Poly(ADP-Ribose) Polymerase Inhibition Reduces Atherosclerotic Plaque Size and Promotes Factors of Plaque Stability in Apolipoprotein E–Deficient Mice
Effects on Macrophage Recruitment, Nuclear Factor-κB Nuclear Translocation, and Foam Cell Death
Background— Poly(ADP-ribose) polymerase (PARP) was suggested to play a role in endothelial dysfunction that is associated with a number of cardiovascular diseases. We hypothesized that PARP may play an important role in atherogenesis and that its inhibition may attenuate atherosclerotic plaque development in an experimental model of atherosclerosis.
Methods and Results— Using a mouse (apolipoprotein E [ApoE]−/−) model of high-fat diet–induced atherosclerosis, we demonstrate an association between cell death and oxidative stress–associated DNA damage and PARP activation within atherosclerotic plaques. PARP inhibition by thieno[2,3-c]isoquinolin-5-one reduced plaque number and size and altered structural composition of plaques in these animals without affecting sera lipid contents. These results were corroborated genetically with the use of ApoE−/− mice that are heterozygous for PARP-1. PARP inhibition promoted an increase in collagen content, potentially through an increase in tissue inhibitor of metalloproteinase-2, and transmigration of smooth muscle cells to intima of atherosclerotic plaques as well as a decrease in monocyte chemotactic protein-1 production, all of which are markers of plaque stability. In PARP-1−/− macrophages, monocyte chemotactic protein-1 expression was severely inhibited because of a defective nuclear factor-κB nuclear translocation in response to lipopolysaccharide. Furthermore, PARP-1 gene deletion not only conferred protection to foam cells against H2O2-induced death but also switched the mode of death from necrosis to apoptosis.
Conclusions— Our results suggest that PARP inhibition interferes with plaque development and may promote plaque stability, possibly through a reduction in inflammatory factors and cellular changes related to plaque dynamics. PARP inhibition may prove beneficial for the treatment of atherosclerosis.
Received October 5, 2006; accepted February 8, 2007.
Atherosclerosis is a multifactorial progressive arterial disease that is increasingly regarded as an inflammatory condition and is the underlying basis of myocardial infarction, stroke, and peripheral artery diseases.1 Fatty streaks, the earliest detectable lesions in atherosclerosis, contain macrophage-derived foam cells that are differentated from recruited blood monocytes.1 The disease is also characterized by infiltration of other immune cells, the deposition of lipids, and smooth muscle cells (SMCs).2 Cells of atherosclerotic plaques can produce a variety of proinflammatory factors such as monocyte chemotactic protein-1 (MCP-1), cytokines, and growth-promoting factors such as macrophage colony-stimulating factor, in addition to oxidants such as H2O2, which aggravate the situation and provide conditions for chronic inflammation.1 Exposure of the various cell types of the arterial wall to these cytokines and oxidants leads to the generation of reactive oxygen species and reactive nitrogen species (RNS),3 which, in turn, induce expression of inflammatory factors and mediate cell death.1,3,4
Clinical Perspective p 2450
Reactive oxygen species and RNS are potent activators of poly(ADP-ribose) polymerase (PARP) as a result of their ability to damage DNA.5,6 Excessive activation of this enzyme results in the intracellular depletion both of its substrate NAD and of the precursor ATP, thereby causing a cellular energy crisis and irreversible cytotoxicity and cell death.5,6 Excessive activation of PARP by reactive oxygen species or RNS has been associated with the pathogenesis of numerous diseases, including energetic failure and vascular collapse in shock, streptozotocin-induced diabetes, cerebral ischemia, glutamate neurotoxicity,7,8 and angiogenesis.9 Endothelial dysfunction has been described as the common underlying mechanism for PARP involvement in cardiovascular diseases in light of the ability of PARP inhibition to provide protection against such dysfunction in shock, hypertension, and heart failure.10,11 Recent studies provided evidence of elevated levels of oxidative DNA damage accompanied by elevated levels of PARP in human atherosclerotic plaques and rat carotid arteries after balloon injury.12,13
PARP has also been suggested to regulate the expression of a variety of key inflammatory genes including MCP-1, inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, all of which are regulated by nuclear factor-κB (NF-κB).14–16 We recently showed that PARP is activated on allergen challenge in an animal model of asthma, and its inhibition, pharmacologically or genetically, prevents infiltration of inflammatory cells into the lungs on allergen exposure.17,18 We correlated such effects with a modulation of Th2 cytokines, namely, interleukin-5 and inflammatory factors such as inducible nitric oxide synthase expression and NF-κB activation. Benko et al19 showed that the early atherosclerotic functional alterations that develop in the endothelium of the apolipoprotein E (ApoE)−/− mice are dependent on the activation of PARP in endothelial cells.
