Poly (ADP-Ribose) Polymerase Inhibition Attenuates

641Journal of Atherosclerosis and Thrombosis　Vol.16, No.5

Original Article

Poly (ADP-Ribose) Polymerase Inhibition Attenuates Atherosclerotic Plaque Development in ApoE－/－ Mice with Hyperhomocysteinemia

Jiang-jiao Xie, Xian Yu, Yu-hua Liao, Jian Chen, Rui Yao, Yong Chen, Meng-yang Liao, Ying-jun Ding, Ting-ting Tang, and Xiang Cheng

Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China

Aim: Hyperhomocysteinemia (Hhcy) is an important and independent risk factor for atherosclerosis. Recent studies have shown that Poly (ADP-ribose) polymerase (PARP) activation may be associated with Hhcy-induced endothelial dysfunction, which is an important mechanism for Hhcy to affect atherosclerotic progress. Thus, we investigated whether PARP inhibitors may attenuate atheroscle-rotic plaque development in an Hhcy-induced experimental animal model with atherosclerosis.Methods: Six-week-old homozygous apolipoprotein E-deficient (ApoE－/－) male mice fed a normal diet or high methionine diet randomly received intraperitoneal injections of 10 mg/kg 3-aminoben-zamide (3-AB, a PARP inhibitor) dissolved in phosphate-buffered saline (PBS), or physiological saline every other day for 12 weeks. Atherosclerotic lesion sizes and PARP activity were measured. Related inflammatory factors in atherogenesis were investigated by real-time quantitative PCR and Western blot analysis.Results: Our data demonstrated that ApoE－/－ mice fed a high methionine diet generated Hhcy, which subsequently increased the atherosclerotic lesion size significantly, promoted oxidative stress-associated DNA damage and PARP activation, then increased the expression of proinflammatory fac-tors within atherosclerotic plaques. Although PARP inhibition by 3-AB did not markedly inhibit plaque development in ApoE－/－ mice with spontaneous hyperlipidemia by feeding a normal diet, it significantly reduced the atherosclerotic lesion size by 40% in Hhcy-induced atherosclerosis without affecting plasma homocysteine levels and lipid contents, effectively suppressed PARP activation, and inhibited nuclear translocation of nuclear factor-κB (NF-κB) and subsequent production of inflam-matory factors, such as vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattactant protein-1 (MCP-1).Conclusion: Our results suggest that PARP inhibition attenuates atherosclerotic plaque development under hyperhomocysteinemic conditions, through the inhibition of PARP activation, nuclear NF-κB translocation and subsequent expression of inflammatory factors.

J Atheroscler Thromb, 2009; 16:641-653.

Key words; Atherosclerosis, Hyperhomocysteinemia, Oxidative stress, Proinflammatory

strated that increased concentration of plasma total homocysteine (tHcy) is an independent risk factor for atherothrombotic diseases, such as ischemic heart dis-ease, stroke, and peripheral vascular disease1). Homo-cysteine is a metabolite of the essential amino acid methionine, which plays a key role in the generation of methyl groups required for the synthesis of DNA. Recent studies suggest that even mild hyperhomocys-teinemia (Hhcy) can impair endothelial cell func-tion2-4), the mechanism relates to oxidative stress. The

Introduction

Numerous epidemiological studies have demon-

Address for correspondence: Xiang Cheng, Laboratory of Cardiovascular Immunology, Institute of Cardiology, Union Hospital, Huazhong University of Science and Technology, Wuhan 430022, ChinaE-mail: [email protected]: January 18, 2009Accepted for publication: April 10, 2009

642 Xie et al. 643PARP Inhibition Attenuates Atherosclerosis

reactive oxygen and nitrogen species associated with oxidative stress not only have significance in the pathogenesis of atherosclerosis and vascular remodel-ing following injury in coordination with lipid metab-olism and inflammation together5-7), but also cause DNA damage, which are effective activators of Poly (ADP-ribose) polymerase (PARP)8).

