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

Abstract

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.

Fig. 1

Drug treatment protocols. Pregnant dams were treated with a nonselective cyclooxygenase (COX) inhibitor [indomethacin (Indo)], a selective COX-1 inhibitor (SC560), and/or a selective COX-2 inhibitor (SC236) on the indicated days of gestation [day (d) 1 = presence of vaginal plug]. Fetal studies: protocol A examined the effects of a single dose of a COX inhibitor (either indomethacin, SC236, SC560, or the combination of SC236 with SC560) on the fetal ductus (tissue collected 4 h after treatment); protocol B examined the effects of prolonged COX-1, COX-2, or combined COX-1 and COX-2 inhibition on the fetal ductus (study drugs were administered at the indicated times, and the tissues were harvested 4 h after the last dose); protocol C examined the effects of a single treatment with both COX-1 and COX-2 inhibitors on the fetal ductus. Pregnant dams were treated at either gestation day 16, 17, 18, or 19, and the fetal tissues were harvested 4 h later; protocol D examined the effects of prolonged COX-1 and COX-2 inhibition on the fetal ductus at day 18 of gestation. Pregnant dams were treated on days 15–18 of gestation, and the fetal tissues were harvested 4 h after the last dose on day 18. Newborn studies: protocol E examined the effects of prolonged COX-1 and COX-2 inhibition (on days 11–14 of gestation) on the newborn ductus. Newborn tissues were harvested 4 h after delivery; protocol F examined the effects of prolonged in utero exposure to either COX-1, COX-2, or combined COX-1 and COX-2 inhibitors (on days 15–19 of gestation) on the newborn ductus. Newborn tissues were harvested 4 h after delivery; protocol G examined whether treatment of newborn littermates with N^G-nitro-l-arginine methyl ester (l-NAME) or U-46619 (or saline) altered patency of the ductus arteriosus (after prolonged in utero exposure to both COX inhibitors on days 15–19 of gestation). PP1, postpartum day 1.

Fig. 2

Physiological closure of the mouse ductus arteriosus. Anatomical views of the open fetal ductus arteriosus on the morning of day 19 of gestation (A) and constricted newborn ductus arteriosus at 4 h of age (B). Serial thoracic sections demonstrate histologic features and dimensions of the open fetal ductus (C) and adjacent transverse aortic arch (E) compared with the closed newborn ductus (D) and corresponding transverse aortic arch (F). aAO, ascending aorta; tAO, transverse aorta; dAO, descending aorta; PA, pulmonary artery; DA, ductus arteriosus; Tra, trachea; Eso, esophagus, VB, vertebral body.

Fig. 3

Response of the fetal ductus arteriosus to acute and chronic COX-1 or COX-2 inhibition. Fetuses of wild-type dams treated with indomethacin, a selective COX-1 inhibitor (SC560), or selective COX-2 inhibitor (SC236) were compared. Vessel dimensions are expressed as a ratio of ductus arteriosus (DA) and transverse aorta (AO) diameters. A: acute pharmacological inhibition with indomethacin (n = 6, 2 litters) or combined COX inhibitors (n = 19, 7 litters) showed similar ductus constriction at term gestation compared with untreated (No Tx) controls (n = 11, 6 litters). Acute COX-1 inhibition (n = 9, 3 litters) caused less ductus constriction than COX-2 (n = 11, 4 litters). B: chronic COX-1 (n = 9, 3 litters), COX-2 (n = 35, 12 litters), or combined COX inhibition (n = 20, 6 litters) did not constrict the fetal ductus. P < 0.05 compared with control (*) and compared with SC560 (§).

Fig. 4

Developmental stage-specific response of the fetal ductus arteriosus to acute and chronic COX inhibition. Fetuses of wild-type dams treated with combined COX-1 (SC560) and COX-2 (SC236) inhibitors were compared. Vessel dimensions are expressed as a ratio of DA and AO diameters. A: acute inhibition of both COX isoforms did not alter the day 16 (n = 9, 3 litters) or day 17 (n = 9, 3 litters) gestation ductus but induced constriction on day 18 (n = 13, 4 litters) and day 19 (n = 19, 7 litters). B: in contrast to the acute responses, chronic exposure to both COX inhibitors, starting on day 15, was not associated with ductus constriction on either day 18 (n = 12, 3 litters) or day 19 (n = 20, 6 litters). *P < 0.05 compared with vehicle-treated dams at each gestation.

Fig. 5

Ductus contractility in the newborn mouse after genetic and/or prolonged pharmacological COX inhibition in utero. A: newborn pups born to dams with chronic COX-1 (SC560), COX-2 (SC236), or combined COX inhibitors were placed in 80% oxygen for 4 h after birth. Vessel dimensions are expressed as a ratio of DA and AO diameters. Chronic COX inhibition on days 11–14 of gestation (n = 20, 7 litters) did not alter ductus closure after birth, whereas pups with COX-1 (n = 12, 4 litters), COX-2 (n = 23, 13 litters), or combined COX inhibition (n = 29, 11 litters) on days 15–19 of gestation had delayed closure of the ductus compared with controls (n = 12, 4 litters). B: targeted deletion of COX-1 (n = 11, 4 litters) had minimal effect on ductus closure after birth, whereas COX-2 deletion (n = 20, 8 litters) and COX-1/COX-2 double-null pups (n = 12, 6 litters) had a significant delay in ductal closure compared with controls (n = 12, 4 litters). Closure of the ductus of COX-1 null pups chronically exposed to COX-2 inhibitor in utero (n = 16, 6 litters) was also significantly delayed. KO, knockout. *P < 0.05 compared with untreated newborns at 4 h of age.

Fig. 6

Responsiveness of the newborn patent ductus arteriosus. Newborn pups born to dams with chronic COX-1 (SC560) plus COX-2 (SC236) inhibition on days 15–19 of gestation were placed in 80% oxygen for 4 h after birth. Vessel dimensions are expressed as a ratio of DA and AO diameters. Ductus closure rates of pups treated with saline (n = 24, 12 litters), l-NAME (n = 21, 6 litters), or U-46619 (n = 24, 7 litters) were significantly delayed compared with untreated controls (n = 12, 4 litters; P < 0.05) but did not differ between the treatment groups. Similarly, there was no difference in ductus caliber between saline-treated (n = 15, 5 litters) and l-NAME-treated (n = 14, 4 litters) COX-1 null pups chronically exposed to COX-2 inhibitor in utero.