Conjugated Estrogens Acutely Abolish Abnormal Cold-Induced Coronary

Vasoconstriction in Male Cardiac Allografts

Steven E. Reis, Vishwajeth Bhoopalam, Kathleen A. Zell, Peter J. Counihan, A. J. Conrad
Smith, Si Pham and Srinivas Murali

Circulation. 1998;97:23-25
doi: 10.1161/01.CIR.97.1.23
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Copyright © 1998 American Heart Association, Inc. All rights reserved.
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 Conjugated Estrogens Acutely Abolish Abnormal
Cold-Induced Coronary Vasoconstriction in Male
Cardiac Allografts
Steven E. Reis, MD; Vishwajeth Bhoopalam, MD; Kathleen A. Zell, BSN; Peter J. Counihan, MD;
A.J. Conrad Smith, MD; Si Pham, MD; Srinivas Murali, MD

Background—Transplant-associated coronary arteriopathy is manifested in its early stages by paradoxical coronary artery
constriction in response to endothelium-dependent vasodilator stimuli such as the cold pressor test (CPT) and is a major
cause of death or retransplantation. Estrogen has vasoactive properties that abolish coronary artery endothelial dysfunction
in native hearts. We hypothesized that estrogen attenuates inappropriate coronary artery constriction in cardiac allografts.
Methods and Results—Coronary artery diameter and systemic hemodynamic responses to a 90-second CPT were measured
before and 15 minutes after double-blind, randomized administration of intravenous conjugated estrogens (1.25 mg) or
placebo in men with male cardiac allografts. Before estrogen, 9 men exhibited an abnormal 15.163.0% CPT-induced
decrease in coronary artery diameter. However, repeat CPT did not induce significant coronary artery constriction when
performed 15 minutes after estrogen. CPT responses before and after estrogen were significantly different (P5.02). Placebo
did not influence coronary artery responses to CPT in 6 men. Systemic hemodynamic responses to CPT were not
influenced by estrogen or placebo. Estrogen was the only significant determinant of changes in coronary artery responses
to CPT.
Conclusions—Conjugated estrogens acutely abolish abnormal CPT-induced coronary artery constriction in male cardiac
allografts. This favorable vasomotor effect suggests that estrogen may prevent inappropriate coronary artery constriction in
men with cardiac transplants. (Circulation. 1998;97:23-25.)
Key Words: transplantation n hormones n endothelium n coronary disease n atherosclerosis

A llograft coronary arteriopathy is a major cause of death or

retransplantation in cardiac transplant recipients.1 Early
transplant-associated arteriopathy is manifested by coronary
informed consent approved by the University of Pittsburgh Biomed-
ical Institutional Review Board.

artery endothelial dysfunction, which precedes the develop-
ment of angiographically documented disease.2 This physio-
logical abnormality may be detected in cardiac allografts by
measurement of coronary artery responses to endothelium-
dependent vasodilator stimuli, such as the cold pressor test
(CPT), that induce dilatation in coronary arteries with intact
endothelium and paradoxical constriction in those with endo-
thelial dysfunction.3
Estrogen has vasomotor properties that prevent in vitro
arterial constriction and inappropriate coronary artery constric-
tion in native human hearts.4 –12 Estrogen-induced attenuation
of abnormal coronary vasoreactivity may provide a clinical
cardioprotective effect because inappropriate coronary artery
constriction has been implicated in the pathogenesis of acute
cardiac syndromes. We hypothesized that estrogen attenuates
inappropriate coronary artery constriction in cardiac allografts.
Methods
Cardiac transplant recipients undergoing routine posttransplant coro-
nary angiography were eligible to participate. All subjects gave written
Assessment of Coronary Hemodynamics
Vasodilator drugs were discontinued for at least 24 hours. Routine
coronary angiography was performed by standard techniques, and a
“normal” coronary artery was selected for assessment. Heart rate and
systemic blood pressure were recorded, and coronary artery diameter
was measured at a fixed anatomic location relative to a branch point by
quantitative angiography (AWOS, Siemens Medical Systems) per-
formed by an investigator (V.B.) blinded to treatment group. The
reliability (ratio of the inherent variance of the measurement between
patients to the variance from all sources) is 0.94, as assessed by analysis
of the variance components.
Effects of Estrogen on Coronary Artery
Responses to the CPT
After baseline hemodynamics and coronary artery diameter were
measured, a CPT was performed with the subject’s hand immersed in
ice water for 90 seconds. At the conclusion of the CPT, systemic
hemodynamics and coronary artery diameter were reassessed. Intra-
venous conjugated estrogens (Premarin 1.25 mg, Wyeth-Ayerst) or
placebo was administered in a double-blind, randomized fashion.
Fifteen minutes later, hemodynamics and coronary artery diameter
were reassessed at baseline and after a repeat CPT. Intrinsic vasodilator

Received September 11, 1997; revision received October 24, 1997; accepted October 27, 1997.
From the Division of Cardiology, Department of Medicine and the Division of Cardiothoracic Surgery, Department of Surgery (S.P.), University of
Pittsburgh (Pa) School of Medicine.
Correspondence to Steven E. Reis, MD, University of Pittsburgh Medical Center, 200 Lothrop St, Pittsburgh, PA 15213.
© 1998 American Heart Association, Inc.

