Research into Specific Modulators of Vascular Sex Hormone Receptors in the Management of Postmenopausal Cardiovascular Disease

Abstract

Cardiovascular disease (CVD) is more common in men and postmenopausal women than premenopausal women, suggesting vascular benefits of female sex hormones. Studies on the vasculature have identified estrogen receptors ERα, ERβ and a novel estrogen binding membrane protein GPR30, that mediate genomic and/or non-genomic effects. Estrogen promotes endothelium-dependent relaxation by inducing the production/activity of nitric oxide, prostacyclin, and hyperpolarizing factor, and inhibits the mechanisms of vascular smooth muscle contraction including [Ca(2+)](i), protein kinase C, Rho kinase and mitogen-activated protein kinase. Additional effects of estrogen on the cytoskeleton, matrix metalloproteinases and inflammatory factors contribute to vascular remodeling. However, the experimental evidence did not translate into vascular benefits of menopausal hormone therapy (MHT), and the HERS, HERS-II and WHI clinical trials demonstrated adverse cardiovascular events. The discrepancy has been partly related to delayed MHT and potential changes in the vascular ER amount, integrity, affinity, and downstream signaling pathways due to the subjects' age and preexisting CVD. The adverse vascular effects of MHT also highlighted the need of specific modulators of vascular sex hormone receptors. The effectiveness of MHT can be improved by delineating the differences in phramcokinetics and pharmacodynamics of natural, synthetic, and conjugated equine estrogens. Estriol, "hormone bioidenticals" and phytoestrogens are potential estradiol substitutes. The benefits of low dose MHT, and transdermal or vaginal estrogens over oral preparations are being evaluated. Specific ER modulators (SERMs) and ER agonists are being developed to maximize the effects on vascular ERs. Also, the effects of estrogen are being examined in the context of the whole body hormonal environment and the levels of progesterone and androgens. Thus, the experimental vascular benefits of estrogen can be translated to the outcome of MHT in postmenopausal CVD, as more specific modulators of sex hormone receptors become available and are used at the right dose, route of administration and timing, depending on the subject's age and preexisting cardiovascular condition.

Fig.1

Sex Steroid Biosynthesis. Biosynthesis of steroid hormones starts with conversion of cholesterol to pregnenolone through the cleavage of the cholesterol side chain by P450scc (Cytochrome P450, family 11, subfamily A, polypeptide 1). Pregnenolone is converted to progesterone though the 3β-DH delta^4,5 isomerase pathway, or testosterone through the P450c17 pathway. Progesterone is converted to androstenedione through the 17,20 desmolase (P450c17) pathway. Androstenedione is then converted to E1 by aromatase in the extragonadal tissue, a pathway that increases with aging. Primary synthesis of E2 comes from a reversible reaction through the 17-ketoreductase pathway from E1. A secondary source of E2 comes from aromatization of testosterone in peripheral adipose tissue. E2 can be converted to E3 through hydroxylation by 16-α hydroxylase or to the highly active 2-methoxy-E2 through catechol-O-methlytransferase. Testosterone can also be converted to the more active dihydrotestosterone through the 5-α-reductase pathway.

Fig 2

Estrogen Receptor Structure. Three estrogen receptors that have been identified: ER-α and ER-β, and GPR30. Classical ERα and ERβ are nuclear receptors that share a common structure with 6 functional domains. The A/B domain is the transactivation domain, containing the activation function −1 (AF-1) and is least conserved domain with 24% homology and accounts for the main difference in size between ERα and ERβ. The C domain is the DNA binding domain (DBD) containing a dimerization interface. The D domain is the hinge region containing a nuclear localization signal and connects the DBD to the ligand binding domain (LBD). The E domain is the LBD containing the AF-2 region, and a dimerization interface that functions with the one present in the C domain. The F domain is the C terminal of ER. GPR30 is a G protein coupled receptor (GPCR) that also binds estrogen. It has an extracellular N terminal, seven transmembrane ( TM ) alpha helices, 3 exo-loops involved in ligand binding, 3 or 4 cyto-loops involved in G protein subunit binding, and a C terminal linked to the membrane through lipid addition, and also involved in binding G protein subunits.

