Estrogen improves vascular function via peroxisome-proliferator-activated-receptor-γ
Vedat Tiyerili, Cornelius F.H. Müller, Stephen Fung, Darius Panek, Georg Nickenig, Ulrich M. Becher ⁎
Medizinische Klinik und Poliklinik II, Innere Medizin, Universitätsklinikum Bonn, Sigmund Freud Str. 25, 53105 Bonn, Germany
a r t i c l e i n f o a b s t r a c t
Article history:
Received 24 March 2012
Received in revised form 4 May 2012 Accepted 12 May 2012
Available online 24 May 2012 Keywords:
Sex hormones Women Vasculature Inflammation Atherosclerosis
The exact mechanism of estrogen in cardiovascular disease is not fully understood. As estrogen receptors (ERs), the peroxisome-proliferator-activated-receptor-γ (PPARγ) belongs to the family of ligand activated nuclear re- ceptors regulating atheroprotective genes. The aim of this project was to investigate whether vascular effects of estrogen are mediated via PPARγ-regulation in the vascular compartment. Estrogen deficient ovariectomized wildtype-mice (OVX) displayed significant reduction of PPARγ-expression in aortic tissue compared to wildtype-mice with intact ovarian function (Sham). Hormone replacement with subdermal 17ß-estradiol pellets significantly increased vascular PPARγ-expression in ovariectomized female wildtype-mice (OVX/E2). Analo- gous to wildtype-mice, estrogen-deficient OVX ApoE-/-‐mice had low vascular PPARγ-expression associated with ROS generation, endothelial dysfunction and atherogenesis. Estrogen replacement (OVX/E2) rescued vascu- lar PPARγ-expression, reduced ROS generation, monocyte recruitment, atherosclerotic lesion formation and im- proved endothelial function. Inhibition of PPARγ by GW9662, a specific PPARγ-antagonist reduced 17ß-estradiol mediated vascular effects (OVX/E2+GW9662). Finally, despite estrogen deficiency treatment with pioglitazone (OVX+pioglitazone), a selective PPARγ-agonist, compensates deterioration of vascular morphology and func- tion. 17ß-estradiol regulates vascular PPARγ-expression in wildtype- and ApoE-/-‐mice. The presented data demonstrate the fundamental relevance of PPARγ as downstream target of 17ß-estradiol-related anti- inflammatory and atheroprotective effects within the vascular wall independent of its cardiovascular risk factor modifications.
© 2012 Elsevier Ltd. All rights reserved.
1.Introduction
The low incidence of vascular disease in premenopausal women and the increase of cardiovascular events after menopause suggest an impor- tant role of 17ß-estradiol in the pathogenesis of atherosclerosis [1,2].
Binding of 17ß-estradiol to the estrogen receptors (ER), members of the sex-steroid-hormone-receptor-superfamily (SSHR), results in a conformational change within the ER leading to specifi c interaction of the receptor with DNA target sequences. Several newer SSHR signaling concepts with implications for cardiovascular physiology have recently emerged. Among these SSHRs interference with non- sex-steroid-hormone-nuclear-receptors (non-SSHR) such as the PPARγ, plays a central role [3]. In addition to its well documented metabolic functions on adipogenesis and glucose metabolism, PPARγ has been recognized to directly infl uence vascular cells. In atherosclerotic mouse models, the inhibitory effects of PPARγ or es- trogen on atherogenesis have been documented [4]. However, both
nuclear receptors have not been linked in regard to atherosclerosis. In most animal studies exogenous administration of 17ß-estradiol leads to beneficial vascular effects primarily on early stages of plaque formation involving modulation of cytokine and cell adhesion molecule expression, release of vasoactive substances and immunoinflammatory components interfering with atherogenesis [5,6]. Other potential targets for early atheroprotection of estradiol were thought to be attributable to changes in plasma lipid levels. In vivo animal studies demonstrated that 17ß-estra- diol treatment was associated with a reduction in total plasma cholesterol [7]. However, reduction of atherosclerosis was not always accompa- nied by a reduction in plasma cholesterol, suggesting that estradiol possesses atheroprotective effects independent of lipid changes. Similar to the benefi cial vascular effects of 17ß-estradiol genetic dis- ruption of PPARγ or treatment with PPARγ-agonists has been previ- ously implicated in the regulation of atherosclerosis. Conditional PPARγ knockout in macrophage increased atherosclerosis under conditions of hypercholesterolemia by modulation of chemokine receptor 2 (CCR2) expression and monocyte recruitment [8]. More- over, knockout of PPARγ in vascular smooth muscle cells promoted vas- cular lesion formation through accelerated VCAM-1 expression while
⁎ Corresponding author. Tel.: +49 228 28715217; fax: +49 228 28711271.
E-mail address: [email protected] (U.M. Becher).
0022-2828/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2012.05.008
pioglitazone stimulation of PPARγ expressing VSMCs reduced vascular lesion formation [9]. Recently, endothelial cell specific PPARγ
disruption in LDLR-knockout mice resulted in severe dyslipidemia, sig- nificant increase in systolic blood pressure and exacerbated vascular in- flammation [10].
