Endothelin in the perinatal circulation

During the fetal period, blood is oxygenated through the placenta, and most of the cardiac output bypasses the lung through the ductus arteriosus. At birth, pulmonary vascular resistance falls with the initiation of ventilation. Coincidentally, the ductus arteriosus constricts. Endothelin-1 (ET-1) appears to play an important role during that transition period and postnatally. ET-1 can dramatically increase resistance in the placental microcirculation and may be involved in blood flow redistribution with hypoxia. At birth, the increase in oxygen tension is important in triggering ductus vasoconstriction. It is proposed that oxygen triggers closure of the ductus arteriosus by activating a specific, cytochrome P450-linked reaction, which in turn stimulates the synthesis of ET-1. On the neonatal heart, ET-1 has a positive chronotropic but negative inotropic effect. In the newborn piglet and the fetal lamb, both term and preterm, ET-1 causes a potent, long-lasting pulmonary vasoconstriction. Furthermore, a transient dilator response has been identified, and it is ascribed to nitric oxide formation. ET receptors are abundant in the piglet pulmonary vasculature. They are predominantly of the ETA constrictor subtype, though ETB2 constrictor receptors may also be present in certain species. The dilator response is linked to the ETB1 receptor, and the number of ETB1 receptors is reduced in hypoxia-induced pulmonary hypertension. ET-1 appears to be a causative agent in the pathogenesis of hypoxia- and hyperoxia-induced pulmonary hypertension as demonstrated by reversal of hemodynamic and morphological changes with treatment with an ETA receptor antagonist. Findings are amenable to practical applications in the management of infants with pulmonary hypertension or requiring persistent patency of the ductus arteriosus.     

Prostanoids in immunity: roles revealed by mice deficient in their receptors

Prostanoids including prostaglandins (PGs) and thromboxanes (TX) are a group of lipid mediators formed and released in response to various, often noxious, stimuli. While the roles of prostanoids in acute inflammatory responses are well known and have been extensively studied, it is generally believed that they play very little in immunity. This is partly because non-steroidal anti-inflammatory drugs that inhibit prostanoid synthesis have little effects on immune processes in vivo. Prostanoids exert their actions by acting on a family of G-protein-coupled receptors. They include PGD receptor, EP1, EP2, EP3 and EP4 subtypes of PGE receptor, PGF receptor, PGI receptor and TX receptor. We generated mice deficient in each of these prostanoid receptors individually, and examined their roles under various pathological conditions. These studies have revealed that prostanoids works at various sites or levels of immune responses and exert many, often opposing, actions. For example, using EP4-deficient mice, we found that stimulation of the PGE(2)-EP4 signaling in dendritic cells facilitates their migration and maturation, while the stimulation of the same pathway in T cells potently suppresses their activation and proliferation. The latter action is evident in PGE(2)-mediated suppression of T cell proliferation in the gut of mice subjected to dextran sodium sulfate-induced colitis, a model of inflammatory bowel disease. Here I summarize our findings obtained by these and other studies. These findings suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of certain immunological disorders.     

Prostanoids in tumor angiogenesis: therapeutic intervention beyond COX-2

Prostanoids regulate angiogenesis in carcinoma and chronic inflammatory disease progression. Although prostanoid biosynthetic enzymes and signaling have been extensively analyzed in inflammation, little is known about how prostanoids mediate tumor-induced angiogenesis. Targeted cyclooxygenase (COX)-2 inhibition in tumor, stromal and endothelial cells is an attractive antiangiogenic strategy; however, the associated cardiovascular side effects have led to the development of a new generation of nonsteroidal anti-inflammatory drugs (NSAIDs) acting downstream of COX. These agents target terminal prostanoid synthases and prostanoid receptors, which may also include several peroxisome proliferator-activated receptors (PPARs). Here, we discuss the role of prostanoids as modulators of tumor angiogenesis and how prostanoid metabolism reflects complex cell-cell crosstalk that determines tumor growth. Finally, we discuss the potential of new NSAIDs for the treatment of angiogenesis-dependent tumor development.     