Fig. 3

ER-activated postreceptor mechanisms. In the genomic pathway, estrogen binds to cytoplasmic ER in the LBD, leading to ER dimerization and localization to the nucleus where the complex interacts with EREs to increase gene transcription. E2 via ER interacts with the SH2 domain of Src and activate Modulator of Nongenomic Action of Estrogen Receptor (MNAR). They interact with p85, a regulatory subunit of the PI3 kinase (PI3K) and lead to activation of the PI3K/Akt pathway). ERs may also activate phospholipase C (PLC) and increase diacylglycerol (DAG) production which leads to activation of MAPK. MAPK translocates to the nucleus where it increases gene transcription. GPR30 also binds E2 leading to activation of matrix metalloproteinase (MMP) with subsequent release of pro-heparan-bound epidermal growth factor (ProHB-EGF) and activation of epidermal growth factor receptor (EGFR). GPR30 also activates adenylyl cyclase (AC) and increases the generation of intracellular cAMP, which inhibits MAPK, thus, acting by different pathways to balance Erk-1/-2 (MAPK) activity.

Fig. 4

Effect of estrogen on endothelium-dependent pathways of vascular relaxation. E2 causes upregulation of COX-1 expression and PGI₂ synthesis in endothelial cells and also increases endothelium derived hyperpolarizing factor (EDHF) production. Estrogen binds to endothelial surface membrane ER and activates phospholipase C (PLC), leading to the generation of inositol 1,4,5- triphosphate (IP₃) and DAG. IP3 stimulates the release of stored Ca²⁺ from the endoplasmic reticulum, followed by influx of extracellular Ca²⁺. Ca²⁺ forms a complex with calmodulin (CAM), which causes initial activation of eNOS, its dissociation from caveolin-1, and its translocation to intracellular sites. Estrogen also activates phosphatidylinositol 3-kinase (PI₃K), leading to transformation of phosphatidylinositol-4,5-bisphosphate (PIP₂) into phosphatidylinositol 3,4,5-trisphosphate (PIP₃), which activates Akt. ER-mediated activation of Akt or MAPK pathway causes phosphorylation of cytosolic eNOS and its second translocation back to the cell membrane where it undergoes myristoylation and palmitoylation to become fully activated. Fully activated eNOS promotes the transformation of L-arginine to L-citrulline and the production of NO, which diffuses through the endothelial cell and causes VSM relaxation.

Fig. 5

Effect of estrogen on the mechanisms of VSM contraction. An agonist (A) activates a specific receptor (R), stimulates membrane phospholipase (PLC), and increases the production of IP3 and diacylglycerol (DAG). IP3 stimulates Ca²⁺ release from the sarcoplasmic reticulum (SR). Also, the agonist stimulates Ca²⁺ entry through Ca⁺² channels. Ca²⁺ binds CAM, activates myosin light chain (MLC) kinase, causes MLC phosphorylation, and initiates VSM contraction. DAG causes activation of PKC. PKC phosphorylates calponin (CaP) and/or activate a protein kinase cascade involving Raf, MAPK kinase (MEK), and MAPK, leading to phosphorylation of caldesmon (CaD) and an increase in the myofilament force sensitivity to Ca²⁺. Estrogen binds to plasma membrane ER, leading to inhibition of agonist-activated mechanisms of VSM contraction. Possible nongenomic effects of estrogen include activation of K⁺ channels, leading to membrane hyperpolarization, inhibition of Ca²⁺ entry through Ca²⁺ channels, and thereby inhibition of Ca²⁺-dependent MLC phosphorylation and VSM contraction. Estrogen may also inhibit PKC and/or the MAPK pathway through activation of plasma membrane ERs and thereby further inhibit VSM contraction. Estrogen-induced NO release from the endothelium 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 Ca⁺² uptake pumps in the sarcoplasmic reticulum and/or decrease the sensitivity of the contractile myofilaments to [Ca²⁺]_i and thereby promote VSM relaxation. Estrogen also induces the release of PGI₂ from the endothelium to activate the PGI₂-cAMP pathway, or EDHF to activate Ca²⁺-activated K⁺ channels and to cause hyperpolarization and relaxation of VSM.