CLINICAL PERSPECTIVE

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(Circulation. 2009;119:2161-2169.)
© 2009 American Heart Association, Inc.

Hypertension

Vascular Smooth Muscle Cell–Selective Peroxisome Proliferator–Activated Receptor- Deletion Leads to Hypotension

Lin Chang, MD, PhD^*; Luis Villacorta, PhD^*; Jifeng Zhang, MS^*; Minerva T. Garcia-Barrio, PhD; Kun Yang, MS; Milton Hamblin, PhD; Steven E. Whitesall, BS; Louis G. D'Alecy, DMD, PhD; Y. Eugene Chen, MD, PhD

From the Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor (L.C., L.V., J.Z., K.Y., M.H., Y.E.C.); Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Ga (M.T.G.-B); and Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor (S.E.W., L.G.D.).

Correspondence to Dr Y. Eugene Chen, University of Michigan Medical Center, 1150 W Medical Center Dr, MSRB III 7301E, Ann Arbor, MI 48105. E-mail echenum{at}umich.edu

Received August 18, 2008; accepted February 17, 2009.

	Abstract

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Background— Peroxisome proliferator–activated receptor- (PPAR) agonists are commonly used to treat diabetes, although their PPAR-dependent effects transcend their role as insulin sensitizers. Thiazolidinediones lower blood pressure (BP) in diabetic patients, whereas results from conventional/tissue-specific PPAR experimental models suggest an important pleiotropic role for PPAR in BP control. Little evidence is available on the molecular mechanisms underlying the role of vascular smooth muscle cell–specific PPAR in basal vascular tone.

Methods and Results— We show that vascular smooth muscle cell–selective deletion of PPAR impairs vasoactivity with an overall reduction in BP. Aortic contraction in response to norepinephrine is reduced and vasorelaxation is enhanced in response to β-adrenergic receptor (β-AdR) agonists in vitro. Similarly, vascular smooth muscle cell–selective PPAR knockout mice display a biphasic response to norepinephrine in BP, reversible on administration of β-AdR blocker, and enhanced BP reduction on treatment with β-AdR agonists. Consistent with enhanced β2-AdR responsiveness, we found that the absence of PPAR in vascular smooth muscle cells increased β2-AdR expression, possibly leading to the hypotensive phenotype during the rest phase.

Conclusion— These data uncovered the β2-AdR as a novel target of PPAR transcriptional repression in vascular smooth muscle cells and indicate that PPAR regulation of β2-adrenergic signaling is important in the modulation of BP.

Key Words: diabetes mellitus • hypertension • lipids • obesity

	Introduction

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Management of type 2 diabetes involves controlling hyperglycemia to maintain blood glucose levels as similar as possible to those in normoglycemic individuals. Several therapeutic approaches are currently in use to achieve this goal.¹ At present, thiazolidinediones stand out among the plethora of therapeutic interventions aimed at lowering blood glucose levels. Thiazolidinediones are ligands for the peroxisome proliferator–activated receptor- (PPAR), a key transcription factor involved in lipid metabolism with a central role in the regulation of insulin sensitivity.²

