Pioglitazone Attenuates Valvular Calcification Induced by Hypercholesterolemia

Abstract

Objective

Development of calcific aortic valve stenosis (CAVS) involves multiple signaling pathways, which may be modulated by peroxisome proliferator-activated receptor-gamma (PPARγ). This study tested the hypothesis that pioglitazone, a ligand for PPARγ, inhibits calcification of the aortic valve in hypercholesteremic mice.

Methods and Results

LDLr^-/-/ApoB^100/100 mice were fed a Western-type diet with or without pioglitazone (20 mg/kg/day) for 6 months. Pioglitazone attenuated lipid deposition and calcification in the aortic valve, but not aorta. In the aortic valve, pioglitazone reduced levels of active caspase-3 and TUNEL staining. Valve function (echocardiography) was significantly improved by pioglitazone. To determine whether changes in gene expression are associated with differential effects of pioglitazone on aortic valves vs. aorta, Reversa mice were fed Western diet with or without pioglitazone for 2 months. Several pro-calcific genes were increased by Western diet, and the increase was attenuated by pioglitazone, in aortic valve, but not aorta.

Conclusions

Pioglitazone attenuates lipid deposition, calcification, and apoptosis in aortic valves of hypercholesterolemic mice, improves aortic valve function, and exhibits preferential effects on aortic valves vs. aorta. We suggest that pioglitazone protects against CAVS, and pioglitazone or other PPARγ ligands may be useful for early intervention to prevent or slow stenosis of aortic valves.

INTRODUCTION

Calcification occurs in atherosclerotic lesions and in the aortic valve.¹ The presence of osteoblasts in atherosclerotic lesions and in calcific aortic valve stenosis (CAVS) implies that calcification is an active, regulated process,^2,3 as first proposed by Demer and colleagues.⁴ If calcification is active, from pro-osteogenic pathways, one might expect that development and progression of calcification could be inhibited.

Several experimental findings suggest that peroxisome proliferator-activated receptor-gamma (PPARγ) may protect against cardiovascular calcification. First, PPARγ in the vascular wall and several cell types protects against development of atherosclerosis.^5-8 Second, PPARγ impairs differentiation of progenitor cells into osteoblasts,⁹ and inhibition of PPARγ increases differentiation of embryonic stem cells to osteoblasts.¹⁰ Third, oxidative stress and inflammation appear to play an important role in vascular calcification and CAVS,^11-16 and PPARγ is anti-inflammatory.^17,18

Multiple signaling pathways appear to be important in the pathophysiology of vascular calcification and CAVS. PPARγ is an attractive intervention to inhibit cardiovascular calcification because, instead of targeting a single mechanism, PPARγ affects a cluster of genes,^19,20 and thus may protect against calcification at multiple levels.

Activation of PPARγ by thiazolidinedione (TZD) ligands is used commonly for treatment of patients with impaired glucose tolerance and type II diabetes.²¹ To minimize an effect on metabolism, we used a relatively low dose of pioglitazone at which no effect on plasma glucose or body weight was observed. The first goal of this study was to test the hypothesis that chronic administration of pioglitazone, a TZD, inhibits calcification of the aortic valve in hypercholesterolemic mice. A unique aspect of this study was to examine calcification in both the aortic valve and aorta, in which mechanisms and functional consequences may differ. The second goal was to examine molecular mechanisms by which pioglitazone may affect calcification in vivo. We examined mechanisms that mediate an osteogenic pathway,^20,22 and measured levels of active caspase-3 (as a reflection of a possible role of cell death in calcification²³). If pioglitazone is effective in slowing CAVS, the findings would imply that a TZD could potentially be clinically useful in slowing the development of CAVS.

METHODS

Animals

Female LDLr^-/-/apoB^100/100 (“LA”) mice were fed normal chow until 2 months of age, and then were fed normal chow, Western diet (Teklad #TD88137) (WD), or WD+pioglitazone (20 mg/ kg/day). At 8 months of age, echocardiograms were performed, plasma was obtained, and aortic valves and ascending aorta were harvested for histological/immunohistological studies (Figure 1A).

Figure 1.