Both, ERs and PPARγ are known to be expressed in endothelial cells (EC), vascular smooth muscle cells (VSMC) and monocytes/macro- phages [3,11,12]. In ECs 17ß-estradiol activated ER and PPARγ improve endothelial function due to ameliorated endothelial-dependent vasodi- latation by increased NO release and induction of NOS genes [13,14]. Moreover, 17ß-estradiol and PPARγ-agonists increase the number and functional activity of bone marrow derived endothelial progenitor cells (EPCs) and reduce EC apoptosis thereby promoting endothelial re- generation [15,16]. Additionally, both receptor types attenuate cytokine activated NF-kB signaling thus inhibiting gene expression of adhesion molecules more specific and limiting immune cell migration [13]. Final- ly, activated ERs and PPARγ prevent VSMC proliferation by blockage of the cell-cycle [13,17]. This coincidence of cellular targets and molecular downstream signaling suggests that 17ß-estradiol may improve vascu- lar function by regulating PPARγ, thereby mediating anti-oxidative and anti-inflammatory actions.
We evaluated the effect of endogenous 17ß-estradiol (Sham), estro- gen deficiency (OVX) or exogenous 17ß-estradiol (OVX/ßE2) on the ex- pression of PPARγ within the vasculature of wildtype- (WT) and ApoE-/-‐mice and investigated endothelial function, monocyte recruit- ment, oxygen species production and atherogenesis in ApoE-/-‐mice fed on a high-cholesterol type diet developing features of endothelial
dysfunction, vascular inflammation and atherosclerosis [18]. Treatment of 17ß-estradiol-deficient ApoE-/–mice with a PPARγ-agonist and non-ovariectomized ApoE-/–mice with a PPARγ-antagonist provided useful insights of the relevance of PPARγ for 17ß-estradiol-dependent ef- fects on vasculature.
2.Material and methods
2.1.Animals and treatment protocols
Fifteen 8-week-old C57-BL6 female WT mice were randomized in 3 groups (Fig. 1A): Sham operated (Sham), bilaterally ovariectomized (OVX) and ovariectomized receiving a 17ß-estradiol hormone replace- ment therapy (OVX/E2) with 17ß-estradiol pellets subcutaneously (containing 1.7 mg 17ß-estradiol each, 60-day release, Innovative Research).
Thirty-five 8-week-old C57-BL6 ApoE-/- animals were randomized in 5 groups (Fig. 3A): The first three groups were operated analogous to the WT-animal groups (Sham, OVX, OVX/E2). The fourth group was ovariectomized receiving 17ß-estradiol replacement therapy and the selective PPARγ-antagonist GW9662 (OVX/E2+GW9662) i.p. at a dose of 1 mg/kg body weight every second day. The fifth group was ovariectomized and received the selective PPARγ-agonist pioglitazone (Actos™) at a dose of 20 mg/kg body weight per day orally via chow but no 17ß-estradiol replacement. ApoE-/–mice were fed with a
A C
B D
Fig. 1. Estrogen‐dependent PPARγ-expression in the aortic wall of WT mice. A, Female WT mice were randomized in three experimental groups and operated as depicted. The fi rst group was sham operated and served as control. The second group was ovariectomized (OVX) and the third group was ovariectomized but received transdermal exogenous 17ß- estradiol supplementation through implantation of a estrogen pellet (OVX/E2). B, PPARγ-mRNA-expression was investigated in aortic tissue by RT-PCR of Sham, OVX and OVX/E2 animals. Values are presented in 2ˉΔΔCT compared to Sham. Estrogen deficiency in OVX animals resulted in a signifi cantly decreased PPARγ-mRNA-expression compared to Sham operated WT mice (*p b 0.05, Sham vs OVX). Transdermal exogenous 17ß-estradiol replacement of ovariectomized WT mice (OVX/E2) rescued the levels of PPARγ-mRNA- expression in the aortic wall (#p b 0.05, OVX/E2 vs. OVX). C, Representative WB showing the PPARγ-protein-expression in aortic tissue of Sham, OVX and OVX/E2 animals (GAPDH served as loading control). D, PPARγ-protein-quantification was evaluated through densitometry and values were presented in percent compared to Sham. Estrogen de- ficiency in OVX animals resulted in a significant decrease of PPARγ-protein-expression compared to Sham operated WT mice (*p b 0.05, Sham vs OVX). Transdermal exogenouse 17ß-estradiol replacement of ovariectomized WT mice (OVX/E2) rescued the levels of PPARγ-protein-expression in the aortic wall compared to OVX animals (#p b 0.05, OVX/E2 vs OVX).
high-fat and cholesterol-rich diet that contained 21% fat, 19.5% casein, and 1.25% cholesterol (Ssniff, Germany). All animal experiments were performed in accordance with institutional guidelines and the German animal protection law.
2.2.Real-time polymerase chain reaction (RT-PCR) and Western blotting (WB)
To assess vascular PPARγ-gene expression, mouse aortas were excised, quickly frozen in liquid nitrogen, and homogenized. RNA was iso- lated with peqGOLD RNAPure (peqLAB Biotechnology). Then, 1 lg of the isolated total RNA was reverse transcribed using random primers and MMLV reverse transcriptase (Invitrogen). The singlestranded cDNA was amplified by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) with the TaqMan system (ABI-7500 fast PCR Sys- tem). For PPARγ, the primers S 5′‐GTC ACG TTC TGA CAG GAC TGT GTG AC-3′ and AS 5′‐TAT CAC TGG AGA TCT CCG CCA ACA GC-3′ were used.
