Garret A. FitzGerald, M.D. - US grants

pregnancy toxemia /hypertension; fatty acid biosynthesis; prostacyclins; human subject;

Aspirin has been postulated to exert a dose dependent, selective inhibition of thromboxane synthesis. We propose to compare the effects of acute and chronic administration of a wide range of aspirin doses on prostanoid generation and platelet function. In particular, we shall focus on the recovery of prostacyclin biosynthesis following aspirin as our preliminary data indicates prolonged suppression after chronic dosing. Aspirin is rapidly converted to salicylate in vivo and salicylate prevents inhibition of platelet thrombosane synthesis by aspirin in vitro. Thus, we also propose to assess the effects of sodium salicylate on endogenous prostanoid generation and the effects of salicylate consumption on aspirin inhibition of prostanoid generation in man. Novel synthesis of a deuterated derivative of aspirin will permit us to relate the pharmacokinetics of a range of aspirin doses to their pharmacodynamic effects and to characterize the potential interactions of aspirin and dipyridamole, two antiplatelet agents often used in combination in clinical practice. In vitro sulfinpyrazone inhibits platelet cyclooxygenase and platelet aggragation. The influence of sulfinpyrazone upon biosynthesis in man is unknown and in vitro studies suggest that metabolites, rather than the parent compound may mediate its antiplatelet effects in vivo. We propose to relate the pharmacokinetics of sulfinpyrazone and its metabolites during acute and chronic dosing to platelet function, and prostanoid production in man. Selective inhibition of thromboxane synthesis would be theoretically attractive, in that depression of prostacyclin synthesis would not occur as with cyclooxygenase inhibitors such as aspirin. We shall test the functional importance of such selectivity by a controlled comparison of the effects of such an inhibitor, dazoxiben, with high and low dose aspirin on indices of venous graft obstruction in patients with peripheral vascular disease. Such a study will also clarify whether diversion of endoperoxide metabolism towards increased biosynthesis of other prostanoids occurs during therapy with a thrombozane synthase inhibitor. Substantial resource has been devoted to clinical trials of antiplatelet drug with inconclusive results. By clarifying their clinical pharmacology, the proposed studies will permit more rational evaluation of these agents in the future.

Thromboxane (Tx) A2 mediates its effects via a G protein coupled receptor, the TP. It is a major product of cyclooxygenase (COX)-1 in platelets and of COX-2, which is induced in monocytes and vascular smooth muscle cells in human atherosclerosis. Additional to its cognate ligand, isoprostanes (iPs), free radical catalyzed prostaglandin isomeric products of arachidonic acid, may also act as incidental ligands at the TP. The present proposals are designed to extend our studies of TP activation to encompass platelet interactions with the vessel wall. In Specific Aim 1, we shall examine the role of TP activation in modulating atherogenesis and the response to vascular injury. The effect of TP antagonism and TP deletion will be examined in the Apobec-1/LDL receptor double knock out (DKO) mouse model of atherosclerosis. The effect of titrated iP suppression with vitamin E in the presence and absence of the TP will be utilized to evaluate the contributions of iPs to TP activation. Similarly, the impact of antagonism and TP deletion on the response to catheter induced injury will be examined. Mice deficient in the prostacyclin receptor (IP) will be used to determine whether depression of PGI2 formation by periprocedural aspirin might limit the efficacy of TP antagonism in the prevention of restonosis after angioplasty. In Specific Aim 2, we shall determine whether intrauterine growth retardation, which results from placental ischemia in pregnant mice with directed vascular over-expression of the TP, is exacerbated by coincidental IP deficiency or by COX activation and oxidant stress induced by exposure to cigarette smoke. Again, To antagonists and titrated doses of vitamin E will be used to examine the contribution from cognate and incidental ligands to TP activation. Finally, in Specific Aim 3, we shall perform a randomized prospective, double blind controlled trial to determine if inhibition of platelet COX-1, COX-2 or both retard the progression of plaque volume, as assessed by EBCT and B mode ultrasonography in patients with coronary atherosclerosis.

prostaglandin receptor; eicosanoid metabolism; blood vessels; prostacyclins; platelets; receptor sensitivity; cyclic AMP; receptor binding; chimeric proteins; phosphorylation; G protein; gene targeting; laboratory mouse; electrospray ionization mass spectrometry; immunologic assay /test; confocal scanning microscopy;

It is hypothesized that the mean reduction baseline in net urinary sodium excretion during the first 72 hours of treatment with oral L-748,731 (50 mg q.d.) will be similar to the mean reduction during the first 72 hours of treatment with oral indomethacin (50 mg t.i.d.) in subjects 60 to 80 years of age consuming a 200 mEq sodium diet (i.e., the difference in mean reduction will be within +/- 90 mEq). Administration of oral L-748,731 mg daily for 2 weeks to subjects 60 to 80 years of age will be sufficiently safe and well tolerated, based on assessment of clinical and laboratory adverse experiences, to permit continued clinical investigation of this drug. The specific aims are: 1) To compare the effects of treatment with oral L-748,731 50 mg q.d., indomethacin 50 mg t.i.d., or placebo on urinary sodium excretion over the initial 72 hours of treatment in subjects 60 to 80 years of age consuming a 200 mEq sodium diet; 2) To compare the safety and tolerability of 2 weeks of treatment with oral L-748,731, 50 mg q.d., oral indomethacin, 50 mg t.i.d., or placebo in subjects 60 to 80 years of age consuming a 200 mEq sodium diet; 3) To compare the effects of treatment with oral L-748,731, indomethacin, or placebo on change in body weight as an index of sodium retention; 4) To compare the effects of treatment with oral L-748,731 indomethacin, or placebo on net urinary sodium excretion over the first week and over the total of 2 weeks of treatment; 5) To compare the effects of 2 weeks of treatment with oral L-748,731, indomethacin, or placebo on iohexol and creatinine clearances; 6) To compare the effects of treatment with oral L-748,731, indomethacin, or placebo over a period of two weeks on blood pressure; 7) To compare the effects of 2 weeks of treatment with oral L-748,731, indometacin, or placebo on other parameters of renal function/prostaglandin metabolism, including urinary potassium excretion, urinary eicosanoids, serum TXB2, and urinary N-acetyl-beta-glucosaminidase (NAG).

SC-58635 is a novel compound that selectively inhibits the inducible form of the enzyme cyclooxygenase (prostaglandin G/H synthase). SC-58635 is being developed by G.D. Searle & Co. as an oral antiinflammatory agent for the treatment of rheumatoid and osteoarthristis, and for the relief of pain of musculoskeletal and soft-tissue origin. The present study, partly supported by G.D.Searle & Co., is a double- blind, randomized, comparator and placebo controlled, parallel group, single dose study. A group of 35 healthy subjects who are 18-55 years of age will receive a single dose of either 100, 400 or 800 mg SC-58635, ibuprofen 800 mg or placebo. The primary objectives of this study are to: 1. determine the effect of SC-58635 on platelet aggregation to collagen, arachidonate and U46619 (Tx agonist) in healthy subjects; and 2. correlate the effects on collagen-, arachidonate and U46619-induced platelet aggregation with plasma concentrations of SC-58635.

