Pioglitazone-Induced Reductions in Atherosclerosis Occur via Smooth Muscle Cell–Specific Interaction With PPARγNovelty and Significance
Rationale: Peroxisome proliferator-activated receptor (PPAR)γ agonists attenuate atherosclerosis and abdominal aortic aneurysms (AAAs). PPARγ, a nuclear receptor, is expressed on many cell types including smooth muscle cells (SMCs).
Objective: To determine whether a PPARγ agonist reduces angiotensin II (Ang II)–induced atherosclerosis and AAAs via interaction with SMC-specific PPARγ.
Methods and Results: Low-density lipoprotein receptor (LDLR)−/− mice with SMC-specific PPARγ deficiency were developed using PPARγ floxed (PPARγf/f) and SM22 Cre+ mice. PPARγf/f littermates were generated that did not express Cre (Cre0/0) or were hemizygous for Cre (Cre+/0). To assess the contribution of SMC-specific PPARγ in ligand-mediated attenuation of Ang II–induced atherosclerosis and AAAs, both male and female Cre0/0 and Cre+/0 mice were fed a fat-enriched diet with or without the PPARγ agonist pioglitazone (Pio) (20 mg/kg per day) for 5 weeks. After 1 week of feeding modified diets, mice were infused with Ang II (1000 ng/kg per minute) for 4 weeks. SMC-specific PPARγ deficiency or Pio administration had no effect on plasma cholesterol concentrations. Pio administration attenuated Ang II–increased systolic blood pressure equivalently in both Cre0/0 and Cre+/0 groups. SMC-specific PPARγ deficiency increased atherosclerosis in male mice. Pio administration reduced atherosclerosis in only the Cre0/0 mice, but not in mice with SMC-specific PPARγ deficiency. SMC-specific PPARγ deficiency or Pio administration had no effect on Ang II–induced AAA development. Pio also did not attenuate Ang II–induced monocyte chemoattractant protein-1 production in PPARγ-deficient SMCs.
Conclusions: Pio attenuates Ang II–induced atherosclerosis via the interaction with SMC-specific PPARγ, but has no effect on the development of AAAs.
Thiazolidinediones (TZDs), including rosiglitazone and pioglitazone (Pio), are used widely to improve insulin sensitivity in patients with type 2 diabetes. Experimentally, TZDs reduce atherosclerosis in both low-density lipoprotein receptor (LDLR)−/− and apolipoprotein (Apo)E−/− mice.1,2 Recent studies have demonstrated that TZDs also reduce Ang II–induced abdominal aortic aneurysm (AAA) development in ApoE−/− mice.3,4 The molecular target for TZD is PPARγ, a nuclear receptor that is highly expressed in all cell types involved in vascular pathologies, including macrophages, endothelial cells, and smooth muscle cells (SMCs).5 Currently, it has not been defined whether the beneficial effects of TZDs are attributable to PPARγ agonism in a specific cell type. Furthermore, it has been suggested that TZDs may exert some of their biological effects through PPARγ-independent mechanisms, although this has not been defined in vascular pathologies.6
TZDs have been demonstrated to regulate important SMC functions, including proliferation and migration.7 SMC-specific genetic manipulations have resulted in changes in both atherosclerosis8,9 and AAAs in mice.9 Furthermore, SMC-specific gene deletion of PPARγ results in changes in blood pressure and injury-induced vascular hyperplasia.10,11 However, no studies have currently determined whether the benefits of TZDs on vascular pathologies are mediated via a SMC-specific PPARγ-dependent mechanism.