PARP is the most abundant nuclear enzyme of eukaryotic cells. When activated by DNA single-strand breaks, PARP initiates an energy-consuming cycle by transferring ADP ribose units from NAD＋ to nuclear protein, resulting in rapid depletion of intracellular NAD＋ and ATP pools, slowing the rate of glycolysis and mitochondrial respiration, and eventually leading to cellular dysfunction and death8, 9). PARP can also regulate the expression of a variety of key inflamma-tory mediators, including inducible nitric-oxide syn-thase (iNOS), tumor necrosis factor-α (TNF-α), monocyte chemoattactant protein-1 (MCP-1), inter-cellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), all of which are regulated by nuclear factor-κB (NF-κB)10-12). Using PARP inhibitors or by knocking out PARP gene in cells or mice, both NF-κB activation and transcrip-tion of NF-κB-dependent inflammatory genes can be reduced11, 13-15). Recent studies provided evidence of elevated levels of oxidative DNA damage accompanied by elevated levels of PARP in human atherosclerotic plaques16). Moreover, Oumouna-Benachour K et al.17) detected apolipoprotein E-deficient (ApoE－/－) mice with high-fat diet-induced atherosclerosis and indicated that PARP inhibition by TIQ-A interfered with plaque development and promoted plaque stability through a reduction in inflammatory factors and cellular changes.

In the present study, we aimed to observe the effect of PARP inhibition on Hhcy-induced athero-sclerosis initially, but because Hhcy induced by methi-onine supplementation did not independently cause atherosclerosis in normal mice model18), we used ApoE－/－ mice with high-methionine diet-induced ath-erosclerosis to investigate the potential role of PARP inhibitor 3-aminobenzamide (3-AB) in the develop-ment of atherosclerosis. We hypothesized that oxida-tive DNA damage induced by Hhcy during the onset of atherosclerosis led to excessive activation of PARP, increased the expression of proinflammatory factors, and promoted plaque development, whereas pharma-cological inhibition of PARP could partly attenuate this process.

Methods

Animals and TreatmentApoE－/－ mice on a C57BL/6J background, pur-

chased from Jackson Laboratory (Bar Harbor, Maine, USA), were bred and maintained in the Animal Cen-ter of Beijing University. This investigation was per-formed according to the principles of the Experimen-tal Animal Ethics Committee. All animal studies were reviewed and approved by the Animal Study Commit-tee of Tongji University. Six-week-old homozygous ApoE－/－ male mice with body weight ranging from 18 to 20 g were used in the present study. The mice were housed in a temperature-controlled (25℃) room with a 12-hour light/dark cycle with free access to food and water. After adapting to this environment for a week, the mice were divided into four groups at random: (1) control group (Con, n=15): received reg-ular chow for 12 weeks; (2) regular chow plus 3-AB treatment group (Con＋3AB, n=15): received regular chow and intraperitoneal injections of 10 mg/kg of the PARP inhibitor 3-AB every other day for 12 weeks; (3) high-methionine diet group (HM, n=15): received a high-methionine diet [regular diet plus 1.7% methionine (wt/wt)] and intraperitoneal injections of physiological saline 0.3 mL per mouse every other day for 12 weeks; (4) high-methionine diet plus 3-AB treatment group (HM＋3AB, n=15): received a high-methionine diet and intraperitoneal injections of 10 mg/kg of the PARP inhibitor 3-AB every other day for 12 weeks.

Plasma tHcy and LipidsAt the end of the study, the mice were anesthe-

tized and exsanguinated by withdrawing the maxi-mum amount of blood from the right ventricle. Non-fasting blood samples were obtained in chilled EDTA-containing microtubes and centrifuged immediately, and the plasma was stored at －20℃. Plasma tHcy was measured by HPLC with electrochemical detection using a commercially available kit (Bioanalytical Sys-tems, Inc., West Lafayette, Indiana, USA). Plasma total cholesterol (TC), high-density lipoprotein cho-lesterol (HDL-C), and triglycerides (TGs) were mea-sured by an autoanalyzer (Hitachi 917).

Tissue Preparation and Atherosclerotic Lesions Evaluation

Paraffin and frozen histological section analysis were used to evaluate atherosclerotic lesions. After blood sampling, the mice were flushed (saline contain-ing St. Thomas’ cardioplegic solution and heparin), perfusion-fixed (phosphate-buffered 4% formalde-


hyde, pH 7.2), and then hearts and aortas were removed rapidly. The thoracic and abdominal aorta was quick-frozen in nitrogen for later extraction of protein and RNA. The heart, including the aortic root, was snap-frozen in OCT compound for cryostat sectioning19). For morphometric analysis, paraffin sec-tions were used, and hematoxylin-eosin staining was performed according to standard protocols. For quan-tification of the lesion area, we prepared serial cross sections (10 μm thick) of the aortic root according to the method described by Paigen et al.20), with a slight modification. In brief, atherosclerotic lesions in the aortic sinus region were examined at 5 locations, each separated by 200 μm, with the most proximal site starting where the first aortic valve appeared. Five serial sections prepared from each location were con-ventionally stained with oil red O21). Lesion areas were quantified by a single observer blinded to the experi-mental protocol. All images were captured and ana-lyzed by computer image analysis software. The aver-age value for the 5 locations for each animal was used for analysis22).