This study is being conducted in patients to determine if carvedilol has properties similar to those of an antioxidant. Antioxidants consist of drugs and vitamins that prevent or correct certain chemical reactions from occuring in the body. Such reactions may be harmful so that their prevention by an antioxidant may be beneficial. The objectives of this study is to explore the antioxidant effects of carvedilol following single and repeat oral dosing at two dose levels as compared to dosing with an antioxidant control (Vitamins C and E) and a hypotensive control (hydrochlorothiazide) in hypertensive, chronic smokers.

The clinical benefits and adverse effects of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) derive from inhibition of the enzyme cyclooxygenase (Cox).Two isoforms of Cox are recognized: Cox-1, which is constitutively expressed in platelets, in the gastric mucosa and in most tissues where it is thought to exert "housekeeping" functions, and Cox-2 which is commonly termed "inducible", because it is transiently expressed in response to inflammatory mediators, tumor promoters and growth factors. Conventional NSAIDs inhibit both Cox-1 and Cox-2 with limited selectivity. It is generally assumed that their anti-inflammatory and analgesic activity is mediated via Cox-2 inhibition. Inhibition of Cox-1, by contrast, is thought responsible for the gastric toxicity and bleeding complications associated with NSAID treatment. It is unclear whether NSAID-induced renal toxicity is attributable to inhibition of Cox-1 or Cox-2. The intrarenal distribution and regulation of Cox-2 by sodium intake in rats, suggest a potential role for this enzyme in salt and water balance. Additionally, inactivation of Cox-2 by gene targeting in mice results in severe developmental nephropathy. Inhibitors of Cox-2 have been developed with the rationale that these compounds would exert anti- inflammatory activity without the adverse gastric effects and increased bleeding risk associated with non-selective NSAID treatment. The present study will be undertaken to assess the effects of L748,731, a selective Cox-2 inhibitor, during chronic administration to elderly subjects, the population most susceptible to NSAID-induced nephrotoxicity. Healthy older adults (59 to 80 years of age; n=36) will be admitted to the Clinical Research Center, placed on a constant diet (200 mEq sodium daily) and randomized under double blind conditions to receive the selective Cox-2 inhibitor, L748,731, 50 mg qd, a non-selective Cox-1/Cox-2 inhibitor, indomethacin 50 mg tid, or placebo for two weeks. Sodium excretion and other indices of renal function will be assessed under conditions of controlled sodium intake. It is hypothesized that selective inhibition of Cox-2 would fail to reduce urinary 11-dehydro TXB2 (TX-M) and serum TXB2, indices of Cox-1 dependent thromboxane biosynthesis by platelets. It is also postulated that Cox-2 inhibition would not affect the urinary excretion of 2,3 dinor 6-keto PGF1a (PGI- M), the major metabolite of prostacyclin in vivo.

The glycoprotein (Gp) IIb/IIIa (alpha-Iib beta-3) is expressed on the platelet surface where it exists in an equilibrium between a resting state, unable to bind adhesive proteins and an active (ligand binding- competent) state. Following platelet activation, the Gp IIB/IIIaa complex undergoes a conformational change (inside-out signaling), leading to binding of divalent fibrinogen with consequent formation of fibrinogen bridges between platelets and, ultimately, to platelet aggregation and thrombus formation. Fibrinogen binding to activated platelets is the final common step in platelet aggregation, regardless of the initiating event. In the last ten years several compounds, monoclonal antibodies and synthetic peptidic and nonpeptidic molecules, have been designed to antagonize the binding of fibrinogen to the platelet complex. Several clinical trials have investigated the efficacy and safety of these compounds in various clinical conditions. Orbofiban is an orally active antagonist of the IIb/IIIa complex. The present study will evaluate the safety and pharmacodynamic response of 4 doses (30 mg bid, 40 mg bid, 50 mg bid and 50 mg qd) of Orfbofiban plus aspirin compared to placebo plus aspirin in patients with recent (<96 hours) acute coronary artery syndromes. Orbofin ban will be given for 30 days. Platelet aggregation and plasma levels of orbofiban will be assessed on day 2, 14 and 28. A total of 150 patients will be enrolled in 15 centers.

gas chromatography mass spectrometry; laboratory mouse; low density lipoprotein

To determine the dose-response relationship for vitamins E and C in healthy volunteers. To determine whether the combination of vitamins E and C augment their effect on urinary 8-epi PGF. To determine the effect of taking the optimant antioxidant dose of vitamins E and C on free radical generation in smokers and nonsmokers. To determine whether reduction in cholesterol in mild to moderately hyperlipidemic patients reduces urinary 8-epi PGF excretion. To determine whether the correction of Hhcy in individuals with the MTHFR mutation with folic acid reduced urinary 8-epi PGF. To determine whether pretreatment with antioxidant vitamins reduced 8-epi PGF in atherosclerotic plaque as well as its excretion in urine.

Additional to its effects on vascular tone and platelet function, prostacyclin (PGI/2) is an important inflammatory mediator which modulates adhesive interactions and proliferative responses in vascular cells. Laminar, but not turbulent shear up-regulates cyclooxygenase (Cox)-2 in endothelial cells (ECs) in vitro. This project addresses the hypothesis that a deficiency of COX-2 dependent PGI/2 formation is of functional relevance to the relevance to the focal nature of atherogenesis. It relates to other projects in the program which examine the interaction of mechanical and humoral signaling pathways (Projects 1-4) and to the examination of the cell to cell heterogeneity in these interactions as outlined in Project 5. We shall utilize the LDLR edit mouse deficient in both the LDL receptor and the ability to edit apoB, as our preliminary model of atherosclerosis. Lesions are developed on a chow diet, are more severe than in traditional models and evolve in a distribution which resembles human atherogenesis. Additionally, their elevated cholesterol resides predominantly in LDL. In Specific Aim 1, we shall utilize both specific COX-2 inhibitors and the COX-2 knock out (KO) mouse to determine the functional importance of products of products of this enzyme in the extent of atherosclerosis, the response to vascular injury, platelet function and thrombosis and aspects of cardiovascular function in atherosclerotic mice. In Specific Aim 2, we shall utilize a similar strategy of comparative analysis of selective inhibitors and gene deletion to address the role of COX-1, the isoform which is usually expressed constitutively in the vasculature. In Specific Aim 3, inhibitors are not available to address the role of PGI/2. However, we have generated receptor (IP) KO to asses the influence of the major COX- 2 product in vascular cells. If the effects of IP deletion differ markedly from our COX studies, we shall explore the relevance of other COX products, such as E prostaglandins, particularly if supported by results of PG profiling analysis, using mass spectrometry. In Specific Aim 4, we shall utilize structurally distinct COX-2 inhibitors to assess the role of this isozyme in the disordered platelet and endothelial function in patients with peripheral vascular disease. Comparative experiments with a platelet selective form of aspirin and a thromboxane receptor antagonist will allow us to investigate the role of TxA2. Integration of these studies of murine and human cardiovascular function will allow us determine if a localized deficiency in shear induced COX-2 production of PGI/2 is of functional relevance to atherosclerosis. As selective COX-2 inhibitors depress biosynthesis of PGI2 without concomitant inhibition of platelet function, this is an issue of clinical importance.

