IL-1 alpha causes lung inflammation and maturation by direct effects on preterm fetal lamb lungs

Intra-amniotic endotoxin induces IL-1, causes chorioamnionitis, lung inflammation, lung injury and lung maturation in preterm lambs. Intra-amniotic IL-1alpha also causes chorioamnionitis, lung inflammation and lung maturation. We asked if IL-1alpha effects on the preterm lung are mediated by direct signaling to the lung rather than by indirect effects from the chorioamnionitis. To study IL-1 effects independently of chorioamnionitis, the lungs and the amniotic fluid were surgically separated in fetal sheep by diverting fetal lung fluid via a tracheostomy tube to a sialastic bag. A mini-osmotic pump delivered an intratracheal infusion of recombinant sheep IL-1alpha (10 microg) or saline (control) over 24 h. Preterm lambs were delivered 1d or 7d after the start of the infusion at 124d gestational age (Term = 150d). IL-1alpha recruited inflammatory cells and increased pro-inflammatory cytokine mRNA expression in the fetal lungs. Compared with controls, IL-1alpha did not alter lung antioxidant enzyme activity or alveolar numbers. IL-1alpha had minimal effects on the mRNA or protein expression of proteins essential for vascular development. IL-1alpha induced large increases in alveolar surfactant saturated phosphatidylcholine and increased lung gas volumes. Lung inflammation and maturation result from direct exposure of the fetal lung to a single cytokine - IL-1alpha.     

Maternal endotoxin-induced preterm birth in mice: fetal responses in toll-like receptors, collectins, and cytokines

Major cause of prematurity is spontaneous preterm birth (PTB) associated with intrauterine inflammation. Our aim was to establish a model of endotoxin Lipopolysaccharide-induced PTB of live-born pups and to study early immune activation in fetal and maternal compartments. Expression of several proteins that bind microbes (Toll-like receptors TLR4, TLR2; surfactant proteins SP-A, SP-D) was analyzed. At 16 or 17 d of gestation, C57BL/6 dams received a single dose of intraperitoneal LPS, leading to PTB within 17 h. Cytokine levels increased in maternal serum, followed by a modest increase in fetal serum and in amniotic fluid. In uterus, placenta, and fetal membranes, LPS mostly increased the expressions of TLR, SPs, and cytokines. The number of TLR2-positive macrophages increased in labyrinthine placenta. In fetal lung, intestine, liver, and brain there were modest changes in cytokine expressions. In fetal lung, SP and TLR mRNAs decreased and TLR2-positive macrophages redistributed around vessels. LPS-induced fetal deaths associated with early age (16 d gestation) rather than with proinflammatory activation. Here we propose that maternal LPS response leads to PTB and acute decrease of immune proteins in epithelial lining of fetal lung. Instead, acceleration of lung maturity has been previously observed in intraamniotic inflammation.     

血管活性肠肽对肺损伤大鼠Toll样受体mRNA表达的影响

目的 探讨血管活性肠肽(vasoactive intestinal peptide,VIP)对内毒素(脂多糖,lipopolysaceberide,LPS)致休克大鼠肺损伤后Toll样受体(Toll-like receptor,TLR)2和TLR4 mRNA表达的影响.方法 40只SD大鼠,随机分为LPS组(16只)、LPS+VIP组(16只)和对照组(8只).LPS组尾静脉注射LPS(E.coli O_(55)B_5)10 mg/kg;LPS+VIP组尾静脉注射LPS 10 ms/kg后注射VIP 5 nmol/kg;对照组尾静脉注射等容量生理盐水.分别于注射后6 h和24 h处死,留取肺标本,RT-PCR检测肺TLR2/4 mRNA表达,并观察24 h时肺组织病理变化.结果 (1)肺组织病理改变:制模24 h时.光镜和透射电镜下,LPS组见肺泡间隔弥漫性增宽、炎性细胞浸润,隔内毛细血管不同程度充血,肺泡壁增厚,肺泡腔结构破坏、炎性细胞浸润、出血、间质水肿、细胞器破坏,LPS+VIP组病变较轻.(2)TLR2/4 mRNA表达:注射LPS后6 h、24 h,肺组织TLR2/4 mRNA表达升高(F=16.638,P=0.000;t=5.876,P=0.000);24 h时LPs+VIP组TLR2/4 mRNA表达低于LPS组(F=16.676,P=0.000;t=3.9,16,P<0.001).结论 LPS致休克大鼠肺损伤时,肺组织TLR2/4 mRNA表达增强.VIP可减轻LPS所致肺损伤,其机制可能与下调重要炎症基因TLR2/4 mRNA表达有关.     