For western blot analysis, aortas were homogenized in ice-cold lysis buffer containing additional leupeptin and aprotinin. Protein aliquots were separated on SDS/PAGE. Western blotting of proteins was performed in a semidry blotting chamber (Pharmacia Biotech). Immunoblotting was performed with a PPARγ-rabbit-polyclonal- IgG-antibody (1:1000 dilution, ab27649 Abcam) for 60 min at 37 °C. Immunodetection was accomplished using a goat-anti- rabbit-secondary-antibody (1:5000 dilutions, Sigma Chemical) and the enhanced chemiluminescence kit (Amersham).
2.3.Measurements of blood pressure, heart rate, cholesterol and body weight
Systolic blood pressure, diastolic blood pressure and heart rate were measured by a computerized tail-cuff system (CODA 6, Kent Sci- entific) in conscious animals. Mice were trained for 3 consecutive days in the prewarmed tail-cuff device to accustom them to the pro- cedure. When blood pressure was determinated, 20 measurements were obtained and averaged for each individual animal. The mean values of all 3 days were used for comparison. Body weights were measured weekly.
2.4.Glucose- and insulin tolerance-test
To determine blood glucose tolerance, an intraperitoneal glucose tolerance test (ipGTT) was executed. Mice were refrained from eating for 18 h and given glucose (G 20 Glucose solution, B. Braun) adjusted to their body weight (2 g/kg body weight) by intraperitoneal injec- tion (i. p.) Blood glucose readings (Accu-Chek®-Sensor, Roche, Mann- heim, Germany) were taken at baseline and after 15, 30, 60, 90 and 120 min. In addition, an intraperitoneal insulin tolerance test (ipITT) was executed after 6 h fasting. Here, the animals were injected with human insulin (Actrapid; Novo-Nordisk; 0.75 U/kg body weight) in- traperitoneally and blood glucose readings were taken at baseline and after 15, 30, 60, 90 and 120 min.
2.5.Aortic ring preparations and tension recording
After excision of the descending aorta, the vessel was immersed in chilled, modified Tyrode buffer containing, in mmol/L, NaCl 118.0, CaCl2 2.5, KCl 4.73, MgCl2 1.2, KH2PO4 1.2, NaHCO3 25.0, Na EDTA 0.026, D(+)glucose 5.5, pH 7.4. Three-millimeter rings were mounted in organ baths filled with the above-described buffer (37 °C; continuously aerated with 95% O2 and 5% CO2) and were at- tached to a force transducer to record isometric tension. Drugs were added in increasing concentrations to obtain cumulative concentra- tion–response curves: KCl 20 mmol/L and 40 mmol/L, phenylephrine 1 nmol/L to 10 mol/L, carbachol 10 nmol/L to 100 mol/L, and
nitroglycerin 1 nmol/L to 10 mol/L. The drug concentration was in- creased when vasoconstriction or vasorelaxation was completed.
2.6.Staining of atherosclerotic lesions and morphometric analysis
For the detection of atherosclerotic lesions, aortic cryosections were fixed with 3.7% formaldehyde and stained with oil red O work- ing solution. Color reaction was accomplished with FastRed (Sigma) as a chromogenic substrate. Nuclei were counterstained with hema- toxylin. Isotype-specific antibodies were used for negative controls. All sections were examined under a Zeiss Axiovert 200M microscope using AxioVision version 4.5.0 software. For quantification of athero- sclerotic plaque formation in the aortic root, lipid-staining area and total area of serial histological sections were measured. Atherosclero- sis data are expressed as lipid-staining area in percent of total surface area. The investigators who performed the histological analyses were blinded to the treatment of the respective animal group.
2.7.Measurement of vascular reactive oxygen species
Superoxide release in intact aortic segments was determined by L- 012 chemiluminescence (Wako Chemicals, Germany). L-012 is a luminol derivative with high sensitivity for superoxide radicals. Aor- tas were carefully excised and placed in chilled, modified Krebs– HEPES buffer (pH 7.4; in mmol/L: NaCl 99.01, KCl 4.69, CaCl2 1.87, MgSO4 1.20, Na HEPES 20.0, K2HPO4 1.03, NaHCO3 25.0, D(+)glucose 11.1). Chemiluminescence was assessed over 15 min in a scintillation counter (Lumat LB 9501, Berthold) at 1-minute intervals. The vessel segments were then dried, and dry weight was determined. Superox- ide release is expressed as relative chemiluminescence per milligram of aortic tissue.
2.8.Immunohistochemical analysis of the monocyte/macrophage marker MOMA-2
For immunohistochemical analysis, cryosections were assessed for the monocyte/macrophage marker MOMA-2 with an indirect immunoenzymatic method. The primary antibody (monoclonal rat anti-mouse-MOMA-2-antibody, Acris) was applied for 1 h at room temperature and thereafter at 4 °C overnight. Slides were then incu- bated with an alkaline phosphatase-conjugated-secondary-antibody (goat anti-rat IgG, Sigma) for 1 h at room temperature. Color reaction was accomplished with FastRed (Sigma). Nuclei were counterstained with hematoxylin. Isotype-specific antibodies were used for negative controls. Monocyte recruitment was quantifi ed by expression of MOMA-2-positive-staining area in percent of total aortic plaque size estimated by the average of 5 sections from each animal. Sections were examined under a Zeiss Axiovert 200M microscope using Axio- Vision version 4.5.0 software.
2.9.Statistical analysis
Data are presented as mean±SEM. Statistical analysis was per- formed using the ANOVA test followed by the Newman–Keuls post- hoc analysis. P b 0.05 indicates statistical significance.