The focal development of atherosclerosis involves the complex interplay of mechanical forces & chemical mediators impinging on endothelial (ECs) & smooth muscle cells (SMC) & their interactions with extracellular matrix (ECM). This program is designed to explore how these factors interact, playing specific attention to two issues; the modulation of signalling responses to humoral mediators by mechanical forces & the cell to regional heterogeneity of such interactions in the vasculature. Several humoral factors influence the adhesive interactions between endothelial & circulating cells, so characteristic of the early stages of activated receptors; PARs) G protein coupled receptors; tyrosine kinase receptors (Eph kinases) & integrins (alphaIIbbeta3 & alphaVbeta3), platelets with ECs, & cholesterol export, properties that may limit atherogenesis. Cyclooxygenase (COX)-2 is a major source of these observations will be addressed in Proj. 1, using selective inhibitors of COX-2 in humans, in conventional & novel mouse models of atherosclerosis & in genetic crosses of these models with mice lacking COX-2 or the PGI2 receptor. We shall investigate specifically how mechanical forces modulate expression of the biosynthetic & signaling pathways of PGI2 in vascular cells. Thrombin, by contrast, enhances adhesive interactions & stimulates cellular proliferation. It is a recognized ligand for some PARs but little is known of the diversity of PARs & their distinct functions in vascular cells. In Proj. 2, the mechanisms of activation, processing & regulation of PARs & the functional importance, distribution & signaling pathways activated by the noel Eph kinases will be investigated. The structural motifs which discriminate affinity for osteopontin amongst beta3 integrins will be examined in Proj. 3. We shall also determine the structural basis for selection by this adhesive ligand amongst its several receptors on platelets, examine the role of mechanical shear on the interaction, & examine the interactions or other signaling pathways outlined above (PGI2, thrombin) on regulating the activation of beta3 integrins. Since the ECM is likely to play a major role in conditioning the response of VSMCs to humoral and mechanical factors, Proj. 4 will explore the role of distinct ECM proteins interacting with mechanical forces on these interactions will also be examine din this project. Finally, Proj. 5 will address the hypothesis that differences in surface geometry amongst apparently similar endothelial cells results in differences in regulated expression of elements of the signaling pathways explored in Projects 1- 4 in response to mechanical forces. These observations will be integrated with comparisons of data derived from regions of flow separation, both in vitro & ex vivo, using mouse models & human atherosclerotic tissue. This program will take an integrated approach to the study of humoral and mechanical signaling in vascular cells. Factors which underlie the cell to cell heterogeneity of this interaction are likely to contribute to the focal nature of atherogenesis.

The purpose of this study is to examine the effects of the administration of a new compound called Celecoxib (SC 58,635) on the body "flu" like response and prostaglandin formation that follows endotoxin injection. We plan to measure these compounds at regular time intervals, in urine and blood, before and after endotoxin infection. We also wish to define the analgesic properties of this medication.

A Planning Grant for the MCRC will allow us to harness the local resources of the University of Pennsylvania which are directly relevant to the assembly of a competitive proposal. The objectives of the planning phase will be to coordinate the individuals and the structures of relevance within a unified body; to institute a seminar series which integrates both Health Services Rearch, Patient Oriented Research and Clinical Epidemiological aspects of musculoskeletal, joint and skin diseases; to constitute and develop a bioanalytical and statistical core; to harness the resources of the health system and the GCRC to facilitate performance and integration of the elements of the clinical research enterprise in this area; to initiate the research projects relevant to the proposal; to visit prior units funded through this mechanism; to constitute and achieve input from both internal and external review boards and finally to hold an offsite retreat to assess the results of the planning process prior to submission of a full MCRC proposal. The P.I. leads three entities within the university, the Department of Pharmacology, the Center for Experimental Therapeutics and the GCRC, which cover the spectrum from very basic through translational to applied clinical research. Furthermore, he has a track record of cooperative grant applications with both Dr. Brian Strom of Clinical Epidemiology and Dr. Sanford Schwartz of Health Services Research. During the planning phase the resources will be assembled to allow Dr. Schwartz assume the leadership of the proposal proper with the assistance of Drs. Albert and Catella-Lawson. Additional to the GCRC, the Center for Epidemiology and Biostatistics and the Leonard Davis Institute other elements within the institution of direct relevance to the core activities of this grant are the Department of Epidemiology and Biostatistics, the Center for Experimental Therapeutics, the Institute for Gene Therapy, the Institute of Aging and the Center for Bioethics. The core departments and divisions - Dermatology, Orthopedic Surgery and the Division of Rheumatology in the Department of Medicine all have strong track records in basic and clinical research. Members of these entities are fully integrated into the planning phase of this application. The University of Pennsylvania is well suited towards the development of an MCRC which will integrate both Health Services Research, Clinical Epidemiology and Patient Oriented Research.

This is a phase II research study will evaluate the effects of RWJ-57504, a new compound under investigation for the treatment of pain and arthritis. RWJ-57504 is similar to other marketed drugs like Naprosyn or Voltaren. Like these other drugs, it works by blocking an enzyme called cyclooxygenase (COX)-2 which can cause pain and swellings. RWJ-57504 may cause less stomach irritation than Naprosyn or Voltaren because, in contrast to these other marketed drugs, it is not expected to block another enzyme called COX-1 which protects the stomach lining. This study will evaluate the effect of RWJ-57504 on COX-1 versus COX-2 by measuring the blood and urinary levels of substances that result from COX-1 and COX-2 activity.