In the present study, we hypothesized that the oxidative DNA damage that results from the generation of reactive species in vascular cells during the onset of atherosclerosis causes excessive activation of PARP, which results in an imbalance of cell survival mechanisms and increased expression of proinflammatory factors that contribute to the progression and instability of atherosclerotic plaques. Inhibition of PARP, pharmacologically or genetically, reduces inflammation and promotes factors of plaque stability. We used a mouse (ApoE−/−) model of high-fat diet–induced atherosclerosis and investigated a potential role for PARP in the process of atherogenesis and examined the effects of PARP inhibition on processes critical for plaque progression and instability. An in vitro system composed of naive peritoneal macrophages or ex vivo–generated foam cells were used to gain additional mechanistic insights.
Animals, Generation of ApoE−/− PARP-1+/− Mice, Experimental Protocol, and Lipid Assessment
C57BL/6 wild-type (The Jackson Laboratory, Bar Harbor, Me), ApoE−/− (The Jackson Laboratory), PARP-1−/−, and ApoE−/− PARP-1+/− mice were housed and bred in a pathogen-free animal care facility at Louisiana State University Health Sciences Center, New Orleans, La, and allowed full access to laboratory rodent chow and water. Experimental protocols were approved by the Louisiana State University Health Sciences Center Animal Care and Use Committee. The generation of the C57BL/6 PARP-1−/− mice has been described.18 ApoE−/− PARP-1+/− mice were generated by crossing the offspring of ApoE+/− PARP-1+/− that resulted from mating their respective homozygous knockouts. All animals were genotyped by polymerase chain reaction (PCR).
Six-week-old ApoE−/− mice received regular chow (n=10) or a high-fat diet (Harlan Teklad, Madison, Wis) containing 21% fat by weight (0.15% cholesterol) and were divided into groups receiving intraperitoneal injections of 3 mg/kg of the PARP-1 inhibitor thieno[2,3-c]isoquinolin-5-one (TIQ-A)20 (Sigma-Aldrich, St Louis, Mo) 2 times (n=19) or 3 times (n=15) per week or vehicle alone (n=30). ApoE+/− PARP-1+/− mice (n=12) were subjected to the same diet protocol without injections. Mice were anesthetized with ketamine/xylazine (60 and 3 mg/kg, respectively), and blood was drawn for sera preparation.
Lipid profiles were assessed in sera by Cardiovascular Specialty Laboratories, Inc (Atlanta, Ga). After euthanasia, aortas were processed for histology or immunohistochemistry. The number and size of lesions were assessed in captured images of hematoxylin-eosin–stained sections of each aorta and analyzed with the use of Image-Pro Plus (Silver Spring, Md).
