Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Local Delivery of 17-beta Estradiol for Preventing vascular
intimal hyperplasia and for improving vascular endothelium function after
vascular injury
FIELD OF THE INVENTION
The present invention relates to the local use of estradiol or
a derivative thereof to improve the outcome of a coronary angioplasty. More
specifically, the present invention is concerned with the local use of
estradiol
or a derivative thereof for decreasing neointima hyperplasia that occurs
during restenosis, and for improving the endothelium function after vascular
injury, both events contributing to the ultimate success of an angioplasty.
BACKGROUND OF THE INVENTION
Restenosis is currently the major limitation of percutaneous
transluminal coronary angioplasty (PTCA), and is seen in up to 30-40 % of
patients.' The most important mechanisms contributing to restenosis are
neointima proliferation, vascular remodelling, and elastic recoil.2 Elastic
recoil
and vascular remodelling can be reduced to a large extent by stenting.3
Although radiation therapy has been reported to show beneficial effeets,4,s
no effective therapy exists yet for neointima proliferation. Vascular smooth
muscle cell (SMC) migration and proliferation have been documented to
occur as early as 36 hours following arterial injury.s In cell culture assays,
17-
beta estradiol inhibited migration and proliferation of rat vascular SMC.'~8
Similar effects have also been shown with human vascular SMC from
saphenous vein.9 Prolonged systemic administration of estrogen has been
shown to inhibit intima hyperplasia in animal studies.'°~" Instead of
SUBSTITUTE SHEET (RULE 26)
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administrating estradiol systematically we here tested how a local
administration of 17-beta estradiol during PTCA could effectively inhibit
neointima proliferation.
The vital role of endothelium in the regulation of vascular
tone of arteries is well-recognized (1). The intact endothelium also has
important inhibitory effects on platelet aggregation, monocyte adhesion, and
vascular smooth muscle cell proliferation (2). Endothelial injury associated
with endothelial dysfunction is known to occur as a consequence of
percutaneous transluminal coronary angioplasty (PTCA) (3), and may play
an important role in restenosis following PTCA (4). Impaired endothelial
function has been demonstrated in porcine coronary arteries as long as 4
weeks following PTCA in pigs (5). Systemically administered 17-beta
estradiol has been reported to accelerate endothelial recovery after arterial
injury (10). Since endothelial injury due to PTCA is a local event, we
hypothesized that local delivery of 17-beta estradiol following PTCA may
enhance endothelial recovery.
SUMMARY OF THE INVENTION
An object of the present invention is therefore to provide efficient methods
by which 17-~i estradiol or a derivative thereof is used locally during PTCA
to improve endothelial function after vascular injury and/or to decrease the
neointima hyperplasia and/or prevent restenosis. Compositions for executing
these methods are also a further object of this invention.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
nonrestrictive description of preferred embodiments thereof, given by way of
examples only, with reference to the accompanying drawings.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 Representative light micrographs (x 40 magnification) of arterial
segments from the same animal, stained with Verhoeffs stain. 17-beta
estradiol (a) treated segment shows markedly less neointima hyperplasia
compared to PTCA only (b), or vehicle alone (c) groups. The extent of injury
is similar in all 3 segments.
Figure 2 Comparison of (A) neointima area, (B) neointima/media area, (C)
restenotic index, and (D) % stenosis between PTCA alone vs vehicle only,
and PTCA only vs 17-beta estradiol groups; * p < 0.05, ** p < 0,01 ***
p < 0.002. Values are expressed as mean t SEM.
Figure 3 Representative coronary angiograms demonstrating the
vasoconstrictive response to intracoronary infusion of acetylcholine (Ach) 10-
4M obtained from the same animal at 4 weeks following percutaneous
transluminal coronary angioplasty (PTCA). Column A = basal, column B =
after Ach, column C = following intracoronary nitroglycerin. Top panel =
treatment with vehicle, mid panel = PTCA only, lower panel 17-beta estradiol
treatment groups respectively.
Figure 4 Representative light micrographs (x 1000) of cross sections of
vessels obtained from the same animal for immunohistochemical staining
with the lectin Dolichos biflorus agglutinin (evident as dark brown staining
of
luminal surface). Vessels treated with 17-beta estradiol (A) demonstrate
reendothelialization to a greater degree as compared to PTCA only (B) and
vehicle (C) groups.
Figure 5 Representative light micrographs (x 1000) of cross sections of
vessels obtained from the same animal, for immunohistochemical analysis
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of endothelial nitric oxide synthase (eNOS) expression. Vessels treated with
17-beta estradiol (A) show greater expression of eNOS (evident as dark
brown staining of luminal surface) as compared to PTCA only (B) and vehicle
(C) groups.