The is a Phase II research study will evaluate the effects of aspirin and clopidogrel, two FDA approved blood thinners that prevent platelet clumping (blood cells sticking together) in the blood and therefore prevent the formation of blood clots in the arteries. Both drugs are prescribed to patients like you to help prevent cardiovascular disease (heart attacks or strokes). This study will establish which is the best dosage of clopidogrel on the first day of treatment and whether the combination of the two drugs is better than each drug alone.

nonsteroidal antiinflammatory agent; aspirin; acetaminophen; ibuprofen; drug interactions; comorbidity; lactones; pharmacokinetics; blood coagulation; thromboxanes; coronary disorder; human therapy evaluation; osteoarthritis; clinical research; human subject;

DESCRIPTION (provided by the applicant): This SCOR will explore the role of the oxidative modification of diverse targets in the atherogenic process. Although oxidant injury has been implicated in pathogenesis, the process is poorly characterized in integrated systems, such as humans. Recent developments offer novel opportunities to integrate assessment of free radical based modification of protein, lipids and DNA in vivo. A central theme of this proposal is the opportunity to assess the effects of potential prooxidants and antioxidants on these novel biomarkers. A second broad feature of this proposal is the close integration of genomic (A) and proteomic (B) analysis. In Project 1, we shall address the hypothesis that nicotine sustains the endothelial dysfunction observed in chronic smokers and accelerates atherogenesis in the Apobec-l/LDL receptor double knock out mouse (DKO) by acting as a prooxidant integrating biomarkers developed in Projects 2, 3 and 4 and utilizing Cores A and B to relate alterations in gene and protein expression in circulating cells to those in the vasculature. In Project 2, the biophysical and cell biological consequences of fibrinogen nitration will be assessed. As polymorphisms that result in elevated fibrinogen interact with cigarette smoking in the prediction of cardiovascular risk, we shall investigate the effects of nitration on the interaction of fibrinogen with platelets and vascular cells. In Project 3, we shall address the possibility that hyaluronan, a major extracellular matrix protein and its CD 44 receptor may be modified by oxidative stimuli in both model systems and in humans in collaboration with Projects 1 and 5. Again, we shall utilize the resources of the cores to elucidate the modifications that are induced in these targets. In Project 4, we shall characterize further the covalent modifications to DNA resulting from hydroperoxide derived electrophiles; investigate the role of oxygenases in this process; determine from studies in Projects 1 and 5 whether vitamin C administration results in such DNA modifications in vivo and, using the Cores, assess their role in the apoptotic pathway, using endothelial cells as a model system. In Project 5, we shall address the possibility that HDL, inversely associated with cardiovascular risk, may have functional relevance, at least in part, by acting as an endogenous antioxidant. We will investigate the antioxidant potential of HDL components in vitro and by using genetic manipulation of ApoA in mice. We shall also follow progression of plaque burden in humans with electron beam computerized tomography relating HDL to the integrated indices of oxidative stress developed in Projects 1, 2, 3 and 4 as well as the genomic and proteomic analyses developed in the Cores to both the studies in humans and mice. This highly integrated SCOR will broaden consideration of oxidative targets of potential relevance to atherogenesis and utilize a highly integrated series of studies in mice and humans to assess the role of pro- and anti-oxidant interventions on a novel series of biomarkers of oxidant stress in vivo.

Products of the cyclooxygenase (COX) have been directly implicated in both inflammatory and thrombotic disease. However, predominant products of the COX enzymes vary between cells and have contrasting biology. A combined genetic and pharmacological approach will be used to elucidate the role of discrete COX products in atherosclerosis, thrombosis and hypertension in vivo. We shall use mutants in a synthesis/response pathway of established significance in inflammation to conduct a genomic and proteomic interrogation of the response to inflammatory and oxidant stimuli in vascular cells. Versatile biomarkers of inflammation and oxidant stress will be combined with lipidomic analyses to integrate the divergent impact of receptor deletion on atherosclerosis in vivo with the mechanistic studies in vitro. In Specific Aim 1 we shall assess the relevance of PGI2 and PGE2, to the cardiovascular consequences of COX-2 inhibition in vivo. The effect of systemic inhibition of COX-2 in modulating the response to thrombogenic and hypertensive stimuli will be compared with the impact of hetero and homozygous deletion alone and together, of the IP, the EP2 and the EP4. In Specific Aim 2 we shall assess the role of PGE2 in the promotion of atherosclerosis. The effects of deletion of microsomal PGE synthase (mPGES) on atherosclerosis progression and regression will be compared with that of EP2 and EP4 deletion. The impact of mPGES and EP deletion on oxidant stress, chemokine release and platelet activation will be compared with that of IP deletion and antagonism. Deletion of PG deydrogenase will be utilized to assess the impact of elevating endogenous PGE2 on atherogenesis. In Specifie Aim 3 we shall address comprehensively the cardiovascular biology of PGs of the D, J and F series in vivo. Mast cells and platelets express predominantly DP1, but the chemoattractant DP2 (CRTH2) is coexpressed with DP1 on Th2 and Tc2 lymphocytes, basophils and eosinophils. While both receptor subtypes express similar affinity for PGD2, only CRTH2 expresses affinity for the COX-2 derivative, 15-deoxy PGJ2 that correspond to the concentrations formed in vivo. Mice deficient in DP1 and DP2 (CRTH2) receptors alone and together will be combined with selective receptor subtype antagonism to clarify the role of PGD2 and 15-deoxy PGJ2 in development and regression of atherosclerosis. We shall also address the role of FP activation in atherosclerosis and the functional redundancy between the FP and the TP in vivo. Finally, in Specific Aim 4, we shall assess the impact of deleting pro- (TP, EP1) and anti- (IP, EP4) inflammatory prostanoid receptors on modulating the integrated genomic and proteomic response to oxidant and inflammatory stimuli and its metabolic consequences. We shall use an array of PG products, cytokines and markers of oxidant stress to integrate these observations with the consequences of deleting the same receptors on atherosclerosis in vivo.

cardiovascular disorder risk; disease /disorder etiology; smoking; oxidative stress; tobacco abuse; heart disorder; dosage; cardiovascular function; clinical research; human subject;

