Richard A Dixon - US grants

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (04) Clinical Research and specific Challenge Topic, 04-HL-110: Treatment of Pulmonary Hypertension and Right Heart Failure. PROJECT SUMMARY/ABSTRACT Pulmonary arterial hypertension (PAH) is an incurable, devastating disease with a high mortality rate. Characterized by progressive increases in pulmonary arterial pressure and pulmonary vascular resistance, PAH ultimately leads to right heart failure and death. Current therapies improve hemodynamic measures but have significant drawbacks, including variable patient responses and continued deterioration in patient status. Because vasoconstriction of the pulmonary vessels characterizes the pathology of PAH, intravenous therapy with the potent vasodilator prostacyclin is often used in patients with PAH. However, this therapy is expensive and tethers the patient to a cumbersome delivery system comprising a continuous-infusion pump with an indwelling catheter. Developing a cell-based therapy that replenishes prostacyclin levels would overcome existing flaws in the treatment of patients with PAH and help stimulate development of this novel technology for other cardiovascular diseases. Our broad, long-term challenge is to develop a cell-based therapy using prostacyclin-secreting transfected cells to treat patients with PAH. Our immediate objectives for this proposal are to develop genetically engineered cells that stably overexpress prostacyclin (Specific Aim 1) and study their therapeutic potential in animal models (Specific Aim 2). For Specific Aim 1, we plan to create and characterize rat and human transgenic cell lines overexpressing a recently developed novel fusion protein (COX-1-10aa-PGIS) that continuously produces and releases prostacyclin. For Specific Aim 2, we will study the efficacy of the genetically engineered rat and human cell lines in treating PAH in animal models. We will use the well-established rat model of monocrotaline- induced PAH to examine the effects of COX-1-10aa-PGIS-overexpressing cells on survival and right ventricular systolic pressure and the right ventricular and left ventricular weight ratio, which are the most significant measures of PAH. In addition to efficacy, we will examine cell trafficking in rats by studying the tissue localization of fluorescently labeled prostacyclin-overexpressing transfected cells. Once we establish the efficacy of our cell lines in rats, we will study them in a large animal model that more closely resembles the human anatomy. In the pig model of shunt- induced PAH, we will similarly examine the in vivo distribution of fluorescently labeled transfected cells and monitor pulmonary pressures (via right heart catheterization) as an indicator of efficacy in treating PAH. We believe our work will provide the preclinical foundation to move this novel cell-based therapy for PAH forward into clinical trials. PUBLIC HEALTH RELEVANCE: Pulmonary arterial hypertension is a rare but fatal disease, and current therapies are not curative and are associated with significant drawbacks. As part of a cell-based therapy approach, the objective of the current proposal is to develop and test in animal models genetically engineered rat and human cells that stably overexpress prostacyclin, a potent vasodilator that is reduced in patients with pulmonary arterial hypertension. These studies should provide sufficient preclinical data to allow this innovative cell therapy to move forward into clinical trials and help usher in a new era in the treatment of this devastating disease.

Lignin is one of earth's most abundant natural polymers, a key component of woody plant material, and a major waste product in bio-refineries that process plant material for production of liquid biofuels. The lead investigator and collaborators have discovered a new type of lignin, apparently restricted to the seed coats of a range of non-crop species. This new type of lignin has significant promise for conversion to carbon fibers. If this type of lignin could be engineered into bioenergy feedstocks, it would represent a high value co-product of biorefining. This project will determine the biochemical pathway by which this new type of lignin is synthesized, and the steps that will need to be taken to make it accumulate in the stems and leaves of plants. Understanding the limitations to the accumulation of different types of lignin in different plant tissue and cell types also addresses a fundamental problem in cell biology. This project uses a range of approaches from genetic mechanisms through metabolic engineering to production and testing of biomaterials. Outreach activities will target two demographically distinct groups; highly talented high school students at the Texas Academy of Mathematics and Science, and mathematics and science public school teachers with primarily Hispanic students who are Limited English Proficient (LEP). Teachers will develop curricular activities in Spanish and English to implement in their classrooms. The project will include informal learning activities through deployment of the projects to encourage K-12 girls into STEM through outreach with the Dallas Society of Women Engineers.