Figure 6 Graph depicting correlation between vasoconstrictive response to
Ach 10~ M and (A) reendothelialization (r = -0.48, p < 0.02), (B) eNOS
expression (r = -0.58, p < 0.005). Note: % vasoconstriction denotes
decrease in diameter following Ach 10'4 M as compared to the basal
diameter.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1: The effect of estradiol on neointima hyperplasia
Methods
Animal preparation
Eighteen juvenile farm pigs (9 female, and 9 castrated male) weighing 20-25
kg were studied. The study was approved by, and conducted in accordance
with, the guidelines of the Animal Care and Ethical Research Committee of
the Montreal Heart Institute. Before the procedure, animals were given 650
mg of acetylsalicylic acid and 30 mg of nifedipine orally, premedicated with
intramuscular injection of 6 mg/kg of a mixture of tiletamine hydrochloride
and zolazepam hydrochloride, and given 0.05 mg of atropine. The invasive
procedure was performed under general anesthesia with a mixture of
isoflurane (1 to 1.5 %) and oxygen enriched air. The right femoral artery was
cannulated percutaneously, and an 8 Fr arterial sheath was introduced. After
arterial access had been obtained, 100 mg of lidocaine and 250 U/kg of
heparin were administered intra-arterially via the sheath. Activated
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coagulation time was maintained at > 300 seconds throughout the
procedure.
Preparation of estradiol formulation
5
Each dose individually administered to the tested animals is composed of at
least 12.5 mg hydroxypropyl-beta-cyclodextrin (HPCD) and 600 pg estradiol
in a 5 ml solution volume.
A smaller or larger dose may be used. Indeed, the tested dose corresponds
to the dose of about 675 Ng formulated in a sublingual pellet and
administered to postmenopausal women.45 Such a dose may be
unnecessarily high if administered locally. Indeed, doses of 200 and 400 Ng
have been tried and they were found to be as performing as the dose of
600 pg. Further, the necessary dose for performing the present invention
may be influenced by the hormonal balance of the individual to be treated.
Species variance is also a factor affecting the dosage regimen. Also, any
derivative of 17-beta estradiol may replace the latter. A derivative is
intended to cover a precursor, an active metabolite, an active analog or a
modulator capable of positively influencing the activity of the receptors) to
estradiol or of enhancing the binding and/or the activity of estradiol towards
its receptor(s). Such derivatives are considered functional equivalents of 17-
beta-estradiol, and therefore within the scope of this invention. A unit dose
of 1 to 5000 pg/Kg of 17-beta-estradiol or an equivalent derivative dose is
within the scope of this invention, preferably 10-50 Ng/Kg, even more
preferably 10-30 pg/Kg.
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Angioplasty and Local Delivery
Standard PTCA equipment was used. An 8 Fr right Amplatz guiding catheter
and right Judkins guiding catheter were used for cannulation of the left and
right coronary arteries, respectively. PTCA was performed with a balloon size
chosen to correspond to a balloon/artery ratio of 1.1-1.3. Three 30-second
inflations at 10 atm pressure were performed with a 30-second interval
between each inflation. Inflations were performed adjacent to major side
branches to facilitate identification during harvesting, taking precaution not
to include any side branch in the intended PTCA site. The left anterior
descending, left circumflex, and right coronary arteries of each animal were
subjected to PTCA. After PTCA, each coronary artery of an animal was
randomized to receive either 600 pg of 17-beta estradiol locally, or vehicle
alone locally, or PTCA only. The chemicals 17-beta estradiol and its vehicle
2-hydroxypropyl-beta-cyclodextrin (HPCD) were purchased from Sigma
Chemical Co. The InfusaSleeve catheter (Local Med, Inc.) was used for local
delivery.'2 Five ml of the designated substance was delivered at a driving
pressure of 10 atm and support balloon pressure of 6 atm.
Of the 18 animals, 2 died a few days after PTCA, and were excluded; thus,
16 animals were analyzed. Twelve animals were euthanized at 28 days, and
4 at 7 days. After premedication and anesthesia, the right internal jugular
vein and common carotid artery were cannulated. Following cross-clamping
of the descending thoracic aorta exposed via a left lateral thoracotomy,
exsanguination was performed, with simultaneous administration of 1 I of
0.9 % NaCI solution. The heart was perfusion-fixed in vivo with 2 I of 10
buffered formalin at 200 mm Hg pressure, removed from the animal, and
placed in 10 % buffered formalin solution. Coronary arteries were then
dissected free from surrounding tissues. The site of PTCA was identified in
relation to adjacent side branches, which served as landmarks. The injured
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segment was harvested with a 1 cm normal segment proximal and distal to
the injured site. Serial sections 3 to 5 mm long were made from the
harvested segment, with a minimum of at least 3 sections (maximum 5) from
each PTCA site. Sections were stored in buffered 10 % formalin and
subjected to dehydration with increasing concentrations of alcohol, followed
by treatment with xylene and paraffin. Each section was then cut to slices of
6 pm thickness with a microtome (Olympus cut 4060 E), and stained with
Verhoeff's stain for morphometric analysis.