3.Results
3.1.Estradiol-dependent PPARγ-expression in the vascular wall of WT mice
Female WT mice were randomized in three experimental groups and operated as depicted (Fig. 1A). PPARγ-expression (Figs. 1B, C and D) in aortic tissue of OVX was significantly reduced on an mRNA-level (0.2±0.052 – ΔΔCT) compared to Sham (1.0±0.072 – ΔΔCT). PPARγ- protein levels were similarly altered with significantly lower levels in
OVX (26±12%) compared to Sham. Estrogen replacement in OVX/E2 animals led to the reconstitution of vascular PPARγ-expression on an mRNA-level (1.3±0.22 – ΔΔCT) and protein-level (146±14%).
3.2.Estradiol-dependent vascular release of superoxide radicals in WT mice
Vascular release of superoxide radicals was measured by an L012- chemiluminescence assay in intact aortic segments of Sham, OVX and OVX/E2 treated WT mice (Fig. 2). Vascular superoxide release was significant elevated in OVX (158±21%) compared to Sham (100± 15%) or OVX/E2 (88±19%).
Table 1
Metabolic parameters, blood pressure and heart rate of WT mice. Parameters of cardio- vascular risk factors (cholesterol levels, fasting blood glucose, body weight, blood pres- sure and heart rate) were analyzed in WT-animal groups. Total cholesterol serum levels were not significantly different compared between Sham, OVX and OVX/E2. Measurements of fasting blood glucose levels showed no significant differences be- tween Sham and OVX. OVX/E2 had significant lower fasting glucose levels than com- pared to OVX and similar levels to baseline values of Sham (*p b 0.05, OVX/E2 vs. OVX). At baseline body weight was identical in animals. After 60 days on a standard chow diet all animals displayed a similar increase in body weight. After 60 days no sig- nificant differences in systolic and diastolic blood pressure or heart rate were observed between the groups.
Wildtype Sham OVX OVX/E2
Total cholesterol (mg/dl) 75.8±18 80.6±17 61.1±13
Fasting blood glucose (mg/dl) 109.5±6 122.3±6 100.1±4#
3.3.Metabolic parameters, body weight, blood pressure and heart rate of WT mice
Body weight (g)
Systolic blood pressure (mm Hg)
24.2±0,3 24.6±0,3 24.0±0,5
132±3 137±3 137±3
Diastolic blood pressure (mm Hg) 92±6 95±4 92±5
Cardiovascular risk factors were measured in WT-animal groups (Table 1). Total cholesterol serum levels were not significantly differ- ent between Sham, OVX and OVX/E2 (Table 1). Fasting blood glucose levels showed no significant difference between Sham and OVX. OVX/
Heart rate (bpm)
798±32
764±26
720±77
E2 had signifi cantly lower fasting glucose levels compared with OVX and similar levels to baseline values of Sham (Table 1). Glucose tolerance (Suppl. Fig. 1A) and insulin tolerance (Suppl. Fig. 1B) were non-significantly impaired in OVX compared to Sham and OVX/E2. Signifi cantly lower glucose tolerance in OVX was detected at 120 min compared to Sham and OVX/E2 (Suppl. Fig. 1A). At baseline, body weight was identical in all animals. After 60 days of standard chow diet, all animals had a similar increase in body weight (Table 1). Blood pressure (mm Hg) and heart rate (bpm) were mea- sured in all groups by tail-cuff analysis. After 60 days, no significant differences in systolic and diastolic blood pressure or heart rate were observed between the groups (Table 1).
3.4.Estradiol-dependent PPARγ-expression in the vascular wall of ApoE-/–mice
Female ApoE-/–mice were operated as depicted (Fig. 3A) and used to analyze 17ß-estradiol-PPARγ-interactions in a mouse model of athero- sclerosis. In analogy to the WT-groups, PPARγ-expression was estrogen- dependent (Figs. 3B and C). In OVX PPARγ-expression (22.4±6.7%) was significantly reduced on a protein-level compared to Sham. OVX/E2 significantly increased PPARγ-protein-expression (92±9.7%) similar to controls. Two additional groups of ApoE-/–mice were investigated. One group of OVX/E2 was treated with GW9662, a specific PPARγ- antagonist (OVX/E2+GW9662) and finally, one group of OVX was treat- ed with the PPARγ-specific agonist pioglitazone (OVX+pioglitazone).
PPARγ-protein-expression in OVX/E2+GW9662 was significantly de- creased (8.7±3.9%) although estrogen was supplemented. In contrast, PPARγ-protein-expression was induced in OVX+pioglitazone treated animals (79.8±4.1%) compared to OVX (22.4±6.7%).
3.5.Estradiol-dependent generation of reactive oxygen species in ApoE-/-‐mice
Vascular release of superoxide radicals was measured by a L012- chemiluminescence assay in intact aortic segments of Sham, OVX, OVX/E2, OVX/E2+GW9662 and OVX+pioglitazone treated ApoE-/-‐ mice (Fig. 4A). Vascular superoxide release was significantly higher in OVX (120±7.6%) and OVX/E2+GW9662 (132±8.6%) compared to Sham (100±11.9%) and OVX/E2 (74±10.1%). Interestingly, OVX+ pioglitazone (67±10.8%) had significantly lower ROS levels although estrogen deficiency prevailed.