Clinical Perspective p 2169

Compelling evidence directly relates PPAR to additional effects on the cardiovascular system.³ Growing data suggest that insulin resistance may be strongly linked to hypertension. For instance, hypertension is associated with substantial insulin resistance, even in patients without diabetes in whom thiazolidinediones have been found to decrease blood pressure (BP).⁴ This effect has been observed in type 2 diabetic patients with hypertension⁵ and in normotensive type 2 diabetic individuals.⁶ In addition, dominant-negative mutations in the human PPAR gene have been associated with severe hypertension at a very early age.⁷ Recent clinical data have shown that treatment of these patients with pioglitazone significantly improved endothelium-dependent dilation in response to bradykinin without affecting the response to sodium nitroprusside.⁸ Rosiglitazone therapy also improved the in vivo reendothelization capacity of endothelial progenitor cells from diabetic patients.⁹ However, although thiazolidinedione treatment lowered BP levels in diabetic patients and in murine models of diabetes and hypertension, a conventional PPAR knockout (PPAR KO) experimental model, in which embryonic lethality was rescued to produce global PPAR KO, surprisingly displayed a systemic hypotensive phenotype despite severe insulin resistance.¹⁰ To better understand the contribution of PPAR to BP regulation in the vasculature, tissue-specific knockout animals have been generated. In that regard, it is noteworthy that endothelium-specific PPAR KO mice did not show an apparent hypertensive phenotype unless otherwise induced by different challenges such as high-salt water or feeding of a high-fat diet.¹¹ Because vascular smooth muscle cells (VSMCs) are essential to vascular tone modulation, we examined the contribution of VSMC-specific PPAR function to regulation of vascular tone and BP. To this aim, we developed highly VSMC-selective PPAR KO (SMPG KO) mice using our SM22-Cre knock-in mice¹² crossed to PPAR^flox/flox.¹³ The results presented here indicate that VSMC-selective PPAR deficiency results in a hypotensive phenotype, apparently at odds with the thiazolidinedione agonistic phenotypes. We provide evidence of PPAR-dependent transcriptional repression of β2-adrenergic receptor (β2-AdR), uncovering a possible link between PPAR deficiency with increased vascular relaxation and reduced BP.

	Methods

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BP Measurements
BP was measured by radiotelemetry (PA-C20, Data Sciences International, St Paul, Minn). The left common carotid artery was cannulated with the implant catheter, and the implant body was secured in the abdominal cavity. Mean systolic and diastolic BPs were recorded after implantation of the devices. Mice were kept on a 12-hour light/dark cycle.

Statistics
Mean±SEM values were analyzed. Statistical comparisons between 2 groups were performed by Student’s t test and among 3 groups by 1-way ANOVA. Aorta ring contraction/relaxation curves were adjusted with nonlinear regression first and then were compared by 2-way ANOVA and Bonferroni posttests. Groups were considered significantly different at values of P<0.05.

Please see the detailed Methods in the online Data Supplement.

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.

	Results

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Generation of VSMC-Selective PPAR-Deletion Mice
We generated SMPG KO mice by crossing PPAR^flox/flox mice¹³ with the SM22-Cre knock-in mice that we previously developed.¹² Currently available VSMC-selective PPAR KO mice do not display a highly efficient Cre-mediated excision of loxP sites, and residual PPAR expression could still be functional in the aortic tissue.^14,15 We used our knock-in mice in which Cre expression is driven by the endogenous SM22 promoter to circumvent that limitation. For the current studies, we used SMPG KO and littermate control (LC) mice (see Methods online). Reverse-transcription polymerase chain reaction analysis of the wild-type PPAR allele (700 bp) and the deleted allele (300 bp) indicates that the functional PPAR transcript in SMPG KO mice was fully abolished in the VSMC layer of aortic tissue and only partially excised in the heart and stomach (Figure IA of the Data Supplement). Of significance, PPAR protein expression in the SMC layers from aorta was completely absent in SMPG KO mice as determined by Western blot analysis using a PPAR antibody (supplemental Figure IB).

VSMC-Selective PPAR Deletion Lowers BP Independently of Changes in Metabolic Index or Heart Function
To analyze the contribution of VSMC-specific PPAR function to the systemic regulation of BP, we determined baseline systolic and diastolic BPs of SMPG KO and LC mice by radiotelemetry (n=6). As depicted in Figure 1A and 1B, LC mice showed the typical circadian regulation of BP in mice, higher during nighttime and lower during the day time. SMPG KO mice displayed a similar pattern of circadian rhythmicity in BP, although these mice showed significantly lower baseline systolic and diastolic BPs during the lights-on period, reaching minimal levels around 6 PM (average BP from 2 to 6 PM: systolic BP: 96 27 mm Hg for SMPG KO versus 123±14 mm Hg for LC, P=0.03; diastolic BP: 84±15 mm Hg for SMPG KO versus 102±14 mm Hg for LC, P=0.025) and recovering to the levels of LC mice during the active, lights-off period (average BP from 8 PM to midnight: systolic BP: 140±17 mm Hg for SMPG KO versus 134±6 mm Hg for LC, P=0.19; diastolic BP: 118±22 mm Hg for SMPG KO versus 111±8 mm Hg for LC, P=0.21) (Figure 1A and 1B, dotted lines). That SMPG KO mice present lower BP was confirmed by measuring systolic BP in mice ranging from 1 to 6 months of age (Figure 1C). Measurements were taken between 2 and 6 PM using the tail-cuff method in both SMPG KO and LC mice (n=16). Consistent with the radiotelemetry data, the SMPG KO mice showed significantly lower BP compared with the LC mice during the less active period (103±11 mm Hg for LC versus 84±10 mm Hg for SMPG KO in the first month of age; 107±10 mm Hg for LC versus 90±10 mm Hg for SMPG KO in the second month; 110±7 mm Hg for LC versus 87±9.6 mm Hg for LC in the third month; and 106±8 mm Hg for LC versus 92±9 mm Hg for SMPG KO in 6-month-old mice; Figure 1C). In addition, SMPG KO female mice have a similar hypotensive phenotype (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 1. Hypotension in SMPG KO mice. Baseline systolic (A) and diastolic (B) BPs of SMPG KO and LC mice were measured by radiotelemetry method. Data are shown as mean±SEM; n=6 in each group. C, Systolic BP was measured in a monthly based interval using the tail-cuff method. SMPG KO () displayed lower BPs than LC (•) mice (n=16 mice in each group).