Morphometric analysis
Measurements were made with a video microscope (Leitz Diaplan, equipped
with a Sony DXC 970 MD color video camera) linked to a 486 personal
computer and customized software. A minimum of 3 sections for each injured
segment were analyzed and results averaged. Analyses were made by a
single observer unaware of the treatment group to which each segment had
bee allocated. Randomly selected sections were viewed by a second
observer (also blinded to protocol) independently; inter-observer variability
was < 5 %. The areas of external elastic lamina (EEL), internal elastic lamina
(IEL), and lumen were measured by digital planimetry; neointima (I) area (IEL
- lumen area) and media (M) area (EEL - IEL area) were obtained. The
neointima was defined as the % of total vessel area occupied by neointima
(% neointima = [I/EEL] x 100). Morphologic % stenosis was calculated as
100 (1 - lumen/IEL area).'3 The restenotic index was defined as
[1/(I + M)]/(F/IEL circumference), where F is the fracture length of internal
elastic lamina.'4 Histologic injury score was determined as previously
defined.'S
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Immunohistochemistry
Following slicing with a microtome and blocking of non-specific antibodies,
the sections were treated with mouse anti-proliferating cell nuclear antigen
(PCNA) antibodies and diluted biotinylated goat anti-mouse antibodies. They
were then incubated with avidin-biotin (Elite ABC Kit, Vector Laboratories),
and developed with 3, 3'-diaminobenzidine (Vector Laboratories). They were
finally counter-stained with hematoxylin. Porcine liver cells were used as a
positive control. For each section, a 6 Nm slice counter-stained with
hematoxylin without treatment with the primary antibody (mouse anti- PCNA)
served as a negative control.
The proliferative response to injury was studied by immunohistochemical
analysis of samples from animals euthanized at 7 days. The % proliferating
SMC was obtained by dividing the number of PCNA - positive SMC by the
total number of SMC in each field; separate measurements were made for
neointima and media layers. The proliferating cells were identified as SMC
by positive staining of parallel sections with a smooth muscle actin antibody.
To standardize comparison among treatment groups, measurements were
obtained at 4 fixed locations separated by 90° sites for each section,
and the
results averaged. For each segment, two sections demonstrating maximal
neointima response were analyzed, and the results averaged.
Statistical Analysis
Values are expressed as mean t standard deviation, except as otherwise
indicated. Kruskal - Wallis analysis was used for comparison of data among
the 3 groups; subsequently, 17-beta estradiol and vehicle alone groups were
separately compared with the PTCA only group using the Mann - Whitney
rank sum test. Chi - square analysis was used for comparison of proportions.
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The Mann - Whitney rank sum test was also used for comparison of data
between male and female animals within the 17-beta estradiol treated group.
Values were considered statistically significant if p < 0.05.
Results
Following PTCA and local delivery, animals were allowed to recover, and
gained weight steadily. Two animals died 48 and 72 hours after procedure
respectively, and were not included; thus 16 animals were studied. Autopsy
of the 2 animals revealed occlusive thrombus at the site of PTCA (in the 17-
beta estradiol treated vessel in one pig, and in the vessel treated with PTCA
only in the other pig).
Injured segments
Balloon/artery ratio and artery diameter were not significantly different
among
the 3 treatment groups (Table 1). Segments with intact IEL in which
discernible injury was absent were excluded from analysis (2 from PTCA only
group, and 1 from vehicle alone group). Two segments were lost during
harvesting and processing (1 of vehicle alone, and 1 of PTCA only group).
Morphometric analysis
Of the 12 animals that underwent morphometric analysis at 28 days, arterial
segments treated with local delivery of 17-beta estradiol showed significantly
less neointima hyperplasia (Figure 1). This beneficial effect was noted in all
parameters of neointima response to injury that were analyzed (Table 1 ). Of
note, the extent of morphologic injury was similar among the 3 groups,
suggesting that the use of the InfusaSleeve catheter was not associated with
an enhanced risk of injury.
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It was important to exclude an inhibitory effect on intima proliferation due
to
the vehicle, and, to confirm that the effect noted was in response to
treatment with 17-beta estradiol. Analyses comparing segments treated with
5 vehicle alone and PTCA only showed a similar response in terms of the
extent of neointima proliferation. On the other hand, significantly less
intima
hyperplasia was observed in 17-beta estradiol treated segments as
compared to segments treated with PTCA only (Figure 2). Compared to
PTCA only, or vehicle alone, 17-beta estradiol decreased neointima
10 formation by 54.6 % and 64.9 % respectively.
To exclude the possibility of influence of sex on response to estrogen, the 7
segments obtained from male pigs treated with 17-beta estradiol, and 5
segments obtained from female pigs treated with 17-beta estradiol were
analyzed. No statistically significant differences were evident (Table 2).
Immunohistochemistry
The number of PCNA - positive SMC was low overall; sacrifice at an earlier
time might have yielded a higher number. However, a statistically significant
decrease in the proliferative response was seen in animals treated with 17-
beta estradiol. Among the different groups, the % of PCNA - positive SMC
in the neointima were 0.43 t 0.52 % in 17-beta estradiol, 4.26 t 2.33 % in
PTCA only, and 4.27 t 2.73 % in vehicle alone groups respectively (p < 0.05
for 17-beta estradiol vs other 2 groups). There were no statistically
significant
differences in % PCNA - positive SMC in the media among the 3 groups:
0.4 t 0.3 %, 1.38 t 1.74 %, and 1.24 t 1.57 % for 17-beta estradiol, PTCA
only, and vehicle alone groups respectively (p = NS).
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Vascular remodeling
To determine the effect on vascular remodeling of the agents used, the EEL
area of the injured segment and of the normal vessel proximal to site of
PTCA were obtained, and their ratio calculated.'3 No significant difference
among the groups was noted: 1.01 t 0.16, 1.16 t 0.28, 1.31 t 0.37
respectively for 17-beta estradiol, PTCA only, and vehicle alone groups
respectively (p = NS).