TUNEL, Immunohistochemistry, and Immunoblot Analysis
Aorta sections were subjected to hematoxylin-eosin and trichrome staining with the use of standard protocols and to terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining according to the manufacturer’s instructions (Roche, Indianapolis, Ind) or to immunohistochemistry with antibodies to poly(ADP-ribose) (Alexis, San Diego, Calif), to murine smooth muscle actin (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif), to nitrotyrosine (Upstate, Lake Placid, NY), or to 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) (Oxis International, Foster City, Calif) as described.17 Thymi were used to assess expression of PARP-1 in ApoE−/− PARP-1+/− mice after euthanasia and protein extraction by immunoblot analysis with antibodies to PARP-1 (BD Biosciences, San Diego, Calif) or actin (Santa Cruz Biotechnology, Inc) as described.21
Macrophage and SMC Isolation, Immunofluorescence Microscopy, Electrophoretic Mobility Shift Assay, Foam Cell Generation, and Viability Assessment
Peritoneal macrophages and thoracic SMCs were isolated with the use of a standard protocol. Macrophages were treated with 1 μg/mL lipopolysaccharide (Alexis) for the indicated time intervals for RNA extraction or immunofluorescence, respectively. Cells were stained with antibodies to murine p65 NF-κB (Santa Cruz Biotechnology, Inc) and with DAPI essentially as described.21 Nuclear extract preparation from macrophages and analysis of NF-κB DNA binding activity by electrophoretic mobility shift assay (EMSA) were performed as described.17 For foam cell preparation, macrophages were serum-starved for 2 hours, after which 5 μg/mL acetylated low-density lipoprotein (Biomedical Technologies, Stoughton, Mass) was added. After 24 hours, most of the macrophages changed their phenotype into foam cells as determined by oil red O staining. Foam cells were then treated with H2O2 for 12 hours, after which cell viability was assessed by calcein-AM (Sigma-Aldrich) staining as described21 or subjected to flourescence-activated cell sorter analysis.
Conventional Reverse Transcription PCR and Real-Time PCR
RNA was extracted from thoracic aortas, macrophages, or SMCs, and cDNA was generated by standard methods. Primers for MCP-1 were as follows: forward, 5′-ACTGAAGCCAGCTCTCTCTTC CTC-3′; reverse, 5′-TTCCTTCTTGGGGTCAGCACAGAC-3′; for tissue inhibitor of metalloproteinase (MMP)-2 (TIMP-2): forward, 5′-CCAGAAGAAGAGCCTGAACCA-3′; reverse, 5′-GTCCATCCAGAGGCACTCATC-3′; for β-actin: forward, 5′-ACCGTGAAAAGATGACCCAGA-TC-3′; reverse, 5′-TAGTTTCATGGATGCCACAGG-3′. The RT2Profiler PCR Array kit from SuperArray Bioscience (Frederick, Md) was used to amplify the different MMPs, collagens, and TIMPs. For real-time PCR, the following primers (Biosearch Technologies, Novato, Calif) were used to amplify specifically a fragment inside collagen type 1: forward primer, 5′-GAGCGGAGAGTACTGGATCG-3′; reverse primer, 5′-GTTCGGGCTGAT- GTACCAGT-3′; or β-actin: forward primer, 5′-AGAGGGAAATCGTGCGTGAC-3′; reverse primer, 5′-CAATAGTGATGACCTGGCCGT-3′; molecular beacon probe. The amplification of TIMP-2 by real-time PCR was performed with the same primers described above in combination with cyber green. Amplification, detection, and data analysis were performed with the iCycler real-time PCR system (Bio-Rad Laboratories, Hercules, Calif).
All data are expressed as mean±SD of values from at least 6 mice per group or from 4 to 6 replicates of the same treatment of the cultured cells. PRISM software (GraphPad, San Diego, Calif) was used to analyze the differences between experimental groups by 1-way ANOVA followed by the Dunnett multiple comparison test.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Association Between Cell Death and Oxidative Stress–Associated DNA Damage, Presence of RNS, and PARP Activation Within Atherosclerotic Plaques
A gross morphological examination of hematoxylin-eosin–stained aortas from ApoE−/− mice fed a high-fat diet for different lengths of time revealed the presence of atherosclerotic lesions. These ranged in different developmental stages from fatty streaks with well-defined foam cells to more advanced necrotic lesions (Figure 1A).
