Mature and immature platelets during the first week after birth and incidence of patent ductus arteriosus.

BACKGROUND: Thrombocytopenia is a risk factor for patent ductus arteriosus. Immature and mature platelets exhibit distinct haemostatic properties; however, whether platelet maturity plays a role in postnatal, ductus arteriosus closure is unknown. METHODS: In this observational study, counts of immature and mature platelets (=total platelet count - immature platelet count) were assessed on days 1, 3, and 7 of life in very low birth weight infants (<1500 g birth weight). We performed echocardiographic screening for haemodynamically significant patent ductus arteriosus on day 7. RESULTS: Counts of mature platelets did not differ on day 1 in infants with (n = 24) and without (n = 45) haemodynamically significant patent ductus arteriosus, while infants with significant patent ductus arteriosus exhibited lower counts of mature platelet on postnatal days 3 and 7. Relative counts of immature platelets (fraction, in %) were higher in infants with patent ductus arteriosus on day 7 but not on days 1 and 3. Receiver operating characteristic curve analysis unraveled associations between both lower mature platelet counts and higher immature platelet fraction (percentage) values on days 3 and 7, with haemodynamically significant ductus arteriosus. Logistic regression analysis revealed that mature platelet counts, but not immature platelet fraction values, were independent predictors of haemodynamically significant patent ductus arteriosus. CONCLUSION: During the first week of postnatal life, lower counts of mature platelets and higher immature platelet fraction values are associated with haemodynamically significant patent ductus arteriosus. Lower counts of mature platelet were found to be independent predictors of haemodynamically significant patent ductus arteriosus.

Vascular endothelial growth factor polymorphism rs2010963 status does not affect patent ductus arteriosus incidence or cyclooxygenase inhibitor treatment success in preterm infants.

BACKGROUND: Vascular endothelial growth factor is critically involved in ductus arteriosus closure. Polymorphisms in the vascular endothelial growth factor gene have been associated with several diseases in neonates and adults. AIM: Herein, we investigated if vascular endothelial growth factor polymorphism rs2010963 status is associated with patent ductus arteriosus incidence and/or pharmacological treatment success. METHODS: We assessed rs2010963 status in 814 preterm infants (<1500 g birth weight) by means of restriction fragment length polymorphism analysis. DNA samples were obtained from dry-spot cards used for the German national newborn screening program. Clinical data were obtained by retrospective chart review. RESULTS: We could not find any statistically significant difference in the incidence of patent ductus arteriosus depending on vascular endothelial growth factor rs2010963 polymorphism status. Furthermore, no statistically significant associations between vascular endothelial growth factor polymorphism rs2010963 status and cyclooxygenase inhibitor treatment success were observed. CONCLUSION: Our results indicate that there is no association between vascular endothelial growth factor polymorphism rs2010963 status and the occurrence of patent ductus arteriosus or the response to cyclooxygenase inhibitor treatment in a large cohort of preterm infants. Additional studies are needed to determine the role of genetic factors on patent ductus arteriosus incidence and treatment response.

Association between Platelet Counts before and during Pharmacological Therapy for Patent Ductus Arteriosus and Treatment Failure in Preterm Infants.

BACKGROUND: The role of platelets for mediating closure of the ductus arteriosus in human preterm infants is controversial. Especially, the effect of low platelet counts on pharmacological treatment failure is still unclear. METHODS: In this retrospective study of 471 preterm infants [<1,500 g birth weight (BW)], who were treated for a patent ductus arteriosus (PDA) with indomethacin or ibuprofen, we investigated whether platelet counts before or during pharmacological treatment had an impact on the successful closure of a hemodynamically significant PDA. The effects of other factors, such as sepsis, preeclampsia, gestational age, BW, and gender, were also evaluated. RESULTS: Platelet counts before initiation of pharmacological PDA treatment did not differ between infants with later treatment success or failure. However, we found significant associations between low platelet counts during pharmacological PDA therapy and treatment failure (p < 0.05). Receiver operating characteristic (ROC) curve analysis showed that platelet counts after the first, and before and after the second cyclooxygenase inhibitor (COXI) cycle were significantly associated with treatment failure (area under the curve of >0.6). However, ROC curve analysis did not reveal a specific platelet cutoff-value that could predict PDA treatment failure. Multivariate logistic regression analysis showed that lower platelet counts, a lower BW, and preeclampsia were independently associated with COXI treatment failure. CONCLUSION: We provide further evidence for an association between low platelet counts during pharmacological therapy for symptomatic PDA and treatment failure, while platelet counts before initiation of therapy did not affect treatment outcome.