We then investigated whether changes in BP in SMPG KO mice correlated with changes in metabolic parameters, insulin sensitivity, and heart function. The results obtained for glucose tolerance and insulin tolerance tests (supplemental Figure IIA and IIB) in SMPG KO did not differ from LC mice. We additionally determined whether the reduction in BP was a result of changes in vasomotor activity, housing the experimental groups in metabolic cages to record O₂ consumption (supplemental Figure IIIA), CO₂ generation (supplemental Figure IIIB), and ambulatory activity (supplemental Figure IIIC). No significant differences among the experimental groups were observed during either the nocturnal or diurnal period. We further demonstrated that heart function was not significantly different between SMPG KO mice and LC mice hearts as determined by echocardiography analysis (supplemental Figure IV and Table I); neither did we observe any differences in heart rate among the different genotypes during the telemetry measurements (data not shown). Histological analysis of aortic, heart, and skeletal muscle tissue did not reveal any morphological changes between SMPG KO mice and LC mice (supplemental Figure V).

SMPG KO Mice Show Increased β2-Mediated Vasorelaxation in Aorta
To determine whether reduced BP was caused by impaired vasoactivity, we tested the response of aortic rings isolated from SMPG KO and LC mice to various vasoconstrictors. Administration of phenylephrine, an -AdR agonist, resulted in similar maximal contraction levels in aortic rings from SMPG KO and LC mice (Figure 2A). However, aortic rings from SMPG KO mice showed significantly lower maximum contraction in response to norepinephrine, a dual - and β-AdR agonist, compared with rings from LC mice (Figure 2B). Of interest, norepinephrine showed a biphasic response in SMPG KO mice. Whereas the lower dose-dependent contraction in response to norepinephrine (10^–9 to 10^–7 mol/L) was significantly reduced in SMPG KO mice, a higher dose (10^–6 mol/L) promoted a robust vasorelaxation in SMPG KO mice, an effect not observed in LC mice (Figure 2B and 2C(, line b). The differential contractile response in the SMPG KO animals indicated a selective enhanced sensitivity to specific β-AdR versus -AdR agonists (eg, norepinephrine versus phenylephrine). This observation prompted us to further explore the β-AdR–dependent vasoactivity of SMPG KO mice. Pretreatment of SMPG KO mice aortic rings with propanolol (Figure 2C, line a), a nonspecific β2-AdR antagonist, completely abolished SMPG KO mice aortic ring relaxation in response to norepinephrine (Figure 2C, line b). On the other hand, treatment of phenylephrine-preconstricted aortic rings with a selective β2-AdR agonist, terbutaline, induced greater relaxation in aortic rings from SMPG KO mice (Figure 2D, line b) compared with LC mice (Figure 2D, line a). In contrast, both the endothelium-dependent aortic relaxation response to acetylcholine (Figure 2E) and endothelium-independent vasorelaxation response to sodium nitroprusside (Figure 2F) were not significantly different in aorta rings of SMPG KO and LC mice, indicating that neither the endothelial response nor the endothelium-independent VSMC relaxation was impaired in the SMPG KO mice.

