Molecular mechanisms regulating the vascular prostacyclin pathways and their adaptation during pregnancy and in the newborn

Abstract

Prostacyclin (PGI(2)) is a member of the prostanoid group of eicosanoids that regulate homeostasis, hemostasis, smooth muscle function and inflammation. Prostanoids are derived from arachidonic acid by the sequential actions of phospholipase A(2), cyclooxygenase (COX), and specific prostaglandin (PG) synthases. There are two major COX enzymes, COX1 and COX2, that differ in structure, tissue distribution, subcellular localization, and function. COX1 is largely constitutively expressed, whereas COX2 is induced at sites of inflammation and vascular injury. PGI(2) is produced by endothelial cells and influences many cardiovascular processes. PGI(2) acts mainly on the prostacyclin (IP) receptor, but because of receptor homology, PGI(2) analogs such as iloprost may act on other prostanoid receptors with variable affinities. PGI(2)/IP interaction stimulates G protein-coupled increase in cAMP and protein kinase A, resulting in decreased [Ca(2+)](i), and could also cause inhibition of Rho kinase, leading to vascular smooth muscle relaxation. In addition, PGI(2) intracrine signaling may target nuclear peroxisome proliferator-activated receptors and regulate gene transcription. PGI(2) counteracts the vasoconstrictor and platelet aggregation effects of thromboxane A(2) (TXA(2)), and both prostanoids create an important balance in cardiovascular homeostasis. The PGI(2)/TXA(2) balance is particularly critical in the regulation of maternal and fetal vascular function during pregnancy and in the newborn. A decrease in PGI(2)/TXA(2) ratio in the maternal, fetal, and neonatal circulation may contribute to preeclampsia, intrauterine growth restriction, and persistent pulmonary hypertension of the newborn (PPHN), respectively. On the other hand, increased PGI(2) activity may contribute to patent ductus arteriosus (PDA) and intraventricular hemorrhage in premature newborns. These observations have raised interest in the use of COX inhibitors and PGI(2) analogs in the management of pregnancy-associated and neonatal vascular disorders. The use of aspirin to decrease TXA(2) synthesis has shown little benefit in preeclampsia, whereas indomethacin and ibuprofen are used effectively to close PDA in the premature newborn. PGI(2) analogs have been used effectively in primary pulmonary hypertension in adults and have shown promise in PPHN. Careful examination of PGI(2) metabolism and the complex interplay with other prostanoids will help design specific modulators of the PGI(2)-dependent pathways for the management of pregnancy-related and neonatal vascular disorders.

Fig. 1.

Eicosanoid and prostanoid biosynthesis and metabolism. Membrane phospholipids such as phosphatidylethanolamine are hydrolyzed by PLA₂ to produce AA. AA is metabolized by COX1 and COX2 to produce various prostanoids, 5-LOX to yield LTs and 12- or 15-LOX to yield 12- or 15-HETE, cytochrome P450 monoxygenases, including epoxygenases to produce EETs and ω-hydroxylases to produce HETEs, or undergo nonenzymatic lipid peroxidation to isoprostanes and 9-HETE. AA metabolism by COX yields PGG₂ then PGH₂. PGH₂ is acted upon by specific PG synthases (PGIS, PGDS, PGES, PGFS, and TXAS) to produce PGI₂, PGD₂, PGE₂, PGF_2α, and TXA₂, respectively. PGI₂ and TXA₂ undergo rapid nonenzymatic hydrolysis to the stable and biologically inactive 6-keto-PGF_1α and TXB₂, respectively. TXB₂ undergoes further and relatively slower enzymatic oxidation by 11-hydroxy-TXB₂ dehydrogenase (11-TXDH) to 11-dehydro-TXB₂. Nonenzymatic dehydration of PGD₂ and PGE₂ leads to the formation of the cyclopentenones PGJ₂ and 15-deoxy-PGJ₂, and PGA₂ and PGB₂, respectively. 6-Keto-PGF_1α, PGD₂, PGE₂, and PGF_2α undergo either oxidation by 15-PG-dehydrogenase (15-PGDH) into the respective 15-keto-PGs, which are then reduced by 13-PG reductase to 15-keto-13,14-dihydro-PGs, or β-oxidation with subsequent loss of two or four carbons to form dinor- or tetranor-PGs. Boxed compounds are biologically active.