An increasing body of evidence suggests a major role for cell death in the progression and instability of atherosclerotic plaques.22 Additionally, numerous studies link excess generation of reactive oxygen species/RNS with cellular damage within atherosclerotic plaques.3 Figure 1B shows the presence of cell death–associated DNA damage, detected as TUNEL positivity, in ApoE−/− mice fed a high-fat diet. The TUNEL positivity was observed in the vicinity of necrotic cores of established plaques but not in cores with apparently healthy foam cells. Using immunohistochemistry with antibodies to PARP, we determine that foam cells within, or in proximity to, necrotic cores exhibit a substantial synthesis of the poly (ADP-ribose polymer) (Figure 1B, second series of panels), indicative of a robust activation of PARP. However, cells distant from the necrotic core and intact and healthy foam cells both exhibited well-determined membranes and intact cell structure without any detectable poly(ADP-ribose) synthesis (bottom panel). Similar patterns of staining were observed when antibodies to 8-oxo-dG were used, an indicator of oxidative stress–associated DNA damage (Figure 1B, third series of panels), or when antibodies to nitrotyrosine were used, an indicator of protein nitration and presence of RNS (Figure 1B, fourth series of panels). These results establish a potential mechanistic link given the colocalization between oxidative stress–caused DNA damage and PARP activation, suggesting an active role for PARP in foam cell death within advanced atherosclerotic plaques.
PARP Inhibition by TIQ-A Reduces Size and Changes Structure of Atherosclerotic Plaques of High-Fat Diet–Fed ApoE−/− Mice Without Significantly Lowering Sera Cholesterol and Triglyceride Levels
We next examined whether administration of a novel and potent inhibitor of PARP, TIQ-A,20 would reduce the development of atherosclerotic plaques in the animal model. ApoE−/− mice receiving a high-fat diet were injected with 3 mg/kg TIQ-A 3 times per week for 12 weeks and compared with mice injected with vehicle alone. No apparent toxicity was detected in wild-type or ApoE−/− mice receiving TIQ-A injections for 12 weeks as assessed by measuring hepatic enzymes (data not shown). A gross examination of the aortic roots of high-fat diet–fed ApoE−/− mice and those receiving TIQ-A 3 times per week revealed that the drug markedly reduced the size and number of plaques (Figure 2A and 2C). Aortas of high-fat diet–fed ApoE−/− mice displayed the typical features of atherosclerotic plaques, with cholesterol-rich lipid cores within the intimal layer containing distinct large macrophage-derived foam cells and SMCs (Figure 2B; also see Figure 1A, third panel, for a lower magnification of the same plaque). In contrast, plaques from mice treated with TIQ-A 3 times per week contained a smaller number of foam cells and exhibited a SMC-rich fibrous cap (Figure 2B). TIQ-A administration twice per week did not achieve the reduction in atherosclerotic plaques observed with administration of the drug 3 times per week (Figure 2A and 2C). Interestingly, however, a close histological examination of hematoxylin-eosin–stained paraffin sections of plaques shows clear differences in the internal structures and cell components of plaques between the experimental groups (Figure 2B, lower panels). Plaques of TIQ-A–treated mice exhibited a thicker SMC-rich fibrous cap and markedly smaller foam cells. It is noteworthy that the latter traits are primary features of stable atherosclerotic plaques,23 which may suggest that PARP inhibition by TIQ-A may reduce plaque size and promote plaque stability.
Examination of the lipid profile in sera of the different experimental groups shows that although mice treated with TIQ-A 3 times per week showed a trend toward lower triglycerides and low-density lipoprotein in comparison to untreated mice, the difference was not statistically significant (Figure 2D). No effect was seen in mice that were administered TIQ-A twice per week (data not shown). Although these results suggest that the observed reduction in plaque number and size caused by PARP inhibition may not be associated with alterations in lipid contents in our experimental model, it is premature to exclude a possible association.
PARP-1 Heterozygosity Reduces Size and Changes Structure of Atherosclerotic Plaques of High-Fat Diet–Fed ApoE−/− Mice
PARP-1 heterozygosity reduces the expression of the protein (Figure 3A) and its activity by ≈50% and was shown to confer partial protection against streptozotocin-induced diabetes.24 To confirm the role of PARP in progression of atherosclerotic plaques using a genetic approach, we generated heterozygous PARP-1 ApoE−/− mice (PARP-1+/− ApoE−/−). ApoE−/− and PARP-1+/− ApoE−/− mice were subjected to a high-fat diet protocol, and their thoracic aortas were examined macroscopically after euthanasia. PARP-1 heterozygosity markedly reduced plaque formation after 12 weeks of a high-fat diet (Figure 3B) and significantly reduced plaque number and size (Figure 3C) to levels similar to those observed in high-fat diet–fed ApoE−/− mice that received TIQ-A 3 times per week. Interestingly, microscopic examination of plaques generated in high-fat diet–fed PARP-1+/− ApoE−/− mice revealed traits very similar to those observed in high-fat diet–fed ApoE−/− mice that received TIQ-A 3 times per week, including SMC-rich intima and a low number of foam cells (Figure 3D). These results clearly support the hypothesis that PARP plays an important role in plaque formation and structure.
