A Novel Role for an Endothelial Adrenergic Receptor
System in Mediating Catecholestradiol-Induced Proliferation
of Uterine Artery Endothelial Cells
Sheikh O. Jobe, Sean N. Fling, Jayanth Ramadoss, Ronald R. Magness
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Abstract—Sequential conversion of estradiol-17␤ to its biologically active catecholestradiols, 2-hydroxyestradiol (OHE2)
and 4-OHE2, contributes importantly to its angiogenic effects on uterine artery endothelial cells (UAECs) derived from
pregnant, but not nonpregnant ewes via an estrogen receptor-independent mechanism. Because catecholestradiols and
catecholamines exhibit structural similarities and have high affinity for ␣- and ␤-adrenergic receptors (ARs), we
investigated whether the endothelial ␣- or ␤-ARs mediate catecholestradiol-induced proliferation of P-UAECs and
whether catecholamines alter these responses. Western analyses revealed expression of specific AR subtypes in
nonpregnant UAECs and P-UAECs, including ␣2-, ␤2-, and ␤3-ARs but not ␣1- and ␤1-ARs. Levels of ␤2-ARs and
␤3-ARs were unaltered by pregnancy, whereas ␣2-ARs were decreased. Norepinephrine and epinephrine increased
P-UAEC, but not nonpregnant UAEC proliferation, and these effects were suppressed by propranolol (␤-AR blocker)
but not phentolamine (␣-AR blocker). Catecholamines combinations with 2-OHE2 or 4-OHE2 enhanced P-UAEC
mitogenesis. Catecholestradiol-induced P-UAEC proliferation was also inhibited by propranolol but not phentolamine.
␤2-AR and ␤3-AR antagonists (ICI 118 551and SR 59230A, respectively) abrogated the mitogenic effects of both
2-OHE2 and 4-OHE2. Stimulation of ␤2-ARs and ␤3-ARs using formoterol and BRL 37344 dose-dependently stimulated
P-UAEC proliferation, which was abrogated by ICI 118 551 and SR 59230A, respectively. Proliferation effects of both
catecholamines and catecholestradiols were only observed in P-UAECs (not nonpregnant UAECs) and were mediated
via ␤2-ARs and ␤3-ARs. We demonstrate for the first time convergence of the endothelial AR and estrogenic systems
in regulating endothelial proliferation, thus providing a distinct evolutionary advantage for modulating uterine perfusion
during stressful pregnancies. (Hypertension. 2011;58:00-00.) ● Online Data Supplement
Key Words: catecholamines 䡲 catecholestradiols 䡲 adrenergic receptors 䡲 endothelial proliferation
stradiol-17␤ (E2␤) has physiological/pathophysiologic
effects on the cardiovascular system via diverse mechanisms, including local conversion to catecholestradiols
2-hydroxyestradiol (OHE2) and 4-OHE2 by cytochrome
P450s.1,2 Catecholestradiols improve endothelial function and
alleviate hypertension in ZSF1 rats, an animal model for
hypertension and metabolic syndrome.2,3 We reported that
E2␤, 2-OHE2, and 4-OHE2 increase proliferation in ovine
uterine artery endothelial cells (UAECs) derived from pregnant (P-UAECs), but not the nonpregnant (NP-UAECs)
ewes.4 Unlike E2␤, 2-OHE2- and 4-OHE2-induced P-UAEC
proliferation were not blocked by ICI-182 780, demonstrating
that catecholestradiol-induced P-UAEC proliferation was estrogen receptor independent.4
Catecholestradiols exhibit close structural similarities (Figure 1) and functional interactions with the norepinephrine and
epinephrine.5 Because of the shared phenolic A ring “catechol” moiety, catecholestradiols interact directly with cate-
cholamine responses, a property not shared by E2␤.5 Catecholestradiols compete with high affinity for binding to
neuroendocrine enzymes, ␣-adrenergic receptors (ARs) and
␤-ARs in the hypothalamus, anterior pituitary, corpus striatum, and liver.5–10 3D structural and functional analyses
demonstrate that the catecholamine catechol moiety is functionally important in AR activation.11,12 Thus, it is conceivable that the estrogen receptor–independent mitogenic effects
of 2-OHE2 and 4-OHE2 on the uterine endothelium may be
mediated by ␣-ARs or ␤-ARs, and they may directly interact
with the catecholamines that endogenously activate ARs.
However, there are no reports on the potential role of an
endothelial AR system in catecholestradiol-induced
proliferation.
We hypothesized that catecholestradiols stimulate
P-UAEC proliferation via ␣-ARs and/or ␤-ARs and that
catecholamines that directly activate these ARs will alter
these responses. We investigated the following: (1) NP-
E
Received June 13, 2011; first decision June 30, 2011; revision accepted August 30, 2011.
From the Departments of Obstetrics and Gynecology (S.O.J., S.N.F., J.R., R.R.M.), Perinatal Research Laboratories, Pediatrics (R.R.M.) and Animal
Sciences (R.R.M.), University of Wisconsin-Madison, Madison, WI; Spelman College (S.N.F.), Atlanta, GA.
Correspondence to Ronald R. Magness, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, Atrium B. Meriter Hospital, 202
S Park St, Madison, WI 53715. E-mail rmagness@ wisc.edu
© 2011 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.111.178046
1
2
Hypertension
November 2011
Catecholamines
Catecholestradiols
Estradiol-17β
OH
2-hydroxyestradiol
OH
4-hydroxyestradiol
Epinephrine
Norepinephrine
OH
C H3
NH
NH 3
OH
OH
HO
HO
HO
HO
HO
HO
HO
HO
HO
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
Receptors
Estradiol-17β
Catecholestradiols
Catecholamines
ER -α/ER -β
YES
NO
NO
α-AR/ β-AR
NO
??