To elucidate a role of SMC-specific PPARγ expression on TZD-induced reductions in atherosclerosis and AAAs, we bred female LDLR−/− mice harboring PPARγ floxed genes to similarly genetically manipulated males that were hemizygous for Cre regulated by the SM22 promoter. This breeding strategy generated littermate controls that were either wild-type or SMC-specific deficient in PPARγ. Using these mice, we determined the contribution of PPARγ expression in SMCs to the effects of Pio on Ang II–induced atherosclerosis and AAAs.12 The results demonstrate that SMC-PPARγ deficiency resulted in increased Ang II–induced atherosclerosis. Furthermore, these data demonstrate that PPARγ expression in SMCs is a major contributor to Pio-induced reduction in atherosclerosis. Contrary to previous studies, we did not discern an effect of Pio on Ang II–induced AAAs.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
Generation of LDLR−/− Mice With SMC-Specific PPARγ Deficiency
To verify the genotype of mice, aortas were dissected free, adventitia and endothelium were removed, and DNA was isolated from SMC-containing media. PCR analyses were performed on DNA isolated from the arch, thorax, suprarenal, and infrarenal aortic regions to determine the uniformity of Cre-based exon excision. These analyses demonstrated the presence of nonfunctional alleles (240-bp amplicon) throughout aortas of Cre-expressing mice. In contrast, aortas from nontransgenic littermates generated 215-bp amplicons derived from intact floxed genes (Figure 1A).
RT-PCR analyses showed complete deletion of PPARγ mRNA in SMC aortic medias of Cre+/0 mice (Figure 1B), indicating that functional PPARγ transcripts were ablated. Western blot analyses demonstrated that PPARγ protein was ablated in aortic SMCs from Cre+/0 mice, while not influencing abundance in liver, kidney, and adipose tissue (Figure 1C and Online Figure II).
SMC-Specific Deficiency of PPARγ Augmented Ang II–Induced Atherosclerosis Without Affecting AAAs
SMC-specific PPARγ deficiency in LDL receptor−/− mice resulted in significant (P<0.05) increases in Ang II–induced atherosclerotic lesion areas in male mice, but had no effect in females (Figure 2, A and B). SMC-specific deletion of PPARγ had no effect on body weight, plasma total cholesterol concentrations (Online Table I), or lipoprotein-cholesterol distributions (data not shown). Ang II infusion significantly increased systolic blood pressure (SBP) in male mice of both groups (Online Table I). SMC-specific PPARγ deficiency had no effect on Ang II–induced AAA formation (Figure 2C) or aortic rupture (Cre0/0, 25%; versus Cre+/0, 28%) in either sex.
Pio Attenuated Ang II–Induced Atherosclerosis Only in the Presence of PPARγ in SMCs
In Ang II–infused mice fed a fat-enriched diet, PPARγ mRNA abundance was not significantly increased in peritoneal macrophages (Online Figure III). Pio administration to these mice induced PPARγ mRNA abundance and activity in selected cell types and tissues, including macrophages, liver, kidney, and adipose. These inductions did not differ between Cre0/0 and Cre+/0 mice (Figure 3A and 3B; Online Figure IV). Increased PPARγ activity was demonstrable by increased mRNA abundance of PPARγ target genes: AP2 and CD36 in macrophages (Online Figure V) and selected tissues (Online Figure VI).
Pio administration profoundly reduced atherosclerosis only in Cre0/0 mice, but not in mice with SMC-specific PPARγ deficiency (Figure 3C and 3D). In contrast, Pio administration significantly attenuated Ang II–increased SBP equivalently in both Cre0/0 and Cre+/0 groups (Online Table II). Pio administration had no effect on body weight, plasma total cholesterol concentrations (Online Table II), or lipoprotein cholesterol distributions (data not shown). AAA formation (Figure 3E) or aortic rupture (Cre0/0, 11%; versus Cre+/0, 20%) was not different between groups.
Immunostaining of atherosclerotic lesions with α-actin demonstrated uniform reactivity throughout the medial intralaminar spaces of all groups, but minimal SMC immunostaining was detected in atherosclerotic lesions from any group. Although PPARγ deficiency increased lesion size, immunostaining for macrophages was dominant in atherosclerosis from both Cre0/0 or Cre+/0 mice.
Ang II Augmented Monocyte Chemoattractant Protein-1 Production in PPARγ-Deficient SMCs
To define potential mechanisms of Pio reducing atherosclerosis, plasma monocyte chemoattractant protein (MCP)-1 concentrations were measured. No significant difference was observed among groups demonstrating no systemic effect on MCP-1 (Online Figure VII).
