Permanent closure of the ductus arteriosus requires loss of cells from the muscle media and development of neointimal mounds, composed in part of proliferating endothelial cells. We hypothesized that postnatal ductus constriction produces hypoxia of the inner vessel wall; we also hypothesized that hypoxia might lead to cell death and the production of vascular endothelial cell growth factor (VEGF), a hypoxia-inducible growth factor that stimulates endothelial proliferation. We mapped the distribution of hypoxia in newborn baboons and correlated it with the appearance of cell death (TUNEL technique), VEGF expression, and endothelial proliferation (proliferating cell nuclear antigen expression). In the full-term baboon (n=10), the ductus was functionally closed on Doppler examination by 24 h after delivery. Regions of the ductus where the lumen was most constricted were associated with moderate/intense hypoxia; VEGF expression was increased in the hypoxic muscle media, and luminal endothelial cells, adjacent to the hypoxic media, were proliferating. Cells in the most hypoxic regions of the ductus wall were undergoing DNA fragmentation. In contrast, regions of the ductus with mild degrees of hypoxia had no evidence of cell death, VEGF expression, or endothelial proliferation. Cell death and endothelial proliferation seemed to be limited to regions of the full-term ductus experiencing moderate/intense hypoxia. In the premature baboon (67% gestation) (n=24), only 29% closed their ductus by Doppler examination before d 6. None of the premature baboons, including those with a closed ductus by Doppler, had evidence of moderate/intense hypoxia; also, there was no evidence of cell death, VEGF expression, endothelial proliferation, or neointima formation by d 6. Therefore, the premature ductus is resistant to developing hypoxia, even when its lumen is constricted; this may make it susceptible to later reopening.

Permanent closure of the full-term newborn ductus arteriosus (DA) occurs only if profound hypoxia develops within the vessel wall during luminal obliteration. We used fetal and newborn baboons and lambs to determine why the immature DA fails to remodel after birth. When preterm newborns were kept in a normoxic range (Pa(O(2)): 50-90 mmHg), 86% still had a small patent DA on the sixth day after birth; in addition, the preterm DA wall was only mildly hypoxic and had only minimal remodeling. The postnatal increase in Pa(O(2)) normally induces isometric contractile responses in rings of DA; however, the excessive inhibitory effects of endogenous prostaglandins and nitric oxide, coupled with a weaker intrinsic DA tone, make the preterm DA appear to have a smaller increment in tension in response to oxygen than the DA near term. We found that oxygen concentrations, beyond the normoxic range, produce an additional increase in tension in the preterm DA that is similar to the contractile response normally seen at term. We predicted that preterm newborns, kept at a higher Pa(O(2)), would have increased DA tone and would be more likely to obliterate their lumen. We found that preterm newborns, maintained at a Pa(O(2)) >200 mmHg, had only a 14% incidence of patent DA. Even though DA constriction was due to elevated Pa(O(2)), obliteration of the lumen produced profound hypoxia of the DA wall and the same features of remodeling that were observed at term. DA wall hypoxia appears to be both necessary and sufficient to produce anatomic remodeling in preterm newborns.

After birth, the full-term ductus arteriosus actively constricts and undergoes extensive histologic changes that prevent subsequent reopening. These changes are thought to occur only if a region of intense hypoxia develops within the ductus wall after the initial active constriction. In preterm infants, indomethacininduced constriction of the ductus is often transient and is followed by reopening. Prostaglandins and nitric oxide both play a role in inhibiting ductus closure in vitro. We hypothesized that combined inhibition of both prostaglandin and nitric oxide production (with indomethacin and N-nitro-L-arginine (L-NA), respectively) may be required to produce the degree of functional closure that is needed to cause intense hypoxia. We used preterm (0.67 gestation) newborn baboons that were mechanically ventilated for 6 d: 6 received indomethacin alone, 7 received indomethacin plus L-NA, and 16 received no treatment (control). Just before necropsy, only 25% of control ductus and 33% of indomethacin-treated ductus were closed on Doppler examination; in contrast, 100% of the indomethacin-plus-L-NA-treated ductus were closed. Control and indomethacin-treated baboons developed negligible-to-mild ductus hypoxia (EF5 technique). Similarly, there was minimal evidence of ductus remodeling. In contrast, indomethacin-plus-L-NA-treated baboons developed intense hypoxia in regions where the ductus was most constricted. The hypoxic muscle strongly expressed vascular endothelial growth factor, and proliferating luminal endothelial cells filled and occluded the lumen. In addition, cells in the most hypoxic regions were undergoing DNA fragmentation. In conclusion, preterm newborns are capable of remodeling their ductus, just like the full-term newborn, if they can reduce their luminal blood flow to a point that produces intense ductus wall hypoxia. Combined prostaglandin and nitric oxide inhibition may be necessary to produce permanent closure of the ductus and prevent reopening in preterm infants. In the full-term infant, closure of the DA occurs in two phases: 1) initial "functional" closure of the DA lumen by smooth muscle constriction, and 2) "anatomic" occlusion of the lumen resulting from endothelial proliferation, neointimal thickening, and loss of smooth muscle cells from the inner muscle media (1, 2). Hypoxia of the DA wall seems to be the required stimulus for irreversible, anatomic closure (3). Anatomic remodeling occurs only in the presence of moderate to intense hypoxia (3).