3.6.Vascular function in ApoE-/-‐mice
Vascular function was assessed in isolated aortic ring preparations and presented as maximal endothelial‐dependent vasorelaxation (Fig. 4B). In contrast to Sham (29±4%), endothelium dependent va- sodilatation was significantly impaired in OVX (39±6%), indicating that in 17ß-estradiol deficiency endothelial dysfunction is increased. OVX/E2 (14±6%) had significantly better endothelial function com- pared to OVX. No significant differences were observed between OVX/E2 and Sham. Vascular function was severely worsened in OVX/E2+GW9662 treated animals (52±5%). In contrast, OVX treat- ed with pioglitazone (10±4%) displayed significant better endotheli- al function although estrogen deficiency prevailed. Endothelium‐ independent vasorelaxation induced by nitroglycerin was similar in all groups (Suppl. Fig. 2).
3.7.Metabolic parameters, body weight, blood pressure and heart rate in ApoE-/-‐mice
Parameters of cardiovascular risk factors were measured in ApoE-/-‐animals. Total cholesterol serum levels were not signifi- cantly different compared between Sham, OVX, OVX/E2 and OVX/
E2+GW9662. Pioglitazone significantly reduced cholesterol levels
Fig. 2. Vascular ROS release investigated in WT-animal groups. The production of ROS was measured via L012 fl uorescence. Values are presented in percent (%) compared with Sham±SEM. Estrogen deficiency resulted in significantly increased ROS release compared to sham operated controls. Exogenouse 17ß-estradiol supplementation sig- nificantly reduced the rate of ROS production compared to OVX (*p b 0.05,Sham vs OVX; #p b 0.05, OVX/E2 vs OVX).
compared with all other groups (Table 2). Highest levels of cholester- ol were found in OVX (Table 2). Fasting blood glucose levels did not differ between Sham, OVX, OVX/E2 and OVX/E2+GW9662. OVX+ pioglitazone had significantly better fasting glucose levels compared with all other groups (Table 2). Glucose tolerance (Suppl. Fig. 1C)
A
B
C
Fig. 3. Estrogen-dependent PPARγ-expression in the aortic wall of ApoE-/-‐mice. A, Analogues to WT mice female ApoE-/-‐mice were randomized and operated as depicted. Ad- ditional to Sham, OVX, OVX/E2 two more groups of ApoE-/-‐mice were investigated. One group was ovariectomized, received transdermal exogenous 17ß-estradiol supplemen- tation and was treated with GW9662, a specific PPARγ-antagonist (OVX/E2+GW9662) and finally, one group of ovariectomized ApoE-/-‐mice was treated with the PPARγ- specific agonist pioglitazone (OVX+pioglitazone) without estrogen supplementation. Animals were fed a high-cholesterol type diet for 60 days to induce endothelial dysfunction, vascular inflammation and atherogenesis before read outs were performed. B, Representative WB showing the PPARγ-protein-expression in aortic tissue of all fi ve groups of ApoE-/-‐mice (GAPDH served as loading control). C, PPARγ-protein-quantification was evaluated through densitometry and values are presented in percent compared to Sham. Estrogen deficiency in OVX animals resulted in significantly decreased PPARγ-protein-expression compared to Sham operated ApoE-/-‐mice (*p b 0.05, Sham vs. OVX). Transdermal exogenouse 17ß-estradiol replacement of ovariectomized ApoE-/-‐mice (OVX/E2) rescued the levels of PPARγ-protein-expression in the aortic wall compared to OVX animals (#p b 0.05, OVX/E2 vs. OVX). In GW9662 treated ovariectomized ApoE-/-‐mice (OVX/E2+GW9662) PPARγ-protein-expression was found to be decreased compared to Sham and OVX/E2 (*p b 0.05, OVX/E2+GW9662 vs. Sham; ‡p b 0.05, OVX/E2+GW9662 vs. OVX/E2) while in contrast PPARγ-protein-expression was elevated in OVX+pioglitazone treated animals compared to OVX (§p b 0.05, OVX+pioglitazone vs. OVX).
and insulin tolerance (Suppl. Fig. 1D) were found to be significantly better in OVX+pioglitazone compared to all other groups. At base- line body weight was identical in all animals. After 60 days on a high-cholesterol diet all animals displayed a similar increase in body weight (Table 2). Blood pressure (mm Hg) and heart rate (bpm)
were measured in all groups by tail-cuff analysis. Signifi cant differ- ences were only found in systolic blood pressure of OVX+ pioglitazone. No signifi cant differences were observed for systolic and diastolic blood pressure or heart rate between the other groups (Table 2).
A B
Fig. 4. Estrogen-dependent generation of vascular ROS and vascular function in ApoE-/-‐mice. A, Vascular release of ROS was measured by a L012-chemiluminescence assay in intact aortic segments of Sham, OVX, OVX/E2, OVX/E2+GW9662 and OVX+pioglitazone treated ApoE-/-‐mice. Values are presented in percent (%) compared with Sham± SEM. Measurements showed that vascular superoxide release was significantly higher in OVX compared to OVX/E2 and OVX+pioglitazone (*p b 0.05 OVX/E2 and OVX+ pioglitazone vs. OVX). On the other hand OVX/E2+GW9662 had signifi cantly higher rates of ROS release compared to OVX/E2 (#p b 0.05 OVX/E2+GW9662 vs. OVX/E2). B, Vas- cular function in ApoE-/-‐mice was assessed in isolated aortic ring preparations. In contrast to Sham, endothelium dependent vasodilatation was impaired in OVX, as assessed by stimulation with carbachol, indicating that estrogen deficiency increases endothelial dysfunction. OVX/E2 and OVX+pioglitazone showed significantly increased endothelial func- tion compared to OVX (*p b 0.05 OVX/E2 and OVX+pioglitazone vs. OVX). Vascular function was severely worsened in OVX/ßE2+GW9662 treated animals compared to OVX/E2 (#p b 0.05 OVX/E2+GW9662 vs. OVX/E2). Endothelium‐independent vasorelaxation induced by nitroglycerin was similar in all groups (Suppl. Fig. 2).
