Jayanth Ramadoss, Ph.D. - US grants

DESCRIPTION (provided by applicant): Efforts to successfully prevent or ameliorate the teratogenic effects of alcohol have been impeded, at least in part, by a limited understanding of the mechanisms by which alcohol damages the developing fetus. In this K99/R00 grant, we will explore the maternal uterine origins of Fetal Alcohol Spectrum Disorders (FASD) and devise a strategy for development of a future proteomic biomarker(s)/unique signature profile for maternal alcohol consumption. Coordinated growth and remodeling of the entire uterine circulation and creation of a placenta are requisites for normal fetal development. These intricate processes are controlled by endothelial-derived nitric oxide (NO) and enzyme activity of endothelial nitric oxide synthase (eNOS). The overall goal of this application is to investigate the direct effects of chronic binge alcohol on: 1) NO and eNOS-related signaling cascades in the uterine artery endothelium during pregnancy;and 2) the caveolae, the natural home for eNOS, and to utilize this knowledge to develop a high throughput proteomic biomarker(s)/unique signature profile for maternal alcohol consumption, a stated goal of NIAAA strategic plan for years 2009-2014. Unique pathways regulate NO and eNOS in the pregnant uterus and these play a distinct role in pregnancy-associated maternal uterine vascular adaptations. In specific aim#1, we will directly compare binge alcohol mediated adaptive responses and specific signaling pathways in the pregnant uterine artery endothelial cells under shear stress via graded pulsatile in vivo-like flow conditions. Data derived from these studies will provide the first mechanistic framework for understanding the interactions between shear stress and alcohol to regulate NO production in pregnant uterine endothelium. Binge alcohol alters the stoichiometric relationship between eNOS and cav-1 and with every bout of alcohol, there are significant rises in [Ca+2]i and in turn eNOS is driven away from the caveolae, its "natural home" which acts as a major stabilizing environment. In specific aim #2, we will investigate alcohol-induced repeated intracellular increases in [Ca+2]i and its effects on repeated depletion of eNOS from caveolae and NO production. In specific aim #3, we will utilize high throughput proteomics to identify a biomarker(s)/unique caveolar signature protein profile that is dependent on the level of alcohol insult. These findings will place us in an excellent position to understand the multimechanistic causes of alcohol damage, especially from the perspective of the mother and the uterus, and to correctly design and propose a comprehensive preventative strategy. PUBLIC HEALTH RELEVANCE: Each year, at least 40,000 babies are born with FASD in the U.S. at an estimated cost of $1.4 million per individual and total cost of at least $6 billion. Efforts to successfully prevent or ameliorate the teratogenic effects of alcohol have been impeded, at least in part, by a limited understanding of the mechanisms by which alcohol damages the developing fetus. In this K99/R00 grant, we will explore the maternal uterine origins of Fetal Alcohol Spectrum Disorders (FASD) and devise a strategy for development a high throughput proteomic biomarker(s)/unique signature profile for maternal alcohol consumption.

