Daniel S. Ory - US grants

The proposed research is a study of the molecular mechanisms involved in atherogenesis and restenosis. Gene transfer provides a means to examine these mechanisms by genetic modification of vascular cell biology. I will examine the feasibility of modifying vascular cells within atheromatous plaque: (1) by resurfacing with genetically altered endothelial cells and (2) by direct retroviral infection in vivo. Heterozygote Watanabe Heritable Hyperlipemic rabbits fed a high cholesterol and high fat diet will serve as the atherosclerotic model. Atheromatous plaques within the iliac arteries will be treated with an atherectomy device. Tissue removal will be performed to varying depths within the vessel wall to examine how this influences endothelial cell adhesion and the efficiency of direct retroviral infection in vivo. The preferred source of autologous rabbit endothelial cells (large vessel vs. microvascular) will be determined. The exposed vascular cells of the atherectomy-treated iliac artery segments will be resurfaced with genetically modified endothelial cells. In parallel experiments the atherectomy-treated iliac artery segments will undergo direct retroviral infection in vivo. Replication-defective retroviruses with amphotropic host range will be used. Initial experiments will use a retroviral construct containing the E. coli lacZ reporter gene to assess the resurfacing efficiency with the modified endothelial cells and the transduction efficiency of vascular cells by direct in vivo infection. Enzymatic histochemical studies will allow determination of the in vivo survival of modified endothelial cells, the vascular cell types involved in direct infection, and the persistence of retroviral gene expression. Retroviral constructs encoding genes for thrombolytic agents, thrombin and platelet-aggregation inhibitors, growth factor antagonists and cytokine receptor antagonists will also be transfected into endothelial cells. Application of these gene transfer methods in an experimental atherosclerotic animal model will permit examination of the role of specific gene products in the pathogenesis of atherosclerosis and restenosis.

The proposed research is a study of the molecular mechanisms involved in atherogenesis and restenosis. Gene transfer provides a means to examine these mechanisms by genetic modification of vascular cell biology. I will examine the feasibility of modifying vascular cells within atheromatous plaque: (1) by resurfacing with genetically altered endothelial cells and (2) by direct retroviral infection in vivo. Heterozygote Watanabe Heritable Hyperlipemic rabbits fed a high cholesterol and high fat diet will serve as the atherosclerotic model. Atheromatous plaques within the iliac arteries will be treated with an atherectomy device. Tissue removal will be performed to varying depths within the vessel wall to examine how this influences endothelial cell adhesion and the efficiency of direct retroviral infection in vivo. The preferred source of autologous rabbit endothelial cells (large vessel vs. microvascular) will be determined. The exposed vascular cells of the atherectomy-treated iliac artery segments will be resurfaced with genetically modified endothelial cells. In parallel experiments the atherectomy-treated iliac artery segments will undergo direct retroviral infection in vivo. Replication-defective retroviruses with amphotropic host range will be used. Initial experiments will use a retroviral construct containing the E. coli lacZ reporter gene to assess the resurfacing efficiency with the modified endothelial cells and the transduction efficiency of vascular cells by direct in vivo infection. Enzymatic histochemical studies will allow determination of the in vivo survival of modified endothelial cells, the vascular cell types involved in direct infection, and the persistence of retroviral gene expression. Retroviral constructs encoding genes for thrombolytic agents, thrombin and platelet-aggregation inhibitors, growth factor antagonists and cytokine receptor antagonists will also be transfected into endothelial cells. Application of these gene transfer methods in an experimental atherosclerotic animal model will permit examination of the role of specific gene products in the pathogenesis of atherosclerosis and restenosis.