View larger version (26K): [in this window] [in a new window]   Figure 2. VSMC-selective PPAR deletion increased responses to AdR agonists in aorta. Phenylephrine (PE; A) and norepinephrine (NE; B) dose-dependent contraction curves of thoracic aorta rings from SMPG KO and LC mice. Results were shown as mean±SEM; n=12 in each group. *P<0.05, **P<0.01 vs LC, Bonferroni posttest. C, Representative tracing of NE-induced thoracic aorta response in SMPG KO mice. NE induced thoracic aorta relaxation (line b), and preincubation of aorta ring 10 minutes with 10–8 mol/L propanolol (line a) completely blocked the NE-induced relaxation effect. The dark circles indicate NE added into the incubation bath in a series of concentrations ranging from 10–11 to 10–6 mol/L (left to right). D, Representative thoracic aorta ring tracing in response to β2-AdR agonist. Terbutaline increased relaxation of the thoracic aorta ring in SMPG KO mice (line b) compared with LC mice (line a) originally preconstricted by 10–7 mol/L PE (indicated by an arrow). Dark circles indicate terbutaline added into the incubation bath in a series of concentrations ranging from 10–12 to 10–6 mol/L (left to right). E, VSMC-selective PPAR deletion did not affect thoracic aorta endothelium-dependent relaxation induced by acetylcholine (Ach) in aortic rings with intact endothelia or (F) endothelium-independent relaxation induced by sodium nitroprusside (SNP) in aortic rings with the endothelium removed. Results are shown as mean±SEM; n=12 in each group.

Hypotension in SMPG KO Mice Is Associated With Enhanced β-Adrenergic Response
We then examined whether there was a direct correlation in BP regulation on treatment with β2-AdR agonists or antagonists in vivo. Infusion of 10^–6 mol/L of norepinephrine at 2 µL/min caused a sustained increase in BP in LC mice (Figure 3A). However, norepinephrine infusion in SMPG KO mice caused an initial increase in BP, followed by a gradual decrease (Figure 3B), recapitulating the response of the aortic rings in vitro. Coinfusion with 10^–7 mol/L propranolol (2 µL/min) and norepinephrine further increased BP in LC mice (Figure 3C) and completely abrogated the biphasic effect of norepinephrine in SMPG KO mice (Figure 3D). Additional experiments using 10^–6 mol/L terbutaline (2 µL/min), a specific β2-AdR subtype agonist, failed to alter BP in LC mice (Figure 3E), whereas the SMPG KO mice displayed higher susceptibility to the BP-lowering effect of terbutaline (Figure 3F). These results confirmed that the β-AdR response was exacerbated in the SMPG KO mice in vivo.

View larger version (27K): [in this window] [in a new window]   Figure 3. VSMC-selective PPAR deletion caused enhanced sensitivity to β2-AdR agonists in vivo. All tracing BP data were collected as described in the supplemental Methods section and are shown as 1 representative mouse of 6 in each group. Agonists and/or antagonists were infused (arrow) at a constant rate of 2 µL/min via the jugular vein as the indicated time. A, Norepinephrine (NE) 10–5 mol/L infusion increased BP in LC mice. B, NE 10–5 mol/L infusion in SMPG KO mice caused an initial increase in BP, returning to baseline levels after 3 minutes of infusion. C, Coinfusion of 10–7 mol/L propranolol with 10–5 mol/L NE elevated BP in LC mice (D) but completely blocked the reduction in BP in SMPG KO mice. E, Terbutaline 10–6 mol/L infusion did not affect BP in LC mice (F) but lowered BP in SMPG KO mice.