DESCRIPTION (provided by applicant): Atherosclerosis is a chronic inflammatory disease of the vasculature that often progresses to debilitating or fatal myocardial and stroke events. Macrophages are key players in progression of this disorder, interacting with lymphocytes and smooth muscle cells. 12/15-Lipoxygenase (12/15-LO) and 5-Lipoxygenase (5-LO) are present in subpopulations of macrophages and can oxygenate accumulating lipids to form hydroperoxides and leukotrienes, which have a variety of potent pro-inflammatory actions. Data generated during the past cycle of this grant established strong evidence for a pro-atherogenic role of 12/15-LO in three distinct mouse models and provided initial insight into the mechanisms involved. Recent data from other labs have shown the presence of 5-LO in atherosclerotic lesions and implicated this gene as a major atherosclerosis susceptibility gene in mice. The overall goal of this proposal is to establish the role and mechanisms for lipoxygenases in atherosclerosis. A central hypothesis is that subpopulations of 12/15-LO and 5-LO expressing macrophages can contribute to atherogenesis via specific pro-inflammatory gene signaling networks. In Specific Aim 1, the expression and roles of 12/15-LO and 5-LO in atherosclerosis will be investigated. Considerable controversy now exists as to the true expression pattern of these enzymes throughout lesion development in humans and mice. We shall investigate expression patterns of 12/15-LO and 5-LO in atherosclerosis prone mice using immunohistochemistry and in specific macrophage populations using laser capture microdissecfion. The role of 5-LO in atherogenesis throughout the lifetime of mice on apoE and LDL-R genetic backgrounds will be examined by en face lesion analysis and potential additive or synergistic actions with 12/15-LO explored. Preliminary data in atherosclerotic models and microarray studies have indicated that lipoxygenase inhibition via gene disruption influences expression of several important cytokine and inflammatory mediators, which may offer an explanation for the roles of lipoxygenases in atherogenesis. In Specific Aim 2, we will employ a microarray approach to examine the alterations in gene expression in both 12/15-LO and 5-LO deficient macrophages compared to C57BL/6 wildtype controls. Candidate gene changes in expression will be verified and functional links to lipoxygenase/cytokine signaling explored. In Specific Aim 3, a small interfering RNA (siRNA) gene silencing approach will be instituted to block macrophage lipoxygenase expression. Overall, these studies will illuminate the importance of lipoxygenase pathways in macrophages in relation to atherosclerotic disease.

cyclooxygenase inhibitors; nonsteroidal antiinflammatory agent; rheumatoid arthritis; drug screening /evaluation; thrombosis; pharmacokinetics; antiarthritic agent; naproxen; arthritis therapy; human therapy evaluation; clinical trials; drug adverse effect; patient oriented research; human subject; clinical research;

blood circulation; human therapy evaluation; aspirin; cardiovascular disorder chemotherapy; hemodynamics; dipyridamole; peripheral blood vessel disorder; anticoagulants; leg; drug screening /evaluation; patient oriented research; clinical research; human subject;

Cyclooxygenase (COX) inhibitors have an established place in the treatment of thrombotic and[unreadable] inflammatory diseases. Although randomized clinical trials have established that low dose aspirin is[unreadable] efficacious in the secondary prevention of stroke and myocardial infarction, it has been suggested[unreadable] that a syndrome of "aspirin resistance" constrains its efficacy in up to a third of aspirin users. We[unreadable] propose to determine the stability, specificity and consequent incidence of the "aspirin resistant"[unreadable] phenotype, to compare the ability of aspirin to prevent transient haemostatic activation by bacterial[unreadable] lipopolysaccharide (IPS) in "resistant" and "responsive" individuals, to define novel lipidomic and[unreadable] proteomic signatures of aspirin resistance and to relate conventional and novel biomarkers of this[unreadable] phenotype to a surrogate of clinical outcome- progression of atherosclerotic plaque burden. Specific[unreadable] Aim 1 addresses the incidence of a stable and specific phenotype of "aspirin resistance" in healthy[unreadable] volunteers given aspirin by measuring platelet aggregation and both ex vivo and in vivo indices of[unreadable] platelet prostaglandin production. Specific Aim 2 will determine the impact of a resistant phenotype[unreadable] on inhibition of evoked haemostatic activation by aspirin. The impact of low dose aspirin on the[unreadable] prevention of hemostatic activation by lipopolysaccharide in "resistant" and "responsive" volunteers[unreadable] will be studied to determine the impact of combined inhibition of COX-1 and COX-2 on haemostatic[unreadable] activation by IPS in these subsets, to identify novel proteomic and lipidomic signatures which[unreadable] identify individuals as "aspirin resistant", and to identify genetic variants of COX-1 which correspond[unreadable] to the resistant phenotype. Specific Aim 3 will relate prospectively individual biomarkers of the[unreadable] stable aspirin-resistant phenotype to carotid and coronary artery plaque progression, relate novel[unreadable] proteomic and lipidomic biomarkers of resistance to this clinical surrogate of cardiovascular disease[unreadable] in patients treated with aspirin, and initiate assessment of the correlation of genetic covariants of[unreadable] resistance with cardiovascular events in patients treated with aspirin.

disease /disorder model; laboratory mouse; tissue /cell preparation

The Clinical and Translational Research Award ,(CTSA) has been greeted enthusiastically by the[unreadable] University of Pennsylvania (Penn). A strategic plan had identified clinical and translational research as a[unreadable] priority, leading to formation of the Institute for Translational Medicine and Therapeutics (ITMAT) in January[unreadable] 2005. ITMAT anticipated some aspects of the CTSA - inclusion of the GCRC, dedicated "dry" and "wet[unreadable] bench" space for translational research and a robust educational program, configured on a Masters in[unreadable] Translational Research (MTR). This CTSA proposal prompted intra and inter-institutional consideration of[unreadable] how to build on this achievement. This has forged a transformational alliance between Penn, the Children's[unreadable] Hospital of Philadelphia (CHOP), the Wistar Institute (Wl) and the University of the Sciences in Philadelphia[unreadable] (USP). Faculty from 9 of the 12 schools at Penn and from the partner institutions are represented in[unreadable] leadership roles in the response to this CTSA.[unreadable] ITMAT, designated as the "academic home" for clinical and translational research, has been broadened[unreadable] to serve a trans-institutional role. Its structure has been transformed to foster interdisciplinary science from[unreadable] discovery of new molecules through to the study of drug action in large populations. This has been[unreadable] accomplished by developing interdisciplinary centers, related cores, innovative interdisciplinary programs of[unreadable] research and strategies to engage and inform communities and their physicians. A particular emphasis has[unreadable] been placed on training and innovative programs, which cover the entire career span, engaging[unreadable] undergraduate students through to mature clinicians, are proposed/These include the flexible use of the[unreadable] MTR and new tracks in the Masters in Clinical Epidemiology (MSCE) with the MD, PhD, VMD, MSN, DM0[unreadable] and MBA degrees; the set aside of places for medical school entrants pursuing MD-MTR/MSCE degrees[unreadable] and the flexible use of our diverse faculty tracks at Penn and CHOP to broaden physician engagement in[unreadable] research. These initiatives will be pursued in partnership with industry (e.g. GSK, Oracle), the State of[unreadable] Pennsylvania (BioAdvance), the FDA and a national network of institutions holding CTSAs. An elaborate and[unreadable] diversified approach to tracking the productivity of this program has been developed and will be integrated[unreadable] into a national plan with similar centers.[unreadable] In summary, this initiative has fostered (i) an integrated strategy to clinical and translational research by[unreadable] Penn, CHOP, the Wl and USP and (ii) the transformation of ITMAT. This will permit the development of[unreadable] interdisciplinary structures designed to. foster and facilitate research and education in this emerging[unreadable] discipline.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Predisposition to clinical syndromes of vascular occlusion reflects the cumulative impact of xenobiotic and environmental insults, immunological susceptibility, an inflammatory response and genetic substrate. Injury may occur in many forms - an angioplasty catheter, a drugj a metabolic insult, an environmental toxin, or an infectious agent. Correspondingly, the genetic makeup of an individual may condition the vascular response by modulating metabolism or target expression for a drug or toxin or immunological response to invasion by a pathogen. Our incomplete understanding of this complex interaction is reflected by the modest repertoire of therapeutic options available to us to control or reverse this process. Rather than focus on a descriptive analysis of vascular injury, the present proposal takes a translational approach to defining mechanism in a domain replete with novel therapeutic targets - the intersection of the nicotinic acid / HM74A and PGD2 biosynthetic/ response systems. We avail of many novel models of gene deletion in mice, the discovery of new targets of drug action, such as HM74A and the DP2, arid the emergence of novel pharmacological probes to project and integrate our explorations of mechanism in mice into corresponding studies of the mechanism of vascular injury in humans. These basic and clinical studies in this highly integrated proposal will deploy a common approach - the use of common lipidomic and proteomic discovery tools, the common use of pharmacological probes and gene depleted mice, and common approaches to evocation of phenotypic responses in mice and humans. This will permit a truly translational approach using hypothesis based and unbiased methodologies to investigating the role of bioactive lipids in the response to vascular injury.