YES
Figure 1. Adding 1 hydroxy (OH) group modifies the estrogenic phenolic A ring forming “catechol” moieties, potentially making it an
adrenergic receptor “(AR)-mimetic” agent for inducing estrogen receptor (ER)-independent angiogenesis. The boxes outline the shared
phenolic A ring catechol moiety among 2-hydroxyestradiol, 4-hydroxyestradiol, norepinephrine, and epinephrine. ?? indicates hypotheses tested.
UAEC versus P-UAEC subtype-specific expression of ␣1-,
␣2-, ␤1-, ␤2-, and ␤3-ARs; (2) whether norepinephrine and
epinephrine stimulate NP-UAEC versus P-UAEC proliferation and the interactive effects of catecholamines and catecholestradiol in mitogenesis; (3) whether catecholestradiols
and catecholamines stimulate P-UAEC proliferation via
␣-ARs or ␤-ARs; (4) whether ARs exhibit subtype specificity
in catecholestradiol-induced P-UAEC proliferation; and (5)
subtype-specific pharmacological activation of ARs on
P-UAEC proliferation. We report for the first time specific
␤2-AR and ␤3-AR subtype-mediated mechanisms for the
novel convergence of the endothelial AR system with estrogen metabolites in regulating P-UAEC, but not NP-UAEC
proliferation. We discovered in P-UAECs a specific codependence via conserved catechol phenolic moieties (derivatives
of both E2␤ and tyrosine) of a unique ␤-AR– coupled system.
This may provide for a distinct gestational evolutionary
advantage by modulating uterine angiogenesis to help/maintain fetal developmental well being during periods of repeated bouts of stress releases of catecholamines as seen
during “fight or flight” responses.
Methods
For complete details, please see the online Data Supplement at
http://hyper.ahajournals.org.
Cell Preparation and Culture
Protocols were approved by the University of Wisconsin-Madison
Animal Care Committee.4,13 NP-UAECs and P-UAECs were isolated and cultured from nonpregnant (n⫽6) and late-gestation (n⫽6)
ewes.4 At passage 4 (⬇70%) confluence, UAECs were lysed for
Western analyses or transferred to 96-well plates for experimental
treatments.
Western Analyses
Western analyses were performed4 using rabbit anti–␣1-AR, anti–
␣2-AR, anti–␤1-AR, anti–␤2-AR, or anti–␤3-AR antibodies (1:500)
and secondary antibodies (1:2000). ␤-Actin and GAPDH were used
as loading controls.
5-Bromodeoxyuridine Cell Proliferation Assays
5-Bromodeoxyuridine assay was performed and validated as described previously.4
Experimental Treatments
Proliferation experiments were performed in quadruplicates and
replicated in ⱖ4 different preparations. NP-UAECs and P-UAECs in
96-well plates were serum starved (24 hours) and washed in
endothelial basal media, and medium was replaced with endothelial
basal media containing 0.0, 0.1, 1.0, 10.0, or 100.0 nmol/L of
norepinephrine or epinephrine (24 hours). Additional P-UAEC studies investigating interactions were performed by combination treatments (0.1 nmol/L) of 2-OHE2 or 4-OHE2 with norepinephrine or
epinephrine. ␣-ARs and/or ␤-ARs were blocked nonselectively by
pretreating P-UAECs (10 ␮mol/L; 1 hour) with phentolamine (␣-AR
blocker) or propranolol (␤-AR blocker) followed by norepinephrine,
epinephrine, 2-OHE2, or 4-OHE2 (0.1 nmol/L; 24 hours). Based on
Western analyses of specific AR subtypes, we conducted AR
subtype-specific blockade using (10 ␮mol/L; 1 hour) yohimbine
(␣2-AR), ICI 118 551(␤2-AR), and SR 59230A (␤3-AR), followed
by catecholestradiol treatments (0.1 nmol/L; 24 hours). We validated
AR subtype-specific inhibition for P-UAEC mitogenic responses
(0.0, 0.1, 1.0, 10.0, and 100.0 nmol/L) using specific ␤2-AR agonist
formoterol and ␤3-AR agonist BRL 37344. ICI 118 551 and SR
59230A (10 ␮mol/L) effects, respectively on, formoterol and BRL
37344 (100 nmol/L) were utilized for specificity validation of ␤2-AR
and ␤3-AR agonists in P-UAECs. For antagonists/agonist specificities, please see the online Data Supplement.
Statistical Analysis
Data means⫾SEM are presented as a fold of untreated control.
Overall group differences were determined by 1-way or 2-way
Jobe et al
A
P-UAECs
Positive
Control
Catecholestradiols and Adrenergic Receptors
B
NP -UAECs
3
P-UAECs
VSM
α1-AR
52 kDa
α2-AR
70 kDa
70 kDa
β2-AR
68 kDa
65 kDa
β3-AR
44 kDa
68 kDa
GAPDH
37 kDa
VSM
α2-AR
CMC
β1-AR
VSM
β2-AR
AT
β3-AR
44 kDa
β-Actin
42 kDa
Figure 2. Adrenergic receptor (AR) subtypes in uterine artery endothelial cells (UAECs). A, Western blots demonstrating expression of
␣2-ARs, ␤2-ARs, and ␤3-ARs but not ␣1-AR or ␤1-AR subtypes in pregnant (P)-UAECs. Positive control lanes are vascular smooth muscle cells (VSM), left ventricle cardiomyocytes (CMC), and adipose tissue (AT). B, Expression ␣2-ARs, ␤2-ARs, and ␤3-ARs in nonpregnant (NP)-UAECs vs P-UAECs. Blots are representative of 2 independent experiments from individual UAEC lines.