A. Summary of experimental protocol. 1B. Effects of pioglitazone on calcification of aortic valve and aorta of LA mice. Calcification of the aortic valve and ascending aorta was measured as percent of area stained with Alizarin Red in the base of aortic valves and the ascending aorta. Pioglitazone attenuated calcification of the aortic valve, but not ascending aorta, produced by Western diet in LA mice. n=9-13. 1C. Effects of pioglitazone on lipid deposition in aortic valve and ascending aorta of LA mice. Lipid deposition was measured as percent of area stained with Oil Red O. Pioglitazone attenuated lipid deposition produced by Western diet in aortic valves, but not in the aorta. n=5-8. 1D. Effects of pioglitazone on collagen in aortic valve and ascending aorta of LA mice. Collagen was measured as percent of area stained with Masson's staining. Pioglitazone, nor Western diet, had an effect on collagen. n=6-8. Values are mean±SE, * =p <0.05 vs. chow, ** = p<0.05 vs. WD.

Another study was performed to examine earlier effects of pioglitazone on gene expression in aortic valves and aorta. We used Reversa mice, which are similar to LA, except with insertion of lox sites flanking the Mttp gene and the Cre gene under the control of the promoter of the interferon-inducible Mx-1 protein.²⁴ (Figure 1A).

Evaluation of aortic valve function

Aortic valve function was evaluated as described previously.^14-16 Briefly, mice were sedated with midazolam 0.15 mg SC and cradled in the left lateral recumbent position while a 30-MHz linear-array probe was applied horizontally to the chest. The imaging probe was coupled to an imager (Vevo 2100^R, VisualSonics, Toronto) generating 200 frames (2D) per second in both short- and long-axis left ventricular planes. Images of the aortic valve were acquired in M-mode, at a nominal sampling rate of 1000 frames per second, with 2D images used for guidance.

Plasma measurements

Total cholesterol was measured using the CHOD-DAOS method (Wako #439-17501). Plasma glucose was measured with AccuChek test strips (Roche). Plasma adiponectin was measured with ELISA (R&D). Serum amyloid A was measured in plasma using a mouse SAA ELISA kit (Invitrogen).

Histology, immunofluorescence, and immunohistochemistry

The aortic valve and aorta, embedded in OCT, were cut in 10 μm thick sections, and stained with Alizarin Red (Sigma) for calcium, Oil Red O (Sigma) for lipid, and Masson's trichrome or picrosirius red for collagen. The number of pixels expressing red (Alizarin Red, Oil Red O, or picrosirius red) or blue (Masson's) staining was quantified as described previously.^14-16 Data are expressed as percent of valve area that displays positive staining.

Immunofluorescence of osterix was performed as described previously^14,16. Immunohistochemistry of osteocalcin (rabbit antibody purchased from ABBIOTECH) and active caspase-3 (rabbit antibody from Cell Signaling) were performed as follows. Sections were fixed with phosphate-buffered paraformaldehyde (4%), and treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. After blockage with 10% BSA in TBS/0.3% Triton X-100, sections were incubated with a primary antibody, or normal rabbit IgG as negative control, at 4°C overnight. After rinses in PBS, sections were incubated with SignalStain Boost IHC Detection Reagent (HRP, Rabbit, Cell Signaling). After rinses in PBS, sections were incubated in DAB solution (Vector) for exactly 3-9 minutes. Slides were dehydrated, and mounted with coverslips. Images were digitally photographed, and brown staining was quantified as described previously.^14,16

Comparison of gene expression in aortas vs. aortic valves

Total RNA of aorta and aortic valves was isolated using TriZol extraction followed by Qiagen miRNeasy mini kit. Aortic valves were harvested from 80-μm-thick OCT-embedded sections under a stereoscope. Reverse transcription was performed as described previously.²⁵ qPCR for a gene of interest (FAM) with TaqMan primers ordered from Applied Biosystems (with a few ordered from IDT) was performed with the normalizer GAPDH (VIC) in the same well.

Statistical analyses

All data are reported as mean±SE. Significant differences (p<0.05) between groups were detected using one-way ANOVA followed by Student-Newman-Keuls tests.

RESULTS

Plasma levels

Total plasma cholesterol and non-fasting plasma glucose were not altered by pioglitazone (20 mg/kg/day) (Table 1). Plasma adiponectin was increased by pioglitazone, as expected. Plasma SAA, a systemic inflammation marker in mice,²⁶ was increased in mice fed Western diet, and pioglitazone attenuated the increase in SAA (Table 1).

Table 1.