Aortic SMCs cultured from either Cre0/0 or Cre+/0 mice were incubated with Pio (20 μmol/L) for 24 hours, and with or without Ang II (1 μmol/L) for a further 18 hours. Ang II significantly increased MCP-1 concentrations from Cre+/0 SMCs but had no significant effect on Cre0/0 SMCs (Figure 4). Coincubation with Pio had no effect on Ang II–induced MCP-1 production in Cre+/0 SMCs.
Consistent with SMCs harvested from Cre0/0 and Cre+/0 mice, Ang II increased MCP-1 concentrations in media of SMCs cultured from mice expressing a dominant-negative mutation of PPARγ P465L (PPARγL+)13 but not in cells isolated from nontransgenic littermates. To determine whether PPARγ has a dominant effect on MCP-1 secretion, SMCs were incubated with interferon (IFN)γ. In contrast to Ang II, IFNγ (300 U/mL) significantly increased MCP-1 concentrations in media of SMCs from both strains (Figure 4B). Pio had no effect on IFNγ-induced MCP-1 (Figure 4C).
To confirm that the effects of Pio on MCP-1 were attributable to interactions with PPARγ, Cre0/0 and Cre+/0 SMCs were incubated with Pio and Ang II as described above. The absence of PPARγ in SMCs significantly lowered AP2 mRNA abundance but failed to affect CD36 (Online Figure VIII). Ang II incubation significantly reduced mRNA abundance of both these target genes in Cre+/0 SMCs. Coincubation of Ang II and Pio significantly attenuated the reduced mRNA abundance of target genes in SMCs from Cre0/0 but not Cre+/0 mice.
In the present study, we demonstrate that SMC-specific PPARγ deficiency augments Ang II–induced atherosclerosis in male LDLR−/− mice. Interestingly, Pio administration attenuates Ang II–induced atherosclerosis only in wild-type mice, but not in SMC-specific PPARγ-deficient mice, which characterizes SMC-specific PPARγ as the key molecular target for the ligand-mediated attenuation of atherosclerosis.
SMC-specific PPARγ deficiency augmented Ang II–induced atherosclerosis only in male mice. This is in agreement with the study by Li et al, in which the attenuation of atherosclerosis by a PPARγ ligand was observed only in male LDLR−/− mice.1 The basis for these sex differences have not been defined.
Pio administration activates PPARγ in both Cre0/0 and Cre+/0 genotypes, which was evidenced by increased PPARγ expression observed in peritoneal macrophages and other tissues. Previous in vitro studies demonstrated that TZDs inhibited SMC proliferation and induced apoptosis through PPARγ-dependent mechanisms.14 In the present study, Pio administration attenuates Ang II–induced atherosclerosis only in Cre0/0 mice, but not in mice with SMC-specific PPARγ deficiency. Considering that SMC proliferation constitutes an important cellular mechanism for atherosclerosis initiation,15 our findings not only demonstrated SMC-specific PPARγ as an endogenous inhibitor of atherosclerosis but also established that TZDs exert antiatherosclerotic effects through this pathway.
Pio administration significantly suppresses Ang II–induced SBP in both genotypes. This result indicates that Pio-mediated SBP-lowering effect is independent of SMC-specific PPARγ. In support of this observation, a recently published study using both SM22-Cre+ and Tie2-Cre+ PPARγ flox mice showed that TZD-mediated the SBP-lowering effects via PPARγ expressed in endothelium.16 Because endothelial PPARγ is intact, Pio administration attenuates Ang II–induced SBP in both Cre0/0 and Cre+/0 groups in our study.
SMC-specific PPARγ deficiency or Pio administration did not influence aneurysm formation in LDLR−/− mice, which is contrary to a recent publication in which Pio reduced suprarenal aortic expansion in Ang II–infused ApoE−/− mice.4 The differences may be attributable to the lower dose used in the present study.4 Our dietary delivery was estimated to be ≈20 mg/kg per day, whereas the drinking water delivery in the study by Golledge et al4 was estimated to be 50 mg/kg per day. In another study, rosiglitazone attenuated Ang II–induced AAA formation in ApoE−/− mice, which was mainly associated with decreased expression of inflammatory mediators.3 The basis for the inconsistent effects of TZDs on Ang II–induced AAAs is unclear.
