Ulrike Weichelt

Prevention of hyperoxia-mediated pulmonary inflammation in neonatal rats by caffeine

In preterm human infants, briefly elevated concentrations of oxygen are associated with a prolonged increase in blood chemokine concentrations and the development of bronchopulmonary dysplasia (BPD). Caffeine given to preterm infants for the prevention or treatment of apnoea has been shown to reduce the rate of BPD. We tested the hypotheses that infant rats exposed to a combination of caffeine and hyperoxia would be less susceptible to lung injury than those exposed to hyperoxia alone and that caffeine decreases the pulmonary tissue expression of chemokines and leukocyte influx following hyperoxia. Using 6-day-old rat pups, we demonstrated that 24 h of 80% oxygen exposure caused pulmonary recruitment of neutrophils and macrophages. High levels of oxygen upregulated the expression of: the CXC chemokines, cytokine-induced neutrophil chemoattractant-1 and macrophage inflammatory protein-2; the CC-chemokine monocyte chemoattractant protein-1; the pro-inflammatory cytokines tumour necrosis factor-&agr; and interleukin-6, as measured by realtime PCR after the administration of caffeine (10 mg·kg−1 body weight); and attenuated chemokine and cytokine upregulation, as well as the influx of CD11b+, ED-1+ and myeloperoxidase+ leukocytes. These experiments suggest that protective effects of caffeine in the neonatal lung are mediated, at least in part, by reduction of pulmonary inflammation.

Erythropoietin attenuates hyperoxia-induced oxidative stress in the developing rat brain.

Oxygen toxicity contributes to the pathogenesis of adverse neurological outcome in survivors of preterm birth in clinical studies. In infant rodent brains, hyperoxia triggers widespread apoptotic neurodegeneration, induces pro-inflammatory cytokines and inhibits growth factor signaling cascades. Since a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), we hypothesized that rEpo would influence hyperoxia-induced oxidative stress in the developing rat brain. The aim of this study was to investigate the level of glutathione (reduced and oxidized), lipid peroxidation and the expression of heme oxygenase-1 (HO-1) and acetylcholinesterase (AChE) after hyperoxia and rEpo treatment. Six-day-old Wistar rats were exposed to 80% oxygen for 2-48 h and received 20,000 IU/kg rEpo intraperitoneally (i.p.). Sex-matched littermates kept under room air and injected with normal saline or rEpo served as controls. Treatment with rEpo significantly reduced hyperoxia-induced upregulation of oxidized glutathione (GSSG) and malondialdehyde, a product of lipid breakdown, whereas reduced glutathione (GSH) was upregulated by rEpo. In parallel, hyperoxia-treated immature rat brains revealed rEpo-suppressible upregulation of synaptic AChE-S as well as of the stress-inducible AChE-R variant, together predicting rEpo-protected cholinergic signaling and restrained inflammatory reactions. Furthermore, treatment with rEpo induced upregulation of HO-1 on mRNA, protein and activity level in the developing rat brain. Our results suggest that rEpo generates its protective effect against oxygen toxicity by a reduction of diverse oxidative stress parameters and by limiting the stressor-inducible changes in both HO-1 and cholinergic functions.

Caffeine protects neuronal cells against injury caused by hyperoxia in the immature brain

Caffeine administered to preterm infants has been shown to reduce rates of cerebral palsy and cognitive delay, compared to placebo. We investigated the neuroprotective potential of caffeine for the developing brain in a neonatal rat model featuring transient systemic hyperoxia. Using 6-day-old rat pups, we found that after 24 and 48h of 80% oxygen exposure, apoptotic (TUNEL(+)) cell numbers increased in the cortex, hippocampus, and central gray matter, but not in the hippocampus or dentate gyrus. In the dentate gyrus, high oxygen exposure led to a decrease in the number of proliferating (Ki67(+)) cells and the number of Ki67(+) cells double staining for nestin (immature neurons), doublecortin (progenitors), and NeuN (mature neurons). Absolute numbers of nestin(+), doublecortin(+), and NeuN(+) cells also decreased after hyperoxia. This was mirrored in a decline of transcription factors expressed in immature neurons (Pax6, Sox2), progenitors (Tbr2), and mature neurons (Prox1, Tbr1). Administration of a single dose of caffeine (10mg/kg) before high oxygen exposure almost completely prevented these effects. Our findings suggest that caffeine exerts protection for neonatal neurons exposed to high oxygen, possibly via its antioxidant capacity.

