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Cardiovascular Research 2007 73(1):217-226; doi:10.1016/j.cardiores.2006.10.024
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Copyright © 2006, European Society of Cardiology

Inhibition of neointima formation by local delivery of estrogen receptor alpha and beta specific agonists

Yvonne D. Kroma,1, Nuno M.M. Piresb,c,1, J. Wouter Jukemac,*, Margreet R. de Vriesb, Rune R. Frantsa, Louis M. Havekesb,c,d, Ko Willems van Dijka,d and Paul H.A. Quaxb,e

aDepartment of Human Genetics, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
bTNO-Quality of Life, Gaubius Laboratory, Zernikedreef 9, 2333 CK Leiden, The Netherlands
cDepartment of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
dDepartment of General Internal Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
eDepartment of Surgery, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands

* Corresponding author. Department of Cardiology, C5-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Tel.: +31 71 526 6695; fax: +31 71 526 6886. Email address: j.w.jukema{at}lumc.nl

Received 17 March 2006; revised 25 October 2006; accepted 26 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Neointima formation is the underlying mechanism of (in-stent) restenosis. 17β-Estradiol (E2) is known to inhibit injury-induced neointima formation and post-angioplasty restenosis. Estrogen receptor alpha (ER{alpha}) has been demonstrated to mediate E2 anti-restenotic properties. However, the role of estrogen receptor beta (ERβ) is not fully elucidated. In the present study, the specific role of vascular ER{alpha} and ERβ in neointima formation is assessed.

Methods and results: Neointima formation was induced by placement of a perivascular cuff around the femoral artery of male C57BL/6J mice. E2-eluting cuffs significantly inhibited cuff-induced neointima formation. To address the specific roles of ER{alpha} and ERβ on neointima formation, the ER{alpha}-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) and the ERβ-selective agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) were applied via a drug-eluting cuff. PPT inhibited neointima formation at low but not at high concentrations. Conversely, DPN inhibited neointima formation dose dependently. To demonstrate the specificity of these responses, an ER{alpha}-selective antagonist, MPP, was also used in combination with E2, PPT, or DPN. While the effect of PPT on neointima formation inhibition was blocked by co-delivery of MPP, E2 and DPN could still inhibit neointima formation.

Conclusions: Our data suggest that, in addition to ER{alpha}, specific ERβ activation inhibits neointima formation in a mouse model of restenosis. These data reveal a yet unidentified protective role of ERβ on neointima formation.

KEYWORDS Estrogens; Hormones; Receptors; Restenosis; Animal model


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
17β-Estradiol (E2) has been shown to have anti-restenotic properties [1–4]. Nevertheless, the anti-restenotic mechanism of action of E2 is not fully understood and controversial results regarding its effects on vascular remodeling have been reported [5–8]. This phenomenon may be attributed to the presence of two distinct estrogen receptors (ERs) in the vasculature, ER{alpha} and ERβ. ERs are ligand-activated transcription factors [9] and, although ER{alpha} and ERβ are highly homologous, activation of either one of them may lead to distinct and even opposite biological activities [10–13]. So far, studies in ERs-null mouse models have revealed a putative involvement of ER{alpha} in the protective effect of E2 on restenosis [14–17]. Nonetheless, the role of ERβ in mediating the anti-restenotic properties of E2 is not fully elucidated.

Recently, ligands that show ER{alpha} or ERβ selectivity have been developed allowing the evaluation of the specific function of each receptor. These molecules are described as ER{alpha}- and ERβ-selective agonists based on their binding capacity to each receptor. ER{alpha}-selective agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)tris-phenol (PPT) binding affinity is 410 times greater (Kd=0.4 nM) for ER{alpha} over ERβ [18]. The ERβ-potency selective agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) binds to ERβ with a 72-fold higher affinity (Kd=2.8 nM) compared to ER{alpha} [19]. Conversely, 1,3-bis(4-hydroxy-phenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazoledihydrochloride (MPP) is an ER{alpha}-selective antagonist (Ki=2.7 nM) with no antagonism to ERβ (>200-fold selectivity for ER{alpha} over ERβ) [20]. Therefore, these compounds provide an attractive pharmacological approach to elucidate the biological role of ERβ on neointima formation.