The β2-AdR Is a Novel Target of PPAR Transcriptional Repression
The results above prompted us to hypothesize that PPAR depletion may directly affect β2-AdR expression. Western blot analysis of protein extracts prepared from aortic vessels of LC and SMPG KO mice demonstrated that β2-AdR protein levels were significantly increased in SMPG KO mice compared with LC mice (Figure 4A), suggesting that β2-AdR could be a novel target of PPAR-dependent transcriptional repression in vivo. These results were further confirmed by gain and loss of PPAR in vitro. Adenovirus-mediated expression of PPAR in VSMCs strongly inhibits β2-AdR protein levels. Conversely, shRNA-mediated PPAR knockdown leads to enhanced expression of the β2-AdR (Figure 4B and supplemental Figure VI), reinforcing the notion of a repressor effect of PPAR on the β2-AdR. The effect of activation of PPAR by thiazolidinediones was tested in SMPG KO mice compared with LC mice, with rosiglitazone added to the animal chow at the dosage of 10 mg · kg^–1 · d^–1. After 2 weeks of rosiglitazone, β2-AdR mRNA expression was determined in isolated aortic SMCs from SMPG KO and LC mice compared with those eating a normal diet. Figure 4C shows again that β2-AdR mRNA is significantly upregulated in SMPG KO mice compared with LC mice on a normal diet but increased after rosiglitazone treatment in both SMPG KO and LC mice. This was surprising but not totally unexpected because thiazolidinediones may depict off-target effects independently of PPAR.^16–18 No differences in -AdR expression were observed in SMPG KO mice with or without rosiglitazone treatment (supplemental Figure VII). Therefore, we tested the effects of the highly selective nonthiazolidinedione PPAR ligand, GW7845, in VSMCs in vitro. Ligand activation by GW7845 and overexpression of PPAR in VSMCs significantly reduce β2-AdR mRNA expression (Figure 4D), suggesting an active role of PPAR on β2-AdR repression. Furthermore, transient transfection with a mutated PPAR construct (PPARDBDmu), in which the DNA-binding domain is deleted, significantly abrogates the inhibitory effect of the GW7845 (1 µmol/L) on β2-AdR mRNA expression. In basal conditions in vitro, upregulation of PPAR in VSMCs causes inhibition of β2-AdR transcription, whereas shRNA-mediated downregulation of PPAR leads to increased β2-AdR mRNA in the vehicle-treated cells (supplemental Figure VI). On the other hand, rosiglitazone treatment (1 µmol/L) results in upregulation of β2-AdR independently of the levels of PPAR, again indicating the possibility of PPAR-independent effects (supplemental Figure VI). Taken together, these results strongly indicate that β2-AdR is a novel target of PPAR transcriptional repression.

View larger version (18K): [in this window] [in a new window]   Figure 4. Ligand-specific activation of PPAR represses β2-AdR expression. A, Western blot analysis of β2-AdR protein expression in aortic tissue homogenates from LC mice vs SMPG KO mice. B, Western blot analysis of β2-AdR protein expression of protein extracts from primary VSMCs by adenoviral expression of human PPAR and small interfering RNA against PPAR (sh PPAR). Adenovirus carrying green fluorescent protein (GFP) was used as control. C, LC and SMPG KO mice were treated with rosiglitazone diet (10 mg · kg–1 · d–1) vs control diet. After 2 weeks, RNA samples were collected from the medial aortic tissue as described in Methods, and β2-AdR mRNA expression was determined for each experimental group. β2-AdR mRNA expression increased in SMPG KO mice vs LC mice, and rosiglitazone enhanced β2-AdR mRNA expression in both experimental groups (n=6). D, Cells were transfected with a Flag-PPAR plasmid or with a PPAR DNA binding domain mutant (PPARDBDmu) and treated with 1 µmol/L of the nonthiazolidinedione PPAR ligand GW7845 overnight. pcDNA3.1 was used as transfection control. Both GW7845 treatment and PPAR expression reduced β2-AdR mRNA levels, but GW7845 did not reduce β2-AdR expression in PPARDBDmu expressing VSMCs. Data are shown as mean±SEM; n=6. DMSO indicates dimethyl sulfoxide.

