to chronically treat transplant recipients and patients with autoimmune diseases. The cardiovascular risk profiles of immunosuppressive drugs used in these patients are extremely relevant, because these individuals are, because of their specific condition, already at increased risk for cardiovascular disease.4,5 Studies on 6-MP in the human population are compromised because factors such as disease duration and severity are major confounding factors. We therefore chose to gain more insight into the effect of 6-MP in atherosclerosis by using cultured cells and an animal model of atherosclerosis.
We initiated our study by investigating the effect of 6-MP in cultured monocytes and macrophages, as these cells are crucial in atherosclerotic lesion formation. To our knowledge, this is the first time that 6-MP has been investigated in these cells with respect to atherosclerosis. We demonstrated that 6-MP induces monocyte apoptosis through reduced expression of the intrinsic antiapoptotic factors Bcl-2 and Bcl-xL, and enhanced caspase activation. In addition, we showed that 6-MP inhibits expression of the adhesion molecules VLA-4 and PECAM-1 in monocytes and disturbs adhesion of these cells to the extracellular matrix protein fibronectin. The relevance of monocyte adhesion in atherosclerosis has been well described, and the importance of VLA-4, most strongly suppressed by 6-MP, is further underlined by a report demonstrating that blocking VLA-4 inhibits atherosclerosis in low-density lipoprotein receptor–deficient mice.4,18 Furthermore, 6-MP is shown to robustly inhibit expression and secretion of MCP-1, a pivotal chemokine in atherosclerosis.19
We verified our data obtained in cultured cells in vivo, using an animal model of atherosclerosis. Consistent with the antiatherogenic effects of 6-MP on cultured monocytes and macrophages, we observed that local application of 6-MP to the vessel wall, using a drug-eluting cuff, inhibits atherosclerotic lesion formation in hypercholesterolemic ApoE*3-Leiden transgenic mice. Moreover, we showed reduced MCP-1 levels in lesions treated with 6-MP, which may explain at least partially the lower number of macrophages in the atherosclerotic vessel wall. However, we propose that monocyte apoptosis and reduced monocyte adhesion are also involved in this decreased macrophage content.
It is conceivable that 6-MP influences atherosclerosis development through actions on several other cell types besides monocytes and macrophages that are relevant in atherosclerosis. For example, proapoptotic effects of 6-MP on T cells will likely also interfere in atherosclerosis development.5,7 Likewise, effects of 6-MP on endothelial cells are described, including effects of 6-MP on purine synthesis,20 but also enhancement of HIF1α activity and angiogenesis partly mediated through activation of Nur77 by 6-MP.21 It will be extremely relevant to study 6-MP in these other vascular cells with respect to atherosclerosis and under inflammatory conditions to gain even further insight into effects of 6-MP on atherosclerosis. We have previously demonstrated that Nur77 is crucially involved in the inhibition of proliferation of venous SMCs by 6-MP.17 Similarly, we have shown that 6-MP inhibits SMC-rich lesion formation through activation of Nur77 involving upregulation of cell cycle inhibitor p27Kip1 in SMCs.12 As atherosclerotic lesions also contain SMCs, it is likely that the inhibition of SMC proliferation contributed to the decreased atherosclerotic lesion development that we observed in our animal model.
The clinical indications for which 6-MP and azathioprine are currently being applied as an immunosuppressive drug include multiple sclerosis, systemic lupus erythematosus, and inflammatory bowel diseases, such as Crohn’s disease, which are all pathologies in which macrophages contribute significantly to disease progression.22–24 Furthermore, MCP-1 is elevated in Crohn’s disease and is associated with the degree of intestinal inflammation.24 Although beyond the scope of this study, we speculate that effects of 6-MP on monocytes and macrophages, including downregulation of MCP-1 expression, may contribute to the beneficial action of 6-MP in these chronic inflammatory diseases.
In conclusion, we provide evidence that local delivery of 6-MP inhibits atherosclerosis development in hypercholesterolemic ApoE*3-Leiden transgenic mice, which is accompanied by reduced MCP-1 levels and decreased lesion macrophage content. This can be explained by an as of yet unrecognized effect of 6-MP on the induction of monocyte apoptosis, attenuation of monocyte adhesion, and the inhibitory effect of azathioprine/6-MP on MCP-1 secretion in macrophages. We propose that our results provide further insight into the action of 6-MP in atherosclerosis.
Sources of Funding
This research forms part of the Project P1.02 NEXTREAM of the research program of the BioMedical Materials institute, cofunded by the Dutch Ministry of Economic Affairs. The financial contribution of the Netherlands Heart Foundation (grants 2003B199 and M93.007) is gratefully acknowledged. Collaborations were formed with the European Vascular Genomics Network within the European Union FP6 Network of Excellence LSHM-CT-2003-503254.
Received on: August 7, 2009; final version accepted on: April 8, 2010.
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