Intracellular cholesterol sorting and transport pathways govern the physiologic utilization of lipoprotein-derived cholesterol, and are central to pathophysiology of inherited disorders such as Niemann-Pick type C (NPC) and of acquired atherosclerotic disease. The NPC1 protein, which functions in these pathways, bears sequence similarity to Patched, HMG- CoA reductase, and SCAP. Mutations in NPC1 result in accumulation of unesterified lysosomal cholesterol, but the specific function of NPC1 in cellular cholesterol metabolism is not well understood. The present proposal is designed to test the hypothesis that NPC1 is a key participant in the post-plasma membrane (PM) trafficking of low density lipoprotein (LDL) cholesterol. We propose that NPC1 resides within an endocytic organelle, whose function is to sort and recycle internalized PM lipids. NPC1 may perform an essential function in the endocytic pathway by facilitating vesicular trafficking of the lipid cargo back to the PM. Moreover, NPC1 may play an important role in cellular cholesterol homeostasis, serving to maintain normal levels of PM cholesterol, as well as to shuttle excess unesterified cholesterol to the endoplasmic reticulum (ER) for esterification. These hypotheses will be tested by the following specific aims: (1) Examination of the role of NPC1 in intracellular trafficking of LDL cholesterol, (2) Use of a functional genetic screen to identify genes required for LDL cholesterol trafficking, (3) Characterization of NPC1- interacting proteins identified by a yeast two-hybrid screen and their roles in cholesterol trafficking, and (4) Characterization of the function of NPC1 and proteins identified by our genetic screens in cell culture models of atherogenesis. These studies will contribute to our understanding of the molecular basis of intracellular cholesterol trafficking under normal conditions and in atherogenesis. The work proposed herein may identify novel targets for pharmacologic therapy within the sterol transport pathway for treatment of coronary and cerebral vascular disease and for inherited metabolic disorders.

DESCRIPTION (Provided by applicant): Hypercholesterolemia is a major risk factor for the development of coronary artery disease (CAD) and cerebral vascular disease (CVD). Increased plasma levels of low-density lipoprotein (LDL) leads to deposition of excess cholesterol in arteries, initiating atherosclerosis. While homeostatic mechanisms that regulate LDL uptake and de novo synthesis of cholesterol are well characterized, the cellular mechanisms that regulate trafficking of LDL cholesterol after internalization are not well understood. Evidence is emerging that the NPC1 and HE1 proteins participate in a common pathway for the efficient trafficking of internalized membrane cholesterol to the plasma membrane (PM) and to the ER. We hypothesize that NPC1 promotes formation of vesicles and/or tubules through the budding of the limiting endosomal membrane. NPC1-containing vesicles then traffic to the PM or ER to deliver their sterol cargo. The purpose of this project is to identify the molecular machinery in the sterol trafficking pathway and to test our hypothesis regarding this role of NPC1 in the sorting and distribution of internalized membrane cholesterol. This will be achieved by the following specific aims: (1) Use of a functional mammalian genetic screen to isolate Chinese hamster ovary (CHO) mutants with impaired intracellular trafficking of cholesterol, (2) Characterization and identification of the cholesterol trafficking defects in the mutant CHO cell lines, and (3) Compositional and functional analysis of proteins in the NPC1-containing late endosomal compartment, and examination of whether NPC1 membrane vesciulation is dependent on sterol concentrations and/or an intact sterol-sensing domain in NPC1. The studies outlined in this proposal will further our understanding of the critical role of NPC1 in cholesterol homeostasis. Furthermore, study of the function of gene products identified by our genetic screens and by compositional analysis of the NPC1-containing endosomal compartment may identify novel targets within the sterol transport pathway for pharmacologic therapy of CAD and CVD.

