Brain inflammation and injury at 48 h is not altered by human amnion epithelial cells in ventilated preterm lambs.

BACKGROUND: Mechanical ventilation of preterm neonates is associated with neuroinflammation and an increased risk of adverse neurological outcomes. Human amnion epithelial cells (hAECs) have anti-inflammatory and regenerative properties. We aimed to determine if intravenous administration of hAECs to preterm lambs would reduce neuroinflammation and injury at 2 days of age. METHODS: Preterm lambs were delivered by cesarean section at 128-130 days' gestation (term is ~147 days) and either ventilated for 48 h or humanely killed at birth. Lambs received 3 mL surfactant (Curosurf) via endotracheal tube prior to delivery (either with or without 90 × 106 hAECs) and 3 mL intravenous phosphate-buffered saline (with or without 90 × 106 hAECs, consistent with intratracheal treatment) after birth. RESULTS: Ventilation increased microglial activation, total oligodendrocyte cell number, cell proliferation and blood-brain barrier permeability (P < 0.05, PBS + ventilation and hAEC + ventilation vs. control), but did not affect numbers of immature and mature oligodendrocytes. Ventilation reduced astrocyte and neuron survival (P < 0.05, PBS + ventilation and hAEC + ventilation vs. control). hAEC administration did not alter markers of neuroinflammation or injury within the white or gray matter. CONCLUSIONS: Mechanical ventilation for 48 h upregulated markers of neuroinflammation and injury in preterm lambs. Administration of hAECs did not affect markers of neuroinflammation or injury. IMPACT: Mechanical ventilation of preterm lambs for 48 h, in a manner consistent with contemporary neonatal intensive care, causes neuroinflammation, neuronal loss and pathological changes in oligodendrocyte and astrocyte survival consistent with evolving neonatal brain injury.Intravenous administration of hAECs immediately after birth did not affect neonatal cardiorespiratory function and markers of neuroinflammation or injury.Reassuringly, our findings in a translational large animal model demonstrate that intravenous hAEC administration to the preterm neonate is safe.Considering that hAECs are being used in phase 1 trials for the treatment of BPD in preterm infants, with future trials planned for neonatal neuroprotection, we believe these observations are highly relevant.

NFAT5 regulates renal gene expression in response to angiotensin II through Annexin-A2-mediated posttranscriptional regulation in hypertensive rats.

The aim was to identify new targets that regulate gene expression at the posttranscriptional level in angiotensin II (ANGII)-mediated hypertension. Heparin affinity chromatography was used to enrich nucleic acid-binding proteins from kidneys of two-kidney, one-clip (2K1C) hypertensive Wistar rats. The experiment was repeated with 14-day ANGII infusion using Alzet osmotic mini pumps, with or without ANGII receptor AT1a inhibition using losartan in the drinking water. Mean arterial pressure increased after 2K1C or ANGII infusion and was inhibited with losartan. Heparin affinity chromatography and mass spectrometry were used to identify Annexin-A2 (ANXA2) as having differential nucleic acid-binding activity. Total Annexin-A2 protein expression was unchanged, whereas nucleic acid-binding activity was increased in both kidneys of 2K1C and after ANGII infusion through AT1a stimulation. Costaining of Annexin-A2 with α-smooth muscle actin and aquaporin 2 showed prominent expression in the endothelia of larger arteries and the cells of the inner medullary collecting duct. The nuclear factor of activated T cells (NFAT) transcription factor was identified as a likely Annexin-A2 target using enrichment analysis on a 2K1C microarray data set and identifying several binding sites in the regulatory region of the mRNA. Expression analysis showed that ANGII increases NFAT5 protein but not mRNA level and, thus, indicated that NFAT5 is regulated by posttranscriptional regulation, which correlates with activation of the RNA-binding protein Annexin-A2. In conclusion, we show that ANGII increases Annexin-A2 nucleic acid-binding activity that correlates with elevated protein levels of the NFAT5 transcription factor. NFAT signaling appears to be a major contributor to renal gene regulation in high-renin states.

Losartan does not decrease renal oxygenation and norepinephrine effects in rats after resuscitated hemorrhage.