Ibuprofen and indomethacin differentially regulate vascular endothelial growth factor and its receptors in ductus arteriosus endothelial cells.

BACKGROUND: Cyclooxygenase inhibitors are widely applied to facilitate ductal closure in preterm infants. The mechanisms that lead to patent ductus arteriosus closure are incompletely understood. Vascular endothelial growth factor plays pivotal roles during ductal closure and remodelling. Aim The aim of this study was to investigate the effects of ibuprofen and indomethacin on the expression of vascular endothelial growth factor and its receptors in a primary rat ductus arteriosus endothelial cell culture. METHODS: Protein expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 and 2 was confirmed in rat ductus arteriosus and aorta by immunofluorescence staining. Fetal rat endothelial cells were isolated from ductus arteriosus and aorta using immunomagnetic cell sorting and treated with ibuprofen or indomethacin. mRNA expression levels were assessed by quantitative polymerase chain reaction analysis. RESULTS: In ductal endothelial cells, ibuprofen significantly induced vascular endothelial growth factor and its receptor 2, but not receptor 1, whereas indomethacin did not alter the expression levels of the vascular endothelial growth factor system. In contrast, ibuprofen significantly induced vascular endothelial growth factor and its receptors 1 and 2 in aortic endothelial cells, whereas indomethacin only induced vascular endothelial growth factor receptor 2. CONCLUSION: Our results indicate differential effects of ibuprofen and indomethacin on the expression levels of the vascular endothelial growth factor system in ductus arteriosus endothelial cells. In addition, vessel-specific differences between ductal and aortic endothelial cells were found. Further in vivo studies are needed to elucidate the biological significance of these findings.

Lung function in very low birth weight infants after pharmacological and surgical treatment of patent ductus arteriosus - a retrospective analysis.

BACKGROUND: The indications and strategies for treatment of patent ductus arteriosus (PDA) are controversial, and the safety and long-term benefits of surgical PDA closure remain uncertain. The aim of this study was to compare the lung function of very low birth weight (VLBW) infants after successful PDA treatment with a cyclooxygenase inhibitor or secondary surgical ligation. METHODS: A total of 114 VLBW infants (birth weight < 1500 g), including 94 infants (82%) with a birth weight < 1000 g, who received treatment for hemodynamically significant PDA (hsPDA), were examined at a median postmenstrual age of 48 weeks. All infants were initially given pharmacological treatment, and 40 infants (35%) required PDA ligation. Lung function testing (LFT) included tidal breathing measurements, measurement of respiratory mechanics assessed by the occlusion test, whole-body plethysmography, SF6 multiple breath washout, forced expiratory flow (V'maxFRC) by the rapid thoracoabdominal compression technique, exhaled NO (FeNO), and arterialized capillary blood gas analysis. RESULTS: On the day of the LFT, the 2 groups had similar postconceptional age and body weight. However, the PDA ligation group was more immature at birth (p < 0.001) and had reduced respiratory compliance (p < 0.001), lower V'maxFRC (p = 0.006), increased airway resistance (Raw) (p < 0.001), and impaired blood gases (p < 0.001). Multivariate analysis showed that PDA surgery was an independent risk factor for increased Raw. CONCLUSION: PDA ligation after failed pharmacological treatment is associated with impaired lung function as compared to successful pharmacological closure in infants at a postmenstrual age of 48 weeks. However, only Raw was independently affected by PDA ligation, while all other differences were merely explained by patient characteristics.

Clinical, Laboratory, and Placental Findings in Perinatal Listeriosis.