Effects of pioglitazone (20 mg/kg/day) on plasma of LA mice after 6 months of treatment

	Chow	Western Diet	Western Diet +Pio
Cholesterol mg/dl	298±16	1233±67*	1167±37*
Non-fasting glucose mg/dl	247±7	254±11	264±10
Adiponectin mg/dl	119±2.6	109±4.0	154±2.8**
Serum Amyloid A μg/ml	8.0±0.03	13.2±0.9*	11.1±0.7**

Values are mean±SE, n= 9-13

^*p<0.05 vs chow

^**p<0.05 vs. Western diet

Calcification, lipid deposition, and fibrosis in aortic valves and aorta

There were small amounts of calcium in the aortic valve of chow-fed LA mice. Western diet significantly increased calcification of the aortic valve (Figure 1B). Pioglitazone attenuated the increase in calcification of the valve (Fig. 1B). In aorta, pioglitazone did not prevent the increase in calcium in LA mice fed Western diet (Fig. 1B) (see Supplement Fig. I for images of staining). Thus, pioglitazone attenuates calcification in aortic valves, but not in aorta.

Lipid deposition in the aortic valve was greater when LA mice were fed Western diet than chow (Fig. 1C). Pioglitazone attenuated the increase in lipid deposition produced by Western diet (Fig. 1C). (see Supplement Fig. II for images of staining). In the aorta, Western diet did not increase lipid deposition significantly, and pioglitazone did not have an effect (Fig. 1C).

Masson's staining indicated no difference in collagen among the three groups in both aortic valves and aortas (Fig. 1D), which suggests that fibrosis is not changed by pioglitazone (see Supplement Fig. III for images of staining). As also studied with picrosirius red staining, fibrosis in aortic valves was not reduced by pioglitazone (Supplement Fig. IV).

Protein expression in aortic valve and aorta

Expression in the aortic valve of osterix, a pro-calcific transcription factor, and osteocalcin, an osteoblast marker, was not increased by Western diet, and was not altered by pioglitazone (Fig. 2A). Active caspase-3, a marker for apoptosis, tended to increase in the aortic valve during Western diet, and pioglitazone prevented the increase (Fig. 2B). (see Supplement Fig. V for images of staining). In contrast, there was no difference in active caspase-3 among the three groups in the aorta (Fig. 2B). Double-staining confocal images suggest that active caspase 3-positive cells colocalized with several markers, and thus could be osteoblast-like cells (positive for Cbfa1), myofibroblasts (positive for α-smooth muscle actin), and macrophages (positive for F4/80) (Fig. 2C). TUNEL staining also indicated that pioglitazone attenuated apoptosis in aortic valves (see Supplement Fig. VI).

Figure 2.

A. Effects of pioglitazone on protein expression of osterix and osteocalcin in aortic valves of LA mice. Protein expression was measured as percent of area stained with antibody. Osterix and osteocalcin were not significantly different among the three groups. n=7-10. 2B. Effects of pioglitazone on protein expression of active caspase-3 in aortic valves and aorta of LA mice. Protein expression was measured as percent of area stained with antibody. Pioglitazone prevented the increase in expression of active caspase-3 produced by Western diet. n=7-10. Values are mean±SE, * = p<0.05 vs. chow; **=p<0.05 vs. WD. 2C. Colocalization (yellow) of active caspase-3 (red) and cell markers (green). Active caspase-3 is colocalized in aortic valves with myofibroblast (α-smooth muscle actin-positive), macrophage (F4/80-positive), and osteoblast-like cells (Cbfa1-positve).

Differences in gene expression in aortic valves vs. aorta

Rbp7 responded similarly in aortic valves and aorta to Western diet and pioglitazone, which suggests that pioglitazone activated PPARγ in both tissues (Fig. 3). Cbfa1 expression, reflecting osteoblastic transformation, increased markedly in response to Western diet in aortic valves but not in aorta. Pioglitazone strongly attenuated the Western diet-induced increase in Cbfa1 expression in aortic valves (Fig. 3). Expression of osteocalcin, an osteoblast-specific gene encoding a bone matrix protein, was significantly increased by the Western diet both in aortic valves and aorta. Pioglitazone eliminated the increase in osteocalcin expression associated with the Western diet (Fig. 3). Expression of the proinflammatory gene, IL-6, was significantly increased by the Western diet, and decreased by pioglitazone, only in aortic valves (Fig. 3). Among profibrotic genes, collagen 1a2 responded to Western diet and pioglitazone only in aortic valves (Fig. 3). Among the 36 genes examined, 18 genes demonstrated different expression patterns in aortic valves vs. aorta (Table 2).