Erythropoietin Attenuates Hyperoxia-Induced Cell Death by Modulation of Inflammatory Mediators and Matrix Metalloproteinases

Oxygen toxicity appears to contribute to the pathogenesis of adverse neurological outcome in survivors of preterm birth. In infant rodent brains, hyperoxia triggers widespread apoptotic neurodegeneration, induces proinflammatory cytokines and inhibits growth factor signaling cascades. Since a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), we hypothesized that rEpo would influence the expression of proinflammatory cytokines and matrix metalloproteinase (MMP)-2 and MMP-9. Six-day-old Wistar rats were exposed to 80% oxygen for 2–48 h and received 20,000 IU rEpo i.p. Sex-matched littermates kept in room air and injected with normal saline or rEpo served as controls. Treatment with rEpo significantly reduced hyperoxia-induced upregulation of the proinflammatory cytokines IL-1β and IL-18 in infant rodent brains on the mRNA and protein levels. In parallel, gelatin zymography in hyperoxia-treated immature rat brains revealed an upregulation of active MMP-2 which was reduced by concomitant rEpo treatment. Furthermore, hyperoxia induced upregulation of MMP-9 following 12 h of oxygen exposure and this was attenuated by rEpo treatment. Our results suggest that rEpo generates its protective effect against oxygen toxicity through a reduction of proinflammatory mediator levels.

Hyperoxia changes the balance of the thioredoxin/peroxiredoxin system in the neonatal rat brain.

Reactive oxygen species (ROS) and intrinsic antioxidant defense systems play an important role in both physiological cell signaling processes and many pathological states, including neurodegenerative disorders and oxygen-toxicity. Here we report that short exposures to non-physiologic oxygen levels change the balance of the ROS-dependent thioredoxin/peroxiredoxin system in the developing rat brain. The aim of this study was to evaluate the expression of peroxiredoxins, thioredoxin 1, sulfiredoxin 1, and DJ-1 on gene and protein level under hyperoxic conditions. Six-days old Wistar rats were exposed to 80% oxygen for 6-48 h while sex-matched littermates were kept in room-air and served as controls. Oxygen-toxicity significantly induced upregulation of peroxiredoxins 1 and 2, peroxiredoxin sulfonic form, thioredoxin 1, and sulfiredoxin 1 in the brains of infant rats. Additionally, hyperoxia reduced the level of DJ-1, a hydroperoxide-responsive protein in the developing rat brain. The pathology of hyperoxia-mediated injury to the developing brain is still elusive and oxygen administration to neonates is often inevitable. These findings may provide evidence for the development of targeted therapeutic strategies to enhance the antioxidative defense of the immature brain.

Neuroprotection by Caffeine in Hyperoxia-Induced Neonatal Brain Injury

Sequelae of prematurity triggered by oxidative stress and free radical-mediated tissue damage have coined the term “oxygen radical disease of prematurity”. Caffeine, a potent free radical scavenger and adenosine receptor antagonist, reduces rates of brain damage in preterm infants. In the present study, we investigated the effects of caffeine on oxidative stress markers, anti-oxidative response, inflammation, redox-sensitive transcription factors, apoptosis, and extracellular matrix following the induction of hyperoxia in neonatal rats. The brain of a rat pups at postnatal Day 6 (P6) corresponds to that of a human fetal brain at 28–32 weeks gestation and the neonatal rat is an ideal model in which to investigate effects of oxidative stress and neuroprotection of caffeine on the developing brain. Six-day-old Wistar rats were pre-treated with caffeine and exposed to 80% oxygen for 24 and 48 h. Caffeine reduced oxidative stress marker (heme oxygenase-1, lipid peroxidation, hydrogen peroxide, and glutamate-cysteine ligase catalytic subunit (GCLC)), promoted anti-oxidative response (superoxide dismutase, peroxiredoxin 1, and sulfiredoxin 1), down-regulated pro-inflammatory cytokines, modulated redox-sensitive transcription factor expression (Nrf2/Keap1, and NFκB), reduced pro-apoptotic effectors (poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis inducing factor (AIF), and caspase-3), and diminished extracellular matrix degeneration (matrix metalloproteinases (MMP) 2, and inhibitor of metalloproteinase (TIMP) 1/2). Our study affirms that caffeine is a pleiotropic neuroprotective drug in the developing brain due to its anti-oxidant, anti-inflammatory, and anti-apoptotic properties.

Loss of SMAD3 Promotes Vascular Remodeling in Pulmonary Arterial Hypertension via MRTF Disinhibition

Rationale: Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR‐II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF‐&bgr; (transforming growth factor &bgr;) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])‐2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH. Objectives: We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin‐related transcription factor), which drives muscle protein expression. Methods: SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats. Measurements and Main Results: SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF‐&bgr; treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF‐&bgr;‐treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR‐II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF‐&bgr;‐induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2‐silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats. Conclusions: Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and—via MRTF disinhibition—hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype.

Transient Receptor Potential Vanilloid 4 and Serum Glucocorticoid-regulated Kinase 1 Are Critical Mediators of Lung Injury in Overventilated Mice In Vivo.