Atheroclerosis is a major cause of morbidity and mortality in insulin-resistant states such as metabolic[unreadable] syndrome and obesity. Defective regulation of macrophage lipid metabolism plays a central role in the[unreadable] development of vascular lesions associated with these dyslipidemic states. During atherogenesis,[unreadable] macrophages respond to the challenge of lipid excess through enzymatic production of oxysterols and[unreadable] activation of feed-forward nuclear receptor pathways that induce transcriptional programs involved in lipid[unreadable] uptake and efflux, and negatively regulate inflammatory responses. In recent studies, we have shown that[unreadable] the products of the Niemann-Pick type C (NPC) disease genes, NPC1 and NPC2, are required for delivery of[unreadable] lipoprotein-derived cholesterol to sites of intracellular oxysterol synthesis. In this proposal, we will test the[unreadable] hypothesis that macrophage-derived oxysterols play a critical role in the regulation of lipid[unreadable] homeostasis in the vascular wall. We propose that impaired synthesis of side-chain oxygenated sterols[unreadable] will reduce cholesterol efflux and increase cholesterol accumulation in macrophages, and, in concert with[unreadable] increased levels of oxidized cholesterol, promote cytotoxicity and lesion formation. Production of specific[unreadable] macrophage-derived cholesterol metabolites additionally may play a critical role in governing plasma[unreadable] lipoprotein levels through regulation of hepatic lipoprotein synthesis and/or clearance of plasma lipoproteins.[unreadable] This hypothesis will be tested by the following specific aims:[unreadable] 1) To determine whether macrophage-specific NPC1 and NPC2 loss of function alters macrophage oxysterol[unreadable] synthesis, thereby disrupting lipid homeostasis and promoting atherosclerosis in a murine model,[unreadable] 2) To determine whether macrophage-derived cholesterol metabolites affect regulation of plasma lipoprotein[unreadable] levels in chimeric mice with macrophage-specific NPC1 and NPC2 loss of function, and[unreadable] 3) To assess the relationship between plasma oxysterol levels and coronary heart disease and determine if[unreadable] oxysterol levels change following weight loss in humans with the metabolic syndrome.[unreadable] These studies will contribute to our understanding of the role of macrophage-derived cholesterol[unreadable] metabolites in atherogenesis and regulation of lipoprotein metabolism. This project has the potential to[unreadable] transform the care of people with the metabolic syndrome by establishing these metabolites as novel[unreadable] biomarkers for detection of subclinical atherosclerotic disease.

DESCRIPTION (provided by applicant): Diabetes is associated with serious cardiovascular complications that include heart failure. Data from studies conducted in animal models and human subjects suggest that alterations in fatty acid metabolism, independent of atherosclerosis, are involved in the pathogenesis of heart failure in diabetic cardiomyopathy. This study will test the overall hypothesis that excessive myocardial fatty acid delivery and utilization is associated with cardiomyopathy in patients who have type 2 diabetes mellitus. Moreover, we hypothesize that alterations in lipid metabolism can be exploited to develop novel biomarkers for early detection of myocardial lipid exposure and diabetic cardiomyopathy. To test these hypotheses, we will study the relationship between fatty acid metabolism, lipid biomarkers and cardiac function in carefully characterized men and women who span physiological and pathophysiological ranges in insulin sensitivity, plasma lipid biomarkers and cardiac function. The goals of this study are to 1) identify novel plasma biomarkers for early detection and management of diabetic cardiomyopathy;and 2) determine whether manipulations of fatty acid delivery to the heart affect the functional manifestations of diabetic cardiomyopathy. This study will provide a better understanding of the relationship between systemic and myocardial fatty acid metabolism and cardiac function, which could also lead to new strategies for diagnosis, prevention, and treatment of diabetic cardiomyopathy. PUBLIC HEALTH RELEVANCE: Diabetes is associated with serious cardiovascular complications including heart failure that is unrelated to coronary artery disease. Scientific evidence suggests that blood fat levels may play a major role in this complication. Our study will investigate the link between blood fat levels and heart function in adults with type 2 diabetes. Our goal is to develop new blood-based biomarkers of heart disease in diabetics and to provide insight into new therapeutic strategies.