23
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24 Estrogens Abolish Vasoconstriction

TABLE 1. Demographic Characteristics P,.01) in response to the initial CPT but no significant
Estrogen Placebo
diameter change in response to repeat CPT performed 15
(n59) (n56) minutes after estrogen (3.160.3 to 3.260.2 mm, P5NS).
Age, y 54.062.8 53.763.9
Coronary artery constriction elicited by the first CPT was
significantly different from the coronary artery response elicited
Time since transplantation, y 4.660.8 4.860.8
by the postestrogen CPT (P5.02).
Hyperlipidemia, % 33.3 20.0
Placebo did not influence coronary artery responses to CPT.
Diabetes, % 11.1 16.7
Before placebo, the studied men exhibited an insignificant
Hypertension, % 100.0 66.7 9.063.8% decrease in coronary artery diameter (from 2.760.4
Rejection, % 33.3 20.0 to 2.560.4 mm, P5.07) in response to CPT. Fifteen minutes
Baseline hemodynamics after placebo, their coronary artery diameters also did not
Coronary diameter, mm 3.360.3 2.760.4 change significantly in response to repeat CPT (2.560.4 to
Mean blood pressure, mm Hg 113.465.9 111.565.0 2.560.4 mm, P5NS). Coronary artery responses to the CPT
Heart rate, bpm 78.863.0 76.065.7 did not differ before and after placebo.
Coronary diameter response to NTG, % 15.166.3 13.464.4 Multivariate analysis demonstrated that estrogen was the
Initial CPT-induced changes only significant determinant of changes in coronary artery
Coronary diameter, mm 15.163.0 9.063.8
diameter responses to CPT. Other variables, including age,
baseline coronary artery diameter, systemic hemodynamic
Mean blood pressure, mm Hg 11.362.2 17.163.0
response to CPT, intrinsic coronary artery vasodilator capacity,
Heart rate, bpm 2.760.7 5.860.8
and degree of rejection did not influence coronary artery
NTG indicates Intracoronary nitroglycerin (200 mg); CPT, cold pressor test. responses to CPT.
There were no statistically significant differences between groups.