Conclusions
The present study demonstrates, for the first time, that locally delivered 17-
beta estradiol decreases neointima proliferation following PTCA in pigs. The
study also shows that the InfusaSleeve catheter can be used to deliver
effectively 17-beta estradiol intramurally in coronary arteries.
Several previous experiments in animals have demonstrated that estrogen
administered subcutaneously for up to 3 weeks inhibited the myointima
response to arterial injury.'°'" Recently, short-term subcutaneous
estrogen
therapy (6 to 17 days) was also shown to be effective in reducing the injury
response in rat carotid artery.'6 Estrogen administered intra-muscularly for
at least 3 weeks has also demonstrated the potential to inhibit vascular
smooth muscle cell proliferation and neointima hyperplasia in rabbits."
However, the efficacy of local delivery of 17-beta estradiol to inhibit intima
hyperplasia has not been previously studied.
The biologic effects of estrogen, like other steroid hormones, involve
intracellular receptors. The first estrogen receptor (ER) to be discovered was
ERa,'$~'9 which was thought to mediate the beneficial effects of estrogen
following vascular injury. ERa was also present in coronary arteries obtained
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from autopsy specimens in both pre and postmenopausal women,2° and in
cell cultures of human saphenous vein and internal mammary artery
specimens.2' Recently, a second estrogen receptor, ER~i, has been identified
in animals and humans.22,Z3 The role of ER[3 in response to vascular injury
was subsequently demonstrated in experiments with ERa deficient mice.24
Normal and ERa deficient mice treated with estrogen, when subjected to
arterial injury, showed the same extent of inhibition of neointima
proliferation
compared to control mice; thereby demonstrating that inhibition of vascular
injury response by estrogen is independent of ERa. Although the present
experiment was not designed to study the mechanism of action of 17-beta
estradiol, evidence exists for multiple potential mechanisms by which 17-beta
estradiol can inhibit the vascular response to injury. Of importance may be
the effect of 17-beta estradiol on nitric oxide (NO) synthesis. In cell
culture
studies with human and bovine endothelial cells, treatment with 17-beta
estradiol stimulated NO synthase and increased NO production.25,2s
Postmenopausal women treated with transdermal 17-beta estradiol showed
enhanced in vivo NO synthesis.2' NO has demonstrated inhibitory effects on
both migration 2$ and proliferation 29 of vascular SMC, and decreased
neointima formation after PTCA.'3 Preliminary reports have shown that
therapy with 17-beta estradiol decreases intercellular and vascular cell
adhesion molecule expression by human coronary SMC.3° Cellular adhesion
molecules are expressed by SMC following arterial injury3' and their
suppression with the use of monoclonal antibodies inhibited intima
hyperplasia after arterial injury in rats.32 The regulatory effect of 17-beta
estradiol on vascular endothelial growth factor expression may also be partly
responsible.33-s5 perhaps the most important mechanism may be a direct
inhibitory effect of 17-beta estradiol on vascular SMC proliferation.36 The
binding of 17-beta estradiol to its intracellular receptor activates DNA
containing "estrogen responsive elements", leading to altered gene
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expression. 17-beta estradiol also reduces platelet derived growth factor-
induced migration and proliferation of vascular SMC.9
The beneficial effects of 17-beta estradiol, the predominant circulating
estrogen in premenopausal women, on vascular injury response may not be
replicated by other kinds of estrogens; for example, conjugated equine
estrogen was found to have no effect on neointima proliferation in
non-human primate models.3' Simultaneous administration of progesterone
may attenuate the vascular injury response to 17-beta estradiol.3$ A sexually
dimorphic response to estrogen in intact rats has been reported following
arterial injury, with male rats deriving no benefit with estrogen therapy .39
This
sexually dimorphic effect was, however, not observed in another experiment
with gonadectomized rats." In the present study, too, no significant
difference in neointima proliferative response to 17-beta estradiol was noted
between the sexes. Increased expression of ER~i mRNA (ER~3 is directly
associated with inhibition of vascular SMC proliferation) following arterial
injury has been demonstrated in intact male rats;4° of additional
interest in
the study is that no increase in ERa was seen following arterial injury.
17-beta estradiol is a lipophilic compound with poor solubility in aqueous
solutions, thereby needing a vehicle for parenteral administration. HPCD is
a starch derivative that has been successfully tested as an effective
excipient
for protein drugs.4' The pharmacokinetics of HPCD are similar to that of
inulin, and the toxic dose (nephrotoxicity) has been estimated to be 200
mg/kg in rats.42 The dose of HPCD used to dissolve 17-beta estradiol in the
present study was 0.63 mg/kg, far below the toxic dose. Furthermore, HPCD
has been used for administration of ophthalmic preparations and intravenous
anaesthetic agents in humans.4s,4a HPCD complexed to 17-beta estradiol has
been used to enhance bioavailability of orally, or, sublingually administered
17-beta estradiol with no untoward effects in humans.4s
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Retrospective studies in humans have shown no benefit of hormonal
replacement therapy on angiographic restenosis following PTCA46 although
one study did show a beneficial effect after directional atherectomy.4'
However, it should be noted that conjugated estrogen (and not 17-beta
estradiol) was the predominant form of estrogen used in many of these
patients, and, no information about concomitant use of progesterone is
available.