Functional Analysis of a PPAR Response Element in the β2-AdR Promoter
Consistent with these observations, we identified a putative PPAR binding site (PPAR element) in the promoter of the β2-AdR located between nucleotides –1371 and –1382 upstream of the transcription start site. To determine the functionality of this putative PPAR element in the β2-AdR promoter, we first performed electrophoretic mobility shift analysis that confirmed the ability of PPAR to effectively bind to this putative PPAR element newly identified in the β2-AdR promoter (–1371 and –1382) (see Methods online and supplemental Figure VIII). Next, chromatin domains in the β2-AdR promoter were scanned for PPAR binding by chromatin immunoprecipitation analysis. As shown in Figure 5A, PPAR detectably binds to the site located between –1371 and –1382 bp upstream of the transcription start site (lanes 7 and 8). An isotypic IgG antibody was included as a negative immunoprecipitation control (lanes 5 and 6). The negative control for the chromatin immunoprecipitation assay detected no binding of PPAR to flanking regions located 15 kb upstream of the β2-AdR promoter, where another putative PPAR binding site was predicted by bioinformatics analysis (lanes 1 through 4). These data strongly suggest that in the context of chromatin, the PPAR site (PPRE) between nucleotides –1371 and –1382 appears to be functional, showing binding of PPAR to the β2-AdR promoter.

View larger version (25K): [in this window] [in a new window]   Figure 5. Functional analysis of the PPRE located in the β2-AdR promoter. A, PPAR binds to the putative PPAR element in the mouse β2-AdR promoter. Cells were transfected with pcDNA3.1 carrying Flag-tagged PPAR for 48 hours. Chromatin immunoprecipitation (ChIP) assay of the mouse β2-AdR promoter was performed with anti-Flag antibody. DNA from immunoprecipitated β2-AdR chromatin, and input was subjected to polymerase chain reaction analysis with 2 pairs of primers covering regions containing the putative PPRE and at –15 kb (control) in the mouse β2-AdR promoter. The experiments were repeated at least 3 times. B, Dual-luciferase reporter assays were performed, transfecting VSMCs with a β2-AdR wild-type promoter (β2-WT) vs a PPRE-mutated β2-AdR promoter (β2-mu) generated by site-directed mutagenesis. VSMCs were cotransfected with pcDNA3.1 and Flag-PPAR and treated with GW7845 overnight. β2-AdR promoter activity was reduced on GW7845 treatment but not in the β2-mu promoter. Expression of Flag-PPAR reduced β2-WT promoter activity but not that of β2-mu. Data are shown as mean±SEM; n=6. DMSO indicates dimethyl sulfoxide.

The transcriptional response of the β2-AdR promoter to PPAR was further characterized using a luciferase reporter in vitro. The specificity of the identified PPRE within the β2-AdR promoter was additionally determined by specific site-directed mutagenesis of this PPAR binding site (see Methods online). VSMCs were transiently transfected with a 1.4-kb promoter (β2-WT) or the corresponding site-directed mutated promoter on the identified PPRE (β2-Mu). Cells were cotransfected with equivalent amounts of the Flag-PPAR and then treated with 1 µmol/L GW7845. As shown in Figure 5B, ligand activation of PPAR and PPAR transient overexpression reduced β2-AdR promoter activity, whereas luciferase activity from the mutated promoter did not show responsiveness to GW7845 treatment or PPAR overexpression, indicating that PPAR activation repressed β2-AdR expression in vitro.

	Discussion

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Results from the present study provide important insight into the role of PPAR on vascular homeostasis by uncovering potential direct antagonistic transcriptional control of the β2-adrenergic signals leading to a hypotensive phenotype. We used our previously described SM22-Cre knock-in mice in which the expression of Cre recombinase is driven from the endogenous SM22 promoter to efficiently deplete PPAR expression in SMCs.¹² The rationale for this strategy is 2-fold: to avoid the use of conventional transgenesis of SM22-driven expression of Cre recombinase, which does not control the location of insertion into the mouse genome, and thus the recombinase expression is highly influenced by the surrounding sequences at the integration sites in the genome,¹⁹ and to ensure that the efficiency of Cre-mediated recombination is fully restricted to the endogenous transcriptional control of the SM22 promoter. In this way, we achieved full absence of PPAR expression in VSMCs, with only partial depletion in heart and stomach, concomitant with partial expression of Cre recombinase in these tissues.

