Tamas Jilling - US grants

[unreadable] DESCRIPTION (provided by applicant): Platelet-activating factor (PAF) has been implicated as a key mediator in the pathogenesis of gastrointestinal diseases such as Crohn's disease, ulcerative colitis and neonatal necrotizing enterocolitis (NEC). The PAF receptor (PAFR) is a member of the G protein-coupled receptor (GPCR) superfamily. Despite the importance of PAF in gastrointestinal diseases, and the high level expression of PAFR in intestinal epithelial cells, PAFR trafficking and signal transduction have been studied only in non-epithelial tissues. Our previous studies have shown that PAF plays a crucial role in experimental NEC, PAFR is localized exclusively in the apical plasma membrane in cultured intestinal epithelial cells and regulates such diverse cellular functions as gene expression, ion transport, intracellular pH, and cell death. Preliminary data presented in this proposal shows that disruption of detergent resistant membrane domains (DRM), blocking palmitoylation, or treatment of intestinal epithelial cells with polyunsaturated fatty acids (PUFA) eliminates, or blunts PAF-induced cellular responses. PUFA are important nutrients, with a broad range of biological effects in development, health and disease. In particular, PUFA play a preventive role in an experimental model of NEC, where PAF is a critical mediator. We hypothesize that PAFR is targeted to DRM via palmitoylation of C317 in its cytoplasmic tail, and that disruption of palmitoylation and DRM targeting by PUFA can inhibit the efficiency of signal transduction by PAFR. This hypothesis will be tested based on the following three specific aims: 1) To identify the mechanisms that target PAFR to DRM in the apical plasma membrane of polarized epithelial cells and to determine the importance of DRM targeting in PAFR signaling. 2) To determine whether PUFA can modulate signaling by affecting PAFR targeting to DRM. 3) To examine the effect of PAFR palmitoylation and PUFA on PAFR targeting and on experimental NEC in vivo. In order to accomplish these goals we will utilize heterologously expressed, tagged wild type and mutant PAFR in polarized epithelial cell lines, in neonatal rat intestine using adenoviral gene transfer and in transgenic mice along with the pharmacological manipulation of DRM and palmitoylation. Targeting of PAFR will be analyzed using imaging, immunologic and biochemical methods, and function will be evaluated using highly reproducible functional assays. These studies will elucidate the mechanisms of PAFR targeting in epithelial cells, and a novel mechanism by which PUFA might affect the function of PAFR, and other GPCR-s. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Industrial accidents, urban environmental disasters and terrorist attacks involving bromine gas (Br2) are of great concern to public safety. Br2 is used extensively in manufacturing and, therefore, needs to be transported and stored in large quantities. Accidental and intentional discharges of Br2 with consequent large number of casualties have been reported. We are only beginning to understand the mechanisms and potential countermeasures of Br2 toxicity and the identification of particularly vulnerable populations is completely lacking. Our preliminary studies show that pregnant mice exposed to 600 ppm Br2 for 30 minutes at gestation day 15 (E15) and returned to room air, exhibit 75% mortality. This mortality rate is considerably higher than what is seen in non-pregnant female or male mice at the same exposure level (25% mortality). Fetuses of surviving mothers are severely growth restricted at E19 and the mothers exhibit symptoms of preeclampsia. Placentae of pregnant mice exposed to Br2 express high levels of the short form of fms-like tyrosine kinase 1 (sFLT1), a known mediator of systemic endothelial dysfunction and preeclampsia. We propose the following hypotheses: 1) Preeclampsia, as a pregnancy-specific condition is responsible for the increased vulnerability of pregnant mothers and their unborn fetuses to Br2 toxicity. 2) Intervention with inhibitors of type 5 cyclic nucleotide-specific phosphodiesterases (PDE5i) will mitigate systemic endothelial dysfunction, which is a shared mechanism of halogen toxicity and preeclampsia; they will save the lives of mothers and improve fetal growth. We are proposing to conduct the following experiments grouped in two well integrated specific aims: SA #1. Identify the mechanisms by which exposure of pregnant mice to Br2 gas (600 or 400 ppm for 30 min) leads to significantly higher mortality than non-pregnant mice and causes fetal growth restriction/fetal demise (FGR/FD) when returned to room air. We propose that maternal mortality and FGR/FD are due to systemic hypoxemia and damage to endothelial NOS (eNOS), causing the placenta to produce anti-angiogenic factors, leading to the development of preeclampsia/eclampsia.SA #2. Investigate the efficacy of post Br2 exposure administration of (PDE5i) to decrease preeclampsia, FGR/FD and mortality in pregnant mice. Following exposure to Br2, tadalafil (1 mg*kg-1*day-1; a PDE5i with a long half-life) or placebo will be administered by gavage every 24 h starting at 1 h post exposure. Maternal death, fetal growth, and hallmark symptoms of systemic endothelial dysfunction and preeclampsia will be measured in pregnant and non-pregnant mice as discussed in SA#1. In summary, this exploratory translational research project will begin delineating the mechanisms of Br2 toxicity in pregnant mice and will test the efficacy of highly promising countermeasures (PDE5i) which are already in clinical trials for the treatment of preeclampsia. Thus, we believe that this project is consistent with the overarching goal of the CounterACT program to design better countermeasures for vulnerable populations.

