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.     

Prostaglandin E2 in the lung lavage fluid of premature newborns before and after surgical or medical closure of a patent ductus arteriosus

To analyze the role of prostaglandin E2 in maintaining ductal patency in premature newborns, we measured the PGE2 concentration in the lung lavage fluid of nine patients within 24 h before and 4-8 h after surgical ligation of a patent ductus arteriosus and in two patients before and after closure of the ductus following intravenous indomethacin. The concentration of PGE2 ranged from 240 to 3770 pg/ml (mean 1666 +/- 1256 pg/ml) before operative intervention and show a significant decrease to 0-300 pg/ml (mean 93 +/- 106 pg/ml, P less than 0.001, Student's two-tailed t-test) within a few hours after ligation of the ductus arteriosus. The same significant decrease could be seen in two patients with successful indomethacin therapy (0.25 mg/kg in three doses/day) with concomitant ductus closure. In contrast, when indomethacin was given in a reduced dose (0.1 mg/kg in three doses/day), only a slight effect on PGE2 synthesis could be seen without closure of ductus arteriosus. We suggest that the fall of PGE2 levels in lung lavage fluid reflects the local synthesis in the ductus arteriosus itself and is responsible for the decrease induced by surgical ligation or pharmacological inhibition by indomethacin.     

15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer

Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.     

Inhibition of prostaglandin delta 13 reductase activity in rabbit kidney cortex by glutathione disulfide.

t-Butyl hydroperoxide and H2O2-Fe(2+)-EDTA-glutathione system which produces hydroxyl radicals did not affect the 15-hydroxy prostaglandin dehydrogenase activity in rabbit kidney cortex. On the other hand, H2O2-Fe(2+)-EDTA-glutathione system inhibited the prostaglandin delta 13 reductase activity. Mannitol, a scavenger of hydroxyl radicals, had no effect on the inhibitory action of this system, indicating that the effect of H2O2-Fe(2+)-EDTA-glutathione system on the prostaglandin delta 13 reductase may not be due to produced hydroxyl radicals. As a result of further investigation, it was shown that glutathione disulfide, which is synthesized concomitantly with hydroxyl radicals from H2O2-Fe(2+)-EDTA-glutathione, inhibited the prostaglandin delta 13 reductase activity. These results suggest that hydroperoxides and hydroxyl radicals may not be likely candidates for the modulator of the catabolism of prostaglandins in the kidney cortex, and that glutathione disulfide has the potential to modulate the prostaglandin catabolism by affecting the prostaglandin delta 13 reductase activity.     

Mechanical stress and prostaglandin E2 synthesis in cartilage

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Prostaglandin biosynthesis and catabolism in the developing fetal sheep kidney

The enzymatic capacity to form and degrade prostaglandins was studied in kidneys from fetal sheep (gestational ages 40,44,49,77,116 and 140 days). The prostaglandin system was detectable at all ages. Only prostaglandin F_2α was formed by renal homogenates at 40 and 44 days gestation; prostaglandin E₂ was first formed by the 77 day kidney and became the major prostaglandin by 116 days (3 fold relative to prostaglandin F_2α). Prostaglandin catabolism took place via the PG 15-hydroxy dehydrogenase and PG 13-reductase pathways. Catabolism was first detected at 40 days gestation and rose with age to an activity (15-PGDH) approximately 80 ng/min/mg protein in the term kidney. Only PG 15-hydroxydehydrogenase activity was detected at 40 days gestation, but PG 13-reductase activity became evident by 116 days and persisted until term. As with fetal sheep lungs (see preceding publication) PG 13-reductase activity was saturated quickly. These results confirm our observations with other tissues that prostaglandin catabolism is variable during ontogeny.     

Prostaglandin I2 is less relaxant than prostaglandin E2 on the lamb ductus arteriosus.

Prostaglandin (PG) I2 and its stable metabolite, 6-keto-PGF1alpha, were tested on the isolated ductus arteriosus from mature fetal lambs. PGI2 relaxed the ductus in high doses (threshold 10(-6)M) and its activity disappeared on standing at room temperature for 30 minutes. 6-keto-PGF1alpha was inactive at all doses. By contrast, PGE2 produced a dose-dependent relaxation over a range between 10(-10) and 10(-6)M. These findings confirm that PGE2 is the most potent ductal relaxant among the known derivatives of arachidonic acid. PGE2 probably maintains ductus patency in the fetus and, together with PGE1, remains the compound of choice in the management of newborns requiring a viable ductus for survival.     

Studies on closure of the ductus arteriosus. X. In vivo effect of prostaglandin.

