Note: Descriptions are shown in the official language in which they were submitted.
CA 02282982 1999-09-21
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TITLE OF THE INVENTION
Local Delivery of 17 - beta Estradiol During Balloon Angioplasty for
Preventing
Restenosis.
BACKGROUND OF THE INVENTION
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 (6). 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.
DESCRIPTION OF INVENTION
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 nifedipine orally, were premeditated 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 percutaneously.
An 8 Fr
arterial sheath was introduced, and 100 mg/kg of lidocaine and 250 U/kg of
heparin were
'CA 02282982 1999-09-21
, _ 2 _
administered intra-arterially. Additional heparin was administered during PTCA
if
needed, to maintain an activated coagulation time of > 300 seconds
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 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
arteries of each animal. For local delivery, the InfusaSleeve catheter
(L,ocaIMed Ine.) 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 pg of
17 - beta estradiol (in 5 ml), vehicle alone (5 ml), or PTCA only. The vehicle
2-
hydroxypropyl-beta-cyclodextrin (f~CD), and 17 - beta estradiol were obtained
from
Sigma Chemical Co. For local delivery with the InfusaSleeve catheter, a
proximal driving
pressure of 10 atm and support balloon pressure of 6 atm wee 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 of a
coronary artery
was performed with a single lumen balloon catheter (TotalCross, Schneider) for
the
administration of vasoactive agents. Acetylcholine (Ach) in increasing
concentrations of
ACA 02282982 1999-09-21
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10'' M, 10~ M, 10'3 M, and 10~ 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
mUmin using an infusion pump. Coronary angiography was performed at the end of
each
dose. After infusion of the highest concentration of Ach (10'~ Ivl) and
angiography, 100
pg 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 (I~-76, Mallinckrodt Medical Inc) was used throughout the experiment.
Images
were captured at a frame speed of 30 frameslsec, 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-511ed
guiding catheter as the reference segment, to avoid error due to
magnification. Coronary
artery diameter measurements were made using a validated computerized edge-
detecxion
system (8). The midpoint of the injured segment was used for calculation of
coronary
artery diameter. For each analysis, coronary artery diameter measurements wen
~.CA 02282982 1999-09-21
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performed in 3 consecutive end-diastolic frames, and the results averaged.
Measurements
were performed by an independent observer blinded to the treatment goup of the
vessels.
Immunohistoc6emistry
The animals were euthanized at 4 weeks. Under general anaesthesia as described
above,
exsanguination was performed with replacement by 1 1 of 0.9 % NaCI solution.
The heart was
perfusion-fixed in vivo with 21 of 10 % bufr'ered 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 pm
thickness were prepared,
and stained with Verhoef~s stain for assessment of tissue response to injury.
For each injured
segment, 2 slices demonstrating maximal neointimal 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 lrm slices were first treated with hydrogen
peroxide and methanol
to block endogenous peroxide, incubated with the Dolichos b~orus agglutinin
(Sigma Chemical
Co.) followed by treatment with 3,3'-diaminobenzidine (Vector Laboratories)
and, subsequently
counter-stained with hematoxylin. For immunohistochemistry of eNOS expression,
after blocking
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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-
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 Kruslcal-
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
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goups respectively, p = 0.4) among the 3 treatment goups. The extent of
morphologic
tissue injury (9) among the goups was similar. No changes in heart rate, ECG,
or blood
pressure were noted during the local delivery or during intracoronary infusion
of
vasoactive agents.
Response of PTCA only group to Ac6
Compared to the basal coronary artery diameter, there were no significant
changes in
coronary artery diameter following intracoronary infusion of 10'' M, and 10~ M
concentrations of Ach (Table). At a concentration of 10'3 M, a significant
vasoconstrictive response was noted (p < 0.02). A marked vasoconstrictive
response was
observed at a concentration of 10'~ M (p < 0.0001) (Figure 1). 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~ 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 goup (Table). A trend towards
significant
vasoconstriction was noted with 10~ M Ach (p = 0.0~. Significant
vasoconstriction was
produced by 10'3 M (p < 0.02), and at 10'~ M (p < 0.001) Ach infusion
respectively
(Figure 1). Nitroglycerin completely reversed the vasoconstriction, returning
the arteries
~
. CA 02282982 1999-09-21
to their basal diameter (from 1.89 t 0.51 mm after 10'~ M Ach, to 2.69 t 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 Ac6
In the vessels treated with local delivery of 17 - beta estradiol, no
significant
vasoconstrictive response to Ach occurred at any concentration used (Table)
(Figure 1).