DESCRIPTION (provided by applicant): Cellular cholesterol homeostasis is regulated at multiple cholesterol transfer steps and through a negative feedback loop that responds to elevations of membrane cholesterol in the endoplasmic reticulum (ER). Alterations in these sterol sensing and trafficking pathways in contribute to human inborn errors of metabolism and to acquired disease states. To elucidate mechanisms governing these critical cholesterol homeostatic pathways, we performed a functional genetic screen that led to isolation of cell lines with intracellular cholesterol trafficking defects that were enriched in mutants with disruption of long non-coding RNA (ncRNA) genes. These genes exhibit evolutionarily conserved exon/intron organization and core promoter regions, lack significant open reading-frames or nucleotide homology within exons, and contain short, highly conserved intronic regions that harbor small nucleolar RNA (snoRNA) species. This class of long ncRNAs appears principally to serve as host genes to facilitate expressing and processing of the orphan snoRNAs. We hypothesize that the processed snoRNAs modulate expression of genes involved in cholesterol homeostasis, possibly through control of splicing events or translation, and thus represent a previously unrecognized mode of regulation for cellular cholesterol homeostasis. The Specific Aims of this proposal are (1) To characterize ncRNA genes identified by our genetic screen that are critical for maintenance of cholesterol homeostasis, (2) To determine the role of the snoRNA host genes in regulation of intracellular cholesterol transfer and cellular cholesterol homeostasis, (3) To determine the molecular mechanism(s) through which the orphan snoRNA elements exert control over cholesterol regulatory pathways, and (4) To examine the physiological role of the orphan snoRNAs and gene pathways identified through the genetic screen by extending our cell-based studies to in vivo animal models. The proposed studies are innovative in that they explore a novel small RNA-dependent pathway not previously implicated in regulation of cellular cholesterol homeostasis. These studies are highly significant because elucidation of this RNA regulatory pathway has the potential to provide new molecular targets for manipulation of the cellular handling of cholesterol. This proposal is highly relevant to atherosclerosis, a common disease characterized by dysregulation of cholesterol homeostasis, as well as to rare, often fatal inborn errors of sterol metabolism.

ABSTRACT Cellular cholesterol levels are tightly regulated by multiple homeostatic pathways that respond to elevations of membrane cholesterol and to enzymatically formed oxygenated cholesterol derivatives (i.e., oxysterols). Alterations in sterol sensing and trafficking pathways contribute to human inborn errors of metabolism (e.g., Niemann-Pick C disease) and to acquired disease states (e.g., atherosclerosis). Under physiological conditions, sterol-regulated transcriptional pathways act in concert to inhibit uptake of exogenous lipoproteins and suppress de novo cholesterol synthesis, resulting in half-maximal suppression of these responses within several hours. By contrast, pathophysiological cholesterol levels, such as those present in disease states, activate transcription-independent mechanisms that respond within minutes to changes in increments in membrane cholesterol. Recent studies with oxysterol enantiomers provide evidence that sterol-membrane interactions underlie these acute cholesterol homeostatic responses. We hypothesize that side-chain oxysterols serve a critical role in acute regulation of cholesterol homeostasis through direct modulation of plasma membrane lipid environment. We propose that side-chain oxysterols trigger transcription-independent regulatory pathways by disordering membrane phospholipid organization and/or increasing the accessibility of cholesterol. This hypothesis will be tested by the following Specific Aims: (1) Characterization of the mechanism by which oxysterols perturb the structure of model cholesterol-phospholipid bilayers, (2) Examination of the efect of oxysterols on cholesterol accessibility and position in physiological membranes, and (3) Examination of the mechanism by which oxysterols promote release of plasma membrane cholesterol to intracellular pools. The proposed studies wil further our understanding of how perturbations in membrane structure relay cholesterol homeostatic regulatory signals and may identify new pharmacological targets for manipulation of the cellular handling of cholesterol in disease states.

