Aldons J. Lusis - US grants

We believe that some fundamental problems of hematopoietic regulation may be answered by combining the concepts and techniques of molecular genetics with those of cell biology. We propose to use this approach to examine the structure, regulation and function of colony stimulating factors (CSFs) that regulate the production of granulocytes and macrophages from precursor cells. Our studies will focus on the mouse as a model organism, but we will also examine human cells. We recently achieved the first translation of mRNA for CSF and we now intend to expand translational studies to other sources and subtypes of CSF. The mRNA will be assayed either by its ability to give rise to biologically active CSF after microinjection into Xenopus laevis oocytes or by immunoprecipitation of CSF after in vitro translation in a rabbit reticulocyte system. The assays will permit us to: (1) examine the levels of CSF in different cellular sources; (2) characterize and compare the mRNA for the two major CSF subtypes; and (3) determine whether induction of CSF production in lymphocytes by lectins occurs at the level of mRNA. We will also undertake the molecular cloning of CSF sequences. The most promising CSF for cloning studies is mouse L cell CSF, which stimulates the production of macrophages. We have access to both purified L cell CSF and a high titer monospecific antibody to the factor. These tools should permit us to utilize the several techniques for isolation of a CSF cDNA, including polysome precipitation, hybrid selection, and protein sequencing combined with construction of synthetic deoxyoligonucleotide probes. The isolation of such a cDNA would likely lead to studies yielding information about (1) the structure and regulation of CSF genes; (2) evolutionary and functional relationships to other growth factors; (3) the structure of CSF at the level of protein; and (4) the expression of CSF genes in bacteria or eukaryotic cells, perhaps resulting in large quantities of the material for biological or clinical studies. Concommitently, we will carry out genetic studies with mice designed to examine the function and expression of CSF in vivo. Preliminary studies have revealed genetic polymorphisms among strains of mice in both the number of precursor cells and their responsiveness to CSF, and these genetic variants may be useful in mapping and characterizing the genes regulating granulopoiesis.

The overall objective of this proposal is to identify, map and characterize genetic factors affecting lipoprotein metabolism using a mouse animal model. During the previous five years, we have examined genes and proteins involved in lipid metabolism in the mouse and have identified and partially characterized a number of genetic variations affecting the levels and structures of lipoproteins. This proposal is directed at the further development of the mouse animal model and the molecular-genetic characterization of several polymorphisms of particular interest that have been identified in surveys of inbred strains of mice. Among these polymorphisms are the following: (i) Mutations of the apo AII structural locus that affect HDL size and apolipo-protein composition. (ii) Mutations of the Ath-1 gene which affect HDL levels in response to a high fat diet and also susceptibility to aortic intimal lipidosis. (iii) Mutations which control the levels and structures of LDL and VLDL particles on both chow and high fat diets. (iv) Mutations which control the tissue-specific expression of lipoprotein lipase. (v)Mutations which determine the levels of apo AIV mRNA and which affect the induction of apo AIV mRNA levels in response to a high fat diet challenge. Several of these naturally occurring polymorphisms in mice may prove to be valuable models for human polymorphisms.

molecular biology; blood lipoprotein metabolism; lipid metabolism; atherosclerosis; biomedical facility;

atherosclerotic plaque; blood lipoprotein metabolism; lipid metabolism; vasculitis; cholesterol; genetic mapping; farnesyl compound; dietary lipid; HMG coA reductases; low density lipoprotein; autoimmune disorder; genetic markers; calcification; gene expression; lipofuscin; pathogenic diet; RNase protection assay; laboratory mouse; genetically modified animals; polymerase chain reaction; enzyme linked immunosorbent assay; northern blottings; nutrition related tag;