Figure 3.

Effects of pioglitazone on mRNA expression in aortic valves and aorta of Reversa mice at 14 months of age. During the last 2 months, mice were fed WD or WD+Pio. The expression pattern of Rbp7 and osteocalcin was similar in aortic valves and the aorta, whereas IL-6, Cbfa1, and collagen 1a2 exhibited dissimilar patterns between aortic valves and the aorta. Values are mean±SE in 11-12 mice for aortic valves, and 8-9 for the aorta. * = p<0.05 vs. Reversed mice; ** = p<0.05 vs. WD. See Table 2 for more genes.

Aortic valve function

In LA mice fed a Western diet beginning at 2 months of age, treatment with pioglitazone for 6 months resulted in about 20% greater aortic cusp separation, a measure of aortic valve function that we have previously validated hemodynamically,¹⁴ compared to LA mice fed Western diet that did not receive pioglitazone (Figure 4). The difference corresponds nominally to ~44% greater systolic valve area in pioglitazone-treated mice.

Figure 4.

Effects of pioglitazone on separation distance of aortic valve cusps during systole of LA mice. Values were obtained in LA mice fed chow, Western diet (WD) without and with pioglitazone (pio) (20 mg/kg/day) for 6 months. Values are mean±SE in 5-6 mice per group. ** = p<0.05 vs. WD.

Pioglitazone did not affect heart rate, cardiac output, or left ventricular mass, but was associated with increased left ventricular end-systolic/diastolic volume and decreased left ventricular ejection fraction in LA mice that were fed a Western diet (Supplement Table II and Fig. VII).

Regarding LV dilation and reduced systolic function, the mid-systolic antero-posterior diameter of the aortic root at the level of the sinuses of Valsalva was 1.65 ± 0.04 mm in LA mice receiving Western diet alone, and 1.70 ± 0.06 mm in LA mice receiving Western diet + pioglitazone (p = NS). The diameters were measured using the same M-mode echo image sets as were used to determine aortic valve cusp separation (Fig. 4). Mid-systole and sinuses of Valsalva were chosen because they represent the time and anatomic location corresponding to maximal separation of aortic valve cusps. Thus, because there was no significant difference between Pio-treated and untreated LA mice, with respect to aortic root diameter, aortic root size does not explain protection of aortic valve function by pioglitazone. In addition, diameter of sinus of Valsalva was greater than aortic cusp separation in both groups, which argues against restraint of cusp excursion by the aortic root, at the stage of disease reported here.

DISCUSSION

The major findings in this study are that, in the aortic valve of hypercholesterolemic LDLr^-/-/apoB^100/100 mice that are susceptible to CAVS, pioglitazone 1) prevented deposition of lipids during Western diet, 2) attenuated apoptosis, and 3) attenuated calcification and impairment of cusp mobility in the aortic valve, but had no effect on fibrosis. The findings suggest that pioglitazone protects against valvular calcification, in part by inhibition of apoptosis. In the aorta, pioglitazone failed to reduce lipid deposition or calcification, despite positive effects on gene expression of Rbp7 (a PPARγ target), proinflammatory cytokines TNFα and IL-6, and BMP2 (a signaling molecule in a pro-osteogenic pathway) (Supplement Table I and Fig. VII).

Comparison of gene expression in the aortic valve vs. aorta indicates that many genes were expressed in a different pattern in the aortic valve vs. aorta, which supports the observation of different responses to Western diet and pioglitazone of the aortic valve and aorta.

Pioglitazone and lipid deposition

Early studies suggested that PPARγ might be pro-atherosclerotic, as oxidized linoleic acids in oxLDL activate PPARγ, which upregulates CD36, which mediates uptake of oxLDL.^27,28 The first in vivo study using rosiglitazone (a TZD) and GW7845 (a non-TZD activator of PPARγ) in Western diet-fed LDLr-deficient mice, however, indicated that activation of PPARγ is anti-atherosclerotic in male, but not female, mice despite upregulation of CD36.²⁹ Although the mechanisms for the sex difference are not known, PPARγ activation produced greater inhibition of TNFα and MMP9 in aortas of male than female mice, and PPARγ activation worsened the lipid profile in female mice.²⁹ Subsequent studies in vivo, using other PPARγ agonists in Western diet-fed LDLr- or apoE-deficient mice, confirmed that PPARγ activation protects against development of atherosclerosis, but does not reverse advanced atherosclerosis.^5,30

We studied female mice as a more stringent test for effects of pioglitazone on the aortic valve. Our findings in the aortic valve that pioglitazone attenuates calcification, lipid deposition, and apoptosis, and improves valve function, therefore, occurred even though one might expect smaller effects of TZDs on the valve, as well as in protection against atherosclerosis, in female mice.²⁹ We have not performed systematic studies to determine whether severity of CAVS differs in male and female mice.