Renin-angiotensin-system blockers are thought to increase the risk of acute kidney injury after surgery and hemorrhage. We found that losartan does not cause renal cortical hypoxia after hemorrhage in rats because of decreased renal vascular resistance, but we did not evaluate resuscitation. We aimed to study losartan's effect on renal cortical and medullary oxygenation, as well as norepinephrine's vasopressor effect in a model of resuscitated hemorrhage. After 7 days of losartan (60 mg·kg-1·day-1) or control treatment, male Wistar rats were hemorrhaged 20% of their blood volume and resuscitated with Ringer's acetate. Mean arterial pressure, renal blood flow, and kidney tissue oxygenation were measured at baseline and after resuscitation. Finally, the effect of norepinephrine on mean arterial pressure and renal blood flow was investigated. As expected, losartan lowered mean arterial pressure but not renal blood flow. Losartan did not affect renal oxygen consumption and oxygen tension. Mean arterial pressure and renal blood flow were lower after resuscitated hemorrhage. A smaller increase of renal vascular resistance in the losartan group translated to a smaller decrease in cortical oxygen tension, but no significant difference was seen in medullary oxygen tension, either between groups or after hemorrhage. The effect of norepinephrine on mean arterial pressure and renal blood flow was similar in control- and losartan-treated rats. Losartan does not decrease renal oxygenation after resuscitated hemorrhage because of a smaller increase in renal vascular resistance. Further, losartan does not decrease the efficiency of norepinephrine as a vasopressor, indicating that blood pressure may be managed effectively during losartan treatment.

Effect of Human Amnion Epithelial Cells on the Acute Inflammatory Response in Fetal Sheep.

Intra-amniotic (IA) lipopolysaccharide (LPS) injection in sheep induces inflammation in the fetus. Human amnion epithelial cells (hAECs) moderate the effect of IA LPS on fetal development, but their influence on the acute inflammatory response to IA LPS is unknown. We aimed to determine the effects of hAECs on the acute fetal inflammatory response to IA LPS. After surgical instrumentation at 116 days' gestation (d) ewes were randomized to 1 of 4 groups at 123 d: IA LPS (10 mg) and intravenous (IV) saline (n = 8), IA LPS and IV hAECs (n = 6), IA saline and IV saline (n = 5) or IA saline and IV hAECs (n = 5). IV injections were administered immediately after IA injections. Serial fetal blood samples were collected. At 125 d, placental, fetal lung and liver samples were collected. IA LPS increased inflammatory cell recruitment in the placenta and lungs, increased IL-1ß and IL-8 mRNA levels in the lungs and increased serum amyloid A3 (SAA3) and C-reactive protein (CRP) mRNA levels in the liver. IV hAECs reduced fetal lung inflammatory cell recruitment but did not otherwise alter indices of placental, fetal lung or liver inflammation. The acute fetal inflammatory response to IA LPS is not substantially altered by IV hAEC treatment.

Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs.

Ventilation of preterm neonates causes pulmonary inflammation that can contribute to lung injury, propagate systemically and result in long-term disease. Modulation of this initial response may reduce lung injury and its sequelae. We aimed to determine the effect of human amnion epithelial cells (hAECs) on immune activation and lung injury in preterm neonatal lambs. Preterm lambs received intratracheal hAECs (90x106) or vehicle, prior to 2 h of mechanical ventilation. Within 5 min of ventilation onset, lambs also received intravenous hAECs (90x106) or vehicle. Lung histology, bronchoalveolar lavage (BAL) cell phenotypes, and cytokine profiles were examined after 2 h of ventilation, and in unventilated controls. Histological indices of lung injury were higher than control, in vehicle-treated ventilated lambs but not in hAEC-treated ventilated lambs. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1ß and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease.

Differential short-term regional effects of early high dose erythropoietin on white matter in preterm lambs after mechanical ventilation.

Inadvertently injurious ventilation of preterm neonates in the delivery room can cause cerebral white matter (WM) inflammation and injury. We investigated the impact of an early high dose of recombinant human erythropoietin (EPO) on ventilation-induced WM changes in preterm lambs. Injurious ventilation, targeting a V(T) of 15 ml kg(-1) with no positive end-expiratory pressure, was initiated for 15 min in preterm lambs (0.85 gestation). Conventional ventilation was continued for a further 105 min. Lambs received either 5000 IU kg(-1) of EPO (EPREX®; Vent+EPO; n = 6) or vehicle (Vent; n = 8) via an umbilical vein at 4 ± 2 min. Markers of WM injury and inflammation were assessed using quantitative real-time PCR (qPCR) and immunohistochemistry and compared to a group of unventilated controls (UVC; n = 4). In Vent+EPO lambs compared to Vent lambs: (i) interleukin (IL)-1ß and IL-6 mRNA levels in the periventricular WM and IL-8 mRNA levels in the subcortical WM were higher (P < 0.05 for all); (ii) the density of microglia within the aggregations was not different in the periventricular WM and was lower in the subcortical WM (P = 0.001); (iii) the density of astrocytes was lower in the subcortical WM (P = 0.002); (iv) occludin and claudin-1 mRNA levels were higher in the periventricular WM (P < 0.02 for all) and (vi) the number of blood vessels with protein extravasation was lower (P < 0.05). Recombinant human EPO had variable regional effects within the WM when administered during injurious ventilation. The adverse short-term outcomes discourage the use of early high dose EPO administration in preterm ventilated babies.