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
ANOVA (SigmaPlot 11 Statistical Software), followed by post hoc
multiple pairwise comparison Student-Newman-Keuls/Bonferroni
tests. Level of significance was established a priori at P⬍0.05.
norepinephrine or epinephrine with 2-OHE2 were 2.22⫾0.07fold and 2.24⫾0.07-fold, respectively, and with 4-OHE2 were
2.15⫾0.07-fold and 2.23⫾0.07-fold, respectively.
Results
␤-ARs but Not ␣-ARs Mediate Catecholamine and
Catecholestradiol-Induced P-UAEC Proliferation
P-UAECs Express Distinct AR Subtypes
Western immunoblotting showed ␣2-AR, ␤2-AR, and ␤3-AR
but not ␣1-AR or ␤1-AR subtypes in P-UAECs (Figure 2A).
Positive controls show protein expressions of ␣1-ARs, ␣2ARs, ␤1-ARs, ␤2-ARs, and ␤3-ARs in vascular smooth
muscle cells, left ventricular cardiomyocytes, and adipose
tissue, thus validating the absence of ␣1-ARs or ␤1-ARs.
Western analyses and densitometric analyses (data not
shown) showed the equal expression of ␤2-ARs and ␤3-ARs
between NP-UAECs and P-UAECs; however, ␣2-ARs were
higher in NP-UAECs versus P-UAECs.
Catecholamines Increase P-UAEC but Not
NP-UAEC Proliferation and Augment
Catecholestradiol-Induced P-UAEC Proliferation
Neither norepinephrine nor epinephrine stimulated NPUAEC proliferation (Figure 3A). Concentration-dependent
norepinephrine responses were observed in P-UAEC (Figure
3B) with maximum proliferation (2.08⫾0.03-fold) observed
at 100 nmol/L. At a low physiological concentration (0.1
nmol/L), norepinephrine significantly elevated proliferation
(1.78⫾0.028-fold). The highest P-UAEC proliferative responses to epinephrine were 1.89⫾0.035-fold observed at 1
nmol/L; however, at 10 and 100 nmol/L, there was no further
increase in proliferation to epinephrine, and this was slightly
less than that observed with norepinephrine.
We then determined their interactive effects using combination treatments (0.1 nmol/L) of norepinephrine or epinephrine with 2-OHE2 or 4-OHE2 (Figure 3C). At this dose, the
magnitude of P-UAEC proliferative responses to catecholamines was similar to catechoestradiols (1.86⫾0.02-fold
versus 1.81⫾0.02-fold, respectively). Combination treatments with norepinephrine or epinephrine with either 2-OHE2
or 4-OHE2 further increased P-UAEC proliferation versus
either catecholamine or catecholestradiol treatments alone
(P⬍0.05). Proliferative responses for combination treatments of
Phentolamine and propranolol antagonism (10␮mol/L) of
P-UAEC proliferation using both 0.1 (Figure 4A) and 100.0
nmol/L (not shown) doses of catecholamines yielded identical results. Neither antagonist alone altered basal P-UAEC
proliferation. Increases in proliferation seen with norepinephrine or epinephrine were unaltered (P⬎0.05) by nonselective
inhibition of ␣-ARs using phentolamine. In contrast, nonselective blockade of ␤-ARs using propranolol completely
abrogated catecholamine-mediated P-UAEC proliferation
(P⬍0.05).
Confirming our previous report,4 the magnitudes of proliferation of P-UAECs in responses to 2-OHE2 (1.89⫾0.02fold) and 4-OHE2 (1.88⫾0.02-fold) were not different (Figure 4B). Increases in P-UAEC proliferation seen with 0.1
nmol/L of 2-OHE2 and 4-OHE2 were unaltered (P⬎0.05) by
nonselective inhibition of ␣-ARs using phentolamine. In
contrast, nonselective blockade of ␤-ARs using propranolol
completely abrogated catecholestradiol-mediated P-UAEC
proliferation (P⬍0.05). To determine the putative role of
adrenergic G-protein– coupled receptors in E2␤-induced
P-UAEC proliferation, we evaluated these ␣-AR and ␤-AR
antagonists on E2␤-induced proliferation of P-UAECs. The
E2␤-induced rise (0.1 nmol/L) in P-UAEC proliferations was
not altered (P⬎0.05) by either phentolamine or propranolol.
␤2-ARs and, to a Lesser Extent, ␤3-ARs Mediate
Catecholestradiol-Induced P-UAEC Proliferation
We then evaluated subtype-specific ␣-AR and ␤-AR inhibition (10 ␮mol/L) on the P-UAEC proliferative responses to
catecholestradiols. None of the inhibitors alone altered basal
control P-UAEC proliferation (Figure 5A). Inhibition of
␣2-AR subtype with yohimbine did not inhibit 2-OHE2- and
4-OHE2-induced P-UAEC proliferation. In contrast, the selective antagonists of ␤2-AR (ICI 118 551) or ␤3-AR (SR
59230A), respectively, either blocked (P⬍0.01) or only
4
Hypertension
November 2011
A
2.5
NP-UAEC Proliferation
(Fold of Control)
partially attenuated (P⬍0.05) the proliferation induced by 0.1
nmol/L of 2-OHE2 and 4-OHE2.