PARP Inhibition by TIQ-A Promotes an Increase in Collagen Content in Part Through an Increase in TIMP-2 in Atherosclerotic Plaques in Our Animal Model
As shown above, the plaques of ApoE−/− mice that received TIQ-A injections twice per week seemed to exhibit traits of plaque stability, including a marked increase in SMCs. Figure 4A shows an increase in smooth muscle actin staining in plaques of TIQ-A–treated mice, confirming the presence of SMC within these plaques. Figure 4B shows an intense blue staining in plaques of TIQ-A–treated ApoE−/− mice, as assessed by trichrome staining, indicative of a rich collagen extracellular matrix compared with the reduced collagen content observed in plaques of ApoE−/− mice that did not receive the drug. Similar results were obtained in plaques of high-fat diet–fed ApoE−/− mice that received TIQ-A injections 3 times per week or those that were heterozygous for PARP-1 (data not shown).
We next attempted to determine whether PARP inhibition affects factors related to atherosclerotic plaque matrix turnover and stability. Figure 4C shows that TIQ-A administration had no significant effect on expression of collagen type 1 in high-fat diet–fed ApoE−/− mice as assessed by real-time PCR. To examine the mechanism by which PARP inhibition increased collagen content within atherosclerotic plaques, we subjected cDNA generated from RNA of primary wild-type or PARP-1−/− SMCs to the RT2Profiler PCR gene expression array that encompassed a number of factors relevant to matrix turnover and stability, including an assessment of gene expression of the different MMPs, collagens, and TIMPs. No significant differences in the expression patterns of collagens or MMPs were observed (data not shown). Interestingly, however, PARP-1 gene deletion was found to markedly increase expression of TIPM-2 and, to a lower extent, expression of TIMP-3 (data not shown). PARP-1 knockout increased TIMP-2 expression in SMCs by close to 5-fold, as assessed by real-time PCR and confirmed by conventional PCR (Figure 4D). These results provide an initial insight on the mechanism by which PARP inhibition may promote atherosclerotic plaque stability.
PARP Inhibition Reduces MCP-1 Expression In Vivo and In Vitro by Preventing NF-κB Nuclear Translocation
MCP-1 is a major cytokine in monocyte/macrophage recruitment to intima of atherosclerotic plaques.1 Figure 5A shows that PARP inhibition by TIQ-A lowered MCP-1 mRNA expression in thoracic aorta from ApoE−/− mice that received a high-fat diet with injections of TIQ-A 3 times per week for 8 weeks compared with those from mice that received the diet without the drug. We then used an in vitro system to examine the role of PARP-1 in MCP-1 expression and the potential mechanism by which such regulation might occur. Peritoneal macrophages isolated from wild-type or PARP-1−/− mice were treated with 1 μg/mL lipopolysaccharide, a known inducer of MCP-1, for 2 or 4 hours, after which total RNA was extracted and subjected to reverse transcription PCR. Figure 5B shows that PARP-1 gene deletion almost completely abrogated MCP-1 expression in response to lipopolysaccharide compared with wild-type controls. This result confirms the data attained with the use of our animal model.
Given that MCP-1 gene expression is critically dependent on NF-κB activation, we speculated that the negative effect of PARP-1 gene deletion on MCP-1 expression was associated with a defect in NF-κB activation. Figure 5C shows that p65 NF-κB is strictly cytoplasmic before stimulation; its localization quickly changed to nuclear when cells were stimulated with lipopolysaccharide (1 to 2 hours) and to cytoplasmic after 4 hours of treatment as assessed by immunofluorescence. In macrophages derived from PARP-1−/− mice, p65 NF-κB remained primarily cytoplasmic during the course of treatment with lipopolysaccharide. These results were confirmed by subjecting nuclear extracts prepared from lipopolysaccharide-treated wild-type or PARP-1−/− macrophages to EMSA with a radioactively labeled DNA sequence specific to NF-κB (Figure 5D). Altogether, these results confirm our observations on the effects of PARP inhibition on MCP-1 expression and subsequent macrophage recruitment and may provide insight on the mechanism by which PARP inhibition reduced plaque formation.