In conclusion, we have shown that, a single dose of 17-beta estradiol
delivered locally during PTCA has the potential to inhibit neointima
proliferation effectively. The delivery of 17-beta estradiol can be performed
easily with the InfusaSleeve catheter, without risk of additional injury. With
this approach, it may be possible to avoid potential undesirable effects of
long term systemic administration of estrogen. ER(3 has been identified in
humans, and inhibition of proliferation of human vascular SMC by 17-beta
estradiol has been demonstrated in cell culture assays. The local
administration of 17-beta estradiol is therefore a promising new approach,
which might be useful in preventing the proliferative response after PTCA in
humans. Its usefulness in preventing restenosis after PTCA is contemplative
in view of the foregoing promising results.
Example 2: The effect of estradiol on vascular endothelial function
Methods
Animal preparation
The study protocol was approved by the Animal Care and Ethical Research
Committee of the Montreal Heart Institute. Juvenile farm pigs weighing
20-25 kg (1 female, and 8 castrated males) were used. On the day of the
experiment, animals received 650 mg of acetylsalicylic acid and 30 mg of
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nifedipine orally, were premedicated with 6mg/kg of tiletamine hydrochloride
and zolazepam hydrochloride, and were given 0.05 mg of atropine
intramuscularly. Under general anesthesia (a mixture of 1-1.5% isoflurane
and oxygen enriched air), the right femoral artery was cannulated
5 percutaneously. An 8 Fr arterial sheath was introduced, and 100 mg/kg of
lidocaine and 250 U/kg of heparin were administered intra-arterially.
Additional heparin was administered during PTCA if needed, to maintain an
activated coagulation time of > 300 seconds.
10 Procedure
An 8 Fr right Amplatz guiding catheter and right Judkins guiding catheter
were used for cannulation of the left and right coronary arteries,
respectively.
A standard balloon catheter (corresponding to a balloon/artery ratio of
15 1.1-1.3: 1) was advanced over a 0.014" floppy guide wire, and 3 successive
30-second inflations at 10 atm pressure were made with a 30 second interval
between each inflation. PTCA was performed on all 3 coronary merles of
each animal. For local delivery, the InfusaSleeve catheter (LocaIMed Inc.)
was used, which permits safe drug delivery with negligible additional injury
(7). After balloon dilatation, each coronary artery of an animal was
randomized to receive either 600 p,g of 17-beta estradiol (in 5 ml), vehicle
alone (5 ml), or PTCA only. The vehicle 2-hydroxypropyl-beta-cyclodextrin
(HPCD), and 17-beta estradiol were obtained from Sigma Chem. Co. For
local delivery with the InfusaSleeve catheter, a proximal driving pressure of
10 atm and support balloon pressure of 6 atm were utilized.
Intracoronary infusion
All 9 animals underwent cardiac catheterization at the end of 4 weeks. After
a baseline coronary angiogram, selective cannulation of the proximal portion
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of a coronary artery was performed with a single lumen balloon catheter
(TotaICross, Schneider) for the administration of vasoactive agents.
Acetylcholine (Ach) in increasing concentrations of 10-' M, 10-6 M, 10-5 M,
10-4 M, was successively infused through the lumen port of the catheter.
Each dose was administered for a duration of 3 minutes at a constant rate
of 1 ml/min using an infusion pump. Coronary angiography was performed
at the end of each dose. After infusion of the highest concentration of Ach
(10-4 M and angiography, 100 ~,g of nitroglycerin was administered via the
lumen port of the catheter, and a coronary angiogram performed. The same
protocol was repeated for the other 2 coronary arteries. Heart rate, blood
pressure, and ECG were monitored continuously throughout the experiment.
Quantitative coronary angiography
Coronary angiography was performed with a single plane imaging system
(Electromed Intl). Images were obtained in predetermined views which best
demonstrated the vessel segment of interest and without overlap of
branches. Care was taken to maintain the same angulation during
angiography of a segment throughout the procedure. Ionic contrast (MD-76,
Mallinckrodt Medical Inc) was used throughout the experiment. Images were
captured at a frame speed of 30 frames/sec, and stored digitally. A segment
of contrast-filled guiding catheter was included in every frame, for the
purpose of calibration. Calibration was performed using the known diameter
of the contrast-filled guiding catheter as the reference segment, to avoid
error due to magnification. Coronary artery diameter measurements were
made using a validated computerized edge-detection system (8). The
midpoint of the injured segment was used for calculation of coronary artery
diameter. For each analysis, coronary artery diameter measurements were
performed in 3 consecutive end-diastolic frames, and the results averaged.
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Measurements were performed by an independent observer blinded to the
treatment group of the vessels.