We further evaluated additive effects of AR subtypes and
putative AR heterodimerization in regulating catecholestradiolmediated P-UAEC proliferation (Figure 5B). Combination of
ICI 118 551 with either yohimbine or SR 59230A inhibited
catecholestradiol-induced proliferation of P-UAECs, demonstrating primary involvement of ␤2-ARs. In contrast, combination of SR59230A and yohimbine only partially decreased
catecholestradiol-induced P-UAEC proliferation, demonstrating only partial ␤3-AR subtype involvement. These combination inhibitor studies support neither dimerization nor
significant cross-talk between these AR subtypes.
Norepinephrine
Epinephrine
2.0
1.5
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Epinephrine
B
P-UAEC Proliferation
(Fold of Control)
*
*
2.0
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λ
τ
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(-) Agonist
(+) Norepinephrine
(+) Epinephrine
C
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(Fold of Control)
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*
2.5
*
**
2.0
ψ
*
*
*
1.5
1.0
0.5
0.0
l
ro
nt
Co
2-
OH
E2
4-
OH
E2
Figure 3. Catecholamines stimulate pregnant (P)-uterine artery
endothelial cell (UAEC) but not nonpregnant (NP)-UAEC proliferation and augment catecholestradiol-induced P-UAEC proliferation. A, Concentration response of NP-UAECs to 0.0, 0.1, 1.0,
10.0, and 100.0 nmol/L of norepinephrine and epinephrine
(2-way ANOVA; concentration ⫻ group; F8,45⫽0.306; P⫽0.960;
n⫽6). B, Concentration response of P-UAECs to 0.0, 0.1, 1.0,
10.0, and 100.0 nmol/L of norepinephrine and epinephrine
(2-way ANOVA; concentration ⫻ group; F8,45⫽10.52; P⬍0.001;
n⫽6). C, Combinations of 0.1 nmol/L of norepinephrine or epinephrine with either 2-hydroxyestradiol (OHE2) or 4-OHE2
Stimulation of ␤2- and ␤3-ARs Promotes
P-UAEC Proliferation
To further evaluate ␤2-ARs versus ␤3-ARs, we tested the
actions of specific ␤-AR agonists. Both of the subtypespecific ␤2-AR (formoterol) and ␤3-AR (BRL 37344) agonists produced concentration-dependent and similar P-UAEC
proliferative responses (Figure 6A). Formoterol and BRL
37344 (100 nmol/L) produced maximal P-UAEC proliferations of 1.89⫾0.07-fold and 1.90⫾0.07-fold, respectively.
We then validated specificities of these agonists on their
respective ARs (Figure 6B). P-UAEC proliferation by formoterol was completely attenuated by ␤2-AR (ICI 118 551)
but not ␤3-AR (SR 59230A) antagonist. ␤3-AR antagonist
completely abrogated responses by BRL 37344 but not by
formoterol.
Discussion
We reported recently that, unlike their parent substrate
hormone E2␤, 2-OHE2 and 4-OHE2 do not stimulate
P-UAEC proliferation via classic estrogen receptors.4 Herein
we hypothesized that catecholestradiols mediate P-UAEC
proliferation via either ␣-ARs or ␤-ARs and that the catecholamines will modify/interact with these proliferative effects. We describe the first report of a complete and coupled
AR system in P-UAECs (not NP-UAECs) that is responsible
for catecholestradiol- and catecholamine-mediated proliferation, a critical process for angiogenic-mediated uterine perfusion during gestation. These data provide a novel previously undescribed model by which estrogen metabolites
function as potential circulating ␤-AR mimetic agonists.
Therefore, modifying the phenolic A ring of estrogens to
catechol moieties generates an endogenous ␤-mimetic agent
with angiogenic and possibly other cardiovascular capabilities. Our key findings are as follows: (1) in NP-UAECs and
P-UAECs, there are distinct AR subtypes expressed, including ␣2-ARs, ␤2-ARs, and ␤3-ARs, but only in P-UAECs do
norepinephrine and epinephrine increase proliferation; (2)
catecholamines play a complementary and a conserved role to
2-OHE2 and 4-OHE2 by acting as positive modulators of
augmented P-UAEC proliferation responses (2-way ANOVA;
group ⫻ agonist; F4,27⫽3.73; P⫽0.015; n⫽4). *Increase vs control. ␭Increase vs 0.1 and 1.0 nmol/L. ␶norepinephrine ⬎ epinephrine. ␺Increase vs catecholestradiols or catecholamines
alone.
Jobe et al
(-) Antagonist
(+) Phentolamine
(+) Proprandol
2.5
P-UAEC Proliferation
(Fold of Control)
*
1.0
0.5
2.0
1.5
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†
1.0
0.5
0.0
l
*
*
*
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†
1.5
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*
2.0
5
(-) Antagonist
(+) Phentolamine
(+) Propranolol
B
P-UAEC Proliferation
(Fold of Control)
A
Catecholestradiols and Adrenergic Receptors
e
e
rin
rin
ph
ph
e
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Ep
re
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2-
OH
E2
4-
OH
β
E2
E2
When compared with NP-UAECs, ␤2-AR and ␤3-AR expressions were unaltered by pregnancy status, whereas ␣2-ARs
were reduced. It is unclear whether coexpression of different
specific ARs within the same endothelial cells represents
unappreciated signaling complexity or just simply a functional redundancy. Using high-throughput proteomic analyses
of P-UAECs, we observed that ␤2-ARs are abundantly
localized in the P-UAEC caveolae domain, a “hub” for
compartmentalizing signal transduction for regulation of
multiple functions (J. Ramadoss, PhD, and R.R. Magness,
PhD, unpublished data, 2011). Therefore, demonstration of
specific AR expression relative to the subcellular localization
of ␣2, ␤2, and ␤3-ARs in NP-UAECs versus P-UAECs needs
P-UAEC proliferation; (3) neither catecholestradiols nor catecholamines induce P-UAEC proliferation via ␣-ARs but
rather solely via ␤-ARs; (4) 2-OHE2 and 4-OHE2 modulate
P-UAEC proliferation primarily via ␤2-ARs and, to a lesserextent, via ␤3-ARs; and (5) pharmacological agonists for
either ␤2-ARs or ␤3-ARs specifically stimulate P-UAEC
proliferation, suggesting similar coupling mechanisms and/or
signaling convergence.