Clinical, laboratory, and placental manifestations of perinatal listeriosis are highly variable. Herein, we retrospectively analyzed all patients treated for neonatal listeriosis at the Charité University Medical Center in Berlin, Germany, 1999-2013. A total of 16 cases were identified. In 14 patients listeriosis was confirmed in neonatal specimens, while in two only the placenta tested positive. Elevated C-reactive protein and/or interleukin-6 levels were only inconsistently found, while a marked white blood cell left shift was present in all infants, if available. All but one infant manifested symptoms on the first day of life. Most patients required respiratory support, while none developed meningoencephalitis as evidenced by clinical or cerebrospinal fluid findings. Two patients died, all other patients survived without sequelae. In conclusion, perinatal listeriosis is still associated with significant morbidity and mortality. Clinical and laboratory findings are highly heterogeneous, but extreme leukocyte left shift seems to be a common feature.

Recent Advances in the Treatment of Preterm Newborn Infants with Patent Ductus Arteriosus.

A patent ductus arteriosus (PDA) is associated with several adverse clinical conditions. Several strategies for PDA treatment exist, although data regarding the benefits of PDA treatment on outcomes are sparse. Moreover, the optimal treatment strategy for preterm neonates with PDA remains subject to debate. It is still unknown whether and when PDA treatment should be initiated and which approach (conservative, pharmacologic, or surgical) is best for individual patients (tailored therapies). This article reviews the current strategies for PDA treatment with a special focus on recent developments such as oral ibuprofen, high-dose regimens, and the use of paracetamol (oral, intravenous).

Natural History of Patent Ductus Arteriosus in Very Low Birth Weight Infants after Discharge.

Data on the natural history of infants discharged with patent ductus arteriosus is sparse. We report on the 36-months follow-up after hospitalization in 68 infants discharged with an open ductus arteriosus. Notwithstanding a high spontaneous closure rate, catheter intervention in 5 infants illustrates a critical need for cardiologic follow-up.

Thrombocytopenia in the first 24 hours after birth and incidence of patent ductus arteriosus.

BACKGROUND: Experimental studies suggest that platelet-triggered ductal sealing is critically involved in definite ductus arteriosus closure. Whether thrombocytopenia contributes to persistently patent ductus arteriosus (PDA) in humans is controversial. This was a retrospective study of 1350 very low birth weight (VLBW; <1500 g) infants, including 592 extremely low birth weight (ELBW; <1000 g) infants. METHODS: All infants who had a platelet count in the first 24 hours after birth and an echocardiogram performed on day of life 4 to 5 were included. The incidence of thrombocytopenia was analyzed in infants with and without PDA, and in those who did or did not undergo PDA intervention. The impact of thrombocytopenia, gestational age, birth weight, gender, and sepsis on PDA was determined by receiver operating characteristic curve, odds ratio, and regression analyses. RESULTS: Platelet numbers within the first 24 hours after birth did not differ between VLBW/ELBW infants with and without spontaneous ductal closure. Platelet numbers were not associated with subsequent PDA treatment. Low platelet counts were not related to failure of pharma-cologic PDA treatment and the need for subsequent surgical ligation. Lower gestational age or birth weight, male gender, and sepsis were linked to the presence of PDA in VLBW infants on day of life 4 to 5. CONCLUSIONS: Thrombocytopenia in the first 24 hours after birth was not associated with PDA in this largest VLBW/ELBW infant cohort studied to date. Impaired platelet function, due to immaturity and critical illness, rather than platelet number, might play a role in ductus arteriosus patency.