SUBPROJECT ABSTRACT NOT AVAILABLE

SUBPROJECT ABSTRACT NOT AVAILABLE

The theme of this proposal is the examination of altered states of triglyceride metabolism contributing to coronary artery disease. The program addresses this theme using the mouse and human homology at the pathological, physiological, biochemical, genomic and molecular levels. Of the 5 projects in this proposal, Projects 3 will mostly involve studies in mice while 2 projects principally involve studies in humans, with 5 cores providing phenotyping, genotyping, sequencing, positional cloning, biostatistical and administrative support. Our major approach will be use genetic defects in mice and humans to identify underlying genes that contribute to altered states of triglyceride metabolism. We will emphasize familial combined hyperlipidemia (FCHL), a genetically complex disease characterized by increased plasma triglyceride metabolism. We will emphasize familial combined hyperlipidemia (FCHL), a genetically complex disease characterized by increased plasma triglyceride and/or cholesterol levels which accounts for up to 20% of premature coronary artery disease. Dr. Lusis' project will extend previous genetic studies in mice to systematically delineate genetic factors contributing to triglyceride metabolisms. Notably, will combine forces with Dr. Peltonen's Project to identify the gene affected by Hyplip1, a mutation in the mouse that causes combined hyperlipidemia and co- localizes with a homologous FCHL locus in humans. Dr. Wong's Project will focus on the structure-function properties of lipoprotein lipase (LPL), an enzyme central to triglyceride metabolism, as well as identifying genetic loci that affect LPL expression and may contribute to the LPL deficiency observed in FCHL. Dr. Reve's Project will isolate the genes, and characterize the function of the corresponding gene products, for two mouse mutations affecting triglyceride metabolism, fatty liver dystrophy (fld) and combined lipase deficiency (cld); these mutations are characterized by insulin resistance (fld) and LPL deficiency (cld) that often associated with FCHL. Dr. Rotter's project will use a combination of genome wide linkage and candidate gene association approaches to characterize genetic risk factors for coronary artery disease in human populations by identifying genes for lipid and lipoprotein variation in FCHL. Dr. Peltonen's project, using the power of a discrete population isolate (the Finn's) will perform fine mapping of a recently identified FCHL locus to isolate the predisposing FCHL gene by a positional cloning approach. Importantly, the Finnish FCHL locus is syntenic with mouse Hyplip1, and Project V will assist in isolating the mouse gene to complement and aid in the identification of the homologous human FCHL gene. Thus, the 5 projects provide a coordinated approach to identifying major genes affecting triglyceride metabolism and predisposing to coronary artery disease.

Specific Aim 1 is to dissect the genetic factors contributing to plasma lipoprotein metabolism in the mouse model. The approach has been to map candidate genes involved in plasma lipoprotein metabolism and to identify quantitative trait loci (QTLs) associated with plasma lipoprotein variations and obesity. In cases where candidate genes coincide with quantitative loci, we will look for variations in sequence or expression of the candidate gene. In selected cases, we will further characterize the QTL by isolating the responsible gene as a congenic mouse strain. In aims 2 and 3, we carry out detailed characterization of loci with important effects on lipid metabolism. Specific Aim 2 will characterize and identify a novel gene, Hyplip1, that causes combined hyperlipidemia in the mouse model. We will biochemically characterize the effects of the Hylip1 gene and perform fine-structure genetic mapping. A particularly exciting aspect of the Hyplip1 locus is its synteny to the QTL, for human familial combined hyperlipidemia studied in Project V. In order to better focus the efforts of both projects on this exciting region; positional cloning of the Hypli1 gene will be carried out in collaboration with Project V. Specific Aim 3 will determine the mechanism by which apolipoprotein AII influences triglyceride metabolism, particularly the insulin resistance phenotype we observe in apolipoprotein AII transgenic mice. Since these mice exhibit increased plasma levels of triglycerides and free fatty acids, as well as increased fat pad mass and insulin resistance, we have begun to investigate the effect of the transgene on adipocyte metabolism. Strikingly, we found that HDL from control mice can simulate hydrolysis of triglycerides from adipose tissue through a direct effect on adipocytes, a pathway not previously identified. By contrast, HDL from apolipoprotein AII transgenic mice, presumably enriched in apolipoprotein AII, has lost this ability. As part of this aim, we will examine the pathway by which HDL affects lipolysis in adipocytes.