A well-defined mouse model of neointima formation consists of placement of a non-constrictive perivascular cuff around the mouse femoral artery [21,22]. Previously, we showed that the non-constrictive perivascular cuff to induce neointima formation can be constructed from a polymeric formulation suitable for controlled drug delivery. This novel drug-eluting cuff simultaneously induces reproducible neointima formation and allows locally confined delivery of drugs to the cuffed vessel segment [23–26]. Due to the hydrophobic character of the compounds to be used in the drug-eluting polymeric cuff, the release from the drug-eluting cuffs will be slow and sustained. Consequently, the concentration within the vessel wall will be relatively low and therefore most likely within the ranges where differences in affinity of the specific agonists really apply.

In the present study, we assessed the respective role of vascular ER{alpha} and ERβ in the anti-restenotic properties of E2 in a mouse model of restenosis. By local delivery of an ER{alpha}-selective agonist (PPT) and an ERβ-selective agonist (DPN), either or not in the presence of an ER{alpha}-selective antagonist (MPP), our data suggest that in addition to ER{alpha}, ERβ-specific activation leads to neointima formation inhibition in a murine model of restenosis. These data reveal a yet unidentified protective role of ERβ on neointima formation.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Drug-eluting cuffs
Poly({varepsilon}-caprolactone)-based drug-eluting cuffs were manufactured as previously described [23]. E2 was purchased from Sigma Diagnostics (St Louis, USA). PPT, DPN and MPP were obtained from Tocris Bioscience (Bristol, UK). Cuffs were made from the different blended molten drug-polymer mixtures and designed to fit around the femoral artery of mice. Drug-eluting cuffs had the shape of a longitudinal cut cylinder with an internal diameter of 0.5 mm, an external diameter of 1.0 mm, a length of 2.0 mm, and a weight of approximately 5 mg.

2.2. In vitro release profiles
Drug-eluting cuffs were loaded with 1% and 5% (w/w) E2, with 1% and 5% (w/w) DPN, with 0.5%, 1%, 2.5%, and 5% (w/w) PPT, and with 2% (w/w) MPP. Double drug-eluting cuffs were loaded with 2% MPP/1% PPT (w/w), 2% MPP/1% E2 (w/w), and 2% MPP/1% DPN (w/w). In vitro release profiles (n=5/group) were performed by UV–VIS absorbance methods (E2: 225 nm, DPN: 235 nm, PPT: 257 nm, MPP: 259 nm) as described elsewhere [23]. Calibration graphs of the different compounds were established by measuring the absorbance of a set of standards of each compound in the 0–50 µg/ml concentration range.

2.3. Femoral artery cuff mouse model
For experiments, 10–12 weeks old male C57BL/6J mice were used. Animals were fed a standard chow diet (R/M-H, ssniff, Soest, Germany). At the time of surgery, mice were anaesthetized with an intraperitoneal injection of 5 mg/kg Midazolam (Roche, Basel, Switzerland), 0.5 mg/kg Medetomidine (Orion, Helsinki, Finland) and 0.05 mg/kg Fentanyl (Janssen, Geel, Belgium). The femoral artery was dissected from its surroundings and loosely sheathed with a non-constrictive cuff [21,22]. Either a control empty cuff, an E2-eluting cuff (1% and 5% w/w), a PPT-eluting cuff (0.5%, 1%, 2.5%, and 5% w/w), a DPN-eluting cuff (1% and 5% w/w), or a 2% (w/w) MPP-eluting cuff was used (n=6/group). For the double drug-eluting cuff experiments, either a 2% MPP/1% PPT (w/w), a 2% MPP/1% E2 (w/w) or a 2% MPP/1% DPN (w/w) was used (n=6/group).

All animal work was approved by TNO institutional regulatory authority and carried out in compliance with guidelines issued by the Dutch government. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.4. Quantification and histological assessment of intimal lesions in cuffed femoral arteries
Animals were sacrificed 21 days after cuff placement. Histological analyses were performed as described previously [22–24]. All samples were routinely stained with hematoxylin-phloxine-saffron (HPS). Weigert's elastin staining was used to visualize elastic laminae.