university

university

disease /disorder model; inhibitor /antagonist; prostaglandin endoperoxide synthase

Award; Clinical; Infrastructure; Pediatric Research; Research Infrastructure; Translational Research; Translational Research Enterprise; Translational Science; translation research enterprise

This subproject represents an estimate of the percentage of the CTSA funding that is being utilized for a broad area of research (AIDS research, pediatric research, or clinical trials). The Total Cost listed is only an estimate of the amount of CTSA infrastructure going towards this area of research, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Clinical and Translational Research Award (CTSA) has been greeted enthusiastically by the University of Pennsylvania (Penn), A strategic plan had identified clinical and translational research as a priority, leading to formation of the Institute for Translational Medicine and Therapeutics (ITMAT) in January 2005. ITMAT anticipated some aspects of the CTSA - inclusion of the GCRC, dedicated "dry" and "wet bench" space for translational research and a robust educational program, configured on a Masters in Translational Research (MTR). This CTSA proposal prompted intra and inter-institutional consideration of how to build on this achievement. This has forged a transformational alliance between Penn, the Children's Hospital of Philadelphia (CHOP), the Wistar Institute (Wl) and the University of the Sciences in Philadelphia (USP). Faculty from 9 of the 12 schools at Penn and from the partner institutions are represented in leadership roles in the response to this CTSA. ITMAT, designated as the "academic home" for clinical and translational research, has been broadened to serve a trans-institutional role. Its structure has been transformed to foster interdisciplinary science from discovery of new molecules through to the study of drug action in large populations. This has been accomplished by developing interdisciplinary centers, related cores, innovative interdisciplinary programs of research and strategies to engage and inform communities and their physicians. A particular emphasis has been placed on training and innovative programs, which cover the entire career span, engaging undergraduate students through to mature clinicians, are proposed. These include the flexible use of the MTR and new tracks in the Masters in Clinical Epidemiology (MSCE) with the MD, PhD, VMD, MSN, DMD and MBA degrees;the set aside of places for medical school entrants pursuing MD-MTR/MSCE degrees and the flexible use of our diverse faculty tracks at Penn and CHOP to broaden physician engagement in research. These initiatives will be pursued in partnership with industry (e.g. GSK, Oracle), the State of Pennsylvania (BioAdvance), the FDA and a national network of institutions holding CTSAs. An elaborate and diversified approach to tracking the productivity of this program has been developed and will be integrated into a national plan with similar centers. In summary, this initiative has fostered (i) an integrated strategy to clinical and translational research by Penn, CHOP, the Wl and USP and (ii) the transformation of ITMAT. This will permit the development of interdisciplinary structures designed, to foster and facilitate research and education in this emerging discipline.

Animals

This Program Project will be administered at the level of the University of Pennsylvania by the Office of Research Administration under the direction of Ms. Pamela Caudill. The Program includes a consortium arrangement with the Wistar Institute, which is a corporate entity separate from the University of Pennsylvania. Similar consortium arrangements presently exist between the two institutions and the financial and administrative officers of each are experienced in the efficient management of such arrangements. The participants in this Program Project are members of the faculty of the Medical School of the University of Pennsylvania. Thus, they have access to all of the resources of the institution.

Project 1: Dynamic Interplay of Eicosanoids in Cardiovascular Biology Products of the cyclooxygenase (C0X)-2 have been directly implicated in both inflammatory and thrombotic disease. However, predominant products of the COX enzymes vary between cells and have contrasting biology. The microsomal PGE synthase (mPGES)-1 has emerged as a drug target alternative to COX-2. A combined genetic and pharmacological approach will be taken to elucidate the roles of these enzymes in discrete cells and to assess the impact of COX-2 perturbation on biological networks. Versatile biomarkers of inflammation and oxidant stress will be combined in lipidomic analyses to integrate the divergent impact of cell specific enzyme disruptions on atherosclerosis in vivo with mechanistic studies in vitro. In Specific Aim 1 we shall assess the importance of COX-2 in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in regulating (i) the response to thrombogenic stimuli in large and small blood vessels and (ii) blood pressure homeostasis. Analysis of these phenotypes in mice deficient in discrete prostanoid receptors and cell specific deletions of COX-2 will be compared with the effects of global disruption of COX -2 function using genetic and pharmacological approaches. In Specific Aim 2 we shall seek to elucidate the controversy surrounding COX-2 inhibition and atherosclerosis. The impact of induced global deletion of the enzyme will be assessed at varied stages of disease evolution, as will the distinct roles of EC, VSMC and macrophage COX-2 on prostanoid biosynthesis, the inflammatory response and lesional progression and morphology. Multiple tissues will be harvested from these and other mice with discrete deletions of COX-2 to determine how enzyme perturbation might impact on biological networks relevant to inflammation and how this might be influenced by age, gender and diet. In Specific Aim 3 we will utilize both a novel selective inhibitor of mPGES-1 and cell specific deletions ofthe enzyme to address the hypothesis that contrasting, functionally relevant patterns of substrate rediversion might ocpur at different stages of disease evolution. Unbiased analyses will be performed of the comparative impact of mPGES-1 vs COX-2 deletion on the response to inflammatory stimuli in discrete cell types. Finally, in Specific Aim 4, we shall assess the effect of deleting COX-2 or mPGES-1 in discrete cells - that differ in their primary prostanoids or rediversion products - on the response to vascular injury. We shall also determine whether systemic hypertension might exacerbate the impact of deleting either enzyme in cardiomyocytes on heart function and rhythm.