The effect of prostaglandins F2alpha, E1 and of 7-oxa-13-prostynoic acid on the newborn rabbit ductus can be studied using the whole-body freezing technique. PGF2alpha and PGE1 were able to re-open the closing ductus arteriosus in adequately oxygenated animals. PGF2alpha administration was accompanied by a strong physical reaction in the rat but less in the rabbit. PGE1 had sedative effects in both animals. A prostaglandin antagonist, 7-oxa-13-prostynoic acid had no effect on normal ductal closure nor did it counteract the effects of PGF2alpha and PGE1. The role of prostaglandins in homeostasis during the fetal and newborn period may be to modify ductal tone.     

Involvement of intramural prostaglandin E2 in prenatal patency of the lamb ductus arteriosus

Release of prostaglandin E2 (PGE2) was studied in isolated ductus arteriosus preparations from immature (103 or 104 days gestation; term, 147 days) and near-term fetal lambs. Mature preparations produced measurable amounts of the compound in most cases and the release rate was 19 +/- 2 pg/(100 mg wet weight X min) at a PO2 of 3-8 Torr (1 Torr = 133.3 Pa). PGE2 release increased with the PO2 of the medium, peak values (about 125 pg/(100 mg X min)) being attained at 106-276 Torr when the oxygen-induced contraction was still submaximal. Experiments in which tissues were either contracted with excess potassium or relaxed with CO proved that PGE2 formation is independent from the contractile state. PGE2 was also released from ductus preparations lacking the adventitia, the intima, or both; however, release values were maximal when the adventitia was preserved. The magnitude of the intrinsic tone in these stripped preparations was inversely related to the rate of PGE2 formation. Reduced glutathione increased PGE2 release from the mature ductus, whole or stripped, and also relaxed hypoxic preparations; both effects were reversed by concomitant treatment with indomethacin. PGE2 synthesis tended to be greater in the immature than the mature ductus, maximal values (115 +/- 27 pg/(100 mg X min)) being observed at 6-8 Torr. We conclude that the ductus arteriosus is endowed with an enzyme system for the synthesis of PGE2 whose function accords with an effector role of the compound in the regulation of tone. These findings, together with the potent relaxation exerted by PGE2 at low PO2, indicate that the locally generated prostaglandin is well suited for keeping the ductus patent in the fetus.     

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.     

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A1, A2, E1, E2, F2alpha and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2. All the prostaglandins (except PGF2alpha and its 15-keto-metabolites) relaxed the tissue. However, only PGE1, E2, and their 13,14-dihydro-metabolites, were effective at concentrations below 10(-8) M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Prostaglandin biosynthesis and catabolism in the lamb ductus arteriosus, aorta and pulmonary artery.

Homogenates of tissues from fetal and neonatal lamb ductus arteriosus, aorta and pulmonary artery have the capacity to convert arachidonic acid as well as the intermediate prostaglandin endoperoxide, prostaglandin H2, into three products: prostaglandins E2, F2alpha and a major product 6-ketoprostaglandin F1alpha. The three tissues also displayed prostaglandin 15-hydroxydehydrogenase and 13-reductase catabolic activities. The catabolishing system showed considerable substrate specificity: prostaglandin E1 was a good substrate whereas prostaglandins F1alpha and F2alpha were completely devoid of catabolism. The complete system was observed in immature as well as mature arterial vessels, in the fetus as well as the neonate (up to 7 days old). These experiments demonstrate the presence of several components of the prostaglandin system in these tissues and offer biochemical evidence for the implication of prostaglandins E2 and I2 in the maintenance of the ductus and neighboring vessels in a relaxed state in the fetus.     

Meclofenamate does not affect lung development in fetal sheep

Prostaglandins may be involved in some aspects of fetal lung development, including surfactant metabolism, tracheal fluid production, and possibly lung growth. In the fetus, during the days before delivery, plasma PGE2 concentration increases and concurrently, tracheal fluid production decreases and surfactant production increases. To determine whether the increase in PGE2, specifically plasma PGE2 concentration, is responsible for these changes, we continuously infused the prostaglandin synthetase inhibitor, meclofenamate (0.7 mg/h per kg), into 8 fetal sheep for 5-13 days before delivery; 5 control fetuses received a continuous infusion of solvent for 5-11 days before delivery. Meclofenamate infusion significantly decreased plasma PGE2 concentrations until the day of delivery. However, meclofenamate did not affect tracheal fluid production or its decrease before delivery, fetal plasma cortisol concentration, surfactant content of tracheal fluid and lung tissue, organ weights, lung weights, or lung DNA and protein content. We conclude that the changes in lung development during the days before delivery are not dependent on the usual high fetal plasma concentration of PGE2 or its increase before delivery.     