Smooth muscle cells (SMC) were visualized with {alpha}-SM actin staining (1:800, Roche). Collagen content was determined using Sirius red stained sections. The amount of medial SMC and collagen content was determined by morphometry (Leica Qwin, Wetzlar, Germany) and expressed as the percentage of total medial area consisting of SMC actin- or Sirius red-positive area in six equally spaced serial cross-sections in all animals [24].

Incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA as a marker of DNA synthesis was used to determine the rate of cell proliferation in cuffed vessel segments. Mice (n=6/group) were injected intraperitoneally with 25 mg/kg BrdU (Sigma) three times at 72, 48, and 24 h prior to sacrifice. Sections were incubated with a mouse monoclonal anti-BrdU antibody (1:50; DakoCytomation, Glostrup, Denmark). Specimens incubated with a mouse isotype-matched IgG diluted to the same concentration as the primary antibody were used as control. The number of BrdU-labeled nuclei per cuffed artery were counted in six equally spaced cross-sections and expressed as a percentage of the total number of nuclei.

2.5. Estrogen receptors in femoral arteries
The presence of ER{alpha} and ERβ in cuffed vessel segments was visualized by immunohistochemistry using a rabbit and goat primary polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, USA) against the mouse ER{alpha} (1:600) and ERβ (1:100), respectively, according to the manufacturer's instructions.

Immunohistochemical analysis were performed in paraffin-embedded femoral artery segments at 0, 1, 7, and 21 days after cuff placement (n=6/timepoint). Specimens incubated with a mouse isotype-matched IgG diluted to the same concentration as the primary antibody were used as control. ER{alpha}- and ERβ-positive cells were counted in six equally spaced cross-sections in all mice and expressed as a percentage of the total number of cells.

2.6. Real time RT-PCR mRNA analysis
Mice underwent femoral artery cuff placement as described. Animals were sacrificed at different timepoints after surgery (0, 1, 7, and 21 days), four mice per timepoint. Femoral arteries were isolated, harvested and snap frozen. Total RNA was isolated using the RNeasy Fibrous Tissue Mini-Kit (Qiagen, Venlo, The Netherlands) according to manufacturer's protocol. Of all RNA samples cDNA was made using Ready-To-Go RT-PCR beads (Amersham Biosciences, Uppsala, Sweden).

Intron-spanning primers and TaqMan® probe were purchased from TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, USA). HPRT (hypoxanthine phosphoribosyltransferase) was assayed to correct for cDNA input. For each timepoint RT-PCR was performed in duplicate. Per timepoint the signals were averaged and the average signal of the housekeeping gene HPRT was subtracted ({delta}Ct). {delta}{delta}Ct was determined as the difference between {delta}Ct values of the control sham-operated arteries (T=0 days) and the cuffed femoral arteries. Data are presented as fold induction (normalized to T=0 days), which was calculated as 2–{delta}{delta}Ct [26].

2.7. Statistical analysis
Data are reported as mean±SEM. Multiple comparisons were made with nonparametric Kruskall–Wallis test, whereas comparisons between groups were made with the nonparametric Mann–Whitney U test. Differences between mean ranks were considered significant if P<0.05. All statistical analyses were performed with the SPSS 14.0 software program (SPSS Inc., Chicago, USA).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Local delivery of E2 using E2-eluting cuffs
3.1.1. E2 in vitro release profiles
In vitro release profiles of drug-eluting cuffs loaded with 1% and 5% (w/w) E2 was determined for a three weeks period (n=5/group). As depicted in Fig. 1, E2 was released in a sustained dose-dependent manner over the 21-day period for both E2 concentrations (1%: 30.9±1.4 µg; 5%: 210.7±13.6 µg).


Figure 1
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  		Fig. 1 In vitro release profile of 1% and 5% (w/w) E2-eluting cuff for 21 days (mean±SEM, n=5).