Cholesterol efflux from macrophages and possibly other cells may be a better marker than high-density lipoprotein (HDL) levels in predicting severity of atherosclerosis.³¹ Pioglitazone attenuates lipid deposition by promoting cholesterol efflux, an effect that is not observed with a statin.³¹ Thus, we measured gene expression of molecules that mediate cholesterol efflux. In aorta of LA mice (Supplement Table 1), ABCA1, ABCG1, and apoE were upregulated by Western diet regardless of treatment of pioglitazone, whereas upregulation of apoAI by Western diet was inhibited by pioglitazone. The effects were similar, with variations, in aortic valve and aorta of Reversa mice (Table 2). The most pertinent change is that CD36 was greatly upregulated by pioglitazone in aortic valves of Reversa mice, and in aortas of LA and Reversa mice. In addition to mediating uptake of oxLDL, CD 36 may be involved in cholesterol efflux.³² Whether this great increase in CD36 expression by pioglitazone contributes to decreased lipid deposition in the aortic valve warrants further investigation.

Pioglitazone, apoptosis, and calcification

Cell death may contribute to initiation of vascular and valvular calcification.^33,34 Apoptosis is common in atherosclerotic arteries and stenotic aortic valves.^35,36 In this study, active caspase 3, a marker for apoptosis, was increased in aortic valves of LA mice receiving Western diet, and was reduced by pioglitazone to a level similar to that in chow-fed LA mice. TUNEL staining also was significantly decreased by pioglitazone. The finding that pioglitazone attenuated apoptosis is consistent with effects of pioglitazone on lipid deposition and calcification, as colocalization studies indicate that cells that express active caspase-3 may be macrophage (positive for F4/80), myofibroblast (positive for α-smooth muscle actin), or osteoblast-like cells (positive for Cbfa1). This pattern of apoptotic cell types suggests that apoptosis may result from a general injury, perhaps by the atherosclerotic environment, including oxLDL and inflammatory cytokines. Pioglitazone reduced apoptosis, perhaps through inhibition of inflammatory cytokines and reduction of intracellular lipid content. A recent study also suggests that activation of PPARγ inhibits shortening of telomeres, and thus inhibits apoptosis in endothelial cells in an injury model.³⁷

The importance of cell death as a mechanism for calcification in CAVS and atherosclerosis is not known. In a study of the time course of calcification in a rabbit balloon injury model, calcium deposits occurred within 2 days after injury, and osteopontin and osteocalcin were detected only after 8 to 14 days, whereas osteonectin was undetectable at all time points.³⁸ This finding suggests that apoptosis may be a primary cause of vascular calcification in that experimental model. Our finding that pioglitazone inhibited both apoptosis and calcium deposits suggests that apoptosis may be important in valvular calcification induced by hypercholesterolemia. It is of interest that pioglitazone increased macrophage apoptosis and plaque necrosis in advanced atherosclerotic lesions of LDLr^-/- mice.³⁹ The explanation for contrasting findings, that pioglitazone is pro- or anti-apoptotic, is not clear. However, the dose of pioglitazone differed (40 mg/kg/d in ref. 39 vs. 20 in our study), the age of mice differed (6 mos of age in ref. 39 vs. 8 in our study), and the strain of mice differed.

Pioglitazone and osteogenic calcification pathways

Our previous studies demonstrated that protein expression of osteogenic calcification molecules (P-Smad1/5/8, Msx2, Osterix, and Cbfa1) increased in Western diet-fed Reversa mice, and was reduced following reversal to normocholesterolemia by a genetic switch.¹⁴ We have found that treatment with osteoprotegerin reduced osterix and osteocalcin, but not Cbfa1 (Runx2) in LA mice.⁴⁰ The findings suggest that osteoprotegerin inhibits an osteogenic signaling step downstream to Cbfa1 and upstream to osterix.⁴¹