Human Amnion Epithelial Cells Modulate Ventilation-Induced White Matter Pathology in Preterm Lambs.

BACKGROUND: Preterm infants can be inadvertently exposed to high tidal volumes (VT) during resuscitation in the delivery room due to limitations of available equipment. High VT ventilation of preterm lambs produces cerebral white matter (WM) pathology similar to that observed in preterm infants who develop cerebral palsy. We hypothesized that human amnion epithelial cells (hAECs), which have anti-inflammatory and regenerative properties, would reduce ventilation-induced WM pathology in neonatal late preterm lamb brains. METHODS: Two groups of lambs (0.85 gestation) were used, as follows: (1) ventilated lambs (Vent; n = 8) were ventilated using a protocol that induces injury (VT targeting 15 ml/kg for 15 min, with no positive end-expiratory pressure) and were then maintained for another 105 min, and (2) ventilated + hAECs lambs (Vent+hAECs; n = 7) were similarly ventilated but received intravenous and intratracheal administration of 9 × 10(7) hAECs (18 × 10(7) hAECs total) at birth. Oxygenation and ventilation parameters were monitored in real time; cerebral oxygenation was measured using near-infrared spectroscopy. qPCR (quantitative real-time PCR) and immunohistochemistry were used to assess inflammation, vascular leakage and astrogliosis in both the periventricular and subcortical WM of the frontal and parietal lobes. An unventilated control group (UVC; n = 5) was also used for qPCR analysis of gene expression. Two-way repeated measures ANOVA was used to compare physiological data. Student's t test and one-way ANOVA were used for immunohistological and qPCR data comparisons, respectively. RESULTS: Respiratory parameters were not different between groups. Interleukin (IL)-6 mRNA levels in subcortical WM were lower in the Vent+hAECs group than the Vent group (p = 0.028). IL-1ß and IL-6 mRNA levels in periventricular WM were higher in the Vent+hAECs group than the Vent group (p = 0.007 and p = 0.001, respectively). The density of Iba-1-positive microglia was lower in the subcortical WM of the parietal lobes (p = 0.010) in the Vent+hAECs group but not in the periventricular WM. The number of vessels in the WM of the parietal lobe exhibiting protein extravasation was lower (p = 0.046) in the Vent+hAECs group. Claudin-1 mRNA levels were higher in the periventricular WM (p = 0.005). The density of GFAP-positive astrocytes was not different between groups. CONCLUSIONS: Administration of hAECs at the time of birth alters the effects of injurious ventilation on the preterm neonatal brain. Further studies are required to understand the regional differences in the effects of hAECs on ventilation-induced WM pathology and their net effect on the developing brain.

Maintenance of human amnion epithelial cell phenotype in pulmonary surfactant.

INTRODUCTION: Preterm newborns often require mechanical respiratory support that can result in ventilation-induced lung injury (VILI), despite exogenous surfactant treatment. Human amnion epithelial cells (hAECs) reduce lung inflammation and resultant abnormal lung development in preterm animals; co-administration with surfactant is a potential therapeutic strategy. We aimed to determine whether hAECs remain viable and maintain function after combination with surfactant. METHODS: hAECs were incubated in surfactant (Curosurf) or phosphate-buffered saline (PBS) for 30 minutes at 37°C. Cell viability, phenotype (by flow cytometry), inhibition of T-cell proliferative responses and differentiation into lung epithelium-like cells (assessed with immunohistochemical staining of surfactant protein (SP)-A) were investigated. RESULTS: Cell viability and apoptosis of hAECs were not altered by surfactant, and hAEC phenotype was not altered. hAECs maintained expression of epithelial cell adhesion molecule (EpCAM) and human leukocyte antigen (HLA)-ABC after surfactant exposure. Expression of HLA-DR, CD80 and CD86 was not increased. Immunosuppression of T cells by hAECs was not altered by surfactant. hAEC differentiation into lung epithelium-like cells was equivalent after exposure to PBS or surfactant, and SP-A expression was equivalent. CONCLUSION: Surfactant exposure does not alter viability or function of hAECs. Thus a combination therapy of hAECs and surfactant may be an efficacious therapy to ameliorate or prevent preterm lung disease.

Renal neurohormonal regulation in heart failure decompensation.

