Samuel M. Poloyac - US grants

DESCRIPTION (provided by applicant): Decreasing tissue infarction in the ischemic penumbra is the primary therapeutic target after stroke. The degree of tissue damage in the ischemic penumbra is a function of the severity of the ischemic insult, which is a function of cerebral microvascular resistance. Recent studies have shown that the monohydroxylated metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), is an important regulator of cerebrovascular resistance and mediates the vascular effects of nitric oxide. Our laboratory has recently shown that inhibiting 20-HETE formation dramatically reduces lesion volume after temporary focal ischemia. Based on this data, we hypothesize that 20-HETE is an important contributor to ischemic damage via its vasoconstrictive effects on the cerebral microvasculature. The goals of this project are: 1) To determine the temporal relationship between 20-HETE formation, lesion volume, and outcome in the rat temporary focal ischemia model;2) To identify the in vivo effect of 20-HETE alterations on cerebral blood flow after temporary focal ischemia;3) To determine the role of 20-HETE on nitric oxide mediated protective effects after temporary focal ischemia. This project will utilize combinations of in vivo blood flow assessment, molecular biology, and analytical chemistry techniques in the rat and transgenic mouse models of temporary focal ischemia. The results of this research are necessary to determine: 1) The role of 20-HETE in the pathogenesis of stroke;2) The effects of 20-HETE on cerebrovascular regulation during ischemia and during post-ischemic hypoperfusion after stroke;3) The role of 20-HETE inhibition in mediating the neuroprotective effects of nitric oxide. Elucidation of these mechanisms will enable future studies to delineate the therapeutic utility of 20-HETE inhibitors as a mechanism to reduce tissue damage after stroke. Furthermore, these studies will provide important information about the role of monohydroxylated arachidonic acid metabolites as a contributing pathway in the pathogenesis of stroke.

DESCRIPTION (provided by applicant): Clinical use of systemic therapeutic hypothermia is being instituted nationally as standard of care for patients suffering a cardiac arrest (CA). Recent clinical trials have demonstrated the safety and neuroprotective benefits, thereby, prompting the American Heart Association to designate systemic hypothermia as a level one therapy in CA patients. In addition, hypothermia provides neuroprotective benefits after traumatic brain injury (TBI). Patients who suffer a CA or TBI receive a wide array of drugs that depend on hepatic metabolism by the cytochrome P450 (CYP450) enzyme system. In fact, several studies have demonstrated that hypothermia increases the plasma concentrations of hepatically eliminated drugs. Despite the increased clinical implementation and the potential for therapy-drug interaction, a paucity of data exists concerning the effects of therapeutic hypothermia on CYP450-mediated drug clearance. The hypotheses of this proposal are that: 1) Mild and moderate hypothermia differentially affect the in vitro functional activity and regulation of the 7 major CYP450 isoforms in rat CA and TBI models. 2) Mild to moderate hypothermia produce alterations in hepatic blood flow, intrinsic enzyme clearance, and plasma protein binding in rat CA and TBI models. 3) Mild and moderate hypothermia alters the disposition of therapeutic agents in vivo in rat CA and TBI models, thereby, affecting the full neuroprotective benefits of therapeutic hypothermia. This project will utilize combinations of in vivo pharmacokinetic assessments, in vitro functional regulation analyses, and analytical chemistry techniques in models of CA and TBI under normothermic (37 degrees), mild hypothermic (33 degrees), and moderately hypothermic (30 degrees) conditions. In addition, this research will lend insight into the cellular mechanisms that produce both acute and chronic changes in CYP450 function. The results of this research are necessary to determine: 1) The implications of hypothermic therapy after traumatic insult on the disposition of drugs that depend on hepatic elimination;2) The in vivo mechanism(s) by which hypothermia and trauma alter hepatic drug elimination, and;3) The effect of hypothermia and trauma on the cellular mechanisms and functional regulation of CYP450 isoform expression. Once completed, this research will provide critical information to design future clinical studies to optimize the benefits of this breakthrough therapy.

