Effects of Advancing Gestation and Non-Caucasian Race on Ductus Arteriosus Gene Expression.

OBJECTIVE: To identify genes affected by advancing gestation and racial/ethnic origin in human ductus arteriosus (DA). STUDY DESIGN: We collected 3 sets of DA tissue (n = 93, n = 89, n = 91; total = 273 fetuses) from second trimester pregnancies. We examined four genes, with DNA polymorphisms that distribute along racial lines, to identify "Caucasian" and "non-Caucasian" DA. We used real time polymerase chain reaction to measure RNA expression of 48 candidate genes involved in functional closure of the DA, and used multivariable regression analyses to examine the relationships between advancing gestation, "non-Caucasian" race, and gene expression. RESULTS: Mature gestation and non-Caucasian race are significant predictors for identifying infants who will close their patent DA when treated with indomethacin. Advancing gestation consistently altered gene expression in pathways involved with oxygen-induced constriction (eg, calcium-channels, potassium-channels, and endothelin signaling), contractile protein maturation, tissue remodeling, and prostaglandin and nitric oxide signaling in all 3 tissue sets. None of the pathways involved with oxygen-induced constriction appeared to be altered in "non-Caucasian" DA. Two genes, SLCO2A1 and NOS3, (involved with prostaglandin reuptake/metabolism and nitric oxide production, respectively) were consistently decreased in "non-Caucasian" DA. CONCLUSIONS: Prostaglandins and nitric oxide are the most important vasodilators opposing DA closure. Indomethacin inhibits prostaglandin production, but not nitric oxide production. Because decreased SLCO2A1 and NOS3 expression can lead to increased prostaglandin and decreased nitric oxide concentrations, we speculate that prostaglandin-mediated vasodilation may play a more dominant role in maintaining the "non-Caucasian" patent DA, making it more likely to close when inhibited by indomethacin.

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.

Association of the calcyon neuron-specific vesicular protein gene (CALY) with adolescent smoking initiation in China and California.

Although previous investigations have indicated a role for genetic factors in smoking initiation, the underlying genetic mechanisms are still unknown. In 2,339 adolescents from a Chinese Han population in the Wuhan Smoking Prevention Trial (Wuhan, China, 1998-1999), the authors explored the association of 57 genes in the dopamine pathway with smoking initiation. Using a conservative approach for declaring significance, positive findings were further examined in an independent sample of 603 Caucasian adolescents followed for up to 10 years as part of the Children's Health Study (Southern California, 1993-2009). The authors identified 1 single nucleotide polymorphism (rs2298122) in the calcyon neuron-specific vesicular protein gene (CALY) that was positively associated with smoking initiation in females (odds ratio = 2.21, 95% confidence interval: 1.49, 3.27; P = 8.4 × 10(-5)) in the Wuhan Smoking Prevention Trial cohort, and they replicated the association in females from the Children's Health Study cohort (hazard rate ratio = 2.05, 95% confidence interval: 1.27, 3.31; P = 0.003). These results suggest that the CALY gene may influence smoking initiation in adolescents, although the potential roles of underlying psychological characteristics that may be components of the smoking-initiation phenotype, such as impulsivity or novelty-seeking, remain to be explored.

Increase in tissue inhibitor of metalloproteinase-2 (TIMP-2) levels and inhibition of MMP-2 activity in a metastatic breast cancer cell line by an anti-invasive small molecule SR13179.

Tissue inhibitor of metalloproteinase-2 (TIMP-2), the endogenous inhibitor of matrix metalloproteinase-2 (MMP-2), regulates tumor invasion by modulating the activity of MMP-2. In addition, TIMP-2 is involved in the direct regulation of cell growth and angiogenesis, independent of MMP inhibition. Therefore, the development of therapeutic agents that increase TIMP-2 levels may offer a novel and broad approach to anti-neoplastic therapy. We report that a novel small molecule synthetic flavanoid SR13179, which inhibits the invasion of a highly metastatic human breast cancer cell line MCF-10CA1a through Matrigel, significantly increases protein and mRNA levels of TIMP-2 in a time- and dose-dependent manner. SR13179 inhibits the gelatinolytic activity of MMP-2 by >50% but has no effect on MMP-2 protein expression. SR13179 also possesses potent anti-tumor activity in several tumor cell lines regardless of their hormone receptor, p53 or multi-drug resistance status. Given the multifunctional inhibitory activity of TIMP-2 on tumor growth and invasion, the observed increase in TIMP-2 expression by SR13179 may play a central role in the anti-tumor and anti-invasive activity of this novel small molecule. Modulation of TIMP-2 protein expression may be a new molecular target for anti-metastatic adjuvant therapy for breast and other cancers.

Postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in bronchopulmonary dysplasia.

RATIONALE: Nitric oxide (NO) plays an important role in lung development and perinatal lung function, and pulmonary NO synthases (NOS) are decreased in bronchopulmonary dysplasia (BPD) following preterm birth. Fetal estradiol levels increase during late gestation and estradiol up-regulates NOS, suggesting that after preterm birth estradiol deprivation causes attenuated lung NOS resulting in impaired pulmonary function. OBJECTIVE: To test the effects of postnatal estradiol administration in a primate model of BPD over 14 days after delivery at 125 days of gestation (term = 185 d). METHODS: Cardiopulmonary function was assessed by echocardiography and whole body plethysmography. Lung morphometric and histopathologic analyses were performed, and NOS enzymatic activity and abundance were measured. MEASUREMENTS AND MAIN RESULTS: Estradiol caused an increase in blood pressure and ductus arteriosus closure. Expiratory resistance and lung compliance were also improved, and this occurred before spontaneous ductal closure. Furthermore, both oxygenation and ventilation indices were improved with estradiol, and the changes in lung function and ventilatory support requirements persisted throughout the study period. Whereas estradiol had negligible effect on indicators of lung inflammation and on lung structure assessed after the initial 14 days of ventilatory support, it caused an increase in lung neuronal and endothelial NOS enzymatic activity. CONCLUSIONS: In a primate model of BPD, postnatal estradiol treatment had favorable cardiovascular impact, enhanced pulmonary function, and lowered requirements for ventilatory support in association with an up-regulation of lung NOS. Estradiol may be an efficacious postnatal therapy to improve lung function and outcome in preterm infants.

Adamantyl-substituted retinoid-derived molecules that interact with the orphan nuclear receptor small heterodimer partner: effects of replacing the 1-adamantyl or hydroxyl group on inhibition of cancer cell growth, induction of cancer cell apoptosis, and inhibition of SRC homology 2 domain-containing protein tyrosine phosphatase-2 activity.

(E)-4-[3-(1-Adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC) induces the cell-cycle arrest and apoptosis of leukemia and cancer cells. Studies demonstrated that 3-Cl-AHPC bound to the atypical orphan nuclear receptor small heterodimer partner (SHP). Although missing a DNA-binding domain, SHP heterodimerizes with the ligand-binding domains of other nuclear receptors to repress their abilities to induce or inhibit gene expression. 3-Cl-AHPC analogues having the 1-adamantyl and phenolic hydroxyl pharmacophoric elements replaced with isosteric groups were designed, synthesized, and evaluated for their inhibition of proliferation and induction of human cancer cell apoptosis. Structure-anticancer activity relationship studies indicated the importance of both groups to apoptotic activity. Docking of 3-Cl-AHPC and its analogues to an SHP computational model that was based on the crystal structure of ultraspiracle complexed with 1-stearoyl-2-palmitoylglycero-3-phosphoethanolamine suggested why these 3-Cl-AHPC groups could influence SHP activity. Inhibitory activity against Src homology 2 domain-containing protein tyrosine phosphatase 2 (Shp-2) was also assessed. The most active Shp-2 inhibitor was found to be the 3'-(3,3-dimethylbutynyl) analogue of 3-Cl-AHPC.

Expression, activity, and function of phosphodiesterases in the mature and immature ductus arteriosus.

A patent ductus arteriosus is due in large part to increased sensitivity of the premature ductus to PGE2. After PGE2 stimulation, cAMP concentrations are higher in the immature than in the mature ductus. cAMP concentrations depend on the rates of adenyl cyclase production and phosphodiesterase (PDE)-mediated degradation. We used ductus from immature (n = 25) and mature (n = 21) fetal sheep to investigate whether a developmental increase in PDE activity could explain the diminished cAMP accumulation that follows PGE2 stimulation in the mature ductus. With advancing gestation, mRNA expression of the smooth muscle PDE isoforms (PDE1A, 1B, 1C, 3A, 3B, 4D, and 5A) increased in the ductus as did their hydrolytic activities. Selective inhibitors of PDE1, PDE3, and PDE4 relaxed the mature and immature ductus in the presence of inhibitors of prostaglandin and nitric oxide production. The mature ductus required higher concentrations of each of the PDE inhibitors to inhibit its tension to the same extent as in the immature ductus. There were no developmental changes in PDE expression in the fetal aorta. In conclusion, we observed a developmental increase in cAMP and cGMP PDE activity that contributes to the decreased sensitivity of the late-gestation ductus arteriosus to vasodilators like PGE2.