PARP-1 Gene Deletion Confers Protection Against H2O2-Induced Death to Ex Vivo–Generated Foam Cells and Switches the Mode of Death From Necrosis to Apoptosis
To test our hypothesis that PARP exacerbates oxidative stress–mediated injury associated with atherosclerosis, we examined the effect of PARP-1 gene knockout on the viability of ex vivo–generated foam cells (Figure 6A). As shown in Figure 6B, PARP-1 inhibition was highly protective against 25 μmol/L H2O2. At the higher concentration of H2O2 (50 μmol/L), PARP-1 knockout not only protected against the cytotoxic effect of H2O2 (Figure 6C) but also switched the death process from necrosis to apoptosis as assessed by fluorescence-activated cell sorter analysis (Figure 6D). These results clearly show that PARP may be participating in plaque formation in our experimental model by promoting cell death in plaques and thereby contributing to progression and possibly rupture of atherosclerotic plaques.
In the present study, we used an experimental model of atherosclerosis and in vitro analyses to examine the role of PARP in atherosclerotic plaque formation. We provide evidence for a relationship between PARP activation, generation of RNS and oxidative stress–associated DNA damage, and cell death within atherosclerotic plaques. This relationship was corroborated with the use of ex vivo–generated foam cells because oxidative stress–induced death of these cells is promoted by PARP. In vivo, we found that PARP inhibition not only reduced plaque size and number but also caused important structural changes within plaques, suggestive of an increase in their stability. The increase in collagen content may be associated with an elevation in TIMP-2 expression. The observed structural changes were also associated with diminution in macrophage recruitment, potentially as a direct result of a reduction in MCP-1 expression. Defective NF-κB nuclear translocation seemed to be key to PARP inhibition–mediated modulation of MCP-1. Our results implicate PARP in atherogenesis and suggest that its inhibition may constitute a viable therapeutic strategy to treat the disease.
Recent studies provide evidence of elevated levels of oxidative DNA damage and elevated levels of PARP-1 in human atherosclerotic plaques and rat carotid arteries after balloon injury.12,13 Obviously, an increase in PARP-1 expression does not necessarily mean an increase in its activity unless the appearance of DNA breaks and PARP-1 expression coincide. Data from our study establish a mechanistic link between the presence of RNS, oxidative DNA damage, and PARP activation and associated cell death within atherosclerotic plaques. Given the complexity of atherosclerotic plaques, cell death of the different cell types may play different roles in the different stages of atherogenesis.22 For instance, cell death of foam cells in early lesions may provide additional space for macrophage recruitment and the ensuing increase in foam cell formation.4,25 On the other hand, death of foam cells in a well-established plaque may contribute to plaque regression and instability based on the surrounding environment. It is important to note that the mode of cell death that occurs within atherosclerotic plaques depends closely on plaque stage and the content(s) of pro-death factors found within plaque environment. Accordingly, death of vascular cells can occur via apoptosis, and it can also occur via necrosis when increasing amounts of oxysterols are present in necrotic cores in addition to when the phagocytic ability of macrophages is defective.25
Macrophages as well as SMCs contribute to inflammation by producing cytokines, reactive species, and inflammatory factors such as MCP-1 and macrophage colony-stimulating factor that are critical for recruitment of additional macrophages and their persistence within plaques.1 These cells also contribute to plaque remodeling and instability by producing proteases that degrade the extracellular matrix, rendering plaques susceptible to rupture.1,25 Our data clearly suggest that PARP may play an important role in several of these steps. The ability of PARP inhibition to modulate the expression of MCP-1 and increase the expression of TIMP-2 could be a direct cause of the observed smaller plaques in high-fat diet–fed ApoE−/− mice that received TIQ-A or that were heterozygous for PARP-1. Given that MCP-1 expression is NF-κB dependent, this study further supports the relationship between PARP and NF-κB. The mechanism(s) by which PARP-1 deficiency reduces the signal transduction of NF-κB and increases the expression of TIMP-2 is yet to be elucidated.
