A novel role for an endothelial adrenergic receptor system in mediating catecholestradiol-induced proliferation of uterine artery endothelial cells

Abstract

Sequential conversion of estradiol-17β to its biologically active catecholestradiols, 2-hydroxyestradiol (OHE(2)) and 4-OHE(2), 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 α- and β-adrenergic receptors (ARs), we investigated whether the endothelial α- or β-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 α(2)-, β(2)-, and β(3)-ARs but not α(1)- and β(1)-ARs. Levels of β(2)-ARs and β(3)-ARs were unaltered by pregnancy, whereas α(2)-ARs were decreased. Norepinephrine and epinephrine increased P-UAEC, but not nonpregnant UAEC proliferation, and these effects were suppressed by propranolol (β-AR blocker) but not phentolamine (α-AR blocker). Catecholamines combinations with 2-OHE(2) or 4-OHE(2) enhanced P-UAEC mitogenesis. Catecholestradiol-induced P-UAEC proliferation was also inhibited by propranolol but not phentolamine. β(2)-AR and β(3)-AR antagonists (ICI 118 551and SR 59230A, respectively) abrogated the mitogenic effects of both 2-OHE(2) and 4-OHE(2). Stimulation of β(2)-ARs and β(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 β(2)-ARs and β(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.

Figure 1

Adding one OH group modifies the estrogenic phenolic A ring forming “catechol” moieties potentially making it an “AR-mimetic” agent for inducing ER-independent angiogenesis. The boxes outline shared phenolic A ring “catechol” moiety between 2-hydroxyestradiol, 4-hydroxyestradiol, norepinephrine and epinephrine. ?? indicates hypotheses tested.

Figure 2. AR subtypes in UAECs

(A) Western blots demonstrating expression of α₂-ARs, β₂-ARs and β₃-ARs; but not α₁-ARs or β₁-ARs subtypes in P-UAECs. Positive control lanes are vascular smooth muscle cells (VSM), left ventricle cardiomyocytes (CMC) and adipose tissue (AT). (B) Expression α₂-ARs, β₂-ARs and β₃-ARs in NP-UAECs versus P-UAECs. Blots are representative of two independent experiments from individual UAEC cell lines.

Figure 3. Catecholamines stimulate of P-UAECs but not NP-UAECs Proliferation and Augement Catecholestradiol-induced P-UAEC Proliferation

(A) Concentration response of NP-UAECs to 0, 0.1, 1, 10 and 100 nmol/L norepinephrine and epinephrine (two-way ANOVA; concentration x group; F_8,45 = 0.306, P = 0.960; n = 6). (B) Concentration response of P-UAECs to 0, 0.1, 1, 10 and 100 nmol/L norepinephrine and epinephrine (two-way ANOVA; concentration x group; F_8,45 = 10.52, P < 0.001; n = 6). (C) Combinations of 0.1 nmol/L norepinephrine or epinephrine with either 2-OHE₂ or 4-OHE₂ augmented P-UAEC proliferation responses (two-way ANOVA; group x agonist; F_4,27 = 3.73, P = 0.015; n = 4) *Increase vs. control. λ Increase vs. 0.1 and 1 nmol/L. τ norepinephrine > epinephrine. ψ Increase vs. catecholestradiols or catecholamines alone

Figure 4. β-ARs, but not α-ARs, mediate Catecholamine and Catecholestradiol-induced P-UAEC Proliferation

(A) Effects of phentolamine or propranolol on P-UAECs proliferation to 0.1 nmol/L norepinephrine or epinephrine. Phentolamine had no effect; whereas propranolol completely abrogated catecholamine-induced P-UAEC proliferation (two-way ANOVA; antagonist x group; F_8,45 = 9.12, P < 0.001; n = 4). (B) Effects of phentolamine or propranolol on P-UAECs proliferative responses to 2-OHE₂, 4-OHE₂ or E₂β (0.1 nmol/L). Phentolamine had no effect; whereas propranolol completely abrogated 2-OHE₂- and 4-OHE₂-, but not E₂β-induced P-UAECs proliferative responses (two-way ANOVA; antagonist x group; F_6,36 = 7.88, P < 0.001; n = 4). *Increase vs. untreated. † Complete inhibition.

Figure 5. β₂-ARs and to a Lesser Extent β₃-ARs Mediate Catecholestradiol-induced P-UAEC Proliferation

(A) Effects of yohimbine, ICI 118,551 or SR59230A on P-UAEC proliferation to 2-OHE₂ or 4-OHE₂ (0.1 nmol/L). Yohimbine had no effect; whereas ICI 118,551 attenuated and SR59230A partially inhibited catecholestradiol-mediated P-UAEC proliferation (two-way ANOVA; antagonist x group; F_8,33= 7.871, P < 0.001; n = 4). (B) Effects of yohimbine, ICI 118,551, and SR59230A combinations on P-UAEC proliferative responses to catecholestradiols. ICI 118,551 in all combinations completely blocked P-UAEC proliferation responses to catecholestradiols (two-way ANOVA; antagonist combination x group; F_8,33 = 9.551, P < 0.001; n = 4). *Increase vs. untreated. † Complete inhibition. τ Partial inhibition.

Figure 6. Stimulation of β₂- and β₃-ARs promotes P-UAEC proliferation

(A) Concentration response of P-UAECs to 0, 0.1, 1, 10 and 100 nmol/L of β₂- and β₃-AR agonists Formoterol and BRL 37344 (two-way ANOVA; concentration x group; F_4,30 = 3.01, P < 0.001; n = 6). (B) Effects of ICI 118, 551 or SR59230A on P-UAEC proliferative responses to Formoterol and BRL 37344 (two-way ANOVA; antagonist x group; F_8,45 = 20.53, P < 0.001; n = 6). *Increase vs. untreated. λ Increase vs. 0.1 and 1 nmol/L. † Complete inhibition.