Fig. 2.

Human COX1 and COX2. A, human COX1 and COX2 polypeptides share 61% primary sequence identity. The aa sequence of the COX protein starts with an N terminus with a signal peptide of 23 aa in COX1 and 17 aa in COX2 that is absent in the mature protein. Each COX monomer consists of three structural domains: dimerization domain composed of d1 (or the epidermal growth factor, EGF domain) and d2, membrane binding domain (MBD), and a large C-terminal globular catalytic domain. The catalytic domain contains a long narrow channel of hydrophobic character comprising the cyclooxygenase active site that binds AA, other fatty acids, and COX inhibitors such as aspirin and NSAIDs, and a peroxidase active site that binds heme. Both COX1 and COX2 contain C-terminal STEL-type sequence of 4 aa (Ser-Thr-Glu-Leu), which may constitute the signal for COX attachment to the ER. The mature COX1 has an 8-aa insertion at the N terminus, whereas COX2 has an 18-aa insertion at the C terminus. COX-1 and COX-2 share several N-glycosylation sites at homologous aa, and COX2 has an additional N-glycosylation site at the carboxyl terminal. The size of the cyclooxygenase active site is approximately 20% larger in COX2 than COX1, primarily due to substitution of Ile522 in human COX1 with Val509 in human COX2 that results in a small “side pocket” in the cyclooxygenase active site channel of COX2. Aspirin and other NSAIDs compete with AA at the cyclooxygenase active site. Ser529 in human COX1 (Ser530 in sheep COX1) and the corresponding Ser516 in COX2 represent the aspirin acetylation sites. The side pocket in the cyclooxygenase active site of COX2 can be accessed by selective COX2 inhibitors such as celecoxib. B, critical transcription factors and corresponding regulatory response elements on inducible COX2 promoter. In the transcription of inducible COX2, the COX2 5′-promoter contains regulatory response elements for various transcription factors starting from the TATA box, CRE (cAMP/PKA/CREB response element), NF-IL6, AP-2, NFκB, SP-1, and glucocorticoid receptor response element. Glucocorticoids inhibit transcription of COX2, but the intermediate transcription factor and response element involved are not clear.

Fig. 3.

Prostanoid receptor structure. PRs have three domains: an extracellular domain consisting of a short N-terminal tail and three extracellular loops, a transmembrane domain (TMD) composed of seven transmembrane-spanning α-helices, and a cytoplasmic domain made of three intracellular loops (in most PRs), and a C terminus. There are 34 amino acid residues conserved in all PRs in different species (white circles), mainly located within the TMDs. A highly conserved Arg residue (Arg279) and a DPW (Asp-Pro-Trp) motif (100% conserved among all PRs) are located in the middle of TMD-VII. In all PRs, a conserved disulfide bond is formed in the extracellular domain between Cys92 at the top of TMD-III and Cys170 in the second extracellular loop of human IP receptor. Another disulfide bond specific for human IP receptor is formed between Cys5 at the top of TMD-I and Cys165 in the second extracellular loop. One or more consensus sequences for N-glycosylation sites, demonstrated as CHO (C₆H₁₂O₆; glucose), are present in the amino terminus extracellular portion (asparagine residues Asn7 and Asn78 in human IP). In the cytoplasmic domain of IP receptor, lipid isoprenylation-palmitoylation anchoring sites starting at Cys308 (Cys-Cys-Leu-Cys) create a fourth intracellular loop.

Fig. 4.

Commonly used prostanoid-type prostacyclin analogs. Prostanoid analogs share a primary structure that determines their binding to the IP receptor, including the essential carboxyl group at C1, and hydrogen-bonding OH groups at C11 and C15. Functional IC₅₀ is the half-maximal inhibitory concentration of platelet aggregation. Functional EC₅₀ is the half-maximal effective concentration of inducing a cAMP response in cultured human pulmonary artery SMCs.

