Anatomic closure of the premature patent ductus arteriosus: The role of CD14+/CD163+ mononuclear cells and VEGF in neointimal mound formation.

Permanent closure of the newborn ductus arteriosus requires the development of neointimal mounds to completely occlude its lumen. VEGF is required for neointimal mound formation. The size of the neointimal mounds (composed of proliferating endothelial and migrating smooth muscle cells) is directly related to the number of VLA4 mononuclear cells that adhere to the ductus lumen after birth. We hypothesized that VEGF plays a crucial role in attracting CD14/CD163 mononuclear cells (expressing VLA4) to the ductus lumen and that CD14/CD163 cell adhesion to the ductus lumen is important for neointimal growth. We used neutralizing antibodies against VEGF and VLA-4 to determine their respective roles in remodeling the ductus of premature newborn baboons. Anti-VEGF treatment blocked CD14/CD163 cell adhesion to the ductus lumen and prevented neointimal growth. Anti-VLA-4 treatment blocked CD14/CD163 cell adhesion to the ductus lumen, decreased the expression of PDGF-B (which promotes smooth muscle migration), and blocked smooth muscle influx into the neointimal subendothelial space (despite the presence of increased VEGF in the ductus wall). We conclude that VEGF is necessary for CD14/CD163 cell adhesion to the ductus lumen and that CD14/CD163 cell adhesion is essential for VEGF-induced expansion of the neointimal subendothelial zone.

Postnatal constriction, ATP depletion, and cell death in the mature and immature ductus arteriosus.

After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1alpha/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1alpha/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age.

ATP depletion and cell death in the neonatal lamb ductus arteriosus.

Postnatal constriction of the full-term ductus arteriosus produces cell death and remodeling of the ductus wall. Using a bioluminescence imaging technique, we found that after birth, the lamb ductus develops ATP, glucose, and glycogen depletion in addition to hypoxia. In vitro studies showed that cell death correlates best with ATP depletion and is most marked when both glucose and oxygen are severely depleted; in addition, the degree of ATP depletion found in vivo is sufficient to account for the extensive degree of cell death that occurs after birth. Under hypoxic conditions, the immature ductus is more capable of preserving its ATP supply than the mature ductus as a result of increased glucose availability, glycogen stores, and glucose utilization. However, the immature ductus is just as susceptible as the mature ductus to ATP depletion when glucose supplies are restricted. The extensive degree of cell death that occurs in the newborn ductus after birth is associated primarily with ATP depletion. The increased glycolytic capacity of the immature ductus may enable it to tolerate episodes of hypoxia and nutrient shortage, making it more resistant to developing postnatal cell death and permanent closure.

The role of monocyte-derived cells and inflammation in baboon ductus arteriosus remodeling.

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-gamma, IL-6, and tumor necrosis factor-alpha] was increased in the ductus wall. We were unable to detect intercellular adhesion molecule (ICAM)-1, ICAM-2, P-selectin, 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+ monocytes/macrophages (CD68+ and CD14+) 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+ 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.

Effects of hypoxia, hypoglycemia, and muscle shortening on cell death in the sheep ductus arteriosus.