Decompensation in heart failure occurs when the heart fails to balance venous return with cardiac output, leading to fluid congestion and contributing to mortality. Decompensated heart failure can cause acute kidney injury (AKI), which further increases mortality. Heart failure activates signaling systems that are deleterious to kidneys such as renal sympathetic nerve activity (RSNA), renin-angiotensin-aldosterone system, and vasopressin secretion. All three reduce renal blood flow (RBF) and increase tubular sodium reabsorption, which may increase renal oxygen consumption causing AKI through renal tissue hypoxia. Vasopressin contributes to venous congestion through aquaporin-mediated water retention. Additional water retention may be mediated through vasopressin-induced medullary urea transport and hyaluronan but needs further study. In addition, there are several systems that could protect the kidneys and reduce fluid retention such as natriuretic peptides, prostaglandins, and nitric oxide. However, the effect of natriuretic peptides and nitric oxide are blunted in decompensation, partly due to oxidative stress. This review considers how neurohormonal signaling in heart failure drives fluid retention by the kidneys and thus exacerbates decompensation. It further identifies areas where there is limited data, such as signaling systems 20-HETE, purines, endothelin, the role of renal water retention mechanisms for congestion, and renal hypoxia in AKI during heart failure.

Prophylactic erythropoietin exacerbates ventilation-induced lung inflammation and injury in preterm lambs.

Ventilation-induced lung injury (VILI) of preterm neonates probably contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Erythropoietin (EPO) has been suggested as a therapy for BPD. The aim of this study was to determine whether prophylactic administration of EPO reduces VILI in preterm newborn lambs. Lambs at 126 days of gestation (term is 147 days) were delivered and ventilated with a high tidal volume strategy for 15 min to cause lung injury, then received gentle ventilation until 2 h of age. Lambs were randomized to receive intravenous EPO (5000 IU kg(-1): Vent+EPO; n = 6) or phosphate-buffered saline (Vent; n = 7) soon after birth: unventilated controls (UVC; n = 8) did not receive ventilation or any treatment. Physiological parameters were recorded throughout the experimental procedure. Samples of lung were collected for histological and molecular assessment of inflammation and injury. Samples of liver were collected to assess the systemic acute phase response. Vent+EPO lambs received higher F IO 2, P aO 2 and oxygenation during the first 10 min than Vent lambs. There were no differences in physiological indices beyond this time. Total lung injury score, airway wall thickness, inflammation and haemorrhage were higher in Vent+EPO lambs than in Vent lambs. Lung inflammation and early markers of lung and systemic injury were elevated in ventilated lambs relative to unventilated lambs; EPO administration further increased lung inflammation and markers of lung and systemic injury. Prophylactic EPO exacerbates VILI, which may increase the incidence and severity of long-term respiratory disease. More studies are required before EPO can be used for lung protection in preterm infants.

The immune consequences of preterm birth.

Preterm birth occurs in 11% of live births globally and accounts for 35% of all newborn deaths. Preterm newborns have immature immune systems, with reduced innate and adaptive immunity; their immune systems may be further compromised by various factors associated with preterm birth. The immune systems of preterm infants have a smaller pool of monocytes and neutrophils, impaired ability of these cells to kill pathogens, and lower production of cytokines which limits T cell activation and reduces the ability to fight bacteria and detect viruses in cells, compared to term infants. Intrauterine inflammation is a major contributor to preterm birth, and causes premature immune activation and cytokine production. This can induce immune tolerance leading to reduced newborn immune function. Intrauterine inflammation is associated with an increased risk of early-onset sepsis and likely has long-term adverse immune consequences. Requisite medical interventions further impact on immune development and function. Antenatal corticosteroid treatment to prevent newborn respiratory disease is routine but may be immunosuppressive, and has been associated with febrile responses, reductions in lymphocyte proliferation and cytokine production, and increased risk of infection. Invasive medical procedures result in an increased risk of late-onset sepsis. Respiratory support can cause chronic inflammatory lung disease associated with increased risk of long-term morbidity. Colonization of the infant by microorganisms at birth is a significant contributor to the establishment of the microbiome. Caesarean section affects infant colonization, potentially contributing to lifelong immune function and well-being. Several factors associated with preterm birth alter immune function. A better understanding of perinatal modification of the preterm immune system will allow for the refinement of care to minimize lifelong adverse immune consequences.

Changes in fetal thymic immune cell populations in a sheep model of intrauterine inflammation.

Intrauterine inflammation is a common antecedent of preterm birth and can alter the development of the fetal thymus, the site of development, and maturation of T lymphocytes. The effects of intrauterine inflammation on specific thymic T lymphocyte populations are largely unknown. We hypothesized that intrauterine inflammation would alter fetal thymic T cell populations. Immunohistochemistry was used to quantitate the relative proportions of thymic cortical and medullary cell populations in fetal sheep 7 days after intra-amniotic lipopolysaccharide (LPS) injection. The proportions of CD8⁺and MHC II⁺ cells in the fetal thymus were reduced in response to LPS. The ratio of CD4:CD8 cells was increased by LPS exposure. No changes were observed in the percentage of CD4⁺, γδ(WC1)⁺, CD45R⁺B cells, or CD44⁺ cells. These studies indicate that intrauterine inflammation impacts thymic composition of CD8 T cells and the development and/or activation of CD4 T cells in the fetal thymus.