In vivo reopening of the neonatal ductus arteriosus by a prostanoid EP4-receptor agonist in the rat

Prostaglandin E1 is used to reopen the constricted ductus arteriosus in neonates with ductus-dependent circulation. To clarify possible prostanoid receptor agonists that can reopen the neonatal ductus with fewer side effects, we studied in vivo reopening of the neonatal ductus arteriosus by AE1-329, a prostanoid EP4-receptor agonist, in the rat. Neonatal rats were incubated at 33 degrees C. The inner diameter of the ductus was measured with a microscope and a micrometer following rapid whole-body freezing. Intraesophageal pressure was measured with a Millar micro-tip transducer. The ductus arteriosus constricted quickly after birth, and the inner diameter was 0.80 and 0.08 mm at 0 and 60 min after birth. PGE1 and AE1-329, injected subcutaneously at 60 min after birth, dilated the ductus dose-dependently. Thirty minutes after injection of 10 ng/g of PGE1 and AE1-329, the ductus diameter was 0.52 and 0.65 mm, respectively. The ductus-dilating effect of PGE1 was maximal at 15-30 min, and disappeared at 2 h. The ductus-dilating effect of AE1-329 was prolonged, the ductus was widely open at 6 h, and closed at 12 h after injection of 10 ng/g AE1-329. AE1-259-01 (EP2 agonist) (100 ng/g) did not dilate the neonatal ductus. Respiration was depressed by PGE1, but not by AE1-329. These results indicate the major role of EP4 in the neonatal ductus and that AE1-329, an EP4 agonist, can be used to dilate the neonatal constricted ductus without the side effects shown by EP3, including apnea.     

Prostanoids receptors signaling in different diseases/cancers progression

Abstract

Prostanoids, that is, prostaglandins (PGs) PGE₂, PGF_2α, PGI₂, PGD₂ and thromboxane A₂(TXA₂), are the oldest members of the eicosanoid family. The PGs are a family of lipid mediators formed in response to various stimuli. They are transported into the extracellular microenvironment by specific multidrug resistance-associated proteins (MRPs) after synthesis. Once exported to the microenvironment, prostanoids bind to G-protein coupled receptors that contain seven transmembrane spanning domains. There are eight types of the prostanoid receptors conserved in mammals from mouse to human. They are the PGD receptor (DP), four subtypes of the PGE receptor (EP₁, EP₂, EP₃ and EP₄), the PGF receptor (FP), PGI receptor (IP) and TXA receptor (TP). Recently, several studies have revealed the roles of PG receptor signaling in various pathological conditions, and suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of the pathological conditions. Here we review these recent findings of roles of prostanoid receptor signaling and their therapeutic implications.     

Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall

Nitric oxide (NO) and cyclooxygenase (COX)-derived prostaglandins are critical regulators of the fetal ductus arteriosus. To examine the interaction of these pathways within the ductus wall, the ductus arteriosus of term and preterm fetal mice was evaluated by pressurized myography. The isolated preterm ductus was more sensitive to NOS inhibition than at term. Sequential NOS and COX inhibition caused 36% constriction of the preterm ductus regardless of drug order. In contrast, constriction of the term ductus was dependent on the sequence of inhibition; NOS inhibition prior to COX inhibition produced greater constriction than when inhibitors were given in reverse order (36 ± 6% versus 23 ± 5%). Selective COX-1 or COX-2 inhibition prior to N(G)-nitro-l-arginine methyl ester (l-NAME) induced the expected degree of constriction. However, NOS inhibition followed by selective COX-2 inhibition caused unexpected ductal dilation. These findings are consistent with NO-induced activation of COX in the ductus arteriosus wall and the production of a COX-2-derived constrictor prostanoid that contributes to the balance of vasoactive forces that maintain fetal ductus arteriosus tone.     

Prostanoid function and cardiovascular disease

Prostanoids, including prostaglandins (PGs) and thromboxanes (TXs) are synthesized from arachidonic acid by the combined action of cyclooxygenases (COXs) and PG and TX synthases. Finally after their synthesis, prostanoids are quickly released to the extracellular medium exerting their effects upon interaction with prostanoid receptors present in the neighbouring cells. These agents exert important actions in the cardiovascular system, modulating vascular homeostasis and participating in the pathogenesis of vascular diseases as thrombosis and atherosclerosis. Among prostanoids, Tromboxane (TX)A(2), a potent platelet activator and vasoconstrictor and prostacyclin (PGI2), a platelet inhibitor and vasodilator, are the most important in controlling vascular homeostasis. Although multiple studies using pharmacological inhibitors and genetically deficient mice have demonstrated the importance of prostanoid-mediated actions on cardiovascular physiology, further analysis on the prostanoid mediated actions in the vascular system are required to better understand the benefits and risks for the use of COX inhibitors in cardiovascular diseases.     