DESCRIPTION (provided by applicant): Niemann-Pick type C1 (NPC1) disease is a rare, progressive neurodegenerative disorder characterized by accumulation of cholesterol and other lipids in the viscera and central nervous system. A barrier to the development of treatments for NPC1 disease is the lack of readily quantifiable outcome measures to evaluate efficacy of therapy in clinical trials. Through broad-based metabolomic efforts, we have discovered in NPC1 subjects cholesterol oxidation product (oxysterol) biomarkers that reflect the unique intersection of oxidative stress and unesterified cholesterol storage - the biochemical hallmark of NPC1 disease. This proposal tests the highly innovative hypothesis that oxysterol biomarkers, together with other cholesterol homeostatic markers, can serve as outcome measures to assess the effect of disease-modifying therapies (e.g., 2-hydroxypropyl-?-cyclodextrin, HP-?-CD) on cholesterol metabolism in the CNS and to monitor disease progression. 24(S)-HC, which is synthesized almost exclusively in large neurons in the CNS, and CSF cholesteryl esters (CE) offer potential quantitative, non-invasive metrics to guide dosing and to monitor drug response. Likewise, cholestane-3?, 5?, 6?-triol (triol), which we have previously shown to be elevated in the CSF of NPC1 subjects, will inform with respect to the effect of HP-?-CD on intraneuronal cholesterol storage. This hypothesis will be tested in NPC1 animal models administered intracerebroventricular (ICV) HP-?-CD (Aim 1), and in NPC1 human subjects enrolled in a natural history study and in a Phase 1 trial for ICV HP-?-CD at the NIH Clinical Center (Aim 2). The proposal will also explore the possibility that the exceptional receiver operating characteristics (ROC) of the triol assay can be harnessed to develop a newborn screen to identify NPC1 patients earlier and thus intervene in pre-symptomatic patients (Aim 3). The proposed newborn screen for NPC1 disease is innovative and would be the first for a non-enzymatic lysosomal disorder, as well as the first for an inborn error of sterol metabolism. While the goal of this project is to develop a prototype newborn screen for NPC1 disease suitable for implementation at the statewide or regional level, the tandem mass spectrometry methods developed for extraction and detection of the oxysterols could be readily extended to metabolites that accumulate in other sterol disorders (e.g., Smith-Lemli-Opitz Syndrome), thereby permitting multiplexed screening for several inherited disorders within the context of a single screen. The studies in this proposal are highly significant because we address the critical unmet therapeutic and diagnostic needs of NPC1 disease.

DESCRIPTION (provided by applicant): Niemann-Pick C (NPC) is a rare, neurodegenerative, lipid storage disease. Approximately 95% of the disease is caused by mutations in NPC1, a late endosomal/lysosomal (LE/Ly) membrane protein that functions in export of lipoprotein-derived cholesterol. Affected individuals typically present in early childhood with ataxia and progressive impairment of motor and intellectual function, and usually die in adolescence. There are currently no FDA-approved therapies for this fatal neurodegenerative disorder. Recently, we found that treatment of human NPC1 mutant cells with certain histone deacetylase inhibitors (HDACi), including Vorinostat (SAHA, Zolinza?)), leads to clearance of excess cholesterol and other lipids from the LE/Ly, and it corrects the overall defect in cholesterol regulation. In other unpublished work, we found that 60 of the 80 NPC1 mutants examined show significant cholesterol clearance upon treatment with the HDACi, indicating the majority or NPC1 patients may benefit from HDACi therapy. Vorinostat is an excellent candidate for clinical testing as an NPC1 therapeutic because it is orally-available, CNS-penetrant, and FDA-approved. The goal of our study is to examine Vorinostat in a Phase 1 clinical trial for the treatment of NPC1 disease. To meet this objective, we will develop a Phase 1, first-in-human, open-label, single-center, dose escalation study of Vorinostat in late adolescents and adults with NPC1 disease to establish the safety of Vorinostat for treatment of this disorder. 12 NPC1 patients (18 years and older) will be recruited for the study. Study participants will initially be dosed with 200 mg po daily for three months, followed by dose escalation to 400 mg po daily for three months. Plasma and CSF pharmacokinetics will be obtained, toxicity monitored, and clinical assessments performed. We will further evaluate the utility of peripheral and CSF disease biomarkers to guide therapy in the Phase 1 Vorinostat dose-escalation study. The primary outcome measure will be CSF 3ß,5?,3ß- cholesten-triol, a cholesterol oxidation product that is specifically elevated in NPC1 disease and decreases in response to alleviation of neuronal cholesterol storage. Secondary outcome measures will include plasma 24(S)-hydroxycholesterol, a CNS-specific oxysterol that is elevated following correction of the neuronal cholesterol trafficking defect; CSF sphingolipid markers; CSF proteins (e.g., Calbindin D and FABP3); and histone acetylation and NPC1 protein levels in circulating mononuclear cells. These outcome measures can potentially serve as surrogate outcome measures in future Phase 2/3 HDACi trials.