This subproject will test hypotheses generated in our Program using transgenic and gene targeting technologies in mice. During the present grant period, our work has focused on aspects of oxidation (paraoxonase 1, myeloperoxidase, heme oxygenase-1, and secretory phospholipase A2) and macrophage function (macrophage colony stimulating factor, scavenger receptor CD68 and the nuclear receptor LXR). We now propose to extend some of these studies of paraoxonase-1 (PON1), heme oxygenase (HO-1) and macrophage colony stimulating factor (M-CSF) and to examine the functions of two additional proteins that have emerged as candidates from studies in this program: paraoxonase 3 (PON3) and 5-lipoxygenase (5-LO). PON1 and PON3 are members of a family of esterases, and both are carried on HDL. Using gene targeting, we provided strong evidence that PON1 protects against atherosclerosis and inhibits lipid oxidation. During the past two years, we and others have obtained evidence for a strong anti-oxidant function of PON3, and we will now test this using gene targeting. HO-1 has proved to be difficult to examine because HO-1 null mice breed poorly, although during the present grant period we did obtain evidence for its protective function in vivo using induction and inhibition with pharmacologic agents. We now propose to utilize transgenic approaches to pursue more detailed investigations of its role in atherosclerosis. M-CSF has been a long-term interest, since we originally cloned it in the mid-1980s. The expression of M-CSF is critical in the development of atherosclerosis in mice, and null mice have more than a 100-fold decrease in lesion development on an LDL receptor null background. But precisely how M-CSF contributes to the disease and which are the relevant sites of expression are unknown. These questions will be addressed using cell-specific knockouts of the M-CSF gene. Finally, 5-LO was identified in the subproject by Lusis as the gene controlling susceptibility to atherosclerosis in a genetic cross between inbred strains CAST and C57BL/6. 5-LO knockout were then shown to exhibit dramatically reduced atherosclerosis. We now propose to examine mechanistic questions pertaining to the role of 5-LO in atherosclerosis, including effects on monocyte proliferation and apoptosis.

This subproject will utilize genetic studies in mice to help identify new genes and pathways that are involved in the development of atherosclerotic lesions and inflammation. During the past grant period, this subproject 6 has focused on the analysis of naturally occurring variations in three separate genetic crosses, involving strains C3H/HeJ (C3H), CAST/Ei (CAST), and DBA/2J (DBA), each crossed to the atherosclerosis-susceptible C57BL/6J (B6) strain. These studies have revealed novel genetic factors for atherosclerosis involving vascular cells, monocyte/macrophages, and lipoprotein metabolism. We now propose to extend these studies in the same 3 sets of strains to address the following 3 mechanistic questions relating to the genetic determinants of atherosclerotic lesion development and lesion composition, with a focus on lipid oxidation, inflammation, and calcification. (1) How do oxidized lipids induce expression of inflammatory genes, and how do genetic variations in these pathways influence the development of atherosclerotic lesions? For these studies, we will focus on the C3H x B6 cross. In this model, we have produced strong evidence that the genes that control response of endothelial cells to oxidized lipids are the primary determinant of atherosclerosis susceptibility in this model. We will now identify the pathways and genes involved using in vitro and in vivo approaches. (2) What genetic factors contribute to the ability of HDL to protect against oxidative stress and inflammation? For this, we will focus on the CAST x B6 cross, for with multiple genetic loci controlling HDL levels and/or function have been isolated as congenic strains. The genes responsible will be identified and the functional properties defined in conjunction with the subproject by Fogelman. (3) What are the genetic factors contributing to vascular calcification, and what are the interactions between atherosclerosis, vascular calcification, myocardial calcification and bone metabolism? These aspects will be studied using the DBA x B6 cross. Loci on chromosomes 2 and 6 have been identified as influencing both bone density and lipid metabolism, a locus on chromosome 7 (Dyscalc) influences cardiovascular calcification and a locus on chromosome 10 influences atherosclerosis. Positional candidate genes in these regions will be investigated, and interactions between the loci will be studied.

This project is a continuation of a project in the original cycle of our PPG two decades ago. The concept is to apply the power of mouse genetics to understand, in vivo, the complex physiologic, cellular and molecular interactions that contribute to cardiovascular disease and the metabolic syndrome. During the current grant, we positionally cloned a gene in mouse that results in a combined hyperlipidemic phenotype. The gene proved to correspond to thioredoxin interacting protein (Txnip), a poorly understood protein that binds to and inactivates thioredoxin. Our recent studies and those of other laboratories have now implicated Txnip in fundamental regulation of both lipid and glucose metabolism. We will now examine the mechanisms involved using our mouse model. Another protein that we identified in genetic studies in mice is apolipoprotein All (apoAII). We have shown that the levels of this protein, ranging from null (apoAII knockout) to a few mg/dl (strain SM) to approximately 20 mg/dl (strain C57BL/6) to approximately 30 mg/dl (strain BALB/c) to approximately 100 mg/dl (apoAII transgenic) have ;a continuous, significant impact on insulin resistance, triglyceride levels, body fat, atherosclerosis and inflammatory properties of HDL. We now propose to further pursue the causal interactions involved. The third aim is to positionally clone a novel gene in mouse that contributes to combined hyperlipidemia and atherosclerosis. The last aim is to study in mouse a gene that was positionally cloned in the current grant in studies of Finnish familial combined hyperlipidemic pedigrees. The gene, USF1, will be examined in knockout and transgenic mouse models on a variety of backgrounds. Project I will interact with each of the other projects and with all the cores.