Increasing evidence indicates that the risk of plaque rupture is critically dependent on both the quantity and quality of the cellular and matrix components of the fibrous cap of an already advanced atherosclerotic plaque and not by its size.1 MMPs play an important role in atherosclerosis through their involvement in the remodeling process of atherosclerotic lesions by degrading the extracellular matrix with a subsequent weakening of the vascular wall.1,25 The specific inhibition of MMPs by TIMPs regulates these effects and reduces the potential vulnerability of plaques to rupture. It is important to note that lesions in the high-fat diet ApoE−/− mouse model typically do not show plaque rupture; nevertheless, such model remains useful for examining factors of plaque stability. Our study shows that PARP inhibition reduced inflammatory cell infiltration and promoted an increase in collagen content and TIMP-2 expression, which may be related to the observed thicker fibrous caps in our experimental model. Furthermore, PARP-1 gene deletion not only protected against oxidant-induced death of foam cells but also switched the mode of death from necrosis to apoptosis. Such effect may be related to intracellular NAD and ATP preservation in response to DNA strand breakage.6,26,27 This becomes highly relevant to the dynamics of atherosclerotic plaques because viability of macrophages is necessary for lipid clearing and apoptosis is an important organized process that allows clearance without additional recruitment of inflammatory cells. Overall, these results suggest that the protective effect of PARP inhibition on foam cell death and its ability to modulate inflammatory cell infiltration may constitute a capability to promote factors of plaque stability.
A number of studies have demonstrated an important connection between PARP expression and activity with the proper function of the endothelium.19,28 Indeed, the early atherosclerotic functional alterations that develop in the endothelium of the ApoE−/− mice were shown to be dependent on the activation of PARP in endothelial cells.19 Furthermore, PARP inhibition was shown to prevent neointimal hyperplasia after endarterectomy.28 Therefore, it is conceivable that the effects of PARP inhibition on plaque number and size observed in the present study may be due in part to protective effects against endothelial dysfunction.
Overall, our results implicate PARP in the inflammation and cell death that are crucial for atherosclerotic plaque progression and rupture and show that PARP inhibition may prove beneficial in blocking these processes.
We thank Dr Alberto Martinez and the Ochsner Clinic Foundation for providing us with laboratory space to pursue our research in the aftermath of Hurricane Katrina. We thank Drs Sampath Parthasarathy and Jack Strong for their guidance and helpful suggestions. We also thank Dr Lisa Harrison-Bernard for her help with Image-Pro Plus.
Sources of Funding
This work was supported in part by grants 1P20RR18766 (overall Principal Investigator, D. Kapusta) and HL072889 from the National Institutes of Health to Dr Boulares.
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Poly (ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is activated in response to DNA damage and participates in DNA repair. Excessive activation of this enzyme results in the intracellular depletion of both its substrate NAD and the precursor ATP, thereby causing a cellular energy crisis and irreversible cytotoxicity. PARP has been associated with the pathogenesis of numerous diseases, including diabetes, cerebral ischemia, and asthma. The findings of the present study establish a close association between oxidative DNA damage, protein nitration, and PARP activation within plaques in an animal model of atherosclerosis, suggesting a potential role for this enzyme in plaque dynamics. Additionally, this study further shows that PARP inhibition, either pharmacologically by thieno[2,3-c]isoquinolin-5-one or genetically, not only reduced plaque number and size but also caused a marked increase in factors of plaque stability. These included an increase in intimal smooth muscle cell content, decreased collagen degradation, potentially through an increase in tissue inhibitors of metalloproteinases-2, and a decrease in monocyte chemotactic protein-1–mediated monocyte/macrophage recruitment. Furthermore, PARP-1 gene deletion conferred protection against H2O2-induced cell death to ex vivo–generated foam cells. Overall, the study establishes a linear association between PARP activation and oxidative stress within the atherosclerotic plaques and provides evidence that PARP inhibition may constitute a viable therapeutic strategy to achieve positive clinical outcome for the treatment of atherosclerosis.
↵*The first 2 authors contributed equally to this work.