? DESCRIPTION (provided by applicant): Niemann Pick C disease is a rare, neurodegenerative, lipid storage disorder. Approximately 95% of the disease is caused by mutations in NPC1, a late endosomal membrane protein that functions in export of lipoprotein-derived cholesterol. The most prevalent NPC1 mutation, I1061T, produces a protein that is misfolded and rapidly degraded. Histone deacetylase inhibitors (HDACi) recently have been shown to reduce the accumulation of cholesterol and other lipids found in patient cells harboring the NPC1I1061T and other mutations. This beneficial effect is associated with decreased endoplasmic reticulum-associated degradation and enhanced delivery of the mutant NPC1 proteins to late endosomes and lysosomes. With the recent generation in our laboratory of a humanized mouse model in which the I1061T mutation knocked into the murine NPC1 locus, it is possible to examine the effect of HDACi on NPC1 stability in vivo. We hypothesize that treatment with an HDACi in the NPC1I1061T knockin model of NPC1 disease will increase levels of the mutant NPC1I1061T protein, slowing progression of neurodegeneration and prolonging survival. The therapeutic potential of HDACi for treatment of NPC1 disease is being explored in through a collaboration involving pharmaceutical partners and an HDACi collaborative involving investigators from NIH (NICHD/NCATS), Weill Cornell Medical College, University of Notre Dame, Albert Einstein College of Medicine, and Washington University, along with the Ara Parseghian Medical Research Foundation. The goals of this proposal are to identify orally-available, CNS-penetrant HDAC-selective compounds using cell-based screens; to evaluate in vivo in the NPC1I1061T knockin model candidate HDACi compounds; and to develop effective therapeutic regimens for testing of the HDACi in clinical trials. The proposed in vivo studies further will provide valuable data for initial dosing protocols and biomarker monitoring in future human trials.

PROJECT SUMMARY/ABSTRACT - Biomolecular Analysis Core: The Biomolecular Analysis Core (BMAC) serves as a key analytical core for NORC investigators and combines the mass spectrometer instrument inventories and expertise of the Washington University (WU) Metabolomics and Mass Spectrometry (MS) facilities to provide centralized, standardized, and cost-effective metabolomics analyses. The major goal of the BMAC is to provide state-of-the-art mass spectrometry (MS) and metabolomics profiling to NORC investigators to study the molecular mechanisms of the pathogenesis of nutrition-related diseases, including obesity and diabetes, and their risk factors and complications. Specific objectives of the BMAC are: 1) To perform MS analytical services to quantify target analytes, obtain spectra for structural identification, and assist in interpreting mass spectra in a reliable, cost-effective manner; 2) To develop new MS methods for NORC investigators; 3) To provide and maintain functional MS systems for use in nutrition- and obesity-related studies; and 4) To provide training to students and fellows in principles of MS and use of MS systems. The services offered by the BMAC reflect the evolving bioanalytical needs of NORC investigators, including targeted metabolomic services to broadly survey multiple metabolic pathways and to quantify pathway metabolites, and high-throughput, quality-controlled measurements of analytes in large sample sets from clincal studies.

ABSTRACT Niemann-Pick type C1 (NPC1) disease is a rare, neurodegenerative cholesterol storage disorder. Affected individuals typically present in early childhood with ataxia and progressive impairment of motor and intellectual function, and usually die in adolescence, though increasingly NPC disease is being recognized among the adult population with cognitive defects. There are currently no FDA-approved therapies for this progressively fatal neurodegenerative disorder. In preclinical studies, treatment with 2-hydroxypropyl-?-cyclodextrin (HP-?- CD) has been shown to reduce both cholesterol and sphingolipid storage and prolong survival, and has been advanced to clinical trials. A Phase 1/2a trial of intrathecal (IT) HP-?-CD was initiated at the NIH Clinical Center in January 2013, and in collaboration with Vtesse, Inc., an international, multisite Phase 2b/3 trial of IT VTS-270 (a specific formulation of HP-?-CD) was launched in September 2015. While IT delivery of VTS-270 in the clinical trials directly addresses the neurodegenerative component of NPC disease, visceral manifestations of the disease are left untreated. In infantile and juvenile forms of NPC disease, patients typically present with neonatal cholestasis or hepatosplenomegaly, and in severe cases may progress to liver failure. In older NPC patients, 2-3-fold chronic elevation of serum transaminases (ALT/AST) and liver inflammation are common. We hypothesize that reduction of hepatic cholesterol storage through intravenous (IV) delivery of VTS-270 will be effective in reducing liver inflammation in NPC1 patients. We will test this hypothesis by (1) performing a Phase 1/2a, open-label, single-center, randomized study of IV VTS-270 in human NPC1 subjects (? 3 years old) at the NIH Clinical Center, and (2) conducting a Phase 1/2a, open-label, multi-center, dose escalation study of IV VTS-270 to establish safety and potential clinical efficacy in treating cholestatic liver disease in infants (< 3 years old) with NPC1 at Johns Hopkins and Washington University sites. Our primary objective is to determine the safety and tolerability of intravenous VTS-270 in NPC1 disease. Secondary objectives will be to evaluate the efficacy of VTS-270 to reduce plasma cholestane-3?,5?,6?-triol, an NPC1-specific pharmacodynamic biomarker, and to normalize liver function tests, as well as the examination of exploratory lipid and protein biomarkers. Clinical efficacy will be evaluated by assessment of liver function tests, determination of liver size, and changes in liver histopathology. Biochemical efficacy will be assessed by measurement of plasma cholestane-3?,5?,6?-triol and other biomarkers. We anticipate that treatment with IV VTS-270 monthly will reduce liver inflammation and restore normal hepatic function. In both NPC groups, long-term treatment with IV VTS-270 would be expected to prevent liver fibrosis and lower risk of hepatocellular carcinoma.