DESCRIPTION (provided by applicant): The theme of our Program Project is the molecular genetic analysis of the metabolic syndrome, particularly hypertriglyceridemia, insulin resistance and obesity. Our approach is to use mouse and human homology at the genetic, physiological, pathological and biochemical levels. During the current grant period, we have utilized this approach to identify three genes relevant to the metabolic syndrome-Txnip (thioredoxin interacting protein), lipin, and USF1 (upstream transcription factor 1). Txnip and lipin were identified via positional cloning in mouse models of combined hyperlipidemia and lipodystrophy, respectively, and function in fundamental processes such as adipose tissue development, and glucose and lipid homeostasis. USF1 is the first major gene identified for familial combined hyperlipidemia (FCHL), the most prevalent familial dyslipidemia predisposing to coronary heart disease. USF1 was identified by genetic analysis of families from the Finnish isolate, and we have recently confirmed its role in FCHL in three additional populations. We propose 4 Projects and 4 Cores that will interact closely. We will continue the strategy of using the mouse model and multiple human populations to search for additional genes and to characterize the metabolic dysregulation underlying the metabolic syndrome. Dr. Wong's Project will investigate mechanisms by which Txnip and apolipoprotein A-II function in glucose and lipid metabolism, identify the Hyplip2 gene which contributes to combined hyperlipidemia and atherosclerosis, and investigate USF1 function in mouse models. Dr. Pajukanta's Project will analyze multiple human FCHL populations to identify additional genes contributing to this disorder. Dr. Aldons' Project will isolate the gene causing combined lipase deficiency in a mouse model, further explore the structure of lipase enzymes, and determine the contribution of lipase genetic polymorphisms to insulin resistance in the metabolic syndrome. Dr. Reue's Project will further elucidate the physiolgical and molecular function of lipin in adipocyte differentiation and energy metabolism and evaluate its role in human disease. These projects will interact closely with Cores providing expertise and resources for Lipoprotein Analysis and Biochemistry, Genotyping and Sequencing, and Statistical Analysis.

DESCRIPTION (provided by applicant): The C57BLKS/J mouse (BKS), when carrying a mutation to the leptin receptor gene (BKS-db) is a classic model of obesity-induced diabetes in the mouse. Interestingly, >70% of the BKS genome is identical to that of C57BL/6J (B6) with the bulk of the remainder deriving from a DBA/2-like strain. And yet, the same leptin receptor mutation induces much less severe diabetes in the B6 than in the BKS mouse suggesting that the regions of introgressed DMA confer diabetes susceptibility. In preliminary work, we show that the 4-week old prediabetic BKS-db mouse is already severely insulin resistant and that hepatic lipogenic genes are generally suppressed compared to B6-db. In addition, we have used ultra-fine SNP mapping to precisely localize the introgressed DMA regions responsible for these phenotypes. Finally, we have developed a comprehensive set of congenic mouse strains with segments of DBA/2 DNA introgressed on a B6 background. These strains will allow us to test the impact of individual DBA regions on diabetes susceptibility in the BKS-db mouse. In this project, we will take a comprehensive approach to analysis of this striking diabetes susceptibility including (1) identifying and characterizing the associated shifts in lipid, glucose and insulin metabolism (2) mapping the responsible chromosomal loci, (3) identifying the underlying genetic variations and (4) characterizing specific shifts in metabolic pathways and networks that result from these variations. To accomplish this, we will take advantage of a number of recent developments in mouse genetics including complete DNA sequence information for several key mouse strains, high-density single nucleotide polymorphism mapping data for BKS and related strains, large scale expression array analysis applied to all animals in a genetic cross and, a set of newly emerging bioinformatics tools that use these data to prioritize candidate genes within each locus and to determine the metabolic networks involved. The results will provide an enhanced understanding of the mechanisms of obesity-induced diabetes.