Intraamniotic endotoxin increases lung antioxidant enzyme activity in preterm lambs

Proinflammatory stimulation resulting from intraamniotic endotoxin improves lung function, increases surfactant protein mRNA expression and protein content, increases alveolar and lung saturated phosphatidylcholine pools, and accelerates lung morphometric maturation in fetal sheep. The mechanism for induction of lung maturation does not involve an increase in fetal cortisol. The effect of endotoxin on the maturation of a different lung system, the antioxidant enzyme (AOE) system, has not been examined. Therefore, we hypothesized that intraamniotic endotoxin would produce acceleration of AOE activity in fetal sheep at similar doses and schedule of administration to those producing lung functional and surfactant maturation. In a dose-response study, intraamniotic injections of 1, 4, 20, or 100 mg of Escherichia coli 055:beta5 endotoxin were administered 7 d before preterm delivery of sheep at 125 d gestation. In a study examining time interval of administration before delivery, 20 mg of endotoxin was injected at either 1-, 2-, 4-, 7-, or 15-d intervals before preterm delivery at 125 d. Doses of 1-100 mg of endotoxin produced significant increases in glutathione peroxidase activity; doses of 4-100 mg significantly increased catalase activity, whereas doses of 20-100 mg resulted in significant increases in total superoxide dismutase activity. Glutathione peroxidase activity was elevated within 2 d, whereas superoxide dismutase was increased by 4 d and catalase activity increased by 7 d after endotoxin. No AOE increases were sustained for 15 d. Endotoxin increased fetal lung AOE activity at similar dosing amounts and intervals to those producing maturation of lung function and surfactant. Thus, mechanisms involving proinflammatory stimulation, unrelated to glucocorticoid hormones, can induce maturation of the AOE system of the fetal lung.     

Lipopolysaccharide induces IL-6 production in respiratory syncytial virus-infected airway epithelial cells through the toll-like receptor 4 signaling pathway

Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis in young children. Microbial agents such as endotoxin and RSV are implicated in airway inflammation during the development of reactive airway disease (RAD) later in childhood. Toll-like receptors (TLRs) are involved in an inflammation cascade through pathogen-associated molecular pattern recognition including lipopolysaccharide (LPS) and viral components. In this study, we investigated the expression of TLRs and cytokine-chemokine production profiles of RSV-infected epithelial cells. In live-RSV infected human tracheal epithelial cell line (9HTEo), TLRs 1-10 mRNA levels were up-regulated in a time-dependent manner compared with ultraviolet (UV)-inactivated RSV. RSV was shown to alter TLR4 membrane and cytosolic location in epithelial cells. Stimulating RSV-infected epithelial cells with TLR4 agonist LPS increased synthesis of IL-6, IL-8, and reduced regulated on activation, normal T cell expressed and secreted (RANTES) production. TLR4 neutralizing antibody HTA125 and TLR4-targeting RNA interference experiments revealed that TLR4 signaling pathway played a predominant role in mediating LPS-induced-IL-6 production of RSV infected epithelial cells. Altogether, our studies indicated that TLR4 play a critical role in leading LPS mediated-IL-6 response in RSV infected-epithelial cells and might be an important factor influencing the cytokine-chemokine profile of epithelial cells interacting with virus and endotoxin, which is correlated with phenotypes of RSV diseases.     