Vascular Endothelial Growth Factor (VEGF) and soluble VEGF receptor 1 (sFlt-1) levels in early and mature human milk from mothers of preterm versus term infants.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors regulate angiogenesis (formation of blood vessels). The soluble VEGF receptor 1 (sFlt-1) binds VEGF as a potent antagonist. OBJECTIVE: The objective of this study was to compare VEGF and sFlt-1 levels in milk from mothers of preterm (n = 50) versus term (n = 49) infants in a longitudinal study. METHODS: Milk samples were collected on days 3 and 28 of lactation. Vascular endothelial growth factor and sFlt-1 were quantified by sandwich-type enzyme-linked immunosorbent assay. RESULTS: Vascular endothelial growth factor and sFlt-1 were found in high concentrations in early milk (lactation day 3) from mothers of preterm and term infants and were lower in mature milk (lactation day 28). On day 3, median VEGF concentration was lower in preterm than in term milk (37.1 vs 53.9 ng/mL, P < .01). Otherwise, VEGF (day 28) and sFlt-1 (days 3 and 28) did not differ in preterm versus term milk. CONCLUSIONS: It was shown for the first time that sFlt-1 is present in human milk. Early human milk contains high concentrations of VEGF and sFlt-1, which decrease over the course of lactation.

The expression of vascular endothelial growth factor and its receptors in congenital bronchopulmonary cystic malformations.

BACKGROUND: Bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM) represent rare hamartomatous abnormalities of the lung. Dysregulation of cytokines that influence pulmonary vasculogenesis and epithelial growth, both known to be altered in BPS and CCAM, may play a role in their pathogenesis. OBJECTIVE: We hypothesized that expression of vascular endothelial growth factor (VEGF) or its receptors might be altered in CCAM and BPS, possibly distinguishing CCAM from BPS, or from controls. METHODS: Lung biopsy specimens obtained from infants who had undergone surgery for BPS (n = 4) or CCAM (n = 5) within the first month of life and normal lung autopsy samples (n = 4) serving as controls were investigated immunohistochemically for the protein expression levels of VEGF and its corresponding receptors. RESULTS: VEGF, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor receptor 3 (VEGFR3) staining was detected in CCAM and BPS specimens, as well as in control samples. VEGFR2 expression increased from controls to CCAM and from CCAM to BPS, the difference between controls and BPS being significant. The expression of VEGF, VEGFR1, and VEGFR3 was similar among the three groups. Consistent with a possible involvement of VEGFR2 in altered vasculogenesis-bronchiogenesis interaction, its expression was predominantly found in bronchial but not alveolar regions. CONCLUSIONS: The data suggest a possible role of VEGF-VEGFR2 interaction in the pathogenesis of congenital bronchopulmonary cystic malformations. However, VEGFR2 does not represent a suitable histochemical marker to distinguish between BPS and CCAM.

Isolation and culture of fibroblasts, vascular smooth muscle, and endothelial cells from the fetal rat ductus arteriosus.

The ductus arteriosus (DA), a fetal arterial shunt vessel between the proximal descending aorta and the pulmonary artery, closes shortly after birth. Initial functional closure as a result of the DA's smooth muscle contraction is followed by definite anatomical closure. The latter involves several complex mechanisms like endothelial cushion formation and smooth muscle cell migration resulting in fibrosis and sealing of the vessel. These complex steps indicate highly specialized functions of the DA vascular smooth muscle cells (VSMCs), endothelial cells, and fibroblasts. Herein, we describe a new reproducible method for isolating VSMCs, endothelial cells, and fibroblasts of high viability from fetal rat DA using immunomagnetic cell sorting. Purity of the different cell cultures was assessed by immunohistochemistry and flow cytometry and ranged between 85 and 94%. The capability of the VSMCs to react to hypoxic stimuli was assessed by intracellular calcium and ATP measurements and by VEGF mRNA expression analysis. VSMCs respond to hypoxia with decreases in intracellular calcium concentrations and ATP levels, whereas VEGF mRNA expression increased 3.2-fold. The purified vessel-specific different cell types are suitable for subsequent gene expression profiling and functional studies and provide important tools for improving our understanding of the complex processes involved in the closure of the DA.

Echocardiography predicts closure of patent ductus arteriosus in response to ibuprofen in infants less than 28 week gestational age.