We report, for the first time, the in vitro expressions of
several specific AR subtypes, ␣2-ARs, ␤2-ARs, and ␤3-ARs,
in NP-UAECs and P-UAECs, findings consistent with reports
demonstrating distinct individual AR subtypes in endothelia
of aorta, choroid, placenta, femoral artery, and retina.14 –19
(-) Antagonist
(+) Yohimbine
(+) ICI 118, 551
(+) SR 59230A
A
2.5
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(+) Yoh + ICI
(+) ICI + SR
(+) SR + Yoh
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B
P-UAEC Proliferation
(Fold of Control)
P-UAEC Proliferation
(Fold of Control)
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Figure 4. ␤-Adrenergic receptors (ARs), but not ␣-ARs, mediate catecholamine and catecholestradiol-induced pregnant (P)-uterine
artery endothelial cell (UAEC) proliferation. A, Effects of phentolamine or propranolol on P-UAEC proliferation to 0.1 nmol/L of norepinephrine or epinephrine. Phentolamine had no effect, whereas propranolol completely abrogated catecholamine-induced P-UAEC proliferation (2-way ANOVA; antagonist ⫻ group; F8,45⫽9.12; P⬍0.001; n⫽4). B, Effects of phentolamine or propranolol on P-UAEC proliferative responses to 2-hydroxyestradiol (OHE2), 4-OHE2, or estradiol-17␤ (0.1 nmol/L). Phentolamine had no effect, whereas propranolol
completely abrogated 2-OHE2- and 4-OHE2- but not E2␤-induced P-UAEC proliferative responses (2-way ANOVA; antagonist ⫻ group;
F6,36⫽7.88; P⬍0.001; n⫽4). *Increase vs untreated. †Complete inhibition.
*
2.0
*
t
*
t
*
†
†
2-OHE2
4-OHE2
1.5
1.0
0.5
0.0
0.0
Control
2-OHE2
4-OHE2
Control
Figure 5. ␤2-Adrenergic receptor (ARs) and, to a lesser extent, ␤3-ARs, mediate catecholestradiol-induced pregnant (P)-uterine artery
endothelial cell (UAEC) proliferation. A, Effects of yohimbine, ICI 118 551, or SR 59230A on P-UAEC proliferation to 2-hydroxyestradiol
(OHE2) or 4-OHE2 (0.1 nmol/L). Yohimbine had no effect, whereas ICI 118 551 attenuated and SR 59230A partially inhibited
catecholestradiol-mediated P-UAEC proliferation (2-way ANOVA; antagonist ⫻ group; F8,33⫽7.871; P⬍0.001; n⫽4). B, Effects of yohimbine, ICI 118 551, and SR 59230A combinations on P-UAEC proliferative responses to catecholestradiols. ICI 118 551, in all of the
combinations, completely blocked P-UAEC proliferation responses to catecholestradiols (2-way ANOVA; antagonist combination ⫻
group; F8,33⫽9.551; P⬍0.001; n⫽4). *Increase vs untreated. †Complete inhibition. ␶Partial inhibition.
Hypertension
November 2011
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Formoterol
BRL 37344
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(Fold of Control)
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0
1 n 10
0.1
10
(-) Antagonist
(+) ICI 118, 551
(+) SR 59230A
B
*
2.0
P-UAEC Proliferation
(Fold of Control)
6
*
1.5
†
†
1.0
0.5
0.0
tro
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to be determined. This may fulfill distinct physiological and
pathophysiologic functional significance for expression relative to localization of multiple AR subtypes in the
endothelium.
Because ARs are present on the endothelium, they are
undoubtedly exposed to circulating endogenous norepinephrine and epinephrine released from the adrenal medulla.
Normal physiological circulating catecholamine concentrations are 1 to 2 nmol/L20 –22 and increase dramatically in
pathological cardiovascular conditions and during fight or
flight stress responses. Hence, we demonstrated that even a
low physiological concentration (0.1 nmol/L) of both norepinephrine and epinephrine significantly increases P-UAEC,
but not NP-UAEC, proliferation, suggesting that catecholamines indeed may play roles in regulating physiological
angiogenesis during gestation. Consistent with these findings,
catecholamines augment in vivo angiogenesis in dopamine
␤-hydroxylase knockout mice deficient in plasma catecholamines.23 Confirming our recent report, a low physiological
concentration (0.1 nmol/L) of 2-OHE2 and 4-OHE2 stimulates P-UAEC proliferation.4 We report herein for the first
time that catecholamine and catecholestradiol combinations
induced significantly higher P-UAEC proliferation. We further demonstrate for the first time that both catecholamines
and catechoestradiols individually elevate P-UAEC proliferation only via ␤-ARs, suggesting that functional ␤-ARs are
likely important for regulating physiological and/or pathological angiogenesis during gestation. These data, therefore,
demonstrate that catecholamines play a complementary and
even an additive role to 2-OHE2 and 4-OHE2 as positive
␤-AR–mediated modulators of physiological angiogenesis.