Effects of Estrogen on CPT-Induced Changes in

capacity was evaluated by measuring coronary artery diameter after
intracoronary nitroglycerin (200 mg).
Statistical Analysis
The data are expressed as mean6SEM. Values of P#.05 are consid-
ered significant. Only subjects who exhibited no change or a para-
doxical decrease in coronary artery diameter in response to CPT were
selected for analysis, because we previously demonstrated that estrogen
does not improve vasoreactivity in coronary arteries that dilate
normally in response to an endothelium-dependent stimulus.8 Baseline
characteristics were compared between groups by x 2 analysis and
Fisher’s exact test and the independent Student’s t test. Within each
group, the influence of CPT on hemodynamics and coronary artery
diameter was assessed by the paired Student’s t test.
To assess the influence of pharmacological intervention (estrogen or
placebo) on coronary artery responses to CPT, we first calculated the
difference between coronary artery diameter after CPT and at baseline
(DD5diameterCPT2diameterbaseline). The effects of intervention on
coronary artery diameter were then quantified by calculation of the
difference between DD before and after intervention [D(DD)5DDafter
intervention2DDbefore intervention] followed by linear regression analysis.
Results
Twenty-seven patients were enrolled and randomized to
estrogen or placebo. Fifteen exhibited abnormal CPT-induced
coronary artery constriction and were included in the analysis.
All subjects were men, because no postmenopausal female
cardiac transplant recipients underwent coronary angiography
during the study. All allografts were from male donors. Nine of
the analyzed subjects received estrogen, and 6 received pla-
cebo. The Table demonstrates no significant differences in
baseline characteristics between groups.
Effects of Estrogen on CPT-Induced Coronary
Artery Constriction
Conjugated estrogens acutely abolished abnormal CPT-in-
duced coronary artery constriction in male cardiac allografts.
Men assigned to estrogen exhibited a 15.163.0% decrease in
coronary artery diameter (from 3.360.3 to 2.860.3 mm,
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Systemic Hemodynamics
CPT-induced endothelium-dependent vasodilatation requires
a cold-induced increase in catecholamines, which may be
assessed indirectly by measurement of changes in blood pres-
sure. In subjects assigned to estrogen, baseline CPT induced a
mean 11.362.2% increase in mean blood pressure (from
113.465.9 to 126.266.9 mm Hg, P,.01), which was not
significantly different from the 12.563.1% increase (from
118.065.4 to 132.967.2 mm Hg, P,.01) induced by repeat
CPT performed 15 minutes after estrogen. In placebo subjects,
baseline CPT induced a 17.163.0% increase in mean blood
pressure (from 111.565.0 to 130.064.3 mm Hg, P,.01),
which did not differ from the 23.467.3% increase (from
107.765.2 to 131.564.9 mm Hg, P5.01) induced by repeat
CPT performed after placebo. These blood pressure responses
to CPT are similar to those observed in patients who did not
receive transplants.10,11
Although our transplant patients exhibited a normal blood
pressure response to CPT, cardiac allografts are denervated and
may not manifest an appropriate chronotropic response to
adrenergic stimulation. In the men assigned to estrogen,
baseline CPT induced a 2.760.7% increase in heart rate (from
78.863.0 to 80.963.1 bpm, P,.01), and postestrogen CPT
induced a similar 3.261.3% increase in heart rate (from
77.163.0 to 79.462.8 bpm, P5.04). Placebo patients in-
creased their heart rate by 5.860.8% (from 76.065.7 to
80.365.9 bpm, P,.01) in response to preplacebo CPT and
did not exhibit a significant change in heart rate (from
79.366.0 to 80.066.3 bpm, P5NS) in response to postpla-
cebo CPT. Overall, the study population had smaller CPT-
induced increases in heart rate than a nontransplant po-
pulation.10,11 However, heart rate is not a determinant of
baseline coronary artery diameter or of coronary artery re-
sponses to CPT.
 Reis et al
Discussion
This study demonstrates that estrogen attenuates abnormal
coronary artery vasoreactivity in male cardiac allografts. Intra-
venous conjugated estrogens acutely abolished the abnormal
15.1% decrease in coronary artery diameter induced by the
CPT in men with cardiac transplants. This finding was
independent of cold-induced changes in systemic hemody-
namics and degree of rejection. In contrast, placebo did not
influence coronary artery responses to exogenous cold
exposure.
Previous studies of transplant recipients have described
abnormal coronary artery constriction in response to exoge-
nous cold exposure that has been attributed to immune
system–mediated graft endothelial dysfunction.13,14 The present
study confirms this finding and demonstrates that it may be
acutely abolished by intravenous conjugated estrogens. Al-
though we used conjugated estrogens because they contain a
potent vasoactive compound (17a-dihydroequilenin), it is
possible that multiple components of this preparation influ-
enced coronary artery responses to CPT. The favorable vaso-
motor effects of estrogen in cardiac allografts may be related to
estrogen-induced inhibition of endothelin-1 and calcium-
mediated vasoconstriction, an increase in arterial endothelial
nitric oxide, and/or alteration of arterial myocyte ATP-
sensitive potassium channels.4 –7
Another possible explanation for our findings is that estro-
gen may lessen the degree of cold-induced sympathetic stim-
ulation of the coronary arteries.15 Although we did not
measure catecholamine responses to each of the two CPTs, we
demonstrated that estrogen does not affect CPT-induced
changes in blood pressure, which is a physiological surrogate
marker of catecholamines. Therefore, it is unlikely that inhi-
bition of cold-induced sympathetic stimulation explains the
observed favorable effect of estrogen.
Our study is limited by our use of the CPT to assess
coronary vasoreactivity. Cardiac allografts are denervated, as
manifested in this study by their attenuated chronotropic
response to CPT. However, a- and b-adrenergic receptors on
graft coronary arteries regulate vasomotor tone and respond to
circulating catecholamines in an endothelium-dependent man-
ner independent of cardiac denervation. In addition, our
findings are consistent with those reported in coronary arteries
of native hearts.10,11
Estrogen modulates the immune system and inhibits in vivo
allograft rejection. Lou and colleagues16 demonstrated that
chronic estradiol treatment abolishes MHC class II antigen
expression, decreases cellular infiltration of the arterial wall,
and inhibits myointimal proliferation in coronary arteries of
New Zealand White rabbits that underwent cardiac transplan-
tation. Local administration of estrogen has also been shown to
inhibit insulin-like growth factor I–induced atherosclerosis in
rat orthotopic abdominal aortic allotransplants.17 These find-
ings suggest that chronic estrogen therapy may be a novel
approach to inhibiting the progression of coronary arteriopathy
in human cardiac allografts. Our results demonstrate that
estrogen also acutely prevents inappropriate coronary artery
constriction. Although it is not known whether these results
may be extrapolated to chronic estrogen therapy, estrogen-
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25
induced improvement in coronary artery vasomotor tone may
also provide a cardiovascular benefit in cardiac transplant
patients. Other studies have demonstrated that nonfeminizing
phytoestrogens have favorable vascular properties,18 suggesting
their potential utility in men. Therefore, estrogen should be
evaluated as a novel antiatherosclerotic therapy in male and
female transplant recipients.
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