Background: Mechanical ventilation can cause lung endothelial barrier failure and inflammation cumulating in ventilator-induced lung injury. Yet, underlying mechanotransduction mechanisms remain unclear. Here, the authors tested the hypothesis that activation of the mechanosensitive Ca2+ channel transient receptor potential vanilloid (TRPV4) by serum glucocorticoid–regulated kinase (SGK) 1 may drive the development of ventilator-induced lung injury. Methods: Mice (total n = 54) were ventilated for 2 h with low (7 ml/kg) or high (20 ml/kg) tidal volumes and assessed for signs of ventilator-induced lung injury. Isolated-perfused lungs were inflated with continuous positive airway pressures of 5 or 15 cm H2O (n = 7 each), and endothelial calcium concentration was quantified by real-time imaging. Results: Genetic deficiency or pharmacologic inhibition of TRPV4 or SGK1 protected mice from overventilation-induced vascular leakage (reduction in alveolar protein concentration from 0.84 ± 0.18 [mean ± SD] to 0.46 ± 0.16 mg/ml by TRPV4 antagonization), reduced lung inflammation (macrophage inflammatory protein 2 levels of 193 ± 163 in Trpv4−/− vs. 544 ± 358 pmol/ml in wild-type mice), and attenuated endothelial calcium responses to lung overdistension. Functional coupling of TRPV4 and SGK1 in lung endothelial mechanotransduction was confirmed by proximity ligation assay demonstrating enhanced TRPV4 phosphorylation at serine 824 at 18% as compared to 5% cyclic stretch, which was prevented by SGK1 inhibition. Conclusions: Lung overventilation promotes endothelial calcium influx and barrier failure through a mechanism that involves activation of TRPV4, presumably due to phosphorylation at its serine 824 residue by SGK1. TRPV4 and SGK1 may present promising new targets for prevention or treatment of ventilator-induced lung injury.

314 Hyperoxia Changes the Balance of the Thioredoxin/Peroxiredoxin System in the Neonatal Rat Brain

Background and Aim As demonstrated previously, oxygen contributes to the pathogenesis of neonatal brain damage and leading to neurocognitive impairment of prematurely born infants in later life. Reactive oxygen species (ROS) and intrinsic antioxidant defense systems play an important role in both physiological cell signaling processes and many pathological conditions, including neurodegenerative disorders and oxygen-toxicity. Beside the glutathione-system several other redox-modulating proteins are known to be involved in redox-homeostases. The aim of this study was to evaluate potential alterations within the thioredoxin/peroxiredoxin system after exposures to nonphysiologic high oxygen levels in the developing rat brain. Methods Six-days old Wistar rats were exposed to 80% oxygen for 6, 12, 24 or 48 hours and littermates kept in room air served as controls (n=6–8). Brains (excluding cerebellum) were evaluated after perfusion with PBS and dissection of both hemispheres for RNA and protein analyses. Results We demonstrate that elevated oxygen concentrations change the balance of the ROS-dependent thioredoxin/peroxiredoxin system. Oxygen-toxicity significantly induced upregulation of peroxiredoxins in infant rat brain. In parallel, hyperoxia reduced the level of DJ-1, a hydroperoxide-responsive protein. Discussion These findings are highly relevant from a clinical aspect because oxygen administration to neonates is often inevitable, and we recommend that every effort should be made in neonatal medicine to limit exposure of these immature babies to high oxygen concentrations. These results may also contribute to receive optimal therapeutical approaches to ameliorate oxygen toxicity.

Transient Receptor Potential Vanilloid 4 Channel Deficiency Aggravates Tubular Damage after Acute Renal Ischaemia Reperfusion

Transient receptor potential vanilloid 4 (TRPV4) cation channels are functional in all renal vascular segments and mediate endothelium-dependent vasorelaxation. Moreover, they are expressed in distinct parts of the tubular system and activated by cell swelling. Ischaemia/reperfusion injury (IRI) is characterized by tubular injury and endothelial dysfunction. Therefore, we hypothesised a putative organ protective role of TRPV4 in acute renal IRI. IRI was induced in TRPV4 deficient (Trpv4 KO) and wild–type (WT) control mice by clipping the left renal pedicle after right–sided nephrectomy. Serum creatinine level was higher in Trpv4 KO mice 6 and 24 hours after ischaemia compared to WT mice. Detailed histological analysis revealed that IRI caused aggravated renal tubular damage in Trpv4 KO mice, especially in the renal cortex. Immunohistological and functional assessment confirmed TRPV4 expression in proximal tubular cells. Furthermore, the tubular damage could be attributed to enhanced necrosis rather than apoptosis. Surprisingly, the percentage of infiltrating granulocytes and macrophages were comparable in IRI–damaged kidneys of Trpv4 KO and WT mice. The present results suggest a renoprotective role of TRPV4 during acute renal IRI. Further studies using cell–specific TRPV4 deficient mice are needed to clarify cellular mechanisms of TRPV4 in IRI.