DESCRIPTION (provided by applicant): Maternal alcohol abuse during pregnancy can result in Fetal Alcohol Spectrum Disorders (FASD), a lifelong disability characterized by a range of neuroanatomical and neurobehavioral deficits. Current estimates of FASD prevalence is about 2-5% among young school children in the United States. Therefore, early detection of alcohol use among pregnant women is critical to prevent FASD. Maternal self-reporting is largely unreliable and the utility of current biomarkers is also limited during pregnancy. Among these markers, glycosylated variants of the protein transferrin (carbohydrate deficient transferrin, CDT), is reported to be the most diagnostically specific biomarker for chronic alcohol abuse. However, the mean sensitivity is about ~65.4% with a wide range depending upon the analytical method employed. Moreover, utilizing the conventional electrophoretic, chromatographic, and immunometric methods, it has so far been difficult to characterize the composition and abundance of all glycoforms. This is critical because alcohol produces complex effects on the glycans including loss of sialic acids, partial loss of sugars, and/or complete loss of oligosaccharide chains. In a widely utilized FASD rat model, we have for the first time utilized state of the art mass spectrometry and obtained preliminary data characterizing the sequences and abundances of maternal plasma transferrin N- linked oligosaccharides preserving the location of the N-glycosite. Based on our novel data, we propose the following Specific Aim: Develop and validate a novel and sensitive high throughput platform utilizing a traditional biomarker (variants of transferrin) for maternal alcohol consumption screening in a FASD rat model. Two sub aims are proposed to characterize and validate the protein's post-translational glycosylation signature abundance profile in the maternal plasma and relating it to the dose and duration of alcohol exposure. We will utilize a pregnant rat model exposed to graded alcohol doses administered for different durations of exposure during gestation. Plasma protein (transferrin) identification, sequence mapping, glycopeptide identification with diagnostic fragmentation spectra, and High Resolution/Accurate Mass quantitation will be performed. Validation studies will include N-linked Glycosite confirmation and lectin-based probing. Based on our preliminary data, we expect the proposed platform to provide a highly sensitive protein post-translational modification signature profile that will greatly enhance the window of detection and also clearly distinguish between the alcohol doses. At the end, we plan to correlate the glycosylation signature profile with FASD neurobehavioral measures. We also believe that these novel methodologies can be in the future extended to pregnant women and form the basis for a high throughput screening tool that will help establish a standardized diagnostic criteria with a real clinical impact not only in diagnosis but also in treatment and prevention.

DESCRIPTION (provided by applicant): Alcohol & Maternal Uterine Vascular Adaptations in Pregnancy Maternal alcohol abuse during pregnancy can result in Fetal Alcohol Spectrum Disorders (FASD), a lifelong disability characterized by a range of growth and developmental deficits. Current estimates of FASD prevalence is about 2-5% among young school children in the United States. Efforts to successfully prevent or ameliorate the teratogenic effects of ethanol have been impeded, at least in part, by a limited understanding of alcohol's complex mechanisms of action involving multiple organ systems. A normal pregnancy is associated with major uterine circulatory adaptations that directly relates to fetal growth, neonatal birth weights and survival. A cardinal feature of fetal alcohol syndrome is growth restriction, but traditionally alcohol studies have focused on brain/behavioral deficits, and little attention has been paid to critical gestational uterine vascular adaptations. We herein show novel preliminary data that alcohol impairs the exquisite regulation of gestational uterine circulatory adaptations in rat bing alcohol model. We herein hypothesize that chronic binge alcohol exposure during pregnancy impairs maternal uterine artery vascular adaptations via endothelial nitric oxide (NO) system dysregulation. Aim#1 will test the hypothesis that binge alcohol exposure in pregnancy leads to impaired endothelium-dependent maternal uterine artery relaxation. Following binge paradigm, we will assess growth indices, and utilize wire myography to study agonist-induced uterine artery relaxation in endothelium-intact/denuded vessels from saline control, pair-fed nutrition control, and alcohol rats. Aim#2 will test if binge alcohol impairs uterine artery relaxation via endothelium-derived NO relative to prostacyclin/endothelium-derived hyperpolarizing factor, decreases uterine artery NO production, endothelial NO synthase (eNOS) expression, and impairs eNOS multi-site phosphorylation. Aim#3 will test if binge alcohol decreases uterine artery NO, decreases excitatory Pser1177eNOS levels, and increases inhibitory Pthr495eNOS levels via ERK/AMPK pathway, and alcohol effects on endothelial [Ca+2]i transients. In Aims#2 and #3, we will assess uterine artery relaxation after blocking major vasodilatory pathways and conduct mechanistic studies with/without eNOS activity/multi-site phosphorylation-regulating pathway antagonists via RT-PCR, immunoblotting, histology, fluorescent imaging and spectrophotometry. Simultaneous [Ca2+]i-NO fluorescent imaging will be performed utilizing high-speed excitation/emission wavelength switching system. Our proposal explores a new frontier of gestational alcohol research by developing the first mechanistic framework for binge alcohol-induced uterine artery adaptations and identifying alcohol targets in an in vivo model. In alignment with NIAAA FY14 Strategic Plan, our proposal utilizes powerful methods and presents a new maternal-inclusive paradigm to the FAS field and predicts that a more effective intervention will require innovative pharmacologic targeting of maternal systems, especially the critical uterine circulation, in order to predict and propose a therapy that will have a real promie as a preventive strategy.