The halogen bromine (Br2) is used as water disinfectant, for bleaching fibers, for manufacturing antiepileptic drugs, dyestuffs, flame-retardants, insecticides, drilling fluids, and gasoline additives. When inhaled, it causes exposure-level-dependent acute and chronic pulmonary and systemic injuries ranging from mild eye and airway irritation, to significant damage to cardiopulmonary system and other organs, which can lead to death. Survivors may develop reactive airway disease syndrome, pulmonary fibrosis as well as restrictive and obstructive pulmonary diseases. Presently, there are no studies evaluating acute and chronic sequelae of Br2 inhalation in pregnant rodent and non-rodent models, even though US census bureau data predicts two of every 100 people in the US being pregnant. Exposure of pregnant mice at gestational day 15 (E15) to Br2 (600 ppm for 30 min.) results in 75% mortality over four days, in contrast to 25% mortality in males or non-pregnant females (p<0001). When delivered at E19, fetuses of surviving Br2-exposed mice exhibit severe fetal growth restriction (FGR) and fetal demise (FD). Placentas are poorly developed and express increased levels of short-FMS-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic mediator and biomarker of both preeclampsia and pulmonary hypertension. When born naturally, none of the fetuses survive. Oral administration of an FDA- approved type 5 cyclic nucleotide-specific phosphodiesterase inhibitor (PDE5i; tadalafil) to the dams post- exposure, dramatically improved maternal survival, fetal growth restriction and neonatal survival. We hypothesize that brominated intermediates, formed by the reaction of Br2 and HOBr with plasmalogens cause injury to the endothelium and the placenta, inducing the release of vasoconstrictor and anti-angiogenic mediators which in turn mediate pulmonary vasoconstriction, increased pulmonary artery pressure and right ventricular dysfunction. Tadalafil restores pulmonary and uterine vasodilation, preserves heart function and improves uterine/placental blood supply resulting in maternal and fetal survival. We will test these proposed mechanisms and we will perform the necessary efficacy studies to identify the optimum therapeutic regimen of tadalafil to decrease maternal morbidity and mortality, improve fetal growth restriction and increase fetal survival when administered orally post exposure. Specific Aim #1. To test the hypothesis that exposure of pregnant mice to Br2 at E15 causes extensive pulmonary injury as well as systemic endothelial injury, placental injury, pulmonary hypertension, right heart failure resulting in maternal mortality, fetal growth restriction and fetal demise/stillbirth. Specific Aim #2: To identify the sequence of events and mechanisms involved in the development of maternal vasoconstriction, pulmonary hypertension and right heart failure. Specific Aim #3. To investigate the efficacy of post halogen exposure administration of tadalafil to decrease maternal and fetal death and morbidity and to develop a rabbit (non-rodent) model of Br2 toxicity in pregnancy.

