Steven R. Seidner

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.

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.

Ibuprofen-Induced Patent Ductus Arteriosus Closure: Physiologic, Histologic, and Biochemical Effects on the Premature Lung

OBJECTIVE. The goal was to study the pulmonary, biochemical, and morphologic effects of a persistent patent ductus arteriosus in a preterm baboon model of bronchopulmonary dysplasia. METHODS. Preterm baboons (treated prenatally with glucocorticoids) were delivered at 125 days of gestation (term: 185 days), given surfactant, and ventilated for 14 days. Twenty-four hours after birth, newborns were randomly assigned to receive either ibuprofen (to close the patent ductus arteriosus; n = 8) or no drug (control; n = 13). RESULTS. After treatment was started, the ibuprofen group had significantly lower pulmonary/systemic flow ratio, higher systemic blood pressure, and lower left ventricular end diastolic diameter, compared with the control group. There were no differences in cardiac performance indices between the groups. Ventilation index and dynamic compliance were significantly improved with ibuprofen. The improved pulmonary mechanics in ibuprofen-treated newborns were not attributable to changes in levels of surfactant protein B, C, or D, saturated phoshatidylcholine, or surfactant inhibitory proteins. There were no differences in tracheal concentrations of cytokines commonly associated with the development of bronchopulmonary dysplasia. The groups had similar messenger RNA expression of genes that regulate inflammation and remodeling in the lung. Lungs from ibuprofen-treated newborns were significantly drier (lower wet/dry ratio) and expressed 2.5 times more epithelial sodium channel protein than did control lungs. By 14 days after delivery, control newborns had morphologic features of arrested alveolar development (decreased alveolar surface area and complexity), compared with age-matched fetuses. In contrast, there was no evidence of alveolar arrest in the ibuprofen-treated newborns. CONCLUSIONS. Ibuprofen-induced patent ductus arteriosus closure improved pulmonary mechanics, decreased total lung water, increased epithelial sodium channel expression, and decreased the detrimental effects of preterm birth on alveolarization.

Prostaglandin E2—Mediated Relaxation of the Ductus Arteriosus Effects of Gestational Age on G Protein-Coupled Receptor Expression, Signaling, and Vasomotor Control

Background—In the preterm newborn, a patent ductus arteriosus is in large part a result of the increased sensitivity of the immature ductus to prostaglandin E2 (PGE2). PGE2 acts through 3 G protein–coupled receptors (EP2, EP3, and EP4) that activate both adenyl cyclase and KATP channels. We explored these pathways to identify the mechanisms responsible for the increased sensitivity of the immature ductus to PGE2. Methods and Results—We measured EP receptor content (mRNA and protein), receptor binding, cAMP production, and isometric tension in rings of ductus taken from immature (65% gestation) and mature (95% gestation) sheep and baboon fetuses. Ductus relaxation and cAMP generation were augmented in response to selective EP receptor agonists in the immature ductus. 8-Br-cAMP, a stable cAMP analogue, produced greater relaxation in the immature ductus. In the presence of a selective protein kinase A inhibitor, Rp-8-CPT cAMPS, the developmental differences in sensitivity to PGE2 could no longer be demonstrated. EP2, EP3, and EP4 receptor densities were higher in immature ductus, despite similar receptor mRNA and protein contents at the 2 gestational ages. In contrast, forskolin and NaF, direct activators of adenyl cyclase and Gs, respectively, elicited comparable increases in cAMP in both age groups. KATP channel inhibition also had similar effects on PGE2-induced relaxation in both age groups. Conclusions—Two mechanisms explain the increased sensitivity of the immature ductus to PGE2: (1) increased cAMP production because of increased binding of PGE2 to the individual EP receptors and (2) increased potency of cAMP on protein kinase A–regulated pathways.

Gestational effects of corticosteroids and surfactant in ventilated rabbits

ABSTRACT: Maternal corticosteroid treatments decreased lung protein leaks and increased the compliance responses to exogenous surfactant in 27-day preterm rabbits. We asked if maternal corticosteroid treatments at different gestational ages might alter these responses. Pregnant does were given 0.1 mg/kg betamethasone 48 and 24 h before study of the rabbits at 27, 28, and 29 days of gestational age and term newborns at 31 days of gestational age. Alternate rabbits at each gestation were treated with 50 mg/kg surfactant after delivery. Alveolar saturated phosphatidylcholine pool sizes increased with gestation similarly in control and corticosteroid-treated groups. Corticosteroids improved compliance relative to control values at 29 days of gestational age in animals not treated with surfactant and improved the compliance response to surfactant treatment at 27 and 28 days of gestational age. Corticosteroids decreased the leak of radiolabeled albumin to the lungs and alveolar washes at all preterm gestations with a maximum decrease to 16% of the control value at 29 days of gestation. Surfactant decreased this protein leak more effectively than did corticosteroids at the earlier gestations. There were potentially beneficial effects of corticosteroids either alone or together with surfactant at all preterm gestations studied. No protein leak or compliance effects of either treatment were noted in the term newborns.