Table 2
Metabolic parameters, blood pressure and heart rate in ApoE-/-‐mice. Parameters of cardiovascular risk factors (cholesterol levels, fasting blood glucose, body weight, blood pressure and heart rate) were measured in ApoE-/-‐animals of all five groups. Total cholesterol serum levels were not significantly different compared between Sham, OVX, OVX/E2 and OVX/E2+GW9662. However, pioglitazone significantly reduced cholesterol levels compared with all other groups (#p b 0.05, all groups vs. OVX+pioglitazone). Measurements of fasting blood glucose levels showed no significant differences between Sham, OVX, OVX/E2 and OVX/E2+GW9662, although the fasting blood glucose levels in OVX and OVX/E2+GW9662 tend to be increased. OVX+pioglitazone had significant better fasting glucose levels compared to all other groups (#p b 0.05, all groups vs. OVX+pioglitazone). At baseline body weight was identical in all animals. After 60 days fed on a high-cholesterol type diet all animals had similar increase in body weight. Significant differences were only found in systolic blood pressure of OVX+pioglitazone (‡p b 0.05, all groups vs. OVX+pioglitazone). No significant differences were observed for systolic and diastolic blood pressure or heart rate between the other groups.
ApoE-/-
Sham OVX OVX/E2 OVX/E2
+GW9662
OVX pioglitazone
Total cholesterol (mg/dl) 1132.6±154 1284.3±117 1088.1±40 1071.1±88 657±142#
Fasting blood glucose (mg/dl) 109.1±5 116.4±7 106.1±7 120.6±6 92.7±9#
Body weight (g) 24.9±0.5 25.4±0.5 28.8±0.5 26.1±0.7 24.5±1.3
Systolic blood pressure (mm Hg) 147.7±2 148.1±3 139.5±6 137.8±4 129.7±5‡
Diastolic blood pressure (mm Hg) 97.1±3 97.9±7.1 96.7±6 89.0±3 89.9±7
Heart rate (bpm) 628.1±11 663.9±16 689.5±20 695.1±23 695.6±17
3.8.Atherosclerotic lesion formation in ApoE-/-‐mice
Development of atherosclerotic lesions was quantified after 60 days using oil red O staining and macroscopic analysis of the aortic sinus. Fig. 5A shows representative aortic root preparations and Fig. 5B displays quantification of plaque area in percent. In contrast to Sham (36±2%) and OVX/E2 (36±6%) animals, OVX (51±4%) displayed severe athero- sclerosis in the aortic root. In OVX/E2+GW9662 atherosclerotic lesions were significantly larger (47±2%) than compared to OVX/2E. In contrast, in OVX+pioglitazone animals atherosclerotic lesions were significantly reduced (14±6%).
3.9.Monocyte recruitment of atherosclerotic lesions in ApoE-/-‐mice For immunohistochemical analysis, cryosections were assessed
for the monocyte/macrophage marker MOMA-2 with an indirect immunoenzymatic method. Fig. 6A shows representative MOMA- 2-stainings (magenta) of aortic root preparations of all animal groups and Fig. 6B quantifi cation of monocyte recruitment in per- cent. In contrast to Sham (22±0.1%) and OVX/E2 animals (33± 0.3%), OVX displayed an increased monocyte recruitment in athero- sclerotic lesions (37±0.2%). In OVX/E2+GW9662 monocyte
content was signifi cantly higher (51±4%) compared with OVX/E2. In contrast, OVX+pioglitazone atherosclerotic lesions displayed sig- nifi cantly reduced monocyte recruitment (25±8%) in atherosclerot- ic plaques.
4.Discussion
Cardiovascular diseases (CVDs) occur uncommonly in premenopausal women, but their incidence increases significantly after the menopausal transition. This difference has been attributed to the loss of female sex‐ steroid hormones at the time of menopause, but the underlying biological mechanisms are complex and only insufficiently understood [19]. Evi- dence suggests that cardiovascular risk factors, including disarrangement of glucose- and lipoprotein-metabolism leading to insulin resistance, met- abolic syndrome and hypertension, are responsible for the increased inci- dence of CVDs in postmenopausal women [20]. Although these metabolic changes occur in postmenopausal women, in large-scale studies with ad- justments for multiple risk factors, only 25–50% of the beneficial effects of estrogen appear to be due to metabolic effects [21].
Besides metabolic changes, 17ß-estradiol has major impact on the molecular and cellular signaling pathways and DNA transcription [3]. Several newer signaling concepts with implications for cardiovascular
A
B
Fig. 5. Atherosclerotic lesion formation in ApoE-/-‐mice. A, Representative oil red O stainings of atherosclerotic lesions within aortic root preparations of ApoE-/-‐mice of each group are depicted. B, Development of atherosclerotic lesion formation was quantifi ed after 60 days in all groups of ApoE-/-‐mice using oil red O stainings and macroscopic anal- ysis of the aortic sinus. Values are presented in percent (plaque area/total area of aortic sinus in %). In contrast to Sham, OVX/E2 and OVX+pioglitazone animals, OVX displayed severe atherosclerosis in the aortic root (*p b 0.05 vs. Sham, #p b 0.05 vs. OVX). In OVX/ßE2+GW9662 atherosclerotic lesions were significantly larger compared with OVX/E2 (‡p b 0.05 vs. OVX/E2).