DESCRIPTION (provided by applicant): Alcohol & Maternal Uterine Vascular Adaptations in Pregnancy Maternal alcohol abuse during pregnancy can result in Fetal Alcohol Spectrum Disorders (FASD), a lifelong disability characterized by a range of growth and developmental deficits. Current estimates of FASD prevalence is about 2-5% among young school children in the United States. Efforts to successfully prevent or ameliorate the teratogenic effects of ethanol have been impeded, at least in part, by a limited understanding of alcohol's complex mechanisms of action involving multiple organ systems. A normal pregnancy is associated with major uterine circulatory adaptations that directly relates to fetal growth, neonatal birth weights and survival. A cardinal feature of fetal alcohol syndrome is growth restriction, but traditionally alcohol studies have focused on brain/behavioral deficits, and little attention has been paid to critical gestational uterine vascular adaptations. We herein show novel preliminary data that alcohol impairs the exquisite regulation of gestational uterine circulatory adaptations in rat bing alcohol model. We herein hypothesize that chronic binge alcohol exposure during pregnancy impairs maternal uterine artery vascular adaptations via endothelial nitric oxide (NO) system dysregulation. Aim#1 will test the hypothesis that binge alcohol exposure in pregnancy leads to impaired endothelium-dependent maternal uterine artery relaxation. Following binge paradigm, we will assess growth indices, and utilize wire myography to study agonist-induced uterine artery relaxation in endothelium-intact/denuded vessels from saline control, pair-fed nutrition control, and alcohol rats. Aim#2 will test if binge alcohol impairs uterine artery relaxation via endothelium-derived NO relative to prostacyclin/endothelium-derived hyperpolarizing factor, decreases uterine artery NO production, endothelial NO synthase (eNOS) expression, and impairs eNOS multi-site phosphorylation. Aim#3 will test if binge alcohol decreases uterine artery NO, decreases excitatory Pser1177eNOS levels, and increases inhibitory Pthr495eNOS levels via ERK/AMPK pathway, and alcohol effects on endothelial [Ca+2]i transients. In Aims#2 and #3, we will assess uterine artery relaxation after blocking major vasodilatory pathways and conduct mechanistic studies with/without eNOS activity/multi-site phosphorylation-regulating pathway antagonists via RT-PCR, immunoblotting, histology, fluorescent imaging and spectrophotometry. Simultaneous [Ca2+]i-NO fluorescent imaging will be performed utilizing high-speed excitation/emission wavelength switching system. Our proposal explores a new frontier of gestational alcohol research by developing the first mechanistic framework for binge alcohol-induced uterine artery adaptations and identifying alcohol targets in an in vivo model. In alignment with NIAAA FY14 Strategic Plan, our proposal utilizes powerful methods and presents a new maternal-inclusive paradigm to the FAS field and predicts that a more effective intervention will require innovative pharmacologic targeting of maternal systems, especially the critical uterine circulation, in order to predict and propose a therapy that will have a real promie as a preventive strategy.

