Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA.

The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.

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.

Caffeine Protects Against Anticonvulsant-Induced Impaired Neurogenesis in the Developing Rat Brain.

In preterm infants, phenobarbital is the first-line antiepileptic drug for neonatal seizures while caffeine is used for the treatment of apnea. Data from experimental animals suggest that phenobarbital and other anticonvulsants are toxic for the developing brain, while neuroprotective effects have been reported for caffeine both in newborn rodents and preterm human infants. To characterize the interaction of phenobarbital and caffeine in the hippocampus of the developing rodent brain, we examined the effects of both drugs given separately or together on postnatal neurogenesis after administration to neonatal rats throughout postnatal day (P) 4 to P6. Phenobarbital treatment (50 mg/kg) resulted in a significant decrease of proliferative capacity in the dentate gyrus. Phenobarbital also reduced expression of neuronal markers (doublecortin (DCX), calretinin, NeuN), neuronal transcription factors (Pax6, Sox2, Tbr1/2, Prox1), and neurotrophins (NGF, BDNF, NT-3) up to 24 h after the last administration. The phenobarbital-mediated impairment of neurogenesis was largely ameliorated by preconditioning with caffeine (10 mg/kg). In contrast, caffeine alone reduced proliferative capacity and expression of the neuronal markers DCX and NeuN at 6 h, but increased expression of neurotrophins and neuronal transcription factors at 6 and 12 h. These results indicate that administration of phenobarbital during the vulnerable phase of brain development negatively interferes with neuronal development, which can be prevented in part by co-administration of caffeine.

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-ß (transforming growth factor ß) 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-ß 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-ß-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-ß-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.

Caffeine Protects Against Anticonvulsant-Induced Neurotoxicity in the Developing Rat Brain.

Phenobarbital is the most commonly used drug for the treatment of neonatal seizures but may induce neurodegeneration in the developing brain. Methylxanthine caffeine is used for the treatment of apnea in newborn infants and appears to be neuroprotective, as shown by antiapoptotic and anti-inflammatory effects in oxidative stress models in newborn rodents and reduced rates of cerebral palsy in human infants treated with caffeine. We hypothesized that caffeine may counteract the proapoptotic effects of phenobarbital in newborn rats. Postnatal day 4 (P4) rats received phenobarbital (50 mg/kg) +/- caffeine (10 mg/kg) for three consecutive days. Brains examined at 6, 12, and 24 h after last injection of phenobarbital showed a drastic increase of apoptotic cell death (TUNEL+), which was attenuated by co-treatment with caffeine at 6 and 24 h but not at 12 h. Phenobarbital also increased protein levels of apoptosis inducing factor (AIF) and cleaved caspase-3, which was reduced by caffeine co-administration at all time points investigated. RNA expression of the pro-inflammatory cytokines TNFα, IFNγ, and IL-1ß, but not IL-18, was upregulated by phenobarbital. Co-treatment with caffeine significantly decreased these upregulations at all time points investigated, while caffeine without phenobarbital resulted in increased expression of TNFα, IL-1ß, and IL-18, but not IFNγ at 6 h. Downregulation of the adenosine A1 and A2a receptors, both of which bind caffeine, by 24 h of phenobarbital exposure was partly antagonized by caffeine. These results raise the possibility that the phenobarbital-induced adverse effects could be reduced by a co-treatment with caffeine.

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.

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 Ca 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.

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.

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-α 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.

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.

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.

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-1beta 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.