Although acute COX inhibition in near-term mice led to fetal ductus constriction (), prolonged COX inhibition during the last 25% of gestation led to an impaired contractile response and an incomplete closure in the newborn (). The effects of prolonged COX inhibition, in utero
, are similar to the effects observed after deletion of both COX genes in utero
). Similar findings have also been observed in larger species, (e.g., humans (2
) and sheep (5
)) following indomethacin exposure in utero
. In larger species, however, the loss of ductus contractility, following COX inhibition, appears to be due to ischemia of the ductus wall secondary to in utero
ductus constriction and loss of vasa vasorum blood flow to the muscle media (5
). Ductus wall ischemia does not appear to be the explanation for our findings in mice. The mouse ductus is so thin that luminal flow sustains all of its nutrient needs. As a result, the mouse ductus has no need for muscle media vasa vasorum (34
). In our study, COX inhibition was started on day 15, when COX inhibitors do not contract the mouse ductus (6
). Chronic COX inhibition (between days 15 and 19) does not affect the in utero
dimensions of the ductus lumen (6
) (). Consistent with these findings, surrogate markers of hypoxia, like HIF1α and HIF2α, whose expression increases during ductus hypoxia (36
), are unaffected by chronic COX inhibition in utero
We hypothesized that PGE2 may play a unique role in the development of ductus contractility that is distinct from its function as a vasodilator. Previous studies found that gene deletion of the PGE2 receptor, EP4, produced a persistent PDA phenotype in newborn mice (37
) that was similar to what occurs after COX gene deletion or chronic in utero
COX inhibition. Our findings are consistent with PGE2 having a direct effect on ductus contractility by increasing the developmental expression of genes that regulate calcium availability. The ductus’ developmentally regulated, oxygen-induced constriction appears to be due in large part to increased expression of CaL channels (11
) and oxygen-sensitive K+
). In our experiments, in vitro
exposure to PGE2 increased the expression of CaL channel (CaLα1c, CaLβ2) and K+
channel (Kir6.1, Kv1.5) genes without affecting genes that regulate Rho-kinase-mediated calcium sensitization (). Conversely, inhibition of prostaglandin production, by chronic in utero
COX inhibition, decreased the expression of the same CaL and K+
channel genes, without affecting Rho-kinase-associated genes ().
The effects of chronic in utero
COX inhibition on gene expression are consistent with the effects we found on ductus contractility. Contractile stimuli, which act primarily on ductus K+
and CaL channels (like O2 and K+
)), have a diminished contractile effect, whereas, U46619, which has been shown to affect both calcium entry and Rho-kinase–mediated calcium sensitization in other vascular tissues (32
), constricts Control and COX-inhibited ductus to a similar degree ().
Several other genes were also affected by chronic COX inhibition and may contribute to the delayed ductus closure after birth (). Prior studies have shown that a developmental increase in phosphodiesterase (PDE 1A and PDE 3A) expression and activity decreases the sensitivity of the late gestation ductus to vasodilators, like PGE2 (38
). We found that the phosphodiesterase genes were down-regulated by chronic COX inhibition in utero
() and up-regulated by PGE2 exposure in vitro
(). This is consistent with endogenous PGE2 having a direct, positive effect on PDE 1A and PDE 3A expression in utero
On the other hand, the effects of chronic COX inhibition on other genes (like caldesmon, myocardin, tropomyosin, tenascin C, TGFβ, hemeoxygenase-1, and VEGF) may not be due to direct effects of PGE2 on the ductus. For example, the genes that regulate actin/myosin interactions (caldesmon, myocardin, tropomyosin) and matrix production (tenascin C, TGFβ) (which were decreased by chronic COX inhibition in utero) were not affected by incubation with PGE2 in vitro; and the hemeoxygenase-1 and VEGF genes, which were increased by chronic COX inhibition in utero, were not decreased by incubation with PGE2 in vitro. The effects of chronic COX inhibition on these genes may be due to distal effects of COX inhibition on other maternal, placental or fetal organs. The fact that PGE2 incubation did not produce the opposite effect as chronic COX inhibition for this set of genes may also be due to differences in experimental design (in vitro versus in vivo) or species used (sheep versus mice).
Increased nitric oxide production has been implicated in delayed ductus closure following prolonged COX inhibition in utero
). Functional coupling of COX and NOS systems is a well-recognized phenomenon (5
). In the mouse ductus arteriosus, eNOS is the predominant isoform for nitric oxide production (35
). We did not observe a change in ductus eNOS expression after chronic COX inhibition; however, we did observe changes in PDE1A and VEGF expression, which could contribute to increased nitric oxide activity or production (). Our contractility experiments were not designed to examine the effects of chronic COX inhibition on the production of nitric oxide or other vasodilators. We were primarily interested in examining the effects of COX inhibition on the contractile apparatus. Therefore, we specifically incubated the isolated ductus with inhibitors of prostaglandin and nitric oxide production to eliminate any differences between the groups in in vitro
prostaglandin and nitric oxide production.
Our studies examine the chronic effects of COX inhibition and PGE2 stimulation on ductus gene expression and contractility. They do not identify the mechanism(s) by which PGE2 is able to affect these changes. cAMP has previously been shown to regulate K+
channel activity and expression in excitable cells (39
); a similar mechanism may mediate the effects of PGE2 on CaL and K+
channel gene expression in the current experiments. Future studies, designed to measure protein expression and intracellular ion fluxes, will be necessary to identify the exact pathways that have been altered by our pharmacologic manipulations.
In summary, we speculate that the paradoxical effects of acute and chronic COX inhibition are consistent with the existence of two complementary roles for PGE2 during ductus development: one that promotes the expression of pathways necessary for its oxygen-induced closure following delivery, and, a second, which maintains ductus patency, for fetal wellbeing. A better understanding of these two processes will be important for the development of new strategies to treat preterm labor without affecting fetal vascular development.