Our data also imply that catecholamines and catechoestradiols should exhibit similar AR subtype-specific signaling
pathways to induce P-UAEC proliferation. Catecholestradiols
have been shown previously to competitively bind to AR
subtypes in rat cerebral cortex, striatum, and anterior pituitary, as well as to guinea pig hypothalamic, membranes.9,10
Therefore, our data show that catechol moieties of catecholestradiols and catecholamines are very important for the
binding and activation of ␤-AR signaling.
l
tro
n
Co
ol
er
ot
rm
Fo
B
RL
37
34
4
Figure 6. Stimulation of ␤2and ␤3-adrenergic receptor
(ARs) promotes pregnant (P)uterine artery endothelial cell
(UAEC) proliferation. A, Concentration response of
P-UAECs to 0.0, 0.1, 1.0, 10.0,
and 100.0 nmol/L of ␤2- and
␤3-AR agonists formoterol and
BRL 37344 (2-way ANOVA;
concentration ⫻ group;
F4,30⫽3.01; P⬍0.001; n⫽6). B,
Effects of ICI 118 551 or SR
59230A on P-UAEC proliferative responses to formoterol
and BRL 37344 (2-way
ANOVA; antagonist ⫻ group;
F8,45⫽20.53; P⬍0.001; n⫽6).
*Increase vs untreated.
␭
Increase vs 0.1 and 1.0
nmol/L. †Complete inhibition.
The lack of alteration of P-UAEC proliferation when the
nonspecific ␣-AR antagonist phentolamine and ␣2-AR specific blocker yohimbine were used shows that ␣2-ARs that
were reduced by pregnancy do not play a role in
catecholestradiol-induced angiogenesis in P-UAECs. There
are no reports showing a role for ␣2-ARs in regulating
endothelial cell proliferation. However, ␣2-ARs have been
closely associated with NO signaling in endothelial cells,24
suggesting functional relevance of ␣2-AR expression in
endothelial-mediated vasodilatation.
Consistent with our novel findings that propranolol abrogated 2-OHE2– and 4-OHE2–induced P-UAEC proliferation
are reports showing that stimulation of ␤-ARs by various
pharmacokinetic compounds stimulates proliferation of endothelial cells.15,19,25 Classically, ␤-ARs are prototypical G
protein– coupled receptors triggering diverse signaling cascades through ␣-, ␤-, and ␥-G protein subunits; adenylate
cyclase; intracellular cAMP; and protein kinase A and C.26
However, new layers of complexity in signaling suggest that
␤-AR activation can induce a myriad of cellular responses via
p38 and p42/44 mitogen-activated protein kinases independent of adenylate cyclase, cAMP, and protein kinase A and
C.27–30 Therefore, signal transduction studies are needed to
further elucidate the potential differences in ␤-AR–mediated
molecular mechanism of action of the catecholestradiol versus catecholamines in endothelial cells.
The current observation that subtype-specific ␤2-AR antagonist ICI 118 551 abolished P-UAEC proliferation stimulated by 2-OHE2 and 4-OHE2 suggests ␤2-AR coupling,
whereas the partial inhibition by ␤3-AR blocker SR 59230A
also implies potential involvement of ␤3-ARs. In contrast,
both the specific ␤2-AR (formoterol) and ␤3-AR (BRL
37344) agonists equally induced P-UAEC proliferation,
which was specifically blocked by their specific antagonists
(Figure 6), suggesting that both ␤-ARs may regulate these
proliferative effects. Thus, the partial inhibitory effects of SR
59230A on catecholestradiol responses (Figure 5A) do not
point to a lack of potency or effective concentration, because
a similar concentration of SR 59230A induced significant
abrogation of ␤3-ARs in response to BRL 37344. Activation
Jobe et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
of either ␤2-AR and/or ␤3-AR has been shown to play a role
in endothelial cell proliferation from human umbilical vein,
retina, and bovine aortas.14 –16,18 However, ours is the first
report to demonstrate that ␤2-AR and ␤3-AR mediate the
catecholestradiol-induced proliferation of endothelial cells.
P-UAECs express similar levels of ␤2-ARs and ␤3-ARs
compared with NP-UAECs, demonstrating that the ARmediated effects are not dependent on expression levels but
rather on other gestational programming factors at the level of
signaling. These data, therefore, provide a broader understanding of the mechanism of action of catecholestradiols in
endothelial cell proliferation. Importantly, these results also
point to the potential relevance of previously unappreciated
complexities of estrogen signaling in the cardiovascular
system via interactions of steroid metabolites and the endothelial AR system.
Overall, the present study indicates that actions of catecholestradiols and catecholamines via endothelial ARs represent an evolutionary conserved and highly versatile signaling mechanism for regulating endothelial proliferation.