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 phosphatidylcholine, 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.

An adamantyl-substituted retinoid-derived molecule that inhibits cancer cell growth and angiogenesis by inducing apoptosis and binds to small heterodimer partner nuclear receptor: effects of modifying its carboxylate group on apoptosis, proliferation, and protein-tyrosine phosphatase activity.

Apoptotic and antiproliferative activities of small heterodimer partner (SHP) nuclear receptor ligand (E)-4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorocinnamic acid (3-Cl-AHPC), which was derived from 6-[3'-(1-adamantyl)-4'-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN), and several carboxyl isosteric or hydrogen bond-accepting analogues were examined. 3-Cl-AHPC continued to be the most effective apoptotic agent, whereas tetrazole, thiazolidine-2,4-dione, methyldinitrile, hydroxamic acid, boronic acid, 2-oxoaldehyde, and ethyl phosphonic acid hydrogen bond-acceptor analogues were inactive or less efficient inducers of KG-1 acute myeloid leukemia and MDA-MB-231 breast, H292 lung, and DU-145 prostate cancer cell apoptosis. Similarly, 3-Cl-AHPC was the most potent inhibitor of cell proliferation. 4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorophenyltetrazole, (2E)-5-{2-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]ethenyl}-1H-tetrazole, 5-{4-[3'-(1-adamantyl)-4'-hydroxyphenyl]-3-chlorobenzylidene}thiazolidine-2,4-dione, and (3E)-4-[3'-(1-adamantyl)-2-chloro-4'-hydroxy-4-biphenyl]-2-oxobut-3-enal were very modest inhibitors of KG-1 proliferation. The other analogues were minimal inhibitors. Fragment-based QSAR analyses relating the polar termini with cancer cell growth inhibition revealed that length and van der Waals electrostatic surface potential were the most influential features on activity. 3-Cl-AHPC and the 3-chlorophenyltetrazole and 3-chlorobenzylidenethiazolidine-2,4-dione analogues were also able to inhibit SHP-2 protein-tyrosine phosphatase, which is elevated in some leukemias. 3-Cl-AHPC at 1.0 microM induced human microvascular endothelial cell apoptosis but did not inhibit cell migration or tube formation.

Regulation of the prepronociceptin gene and its effect on neuronal differentiation.

Nociceptin/orphanin FQ (NOP/OFQ) is the endogenous ligand for the NOP receptor and is processed from a precursor protein in the family of opioid peptides. Prepronociceptin (ppN/OFQ) mRNA has been shown to be upregulated by an increase in cAMP, a treatment that leads to differentiation of NS20Y neuroblastoma cells. Although a large increase in endogenous ppN/OFQ mRNA upon cAMP stimulation can be shown in cellular systems, a similar increase cannot be expressed in pGL3 luciferase vector containing 1.3 kb proximal promoter, suggesting that a larger portion of the sequence or a different chromatin structure is necessary for a fully functional promoter. The induction of ppN/OFQ mRNA by cAMP appears to be mediated by a cAMP-response element. Chromatin immunoprecipitation (ChIP) assays show that CREB is recruited to the promoter region upon treatment of NS20Y cells with dibutyryl cAMP. In addition, the production of ppN/OFQ mRNA is regulated by histone acetylation, also through CREB, as the histone deacetylase (HDAC) inhibitor trichostatin A increases both CREB binding to the promoter and ppN/OFQ mRNA expression. In rat progenitor and mouse neuroblastoma cell lines, agents that increase ppN/OFQ mRNA expression also induce neurite outgrowth, suggesting a close relationship between ppN/OFQ and cellular differentiation.

Improved lung growth and function through hypoxia-inducible factor in primate chronic lung disease of prematurity.