During lignin biosynthesis, the monolignol precursors are functionalized by aromatic hydroxylation and O-methylation to generate, successively, hydroxyphenyl (H), catechyl (C), guaiacyl (G), 5-hydroxyguaiacyl (5-OH-G), and syringyl (S) units. Natural lignins in angiosperms consist of approximately equal amounts of G and S units, with less than 2% of H units and no C or 5-OH-G units; gymnosperm lignins are similar but lack S units. 5-OH-G units are incorporated into lignins in transgenic plants in which the second methylation step is blocked, but C-units have not been reported to accumulate in the lignin of angiosperms in which the first methylation step is similarly blocked. The Principal Investigators have shown that the seed coats of a wide range of dicot and monocot plants contain a novel type of lignin (C-lignin) derived totally from catechyl units. This linear polymer has advantageous properties for conversion to carbon fibers. However, C-lignin appears to be restricted to seed coats in nature. This award will address two major questions: what is the mechanism underlying the biosynthesis of C-lignin in plant seed coats, and why does C-lignin not occur naturally in the vegetative tissues of the plant? The researchers hypothesize that C-lignin biosynthesis involves loss of function of one or both methylation steps in classical monolignol biosynthesis, and will determine how this happens using transcriptome profiling in developing seeds of Cleome hassleriana, in which there is an abrupt transition from G lignin to C lignin biosynthesis at around 13 days post-pollination. Addressing the second question will involve attempting to introduce the C-lignin polymer into different tissues of the model legume Medicago truncatula, utilizing existing mutants in which enzymes of lignin methylation have been functionally disrupted. These studies aim to determine the potential for engineering accumulation of C-lignin in vegetative tissues in sufficient yields to enable industrial processing, and, in this context, improved methods for extraction of C-lignin from both seeds and vegetative tissues will be developed, and the physical properties of the polymer further analyzed.

? DESCRIPTION (provided by applicant): We recently identified, in preclinical studies, specific polyphenol-rich botanical dietary supplements [e.g., grape seed polyphenol extract (GSPE), Concord grape juice (CGJ), and resveratrol (RSV)] that are effective in promoting cognitive and psychological resilience under stress conditions. The overall goal of the proposed Botanical Dietary Supplement Research Center (BDSRC) is to identify the specific polyphenol components from these bioactive botanical supplements that underlie their bioactivities and to characterize specific cellular/molecular mechanisms contributing to the attenuation of physiological stress, such as that associated to stressful life events, that have detrimental impact on psychological health, cognitive functions, and ultimately wellbeing (Projects 1-2). In addition the proposed studies will, for the first time, clarify the role of the gastrointestinal (G) microbiome, particularly the human GI microbiome, in modulating the bioavailability of polyphenol components responsible for the cognitive and psychological health benefits of dietary polyphenol botanical supplements using humanized gnotobiotic mice (Project 3). Thus, the proposed studies, which (1) characterize botanicals, (2) assess, pre-clinically, their bioavailability and bioactivity in the periphery and in the brain, while defining molecular mechanisms of resilience, and (3) identify safety issues essential for future development into clinical application, are in full alignment with the goals of NCCAM and ODS, as indicated in the Research Objectives of the RFA. The proposed studies in this BDSRC will improve scientific knowledge and technical capabilities through the development of a rigorous scientific program, will provide training and career development opportunities, and, critically, will provide an intellectually rigorous examination of the role of dietary polyphenol botanical supplements in the maintenance and promotion of psychological and cognitive resilience.

Summary/Abstract The purpose of Research Core B is to support the overall effort of the ODS/NCCAM Botanical Dietary Supplement Research Center in two specific areas: (1) by centralizing the procurement, characterization, archiving, quality control and shelf-life/stability assessment of polyphenolic materials used by individual Center projects; and (2) by centralizing analytical and biosynthesis support for all center projects and cores. Our previous studies have demonstrated the remarkable efficacy of dietary supplementation from a combination of grape seed polyphenolic extract (GSPE), Concord grape juice (CGJ), and resveratrol (RSV) in promoting preservation of cognitive wellness and psychological resiliency under select stress conditions. We have also shown that a number of metabolites arising from the above botanicals can be detected in blood and brain tissues including anthocyanins and methylated and/or glucuronidated derivatives of the various classes of flavonoids (flavan-3-ols such as catechin and epicatecin; flavonols (quercetin); stilbenes (resveratrol)). A number of these compounds are not commercially available, and will be generated by a combination of synthetic and biosynthetic schemes in quantities sufficient for mechanism of action studies in Projects 1 and 2, and as standards for studies in Projects 1-3. Other compounds will be obtained commercially and their purity validated. The aim of the Biosynthetic Component of Core B is therefore the provision of authenticated and chemically characterized samples of unlabeled or, where requested, radiolabeled, brain-bioavailable phenolic metabolites for mechanism of action testing. The Bioanalytical Component of Core B will be responsible for the quantitation of the original and derived metabolites in animal tissues, using a suite of analytical techniques including liquid chromatography coupled to UV detection and mass spectrometry, 1D nuclear magenetic resonance (NMR) techniques including 1H NMR and 13C NMR, and various 2D NMR techniques including 1H- 1H TOCSY (total correlated spectroscopy), 1H-1H NOESY (nuclear Overhauser enhancement spectroscopy), gradient enhanced 1H-1H COSY (correlated spectroscopy), 1H-13C HSQC (heteronuclear single quantum coherence), and 1H-13C HMBC (heteronuclear multiple bond coherence). In addition to these bioavailability studies, Core B will serve as an archived repository of all polyphenolic compounds used throughout the Center, and perform quality control analyses and stability testing in support of this function. The complementary nature of individual investigators within Core B creates a strong synergistic team to support individual projects and collaborative efforts focused on achieving center objectives.