A
B
Fig. 6. Monocyte/macrophage recruitment to atherosclerotic lesions in ApoE-/-‐mice. A, Representative MOMA-2-stainings (magenta color) of monocyte/macrophage recruitment to atherosclerotic lesions within aortic root preparations of ApoE-/-‐mice of each group are depicted. B, Monocyte/macrophage recruitment to aortic lesions was quantified by expression of MOMA-2-positive-staining-area in percent of total aortic plaque size estimated by the average of 5 sections from each animal. Values are presented in percent (Mono- cyte/macrophage burden/plaque area in %). In contrast to Sham animals, OVX displayed increased monocyte/macrophage burden in atherosclerotic lesion of the aortic root (*p b 0.05 OVX vs. Sham). In OVX/E2+GW9662 monocyte/macrophage content was significantly higher compared with OVX/2E (#p b 0.05 vs. OVX/E2).
physiology have emerged recently, adding substantial complexity to the physiological effects of SSHRs in target tissues. One concept en- compasses that 17ß-estradiol receptors regulate non-SSHRs, such as PPARγ, which, as well govern molecular, cellular and metabolic path- ways directly relevant to CVD [3].
The anti-inflammatory and cardiovascular protective action of 17ß-estradiol and PPARγ have been demonstrated [3–17,22]. Howev- er, the relevance of 17ß-estradiol interference with PPARγ in the vas- cular compartment has not been analyzed so far.
Using Western blot and quantitative RT-PCR we could show, that vas- cular PPARγ-expression is estrogen-dependent, implicating a regulative crosstalk between PPARγ and the 17ß-estradiol. Increased ROS genera- tion was found to be present in OVX of WT- and ApoE-/-‐mice, com- pared with Sham or OVX/E2-mice. These results of 17ß-estradiol mediated reduction of ROS are consistent with previous observations in which treatment with estrogen was found to protect against production of ROS by induction of radical scavenging enzymes [23,24]. Compared to OVX 17ß-estradiol induced up-regulation of PPARγ in the aorta of OVX/
E2 ApoE-/-‐mice and reduced ROS generation and monocyte recruit- ment subsequently restoring endothelial function and attenuation in atherogenesis. Adding the selective PPARγ-inhibitor GW9662 to OVX/E2 ApoE-/-‐mice reduced PPARγ-expression and induced increased production of vascular free radicals promoting endothelial dysfunction and atherogenesis, demonstrating that the anti-oxidative effects of 17ß- estradiol supplementation are, at least in part, mediated by PPARγ. These findings suggest that direct atheroprotective effects of 17ß-estradi- ol within the vascular wall are mediated by PPARγ-regulation. No signif- icant difference in cholesterol and glucose metabolism between Sham, OVX, OVX/E2 and OVX/E2+GW9662 treated animals was observed. Therefore, significant interference or bias of metabolic factors or genetic background were excluded between Sham, OVX, OVX/E2 and OVX/E2+ GW9662, making this animal model very useful to analyze nonmetabolic effects of 17ß-estradiol and estrogen deficiency on PPARγ-expression and
-inhibition on vascular inflammation, function and morphology.
More importantly, application of pioglitazone to OVX ApoE-/-‐ mice rescued these detrimental effects of 17ß-estradiol shortage leading to normalized PPARγ-protein levels, reduced production of
vascular free radicals, decreased monocyte recruitment, and attenuat- ed endothelial dysfunction and atherogenesis. Only within the group of OVX+pioglitazone treated animals we found systemic metabolic benefits, such as a significant lower cholesterol levels and improved glucose metabolism as well as lower systolic blood pressure values.
Our fi ndings that PPARγ-agonism significantly reduces endotheli- al dysfunction and plaque area even when 17ß-estradiol was lacking and that PPARγ-antagonism impaired vascular function despite es- trogen supplementation demonstrating the relevance of PPARγ as downstream target of 17ß-estradiol in mediating beneficial vascular effects. These data indicate that impaired vascular function and in- creased vascular inflammation in postmenopausal conditions are sub- stantially caused by reduced PPARγ-expression and -activation and might be rescued by PPARγ-activators, thereby reducing ROS genera- tion and monocyte recruitment and preserving endothelial function and vascular morphology.
A vast amount of data from the last 15 years provides insights that cel- lular protection by PPARγ-activation is based on increased endothelial proliferation and regeneration, reduced VSMC proliferation and migra- tion, and decreased macrophage cytokine production [3–17,22] However, HRT and PPARγ-agonists share undesirable side effects, such as peripheral oedema, anaemia due to plasma volume expansion and weight gain due to the development of subcutaneous adipose tissue [25]. Future develop- ments of synthetic non-thiazolidinedione-PPARγ-agonists are currently under investigation. These newer ligands should cause fewer side effects and at the same time improve metabolic factors and vascular health [26]. Future research is needed to clarify the 17ß-estradiol and PPARγ- interference translating into beneficial anti-atherosclerotic properties but the idea of 17ß-estradiol vasculoprotection mediated by PPARγ is al- ready underpinned by new insights of molecular crosstalk between 17ß- estradiol activated SSHR and the non-SSHR PPARγ. However, on the mo- lecular and cellular level several other signaling pathways might govern the here demonstrated 17ß-estradiol-PPARγ-axis resulting in direct rele- vance for maintenance of vascular health and effective regeneration [3,27]. How the expression pattern of these proteins in cardiovascular cells depends on gender, vascular bed, and the presence of cardiovascular risk factors or CVD is unknown. Moreover, little is known about the
differential expression and function of co-regulatory molecules and the physiological consequences of genetic receptor variants on nuclear- receptor-crosstalk and their cofactor-interactions in vascular cells. This molecular and cellular bases of cardiovascular health and gender differ- ences requires great attention in future research offering new diagnostic and therapeutic options to maintain vascular health, not only in postmen- opausal women but for both genders.