Bronchopulmonary dysplasia (BPD), a chronic lung disease affecting preterm neonates, is associated with significant childhood and adult health problems. Histopathologic features of BPD include impaired vascular and distal airway development. We previously showed that activation of hypoxia-inducible factors (HIFs) by inhibition of prolyl hydroxylase domain-containing proteins (PHDs) is feasible and that it stimulates vascular endothelial growth factor (VEGF)-dependent angiogenesis in vitro. We tested the hypothesis that enhancement of angiogenesis by activation of HIFs improves lung growth and function in prematurely born neonates in vivo. Preterm baboons (125 day+14 day pro re nata O2 model, corresponding to 27 human gestational weeks) were treated for 14 days with intravenous (i.v.) FG-4095, a PHD inhibitor. Notably, 77% of diminished total alveolar surface area in untreated controls was recovered by FG-4095 treatment. Functional significance of the structural changes was indicated by improved oxygenation and lung compliance in FG-4095-treated newborns. Surfactant proteins B and C and saturated phosphatidylcholine were unchanged. Incidence of spontaneous ductus arteriosus closure was increased, likely contributing to lower ratio of pulmonary to systemic blood flow in FG-4095 group. These findings indicate that HIF stimulation by PHD inhibition ameliorates pathological and physiological consequences of BPD.

Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo.

Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days+14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1alpha, but not HIF-2alpha, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD.

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.

Activation of hypoxia-inducible factors in hyperoxia through prolyl 4-hydroxylase blockade in cells and explants of primate lung.

Preterm neonates with respiratory distress syndrome (RDS) often develop a chronic form of lung disease called bronchopulmonary dysplasia (BPD), characterized by decreased alveolar and vascular development. Ventilator treatment with supraphysiological O2 concentrations (hyperoxia) contribute to the development of BPD. Hyperoxia down-regulates and hypoxia up-regulates many angiogenic factors in the developing lung. We investigated whether angiogenic responses could be augmented through enhancement of hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and -2alpha, respectively) via blockade of prolyl hydroxylase domain-containing proteins (HIF-PHDs) in human microvascular endothelial cells from developing and adult lung, in epithelial A549 cells, and in fetal baboon explants in relative or absolute hyperoxia. PHD inhibitor (FG-4095) and positive control dimethyloxaloylglycine (DMOG), selective and nonselective HIF-PHD inhibitors, respectively, enhanced HIF-1alpha and -2alpha, vascular endothelial growth factor (VEGF), and platelet-endothelial cell adhesion molecule 1 expression in vitro in 95% and 21% O2. Furthermore, VEGF receptor fms-like tyrosine kinase 1 (Flt-1) was elevated, whereas kinase insert domain-containing receptor/fetal liver kinase 1 (KDR) was diminished in endothelial, but not epithelial, cells. Intracellular Flt-1 and KDR locations were unchanged by PHD blockade. Like VEGF, FG-4095 and DMOG increased angiogenesis in vitro, both in 95% and 21% O2, an effect that could be blocked through either Flt-1 or KDR. Notably, FG-4095 was effective in stimulating HIFs and VEGF also in fetal baboon lung explants. FG-4095 or DMOG treatment appeared to stimulate the feedback loop promoting HIF degradation in that PHD-2 and/or -3, but not PHD-1, were enhanced. Through actions characterized above, FG-4095 could have desirable effects in enhancing lung growth in BPD.

Novel D-ring analog of epigallocatechin-3-gallate inhibits tumor growth and VEGF expression in breast carcinoma cells.

The cancer chemopreventive activity of green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG) have been attributed to its antioxidant, antiproliferative and antiangiogenic effects. Several new molecular targets for EGCG's anticarcinogenic activity have been proposed in the recent literature. However, the understanding of the molecular mechanisms of EGCG's activity in vivo have been confounded by its low oral bioavailability and low plasma levels. Studies of EGCG would be greatly aided by the availability of synthetic analogs of EGCG designed to understand the contributions of the A, B, and D-rings and the phenolic hydroxyl groups of EGCG to its molecular mechanisms of action. We recently reported the de novo synthesis of a D-ring analog of EGCG, with the objective of using such analogs to understand the molecular mechanisms of EGCG action. We report here the first studies with a synthetic D-ring analog of EGCG. We examined the ability of the synthetic D-ring analog to inhibit tumor cell proliferation in breast carcinoma cells. We also investigated the effect of the analog on stress-induced VEGF production in breast carcinoma cells using Northern analysis and quantitative RT-PCR. We report here that the synthetic D-ring analog inhibits breast cancer cell growth in vitro with potencies equivalent to those of EGCG. Our results also show that, like EGCG, the synthetic analog inhibits hypoxia- and serum starvation-induced production of VEGF mRNA in breast cancer cells. Such synthetic analogs are valuable for understanding the structure-function relationship of EGCG and identifying relevant mechanisms of the chemopreventive action of EGCG.

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.

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 K(ATP) 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.

VEGF regulates remodeling during permanent anatomic closure of the ductus arteriosus.

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.