In the present study, osterix, a critical transcription factor for differentiation to osteoblast,⁴² and osteocalcin, a marker protein produced by osteoblast-like cells,⁴³ did not change with Western diet or pioglitazone in aortic valves or aorta. Thus, the findings suggest that reduction of calcification in aortic valves by pioglitazone in the present study may not be mainly through reduction of osteogenic signaling, as in our previous studies with reduction of cholesterol¹⁴ or administration of exogenous osteoprotegerin⁴⁰. We cannot conclude that pioglitazone does not affect osteogenic signaling, however, because pioglitazone reduced expression of an important osteogenic molecule, BMP2, in the aorta, even though reduction of calcification was not observed. Pioglitazone also may have indirect effects on osteogenic signaling, through reduction of inflammation and oxidative stress (TNFα, IL-6, SAA, adiponectin, Emr1, Nox2), as demonstrated in Table 2 and previously.^11,13

One explanation for our finding that pioglitazone did not alter expression of osterix and osteocalcin could be that osteogenic signaling molecules changed early in treatment with pioglitazone, but not after 6 months. Thus, we measured gene expression in aortic valves and the aorta after only 2 months of treatment with pioglitazone in Reversa mice. Indeed, expression in aortic valves of Cbfa1 (or Runx2), a transcription factor required for early differentiation to osteoblasts,⁴² was upregulated 9-fold by Western diet, and decreased significantly by pioglitazone. Cbfa1 expression, however, did not change in aorta. In contrast, expression of osteocalcin was increased by Western diet and reduced by pioglitazone in both aortic valves and the aorta. These findings clearly indicate that pioglitazone inhibits osteogenic molecules. Thus, our finding of reduction of calcification in aortic valves after 6 months of treatment with pioglitazone may have resulted, at least in part, from inhibition of osteogenic signaling by pioglitazone at an earlier time.

Comparison of gene expression revealed that, among the genes examined, ~50% have distinct expression patterns in aortic valves vs. aorta in the three groups of mice (Table 2). In general, aortic valves are more sensitive than aorta in responses to Western diet and pioglitazone. The changes in gene expression are consistent with structural changes (lipid deposition and calcification) in the younger LA mice. From these experiments with LA and Reversa mice, we conclude that pioglitazone attenuates calcification of aortic valves by reduction of both apoptosis and osteogenic signaling.

There are important limitations in our studies. Previous studies with porcine and human aortic valves demonstrated that gene expression patterns in endothelial cells are different on the aortic and ventricular sides.^43-45 The mouse aortic valve is too small to allow isolation of cells on the two sides. Thus, our findings in both aortic valve and aorta represent an average of all cells. Although we found a detectable difference in many genes, it is likely that there are even greater differences on a particular side and in some regions.

PPARγ as a therapeutic approach to CAVS

Diabetes and metabolic syndrome are risk factors for CAVS.^46,47 We chose a dose of pioglitazone that does not affect plasma glucose levels, to focus on effects of PPARγ and to minimize effects of changes in plasma glucose. We cannot, however, exclude the possibility that the dose of pioglitazone may have affected insulin resistance and plasma levels of insulin.

Studies in vitro and in vivo suggest that PPARγ modulates cardiovascular calcification by many mechanisms, including regulation of genes that modulate expression of osteoblasts,^9,20 or are antiinflammatory.^17,18 It is beyond the scope of this study to examine all of the mechanisms in depth, but we have examined several mechanisms by which pioglitazone may modulate cardiovascular calcification.

First, pioglitazone reduced expression of BMP2, and Cbfa1 and osteocalcin at an earlier time, which implies that pioglitazone inhibits calcification, at least in part, by an effect on an osteogenic pathway, which is similar to pathways in bone.⁹ Second, pioglitazone attenuated increases in serum amyloid A in plasma, and prevented increases in TNFα and IL-6 in aortic valves and the aorta. Thus, pioglitazone attenuated inflammation, which may have contributed to inhibition of calcification.

One of the goals of this study was to test the hypothesis that a novel therapeutic approach might prevent, or slow the progression of, CAVS. Since three clinical trials were published^48-50 and failed to show that a statin slows the progression of CAVS, there is skepticism that any non-surgical therapy will be beneficial in treatment of CAVS. We have shown that reduction of hypercholesterolemia in mice prevents the development of CAVS,¹⁴ but does not produce reversal of established CAVS.¹⁶

The finding that pioglitazone prevents calcification of the aortic valve could lead to a novel approach to therapy. Administration of pioglitazone to patients with impaired glucose tolerance²¹ delays the onset of diabetes. We speculate that a PPARγ ligand might also be useful in a high risk population, such as patients with bicuspid aortic valve⁵¹ or impaired glucose tolerance, in preventions of CAVS.