To further understand the mechanism by which Pio mediates its effect via SMC-PPARγ on atherosclerosis, we examined the effect of Ang II on MCP-1 production in cultured Cre+ and PPARγL+ SMCs. Interestingly, Ang II activates MCP-1 production only in Cre+/0 and PPARγL+ SMCs, but not in control SMCs, suggesting that endogenous SMC-PPARγ regulates Ang II–induced MCP-1 production. In addition, Pio had no effect on Ang II–induced MCP-1 production in Cre+/0 SMCs, which is consistent with this TZD requiring interaction with PPARγ to reduce Ang II–induced atherosclerosis. The specificity of this pathway was demonstrated by the continued induction of MCP-1 secretion in PPARγL+ cells during IFNγ incubation that signals via CD74 pathway in SMCs.17 This SMC-PPARγ–dependent effect of Ang II is localized to SMCs, which is not reflected by plasma concentrations of MCP-1.
In summary, this study provides evidence that lack of PPARγ in vascular SMCs results in significant increases in atherosclerosis associated with increased MCP-1 production. Furthermore, the study reveals that SMC-specific PPARγ expression is a novel mediator of ligand-mediated attenuation of atherosclerosis.
Sources of Funding
This work was supported by National Heart, Lung, and Blood Institute grants HL80010 (to J.G.) and HL80100 (to A.D.) and American Heart Association Great Rivers Affiliate Postdoctoral Fellowship 0825592D (to V.S.).
We acknowledge Deborah Howatt, Jessica Moorleghen, Debra Rateri, and Anju Balakrishnan for technical assistance; Richard Charnigo Jr for assistance with statistics; and Takeda Pharmaceuticals for providing pioglitazone. We thank Manikandan Panchatcharam, Susan Smyth, and Nobuyo Maeda (University of North Carolina, Chapel Hill) for providing PPARγL+ cells.
In July 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.9 days.
This manuscript was sent to Peter Libby, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Non-standard Abbreviations and Acronyms
- abdominal aortic aneurysm
- Ang II
- angiotensin II
- apolipoprotein E
- low-density lipoprotein receptor
- monocyte chemoattractant protein
- peroxisome proliferator-activated receptor
- systolic blood pressure
- smooth muscle cell
- Received February 23, 2010.
- Revision received August 6, 2010.
- Accepted August 16, 2010.
- © 2010 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Peroxisome proliferator-activated receptor (PPAR)γ, a nuclear receptor, is a target of therapeutic interventions to augment insulin sensitivity.
PPARγ expression in macrophages moderates the development of experimental atherosclerosis.
Activation of PPARγ by thiazolidinediones (TZDs) suppresses smooth muscle cell (SMC) proliferation.
TZDs, PPARγ agonists, attenuate atherosclerosis in male mice.
What New Information Does This Article Contribute?
Pioglitazone-induced attenuation of atherosclerosis depends on PPARγ in SMC.
Selective deficiency of PPARγ in SMC augments Ang II–aggravated atherosclerosis.
PPARγ is a nuclear receptor that is highly expressed in many of cell types involved in vascular pathologies, including macrophages, endothelial cells, and SMCs. The TZDs (agonists of PPARγ) have been shown to inhibit the development of atherosclerosis in male animals. Currently, it is unclear whether the beneficial effects of TZDs could be attributed to PPARγ agonism in a specific cell type. In vitro, TZDs inhibit SMC proliferation and migration, the key events that promote intimal hyperplasia during atherogenesis; however, the contribution of SMC PPARγ to the antiatherogenic effects of TZD has not been assessed. Because TZDs regulate SMC proliferation, which is a key step in the development of atherosclerosis, we hypothesized that SMC-specific PPARγ is responsible for the beneficial effects of TZD on atherosclerosis. By generating SMC-specific PPARγ-deficient mice, we show that SMC-specific PPARγ plays a critical role in the development of Ang II–induced atherosclerosis. We demonstrate that PPARγ expression in SMCs is required for the reduction in Ang II–induced atherosclerosis by pioglitazone. This is the first study to report that pioglitazone exerts its beneficial effect on atherosclerosis via a SMC-specific PPARγ-dependent mechanism.