Immunohistochemistry
The animals were euthanized at 4 weeks. Under general anesthesia as
described above, exsanguination was performed with replacement by 1 I of
0.9 % NaCI solution. The heart was perfusion-fixed in vivo with 2 I of 10
buffered formalin at 200 mm Hg pressure. The heart was then removed, and
the coronary arteries were harvested immediately. From the injured segment
(identified in relation to side branches), serial sections of 3-5 mm were
made,
and stored in 10 % buffered formalin solution. The sections were then treated
with incremental concentrations of alcohol followed by treatment with xylene
and paraffin. Slices of 6 ~m thickness were prepared, and stained with
VerhoefFs stain for assessment of tissue response to injury. For each injured
segment, 2 slices demonstrating maximal neointima response were selected
for immunohistochemistry, and the results obtained from analysis of the
cross sections were averaged. The % of reendothelialization and, the % of
endothelial nitric oxide synthase (eNOS) expression were calculated as
follows: (the total length of the luminal surface staining positively / the
perimeter of the lumen) x 100, respectively. Analysis was performed by an
independent examiner with no knowledge of the treatment groups to which
the sections belonged. For lectin immunohistochemistry, the 6 p,m slices
were first treated with hydrogen peroxide and methanol to block endogenous
peroxide, incubated with the Dolichos biflorus agglutinin (Sigma Chemical
Co.) followed by treatment with 3,3'-diaminobenzidine (Vector Laboratories)
and, subsequently counter-stained with hematoxylin. For immunohisto-
chemistry of eNOS expression, after blocking of endogenous peroxide and
non-specific antibodies, the slices were treated serially with the primary
mouse anti-eNOS antibody (Bio/Can Scientific), the secondary goat anti-
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mouse antibody (Vector Laboratories), incubated with avidin-biotin (Vector
Laboratories), treated with 3,3'-diaminobenzidine (Vector Laboratories) and
finally counter-stained with hematoxylin. For both immunohistochemical
examinations, normal porcine carotid artery slices were used as positive
controls; whereas slices obtained from the injured coronary arteries and
stained only with hematoxylin were used as negative controls.
Statistical analysis
Values are expressed as mean t SD. Comparison of basal coronary artery
diameter among the 3 groups was made using the one-way analysis of
variance test. Comparisons between basal coronary artery diameter and
coronary artery diameter following infusion of vasoactive agents were made
with two-tailed Student's t-tests. The Kruskal-Wallis test was used for
comparison of lectin and eNOS expression among the 3 treatment groups.
Linear relationships between lectin expression and response to Ach, and
between eNOS expression and response to Ach were analyzed with Pearson
correlation coefficients. Values were considered to be statistically
significant
if p < 0.05.
Results
There were no significant differences in basal coronary artery diameter
(2.53 t 0.6 mm for 17-beta estradiol, 2.79 t 0.35 mm for PTCA only, and
2.77 t 0.44 mm for vehicle groups respectively, p < 0.4) among the 3
treatment groups. The extent of morphologic tissue injury (9) among the
groups was similar. No changes in heart rate, ECG, or blood pressure were
noted during the local delivery or during intracoronary infusion of vasoactive
agents.
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Response of PTCA only group to Ach
Compared to the basal coronary artery diameter, there were no significant
changes in coronary artery diameter following intracoronary infusion of 10-' M
and 10-6 M concentrations of Ach (Table). At a concentration of 10-4 M a
significant vasoconstrictive response was noted (p < 0.02). A marked
vasoconstrictive response was observed at a concentration of 10'4 M
(p < 0.0001) (Figure 3). The vasoconstriction was completely reversed upon
administration of the endothelium-independent vasodilator nitroglycerin.
Coronary diameter increased from 1.8 t 0.48 mm after 10-4 M Ach, to
2.5 t 0.28 mm following nitroglycerin (p < 0.01; p = 0.2 for post-
nitroglycerin
vs basal diameter).
Response of vehicle treatment group to Ach
Compared to the basal coronary artery diameter, 10-' M Ach did not change
coronary artery diameter in the vehicle treatment group (Table 3). A trend
towards significant vasoconstriction was noted with 10-6 M Ach (p = 0.06).
Significant vasoconstriction was produced by 10-5 M (p < 0.02), and at 10~ M
(p < 0.001) Ach infusion respectively (Figure 3). Nitroglycerin completely
reversed the vasoconstriction, returning the arteries to their basal diameter
(from 1.89 ~ 0.51 mm after 10-4 M Ach, to 2.69 ~ 0.52 mm following
nitroglycerin [p < 0.004; p = 0.7 for post-nitroglycerin vs basal diameter]).
Response of 17-beta estradiol treated group to Ach
In the vessels treated with local delivery of 17-beta estradiol no significant
vasoconstrictive response to Ach occurred at any concentration used (Table)
(Figure 3). A mild and statistically nonsignificant increase in coronary
artery
diameter was observed following administration of nitroglycerin: from 2.28 t
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0.61 mm after 10~ M Ach to 2.61 t 0.48 mm after nitroglycerin (p = 0.4;
p = 0.8 for post-nitroglycerin vs basal diameter).
Immunohistochemistry
5
Immunohistochemical analyses were performed 4 weeks after PTCA on all
9 animals. Three arterial segments were lost/damaged during harvesting of
the samples (2 of PTCA only group, and I of vehicle group). Significant
differences were seen among the 3 treatment groups in the extent of reendo-
10 thelialization, as assessed by immunohistochemical analysis with the lectin
Dolichos biflorus agglutinin (Figure 4). Reendothelialization was noted to a
greater extent in vessels treated with .local delivery of 17-beta estradiol
compared to the other 2 groups (90.6 t 5.5 % for 17-beta estradiol
71 t 6.8 % for PTCA only, and 72.8 t 4.9 % for vehicle, p < 0.0005).