High-field diffusion tensor imaging characterization of cerebral white matter injury in lipopolysaccharide-exposed fetal sheep

Background:In gyrencephalic species such as sheep, precise anatomical and microstructural characterization of the consequences of fetal inflammation remains scarce. The goal of this study was to characterize changes in white matter (WM) structure using advanced magnetic resonance imaging (MRI) following lipopolysaccharide (LPS) exposure in the preterm-equivalent fetal sheep.Methods:Preterm (0.7 gestation) fetal sheep received vehicle (Sham group) or LPS (LPS group), and fetal brains were collected 10 d later for subsequent ex vivo MRI. T(1)-weighted (T(1)W), T(2)-weighted (T(2)W), and diffusion tensor imaging (DTI) data were collected.Results:Fetuses exposed to LPS exhibited reductions in WM volume and corpus callosum thickness at 10 d recovery. Characteristic patterns of diffuse and focal WM lesions (necrosis or cysts) could be identified by various T(1), T(2), and DTI signal changes.Conclusion:Fetal LPS exposure induces a pattern of injury characterized by diffuse and focal WM injury that closely reproduces that observed clinically in preterm infants. This work provides anatomical and microstructural MRI assessment, as well as histopathological correlates, of the consequences of LPS exposure in an animal model with a WM structure similar to that of the human brain. This work will help to further our understanding of MRI changes in preterm infants.     

Prenatal inflammation and lung development

Prenatal exposure of very low birth weight infants to chronic indolent chorioamnionitis with organisms such as mycoplasma and ureaplasma is frequent. Chorioamnionitis is inconsistently associated with changed risks of respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), probably because the diagnosis of chorioamnionitis does not quantify the extent or duration of the fetal exposures to infection and inflammation. The correlations between prenatal exposures and postnatal lung disease also are confounded by the imprecision of the diagnoses of RDS and BPD. In animal models, chorioamnionitis caused by pro-inflammatory mediators or live ureaplasma induces lung maturation, but also causes alveolar simplification and vascular injury. Intra-amniotic endotoxin administration also modulates the fetal innate immune system, resulting in maturation of monocytes to alveolar macrophages and the induction or paralysis of inflammatory responses depending on exposure history. Prenatal inflammation can have profound effects on the fetal lung and subsequent immune responses.     

Postnatal lung inflammation increased by ventilation of preterm lambs exposed antenatally to Escherichia coli endotoxin

Chorioamnionitis resulting in exposure of the fetal lung to inflammation is frequent before preterm delivery. The initiation of mechanical ventilation in the preterm recruits granulocytes to the lungs and increases proinflammatory cytokine expression in the lungs. We hypothesized that when the prematurely born newborn with chorioamnionitis was ventilated, inflammation would increase. Therefore, we asked whether inflammatory exposure to the fetal lung caused by intra-amniotic endotoxin (10 mg, Escherichia coli 055:beta 5) given at 100 d gestation would alter the inflammatory responses to the mechanical ventilation in surfactant-treated preterm lambs delivered at 130 d gestation. Cells in alveolar washes, proinflammatory cytokine expression, and surfactant protein mRNA expression were not different for saline and endotoxin exposed lambs that were not ventilated. The endotoxin- and saline-exposed control animals had similar lung function for 6 h of ventilation. Bronchoalveolar lavage fluid from the ventilated and antenatal endotoxin-exposed animals contained 5.7 times more monocytes, 12 times more lymphocytes, and a nonsignificant increase in neutrophils. Cells from the bronchoalveolar lavage fluid expressed 3-fold more IL-6 and IL-8 mRNA than did cells from the saline exposed comparison animals. An antenatal exposure of the fetal lung to endotoxin enhanced the subsequent inflammatory response of the ventilated preterm lung.     

Oxidative stress in fetal lambs exposed to intra-amniotic endotoxin in a chorioamnionitis model