Involvement of the constitutive prostaglandin E synthase cPGES/p23 in expression of an initial prostaglandin E2 inactivating enzyme, 15-PGDH

We previously showed that cytosolic prostaglandin (PG) E synthase (cPGES/p23) which isomerizes PGH(2) to PGE(2), is essential for fetal mouse development. Embryonic fibroblasts derived from cPGES/p23 knockout mice generated higher amounts of PGE(2) in culture supernatants than wild-type-derived cells. In order to elucidate this apparent conflict that absence of PGE(2) synthetic enzyme caused facilitation of PGE(2) biosynthesis, we examined expression of the PGE(2) degrading enzyme in embryonic fibroblasts. We report here that embryonic fibroblasts deficient in cPGES/p23 decreased the expression of the PGE(2) degrading enzyme, 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which catalyzes the inactivating conversion of the PGE(2) 15-OH to a 15-keto group, compared with that of wild-type. In addition, rat fibroblastic 3Y1 cells harboring cPGES/p23 siRNA exhibited lower 15-PGDH expression than mock-transfected cells. Furthermore, forcible expression of cPGES/p23 in 3Y1 cells resulted in facilitation of 15-PGDH promoter activity. These results suggest that the PGE(2)-inactivating pathway is controlled by the PGE(2) biosynthetic enzyme, cPGES/p23.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGF1alpha.

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE2, PGF2alpha, 6 keto PGF1alpha (and its unstable precursor PGI2). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO2 and indomethacin. All the prostaglandins (except PGF2alpha) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE2, however, relaxed the vessel at concentrations below 10(-8)M. PGI2 and 6 keto PGF1alpha had approximately 0.001 and 0.0001 times the activity of PGE2. Although PGE2 has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE2 might make it the most significant prostaglandin in regulating the patency of the vessel.     

15-Hydroxyprostaglandin-dehydrogenase is involved in anti-proliferative effect of non-steroidal anti-inflammatory drugs COX-1 inhibitors on a human medullary thyroid carcinoma cell line

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin (PG) synthesis enzymes, the cyclooxygenases (COX-1 and 2). It is suggested that these enzymes are not their only targets. We reported that in tumoral TT cell, indomethacin, in vivo and in vitro, decreases proliferation and increases activity of 15-hydroxyprostaglandin-dehydrogenase (15-PGDH), the PG catabolism key enzyme. Here, we show that the COX-1 inhibitors, selective or not, and sulindac sulfone, a non-COX inhibitor, increased 15-PGDH activity and reduced PGE2 levels. This increase was negatively correlated to the decrease in cell proliferation and suggested that 15-PGDH could be implicated in NSAIDs anti-proliferative effect. Indeed, the silencing of 15-PGDH expression by RNA interference using 15-PGDH specific siRNA enhanced TT cell proliferation and abolished the anti-proliferative effect of a representative non-selective inhibitor, ibuprofen. Moreover, a specific inhibitor of 15-PGDH activity, CAY 10397, completely reversed the effect of ibuprofen on proliferation. Consequently our results demonstrate that, at least in TT cells, 15-PGDH is implicated in proliferation and could be a target for COX-1 inhibitors specific or not. NSAIDs defined by their COX inhibition should also be defined by their effect on 15-PGDH.     

Understanding human 15-hydroxyprostaglandin dehydrogenase binding with NAD+ and PGE2 by homology modeling, docking and molecular dynamics simulation

Homology modeling, molecular docking, and molecular dynamics simulation have been performed to determine human 15-hydroxyprostaglandin dehydrogenase (15-PGDH) binding with its NAD+ cofactor and prostaglandin E2 (PGE2) substrate. The computational studies have led to a three-dimensional (3D) model of the entire 15-PGDH-NAD+-PGE2 complex, demonstrating the detailed binding of PGE2 with 15-PGDH for the first time. This 3D model shows specific interactions of the protein with the cofactor and substrate in qualitative agreement with available experimental data. Our model demonstrates the PGE2-binding cavity of the protein for the first time. The model further leads to an interesting prediction that the catalytic activity of 15-PGDH should also significantly be affected by Gln148, in addition to the previously known three catalytic residues (Ser138, Tyr151, and Lys155). The reported 3D model of 15-PGDH-NAD+-PGE2 complex might be valuable for future rational design of novel inhibitors of 15-PGDH.