 
3.1.2. Effect of perivascular delivery of E2 on neointima formation
To assess the effect of local perivascular E2 delivery on cuff-induced neointima formation, drug-eluting cuffs were loaded with 1% and 5% E2 and placed around the femoral artery of male C57BL/6J mice for a 21-day period. Microscopic analysis of the cuffed femoral artery segments revealed that, after three weeks, a concentric neointima had been formed in mice receiving a control drug-eluting cuff. Animals receiving a 1% and 5% E2-eluting cuff showed a strongly reduced cuff-induced neointima formation development (Fig. 2A). Morphometric analysis revealed a significant inhibition of cuff-induced neointima formation between mice receiving a control drug-eluting cuff and animals receiving an E2-eluting cuff (Fig. 2B). Likewise, E2 perivascular treatment resulted in a significant decrease in intima/media ratio for both E2 loading dosages (Control cuff: 0.43±0.07; 1%: 0.17±0.04, P=0.005; 5%: 0.18±0.02, P=0.003) as compared to control drug-eluting cuff. In addition, no toxic effects of local perivascular delivery of increasing E2 concentrations on vascular integrity were found as determined by quantification of medial SMC and collagen content (Fig. 2C and D).


Figure 2
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  		Fig. 2 A: Representative cross-sections of cuffed murine femoral arteries treated with increasing concentrations of E2 21 days after cuff placement. HPS staining, magnification 400x (arrow indicates the internal elastic lamina; arrowhead indicates the external elastic lamina). B: Total intimal area of cuffed murine femoral arteries 21 days after E2-eluting cuff placement. Total intimal area was quantified by image analysis using ten sections in each cuffed artery and expressed in µm2 (mean±SEM, n=6). NS, P>0.05 (NS, not significant); *P<0.05. C: Representative micrographs of cuffed femoral arteries 21 days after placement of either a control empty cuff (Control cuff) or a 5% (w/w) E2-eluting cuff (5% E2). Alpha-SM actin staining for SMC; similar {alpha}-SMC content is observed in both control- and E2-treated cuffed segments. Sirius red stain for collagen; comparable collagen-positive area is present in both treated and untreated cuffed vessel segments. Magnification 400x (arrow indicates internal elastic lamina). D: Percentage of medial SMC- (close bars) and collagen-positive area (open bars) of cuffed femoral arteries treated with increasing concentrations of E2 at 21 days after drug-eluting cuff placement. Medial SMC- and collagen-positive area was quantified by image analysis using six sections in each cuffed artery and expressed in µm2. Mean±SEM, n=6. NS, P>0.05 (NS, not significant).

 
3.2. ER{alpha} and ERβ expression in cuffed femoral arteries
E2 may exert its inhibiting effects on neointima formation via both vascular ER{alpha} and ERβ. As depicted in Fig. 3A, both ERs mRNA levels were upregulated time-dependently after the induction of the stenotic process. ERs mRNA levels showed a peak expression seven days after cuff placement (59.5±3.9-fold increase for ER{alpha} vs. 11.4±4.2-fold increase for ERβ, both P<0.05) compared with control sham-operated arteries (T=0 days), after which the signal declined. In addition, immunohistochemical analyses showed that both ER subtypes are present in murine femoral arteries (ER{alpha}: 19.2±0.5%; ERβ: 48.4±6.8%, Fig. 3B and C). ER{alpha}- and ERβ-positive cells were present both in the tunica media, co-localized with SMC, and endothelium monolayer. Moreover, during the cuff-induced neointima formation process, cuffed femoral arteries contain both ER{alpha}- and ERβ-positive cells also in intimal tissue, co-localizing with smooth muscle and endothelial cells (Fig. 3B and C).


Figure 3
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  		Fig. 3 A: Fold induction of ER{alpha} and ERβ mRNA in cuff-induced neointima formation in time (mean±SEM, n=4; *P<0.05 as compared to T=0 days). B: ER{alpha} and ERβ localization in cuffed murine femoral artery on diverse timepoints. Both ER{alpha} and ERβ were present on medial tissue and endothelial cell monolayer in intact arteries (0 days). During the process of neointima formation development (1, 7, and 21 days) ERs expression was also present in intimal tissue. Magnification 400x. Arrowhead indicates internal elastic lamina; arrow indicates ERs positive cells. C: Percentage of total ER{alpha}- and ERβ-positive cells of cuffed femoral arteries after cuff placement. ER{alpha}- and ERβ-labeled cells were counted in six equally spaced cross-sections from each cuffed artery and expressed as a percentage of the total number of cells (mean±SEM, n=6; *P<0.05 as compared to T=0 days).