Chorioamnionitis is a risk factor for the development of bronchopulmonary dysplasia. Endotoxin-induced oxidative stress to the fetus in the uniquely hypoxic intrauterine environment has not been reported. Using a model of chorioamnionitis, we measured markers of pulmonary and systemic oxidant exposures in fetal lambs at 124 d gestation (term = 150 d) exposed to 10 mg intra-amniotic endotoxin 2 d (n = 6) or 7 d (n = 6) before delivery, or saline as controls (n = 9). The 7 d endotoxin-exposed animals had 3-fold higher protein carbonyls (0.66 +/- 0.46 versus 0.23 +/- 0.14 nmol/mg protein) and 10-fold greater myeloperoxidase activity (2.38 +/- 1.87 versus 0.27 +/- 0.18 nM) in the bronchoalveolar lavage fluid (BALF), suggestive of neutrophil-derived oxidant activity. However, in the lung tissue, protein carbonyls, superoxide dismutase, and peroxiredoxin 1 were not different between groups. The expression of peroxiredoxin 1 was prominent, primarily in the peri-bronchiolar epithelium. Notably, evidence of oxidant exposure was minimal at 2 d when BALF inflammatory cells, lung IL-1beta, and IL-8 were highest. Intra-amniotic endotoxin induced systemic oxidative stress as plasma protein carbonyl was elevated at 7 d (0.14 +/- 0.04 nmol/mg protein; p = 0.005). Surfactant protein A and B mRNAs were highest at 2 d, suggesting that oxidative stress did not contribute to the lung maturation response. A modest lung oxidative stress in chorioamnionitis could contribute to bronchopulmonary dysplasia.     

Antenatal betamethasone changes cord blood monocyte responses to endotoxin in preterm lambs

Corticosteroids are routinely administered to women at risk for preterm delivery to induce fetal lung maturation. Antenatal corticosteroids have immunomodulatory effects on fetal immune cells that are poorly understood. We hypothesized that maternal betamethasone would alter in fetal monocytes both the initiation of inflammation in response to pro-inflammatory stimuli and the resolution of inflammation by phagocytosis of apoptotic neutrophils. Preterm lambs at 124 d gestation were delivered 15 h, 1 d, 2 d, or 7 d after 0.5 mg/kg maternal intramuscular betamethasone. Monocytes from cord blood were isolated and cultured and results were compared with monocytes from preterm lambs exposed to maternal saline or monocytes from adult sheep. Phagocytosis of Escherichia coli was not changed, however, phagocytosis of apoptotic neutrophils was low in fetal monocytes but increased after 7 d exposure to maternal betamethasone to the level found in adult monocytes. Hydrogen peroxide production after endotoxin stimulus was significantly reduced to 7.1 +/- 2.2 micromol at 5 h, 8.7 +/- 2.9 micromol at 24 h, and 4.1 +/- 1.9 micromol at 48 h versus 16.4 +/- 3.6 micromol in control animals; at 7 d, the hydrogen peroxide production increased to 74.3 +/- 19.7 micromol (p < 0.05, per 10(6) monocytes). IL-6 production was reduced at 15 h after maternal betamethasone but at no other time point. Maternal betamethasone initially suppressed several fetal monocyte functions, however, at 7 d, measurements of initiation and resolution of inflammation were increased to levels similar to monocytes from adult sheep. The time-dependent changes in maternal betamethasone modulation of the responses of fetal monocytes may influence immune function of the preterm lamb after delivery.     

Inflammatory and anti-inflammatory responsiveness of surfactant proteins in fetal and neonatal rabbit lung

Spontaneous preterm birth due to intrauterine infection is associated with increased concentrations of cytokines in amniotic fluid and in the airways at birth. Intra-amniotic IL-1 induces fetal lung maturity, consistent with the decrease in the incidence of respiratory distress syndrome (RDS) in intrauterine inflammation. On the other hand, antenatal corticosteroid decreases the incidence of RDS in infants born prematurely. The aim of the present study was to investigate the interaction between IL-1 and glucocorticoid in the expression of the surfactant proteins SP-A, -B, and -C. Lung explants from rabbit fetuses at 22 (immature), 27 (transitional), and 30 (mature) d of gestation (term, 30-31 d) and on d 1 after term birth were cultured with dexamethasone (Dx), IL-1alpha, or vehicle in the presence or absence of actinomycin D. According to the present results, IL-1alpha and Dx additively increased the expression of SP-A and SP-B on d 22. Later in gestation, SP-B and SP-C were suppressed by IL-1, whereas glucocorticoid tended to increase the expression of SP-B and SP-C and prevented the IL-1-induced suppression of SP. IL-1alpha and steroid interactively increased the stability of SP mRNA compared with the single agonist, possibly explaining the additive effects on the SP mRNA levels. The present results reveal beneficial additive effects of glucocorticoid and cytokine on lung surfactant. They may explain some of the acute beneficial effects of glucocorticoid therapy in chorioamnionitis before premature birth and in inflammatory lung disease after birth.     