15 Endothelial nitric oxide synthase expression was also higher in vessels
treated with 17-beta estradiol (35.6 t 11.8 % for 17-beta estradiol
9.4 t 3.9 % for PTCA only, and 9.2 t 4.0 % for vehicle, p < 0.0005) (Figure
5). No significant differences in immunohistochemical analyses were
observed between vessels treated with vehicle or PTCA only.
We proceeded further to analyze whether a linear relationship between
reendothelialization and the response to Ach could be demonstrated. A
significant inverse correlation was noted between reendothelialization as
assessed by immunohistochemistry with the lectin Dolichos biflorus
agglutinin and the response to Ach (r = 0.48, p < 0.02) (Figure 6). An even
stronger inverse linear correlation was observed between eNOS expression
and the response to Ach (r = -0.58, p < 0.005).
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Conclusions
This study demonstrates for the first time that local delivery of 17-beta
estradiol immediately following PTCA enhances subsequent
reendothelialization and endothelial function at the site of injury. Besides
its
critical role in the regulation of vascular tone, the normal endothelium
functions as an effective barrier between blood elements and underlying
vascular smooth muscle cells. Endothelium-derived nitric oxide (NO) is a
potent vasodilator, inhibits monocyte adherence and platelet aggregation
and adhesion (10), vascular smooth muscle cell migration (11) and
proliferation (12).
PTCA is associated with arterial injury and damage to the endothelium (3).
Following arterial injury, varying rates of reendothelialization have been
reported. Reendothelialization rates of 81 % (13), and even lower rates of
< 50 % (14) following arterial injury have been observed. In a study of
specimens of restenotic lesions obtained by atherectomy in humans, no
endothelial cells could be demonstrated (15). In the present study, local
treatment with 17-beta estradiol was followed by nearly complete
reendothelialization (90.6 t 5.5 %), which was significantly greater than that
observed in the groups not treated with 17-beta estradiol. Estrogen receptors
have been identified in human coronary artery and umbilical vein endothelial
cells (16), and when bound to estrogen are capable of regulating protein
synthesis by altering transcription rates (17). In cell culture assay of human
umbilical vein endothelial cells, treatment with 17-beta estradiol markedly
increased both cell migration and proliferation (18). Therapy with
subcutaneously implanted 17-beta estradiol pellets significantly enhanced
reendothelialization following arterial injury (6). The capacity of 17-beta
estradiol to increase vascular endothelial growth factor synthesis (19) and
the effort of 17-beta estradiol on basic fibroblast growth factor may be
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22
responsible for the enhanced reendothelialization. Vascular endothelial
growth factor treatment is known to promote reendothelialization in vivo (20).
In human umbilical vein and coronary artery endothelial cell culture
experiments, treatment with 17-beta estradiol enhanced the release and
phosphorylation of basic fibroblast growth factor (21,22). It has been shown
that administration of basic fibroblast growth factor in vivo stimulates
reendothelialization following arterial injury in rats (23). Another mechanism
by which 17-beta estradiol could possibly influence extent of
reendothelialization is by inhibition of apoptosis of injured endothelial
cells:
a 50 % decrease in apoptosis was seen with 17-beta estradiol treatment of
human umbilical vein endothelial cells exposed to tumor necrosis factor-a
(24). It is noteworthy that increased expression of tumor necrosis factors is
known to occur following balloon injury (25)-
Impaired endothelial function, as in atherosclerosis (26) or following
experimental inhibition of NO (27), has been associated with a paradoxical
constrictive response to Ach. This paradoxical response to Ach could be
modified by treatment with estrogen. In humans, 17-beta estradiol
administered intravenously (28) or by continuous intracoronary infusion (29),
attenuated the vasoconstrictive response to Ach and also inhibited the Ach-
induced increase in coronary resistance and decrease in coronary blood
flow. The regulatory effect of 17-beta estradiol on eNOS that we observed
may be responsible for the beneficial effects on endothelial function, as
vascular response to Ach is closely related to eNOS expression (30,31). In
support of this notion, a strong inverse linear relationship was seen between
the vascular response to Ach and eNOS expression (Figure 4). The ability
of estrogen to induce nitric oxide synthase was first identified during
gestation in guinea pigs (32). Induction of eNOS function by 17-beta estradiol
has been subsequently demonstrated to be accompanied by increased
eNOS protein and mRNA expression (33,34). Increased circulating NO levels
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23
have been observed in postmenopausal women treated with 17-beta
estradiol (35). Following arterial injury, the regenerated endothelium is
often
functionally abnormal (5). Abnormal vasomotion as a result of persistent
endothelial dysfunction at the site of angioplasty has been demonstrated in
patients undergoing PTCA, and is postulated to be responsible for the
symptom of angina noted in patients with nonsignificant stenosis following
PTCA (36). We have shown that functional abnormalities could be improved
significantly by treatment with locally delivered 17-beta estradiol. A
unifying
hypothesis for the responses we observed is that eNOS downregulation
following PTCA prevents the vasodilatory response to Ach mediated by
endothelial NO production. By improving eNOS expression, 17-beta estradiol
allows the vasodilatory response of Ach to counteract its direct
vasoconstricting action, preventing Ach-induced vasoconstriction at the site
of local injury. The vasodilatory response to nitroglycerin in Ach-constricted
arteries post-PTCA is consistent with this concept, since exogenous
nitroglycerin (which is a NO donor) simply provides a local NO-related
dilation that the eNOS deficient angioplastied segment cannot provide for
itself.