Abstract The circadian clock regulates many aspects of physiology including metabolism and cardiovascular function. The past decade of research has seen the development of a scaffold model of oscillator function in which the suprachiasmatic nucleus of the hypothalamus harbors a master clock and orchestrates peripheral oscillators present in most major organ systems. These central and peripheral oscillator systems generate a cascade of circadian transcriptional rhythms that ultimately culminate in observed physiological and behavioral oscillations. Genetic disruption of this organization in animal models results in pathophysiological consequences such as glucose intolerance and insulin resistance, components of the metabolic syndrome seen in people at high risk for cardiovascular disease. We present compelling evidence that communication between clocks is more sophisticated and can involve peripheral-to-peripheral and peripheral-to-central clock communication. Here we test the central hypothesis that communication between peripheral and central oscillators is bidirectional and that peripheral oscillators may directly influence each others' function. Using cell type specific conditional mouse models in which Bmal1, a required component of the oscillator, is deleted, we will use physiological and systems approaches to test the hypothesis that oscillator function in endothelial cells regulates vascular smooth muscle function (and vice versa) and influences diurnal variation in blood pressure, thrombogenesis, and locomotor activity (Specific Aim 1). We will also test the hypothesis that oscillator function in adipocytes regulates macrophage function (and vice versa) and influences glucose homeostasis, response to inflammatory stimuli, and feeding rhythms (Specific Aim 2). Furthermore, we propose testing a mechanistic hypothesis that cell type specific disruption of oscillator function results in oscillator abnormalities in nearby cells, and that this disruption propagates to the liver, adrenal, kidneys, and the brain (Specific Aim 3). Finally, using systems approaches we will examine network level changes provoked by genetic disruption of oscillator function in specific cell types and begin to probe network to network conveyance of circadian information as well as identify candidate signaling molecules (Specific Aim 4).

DESCRIPTION (provided by applicant): The Institute for Translational Medicine and Therapeutics (ITMAT) was founded by the Medical School of the University of Pennsylvania in January 2005. It became the academic home of the Clinical and Translational Science Award (CTSA) after the first round of funding by the National Center for Research Resources (NCRR). The CTSA transformed ITMAT to embrace 9 schools at Penn, the Children's Hospital of Philadelphia, the Wistar Institute and the University of the Sciences in Philadelphia. Each year ITMAT has held an international conference, attracting speakers and attendees from academia, pharma, biotech, venture, politics, the media and regulatory bodies. Topics have included the Personalization of Medicine and the Role of Academia in Drug Development. The meeting to be held on April 14 and 15, 2009 is entitled Global Approaches to Translational Research. Speakers are drawn from the United States, Europe, Asia and Australia several entities including the Wellcome Trust and the United Kingdom Medical Research Council are supporting their constituents to attend the meeting. This application is to seek funds to discount the cost to CTSA leaders to attend the meeting. This will afford the chance for them to develop collaborations with their international peers, share best practices and discuss common challenges. PUBLIC HEALTH RELEVANCE: This proposal will foster the interaction of those leading Clinical and Translational Science Awards (CTSAs) with their international peers at a meeting that considers challenges and opportunities confronting this emerging discipline. It has attracted enthusiastic support from the leadership of NCRR and the Director, together with several PIs of CTSAs featured on the program.

DESCRIPTION (provided by applicant): Nonsteroidal anti-inflammatory drugs (NSAIDs) are consumed by tens of millions worldwide. Although they relieve pain and inflammation, they also cause serious gastrointestinal and cardiovascular adverse effects and are thought to have caused hundreds of thousands of deaths. Despite enrolling more than 100,000 patients in randomized trials, we still do not know the NSAID of choice for patients with arthritis and heart disease or if NSAIDs differ in clinical efficacy. Here we propose a paradigm shifting, strategic approach to harvest benefit and manage risk by personalizing therapy with NSAIDs. An exploratory clinical study at scale will inform translational studies in which data from 5 systems - yeast, mammalian cells, zebrafish, mice and humans - will be integrated to create biochemical networks that inform and are refined by studies relating pharmacokinetics to pharmacodynamics. Hypotheses deriving from these studies will then be tested at scale in randomized prospective trials. This interdisciplinary strategy will deliver innovative tools and technologies, quantitative models and biomarkers of drug response and if successful will allow more rational prescription of NSAIDs to minimize risk and maximize benefit to individuals, creating a novel paradigm for the development and approval of drugs, the design of randomized trials and the treatment of chronic disease. PUBLIC HEALTH RELEVANCE: Drugs are prescribed based on detection of large average signals of effectiveness and hazard. This proposal attempts to refine the use of nonsteroidal anti-inflammatory drugs so that they are used in patients individually most likely to benefit and least likely to suffer adverse effects.

This subproject represents an estimate of the percentage of the CTSA funding that is being utilized for a broad area of research (AIDS research, pediatric research, or clinical trials). The Total Cost listed is only an estimate of the amount of CTSA infrastructure going towards this area of research, not direct funding provided by the NCRR grant to the subproject or subproject staff. DESCRIPTION (provided by applicant): During the past cycle, following consultation with our internal and external advisory boards, organizational governance structure and the leadership of NCRR, we have focused our efforts on two overarching themes - Translational Therapeutics and Bridging the Pediatric to Adult Divide, taking advantage of resources particular to our institutions. We have fostered interdisciplinary and translational research by a series of "top down" and "bottom up" funding initiatives. CTSA support and institutional investment has increased dramatically the footprint of ITMAT more than 7 fold to ~150,000 nsf comprising an expanded, integrated and re-purposed Clinical and Translational Research Center (CTRC) that succeeds the Penn and CHOP GCRCs, dry and wet bench laboratory space, a series of cores and a new home for ITMAT educational programs. ITMAT also supports seminars, workshops and an annual international meeting highlighting the opportunities that the CTSA affords on our campus to pursue clinical and translational research. Interdisciplinary research has increased dramatically on, between, and within the partner institutions during the past funding cycle and these trends have been most pronounced amongst members of ITMAT. During the coming cycle we envisage the continued growth and development of our educational programs;the continued expansion - via recruitment and education - of the critical mass of investigators pursuant of clinical and translational research;enhancement and expansion of our core infrastructure and the development and expansion of programs focused on our two overarching themes. In particular we hope to impact patient care in the community by projecting our two strategic goals to alter the current paradigm of comparative effectiveness research by integrating expertise in basic, pediatric and adult pharmacology and to contribute to predictive strategies in the personalization of medicine. RELEVANCE (provided by applicant): PENN, CHOP, Monell, Wistar Institute, and the University of the Sciences of Philadelphia have benefited greatly from the first cycle of funding of the CTSA, which allowed the development of new centers, research programs, cores and a robust educational program which involve more than 800 investigators across campus and from partner institutions, as well as adding to consortial activities nationally.