One potential concern in consideration of using a TZD for prevention of CAVS is that TZDs in animals and post-menopausal women reduce bone density.⁵² It is possible that new PPARγ agonists⁵³, which do not have side effects on bone, may be developed. Another concern is that, although PPARγ ligands inhibit development of atherosclerosis,^29,30 TZDs fail to slow progression of moderately severe atherosclerosis.^30,39 Thus, it is possible that, although pioglitazone may prevent development of CAVS, it may not slow progression after moderate CAVS has developed.

We found that left ventricular (LV) ejection fraction was decreased in mice treated with pioglitazone for 6 months, compared to mice that did not receive pioglitazone. This decrease was associated with increased LV end-systolic and end-diastolic volumes, with preserved stroke volume. The findings suggest that pioglitazone treatment is associated with development of dilated cardiomyopathy in mice. Studies of diabetic patients treated with TZDs (pioglitazone or rosiglitazone) have demonstrated increased risk of heart failure without concomitant increase in cardiovascular deaths ⁵⁴. Our finding that pioglitazone increased LV end-diastolic volume in mice is consistent with observations in patients treated with TZDs ⁵⁵. In humans, however, TZDs have no effect on LV ejection fraction ⁵⁶. Our finding that pioglitazone reduced LV ejection fraction in mice thus may be an idiosyncratic effect in mice or due to non-equivalency of doses in humans and mice.

Another consideration, in potential use of a TZD to prevent development of CAVS, relates to concerns about safety of TZDs. Rosiglitazone, a TZD, has been reported to paradoxically increase the risk of cardiovascular diseases.^57,58 PROactive studies,^59,60 however, suggest that pioglitazone does not increase the risk of myocardial infarction. The risk of cardiovascular events appears to be less with pioglitazone than rosiglitazone.⁶¹ Thus, we speculate that pioglitazone may be a better choice than rosiglitazone in testing the hypothesis that a PPARγ ligand protects against development of CAVS.

Table 2A.

Gene expression in aortic valves of Reversa mice after 2 months of WD±pioglitazone

	Chow	Western Diet	Western Diet +Pio
Plasma Cholesterol (mg/dl)	46±6	825±45*	701±45*,**
PPARγ-related
PPARγ	ND	ND	ND
Rbp7	1±0.65	1.22±0.22	4.5±1.3*,**
ABCA1	1±0.23	6.7±0.7*	6.0±0.6*
ABCG1	1±0.33	25.5±2.9*	18.0±3.8*
CD36	1±0.34	45±9*	72±15*,**
Caveolin-1	1±0.49	0.32±0.09	0.44±0.16
ApoAI	ND	ND	ND
ApoE	1±0.23	5.1±0.9*	3.7±0.8*
Inflammation
TNFα	1±0.34	1.31±0.26	1.80±0.36
IL-6	1±0.26	3.25±0.41*	1.64±0.49**
IL-10	ND	ND	ND
Calcification-related
BMP2	1±0.27	1.24±0.26	1.16±0.23
Cbfa1	1±0.24	9.1±1.5*	4.5±1.2**
Alkaline phosphatase	1±0.59	6.3±1.5*	5.5±2.0*
Osteopontin	1±0.28	79±11*	95±14*
Osteocalcin	1±0.35	5.4±1.4*	0.66±0.27**
Osteoprotegerin	1±0.29	5.32±0.87*	1.43±0.56**
RANK	1±0.36	2.79±0.30*	2.98±0.51*
RANK ligand	1±0.56	2.09±0.48	1.83±0.90
α-smooth muscle actin	1±0.23	2.56±0.39*	2.19±0.38*
Fibrosis-related
TGFβ1	1±0.23	1.41±0.11	1.28±0.18
CTGF	1±0.16	3.13±0.52*	3.33±0.52*
Collagen 1A2	1±0.23	5.65±0.91*	3.97±0.79*
Collagen3A1	1±0.25	3.14±0.46*	3.00±0.70*
Macrophage
Emr1 (F4/80)	1±0.26	2.29±0.28*	2.66±0.42*
CD68	1±0.54	5.6±0.8*	4.0±0.4*
Arginase 1	1±0.40	8.0±2.0*	3.1±1.2**
Mrc1	1±0.34	0.94±0.11	0.79±0.15
Oxidative stress
SOD1 (CuZnSOD)	1±0.24	0.72±0.08	1.00±0.18
SOD2 (MnSOD)	1±0.26	1.37±0.14	0.98±0.13
SOD3 (ECSOD)	1±0.12	0.82±0.08	0.68±0.07*
Catalase	1±0.16	2.11±0.18*	1.42±0.18**
Nox2	1±0.23	4.1±0.6*	3.1±0.5*
Nox4	1±0.23	1.64±0.10	1.32±0.29
p22phox	1±0.17	3.97±0.45*	3.04±0.29*,**
NOS3 (eNOS)	1±0.21	1.60±0.29	1.12±0.31