 
Altogether, ERβ is more abundantly present in arterial tissue as shown by immunohistochemistry analysis. On the other hand, upregulation of ER{alpha} expression is more prominent upon vascular injury. Thus, both ER{alpha} and ERβ are present and have the potential to contribute to the anti-restenotic properties of E2.

3.3. Local activation of ER{alpha} and ERβ in femoral arteries
3.3.1. PPT and DPN in vitro release profiles
To examine whether PPT and DPN were suitably loaded and released from our drug delivery device, the in vitro release profiles of 0.5%, 1%, 2.5% and 5% PPT- and 1% and 5% DPN-eluting cuffs were assessed (n=5/group). PPT showed a sustained and dose-dependent release for the 21-day period (0.5%: 15.8±0.4 µg; 1%: 35.8±1.7 µg; 2.5%: 68.5±1.3 µg; 5%: 159.7±5.6 µg; Fig. 4A). DPN was also released from the drug-eluting cuffs in a dose-dependent manner over time. As shown in Fig. 4B, DPN release from the drug-eluting cuffs showed an initial burst (first week) followed by a plateau. In total, 32.6±1.2 µg was released from the 1% and 83.3±3.3 µg from the 5% DPN-eluting cuffs, respectively.


Figure 4
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  		Fig. 4 In vitro release profiles of (A) PPT- and (B) DPN-eluting cuffs loaded with increasing concentrations for a 3-week period (mean±SEM, n=5).

 
3.3.2. Effect of PPT- and DPN-selective ER subtypes activation on neointima formation
To assess the role of ER{alpha} in the E2-mediated inhibition of cuff-induced neointima formation, drug-eluting cuffs were loaded with 0.5%, 1%, 2.5%, and 5% PPT, an ER{alpha}-selective agonist, and placed around the femoral artery of mice for three weeks. It should be noted that a broader concentration range of PPT was used as compared to E2 and DPN. This was due to the seemingly contrasting data observed with the 1% and 5% PPT-eluting cuffs on neointima formation, as discussed below.

In animals receiving a control drug-eluting cuff a neointima had been formed. Remarkably, morphometric quantification revealed only a significant inhibition of cuff-induced neointima formation in the cuffed segments treated with the lowest PPT concentrations. Cuffed arteries locally treated with higher PPT concentrations (2.5 and 5%) did not show an inhibitory effect on neointima formation as compared with control cuffed arteries (Fig. 5A and C). Likewise, only intima/media ratio of the PPT-treated arteries with the lowest concentrations were significantly decreased (Control cuff: 0.42±0.07; 0.5%: 0.13±0.01, P=0.001; 1%: 0.20±0.03, P=0.03; 2.5%: 0.34±0.05, P=1.0; 5%: 0.56±0.05, P=0.5) as compared to controls.


Figure 5
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  		Fig. 5 Representative cross-sections of cuffed murine femoral arteries treated with increasing (A) PPT and (B) DPN concentrations 21 days after cuff placement. HPS staining, magnification 400x (arrow indicates the internal elastic lamina; arrowhead indicates the external elastic lamina). Total intimal area of cuffed femoral arteries 21 days after (C) PPT- or (D) DPN-eluting cuff placement. Total intimal area was quantified by image analysis using ten sections in each cuffed artery and expressed in µm2 (mean±SEM, n=6). NS, P>0.05 (NS, not significant); *P<0.05; **P<0.01. E: Percentage of BrdU-positive cells in cuffed femoral arteries treated with increasing concentrations of PPT (0.5 and 2.5%) and DPN (1 and 5%) 21 days after drug-eluting cuff placement. BrdU-labeled nuclei were counted in six equally spaced cross-sections from each cuffed artery and expressed as a percentage of the total number of nuclei. Mean±SEM, n=6. NS, P>0.05 (NS, not significant); *P<0.05.