ABSTRACT: Metabolomics Core The DRC Metabolomics Core provides rigorous mass spectrometry (MS) analyses to DRC Investigators that include quantification as well as structural characterization of diabetes-related biomolecules. The Core increases efficiency and cost effectiveness by providing centralized, standardized analyses to study molecular mechanisms of the pathogenesis of diabetes, its risk factors, and its complications. A major goal of the core is to promote use of MS methods in diabetes research by efforts in training, collaboration, development, service, and dissemination. Specific objectives of the Core are: 1) to provide and maintain functional MS systems for diabetes-related studies; 2) to consult with DRC investigators on application of MS to advance their research programs; 3) to perform service-related MS analyses for diabetes investigators, such as quantifying target analytes, obtaining spectra for structural identification, and assisting with mass spectra interpretation; 4) to develop new MS methods; and 5) to provide training to students and fellows in principles and use of MS systems. The services offered by the Metabolomics Core reflect the evolving bioanalytical needs of DRC investigators, including targeted metabolomic services to broadly survey multiple metabolic pathways and quantify pathway metabolites, in addition to high-throughput, quality-controlled measurements of analytes in large sample sets from clincal studies.

Cholesterol is an essential component of cellular membranes, a regulator of membrane protein function, and an obligate metabolic precursor. Exogenous cholesterol is acquired principally through receptor-mediated endocytosis of cholesteryl ester-laden low-density lipoproteins and trafficked to the lysosome. Lysosomal acid lipase liberates unesterified cholesterol, which is transferred by the Niemann-Pick C1 and C2 (NPC1, NPC2) proteins to the limiting lysosomal membrane and subsequently to other cellular organelles. Cholesterol exiting the lysosome is distributed to multiple compartments ? including the plasma membrane, ER, Golgi, mitochondria and peroxisomes ? and serves to regulate key cholesterol homeostatic responses. While several proteins have been implicated in post-lysosomal cholesterol trafficking, the components and relative contribution of each pathway and specific trafficking itinerary of the lipoprotein-derived cholesterol are poorly understood. This represents an important gap in our knowledge and an area in which advances have the potential to identify new therapeutic targets for treating both atherosclerotic and neurodegenerative diseases. We have generated novel cholesterol probes that can substitute for cholesterol and allow for the selective capture of cholesterol interacting proteins for unbiased identification by mass spectrometry. These probes provide a powerful new tool to study previously elusive post-lysosomal cholesterol trafficking pathways. We hypothesize the specific itinerary and metabolic fate of lipoprotein cholesterol involves both NPC1-dependent and -independent distribution pathways that can be identified based on unique cholesterol cargo interactomes. Elucidation of these protein networks through quantitative proteomics will identify major nodes through which lipoprotein cholesterol traffics and novel therapeutic targets..