No abstract

We hypothesized that nitric oxide (NO) production by the fetal ductus arteriosus is limited because of low fetal PO2, but that at neonatal PO2, NO might be an important regulator of ductus arteriosus tone. We exposed isolated rings of fetal lamb ductus arteriosus to elevated PO2. L-NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS), and methylene blue and 6-anilino-5,8-quinolinedione (LY83583), inhibitors of guanylate cyclase, produced constriction of the ductus arteriosus. When ductus arteriosus rings were exposed to low PO2, L-NAME had no effect, and methylene blue and LY83583 had only a small effect on ductus arteriosus tone. Sodium nitroprusside and calcium ionophore A23187 relaxed ductus arteriosus rings more than aortic rings, and relaxed ductus arteriosus rings from immature fetuses more than those from late gestation fetuses. In contrast, ductus arteriosus rings from both early and late gestation were equally sensitive to 8-bromo-cGMP. By both reverse transcriptase-polymerase chain reaction and immunohistochemistry, endothelial cell NOS and inducible calcium-independent NOS, but not nerve cell NOS, were detected in the ductus arteriosus. Inducible NOS was expressed only by endothelial cells lining the ductus arteriosus lumen; in contrast, endothelial cell NOS was expressed by both luminal and vasa vasorum endothelial cells. The role of inducible NOS in the ductus arteriosus is uncertain because the potency of a specific inducible NOS inhibitor in constricting the ductus arteriosus was negligible compared with that of an endothelial cell NOS inhibitor. We speculate that NO may be an important regulator of ductus arteriosus tone at high but not low PO2. The endothelial cell NOS isoform found in vasa vasorum may be an important source of NO because removal of ductus arteriosus luminal endothelium only partially blocks the effects of L-NAME, methylene blue, and LY83583.

No abstract

Anatomic remodeling and permanent closure of the newborn ductus arteriosus appears to require the development of intense hypoxia within the constricted vessel wall. Hypoxic ductus smooth muscle cells express vascular endothelial cell growth factor (VEGF). We studied premature baboons and sheep to determine the effects of VEGF inhibition (in baboons) and VEGF stimulation (in sheep) on ductus remodeling in vivo. For study of VEGF inhibition, 13 premature newborn baboons (68% gestation) were treated with inhibitors of both prostaglandin and nitric oxide production to constrict the ductus and induce ductus wall hypoxia. Six received a neutralizing monoclonal antibody against VEGF (A.4.6.1, mAbVEGF), while seven did not. Both groups developed the same degree of ductus constriction, tissue hypoxia, and VEGF expression. The mAbVEGF treatment produced a significant ( P < 0.05) reduction in ductus vasa vasorum ingrowth and neointima formation (due to both a decrease in luminal endothelial cell proliferation and a decrease in smooth muscle cell migration into the neointima). For study of VEGF stimulation, nine sheep fetuses (70% gestation) had their ductus wall injected with either VEGF ( n = 6) or vehicle ( n = 4) in vivo. VEGF administration produced a significant ( P < 0.05) increase in vasa vasorum ingrowth and neointima formation. We conclude that VEGF plays an important role in the formation of neointimal mounds and vasa vasorum ingrowth during permanent ductus closure.

Inflammatory processes play a crucial role in the pathogenesis of atherosclerosis and other vascular disorders. We hypothesized that ischemia of the ductus arteriosus might initiate an active inflammatory response that could play a role in ductus remodeling and permanent closure. To test this hypothesis, we studied effects of postnatal ductus construction on inflammatory processes and remodeling in late-gestation fetal and newborn baboons, and preterm newborn baboons. After postnatal ductus constriction, the expression of several genes known to be essential for atherosclerotic remodeling [vascular cell adhesion molecule (VCAM)-1, E-selectin, IL-8, macrophage colony stimulating factor-1, CD154, interferon-␥, IL-6, and tumor necrosis factor-␣] was increased in the ductus wall. We were unable to detect intercellular adhesion molecule (ICAM)-1, ICAM-2, Pselectin, monocyte chemoattractant protein-1, or IL-1 by either real-time PCR or immunohistochemistry. VCAM-1, which is newly expressed by luminal cells of the closed ductus, is an important ligand for the mononuclear cell adhesion receptor VLA4. After postnatal constriction, VLA4 ϩ monocytes/ macrophages (CD68 ϩ and CD14 ϩ ) and, to a lesser extent, T-lymphocytes adhered to the ductus wall. Neutrophils and platelets were not observed. The extent of postnatal neointimal remodeling (both endothelial cell layering and subendothelial space thickening) was associated with the degree of mononuclear cell adhesion. Similarly, the extent of vasa vasorum ingrowth correlated with the invasion of CD68 ϩ cells, from the adventitia into the muscle media. Based on these data, we conclude that the inflammatory response following postnatal ductus constriction may be as necessary for ductus remodeling as it is for atherosclerotic remodeling. Closure of the full-term ductus arteriosus occurs in two phases. First, smooth muscle constriction obstructs the ductus' lumen. Then, anatomic remodeling permanently occludes the lumen. The initial constriction appears to be the required stimulus for anatomic closure. During constriction, loss of luminal and vasa vasorum flow produce a zone of ischemichypoxia in the ductus' muscle media that induces the following anatomic changes: luminal endothelial proliferation, subendothelial thickening, ingrowth of vasa vasorum, and cell death (1). The preterm newborn is capable of remodeling its ductus, just like the full-term newborn, if it can impede its luminal flow and develop the same degree of ischemic-hypoxia as found at term (2).Although the cell death and ingrowth of vasa vasorum in the ductus wall appear to be due to ATP depletion (3,4) and VEGF induction (5), respectively, the mechanism(s) responsible for the neointimal changes are still unknown. It is now clear that inflammatory processes play a crucial role in the pathogenesis of several vascular disorders (6 -9). The most studied model of