PARP Activity AssayPARP activity was measured using a Universal

Colorimetric PARP Assay Kit (Trevigen, Inc., Gaith-ersburg, MD, USA) according to the manufacturer’s instructions. Briefly, aortas (five arteries for each group) were minced on ice and lysed (1×PARP buffer, 0.4 mM PMSF, 0.4 M NaCl, 1%NP-40 or 1%Triton X-100). After protein extraction and quantification, we used at least 20 μg protein per well in the assay. 1×PARP buffer and the sample were added into each of the designated 3 wells to a total volume of 25 μL. PARP-HSA standard was serially diluted in cold microtubes with 1×PARP buffer to design the PARP Standard Curve and then 25 μL 1×PARP Cocktail was placed into each well. After incubation at room temperature for 90 minutes, strip wells were washed 4 times. Then, 50 μL diluted strep-HRP were added per well and incubated at room temperature for 60 min-utes, followed by another 4 washes, after which 50 μL per well of TACS-SapphireTM Colorimetric substrate were added and incubated in the dark for 30 minutes. The reaction was stopped by the addition of 50 μL per well of 0.2 M HCl and absorbance was detected at 450 nm.

Immunohistochemistry and Apoptosis DetectionFor immunohistochemistry, paraffin-embedded

aortic sections were analyzed by standard procedures with antibodies to murine VCAM-1 (R & D Systems) and murine MCP-1 (Abcam). Apoptosis was detected

by the ApopTag Peroxidase In Situ Oligo Ligation (ISOL) Apoptosis Detection Kit (Chemicon) accord-ing to the manufacturer’s instructions.

Western Blot AnalysisTotal proteins, cytoplasmic extracts and nuclear

extracts were prepared from pooled arteries (five arter-ies for each group). The concentrations of proteins were determined by the BCA protein assay (Pierce). Protein samples (20−50 μg) were boiled for 10min in sample buffer [250 mM Tris-Hcl (pH=6.8), 4% SDS, 10% glycerol, 2% β-mercaptoethanol, and 0.003% bromophenol blue], separated on denaturing 7.5%, 10% or 15% SDS-polyacrylamide gels, and then elec-tro-transferred onto nitrocellulose membranes. After being blocked with 5% nonfat dry milk in TBS with 0.05% Tween 20 (TBS-T) for 3 h at room tempera-ture, membranes were washed three times, and then incubated at 4℃ overnight with primary antibodies in their blocking solution. These primary antibodies include anti- phospho-p47phox (1:1000; Upstate Cell Signaling Solutions), anti-poly (ADP-ribose) poly-merase-1 (1:1,000; Alexis), anti-NF-κB p65, anti-inhibitors of κB (IκB), anti-phospho-inhibitors of κB (p-IκB) (1:1,000; Santa Cruz Biotechnology), anti-MCP-1 (1:2,000; Abcam), anti-VCAM-1 (1:1,000; R & D Systems), anti-caspase-3 (1:1,000; Cell Signal-ing), anti-β-actin (1:1500; Abcam), and anti-histone H3 (1:1000; BioLegend). Membranes were washed three times in TBS-T for 10 min and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies in TBS for 2 h. After another three washes with TBS-T for 10 min, membranes were reacted with the enhanced chemiluminescence system (Pierce) and exposed to films23). Protein levels were quantified by scanning densitometry using image-anal-ysis systems.