In the present experimental model, PPAR deletion in VSMCs has no effect on insulin sensitivity and causes neither overt metabolic nor cardiac dysfunction in the conditions used here, although our results clearly indicate that lack of PPAR in VSMCs reduces systemic BP during the rest phase in these animals. Therefore, this BP-lowering effect could be a consequence of a direct role of VSMC-dependent PPAR function in the vasculature. Aortic relaxation was enhanced in response to β-adrenergic agents in SMPG KO mice; consequently, BP was significantly reduced. Infusion with the β-AdR antagonist propranolol increased BP mediated by norepinephrine in SMPG KO mice. With terbutaline, a β2-AdR–specific agonist, BP was reduced in SMPG KO mice, but no significant effect was observed in LC mice, indicating an enhanced sensitivity to β2-AdR agonists in SMPG KO mice. The lack of appreciable different effects of -adrenergic agents in terms of both aortic contraction and BP lowering indicates that the -adrenergic response is not contributing to the observed hypotension in SMPG KO mice. This is consistent with the mRNA levels of -AdRs not being affected in the SMPG KO animals.

These results provided insights into a novel PPAR-dependent physiological response leading to the reduction in BP in these mice. The biphasic effect observed in the SMPG KO mice in terms of aortic relaxation and BP indicates the possibility that these animals may have an enhanced β2-AdR response, which is known to mediate vasorelaxation without affecting that of the -AdR response.²⁰ This prompted us to evaluate whether this was the result of a specific PPAR-dependent transcriptional regulation of the components of the β-adrenergic signaling, and we uncovered β2-AdR as a novel target of PPAR subjected to transcriptional repression by this nuclear receptor in the vessels in SMCs in vivo. Increased levels of β2-AdR expression in SMPG KO mice render them further susceptible to β2-AdR agonist–mediated relaxation compared with control mice. These observations are consistent with a previously proposed model for the BP-lowering effects of PPAR KO mice. PPAR depletion and activation with thiazolidinediones may lead to the release of as-yet unidentified corepressors located on the promoter of genes involved in the control of the vascular tone.^10,16 In this report, we have identified the β2-AdR as such a target that could account, at least in part, for the enhanced hypotensive phenotype in PPAR KO mice. Whether the PPAR repressive effects result from recruitment of specific corepressors and the dynamics of the transcriptional events modulating β2-AdR expression remain to be determined. Furthermore, the observed increased expression of β2-AdR by rosiglitazone in a manner that is independent of PPAR uncovered an off-target effect of thiazolidinediones.

The occurrence of human mutations in the PPAR gene with dominant-negative function was first described by Barroso et al.⁷ Patients with such mutations develop insulin resistance, early-onset diabetes, and premature hypertension with the consequence of severe cardiovascular dysfunction.⁷ Endothelial dysfunction appears to be the primary cause of the development of an early hypertensive phenotype as experimentally determined through impaired acetylcholine-mediated vasorelaxation in animal models expressing such PPAR dominant-negative mutations in mice.^21–23 Moreover, thiazolidinedione treatment significantly decreases BP in experimental models of hypertension (eg, angiotensin II infusion, spontaneous and DOCA-salt hypertensive rats, transgenic mice overexpression of the renin-angiotensin-aldosterone system).^24–29 Specifically, however, lack of PPAR in endothelial cells exacerbates hypertension, a phenotype that could not be reversed on rosiglitazone treatment,¹¹ indicating that interference with PPAR function may primarily cause endothelial damage, a key step for the occurrence of a hypertensive phenotype. In our SMPG KO mice, endothelial function is preserved, as indicated by the fact that acetylcholine-mediated vasorelaxation is not altered in these mice compared with LC mice, which argues that the differential phenotype of hypotension in these animals depends primarily on VSMC-mediated vascular tone.