[unreadable] DESCRIPTION (provided by applicant): Quantitative measurements of small molecules are essential to pharmaceutical sciences research. Discovery of the mechanisms of disease pathogenesis for development of new therapeutic strategies requires the assessment of post-translational modifications of peptides and nucleic acids and quantitative measurements of small molecules in complex biological matrices. The most specific and sensitive method of quantification of these targets is through triple quadrupole mass spectrometry. There are currently no triple quadrupole mass spectrometers available for use by the fourteen PHS-funded University of Pittsburgh investigators listed as the user group in this proposal. In fact, there are only four such systems at the University, of which only one is less that seven years old and all are very heavily utilized and thus not available to meet the needs of our group. The lack of such instrumentation is a particular problem for investigators conducting clinical studies due to the large number of samples that accompanies such work. The specific aim of this proposal is to request funds from the National Center for Research Resources to acquire a Dionex UltiMate 3000 HPLC with an API 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer to meet the critical needs of our user group for such an instrument. This equipment will reside in our Proteomics Core Laboratory where resources available for administering use of the instrument include: 1) system management, maintenance, and cost recovery; and 3) skilled personnel with substantial experience with mass spectrometric methods. Availability of such instrumentation would allow our PHS-funded user group to pursue innovative research that currently is severely limited due to lack of such state-of-the-art instrumentation. II. Relevance: Our user group comprises elements of both basic and clinical research whose work is aimed at improving human health through discovery in such areas as HIV/AIDS, stroke, traumatic brain injury, cardiac arrest, mental health disorders, cancer, pulmonary hypertension, and transplantation. The acquisition of this hybrid triple quadrupole/linear ion trap mass spectrometer would provide essential instrumentation for this research and thereby remove impediments to research progress. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a competitive renewal of our two prior studies: Methods of Predicting DCI in aSAH (R01- NR004339-01), and Role of 20-HETE on vasospasm-induced ischemia after aSAH (R01NR004339-05). It is the long-term goal of this research to improve patient outcomes by utilizing biomarker-directed early- intervention strategies to reduce ischemic complications after aSAH. In the previous support period, our team identified key vasoactive metabolites, ET-1 and 20-HETE, which were predictive of cerebrovascular complications and long term outcomes. As the logical extension of this work, we propose to examine the key gene variants that regulate the production, degradation, and ion channel effects of these metabolites as they relate to DCI and neuropsychological outcomes. We hypothesize that variants in biomarker pathway genes will significantly contribute to the variability in biomarker concentrations, complications, and outcomes after aSAH. The specific aims of this proposal are to determine the relationship between elevated cerebrospinal fluid (CSF) biomarkers (ET-1, 20-HETE, and EETs) and specific gene variants that lead to variation in their production and/or degradation; to examine the direct and mediated effects of gene variants in ET-1, 20-HETE, and EET pathways through CSF biomarkers on DCI, and to examine the direct and mediated effects of gene variants in ET-1, 20-HETE, and EET pathways through CSF biomarkers on functional and neuropsychological (NP) outcomes. We will recruit 192 patients with SAH (ages 18-75 years) in order to achieve a final sample of 145 patients available at twelve month follow up assessment. These subjects coupled with the 372 patients recruited to date will allow for the identification of the key genetic variants and the mediating effects of biomarker concentrations on DCI and NP outcomes. Elucidation of these relationships will allow for future development and evaluation of predictive models for identification of patients at high risk for cerebrovascular complications and poor outcomes after aSAH, thereby, allowing for directed intervention strategies to improve outcomes in this highly heterogeneous, understudied patient population. PUBLIC HEALTH RELEVANCE: Data from the proposed study, currently unexplored in persons with aneurysmal subarachnoid hemorrhage, will impact public health by serving as the basis for both therapeutic interventions designed to decrease the cascade of events that ultimately result in delayed cerebral ischemia and interventions to help patients compensate for long term deficiencies in physical and neuropsychological function.

PROJECT SUMMARY Quantitative measurements of small molecules are essential to pharmaceutical sciences research. Discovery of the mechanisms of disease pathogenesis for development of new therapeutic strategies requires the quantitative measurements of small molecules in complex biological matrices. The most specific and sensitive method of quantification of these targets is through triple quadrupole mass spectrometry system. Thanks to funds from a share instrument grant (S10RR023461), we purchased a Waters Acquity UPLC with a Thermo triple-stage quadrupole (TSQ) Quantum Ultra mass spectrometry and established our Small Molecular Biomarker Core (SMBC) in 2009. Since then our core has developed sensitive and robust quantitation methods for multiple panels in the analysis of biomarkers and endogenous products, drugs and metabolites, and new drug discovery. The highly sensitive and specific techniques employed have allowed preclinical and clinical researchers to accurately measure multiple small molecule drug and biomarker concentrations in the same sample, maximizing the ability to link basic science with clinical outcomes. We have supported over 40 principal investigators with their ongoing NIH-funded research projects, as well as provided preliminary data to support NIH proposals subsequently selected for funding. SMBC has serviced at least 15 NIH funded grants in the last five years. Our core service has been instrumental to support the funded research, securing new funding, and peer-reviewed publication of research results. Despite its essential role in supporting human health related research, the SMBC core cannot continue to fulfill its mission without replacing the aging instrumentation. The specific aim of this proposal is to request funds from NIH to acquire an integrated system including a Vanquish H UHPLC with a TSQ Altis mass spectrometer from Thermo to replace our 12-year old instrumentation, which will soon cease to be supported by the manufacturers. The new LC-MS/MS system will reside in our SMBC core where resources are available for administering use of the instrument, which include: 1) track record of collaborative research with investigators from all of the health sciences; 2) a well-developed administrative plan for system management, maintenance, and cost recovery; and 3) skilled personnel with substantial experience with mass spectrometric and liquid chromatographic methods. Availability of such instrumentation would allow our NIH-funded user group to pursue innovative research that would otherwise be severely limited due to lack of such state-of-the-art instrumentation.