Antenatal endotoxin and glucocorticoid effects on lung morphometry in preterm lambs

In utero inflammation may accelerate fetal lung maturation but may also play a role in the pathogenesis of chronic lung disease. We examined the impact of endotoxin, a potent proinflammatory stimulus, on structural and functional maturation of preterm sheep lungs. Date bred ewes received 20 mg Escherichia coli endotoxin or saline by ultrasound guided intra-amniotic injection at 119 d gestation. A comparison group of animals received 0.5 mg/kg betamethasone, a known maturational agent, at 118 d gestation. Lambs were delivered by cesarean section at 125 d (term = 150 d) and ventilated for 40 min. Lung function data are reported elsewhere. Total and differential white cell counts were performed on amniotic fluid and fetal lung fluid samples. Morphometric analyses were performed on inflation fixed right upper lobes. Total cell count increased slightly but not significantly in both amniotic fluid and fetal lung fluid. Both endotoxin and betamethasone had similar effects on alveolarization: average alveolar volume increased by approximately 20% and total alveolar number decreased by almost 30%. Both treatments led to thinning of alveolar walls, although this was statistically significant in the betamethasone-treated group only. Although antenatal endotoxin leads to striking improvements in postnatal lung function, this may be at the expense of normal alveolar development.     

Glucocorticoids,inflammation and the perinatal lung

Many women delivering preterm infants at less than 30 weeks gestation have subclinical chorioamnionitis. Based on current guidelines, maternal glucocorticoid treatment is given to induce lung maturation. Fetal exposure to proinflammation can cause acute and chronic injury, but inflammation also can induce fetal lung maturation. Both antenatal glucocorticoids and inflammation modulate lung development, by inducing the surfactant system, inducing structural maturation, and inhibiting alveolarization. The opportunities for the future are to develop new safer strategies to mature the preterm foetus, and the risks are potential adverse interactions of repetitive glucocorticoid exposures and unrecognized fetal exposure to inflammation.     

Vessel-specific regulation of angiotensin II receptor subtypes during ovine development

Umbilical and systemic responses to angiotensin II differ in term fetal sheep, and peripheral vascular responses are attenuated or absent before and after birth. These observations may reflect developmental differences in angiotensin II receptor (AT) subtypes in vascular smooth muscle (VSM). Studies of AT subtype ontogeny and regulation are generally limited to the aorta, which may not be extrapolated to other arteries, and neither is completely described during ovine development. We therefore characterized VSM AT subtype expression and regulation throughout an extended period of development in umbilical and carotid artery and aorta from fetal (85-146 d gestation), postnatal (5-23 d), and adult sheep, measuring AT(1) and AT(2) mRNA and protein and performing immunohistochemistry. Parallel increases in umbilical AT(1) mRNA and protein began early in gestation and continued to term, and although AT(2) mRNA was unchanged, protein levels decreased >90% at term. Fetal carotid AT(1) mRNA was <40% of adult values and unchanged before birth; however, AT(1) protein rose >2-fold at term. After birth, AT(1) mRNA increased to 85% of adult values and was associated with another 2-fold rise in protein. In contrast, carotid AT(2) mRNA and protein fell in parallel throughout development and were barely detectable in the newborn and the adult. Immunostaining was consistent with observations in both arteries. A third pattern occurred in aortic VSM. The ontogeny of AT subtype expression and regulation is vessel specific, with changes in umbilical VSM beginning very early in development. Although the mechanisms that regulate mRNA and protein expression are unclear, these changes parallel differences in VSM maturation and function and local blood flow.     