BACKGROUND: Patent ductus arteriosus (PDA) is a frequent problem in preterm infants, and its incidence is inversely correlated with gestational age. The efficacy of medical treatment decreases with decreasing gestational age (GA), and failure rates as well as ductus ligation rates of 40% have been reported in <28 week GA newborns. The aim of this study was to determine whether echocardiographic parameters can predict response to ibuprofen treatment of PDA. STUDY DESIGN: In a longitudinal study, 29 infants born <28 week GA were screened for a significant PDA (left atrial to aortic root ratio>1.4, anterior cerebral artery resistance index>0.8, and oxygen requirement>35%) at 24-72 h of life and, if a PDA was found, treated with 10-5-5mg/kg ibuprofen intravenously every 24h. Ductal parameters were monitored by serial echocardiography. Infant neurodevelopmental outcomes were assessed at 24 month corrected age. RESULTS: All 15 infants with significant PDA responded to the ibuprofen loading dose indicated by reduced PDA diameters or increased PDA maximum flow velocities (PDA V(max)), and 7 patients showed an ongoing response resulting in a closed PDA after the 1st cycle (47%). Of the 8 non-responders, 7 received a 2nd cycle with 2 further responders (29%). All non-responders to the 2nd course had a PDA V(max)

Vascular endothelial growth factor and its soluble receptor in infants with congenital cardiac disease.

Patients with cyanotic congenital cardiac disease often develop major aortopulmonary collaterals. Vascular endothelial growth factor is a key promoter of angiogenesis. Its soluble receptor-1 acts as a potent antagonist. We studied 30 infants with cyanotic congenital cardiac disease and 27 infants with acyanotic congenital cardiac disease. Central venous plasma vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 levels were measured before, and 24 and 96 hours after surgery. There was no difference between plasma vascular endothelial growth factor levels in infants with cyanotic and those with acyanotic congenital cardiac disease. In cyanotic infants, the soluble vascular endothelial growth factor receptor-1 levels tended to be higher than in the acyanotic infants. In conclusion, there is no significant difference in the plasma levels of vascular endothelial growth factor and its soluble receptor-1 between infants with cyanotic and those with acyanotic congenital cardiac disease.

Vascular endothelial growth factor as marker for tissue hypoxia and transfusion need in anemic infants: a prospective clinical study.

OBJECTIVE: Oxygen-carrying capacity of blood is reduced in anemic infants because of low hemoglobin levels. Red blood cell transfusions become necessary if low hematocrit causes tissue hypoxia. No reliable parameters exist for detecting chronic tissue hypoxia. Vascular endothelial growth factor is upregulated by hypoxia; hence, elevated vascular endothelial growth factor levels may be a marker for tissue hypoxia and may indicate the need for red blood cell transfusions. METHODS: In a prospective study, plasma vascular endothelial growth factor levels were measured in 3 groups of infants suspected of requiring red blood cell transfusions to find a vascular endothelial growth factor cutoff value indicative of tissue hypoxia. The 3 groups were acute anemic (an episode of acute bleeding [hematocrit drop > 5%] per day); chronic anemic (hematocrit drop < 5% per day); and nontransfused (hematocrit drop < 5% per day) but not meeting clinical criteria for a transfusion. Blood was sampled before transfusion and again 48 hours after transfusion if required. Plasma vascular endothelial growth factor and erythropoietin concentrations were measured. RESULTS: Vascular endothelial growth factor concentrations were lower in acutely anemic compared with chronically anemic infants, whereas erythropoietin levels did not differ between these groups. The vascular endothelial growth factor concentration was <140 pg/mL in all acutely anemic infants, and this was deemed the threshold level indicating sufficient tissue oxygenation in subsequent analysis. We found that 30% of chronically anemic and 43% of nontransfused infants had vascular endothelial growth factor levels of >140 pg/mL. In transfused infants, with elevated vascular endothelial growth factor levels, red blood cell transfusion resulted in lowering of vascular endothelial growth factor concentrations. CONCLUSIONS: Vascular endothelial growth factor concentrations of >140 pg/mL may indicate insufficient oxygen delivery to tissues and may serve as a marker of the need for transfusion or of tissue hypoxia in other diseases.

Ibuprofen augments bilirubin toxicity in rat cortical neuronal culture.

Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.