During gestation, angiogenic processes are, to a great extent,
responsible for the dramatic 30- to 50-fold rises in uterine
blood flow, such that, by term, the uterine vascular bed
receives ⬇20% of the also greatly expanded cardiac output
and blood volume.31,32 Furthermore, maternal uterine perfusion is maintained 1- to 2-fold in excess of the needs of the
parallel, but separate, fetoplacental circulation.33 We suggested previously that, during an acute gestational flight or
fight response, when catecholamines are greatly elevated, far
in excess of the efficacious levels used herein, cardiac output
redistributes away from the uterine vascular bed (␣-AR
mediated) to the muscles and other tissues (␤-AR mediated)
for survival of the mother and her fetus, thus providing a
distinct short-term survival advantage for placental
mammals.34,35
Perspectives
The current study sheds new light on the existence of a
previously unrecognized 2-ligand system for a single AR
family representing a mechanism by which the same physiological regulators of the flight or fight responses that protect
the mother during a state of acute, but repeated, physiological
stress will indeed act as an angiogenic switch to subsequently
induce maintenance in uterine relative to fetoplacental blood
flows. This provides for a marked evolutionary advantage of
maintaining delivery of oxygen and nutrients through the
uteroplacental circulation, thus protecting the growing fetus
from subsequent stress-induced profound reductions in uterine blood flow. These data also uncover novel complexities of
estrogen signaling in the cardiovascular system via ARs and
necessitate the further investigation of estrogen metabolites,
such as catecholestradiols, in the vascular system, which do
not signal via the classic estrogen receptors.
Acknowledgments
We thank Timothy J. Morschauser, Mayra B. Pastore, Mary Y. Sun,
Jason L. Austin, Gladys E. Lopez, Terrance M. Phernetton, and
Cindy L. Goss.
Catecholestradiols and Adrenergic Receptors
7
Sources of Funding
This work was supported by National Institutes of Health grants
HL49210, HD38843, and HL87144 (to R.R.M.); AA19446 (to
J.R.); R25-GM083252 (to M.L. Carnes); and T32-HD041921-07
(to I.M. Bird).
Disclosures
None.
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A Novel Role for an Endothelial Adrenergic Receptor System in Mediating
Catecholestradiol-Induced Proliferation of Uterine Artery Endothelial Cells
Sheikh O. Jobe, Sean N. Fling, Jayanth Ramadoss and Ronald R. Magness
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Hypertension. published online September 26, 2011;
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ONLINE SUPPLEMENT
A NOVEL ROLE FOR AN ENDOTHELIAL ADRENERGIC RECEPTOR SYSTEM IN
MEDIATING CATECHOLESTRADIOL-INDUCED PROLIFERATION OF UTERINE
ARTERY ENDOTHELIAL CELLS
Sheikh O Jobe1, Sean N Fling1,4, Jayanth Ramadoss1, Ronald R Magness1,2,3
1
Depts. of Ob/Gyn. Perinatal Research Laboratories, 2Pediatrics and 3Animal Sciences,
University of Wisconsin-Madison, 4Spelman College
Address correspondence and reprint request to:
Ronald R. Magness, PhD,
Department of Obstetrics and Gynecology,
Perinatal Research Laboratories,
Atrium B Meriter Hospital,
202 S. Park Street,
Madison, WI, 53715
Phone: (608) 417-6314
Fax: (608) 257-1304
E-mail: rmagness@ wisc.edu
Expanded Materials and Methods:
Materials:
E2β, 2-OHE2 and 4-OHE2 were purchased from Steraloids Inc., Newport, RI. BrdU Cell
Proliferation Assays was obtained from EMD Chemicals Inc., Gibbstown, NJ. Propranolol,
Yohimbine, ICI 118,551, SR 59230A, Formoterol and BRL 37344 were purchased from Tocris
Bioscience, Ellisville, MO. Norepinephrine, epinephrine and phentolamine was purchased from
Sigma-Aldrich, St. Louis, MO. Mouse anti-α1-AR, rabbit anti-α2-AR, rabbit anti-β1-AR, rabbit
anti-β2-AR and rabbit anti-β3-AR were obtained from Santa Cruz Biotechnology Inc., Santa
Cruz, CA.
Cell Preparation and Culture:
All procedures and protocols for animal use were approved by the University of WisconsinMadison School of Medicine and Public Health Research Animal Care and Use Committee.
UAECs were isolated from late gestation ewes (P); (120-130 days; term= 147 days; n=6) and
nonpregnant (NP; luteal n= 3 and follicular n=3) ewes by collagenase digestion, cultured in
growth media (DVal MEM with 20% FCS, 100 mg/ml penicillin, and 100 mg/ml streptomycin)
as previously described.1,2 Validations were conducted on each cell preparation for functional
endothelial cell markers, PECAM-1, eNOS, LDL-uptake and smooth muscle myosin (negative
control) as previously described.1,2 UAECs (passages 3) were plated in T75 flasks containing
phenol free Endothelial Basal Medium (EBM) serum free without growth factors (Lonza,
Walkersville, MD), 20% FBS and 1% penicillin-streptomycin. Cells were grown to ~ 70%
confluence and were at passage 4 when lysed for protein extraction/Western blotting or
transferred to 96 well plates as needed for the respective experiments described below.
Protein Extraction and Western Immunoblotting:
Protein extraction was performed on NP-UAECs or P-UAECs by lysing them in 400 μl of lysis
buffer (0.5 M Tris + 0.1 M EDTA + 0.15 M NaCl + 0.1% Tween-20 + 5 mg/ml aprotinin + 5
mg/ml leupeptin + 0.001 M PMSF). Total protein content was determined using BCA Protein
Assay (Thermo Scientific, Rockford, IL). For Western blotting, 20 μg protein/lane were boiled in
SDS sample buffer for 5 min and electrophoresed on 4-20% gradient SDS-PAGE gels (Bio-Rad,
Hercules, CA) for 100 min at 150 V. Separated proteins were then electrically (100 V, 30 min)
transferred to a PVDF membrane. Non-specific binding was blocked with 5% fat-free milk in
TBST (50 mm Tris-HCl, pH 7.5, 0.15 m NaCl, 0.05% Tween-20) for 120 min and incubated
with primary antibodies (1 μg/ml; 1:500) in TBST + 1% BSA for 120 min. α1-AR, α2-AR, β1AR, β2-AR and β3-AR proteins were detected using mouse anti-α1-AR, rabbit anti-α2-AR, rabbit
anti-β1-AR, rabbit anti-β2-AR and rabbit anti-β3-AR. GAPDH and/or β-actin were utilized as a
loading control. After washing, the membrane was incubated with the corresponding peroxidaseconjugated IgG for 60 min and detected with the Pierce ECL detection kit (Thermo Scientific,
Waltham, MA).