Values are mean±SE, n= 11-12, ND: not detectable

^*p<0.05 vs chow

^**p<0.05 vs. Western diet.

p>0.05 by ANOVA in the groups without asterisk. Genes in bold face have different expression patterns in aortic valve vs. aorta.

Table 2B.

Gene expression in the aorta of Reversa mice after 2 months of WD±pioglitazone

	Chow	Western Diet	Western Diet +Pio
Plasma Cholesterol (mg/dl)	46±6	825±45*	701±45*,**
PPARγ-related
PPARγ	1±0.26	0.59±0.21	0.63±0.20
Rbp7	1±0.26	0.70±0.30	5.5±3.0*,**
ABCA1	1±0.10	4.0±1.1*	5.4±1.3*
ABCG1	1±0.24	9.3±3.4	26.3±6.9*,**
CD36	1±0.28	1.61±0.77	9.1±3.5*,**
Caveolin-1	1±0.16	0.70±0.16	0.79±0.13
ApoAI	1±0.33	1.73±0.51	1.33±0.51
ApoE	1±0.10	4.0±1.1	8.9±2.3*,**
Inflammation
TNFα	1±0.28	1.62±0.50	2.08±0.56
IL-6	1±0.29	7.5±2.4*	6.1±1.5*
IL-10	1±0.17	1.07±0.44	1.01±0.27
Calcification-related
BMP2	1±0.08	0.85±0.14	0.84±0.09
Cbfa1	1±0.21	1.66±0.46	1.44±0.38
Alkaline phosphatase	1±0.21	0.75±0.16	0.60±0.11
Osteopontin	1±0.35	23.5±9.8*	30.5±8.5*
Osteocalcin	1±0.17	1.79±0.44*	0.72±0.09
Osteoprotegerin	1±0.25	2.41±0.79	2.26±0.46
RANK	1±0.03	0.93±0.12	0.84±0.08
RANK ligand	1±0.20	1.04±0.34	0.83±0.28
α-smooth muscle actin	1±0.12	0.69±0.14	0.53±0.07*
Fibrosis-related
TGFβ1	1±0.06	1.55±0.20*	1.67±0.17*
CTGF	1±0.11	1.16±0.12	0.77±0.12*
Collagen 1A2	1±0.08	0.96±0.14	0.83±0.11
Collagen3A1	1±0.09	1.18±0.13	1.21±0.19
Macrophage
Emr1 (F4/80)	1±0.19	1.66±0.57	1.38±0.31
CD68	1±0.23	6.4±2.2*	10.3±2.7*
Arginase 1	1±0.20	1.63±0.43	4.16±2.41
Mrc1	1±0.07	1.07±0.16	1.20±0.16
Oxidative stress
SOD1 (CuZnSOD)	1±0.06	1.21±0.23	1.39±0.25
SOD2 (MnSOD)	1±0.09	1.09±0.22	1.14±0.09
SOD3 (ECSOD)	1±0.12	0.72±0.12	0.83±0.07
Catalase	1±0.07	0.94±0.12	1.19±0.12
Nox2	1±0.19	3.3±1.0*	3.3±0.7*
Nox4	1±0.06	1.32±0.16*	0.85±0.08**
p22phox	1±0.04	2.92±0.81*	3.87±0.75*
NOS3 (eNOS)	1±0.16	0.93±0.11	0.85±0.05

Values are mean±SE, n= 8-9

^*p<0.05 vs chow

^**p<0.05 vs. Western diet.

p>0.05 by ANOVA in the groups without asterisk. Genes in bold face have different expression patterns in aorta vs. aortic valve.