A recent report indicated that VSMC-specific PPAR KO mice obtained by crossing PPAR^flox/flox mice with SM22-Cre transgenic mice developed spontaneous pulmonary artery hypertension associated with right ventricular hypertrophy.¹⁴ Deregulation of a bone morphogenetic protein/PPAR/apolipoprotein E signaling axis is proposed as the mechanism behind pulmonary hypertension. Consistent with this, PPAR activation by rosiglitazone reduces pulmonary hypertension in apolipoprotein E KO mice in a high-fat diet and reduces pulmonary artery atherosclerosis in insulin-resistant animals. Yet, systemic BP in that experimental model was reported as unaffected.³⁰ Loss of PPAR expression also is observed in the lungs of patients with severe pulmonary hypertension.³¹ Recently, SM22-Cre-driven PPAR KO mice were reported to show loss of circadian rhythmicity in BP regulation with increased BP and heart rate during the light cycle.¹⁵ This animal model shows notable differences from ours on several levels. First, the mechanism of transgenesis is essentially different: Cre knock-in model versus transgenic SM22-Cre expression. It is well described that the expression of the endogenous SM22 gene is different when in its native genomic context and in transgenic models. Recently, Long and Miano³² summarized some molecular clues for the regulation of several cis-regulatory elements for the spatial and temporal control of gene expression, emphasizing SMC-specific markers, including SM22. These effects may translate into differential recombinase activity, leading to significant PPAR depletion in other tissues, including heart and kidney, with increased heart rate and evident sympathoadrenal dysfunction in those transgenic animals,¹⁵ all of which may contribute to the differences with our model, in which no altered heart function was observed. Further differences include a loss of BMAL1 circadian expression in SM22-Cre-driven PPAR KO mice, identifying this circadian gene as a target of PPAR in both endothelial cells and VSMCs.¹⁵ In our experimental model, SMPG KO mice displayed shifted circadian BMAL1 expression compared with the LC mice (supplemental Figure IX). BMAL1 KO mice displayed altered circadian rhythmicity of BP with an overall reduction of the mean arterial pressure,³³ which is in disagreement with the data showing that increased BP was observed in SM22-Cre-driven PPAR KO mice with impaired BMAL1 expression.¹⁵ BP regulation is a multifactorial trait with combinatorial contributions of different factors in response to changing metabolic conditions. These conflicting observations suggest that it is likely that neither BMAL1 nor the β2-AdR described here alone will be the sole contributing factor to the changes in BP regulation observed in these intrinsically different PPAR KO models. Thus, in our SMPG KO mice, loss of PPAR function in VSMCs did not affect the circadian periodicity of BP regulation, whereas enhanced β2-AdR expression contributed to the observed diurnal decrease of BP. Interestingly, a recent report did not show a loss of BP circadian rhythmicity in the β1-AdR and β2-AdR double-knockout mice despite clear effects on vascular tone.³⁴ Therefore, further studies aimed at dissecting the molecular mechanisms and signal transduction pathways linking PPAR with β-AdR, sympathoadrenal function, and BP are required.

The data presented here clearly indicate that lack of PPAR in VSMCs results in a significant reduction in systemic BP associated with increased expression of the β2-AdR concomitant with increased sensitization to β-adrenergic agonists.

	Acknowledgments

 
Sources of Funding

This work was partially funded by the National Institutes of Health (HL68878, HL75397, and HL89544 to Dr Chen). Dr Chang and J. Zhang were supported by an American Heart Association Midwest Affiliate Fellowship (0625705Z) and a National Career Development Grant (0835237N), respectively. Dr Chen is an established investigator of AHA (0840025N).

Disclosures

None.

	References

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CLINICAL PERSPECTIVE

Hypertension control is one of the major goals in the management of cardiovascular disease. The occurrence of hypertension in diabetic patients further aggravates their cardiovascular outcomes. In clinical practice, thiazolidinediones, agonists of peroxisome proliferator–activated receptor- (PPAR), stand out as therapeutic drugs for diabetes control. They act as insulin sensitizers but also improve hypertension in diabetic patients. Experimental models have been developed to elucidate the molecular mechanisms by which PPAR mediates these processes. Endothelium-, cardiac-, and smooth muscle tissue–specific gain- and loss-of-PPAR-function animal models display a considerable range of effects on various aspects of the cardiovascular pathophysiology such as pulmonary hypertension, cardiac remodeling, or high-fat diet–induced atherosclerosis. Here, we describe that vascular smooth muscle cell–selective PPAR deletion leads to systemic hypotension with a circadian component, and we specifically identify the β2-adrenergic receptor as a novel gene subjected to PPAR-dependent repression in the vasculature, which will undoubtedly open new paradigms in the regulation of blood pressure and vascular tone.

	Footnotes

 
*The first 3 authors contributed equally to this work.

The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.815803/DC1.

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Clinical Summaries
Circulation 2009 119: 2125-2126. [Extract] [Full Text]

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