Both rapid non-genomic and genomic effects have been postulated to be
involved in the influence of 17-beta estradiol on coronary vasculature
(37,38). Although increased protein synthesis was not quantified in the
present study, the enhanced eNOS expression and the response to Ach
observed as late as 28 days following a single dose of 17-beta estradiol
appears to be consistent with a genomic effect. This is the first study to
suggest the existence of a genomic effect following local therapy with 17-
beta estradiol in coronary circulation in vivo.
Gender differences in the endothelium-dependent vasodilation by 17-beta
estradiol have been noted (39). In our study, a majority of animals were
CA 02381031 2002-03-18
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24
mates and a significant beneficial effect of 17-beta estradiol was noted in
all
the animals studied, irrespective of sex. Thus, local delivery of 17-beta
estradiol appears to be effective in males as well as females. There is
evidence to suggest that the simultaneous administration of progesterone
reduces NO levels induced by 17-beta estradiol (35), this issue was,
however, beyond the scope of the present study.
We conclude that a single dose of 17-beta estradiol delivered locally
following balloon injury can significantly improve reendothelialization and
enhance endothelial function at the injured site as late as 1 month following
injury. Besides the beneficial vascular effects of improved endothelial
function, this observation may be of particular importance following balloon
angioplasty as improved endothelial function is known to be associated with
decreased neointima formation in the injured area (20,40). This approach
merits further study, with a view to potential clinical value in the
prevention
of vascular dysfunction and restenosis following PTCA.
Formulations
The formulations may include estradiol or a derivative thereof and any
pharmaceutically acceptable vehicle. Since estradiol is a lipophilic molecule,
such vehicle would ideally include a solvent component. Such a solvent
component includes molecules such as propylene glycol, ethanol, and
detergents, for example PluronicsT"". The formulations may take the form of
a liquid, a suspension, a semi-solid or a thermoreversible composition which
may form a layer over the endothelium. The formulations may further be
included in or used as a coating for a device such as a stent, or be part of
any similar device that can be left in-situ upon angioplasty or vascular
surgery.
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Although the present invention has been described hereinabove by way of
preferred embodiments thereof, these embodiments can be modified at will,
without departing from the spirit and nature of the subject invention. Such
modifications are within the scope of the present invention as defined in the
5 appended claims.
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26
Table 1: Morphometric Analysis
Characteristics 17-beta estradiolPTCA only Vehicle p value*
alone
Segments analyzed12 9 10 NS
Artery size (mm)2.86 0.35 2.94 0.24 2.94 0.41 NS
Balloon/Artery 1.22 0.09 1.2 t 0.06 1.17 t 0.11NS
ratio
EEL~ef/EEL;~~ 1.01 0.16 1.31 0.37 1.16 0.28 NS
t
Neointima area 0.4 t 0.3 0.88 0.61 1.14 t 1.03< 0.05
(mmz)
neointima 12.16 t 8.8923.02 t 11.9125.46 14.96< 0.025
Neointima/Media 0.59 t 0.48 1.67 t 1.29 1.75 t 1.29< 0.01
area
stenosis 15.67 11.1327.51 13.1730.34 17.05< 0.025
Restenotic index1.3 t 0.5 2.4 0.68 2.42 0.71 < 0.005
Injury score 1.64 0.34 1.7 0.43 1.77 0.47 NS
* 17-beta estradiol vs other 2 groups; tEEL,~ = proximal reference segment
external elastic
lamina area, EEL;~~ = injured segment external elastic lamina area (averaged).
Table 2: Response to 17-beta estradiol According to Sex of the Animal
Characteristics Male Female p value
Restenotic index 1.2 t 0.59 1.37 0.45 > 0.1
Neointima area (mm2)0.51 0.34 0.25 0.15 > 0.1
Neointima/Media 0.78 0.55 0.32 0.16 > 0.1
area
neointima 14.93 10.68 8.29 3.72 > 0.1
stenosis 18.93 t 13.39 11.09 t 5.16> 0. I
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27
Table 3: Response to Intracoronary Acetylcholine
Ach* Diameter-basal Diameter-post value
Ach p
(mm) (mm)
PTCA group
10-'M 2.79 0.35 2.65 0.35 0.4
10-6M 2.79 0.35 2.54 0.32 0.1
10-5M 2.79 0.35 2.3 0.35 0.02
10~M 2.79 0.35 1.8 0.48 0.0001
Vehicle group
10-'M 2.770.44 2.60.41 0.4
10-6M 2.77 0.44 2.33 0.5 0.06
10-5M 2.77 0.44 2.24 0.47 0.02
10-4M 2.77 0.44 1.89 0.51 0.001
17-beta estradiol
group
10-'M 2.53 0.6 2.46 0.58 0.8
10-6M 2.53 0.6 2.38 0.58 0.6
10-5M 2.53 0.6 2.36 0.59 0.6
10-4M 2.53 0.6 2.28 0.61 0.4
* acetylcholine
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28
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