Fetal Alcohol Spectrum Disorders (FASD) affects an estimated 2?5% of young school age children in the U.S., with an estimated cost of $1.4 million per individual. Two cardinal outcomes of FASD are fetal growth restriction and neurodevelopmental deficits. Efforts to successfully prevent or ameliorate these teratogenic effects of alcohol abuse have been impeded by a limited understanding of alcohol?s complex mechanisms of action, which impact multiple organ systems. In addition, etiological reports on FASD outcomes have been mostly limited to investigating indirect downstream mediators. We propose the molecular pathway governing phosphatidylethanol (PEth; 100% specific, most sensitive biomarker for gestational alcohol exposure) formation can yield novel insights into FASD etiology, as during alcohol metabolism, phosphatidylcholine is hydrolyzed to PEth instead of phosphatidic acid (PA, an essential nutrient for growth/neuron development). In an established FASD model, our novel preliminary data shows alcohol decreases PA bioavailability and concurrently increases PEth levels in maternal and fetal compartments. Our data also show alcohol-induced impairment of maternal uterine artery (related to fetal growth) and fetal brain vascular (related to neurodevelopmental outcomes) adaptations. Interestingly, PA addition in vitro to the uterine and middle cerebral arteries reverses alcohol-induced dysfunction in these vessels, and in vivo PA administration reverses FASD growth deficit. Our data also identify a role for endothelial nitric oxide (NO) synthase (eNOS) and mTORC1 signaling in this alcohol/PEth/PA framework. In Aim #1, we hypothesize that in our FASD model, PA plays a major role in alcohol-mediated vasodilatory deficits and the related eNOS pathway in maternal uterine and developing cranially directed arteries, and that alcohol impairs the NO system via PA-mediated mTORC1 system alteration. Following mechanistic in vitro blockade of PA, mTORC1, and related signaling, we will assess uterine and developing cranially directed arterial adaptations using arteriography, LC-MS/MS, immunoblotting, immunofluorescence, RNA-seq, RT-PCR, and patch clamp. In Aim#2, we hypothesize PA administration in vivo reverses alcohol-induced decreases in uterine artery and fetal cranially directed blood flow, and improves fetal nutrient delivery, growth phenotypes, and deficits in alcohol-sensitive neurobiological outcomes. We will measure growth indices, uterine blood flow, uterine O2/nutrient delivery, fetal cranially directed blood flow, and neuronal function/morphology to assess the role of PA in the etiology of two cardinal FASD outcomes. We anticipate that the proposed experiments will provide a much-needed breakthrough in the FASD field by identifying a promising etiological molecular pathway(s) for FASD growth and/or neurodevelopmental deficits. These studies will pave way for future novel prevention/treatment studies strategically aimed at rescuing FASD cardinal outcome phenotypes through manipulation of direct alcohol targets.

ELECTRONIC CIGARETTE VAPING & VASCULAR SEQUELAE IN THE UTERUS DURING PREGNANCY Despite serious pregnancy complications, 50% of women who use tobacco products will continue to do so during pregnancy. A recent 2017 study estimates as many women use electronic-cigarettes (e-cigs) as conventional cigarettes during pregnancy. One major reason for this alarming data is that traditional tobacco users view vaping during pregnancy as a ?safer? alternative. However, e-cig vapor reveals a myriad of chemicals which may be harmful to both the mother and the fetus. Using our well-established pregnant rat model, we obtained preliminary data utilizing a state-of-the-art custom-engineered e-cig atomizer that offered a translational inhalation delivery method and generated vapor profiles directly comparable to human vaping. The preliminary data demonstrated e-cig-induced major fetal growth deficit and provides the first evidence for impaired gestational circulatory adaptations including uterine blood flow, the prime regulator of gas and nutrient delivery from mother to fetus. Aim#1 will test if vaping e-cig base (propylene glycol: glycerin (80:20)) alone or with increasing doses of nicotine produce a dose-dependent effect on uterine blood flow (UBF) and fetal growth response. Further, we will test if there are e-cig-induced systemic cardiovascular adaptations during pregnancy. We will assess reproductive vascular and systemic cardiovascular adaptations of e-cig base alone