Ontogeny of surfactant apoproteins in the rat

Content of the 26-38-kD surfactant apoprotein (SP-A) was determined in lung homogenates from fetal (17-21 d gestation), postnatal (1-28 d of life), and adult male and female rats by a double sandwich ELISA. Expression of mRNA for SP-A as well as the hydrophobic apoproteins, SP-B and SP-C, were also determined in lung homogenates from fetal and adult rats of both sexes by Northern blot analysis. SP-A was undetectable in fetal lungs on d 17 (day of birth = d 22) and barely detectable on d 18. On d 19 there was a 3- to 4-fold increase in SP-A content above d 18 levels. Between d 19 and 21 SP-A content significantly increased another 6- to 9-fold. SP-A content on the day of birth was not significantly different from that seen on gestational d 21. SP-A content decreased 35-40% between the day of birth and postnatal d 7. After the second postnatal week SP-A content gradually increased, reaching adult levels after d 28. No sex differences in SP-A content were observed during fetal or postnatal lung maturation. SP-A mRNA was first detected in fetal lungs on d 18 and increased in relative abundance until d 21, but remained below adult levels. Developmental changes in fetal lung SP-A content closely paralleled changes in fetal expression of SP-A mRNA. SP-B mRNA was also first detected on d 18, then increased in relative abundance to adult levels by d 20. SP-C mRNA was clearly detectable on d 17, then increased in relative abundance to adult levels by d 20-21. Unlike surfactant phospholipids, there are no apparent sex differences in the expression of any of the surfactant apoproteins during late gestation. The differences observed during fetal lung maturation in the time of onset and changes in relative abundance among the three apoprotein mRNA imply that their genes may be differentially regulated in the developing rat lung.     

Developmental regulation of angiotensinogen gene expression in sheep

It has been suggested that the liver is not the main source of angiotensinogen during fetal life in rats, but that the kidney is an important site of fetal angiotensinogen synthesis. In an effort to determine if this phenomenon is specific to the rat or applicable to other species, we compared the ontogenic changes in hepatic and renal angiotensinogen mRNA expression in fetal (60, 90, 118, and 138 d of gestation, term being 145 d), newborn (7 d postnatal), and adult sheep. Total RNA was extracted, subjected to Northern blotting and hybridized using a full-length rat radiolabeled antisense RNA. Angiotensinogen mRNA sequences were detected in all fetal liver samples and appeared to increase 3-fold from 60 to 138 d gestation and then to decrease after birth. In contrast, angiotensinogen mRNA could not be detected in renal cortical tissue of 118 or 138 d fetuses, or newborn or adult sheep. We conclude that, unlike in the rat, liver angiotensinogen gene expression is detectable during the 2nd trimester of gestation in sheep and is developmentally regulated. Furthermore, in contrast to the fetal rat, angiotensinogen mRNA sequences were undetectable in fetal sheep kidney.     

宫内注射脂多糖对围产期大鼠肺Toll样受体4信号转导通路的影响

目的 观察宫内注射脂多糖(LPS)对围产期大鼠肺内天然免疫相关的Toll样受体4(TLR4)信号转导通路的影响,探讨天然免疫在宫内感染中的免疫调节能力及对肺发育的影响.方法 将30只孕17d的SD大鼠随机分为LPS组和生理盐水对照组,LPS组宫内注射LPS 10μl(40μg/ml),对照组宫内注射等体积的灭菌生理盐水.分别留取胎龄18、20、22 d(E18、E20、E22)的胎鼠肺组织、胎盘组织标本以及生后1、3、7d(P1、P3、P7)新生鼠肺组织标本,HE染色观察病理改变,RT-PCR技术检测TLR4、髓样分化因子88(MyD88)和白介素1 β(IL-1β)mRNA表达,免疫组织化学技术检测肺组织TLR4、MyD88的表达分布情况.实验数据采用单因素方差分析和q检验进行统计学分析.结果 (1)LPS组孕鼠胎盘组织有大量中性粒细胞浸润,宫内感染模型建立成功;(2)在E18、E20和E22时,LPS组胎鼠肺组织无明显病理学改变,以后逐渐出现改变,于P7时可见肺泡数量减少,肺泡腔变大,间隔变薄,但未见明显结构紊乱;(3)LPS组TLR4、MyD88和IL-1β mRNA水平于E20和E22均高于对照组,差异有统计学意义(P<0.05),且均于E22表达达高峰,后缓慢下降;(4)免疫组织化学结果显示E18时两组肺组织内均未见明显TLR4和MyD88阳性染色,后均逐渐表达增加,且主要在细支气管和肺泡上皮细胞表达.结论 (1)宫内注射LPS可导致胎鼠和早产鼠肺组织TLR4、MyD88表达在一定范围内增加,后逐渐回复正常水平,同时肺组织的病理改变和炎症反应较为温和,推测在围产期胎肺天然免疫系统可以调节LPS诱导的炎症反应强度;(2)该实验在一定程度上证实宫内感染激活的信号转导通路是MyD88依赖性途径.     