Cell Proliferation Assays:
5-Bromodeoxyuridine was added after 4 hours (i.e. 20 hours BrdU incubation) during the 24
hours of treatment and this in vitro index of proliferation was evaluated. Plates were read using
Synergy HT Multi-Mode Microplate Reader. Results are expressed as the fold increases over
untreated control after subtracting the "blank"(wells incubated without 5-bromodeoxyuridine).
Validation of cell number increase and cytoxicity after treatment was performed using ViaLight
Plus High Sensitivity Cell Proliferation and Cytotoxicity Kit (Lonza Inc., Rockland, ME)
according to manufacturer’s instructions.2 After 24 hour starvation and subsequent treatment in
white opaque 96-well plates (24-hours), cells were lysed with Lysis Reagent (10 mins) to extract
ATP from cells. Then the appropriate amount of ATP Monitoring Reagent Plus was added (2
mins) in each well to generate luminescent signal. Plates were read using Synergy HT MultiMode Microplate Reader to determine luminescence and results expressed in Relative Light
Units as fold increases over untreated control after subtracting the value of the blank against an
ATP standard curve.
Experimental Treatments: Blockade and Activation of α-ARs and/or β-ARs:
P-UAEC proliferation experiments were performed in quadruplicates and replicated in ≥ four
different P-UAEC preparations. For concentration-response studies, P-UAECs in 96-well plates
were serum starved (24 hours) in EBM, washed with serum free EBM and medium was replaced
with EBM or EBM containing 0.1, 1, 10 or 100 nmol/L of norepinephrine or epinephrine (24
hours) or 0.1 nmol/L of 2-OHE2 or 4-OHE2. We specifically chose to study the concentration of
0.1 nmol/L for the catecholestradiols based on the dose-response curves from our previous
study.2 For specificities of all antagonists and agonist used in this study, please see table S1.
The α-ARs and/or β-ARs were blocked nonselectively by pretreating P-UAECs (10 µmol/L; 1
hour) with either the α-AR blocker phentolamine or the β-AR blocker propranolol followed by
treatments with 0.1 nmol/L of norepinephrine and epinephrine or 2-OHE2 or 4-OHE2 (24 hours).
Additional concentration studies to investigate AR activation with catecholamines alone or their
interactive effects with catecholestradiols were evaluated by combining treatments of 0.1 nmol/L
2-OHE2 or 4-OHE2 with 0.1 nmol/L norepinephrine or epinephrine. Based on Western analyses
expression of specific AR subtypes in P-UAECs, we conducted α-ARs or β-ARs subtype specific
blockade by selectively blocking (10 μmol/L; 1 hr) with the α2-AR inhibitor yohimbine, β2-AR
antagonist ICI 118,551, and β3-AR inhibitor SR 59230A followed by catecholestradiol
treatments (0.1 nmol/L for 24 hours). We then performed validation studies of AR-subtype
specific inhibition by evaluating P-UAEC mitogenic concentration responses using 0, 0.1, 1, 10,
100 nmol/L of the specific β2-AR agonist Formoterol as well as the β3-AR agonist BRL 37344.
We also studied the effects of 1 µmol/L of ICI 118,551 or SR 59230A on 100 nmol/L of
Formoterol and BRL 37344 in order to further evaluate the mitogenic effects of receptor
activation and specificity of β2 and β3-AR selective agonists in P-UAECs.
References:
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production in uterine artery endothelial cells. Endocrinology. 2000; 141:1107-1117.
2. Jobe SO, Ramadoss J, Koch JM, Jiang Y, Zheng J, Magness RR. Estradiol-17β and its
cytochrome P450- and catechol-O-methyltransferase-derived metabolites stimulate
proliferation in uterine artery endothelial cells: Role of estrogen receptor-alpha versus
estrogen receptor-beta. Hypertension. 2010; 55:1005-1011.
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Marked Adrenolytic Properties. Proc Soc Exp Biol Med. 1949; 71: 70-72.
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The selectivity in vitro of the stereoisomers of the beta-3 adrenoceptor agonist BRL
37344. J Pharmacol Exp Ther. 1996; 277: 22-27.
Table S1:
Ligand
Compound
α-AR/β-AR
Agonist/Antagonist
Binding Selectivity
Relative to other ARs
Reference
Phentolamine
α-ARs
Antagonist
N/A
Meier et al,
1949.3
Propranolol
β-ARs
Antagonist
N/A
Stoschitzky et
al, 1995.4
Yohimbine
α2-ARs
Antagonist
N/A
Doxey et al,
1984.5
ICI 118,551
β2-ARs
Antagonist
≥ 100-fold
Bilski et al,
1983.6
SR59230A
β3-ARs
Antagonist
≥ 10-fold
Manara et al,
1996.7
Formoterol
β2-ARs
Agonist
≥ 330-fold
Decker et al,
1982.8
BRL37344
β3-ARs
Agonist
≥ 4-fold
Oriowo et al,
1996.9
N/A means negligible binding selectivity and/or complete binding affinity to AR subtype