Developmental regulation of prostaglandin E2 synthase in porcine ductus arteriosus

The synthesis of PGE(2), the major vasodilator prostanoid of the ductus arteriosus (DA), is catalyzed by PGE(2) synthases (PGES). The factors implicated in increased PGE(2) synthesis in the perinatal DA are not known. We studied the developmental changes of PGES along with that of cyclooxygenase (COX)-2 and cytosolic phospholipase A(2) (cPLA(2)) in the DA of fetal (75-90% gestation) and immediately postnatal newborn (NB) piglets. Levels of microsomal PGES (mPGES), COX-2, and PGE(2) in the DA of NB were approximately 7-fold higher than in fetus; activities of cytosolic PGES (cPGES) and cPLA(2) in DA of the fetus and NB did not differ. Because platelet-activating factor (PAF) could regulate COX-2 expression, the former was measured and found to be more abundant in the DA of the NB than of fetus. PAF elicited an increase in mPGES, COX-2, and PGE(2) in fetal DA to levels approaching those of the NB; cPGES, cPLA(2), and COX-1 were unaffected. In perinatal NB DA, PAF receptor antagonists BN-52021 and THG-315 reduced mPGES, COX-2, and PGE(2) levels and were associated with increased DA tone. It is concluded that PAF contributes in regulating DA tone by governing mPGES, COX-2, and ensuing PGE(2) levels in the perinate.     

Developmental response to indomethacin: a comparison of isometric tension with PGE2 formation in the lamb ductus arteriosus

We studied the effects of oxygen and indomethacin on the isometric contractile response and the production of PGE2 in isolated rings of lamb ductus arteriosus from animals of different gestational ages (100 to 144 days; term is 150 days). Rings of ductus arteriosus from animals less than 110 days released significantly less PGE2 than did rings from animals greater than 120 days. The indomethacin-induced increase in muscle tension in relation to the decrease in endogenous PGE2 production in preparations from animals less than 110 days gestation was greater than in animals older than 120 days. These findings do not support the hypothesis that immature animals have a larger indomethacin-induced contraction due to an increased production of PGE2 earlier in gestation. They are, however, consistent with a decreased sensitivity to PGE2 in the more mature animals; they also support the hypothesis that the decreased effectiveness of indomethacin on the ductus arteriosus from later gestation animals reflects primarily a decrease in the sensitivity of the vessel to PGE2 during development.     

Altered plasma proteins released from platelets and endothelial cells are associated with human patent ductus arteriosus

Patent ductus arteriosus is the third most common congenital heart disease and resulted from the persistence of ductal patency after birth. Ductus arteriosus closure involves functional and structural remodeling, controlled by many factors. The changes in plasma protein levels associated with PDA closure are not known. Here we for the first time demonstrate six key differential plasma proteins in human patent ductus arteriosus patients using proteomic technology and present a model to illustrate the constriction and closure of ductus arteriosus. Differentially expressed proteins were analyzed by using isobaric tags for relative and absolute quantification and validated by enzyme-linked immunosorbent assay in new samples. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD008568. We found 74 upregulated and 98 downregulated proteins in the plasma of patients with PDA. Five decreased proteins (platelet factor 4, fibrinogen, von Willebrand factor, collagen, and mannose binding lectin-associated serine protease-2) and one increased protein (fibronectin) may increase the risk of patent ductus arteriosus. Those proteins are closely related to platelet activation and coagulation cascades, complement mannan-binding-lectin, and other systemic signaling pathways. Our findings for the first time indicate that the differential proteins involved in different pathways may play key roles in the nonclosure of the ductus arteriosus in humans and may be developed as biomarkers for diagnosis. All those findings may be served as the basis of understanding the etiology and pathogenesis of patent ductus arteriosus.     