Fig. 5.

PGI₂ signaling pathways in VSMCs. PGI2/IP cell surface interaction is coupled primarily to G_s to activate cAMP/PKA leading to Ca²⁺ extrusion via cell surface and sarcoplasmic reticulum (SR) Ca²⁺ pumps, and activation of different K⁺ channels (including ATP-sensitive K⁺ channels and MaxiK channels), which in turn cause VSMC hyperpolarization and relaxation. In contrast, TXA₂/TP interaction causes stimulation of G_q, activation of phospholipase C (PLC), and increased production of inositol-1,4,5-trisphosphate (IP₃), which stimulates Ca²⁺ release from SR, and DAG, which activates PKC. Increased [Ca²⁺]_i causes activation of Ca²⁺/calmodulin/myosin light chain kinase (MLCK) pathway and stimulation of VSM contraction. TP-mediated stimulation of Gα_12/13 activates Rho signaling and the RhoA/Rho-associated protein kinase (ROCK) pathway, which inhibits myosin-light chain phosphatase (MLCP) and causes Ca²⁺ sensitization and enhancement of VSMC contraction. PGI₂/IP signaling via cAMP/PKA can inhibit TXA₂-induced VSMC contraction by PKA-mediated TPα phosphorylation and thereby inhibition of both G_q/PLCβ/Ca²⁺-dependent and G_12/13/RhoA Ca²⁺-independent signaling pathways. PGI₂ also activate genomic pathways and cellular processes including, as shown from left to right, PGI₂-induced PGI₂ release, PPAR, VSMC hypertrophy, proliferation, differentiation, and migration. PGI₂/IP signaling can induce COX2 expression in VSMCs to metabolize AA and produce PGI₂, which in turn may act in an intracrine fashion on the same VSMC or paracrine on nearby VSMCs (feedback loop). PGI₂ intracrine signaling may involve direct binding to PPAR nuclear receptors and gene transcription. PGI₂/IP signaling can inhibit Shc/GRB2 complex formation and subsequent ERK1/2 activation by TXA₂/TP signaling, thus inhibiting VSMC hypertrophy. PGI₂ also inhibits VSMC proliferation by inhibiting G₁-to-S phase progression through inhibition of cyclin E-cyclin-dependent kinase (cdk2) as well as activation of p27^kip1, which keeps cyclin E-cdk2 in an inactive state, either directly or via inhibition of the gene for S-phase kinase-associated protein (Skp2), which causes p27^kip1 degradation. PGI₂ can also act through IP/cAMP/PKA-mediated activation of CREB or other SM-specific transcription factors to increase the expression of SM-specific differentiation markers and cause VSM differentiation. PGI₂ via cAMP-dependent activation of protein tyrosine phosphatase (PTP) causes inhibition of focal adhesion kinase (FAK) and disruption of focal adhesion formation, leading to inhibition of cell migration.

Fig. 6.

Vascular effects of PGI₂. PGI₂ interact with multiple stimulatory and inhibitory target molecules in circulating platelets, leukocytes, EPCs, and cells of the vascular wall, including ECs, VSMCs, and adventitial fibroblasts. The interaction of PGI₂ with the stimulatory and inhibitory target molecules lead to corresponding stimulation or inhibition of cell signaling or function. cdk, cyclin-dependent kinase; FAK, focal adhesion kinase; ICAM, intercellular adhesion molecule; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule.

Fig. 7.