This subproject will utilize genetic studies in mice to help identify new genes and pathways that are involved in the development of atherosclerotic lesions and inflammation. During the past grant period, this subproject 6 has focused on the analysis of naturally occurring variations in three separate genetic crosses, involving strains C3H/HeJ (C3H), CAST/Ei (CAST), and DBA/2J (DBA), each crossed to the atherosclerosis-susceptible C57BL/6J (B6) strain. These studies have revealed novel genetic factors for atherosclerosis involving vascular cells, monocyte/macrophages, and lipoprotein metabolism. We now propose to extend these studies in the same 3 sets of strains to address the following 3 mechanistic questions relating to the genetic determinants of atherosclerotic lesion development and lesion composition, with a focus on lipid oxidation, inflammation, and calcification. (1) How do oxidized lipids induce expression of inflammatory genes, and how do genetic variations in these pathways influence the development of atherosclerotic lesions? For these studies, we will focus on the C3H x B6 cross. In this model, we have produced strong evidence that the genes that control response of endothelial cells to oxidized lipids are the primary determinant of atherosclerosis susceptibility in this model. We will now identify the pathways and genes involved using in vitro and in vivo approaches. (2) What genetic factors contribute to the ability of HDL to protect against oxidative stress and inflammation? For this, we will focus on the CAST x B6 cross, for with multiple genetic loci controlling HDL levels and/or function have been isolated as congenic strains. The genes responsible will be identified and the functional properties defined in conjunction with the subproject by Fogelman. (3) What are the genetic factors contributing to vascular calcification, and what are the interactions between atherosclerosis, vascular calcification, myocardial calcification and bone metabolism? These aspects will be studied using the DBA x B6 cross. Loci on chromosomes 2 and 6 have been identified as influencing both bone density and lipid metabolism, a locus on chromosome 7 (Dyscalc) influences cardiovascular calcification and a locus on chromosome 10 influences atherosclerosis. Positional candidate genes in these regions will be investigated, and interactions between the loci will be studied.

This subproject will test hypotheses generated in our Program using transgenic and gene targeting technologies in mice. During the present grant period, our work has focused on aspects of oxidation (paraoxonase 1, myeloperoxidase, heme oxygenase-1, and secretory phospholipase A2) and macrophage function (macrophage colony stimulating factor, scavenger receptor CD68 and the nuclear receptor LXR). We now propose to extend some of these studies of paraoxonase-1 (PON1), heme oxygenase (HO-1) and macrophage colony stimulating factor (M-CSF) and to examine the functions of two additional proteins that have emerged as candidates from studies in this program: paraoxonase 3 (PON3) and 5-lipoxygenase (5-LO). PON1 and PON3 are members of a family of esterases, and both are carried on HDL. Using gene targeting, we provided strong evidence that PON1 protects against atherosclerosis and inhibits lipid oxidation. During the past two years, we and others have obtained evidence for a strong anti-oxidant function of PON3, and we will now test this using gene targeting. HO-1 has proved to be difficult to examine because HO-1 null mice breed poorly, although during the present grant period we did obtain evidence for its protective function in vivo using induction and inhibition with pharmacologic agents. We now propose to utilize transgenic approaches to pursue more detailed investigations of its role in atherosclerosis. M-CSF has been a long-term interest, since we originally cloned it in the mid-1980s. The expression of M-CSF is critical in the development of atherosclerosis in mice, and null mice have more than a 100-fold decrease in lesion development on an LDL receptor null background. But precisely how M-CSF contributes to the disease and which are the relevant sites of expression are unknown. These questions will be addressed using cell-specific knockouts of the M-CSF gene. Finally, 5-LO was identified in the subproject by Lusis as the gene controlling susceptibility to atherosclerosis in a genetic cross between inbred strains CAST and C57BL/6. 5-LO knockout were then shown to exhibit dramatically reduced atherosclerosis. We now propose to examine mechanistic questions pertaining to the role of 5-LO in atherosclerosis, including effects on monocyte proliferation and apoptosis.