A mild and statistically nonsignificant increase in coronary artery diameter
was observed
following administration of nitroglycerin: from 2.28 t 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
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 goup, and 1 of vehicle goup). Significant differences were seen among the
3
treatment goups in the extent of re-endothelialization, as assessed by
immunohistochemical analysis with the lectin Dolichos b~orus agglutinin
(Figure 2).
Re-endothelialization was noted to a Beater 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).
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
~
.CA 02282982 1999-09-21
_ g _
t 4.0 % for vehicle, p < 0.0005) (Figure 3). 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 b~orus agglutinin and the response to Ach (r = -
0.48, p < 0.02)
(Figure 4). An even stronger inverse linear correlation was observed between
eNOS
expression and the response to Ach (r = -0.58, p < 0.005).
Discussion
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
'CA 02282982 1999-09-21
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arterial injury have been observed. In a study of specimens of restenotic
lesions obtained
by atherectomy in humans, no endothelial cells could be demonstrated (1~. 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 (1~, 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 (~. The capacity of 17 - beta
estradiol to
increase vascular endothelial growth factor synthesis (19) and the effect of
17 - beta
estradiol on basic fibroblast growth factor may be 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
'CA 02282982 1999-09-21
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(24). It is noteworthy that increased expression of tumor necrosis factor~c is
known to
occur following balloon injury (25).
Impaired endothelial function, as in atherosclerosis (2~ 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 have been observed in
postmenopausal women treated with 17 - beta estradiol (35). Following arterial
injury,
the regenerated endothelium is often functionally abnormal (~. 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 (3~.
We have shown that functional abnormalities could be improved significantly by
treatment with locally delivered 17 - beta estradiol. A unifying hypothesis
for the
~
. CA 02282982 1999-09-21
- 11 -
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 males, 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.
CA 02282982 1999-09-21
_12_
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.
Although the present invention has been described hereinabove by way of a
preferred
embodiment thereof, this embodiment can be modified at will, within the scope
of the
appended claims, without departing from the spirit and nature of the subject
invention.
'CA 02282982 1999-09-21
- 13 -
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CA 02282982 1999-09-21
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'-CA 02282982 1999-09-21
- 18 -
Legends
Figure 1: Representative coronary angiograms demonstrating the
vasoconstrictive
response to intracoronary infusion of acetylcholine (Ach) 10'~ M, 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 2: Representative light micrographs (x 1000) of cross sections of
vessels obtained
from the same animal, for immunohistochemical staining with the lectin
Dolichas
b~orus 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) goups.
Figure 3: Representative light micrographs (x 1000) of cross sections of
vessels obtained
from the same animal, for immunohistochemical analysis 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 4: 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 <
~
CA 02282982 1999-09-21
- 19 -
0.005). Note: % vasoconstriction denotes % decrease in diameter following Ach
10'~ M
as compared to the basal diameter.
ACA 02282982 1999-09-21
- 20 -
Table: Response to Intracoronary Acetylcholine
Ach* Diameter-basal Diameter-post Ach p value
(mm) (mm)
PTLA group
10'~M 2.7910.35 2.6510.35 0.4
10~M 2.7910.35 2.5410.32 0.1
10'sM 2.79 t 0.35 2.3 t 0.35 0.02
10'4M 2.79 t 0.35 1.8 t 0.48 0.0001
Vehicle group
10'~M 2.77 t 0.44 2.6 t 0.41 0.4
lO~IVI 2.77 f 0.44 2.33 t 0.5 0.06
10'sM 2.77 f 0.44 2.24 t 0.47 0.02
10"*M 2.77 t 0.44 1.89 t 0.51 0.001
17 - beta estradiol
Sr~P
10'~M 2.53 t 0.6 2.46 t 0.58 O.g
10'6M 2.5310.6 2.3810.58 0.6
10'sM 2.53 t 0.6 2.36 t 0.59 0.6
10'4M 2.5310.6 2.2810.61 0.4
* acetylcholine