DESCRIPTION (provided by applicant): One of the key challenges in biomedical research and late-stage drug discovery is the lack of appropriate pre-clinical animal models of human disease. The conference Animal Models and Drug Discovery will be jointly presented by Imperial College London, King's College London, and the New York Academy of Sciences (NYAS) at the NYAS Conference Center in New York on September 15-16, 2011, and will critically examine the traditional role of animal models in drug discovery, how these models most effectively contribute to translational medicine, changes needed to increase the predictive power of models for drug efficacy in humans, and ways in which to further refine, reduce, and replace animal models in translational biomedical research. The multidisciplinary scientific organizing committee comprises experienced scientific researchers and clinicians from diverse fields that employ pre-clinical animal models for therapeutic development, including the PI for this grant: Garret A. FitzGerald, MD, Professor of Medicine and Pharmacology;McNeil Professor in Translational Medicine and Therapeutics;Associate Dean for Translational Research;Chair, Department of Pharmacology;Director, Institute for Translational Medicine and Therapeutics, The University of Pennsylvania. The 2-day program, anticipated to attract up to 250 attendees across a wide span of medicine and health, will be especially timely and exciting given the recent explosion of broadly applicable new technologies in bioimaging, biosimulation, and bioinformatics;the generation of genetically modified animals and emerging non-rodent models;and the use of embryonic stem cells, patient-specific induced pluripotent stem cells, and humanized animal models. Thematically, the conference goals align well with NCRR's mission to provide clinical and translational researchers with the training and tools they need to transform basic discoveries into improved human health. This program will also showcase research tools and techniques that serve as the foundation for disease diagnosis, treatment, and prevention, thus also fitting the mission of NIGMS. Lectures, workshops, interactive discussions, networking breaks, and a conference reception and dinner will provide a neutral forum for interaction among multidisciplinary participants who may not otherwise interact at a single scientific meeting and ensure that recent improved pre-clinical animal models with good predictive validity readily become available to the pharmaceutical industry and clinical researchers so as to aid the discovery and development of new disease treatments as quickly as possible. Poster sessions and travel awards will encourage participation of junior/minority investigators and trainees. We expect that the discussions originating from the conference and their dissemination via an open access online multimedia report will ultimately foster advancement in the diagnosis, management, and treatment of a multitude of human diseases. PUBLIC HEALTH RELEVANCE (provided by applicant): This 2-day scientific conference, jointly presented by Imperial College London, King's College London, and the New York Academy of Sciences, will provide a neutral forum to critically examine the traditional role of pre-clinical animal models in drug discovery, how these models most effectively contribute to translational medicine and drug discovery, changes needed to increase the predictive power of various models for drug efficacy in humans, and ways in which to further refine, reduce, and replace animal models in biomedical research. By convening multi-disciplinary clinical and basic science investigators we hope to identify common hurdles and pathways forward to improving model systems for the evaluation of therapeutic efficacy in the areas of metabolic and cardiovascular disease, inflammation, and pain, among others. Sessions on the recent explosion of broadly applicable new technologies in bioimaging, biosimulation, bioinformatics, generating genetically modified animals and phenotype screening along with emerging non- rodent models, the use of embryonic stem cells, patient-specific induced pluripotent stem cells, and humanized animal models will ensure that recent advances in basic science knowledge gained from improved pre- clinical animal models with good predictive validity readily become available to the pharmaceutical industry and clinical researchers so as to aid the discovery and development of new disease treatments as quickly as possible.

Advisory Committees; Collaborations; Communication; Communities; Ensure; Fostering; meetings; member; outreach; programs; Research Personnel; Risk; Treatment Efficacy;

Project Summary Checkpoint blockade targeting the Programmed cell Death (PD-1) / PD-Ligand1 pathway has proven effective in a range of cancers by releasing a restraint on cytotoxic T lymphocytes (CTLs). However, a concern has been that this very mechanism might predispose to cardiovascular and autoimmune diseases. Anecdotal evidence of a cardiovascular hazard has emerged and abundant data point to exacerbation of atherosclerosis in mice consequent to Pd-1 deletion. Recently, the generation of prostaglandin (PG) E2 by the sequential action of the cyclooxygenase (COX)-2 and microsomal PGE synthase enzymes, acting via its E prostanoid (EP) receptors, EP2 and EP4, has been shown also to promote lymphocyte exhaustion. This raises the possibility of a combinatorial approach to cancer with nonsteroidal anti-inflammatory drugs (NSAIDs) and checkpoint inhibitors. However, COX-2 inhibition alone confers a cardiovascular risk and may exacerbate one consequent to checkpoint blockade. Here, we address this possibility, first seeking to build on our preliminary data in mouse and human cells that there appears to be a bidirectional regulatory interaction between the PD-1 and PG pathways. Using pharmacological and genetic approaches, we will determine whether modulation of PGE2 alters lymphocyte phenotype and function and how regulation of PD-1 may influence the PGE2 biosynthetic response pathway both in mouse and human cells. We will use atherogenesis in the mouse as a surrogate for cardiovascular risk in humans (as it proved to be for NSAIDs and as it correlates to date with checkpoint inhibitors) and determine if deletion of Cox-2 accelerates and exacerbates the immuno- inflammatory atherosclerotic phenotype consequent to Pd-1 deletion. We will then determine whether alternative approaches to PGE2 suppression (deletion of the microsomal PGE Synthase [mPGES] ? 1; EP blockade or inhibition of either enzyme restricted to myeloid cells) might limit or avoid the acceleration of atherosclerosis consequent to deletion of Pd-1. These studies will combine differential perturbations of the PG pathway in cells obtained from melanoma patients receiving PD-1 blockade, novel mouse models, state of the art immunophenotyping, single cell transcriptomics and mass-spectrometry based lipidomic substrate imaging and product analysis defining atherosclerotic lesions to understand a potentially serious risk of combining NSAIDs with checkpoint inhibitors. We shall also explore novel alternative approaches to suppressing PGE2 that might conserve the anti-cancer efficacy and minimize the cardiovascular risk of combinatorial therapy.

ABSTRACT OF THE FUNDED PARENT AWARD The Institute for Translational Medicine and Therapeutics is the academic home for the CTSA in Penn. For the past decade the specific aims of ITMAT have been and remain (i) to increase the number of investigators capable of pursuing their research between proof of concept in cells and model systems and (ii) elucidation of the mechanisms of human physiology, disease or drug action through intense phenotyping in small numbers of people. The two priorities of our CTSA are to foster the field of translational therapeutics and to bridge the pediatric to adult divide across the entire spectrum of clinical and translational science. This section describes how these priorities have influenced the growth of this CTSA Hub and outlines our plans for the coming funding period.