Real-Time Quantitative Polymerase Chain ReactionTotal RNA was harvested from mouse aorta (from

the beginning of the aortic arch, just after the aortic roots, to the iliac bifurcation) using Trizol reagent (Invitrogen, Carlsbad, Calif ). After reverse transcrip-tion reaction, real-time quantitative polymerase chain reaction (PCR) was performed with the SYBR Green real-time PCR master mix kit (Toyobo Co., Osaka Japan). Each sample was analyzed in triplicate, and normalized to GAPDH. Fold changes in mRNA expres-sion relative to the control group (n =5 mice per group) were analyzed by the comparative computed tomography method. PCR primers were as follows:

MCP-1: forward, 5’-CTCACCTGCTGCTA-CTCAT TCAC-3’


reverse, 5’-GATTTACGGGTCAAC-TTCACATTC-3’

VCAM-1: forward, 5’-TGAACCCAAACAGAG-GCAGAG-3’

reverse, 5’-GGTATCCCATCACTT-GAGCAG-3’

PARP-1: forward, 5’-GGACGAAGAGGCAG-TAAAGAAG-3’

reverse, 5’-CTCGCTGAGGTAAGA-GTAGGC-3’

GAPDH: forward, 5’-AGCAATGCCTCCTG-CACCACCAAC-3’

reverse, 5’-CCGGAGGGGCCATC-CACAGTCT-3’

Statistical AnalysesValues are expressed as the mean±SD in the text

and figures. The data were analyzed by ANOVA. If significance was found, the Newman-Keuls test was performed for post-hoc analysis to detect differences among groups. A probability value of p＜0.05 was considered significant.

Results

Effect of 3-AB Treatment on Plasma Levels of tHcy and Lipids

As shown in the Table 1, ApoE－/－ mice fed a regular diet or high methionine diet developed spon-taneous hyperlipidemia in all four groups. A high methionine diet significantly elevated plasma tHcy levels (p＜0.01 vs. Con group), but 3-AB treatment did not affect plasma homocysteine levels and lipid contents (p＞0.05 vs. HM group).

Effect of 3-AB Treatment on Atherosclerosis Lesion Area

The atherosclerotic lesion in the aorta sinus was measured as described in the Methods section. As

shown in Fig.1, only a regular diet plus 3-AB treat-ment (Con＋3AB group) tended to decrease plaque size by 15.6%, but was not significant (9.24±0.61%: 10.95±0.81%, p＞0.05); whereas after twelve weeks of a high methionine diet, ApoE－/－ mice displayed a marked increase in atherosclerotic lesion size in the HM group (23.62±6.2%: 10.95±0.81%, p＜0.05), and 3-AB treatment (HM＋3AB group) significantly reduced atherosclerotic lesion size by 40% (14.37±4.5%: 23.62±6.2%, p＜0.05). Longitudinal analysis of lesions demonstrated that the largest lesion exten-sion was located at 600 μm, and our measurements were performed accurately at the same levels of the root in each group.

Effect of 3-AB Treatment on PARP Activation within Atherosclerosis Lesions

Homocysteine-stimulated superoxide anion pro-duction (oxidative stress) stimulates the phosphoryla-tion of p47phox and p67phox subunits of NADPH oxi-dase through a PKC-dependent path24). The effect of homocysteine may involve increased oxidative stress in vessels and related direct damage on the DNA, which causes strand breaks in the DNA and activation of PARP25). Fig.2A showed that the level of phospho-p47phox expression, which demonstrated the level of oxidative stress, was significantly increased in ApoE－/－ mice fed high methionine diet, but 3-AB treatment did not change the expression significantly compared with the diet-matched group. PARP expression was measured by real-time quantitative PCR and Western Blot analysis with antibodies to poly (ADP-ribose) polymerase-1 (PARP-1, classical zinc-finger-contain-ing PARP, referred to as PARP for simplicity). Only a regular diet plus 3-AB treatment (Con＋3AB) did not impair the PARP expression and activity clearly. High methionine diet treatment resulted in increased PARP activity and expression (p＜0.05 vs. Con, Fig.2B, C). Though 3-AB treatment did not effect the expression

Table 1. Plasma tHcy levels and amount of lipids

Group (n) Plasma tHcy (μmol/L) TC (mmol/L) TGs (mmol/L) HDL-C (mmol/L)

Con (15)Con＋3AB (15)HM (15)HM＋3AB (15)

9.0±1.058.23±0.83

48.44±8.20＊＊

43.57±5.33＊＊

13.05±2.6312.34±2.0912.92±3.0414.95±3.99

1.33±0.441.26±0.531.45±0.491.11±0.16

0.59±0.120.62±0.170.52±0.130.56±0.10

Values are expressed as the mean±SDtHcy, total homocysteine; TC, total cholesterol; TGs, triglycerides; HDL-C, high-density lipoprotein cholesterol; Con, normal stan-dard diet; Con＋3AB, normal standard diet plus 3-AB treatment; HM, high-methionine diet plus physiological saline treatment; HM＋3AB, high-methionine diet plus 3-AB treatment. ＊＊p＜0.01 vs. Con group.


of PARP-1 (p＞0.05 vs. HM, Fig.2A, C), it inhibited PARP activity (p＜0.05 vs. HM, Fig.2B) compared with the HM group. In addition, Fig.2A showed that caspase-3-mediated cleavage of the 116-kD polypep-tide PARP to its characteristic 89-kD fragment pre-vented futile DNA repair cycles and was considered a distinct marker of apoptosis. Its expression increased significantly in the HM＋3AB group.