 
By placing a 1% and 5% (w/w) DPN-eluting cuff around the femoral artery of male C57BL/6J mice for 21 days, the role of ERβ on neointima formation was assessed. Morphometric analysis of the cuffed arteries of both DPN-treated groups showed a significant inhibition of neointima formation as compared to control cuffed segments (Fig. 5B and D). Moreover, intima/media ratio of the DPN-treated groups were also significantly decreased (Control cuff: 0.42±0.07; 1%: 0.22±0.05, P=0.02; 5%: 0.15±0.03, P=0.003) as compared to controls.

To further investigate the apparent discrepancy on cuff-induced neointima formation between animals perivascularly treated with either an ER{alpha} or an ERβ specific agonist, DNA synthesis was evaluated. Cellular proliferation was assessed by examining incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA at 21 days after cuff placement in mice receiving either a control drug-eluting cuff, a PPT- (0.5% and 5%) or a DPN-eluting cuff (1% and 5%). As depicted in Fig. 5E, a profound incorporation of BrdU was observed 21 days after surgery in cuffed vessel segments receiving a control drug-eluting cuff (3.45±0.25%). In line with the morphometric analysis, only the 0.5% PPT-eluting cuff showed a decreased cellular proliferation but not the higher PPT dosage (0.5%: 1.62±0.43%, P=0.02; 2.5%: 2.95±1.01%, P=0.2). Conversely, cuffed artery segments of mice receiving either a 1% or a 5% DPN-eluting cuff showed a significantly decreased cellular proliferation as compared to control cuffed arteries (1%: 1.71±0.50%, P=0.02; 5%: 1.27±0.43%, P=0.02). BrdU-labeled nuclei were present both in the medial and intimal region.

3.4. ERβ-specific activation by DPN
Since the difference of DPN for ERβ over ER{alpha} is only a factor 40, the effects of DPN may also be conveyed via an effect on the ER{alpha} due to the local concentration in the vessel wall of this compound. To exclude this effect of DPN on the ER{alpha}, a set of experiments was performed using a highly specific ER{alpha} antagonist (MPP) in combination with the various ER agonists (E2, PPT and DPN). For this, a set of (double) drug-eluting cuffs were designed and loaded either with the ER{alpha}-selective antagonist (2% MPP), the ER{alpha}-selective antagonist together with the ER{alpha}-selective agonist (2% MPP/1% PPT), the ER{alpha}-selective antagonist together with E2 (2% MPP/1% E2), or the ER{alpha}-selective antagonist together with the ERβ-selective agonist (2% MPP/1% DPN).

3.4.1. Double drug-eluting cuffs in vitro release profiles
The in vitro release profiles of these double drug-eluting cuffs were assessed for a 21-day period (n=5/group) as depicted in Fig. 6 and Table 1. It should be noted that the selected dosages of the different drugs were based on the in vitro release profiles of single drug-eluting cuffs aiming at a sustained release of similar amounts of the different compounds over the experimental period.


Figure 6
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  		Fig. 6 In vitro release profiles of 2% (w/w) MPP-eluting cuff and for double drug-eluting cuffs for a 3-week period (mean±SEM, n=5).

 

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  		Table 1 In vitro total drug released from MMP-eluting cuffs and double drug-eluting cuffs at 21 days.

 
3.4.2. ERβ-specific activation effect on neointima formation inhibition
Double drug-eluting cuffs were designed, as described above, to assess DPN specificity on ERβ-selective cuff-induced neointima formation inhibition. Drug-eluting cuffs were loaded with the several drug combinations and placed around the femoral artery of male C57BL/6J mice for a 21-day period. Morphometric analysis of the cuff-induced neointima formation revealed that arteries locally treated with the ER{alpha}-selective antagonist (MPP), as well as co-delivery of MPP and the ER{alpha}-selective agonist PPT, developed a comparable neointima to control empty drug-eluting cuffs (both P>0.05). Likewise, both MPP and MPP/PPT co-delivery had no effect on intima/media ratio (Control cuff: 0.46±0.06; 2% MPP: 0.52±0.10, P=0.8; 2%MPP/1%PPT: 0.37±0.04, P=0.1) as compared to control drug-eluting cuff. This indicates that MPP is able to fully block the ER{alpha}-mediated anti-restenotic properties of PPT (Fig. 7). Conversely, locally co-delivery of either MPP together with E2 and of MPP together with the ERβ-potency selective agonist DPN, by means of a double drug-eluting cuff, significantly inhibits cuff-induced neointima formation (both P<0.05, Fig. 7). Intima/media ratio for both drug combinations were also significantly decreased (Control cuff: 0.46±0.06; 1% MPP/1% E2: 0.24±0.09, P=0.02; 5%: 0.18±0.04, P=0.003) as compared to control. Taken together, this set of data suggests that DPN inhibits cuff-induced neointima formation via ERβ.


