Estrogen, vascular estrogen receptor and hormone therapy in postmenopausal vascular disease

Abstract

Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERβ and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women's Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject's age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and 'timing' could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD.

Keywords: 17β-estradiol; 27-hydroxycholesterol; 27HC; Akt; AngII; C-reactive protein; CEE; CRP; CVD; E2; EC; ECM; ELITE; ER; Early versus Late Intervention Trial with Estradiol; Endothelium; Extracellular matrix; FMD; G protein-coupled receptor 30; GPR30; HERS; HSP90; Hypertension; IL-6; KEEPS; Kronos early estrogen prevention study; MAPK; MHT; MI; MMP; MPA; NHS; NO; Nurses’ Health Study; OVX; P4; PCOS; PI(3)K; Post-MW; Pre-MW; Progesterone; RCT; SHR; Sex hormones; T; TMF-α; TXA2; Testosterone; VSM; VSM cell; VSMC; VTE; Vascular smooth muscle; WHI; Women's Health Initiative; angiotensin II; cardiovascular disease; conjugated equine estrogen; eNOS; endothelial cell; endothelial nitric oxide synthase; estrogen receptor; extracellular matrix; flow mediated dilation; heart and estrogen/progestin replacement study; heat shock protein-90; interleukin-6; matrix metalloproteinase; medroxyprogesterone acetate; menopausal hormone therapy; mitogen-activated protein kinase; myocardial infarction; nitric oxide; ovariectomized; phosphatidylinositol 3-kinase; polycystic ovary syndrome; postmenopausal women; premenopausal women; progesterone; protein kinase B; randomized clinical trial; spontaneously hypertensive rat; testosterone; thromboxane A2; tumor necrosis factor-α; vascular smooth muscle; venous thrombo-embolism.

Fig. 1

Structure of ERα, ERβ, and GPR30. ERα and ERβ share a common structure with five functional domains A/B, C, D, E and F. The A/B region is involved in protein-protein interaction and transcriptional activation of target-genes. The A/B region harbors activation function-1 (AF-1), located toward the N-terminal end, that is ligand-independent and shows promoter and cell-specific activity. Human ERα and ERβ share <20% amino acid identity in A/B region, suggesting that it may contribute to ER-specific actions on target genes. The central C-domain is the DNA-binding domain (DBD), which contains 4 cysteines arranged in 2 zinc fingers, and is involved in DNA binding and receptor dimerization. It is highly conserved between ERα and ERβ and shares 95% amino acid identity, suggesting that both ERs recognize similar DNA sequences and hence regulate many of the same target genes. D-domain is not well-conserved between ERα and ERβ (30%) and works as a flexible hinge between DBD and ligand-binding domain (LBD). D- domain also functions in promoting the association of ER with heat shock protein 90 (HSP90) and nuclear localization of ER. E-domain is the LBD, and ERα and ERβ share ~55% amino acid identity in this region. LBD contains a ligand-dependent AF-2, and is important for ligand binding and receptor dimerization. F-domain has <20% amino acid homology between ERα and ERβ, includes co-factor recruitment regions, but its function is unclear. Full transcriptional activation by ERs is mediated by synergism between AF-1 and AF-2. AF-1 and AF-2 are also required for ligand-independent receptor functions, including growth factor activation by AF-1 and cAMP activation by AF-2. GPR30 is a G protein-coupled receptor that has estrogenic activity and shares little homology with the classical ERs. GPR30 has an extracellular N terminal, 7 transmembrane α helices, 3 exo-loops involved in ligand binding, 3 or 4 cyto-loops involved in G protein binding, and a C terminal linked to the membrane through lipid addition, and also involved in G protein binding.

Fig. 2

Estrogen-induced ER-mediated pathways of vascular relaxation. Estrogens such 17β-estradiol (E2) binds to endothelial ERα, ERβ or GPR30 and activates phospholipase C (PLC), leading to the generation of inositol 1,4,5- triphosphate (IP₃) and diacylglycerol (DAG). IP₃ causes the release of Ca²⁺ from the endoplasmic reticulum (ER). Ca²⁺ forms a complex with calmodulin (CAM), which causes initial activation of eNOS. E2 also activates phosphatidylinositol 3-kinase (PI₃K), which transforms phosphatidylinositol-4,5-bisphosphate (PIP₂) into phosphatidylinositol 3,4,5-trisphosphate (PIP3), and activates Akt. ER-mediated activation of Akt or MAPK causes phosphorylation and full activation of eNOS. Fully activated eNOS transforms L-arginine to L-citrulline and produces NO, which diffuses through ECs and activates guanylate cyclase in VSM leading to increased cGMP and stimulation of cGMP-dependent protein kinase (PKG). PKG decreases [Ca²⁺]_i by stimulating Ca²⁺ extrusion pumps in the plasma membrane and Ca²⁺ uptake pumps in SR and/or decreases the sensitivity of the contractile myofilaments to [Ca²⁺]_i and thereby promotes VSM relaxation. E2 also activates COX to produce prostacyclin (PGI₂) which activates cAMP-dependent pathway, protein kinase A (PKA), and promotes relaxation pathways similar to cGMP/PKG. E2 also induces the release of EDHF which activates K⁺ channels and causes hyperpolarization and relaxation of VSM. E2 could also decrease endothelial ET-1 expression and production via genomic pathway. In VSM, agonists such as ET-1, TXA2 and AngII activate specific VSM receptors, stimulate PLC, and increase the production of IP₃ and DAG. IP₃ stimulates Ca²⁺ release from the sarcoplasmic reticulum. Agonists also stimulate Ca²⁺ entry through Ca²⁺ channels. Ca²⁺ binds CAM, activates myosin light chain kinase (MLCK), causes MLC phosphorylation, and initiates actin-myocin interaction and VSM contraction. DAG activates PKC, which in turn phosphorylates calponin (CaP) and/or activate a protein kinase cascade involving Raf, MAPK kinase (MEK) and MAPK, leading to phosphorylation of caldesmon (CaD) and increased myofilament force sensitivity to Ca²⁺. E2 binds to VSM ERs, leading to inhibition of agonist-activated mechanisms of VSM contraction. E2 activates K⁺ channels, leading to membrane hyperpolarization and inhibition of Ca²⁺ entry through Ca²⁺ channels. E2 may also inhibit PKC, MAPK or the RhoA/Rho-kinase (Rho-K) [Ca²⁺]_i sensitization pathway. Dashed lines indicate inhibition.