Effect of 3-AB Treatment on Nuclear Translocation of NF-κB and the Expression of Inflammatory Factors

As a key regulator of AS, NF-κB regulates the expression of a variety of inflammatory factors, includ-ing MCP-1, TNF-α, VCAM-1 and ICAM-1, aggra-vating the chronic inflammatoty progress. We mea-sured the expression of NF-κB p65 in nuclear and

Fig.1. A. Atherosclerotic lesion in ApoE－/－ mice.

a→d, cross-sectional section stained with hemotoxylin-eosin (magnification ×100). e→h, cryostat section of aortic root stained with oil red O (magnification ×40). a, e: Con; b, f: Con＋3AB; c, g: HM; d, h: HM＋3AB.

B. B1: The percentage of atherosclerotic lesion area/lumina in aortic root. B2: Lesion distri-bution according to distance from appearance of first cusp.

Each bar represents the mean±SD (n =8−10 per group). Con, normal standard diet; Con＋3AB, normal standard diet plus 3-AB treatment; HM, high-methionine diet plus physiological saline treatment; HM＋3AB, high-methionine diet plus 3-AB treatment. ＊p＜0.05 vs. Con group, #p＜0.05 vs. HM group.

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cytoplasmic extracts of aortas independently. As shown in Fig.3, NF-κB nuclear translocation was determined by its expression in nuclear extracts. Although there

was no statistical significance between Con and Con＋3AB groups, 3-AB treatment decreased the expression of NF-κB p65 within nuclear extracts under hyperho-

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Fig.2. Oxidative stress-related PARP activation within atherosclerotic plaques of ApoE－/－ mice.

A. Protein levels of phospho-p47phox and PARP-1 were measured by Western blots. A (1): One representative result of Western blots. A (2): Densitometric measurement from Western blots. Each bar represents the mean±SD (n =3). B: PARP activity was measured as described in Methods. C. Levels of mRNA for PARP-1 were assayed by real-time quantitative PCR. Each bar represents the mean±SD (n =5 per group). Con, normal standard diet; Con＋3AB, normal standard diet plus 3-AB treatment; HM, high-methionine diet plus physi-ological saline treatment; HM＋3AB, high-methionine diet plus 3-AB treatment. ＊p＜0.05 vs. Con group, #p＜0.05 vs. HM group. ＋p＞0.05 vs. HM group.


mocysteinemic conditions (p＜0.05 vs. HM, Fig.3A, B). We also measured the phosphorylation of IκB in total proteins of aortas, and 3-AB treatment decreased the phosphorylation of IκB under hyperhomocysteinemic

conditions (p＜0.05 vs. HM, Fig.3C).Fig.4A showed that the expressions of VCAM-1

and MCP-1 were highly increased by high methio-nine diet treatment measured by immunohistochem-

Fig.3. Effect of 3AB on the nuclear translocation of NF-κB in atherosclerotic plaques.

A.B. the protein level of p65 was measured by Western blots in cytoplasmic extracts and nuclear extracts. A (1) B (1): One representative result of Western blots of p65. A (2) B (2): Densitometric measurement of p65 from Western blots. C. Protein levels of IκB and p-IκB were measured by Western blots in total protein. C (1): One representative result of Western blots of IκB and p-IκB. C (2): Densitometric mea-surement of IκB and p-IκB showed as p-IκB/ IκB. Each bar represents the mean±SD (n =3). Con, normal standard diet; Con＋3AB, normal standard diet plus 3-AB treatment; HM, high-methionine diet plus physiological saline treatment; HM＋3AB, high-methionine diet plus 3-AB treatment. CE, cytoplas-mic extracts; NE, nuclear extracts. ＊p＜0.05 vs. Con group, #p＜0.05 vs. HM group.

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Fig.4. Effect of 3AB on the expression of inflammatory factors.