Figure 7
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  		Fig. 7 Total intimal area of cuffed murine femoral arteries 21 days after MPP-eluting cuff and double drug-eluting cuff placement. Total intimal area was quantified by image analysis using ten sections in each cuffed artery and expressed in µm2 (mean±SEM, n=6). *P<0.05 as compared to control cuff, 2% MPP and 2% MPP/1% PPT groups.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study evaluates the respective roles of vascular ER{alpha} and ERβ on neointima formation. Here we show the effects of ER subtype specific ligands on cuff-induced neointima formation in the mouse femoral artery. Local E2 treatment resulted in a substantial and significant inhibition of cuff-induced neointima formation (Fig. 2). Surprisingly, mice receiving the PPT-eluting cuffs displayed a significant reduction on neointima formation only for the lowest PPT concentrations (0.5%: 78±3%; 1%: 56±8%) but not for the 2.5% and 5% PPT-eluting cuffs (Fig. 5). Conversely, perivascular delivery of DPN displayed an inhibitory effect on cuff-induced neointima formation at both low and high concentrations (1%: 50±10%; 5%: 67±7%, Fig. 5). The inhibitory role of ERβ was confirmed by the use of MPP, an ER{alpha}-selective antagonist. MPP in combination with PPT did not show an effect on neointima formation while mice receiving E2 or DPN, in combination with MPP, neointima formation was still inhibited (Fig. 7).

E2 has been shown to have vasoprotective properties. In rats, systemic E2 therapy resulted in reduced vascular SMC proliferation and migration, which are fundamental steps in restenosis development [5]. In porcine coronary arteries, it has been shown that local delivery of E2 decreases post-angioplasty restenosis due to endothelial function improvement [27–29]. Furthermore, the first short-term clinical study using E2-eluting stents showed low rates of restenosis [30].

The specific role of vascular ER subtypes in neointima formation is not fully elucidated. To dissect the contribution of ER{alpha} and ERβ in neointima formation inhibition, the ER{alpha}-selective agonist PPT and the ERβ agonist DPN were used. The selectivity of PPT and DPN for both receptor subtypes enables detailed analysis of the contribution of both ERs on neointima formation. PPT induces exclusively ER{alpha}-mediated transcription and not ERβ (Kd for ER{alpha}=0.4 nM and for ERβ=417 nM) [18]. Thus, PPT can be stated as a specific ER{alpha} agonist. In vivo, we demonstrated that local release of PPT led to either anti-restenotic effects or no effect on restenosis, as low dosages inhibited neointima formation whereas high concentrations did not. The observation that PPT does not have a protective effect on cuff-induced neointima formation at higher dosages might suggest a disturbed balance in the induction of the diverse signaling cascades. Alternatively, different routes could be induced e.g. due to the presence of truncated ER{alpha} products (ER{alpha}46) [31,32] and/or the ER-related receptor {alpha}1 [33]. Although further research is required to analyze this process in more detail, our current data suggest a role for ER{alpha} in neointima formation.

DPN displays ERβ-potency specificity (Kd for ER{alpha}=80 nM and for ERβ=2.8 nM) [19]. Also, in vivo, DPN seems to act like a specific ERβ agonist. For example, systemic administration of the relatively high dose of 1 mg/kg/day DPN to rats does not alter uterine weight, which is regarded as a true ER{alpha} target tissue [34]. In the present study, both low and high concentrations of DPN led to an inhibition of neointima formation. Therefore we can state that, in this model, activation of ERβ seems to have a protective effect on cuff-induced neointima formation.