Cyclooxygenase-2 inhibitors constrict the fetal lamb ductus arteriosus both in vitro and in vivo

Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents; however, they also have adverse fetal effects such as constriction of the fetal ductus arteriosus. Recently, selective COX-2 inhibitors have been used in the management of preterm labor in the hope of avoiding fetal complications. However, both COX-1 and -2 are expressed by cells of the ductus arteriosus. We used fetal lambs (0.88 gestation) to assess the ability of selective COX-2 inhibitors celecoxib and NS398 to affect the ductus arteriosus. Both selective COX-2 inhibitors decreased PGE(2) and 6ketoPGF(1alpha) production in vitro; both inhibitors constricted the isolated ductus in vitro. The nonselective COX-1/COX-2 inhibitor indomethacin produced a further reduction in PG release and an additional increase in ductus tension in vitro. We used a prodrug of celecoxib to achieve 1.4 +/- 0.6 microg/ml, mean +/- standard deviation, of the active drug in vivo. This concentration of celecoxib produced both an increase in pressure gradient and resistance across the ductus; celecoxib also decreased fetal plasma concentrations of PGE(2) and 6ketoPGF(1alpha). Indomethacin (0.7 +/- 0.2 microg/ml) produced a significantly greater fall in ductus blood flow than celecoxib and tended to have a greater effect on ductus resistence in vivo. We conclude that caution should be used when recommending COX-2 inhibitors for use in pregnant women, because COX-2 appears to play a significant role in maintaining patency of the fetal ductus arteriosus.     

Constriction of fetal ductus arteriosus by non-steroidal anti-inflammatory drugs:study of additional 34 drugs

Our study on transplacental effects of 24 non-steroidal anti-inflammatory drugs (NSAID) on the fetal ductus arteriosus of full-term pregnant rats was extended to 34 other NSAID using the same whole-body freezing technique (1). In total, 58 NSAID were evaluated, and their potency in usual clinical doses was classified into 4 grades. Indomethacin and 15 other NSAID caused strong fetal ductal constriction, phenylbutazone and 14 other NSAID caused moderate, and aspirin and 16 other NSAID caused mild constriction of the fetal ductus arteriosus. Salicylamides, and six out of eight basic NSAID did not constrict the fetal ductus arteriosus. Further clinical implications of these results are discussed.     

Semiquantitative RT-PCR method coupled to capillary electrophoresis to study 4α-reductase mRNA isozymes in rat ventral prostate in different androgen status

Isoprostanes are members of a family of prostaglandin isomers that are produced by free radical-catalysed mechanisms. They have become well-recognized indicators of oxidant-induced cell damage in a variety of pathophysiological conditions. Several isoprostanes have been shown to possess biological activity in whole-animal, isolated tissue and cell-based systems. Their actions include vasoconstriction, platelet aggregation and cardiac hypertrophy. Current evidence suggests that these effects are mediated by prostanoid receptors through a complex set of interactions that involve agonism, partial agonism, desensitization and co-operative behaviors. It is likely that other mechanisms of action are waiting to be discovered. Based on a consideration of these biological effects, we argue that isoprostanes are more than mere markers and may serve as active participants in promoting and exaggerating pathophysiological changes. To tease out their roles requires considerable more work and a willingness to suspend disbelief based on limited evidence.     

Constriction of fetal ductus arteriosus by non-steroidal anti-inflammatory drugs: Study of additional 34 drugs

Our study on transplacental effects of 24 non-steroidal anti-inflammatory drugs (NSAID) on the fetal ductus arteriosus of full-term pregnant rats was extended to 34 other NSAID using the same whole-body freezing technique (1). In total, 58 NSAID were evaluated, and their potency in usual clinical doses was classified into 4 grades. Indomethacin and 15 other NSAID caused strong fetal ductal constriction, phenylbutazone and 14 other NSAID caused moderate, and aspirin and 16 other NSAID caused mild constriction of the fetal ductus arteriosus. Salicylamides, and six out of eight basic NSAID did not constrict the fetal ductus arteriosus. Further clinical implications of these results are discussed.     