After birth, constriction of the full-term ductus arteriosus produces ischemic hypoxia, caspase activation, DNA fragmentation (>70% of cell nuclei are positive by the terminal deoxynucleotidyl transferase nick-end labeling [TUNEL] technique), and permanent ductus closure. In contrast, the preterm ductus frequently fails to develop these changes. We used the TUNEL technique to examine rings of fetal ductus arteriosus (incubated for 24 h at different oxygen and glucose concentrations) to determine the roles of 1) constriction and shortening, 2) hypoxia, and 3) hypoglycemia in producing cell death. Under controlled conditions, late-gestation ductus rings had a low rate of TUNEL-positive staining (0.6 +/- 0.9%) that did not change during muscle shortening. Although hypoxia (6.9 +/- 3.5%) and hypoglycemia (2.4 +/- 1.9%) increased the incidence of TUNEL-positive staining, only the combination of hypoxia-plus-hypoglycemia increased the incidence to the range found in vivo (83 +/- 9.5%). The combination of hypoxia-plus-hypoglycemia was associated with an oligonucleosomal pattern of DNA fragmentation. Under the same experimental conditions, the preterm ductus was capable of developing a similar degree of TUNEL-positive staining as found at term. Although caspase-3 and caspase-7 were activated in rings exposed to hypoxia-plus-hypoglycemia, a nonselective caspase inhibitor, Z-VAD.FMK (which inhibited caspase-3 and caspase-7 cleavage in the rings), did not diminish the degree of TUNEL-positive staining. We hypothesize that the preterm ductus is capable of developing an extensive degree of cell death, if it can develop the same degree of hypoxia and hypoglycemia found in the full-term newborn ductus. We also hypothesize that cell death in the ductus wall may involve pathways that are not dependent on caspase-3 or -7 activation.

Vasa vasorum hypoperfusion is responsible for medial hypoxia and anatomic remodeling in the newborn lamb ductus arteriosus.

Postnatal constriction of the full-term ductus arteriosus produces hypoxia of the muscle media. This is associated with anatomic remodeling (including smooth muscle death) that prevents subsequent reopening. We used late-gestation fetal and neonatal lambs to determine which factors are responsible for the postnatal hypoxia. Hypoxia [measured by 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide technique] and cell death (measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique) were observed in regions of the constricted ductus wall within 4 h after delivery. Although there was a decrease in ductus luminal flow during the first 6 h after delivery (measured by Doppler transducer), the amount of oxygen delivered to the ductus lumen (3070 +/- 1880 micromol O2 x min(-1) x g(-1)) far exceeded the amount of oxygen consumed by the constricted ductus (0.052 +/- 0.021 micromol O2 x min(-1) x g(-1), measured in vitro). Postnatal constriction increased the effective oxygen diffusion distance across the ductus wall to >3x the limit that can be tolerated for normal tissue homeostasis. This was owing to both an increase in the thickness of the ductus (fetus, 1.12 +/- 0.20 mm; newborn, 1.60 +/- 0.17 mm; p < 0.01) and a marked reduction in vasa vasorum flow (fetus, 0.99 +/- 0.44 mL x min(-1) x g(-1); newborn, 0.21 +/- 0.08 mL x min(-1) x g(-1); p < 0.01). These findings suggest that hypoxic cell death in the full-term ductus is caused primarily by changes in vasa vasorum flow and muscle media thickness and can occur before luminal flow has been eliminated. We speculate that in contrast with the full-term ductus, the preterm ductus is much less likely to develop the degree of hypoxia needed for vessel remodeling inasmuch as it only is capable of increasing its oxygen diffusion distance to 1.3x the maximally tolerated limit.

In utero indomethacin alters O2 delivery to the fetal ductus arteriosus: implications for postnatal patency.

Indomethacin produces constriction and hypoxia of the fetal ductus arteriosus. This is associated with death of smooth muscle cells in the ductus wall and an increased incidence of patent ductus arteriosus in the newborn period. We used fetal sheep to determine which factors are responsible for indomethacin-induced hypoxic cell death. Cell death in the ductus wall is directly related to the degree of indomethacin-induced ductus constriction and is present at both moderate and marked degrees of constriction. Both moderate and marked degrees of ductus constriction reduce vasa vasorum flow to the ductus (moderate = 69 +/- 25%; marked = 30 +/- 16% of preinfusion values) and increase the thickness of the ductus wall. In contrast, ductus luminal blood flow is not affected by moderate degrees of constriction and is reduced only after marked constriction. Although indomethacin increases ductus tone, it has no effect on ductus oxygen consumption. These findings suggest that the hypoxic cell death that occurs during the early stages of indomethacin-induced constriction is primarily due to changes in vasa vasorum blood flow and muscle media thickness.