From a clinical perspective it remains to be determined if 17ß- estradiol induced PPARγ-expression indeed plays a dominant role in mediating anti-atherogenic effects in humans. Our results may im- plicate a role for PPARγ-agonism in the presence of CVD, especially in estrogen defi ciency because anti-atherosclerotic and anti- infl ammatory effects may have important clinical ramifi cations. PPARγ-ligands might thus not only be indicated in insulin resistance and diabetes, but also in postmenopausal women prone to CVD. Ob- servational studies have consistently demonstrated a reduction in mortality and cardiovascular disease incidence in women receiving HRT. However, the Heart and Estrogen/Progestin Replacement Study (HERS) [28], a randomized secondary prevention trial, investi- gated a total of 2763 women with established CHD randomly assigned to either placebo or HRT found no cardiovascular benefi t of conjugated equine estrogen (CEE) plus medroxyprogesterone ac- etate (MPA) use but demonstrated a surprising increase in the risk of CHD. The authors reported a trend toward greater mortality among HRT users, particularly in the fi rst year. These fi ndings were supported by the Estrogen Replacement Atherosclerosis (ERA) trial [29], a second randomized secondary prevention trial, in which post- menopausal HRT did not alter the progression of existing coronary artery disease as assessed by angiography. Finally, the fi rst random- ized primary prevention trial of postmenopausal HRT, the Women’s Health Initiative (WHI) [30], was terminated when women receiving active drug had an increased risk of CHD events, stroke, and invasive breast cancer.
Several misunderstandings have contributed to the controversy about the long‐term use of HRT, leading to the estimation that it con- fers more health risks that outweigh the benefits of the effects of es- trogens on the vasculature [31]. First, the coadministration of certain types of progestins, at a dose that attained the desired antagonist ef- fect on the endometrium, may oppose the cardioprotective effects of estrogens. It is important to note that the estrogen-alone arm of the WHI trial has not been halted, suggesting that estrogen alone may be safer than a combination of estrogen and progestin. Second, the re- sults of the HERS, ERA, and WHI trials refer to the use of CEE, a mix- ture of sulphate esters of estrone and other related steroids in which the exact composition is not known. Therefore, these findings cannot be generalized to treatments with 17ß-estradiol or other regimens. Third, the route of administration may be important for the effects of estrogen on CHD risk. Unlike oral formulation, trans- dermal administration avoids the induction of angiotensinogen [32]
and C-reactive protein, an inflammatory marker associated with atherosclerosis [33]. Fourth, the main difference between the positive observational and negative randomized study results was the time elapse since menopause before starting HRT. In the observational studies women choose to take ovarian hormones initially when menopausal symptoms occurred, while in the randomized studies the absence of menopausal symptoms was a pre-requisite for inclusion in the study. This apparently small differ- ence might have important implications because younger symptom- atic menopausal women taking HRT did not appear to have the same related risk as older postmenopausal women using HRT. Indeed, there was evidence that women in their early menopausal years tak- ing HRT had reduced risk of heart disease which might be due to a preserved vascular responsiveness to ovarian hormones. In general, it appears that HRT may be beneficial for women less than 10 years into menopause or less than 60 years of age. In older women adding HRT seemed to produce more cardiovascular events during the fi rst
two years of HRT therapy. However, long-term data suggested, that even older women, who were able to stay on HRT for a longer period of time, may end up with a reduced cardiovascular risk [34].
The divergent results of observational studies, randomized control trials and studies using animal models have not only highlighted the need to understand the differences in replacement therapy formula- tions, route of administration, and timing of pharmacological inter- vention, but also to evaluate alternative substances utilizing the vascular protective effects mediated by estrogens at the cellular and molecular level. Because of this controversy concerning HRT, PPARγ-agonists might offer an alternative to 17ß-estradiol substitu- tion in postmenopausal women prone to CVD. In addition to their well documented metabolic function in adipogenesis and increasing insulin sensitivity, we show that PPARγ-agonism slows down vascu- lar deterioration caused by 17ß-estradiol deficiency. Further studies are required to extend this proof of principle in humans and fully elu- cidate the mechanism of interactions of 17ß-estradiol and PPARγ. Nevertheless PPARγ-agonists have been shown to provide vascular benefits in 17ß-estradiol defi ciency synergistically to their metabolic effects on glucose and lipid homeostasis highlighting these agents as useful in different settings than insulin resistance and diabetes.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.yjmcc.2012.05.008.
Sources of funding
Ulrich M. Becher (O-109.0028) and Vedat Tiyerili (O-109.0033) were supported by the BONFOR program of the University Hospital of Bonn.
Disclosure statement
None declared.
Acknowledgment
We thank Dr. D. Lütjohann, Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn for providing cholesterol measurements.
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