To further dissect the specificity of DPN on ERβ-dependent cuff-induced neointima formation inhibition we designed double drug-eluting cuffs which allow local co-delivery of drugs to the cuffed vessel segment. We made use of an ER{alpha}-selective antagonist, MPP (Ki for ER{alpha}=2.7 nM and for ERβ=1800 nM) to assess the role of DPN on ERβ-induced neointima formation inhibition in vivo [20]. Local delivery of MPP or co-delivery of MPP/PPT had no effect on neointima formation indicating that MPP is able to fully block the PPT-mediated effects on neointima formation inhibition. In contrast, local co-delivery of MPP/E2 and MPP/DPN to the vasculature, by means of a double drug-eluting cuff, significantly decreased cuff-induced neointima formation. These results suggest that both E2 and DPN can inhibit cuff-induced neointima formation in the mouse femoral artery exclusively via ERβ (Fig. 7). Surprisingly, the data of Figs. 2B and 7Go indicate involvement of ERβ only. No difference on neointima formation inhibition has been observed between the E2 and the MPP/E2 treated mice. In contrast, the use of PPT (ER{alpha}-specific agonist) clearly suggest a role for ER{alpha} as well (Fig. 5C), which is in accordance with literature. Since PPT-mediated ER{alpha} activation results in a reverse dose-dependent effect, interpretation of the results where ER{alpha} is involved is rather complex. Thus, we believe that our data implicate involvement of both ER{alpha} and ERβ, and that further research is required to investigate their intriguing complex role.

Thus far, the current knowledge of the respective role of vascular ER subtypes has been derived almost exclusively from ER{alpha}- and ERβ-null mouse models [14–17]. These models have demonstrated an almost exclusive protective role for ER{alpha} and not for ERβ. The discrepancy within the current results, which suggest a role for ERβ as well, could be related to the animal model used. In the current murine model of restenosis the ERs were modulated locally, which circumvents the interference of systemic ER-mediated effects present in the whole body ER-null mice. Since ERβ has a lower affinity for E2, it is also possible that systemic E2 concentrations are not sufficiently high to affect ERβ signaling. Local administration can, of course, result in much higher and/or more sustained local concentrations. Furthermore, due to intervention at a certain time point in adulthood also potential compensatory mechanisms which may have occurred during development in ER-null mice are excluded. In addition, differences in expression of ER{alpha} and/or ERβ and their splice variants between the femoral and the carotid artery may occur which possibly affects the action of the agonist. The same holds true for gender and age, which affect expression levels of the ERs. Furthermore, the degree of vascular injury differs between the models used in our study and the studies in literature. While in the cuff-induced injury model used in our studies the endothelial layer is not removed and remains fairly intact, although cells are activated, the studies using the ER-null mice employed the denudation of the carotid artery. Since ER{alpha} has been demonstrated to have a clear role in re-endothelization, this may be an important issue in explaining the observed differences between our study and the previous reports.

In conclusion, while literature proposes ER{alpha} as the major receptor involved in the anti-restenotic and anti-atherosclerotic effects of E2, our data provide evidence for a yet unidentified protective role of ERβ in neointima formation inhibition as well. Nevertheless, there seem to be complex and dose-dependent opposite roles for ER{alpha} and ERβ in vascular tissue. For future studies it is of pivotal importance to disclose the role of vascular origin, gender, and relative expression levels of ERs and their isoforms as well as their impact on the role of the two receptors.


   Acknowledgements
 
This work was performed in the framework of the Leiden Center for Cardiovascular Research LUMC-TNO. Dr. Y.D. Krom is supported by grants from the Dutch Organization for Scientific Research (NWO 902-26-220). N.M.M. Pires is supported by a Netherlands Heart Foundation grant, 2001-T-32. Dr. K.W. van Dijk is supported by a Netherlands Heart Foundation grant, NHS 2001-141. Dr. P.H.A. Quax (Established Investigator) is supported by the Molecular Cardiology Program of the Netherlands Heart Foundation (M 93.001). Professor J.W. Jukema is a Clinical Established Investigator of the Netherlands Heart Foundation, 2001-D0-32.


   Notes
 
1 Both authors contributed equally to this work. Back

Time for primary review 29 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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D. Xing, S. Nozell, Y.-F. Chen, F. Hage, and S. Oparil
Estrogen and Mechanisms of Vascular Protection
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