A. Immunohistochemistry analysis with antibodies to VCAM-1 (a→d) and MCP-1 (e→h). a, e: Con; b, f: Con＋3AB; c, g: HM; d, h: HM＋3AB (magnification ×400). B. Levels of mRNA for VCAM-1 and MCP-1 were assayed by real-time quantitative PCR. Each bar represents the mean±SD (n =5 per group). C. The protein level of VCAM-1 and MCP-1 were measured by Western blots. C (1): Representa-tive result of Western blots of VCAM-1 and MCP-1. C (2): Densitometric measurement of VCAM-1 and MCP-1 from Western blots. Each bar represents the mean±SD (n =3). Con, normal standard diet; Con＋3AB, normal standard diet plus 3-AB treatment; HM, high-methionine diet plus physiological saline treat-ment; HM＋3AB, high-methionine diet plus 3-AB treatment. ＊p＜0.05 vs. Con group, #p＜0.05 vs. HM group.

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istry analysis. In addition, real-time reverse-transcrip-tion PCR and Western Blot analysis demonstrated increased VCAM-1 and MCP-1 in the HM group, which were significantly reduced by 3-AB treatment (p＜0.05 vs. HM, Fig.4B, C).

Effect of 3-AB Treatment on ApoptosisFig.5A showed that apoptotic cells were highly

increased by high methionine diet treatment, which

was detected by the Apoptosis Detection Kit (Chemi-con). Caspase-3 was an important apoptotic regulator within atherosclerotic plaques, and active forms of cas-pase-3 could yield ≈19-kD and ≈17-kD subunits. Depletion of the ≈35-kD procaspase-3 also showed the later apoptotic extent and appeared to correspond with the induction of caspase-3 activity26). Fig.5B showed significant depletion of naïve caspase-3 by 3-AB treatment under hyperhomocysteinemic condi-

Fig.5. Effect of 3AB on apoptosis within atherosclerotic plaques.

A. Apoptosis detection of aorta sections (magnification ×400). B. The protein level of caspase-3 was measured by Western blots. B (1): Representative result of Western blots of caspase-3. B (2): Densitometric measurement of caspase-3 from Western blots. Each bar represents the mean±SD (n =3). Con, normal standard diet; Con＋3AB, normal standard diet plus 3-AB treatment; HM, high-methionine diet plus physiological saline treatment; HM＋3AB, high-methionine diet plus 3-AB treatment. ＊p＜0.05 vs. Con group, #p＜0.05 vs. HM group.

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tions (p＜0.05 vs. HM).

Discussion

Hhcy is an independent risk factor for athero-sclerosis; studies using animal models of genetic- and diet-induced Hhcy have demonstrated a causal rela-tionship between Hhcy, endothelial dysfunction, and accelerated atherosclerosis27-30). In the present study, we used ApoE－/－ mice with methionine supplemen-tation to investigate the role of PARP in atheroscle-rotic development. We demonstrated that although a regular diet plus 3-AB treatment tended to interfere with plaque development, the results were not statisti-cally significant, whereas in Hhcy-induced atheroscle-rosis in ApoE－/－ mice, PARP inhibitor 3-AB 10 mg/ kg/2d treatment significantly reduced atherosclerotic lesion size by 40% and effectively inhibited the activa-tion of PARP, thereby reducing the levels of nuclear translocation of NF-κB and subsequent expression of inflammatory factors, without affecting plasma tHcy levels and lipid contents in vivo. The results indicated that PARP inhibition could attenuate the develop-ment of atherosclerosis under hyperhomocysteinemic conditions.

It was reported that PARP inhibition by TIQ-A interfered with plaque development and promoted plaque stability in high-fat diet-induced atherosclero-sis17), which seemed contradictory to our data. The reasons may include different inhibitors and doses, diet (with or without high-fat diet), and methionine supplementation-induced Hhcy.