or with increasing doses of nicotine utilizing high frequency ultrasonography, TTE, ECG, Luminex xMAP technology, surgical catheterization for blood pressure, and microsphere-based flow assessment. Aim#2 will test if vaping e-cig will impair uterine artery relaxation via the endothelium-derived NO vs. EDHF vs. PGI2 pathways (the three vasodilators that entirely regulate primary uterine artery blood flow in pregnancy). We will pharmacologically block combinations of endothelial-derived vasodilator pathways using pressure arteriography, and dissect impaired cell signaling utilizing HPLC, histological approaches, immunoblotting, and other molecular tools. Aim#3 will test if re-capitulating e-cig- induced decreased UBF in the absence of e-cig produces fetal growth restriction, and if restoring e-cig- impaired-UBF rescues the growth restriction phenotypes of the e-cig-treated animals. We will phenocopy using RNA interference-mediated suppression of mRNA transcripts of endothelium-dependent vasodilatory mediators, and re-create e-cig induced decreases in UBF in the absence of e-cig vaping. Using lentiviral expressing transgene(s) to restore UBF, we will determine if this can rescue the growth restriction phenotypes of e-cig-treated animals. Our proposal explores a new frontier of gestational research developing the first mechanistic framework for e-cig vaping-induced uterine circulatory adaptations in a model that offers a translational inhalation delivery and vapor profiles comparable to human vaping. The proposed studies on the health effects of e-cigs during pregnancy will facilitate applicable policy implementation on potential risks these devices pose to the public.

ELECTRONIC CIGARETTE VAPING & VASCULAR SEQUELAE IN THE UTERUS DURING PREGNANCY Despite serious pregnancy complications, 50% of women who use tobacco products will continue to do so during pregnancy. A recent 2017 study estimates as many women use electronic-cigarettes (e-cigs) as conventional cigarettes during pregnancy. One major reason for this alarming data is that traditional tobacco users view vaping during pregnancy as a ?safer? alternative. However, e-cig vapor reveals a myriad of chemicals which may be harmful to both the mother and the fetus. Using our well-established pregnant rat model, we obtained preliminary data utilizing a state-of-the-art custom-engineered e-cig atomizer that offered a translational inhalation delivery method and generated vapor profiles directly comparable to human vaping. The preliminary data demonstrated e-cig-induced major fetal growth deficit and provides the first evidence for impaired gestational circulatory adaptations including uterine blood flow, the prime regulator of gas and nutrient delivery from mother to fetus. Aim#1 will test if vaping e-cig base (varying ratios of propylene glycol: glycerol) alone or with increasing doses of nicotine produce a dose- dependent effect on uterine blood flow (UBF) and fetal growth response. Further, we will test if there are e-cig- induced systemic cardiovascular adaptations during pregnancy. We will assess maternal reproductive vascular, and e-cig-induced fetal cardiovascular adaptations, accompanied by effects on the pulmonary system utilizing high frequency ultrasonography, flexiVent, TTE, ECG, Luminex xMAP technology, and surgical catheterization for blood pressure and microsphere-based flow assessment. Aim#2 will test if vaping e-cig will impair uterine artery relaxation via the endothelium-derived NO vs. EDHF vs. PGI2 pathways (the three vasodilators that entirely regulate primary uterine artery blood flow in pregnancy). We will pharmacologically block combinations of endothelial-derived vasodilator pathways using pressure arteriography, and dissect impaired cell signaling utilizing HPLC, histological approaches, immunoblotting, and other molecular tools. Our proposal explores a new frontier of gestational research developing the first mechanistic framework for e-cig vaping-induced uterine circulatory adaptations in a model that offers a translational inhalation delivery and vapor profiles comparable to human vaping. The proposed studies on the health effects of e-cigs during pregnancy will facilitate applicable policy implementation on potential risks these devices pose to the public. Proposed studies directly address a key consensus recommendation of the most recent NIH/NHLBI workshop on cardiovascular disease and the emergence of e-cigs, i.e. investigating effects of e-cigarette aerosol exposure in pregnant women.