The halogen bromine (Br2) is used as water disinfectant, for bleaching fibers, for manufacturing antiepileptic drugs, dyestuffs, flame-retardants, insecticides, drilling fluids, and gasoline additives. When inhaled, it causes exposure-level-dependent acute and chronic pulmonary and systemic injuries ranging from mild eye and airway irritation, to significant damage to cardiopulmonary system and other organs, which can lead to death. Survivors may develop reactive airway disease syndrome, pulmonary fibrosis as well as restrictive and obstructive pulmonary diseases. Presently, there are no studies evaluating acute and chronic sequelae of Br2 inhalation in pregnant rodent and non-rodent models, even though US census bureau data predicts two of every 100 people in the US being pregnant. Exposure of pregnant mice at gestational day 15 (E15) to Br2 (600 ppm for 30 min.) results in 75% mortality over four days, in contrast to 25% mortality in males or non-pregnant females (p<0001). When delivered at E19, fetuses of surviving Br2-exposed mice exhibit severe fetal growth restriction (FGR) and fetal demise (FD). Placentas are poorly developed and express increased levels of short-FMS-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic mediator and biomarker of both preeclampsia and pulmonary hypertension. When born naturally, none of the fetuses survive. Oral administration of an FDA- approved type 5 cyclic nucleotide-specific phosphodiesterase inhibitor (PDE5i; tadalafil) to the dams post- exposure, dramatically improved maternal survival, fetal growth restriction and neonatal survival. We hypothesize that brominated intermediates, formed by the reaction of Br2 and HOBr with plasmalogens cause injury to the endothelium and the placenta, inducing the release of vasoconstrictor and anti-angiogenic mediators which in turn mediate pulmonary vasoconstriction, increased pulmonary artery pressure and right ventricular dysfunction. Tadalafil restores pulmonary and uterine vasodilation, preserves heart function and improves uterine/placental blood supply resulting in maternal and fetal survival. We will test these proposed mechanisms and we will perform the necessary efficacy studies to identify the optimum therapeutic regimen of tadalafil to decrease maternal morbidity and mortality, improve fetal growth restriction and increase fetal survival when administered orally post exposure. Specific Aim #1. To test the hypothesis that exposure of pregnant mice to Br2 at E15 causes extensive pulmonary injury as well as systemic endothelial injury, placental injury, pulmonary hypertension, right heart failure resulting in maternal mortality, fetal growth restriction and fetal demise/stillbirth. Specific Aim #2: To identify the sequence of events and mechanisms involved in the development of maternal vasoconstriction, pulmonary hypertension and right heart failure. Specific Aim #3. To investigate the efficacy of post halogen exposure administration of tadalafil to decrease maternal and fetal death and morbidity and to develop a rabbit (non-rodent) model of Br2 toxicity in pregnancy.

We have shown that pregnant mice are particularly susceptible to injury caused by Br2 exposure and develop symptoms that are reminiscent of preeclampsia. We identified placental production of a short splice variant of FMS-like tyrosine kinase 1 (sFLT-1) and consequent inhibition of signaling via vascular endothelial growth factor (VEGF) as an underlying mechanism. Additionally, treatment with the type V phosphodiesterase inhibitor (PDE5i) tadalafil or treatment with recombinant VEGF mitigate pregnancy-specific Br2 toxicty by reducing blood pressure, restoring heart function, reducing respiratory and metabolic acidosis and reducing lung edema. In preliminary data, we demonstrate that Cl2 exposure is highly toxic to pregnant mice, resulting in severe maternal weight loss, high rate of maternal mortality and severe fetal growth restriction. Additional preliminary data show that treatment with the proton pump inhibitor (PPI) esomeprazole (ESO; 30 mg/kg/day by gavage), which is the active ingredient of Nexium®, results in improved maternal weight gain and fetal growth in Cl2 exposed pregnant mice. ESO is a PPI, but was found to stimulate nuclear factor (erythroid-derived 2)?like 2 (Nrf2), resulting in the increased expression of antioxidant genes including HO-1 as well. HO-1 is critical for the development and maintenance of normal pregnancy. Stimulation of HO-1 expression has been shown to antagonize mechanisms that cause preeclampsia and ameliorate diagnostic signs of preeclampsia in animal models. Our specific aims were designed to test the hypothesis that ESO (30 mg/kg/day by gavage) improves survival and fetal growth by inducing HO-1 via activation of nuclear factor (erythroid-derived 2)?like 2 (Nrf2) and the protective effects will be accompanied by reduced mediators and symptoms of preeclampsia that occur in Cl2 exposed pregnant mice. To test these proposed mechanisms, we will compare the efficacy of ESO to another, FDA-approved Nrf2 activator dimethyl fumarate (DMF; Tecfidera®) and we will assess the effects of the chemical inhibitors of Nrf2 (ML385) and HO-1 (tin proto porphyrin IX; SnPP) on the efficacy of ESO. SA#1: To compare the efficacy of ESO (30 mg/kg/day by gavage) with DMF (100 mg/kg/day by gavage) to reduce maternal mortality, fetal growth restriction and symptoms of preeclampsia in Cl2 exposed pregnant mice. DMF is an FDA approved drug for the treatment of multiple sclerosis. Similar to ESO, DMF is known to activate Nrf2, but it is not a proton pump inhibitor. Comparing the efficacy of ESO to DMF will allow to dissociate the mechanistic contribution of Nrf2 activation from the PPI function of ESO. SA#2 To test the mechanistic roles of Nrf2 activation and HO- 1 activity in protection from Cl2 exposure toxicity by ESO. We will use the small molecule inhibitors of Nrf2 (ML385) and HO-1 (tin protoporphyrin IX; SnPP) for mechanistic testing. Success of this research plan will be the basis of a full application (UO1) for the testing of dose-dependent efficacy of ESO and/or DMF against both Cl2 and Br2 induced pathology in pregnant mice, including the continuous monitoring of blood pressure with telemetry, and additional mechanistic studies using Nrf2 and HO-1 gene-targeted murine models.