Homocysteine is a thiol-containing amino acid with pro-oxidant activity. It is readily oxidized, and reactive oxygen species, such as superoxide anion (O2

－) and hydrogen peroxide (H2O2), are formed during auto-oxidation of homocysteine31, 32). The catalytic activity of the nuclear enzyme PARP, induced by sin-gle-strand DNA breaks, is a direct result of oxidant injury33). Upon encountering DNA strand breaks, PARP catalyzes the cleavage of NAD＋ into nicotin-amide and ADP-ribose and then uses the latter to syn-thesize polymers of ADP-ribose, covalently attached to nuclear proteins, including PARP itself. When DNA damage is mild, poly (ADP-ribosyl) ation facili-tates cell survival. When DNA damage is severe, PARP activation can induce cellular energetic distur-bances, leading to cell dysfunction or death33, 34). Stud-ies have reported that oxidant-mediated PARP activa-tion plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovas-cular diseases; meanwhile, it also demonstrated that the protective effect of PARP inhibition in preventing

vascular dysfunction was associated with oxidative stress in shock, diabetes, and aging35-37). In the present study, our results confirmed that PARP inhibition by 3-AB suppressed PARP activation accelerated by Hhcy; moreover, there was an important correlation between the inhibition of PARP activation and the improve-ment of endothelial dysfunction induced by Hhcy in our previous study38).

Hhcy-induced superoxide anion production pro-motes the activation of NF-κB in the early stages of atherosclerosis in the vascular wall via activation of IκB kinases39). Under resting conditions, NF-κB con-sists of a heterotrimer of p50, p65, and IκBα in the cytoplasm. The phosphorylation, ubiquitination, and degradation of IκBα and phosphorylation of p65 leads to the translocation of NF-κB to the nucleus where it binds to specific response elements in the DNA40), consequently regulating the expressions of a variety of NF-κB-regulated inflammatory mediators, such as MCP-1, VCAM-1, TNF-α and ICAM-1. Many reports have suggested that PARP-1 (classical zinc-finger-containing PARP, referred to as PARP for simplicity) is also involved in the regulation of gene expression at the transcriptional step41, 42). Recent reports have also shown that PARP-1 is required for specific NF-κB-dependent gene expression and acts as a coactivator for NF-κB in vitro15, 43). Accordingly, PARP can modulate the expressions of the inflamma-tory factors mentioned above through modulation of the activation of NF-κB. Oumouna-Benachour K et al.17) showed that in PARP－/－ macrophages, MCP-1 expression was severely inhibited because of defective NF-κB nuclear translocation in response to lipopoly-saccharide. In the present study, the nuclear translo-cation of NF-κB that represented the activation of NF-κB was determined by its expression in nuclear extracts by Western blot analysis. Our results indicated that PARP inhibition by 3-AB inhibited nuclear trans-location of NF-κB and subsequent production of inflammatory factors, such as VCAM-1 and MCP-1, under hyperhomocysteinemic conditions.

Recent reports have shown that 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 apoptosis17). NF-κB modulates the expression of genes involved in cell pro-liferation and programmed cell death, generally inhib-iting apoptosis induced by cytokines and chemothera-peutic agents26). As PARP-1 is an essential and novel transcription coactivator for NF-κB-dependent gene expression, its effective inhibition may also induce apoptosis. It was also reported that PARP regulated several genes of apoptotic regulators, including cas-


pase-1 and -344), whose activation was essential for homocysteine-induced apoptic cell death45). Caspase-3 is a crucial enzyme that shows a connection between the inhibition of NF-κB activation and the promo-tion of apoptosis. Caspase-3-mediated cleavage of 116-kD PARP to its characteristic 89-kD fragment prevents futile DNA repair cycles and is considered a distinct marker of apoptosis46). Although apoptotic cell death is a feature of both animal and human ath-erosclerotic lesions47-49), it is still important to note that given the complexity of atherosclerotic plaques, cell death of the various cell types may play different roles in the different stages of atherogenesis50), not only aggravating the development of atherosclerotic plaques, but also contributing to plaque regression. Our study demonstrated that Hhcy promoted apopto-sis within atherosclerotic lesions and PARP inhibition by 3-AB inhibited NF-κB activation in plaques, aggravating the caspase-3 activation, which made the cells prone to apoptosis.

In summary, reactive oxygen species and reactive nitrogen species produced by oxidative stress are potent activators of PARP as a result of their ability to damage DNA under hyperhomocysteinemic condi-tions51, 52), and pharmacological inhibition of PARP by 3-AB attenuates plaque development. Hyperlipid-emia combined with Hhcy is considered to be a risk factor in clinical patients with atherosclerosis. Our study confirms that PARP inhibitor 3-AB interferes with atherosclerotic progress influenced by multiple factors, indicating 3-AB may prove beneficial for the treatment of atherosclerosis.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (No. 30600234; 30871067) and National Basic Research Program of China (973 Program: 2007CB512000; 2007CB512005).

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