Developmental response to indomethacin: A comparison of isometric tension with PGE2 formation in the lamb ductus arteriosus

We studied the effects of oxygen and indomethacin on the isometric contractile response and the production of PGE₂ in isolated rings of lamb ductus arteriosus from animals of different gestational ages (100 to 144 days; term is 150 days). Rings of ductus arteriosus from animals less than 110 days released significantly less PGE₂ than did rings from animals greater than 120 days. The indomethacin-induced increase in muscle tension in relation to the decrease in endogenous PGE₂ production in preparations from animals less than 110 days gestation was greater than in animals older than 120 days. These findings do $\text{not}$ support the hypothesis that immature animals have a larger indomethacin-induced contraction due to an increased production of PGE₂ earlier in gestation. They are, however, consistent with a decreased sensitivity to PGE₂ in the more mature animals; they also support the hypothesis that the decreased effectiveness of indomethacin on the ductus arteriosus from later gestation animals reflects primarily a decrease in the sensitivity of the vessel to PGE₂ during development.     

The control of cardiovascular shunts in the fetal and perinatal period

The fetal circulation has two major vascular shunts, the ductus arteriosus and the ductus venosus. The ductus arteriosus connects the pulmonary artery with the descending portion of the aortic arch, hence shunting most of the right ventricular output away from the unexpanded lungs. The ductus venosus connects instead the portal sinus with the inferior vena cava and allows well-oxygenated umbilical vein blood to bypass the liver and reach the central circulation rapidly. Both blood vessels cease their function after birth and undergo permanent closure. It is now well established that prenatal patency of the ductus arteriosus is an active state sustained by a prostaglandin. A similar mechanism has been recently recognized in the fetal ductus venosus. Evidence is presented indicating that prostaglandin E2 and prostaglandin I2 are natural relaxants, respectively, for the ductus arteriosus and the ductus venosus. In addition, both vascular shunts share the dependence on an endogenous cytochrome P-450 mechanism to develop their contractile tone. This mechanism may be important in the normal process of shunt closure at birth. While broadening the knowledge of fetal cardiovascular homeostasis, advances in this field have important implications for the prevention and management of certain pathological conditions affecting the newborn.     

Interactions between the right ventricle and pulmonary vasculature in the fetus.

A midsystolic plateau differentiates the pattern of fetal pulmonary trunk blood flow from aortic flow. To determine whether this plateau arises from interactions between the left (LV) and right ventricle (RV) via the ductus arteriosus or from interactions between the RV and the lung vasculature, we measured blood flows and pressures in the pulmonary trunk and aorta of eight anesthetized (ketamine and alpha-chloralose) fetal lambs. Wave-intensity analysis revealed waves of energy traveling forward, away from the LV and the RV early in systole. During midsystole, a wave of energy traveling back toward the RV decreased blood flow velocity from the RV and produced the plateau in blood flow. Calculations revealed that this backward-traveling wave originated as a forward-traveling wave generated by the RV that was reflected from the lung vasculature back toward the heart and not as a forward-traveling wave generated by the LV that crossed the ductus arteriosus. Elimination of this backward-traveling wave and its associated effect on RV flow may be an important component of the increase in RV output that accompanies birth.     

Inhibition of cyclooxygenase isoforms in late- but not midgestation decreases contractility of the ductus arteriosus and prevents postnatal closure in mice

Use of cyclooxygenase (COX) inhibitors to delay preterm birth is complicated by in utero constriction of the ductus arteriosus and delayed postnatal closure. Delayed postnatal closure has been attributed to loss of vasa vasorum flow and ductus wall ischemia resulting from constriction in utero. We used the murine ductus (which does not depend on vasa vasorum flow) to determine whether delayed postnatal closure may be because of mechanisms independent of in utero constriction. Acute inhibition of both COX isoforms constricted the fetal ductus on days 18 and 19 (term) but not earlier in gestation; COX-2 inhibition constricted the fetal ductus more than COX-1 inhibition. In contrast, mice exposed to prolonged inhibition of COX-1, COX-2, or both COX isoforms (starting on day 15, when the ductus does not respond to the inhibitors) had no contractile response to the inhibitors on days 18 or 19. Newborn mice closed their ductus within 4 h of birth. Prolonged COX inhibition on days 11-14 of gestation had no effect on newborn ductal closure; however, prolonged COX inhibition on days 15-19 resulted in delayed ductus closure despite exposure to 80% oxygen after birth. Similarly, targeted deletion of COX-2 alone, or COX-1/COX-2 together, impaired postnatal ductus closure. Nitric oxide inhibition did not prevent the delay in ductus closure. These data show that impaired postnatal ductus closure is not the result of in utero ductus constriction or upregulation of nitric oxide synthesis. They are consistent with a novel role for prostaglandins in ductus arteriosus contractile development.