PGI₂ metabolism and pregnancy-associated vascular disorders. Maternal genetic, immunologic, and environmental factors cause defective placentation early in pregnancy, leading to placental hypoperfusion/hypoxia and the release of cytotoxic factors such as sFlt1, soluble endoglin, ROS, cytokines, and hypoxia-inducible factor (HIF). The circulating cytotoxic factors cause EC dysfunction, with decreased vasodilator NO and PGI₂ and increased vasoconstrictor ET-1 and TXA₂, leading to the development of preeclampsia and IUGR. Defective placentation and other maternal factors such as deficient IGF1 may also lead to IUGR independent of preeclampsia. Maternal diabetes and hyperglycemia lead to increased ROS and DAG in the placental circulation. ROS decreases PGIS activity and leads to increased TXA₂ synthesis over PGI₂, whereas increased DAG increases PKC and TXA₂ activity. This leads to decreased placental perfusion and nutrient transport to the developing fetus, which, together with decreased expression of IP receptors in fetal blood vessels, increases the risk for fetal programming and the development of later adult vascular disease. In addition, in maternal diabetes, hyperglycemia in the embryo leads to increased ROS that decreases PGIS activity, in addition to decreased inositol, PKC, and PLA₂ activity resulting in decreased AA availability and PG synthesis and net decrease in PGE₂ and PGI₂ that are necessary for organogenesis, thereby increasing the risk for diabetic embryopathy. Modulators of PGI₂ or TXA₂ production or activity could affect the course of maternal/fetal vascular disease. Low-dose aspirin and TXAS inhibitors could decrease TXA₂ in preeclampsia, antioxidants decrease ROS production and thereby PGIS inactivation in maternal diabetes, IGF-1 may counter TXA₂ overproduction in IUGR, and carbacyclin can overcome the deficient fetal PGI₂ activity in diabetic embryopathy.

Fig. 8.

PGI₂ metabolism and vascular disorders in premature and full-term newborn. In the premature newborn, PRs and endogenous modulators of PG metabolic pathways are not fully developed and thereby predispose to certain neonatal vascular disorders. In ductus arteriosus (DA) of premature newborn, the increased PGIS, decreased phosphodiesterase (PDE) and cAMP metabolism, and increased vasodilator EP4 receptor, together with decreased PGE₂ catabolism in the lung, lead to increased PGI₂ and PGE₂ availability and vasodilator sensitivity, impaired DA constriction at birth, and patent DA. In the brain, prematurity is associated with increased COX2, decreased PDE, and decreased vasoconstrictor EP1 and EP3 receptors, which, together with decreased PGE₂ catabolism in the lung, lead to increased PGI₂ and PGE₂ production and sensitivity in the cerebral circulation, narrow cerebral BP autoregulation, and increased blood flow to immature cerebral vasculature and result in intraventricular hemorrhage. In addition, in the brain, perinatal cerebral hypoxia and ischemia increase COX2 expression and PGI₂ and PGE₂ production, leading to increased CBF to ischemic tissue, reperfusion injury, neurovascular cell damage, and hypoxic ischemic encephalopathy. Increased COX2 activity also increases ROS that favors increased TXAS activity over PGIS, leading to increased TXA₂ production and further increase in neurovascular cell injury. In the retina, similar to the brain, prematurity could affect the PG pathway with increased COX2, decreased PDE, decreased vasoconstrictor EP1 and EP3 receptors, increased PGI₂ and PGE₂ production and vascular sensitivity to vasodilating PGE₂, resulting in narrow retinal BP autoregulation, and reperfusion injury, leading to retinopathy of prematurity. Because of the immature antioxidant system in the premature retina, the increased oxygen saturation accompanying the increase in retinal blood flow results in increased ROS that leads to increased TXA₂ synthesis, and further retinal vascular damage and aggravation of retinopathy of prematurity. In an ill newborn, perinatal hypoxia or sepsis causes pulmonary vascular damage and endothelial dysfunction, leading to deficient PGI₂ and increased TXA₂ production in pulmonary vessels, increased PVR, and PPHN. Modulators of PG synthesis/activity such as indomethacin and EP4 antagonist inhibit the increase in the vasodilator PGI₂ and PGE₂ production and activity in PDA. Indomethacin could decrease the increased cerebral production of PGI₂ and PGE₂, contributing to intraventricular hemorrhage. A specific IP2 analog 15R-TIC is cytoprotective in hypoxic ischemic encephalopathy. Antioxidants and TXAS inhibitors decrease ROS and TXA₂ in reperfusion injuries of the neonatal retina. IP receptor agonists can compensate for the deficient PGI₂ activity in PPHN.