Effects of ibuprofen treatment on the developing preterm baboon kidney

Preterm neonates are commonly exposed postnatally to pharmacological treatments for a patent ductus arteriosus. Exposure of the developing kidney to nephrotoxic medications may adversely impact renal development. This study aimed to determine the effect of early postnatal ibuprofen treatment, both alone and in combination with a nitric oxide synthase inhibitor (NOSi), on renal development and morphology. Baboon neonates were delivered prematurely at 125-day (125d) gestation (term = 185d) and were euthanized at birth or postnatal day 6. Neonates were divided into four groups: 125d gestational controls (n = 8), Untreated (n = 8), Ibuprofen (n = 6), and ibuprofen (Ibu)+NOSi (n = 4). Animals in the Ibuprofen and Ibu+NOSi groups received five doses of ibuprofen, with the Ibuprofen+NOSi animals additionally administered a NOS inhibitor (N(G)-monomethyl-l-arginine). There was no difference among groups in body weight, kidney weight, or glomerular generation number. Nephrogenic zone width was significantly reduced in the Ibuprofen group (123.5 ± 7.4 μm) compared with the 125d gestational control (176.1 ± 6.9 μm) and Untreated animals (169.7 ± 78.8 μm). In the Ibu+NOSi group, nephrogenic zone width averaged 152.7 ± 3.9 μm, which was not significantly different from any other group. Morphologically abnormal glomeruli were present at a range of 0.0-22.9% in the Untreated group, 0.0-6.1% in the Ibuprofen group, and 0.0-1.4% in the Ibu+NOSi group. In conclusion, early postnatal ibuprofen exposure is associated with a reduced nephrogenic zone width, which may suggest the early cessation of nephrogenesis following treatment. Ultimately, this may impact the number of nephrons formed in the preterm kidney.

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-γ, IL-6, and tumor necrosis factor-α] 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 continuous bed rotation and prolonged mechanical ventilation on healthy, adult baboons

OBJECTIVE To study, in a model of prolonged mechanical ventilation, the role of continuous bed rotation on lung function and pathology. DESIGN Prospective animal study. SETTING Animal research laboratory. SUBJECTS Healthy adult baboons (Papio cynocephalus), anesthetized with ketamine, sedated, paralyzed, mechanically ventilated for 11 days, and monitored with pulmonary and peripheral arterial catheters. INTERVENTIONS Animals were divided into two experimental groups: a) mechanical ventilation alone (control, n = 7); and b) mechanical ventilation with continuous bed rotation therapy to 45 degrees (continuous rotation group, n = 5). Mechanical ventilation was provided for 11 days with an FIO2 of 0.21 and tidal volume of 12 mL/ kg. Bronchoalveolar lavage was performed through a fiberoptic bronchoscope. Nursing care procedures, antacids, enteral feeding, and prophylactic antibiotics were administered. MEASUREMENTS AND MAIN RESULTS Measurements of hemodynamics, pulmonary functions, lung volumes, arterial blood gases, and chest radiographs were done daily. Bronchoalveolar lavage was performed at days 0, 7, and 11. There were no significant changes in hemodynamics, gas exchange, or pulmonary functions during the study period in either group. Microbiological surveillance cultures were negative in both experimental groups. In the control group after 7 days, six of seven animals developed patchy atelectasis; by day 11, two of seven animals demonstrated persistent radiologic abnormalities. Bronchoalveolar lavage neutrophils were significantly increased in control animals at days 7 and 11. Lung pathology in the control group showed areas of bronchiolitis, with surrounding bronchopneumonia in five of seven animals. None of the continuous rotation animals showed any radiologic or morphologic abnormalities. CONCLUSIONS Prolonged mechanical ventilation in the control group resulted in atelectasis, increased concentrations of bronchoalveolar lavage neutrophils, and mild pneumonitis. These effects were not associated with changes in lung volumes, oxygenation, or hemodynamic parameters. Continuous bed rotation helped to prevent these abnormalities.

Patterns of Gene Expression in the Ductus Arteriosus Are Related to Environmental and Genetic Risk Factors for Persistent Ductus Patency

Three independent risk factors (immature gestation, absence of antenatal glucocorticoid exposure, and presence of the rs2817399(A) allele of the gene TFAP2B) are associated with patent ductus arteriosus (PDAs) that fail to close during prostaglandin inhibition. We hypothesized that these three factors may affect a common set of genes that increase the risk of persistent PDA after birth. We studied baboon ductus from term, preterm, and glucocorticoid-treated preterm fetuses and found that both immature gestation and absence of antenatal glucocorticoid exposure decreased RNA expression of calcium- and potassium-channel genes involved in oxygen-induced constriction, and phosphodiesterase genes (that modulate cAMP/cGMP signaling). Ductus obtained from second trimester human pregnancies were genotyped for TFAP2B polymorphisms. When present, the rs2817399(A) allele also was associated with decreased expression of calcium- and potassium-channel genes. In contrast, alleles of two other TFAP2B polymorphisms, rs2817419(G) and rs2635727(T), which are not related to the incidence of PDA after birth, had no effect on RNA expression. In conclusion, three calcium- and potassium-channel genes (CACNA1G/ alpha1G, CACNB 2/CaL-beta2, and KCNA2/ Kv1.2) were similarly affected by each of the PDA risk factors. We speculate that these channels may play a significant role in closing the preterm ductus during prostaglandin inhibition and may be potential targets for future pharmacologic manipulations.

Smad7 inhibits autocrine expression of TGF-β2 in intestinal epithelial cells in baboon necrotizing enterocolitis

Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-β 2 (TGF-β(2)) in the developing intestine. We hypothesized that low epithelial TGF-β(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-β(2) in these cells. Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-β(2), Smad7, Ski, and strawberry notch N (SnoN)/Ski-like oncoprotein (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. NEC was recorded in seven premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-β(2) than term intestine. TGF-β(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-β(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-β(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. Smad7 inhibits autocrine expression of TGF-β(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.