Yao Qi Chen

Permanent Anatomic Closure of the Ductus Arteriosus in Newborn Baboons: The Roles of Postnatal Constriction, Hypoxia, and Gestation

Permanent closure of the ductus arteriosus require 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.

Regulation of Ductus Arteriosus Patency by Nitric Oxide in Fetal Lambs: The Role of Gestation, Oxygen Tension, and Vasa Vasorum

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.

Combined Prostaglandin and Nitric Oxide Inhibition Produces Anatomic Remodeling and Closure of the Ductus Arteriosus in the Premature Newborn Baboon

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, indomethacin-induced 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.

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 μmol O2 · min−1 · g−1) far exceeded the amount of oxygen consumed by the constricted ductus (0.052 ± 0.021 μmol O2 · min−1 · g−1, measured in vitro). Postnatal constriction increased the effective oxygen diffusion distance across the ductus wall to >3× 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 · min−1 · g−1; newborn, 0.21 ± 0.08 mL · min−1 · 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 1.3× the maximally tolerated limit.

In Utero Remodeling of the Fetal Lamb Ductus Arteriosus: The Role of Antenatal Indomethacin and Avascular Zone Thickness on Vasa Vasorum Proliferation, Neointima Formation, and Cell Death

BackgroundThe ductus arteriosus (DA) of newborn infants exposed in utero to indomethacin is resistant to postnatal indomethacin; we hypothesized that this is due to ductus constriction in utero, with subsequent remodeling of the vessel. Methods and ResultsInfusion of fetal lambs with indomethacin for 48 hours constricted the DA and increased the thickness of the avascular zone of the DA, which in turn induced the expression of vascular endothelial growth factor, endothelial nitric oxide synthase (due to ingrowth of vasa vasorum), neointima formation, and loss of smooth muscle cells; moderate degrees of DA constriction in utero increased NO production, which inhibited DA contractility. Marked degrees of DA constriction decreased tissue distensibility and contractile capacity. ConclusionsDA patency is no longer controlled primarily by prostaglandins once it has been exposed to indomethacin in utero.

Cyclooxygenase-2 plays a significant role in regulating the tone of the fetal lamb ductus arteriosus

Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents but have adverse effects on the fetal ductus arteriosus. We hypothesized that COX-2 inhibitors may not affect the ductus if the predominant COX isoform is COX-1. To examine this hypothesis, we used ductus arteriosus obtained from late-gestation fetal lambs. In contrast to our hypothesis, fetal lamb ductus arteriosus expressed both COX-1- and COX-2-immunoreactive protein (by Western analysis). Although COX-1 was found in both endothelial and smooth muscle cells, COX-2 was found only in the endothelial cells lining the ductus lumen (by immunohistochemistry). The relative contribution of COX-1 and COX-2 to PGE2 synthesis was consistent with the immunohistochemical results: in the intact ductus, PGE2 formation was catalyzed by both COX-1 and COX-2 in equivalent proportions; in the endothelium-denuded ductus, COX-2 no longer played a significant role in PGE2 synthesis. NS-398, a selective inhibitor of COX-2, was 66% as effective as the selective COX-1 inhibitor valeryl salicylate and the nonselective COX inhibitor indomethacin in causing contraction of the ductus in vitro. At this time, caution should be used when recommending COX-2 inhibitors for use in pregnant women.Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents but have adverse effects on the fetal ductus arteriosus. We hypothesized that COX-2 inhibitors may not affect the ductus if the predominant COX isoform is COX-1. To examine this hypothesis, we used ductus arteriosus obtained from late-gestation fetal lambs. In contrast to our hypothesis, fetal lamb ductus arteriosus expressed both COX-1- and COX-2-immunoreactive protein (by Western analysis). Although COX-1 was found in both endothelial and smooth muscle cells, COX-2 was found only in the endothelial cells lining the ductus lumen (by immunohistochemistry). The relative contribution of COX-1 and COX-2 to PGE2 synthesis was consistent with the immunohistochemical results: in the intact ductus, PGE2 formation was catalyzed by both COX-1 and COX-2 in equivalent proportions; in the endothelium-denuded ductus, COX-2 no longer played a significant role in PGE2 synthesis. NS-398, a selective inhibitor of COX-2, was 66% as effective as the selective COX-1 inhibitor valeryl salicylate and the nonselective COX inhibitor indomethacin in causing contraction of the ductus in vitro. At this time, caution should be used when recommending COX-2 inhibitors for use in pregnant women.

Phospholipase Cγ Activation, Phosphotidylinositol Hydrolysis, and Calcium Mobilization are Not Required for FGF Receptor-Mediated Chemotaxis

Basic fibroblast growth factor (FGF) is a potent angiogenic factor that stimulates several cell types to migrate along a chemotactic gradient. Most chemoattractant receptors appear to share a common mechanism that involves activation of phospholipase C (PLC), hydrolysis of phosphotidylinositol, and mobilization of intracellular calcium. We transfected two different cell lines with either human FGF receptor-1 cDNA or chimeric FGF receptor cDNA. Ligand stimulation induced chemotaxis, activation of PLC gamma, phosphotidylinositol hydrolysis, and calcium mobilization in both wild-type receptor cell lines. No such response was elicited in control cells. Mutation of the two fibroblast growth factor receptors at residue 766, replacing tyrosine with phenylalanine, made the receptors incapable of associating with and activating PLC gamma following ligand stimulation. These mutant receptors also failed to mediate phosphotidylinositol hydrolysis and calcium mobilization. However, cells transfected with the mutant fibroblast growth factor receptors were as chemotactically responsive to the appropriate ligand as were cells transfected with the wild-type receptors. These findings demonstrate that the ability of the fibroblast growth factor receptor to promote chemotaxis is not dependent on increased activation of PLC gamma, increased hydrolysis of phosphotidylinositol, or increased global mobilization of calcium.

Changes in Endothelial Cell and Smooth Muscle Cell Integrin Expression during Closure of the Ductus Arteriosus: An Immunohistochemical Comparison of the Fetal, Preterm Newborn, and Full-Term Newborn Rhesus Monkey Ductus

Anatomical closure of the ductus arteriosus requires normally quiescent luminal endothelial cells and medial smooth muscle cells to migrate into the subendothelial space forming intimal mounds that eventually coalesce and occlude the vessels lumen. The migration of endothelial cells and smooth muscle cells requires the presence of integrin receptors that interact with the surrounding matrix. We used immunohistochemical staining to examine the repertoires of integrins expressed by endothelial cells and smooth muscle cells during postnatal closure of the ductus arteriosus in full-term and preterm rhesus monkeys. In the fetal ductus, luminal endothelial cells have a limited repertoire of integrins. During postnatal ductus closure, luminal endothelial cells, of both term and preterm monkeys, change their phenotype and express the full repertoire of integrins found on growing capillary endothelial cells (α1β1,α2β1, α3β1,α6β1, αvβ1,α6β4, and αvβ5). Similarly, during ductus closure, smooth muscle cells of both term and preterm monkeys expand their integrin repertoire to include the α5β1 and αvβ3 integrins; these two integrins have been shown to be essential for smooth muscle cell migration in vitro. These changes in integrin profile occur at the same time the endothelial and smooth muscle cells invade their neighboring compartments. In contrast, preterm monkeys with a persistently patent ductus lumen fail to develop these changes in integrin expression and fail to develop neointimal mounds. No evidence of intimal thickening occurs in the absence of changes in integrin expression. Therefore, endothelial cells and smooth muscle cells change phenotypes to produce the intimal thickening required for ductus closure.