Fetal cardiac natriuretic peptide expression and cardiovascular hemodynamics in endotoxin-induced acute cardiac dysfunction in mouse

We hypothesized that, in acute endotoxin-induced fetal cardiac dysfunction, atrial (ANP) and B-type (BNP) natriuretic peptide mRNA expressions are increased in proportion to the severity of fetal cardiovascular compromise in mouse. To investigate in vitro the effect of endotoxin-induced inflammation on cardiac natriuretic peptide expression, fetal hearts were harvested at 15-16 d of gestation and incubated for 6 h with lipopolysaccharide (LPS). To examine the relationship between fetal cardiovascular compromise and cardiac natriuretic peptide expression in endotoxin-induced cardiac dysfunction in the in vivo model, fetuses received intra-amniotically 25 microL LPS (10 microg/mL) or 25 microL of 0.9% saline. Fetal Doppler ultrasonography was performed before and six hours after the injections. In in vitro cultured fetal hearts, LPS induced the production of proinflammatory cytokines without affecting the basal expressions of natriuretic peptides. In the in vivo model, Doppler ultrasonography revealed severe cardiac dysfunction after LPS injection. No significant changes in ANP or atrial BNP mRNA were found. The fetal ventricular BNP mRNA levels were about 2.6-fold in the LPS group compared with the control group. Decreased fetal cardiac outflow mean velocity, increased proportion of isovolumetric contraction time of the cardiac cycle, and increased pulsatility indices of the descending aorta and inferior vena cava were related to elevated ventricular BNP mRNA levels. Our results show that LPS did not increase the mRNA expression of natriuretic peptides in cultured fetal hearts. In contrast, fetal ventricular BNP gene expression was increased in proportion to the severity of the hemodynamic compromise in vivo.     

IGF-II and IGF binding protein (IGFBP-1, IGFBP-3) gene expression in fetal rhesus monkey tissues during the second and third trimesters

The IGF system is a key modulator of somatic fetal growth. Studies with human fetal tissues have shown a specific spatial and temporal pattern of expression of IGF and IGF binding protein (IGFBP) mRNAs, but have been limited to defined periods during gestation (i.e. 8-20 wk gestation) because of tissue availability. To fully assess the role of these peptides in the primate growth process, a longitudinal study was conducted that focused on the expression of IGF-II and IGFBP-1 and IGFBP-3 genes in the rhesus monkey (Macaca mulatta). Liver, kidney, brain, and lung were collected from rhesus monkey fetuses approximately every 2 wk from 65 (early second trimester) through 150 d gestation (term 165 +/- 10 d) (n = 50), then processed for in situ hybridization using radiolabeled human cDNAs. IGF-II mRNA was abundantly expressed in fetal kidney (maturing glomerulus, supporting mesenchyme, cells of the developing nephrons), liver (hepatocytes), cerebral cortex (choroid plexus, capillaries), and lung (blood vessels, connective tissues, lamina propria, cartilage framework). IGFBP-1 was expressed only in the hepatocytes and IGFBP-3 mRNA was modestly expressed within the kidney (developing nephrons, collecting system mesenchyme), and liver (hepatocytes). These studies have shown that (1) IGF-II, IGFBP-1, and IGFBP-3 are expressed in specific cell types of the fetal monkey indicating a paracrine/autocrine role during development; (2) changes in IGF-II and IGFBP mRNA expression occur with advancing gestation; and (3) fetal monkey tissues express IGF-II and IGFBPs in a similar manner when compared with the human fetus.