Common human diseases result from the interplay of many genes and the environment. During the past few years, genome-wide association studies (GWAS) have succeeded in identifying many novel loci underlying cardiovascular disease traits. However, the genes are generally identified out of context and, in most cases, a very small fraction of the genetic component has been identified, even in very large studies. Our laboratory is employing a systems genetic approach to understand the higher order interactions in complex diseases. This involves the integration of DNA variation, global gene expression, and clinical phenotypes. In systems genetics, transcript levels are examined as a function of genetic variation, not simply in cases versus controls. We propose to apply this systems genetics approach to attempt to better understand the association between a haplotype on human chromosome 9p21 and coronary artery disease susceptibility. In Aim 1, we propose to characterize the patterns of expression of the genes in the 9p21 region in tissue culture in human cells and in mouse genetic crosses. In Aim 2, we analyze in detail the effects of the susceptibility alleles on RNA splicing and we examine the possibility the locus encodes microRNAs capable of regulating gene expression. In Aim 3 we utilize a series of transgenic/knockout models to test the role of genes at the locus and to validate findings from Aims 1 and 2.

DESCRIPTION (provided by applicant): Most epidemiologic studies of the role of HDL in atherosclerosis have emphasized HDL levels, but there is increasing evidence that HDL functioning may be critically important as well. It is clear that HDL from different individuals can differ strikingly in both structural and functional characteristics. In particular, there is evidence that HDL can lose its well-documented atheroprotective characteristics and even become pro-inflammatory. Studies of HDL function in humans are complicated by genetic heterogeneity and environmental factors. We propose to identify novel genes and pathways contributing to HDL functions using naturally occurring variations among inbred strains of mice. Two broad classes of HDL functioning will be examined. The first has to do with lipid transport, and particularly "reverse cholesterol transport", and the second with anti-inflammatory and antioxidant properties of HDL. In preliminary studies, we have discovered large variations among inbred strains in both lipid transport functions and anti-inflammatory functions. The underlying genes and pathways will be identified using novel strategies capable of high-resolution genetic mapping and a systems-based approach that integrates genetics, gene expression and clinical traits. In contrast to classical linkage mapping in mice, our novel, association- based approach allows direct identification of likely candidate genes. We also utilize mathematical analyses to model causal relationships and entire gene networks. To validate candidate genes and pathways, we will employ a variety of in vitro and in vivo approaches, including cultured hepatocytes, transgenic mice, and gene targeted mice. PUBLIC HEALTH RELEVANCE: Relevance to Public Health: HDL functions as well as levels are important in conferring protection against atherosclerosis. This study will define the genetic functions and pathways affecting the HDL functions using experimental mouse models.

PROJECT SUMMARY (See Instructions): During the present cycle of our PPG, we have developed a novel approach, which we term integrative genetics, to help identify genes and pathways associated with the Metabolic Syndrome (MetSyn). We have also developed a new mapping tool in mice, which we term a mouse diversity panel (MDP), which allows high resolution mapping of traits such as gene-by-environment interactions. In the present proposal, we will apply these tools to two basic questions concerning the metabolic syndrome. The first question has to do with the nature ofthe molecular networks underlying human MetSyn traits. In our previous integrative genetics studies, we examined both molecular phenotypes (transcript levels) and clinical phenotypes in segregating mouse populations. This allowed us to identify genetic loci controlling transcript levels and model co-expression networks. We will now extend this approach to human populations. In collaboration with Dr. Markku Laakso, we will examine DNA variation and transcript levels in fat biopsies from 1,000 individuals in a MetSyn study population that has been typed for the major MetSyn traits. We will then identify genes and co-expression networks related to clinical traits. Gene-by-diet interactions are critically important in MetSyn, but they are notoriously difficult to study directly in human populations. We will use our mouse diversity panel to examine differences in biologic networks and clinical traits in mice maintained on either a chow diet or a high fat diet for 8 weeks. This will allow us to map genes controlled dietary responsiveness of MetSyn traits and to medol to co-expression networks perturbed by these genes. The mouse and human data will be integrated and aspects relevant to this program validated in collaborative studies with other Projects and the Cores.

Dr. Lusis will be responsible for the day-to-day operation ofthe Program Project, with frequent consultation with the Co-Principal Investigator, Dr. Karen Reue. The Administrative Core has been organized to assist the Principal Investigators in this task. One Administrative Assistant, Melenie Resales is located at UCLA. A committee composed of Dr. Lusis, Dr. Davis and Dr. Pajukanta assisted by Ms. Resales will be responsible for the preparation and submission ofthe human use protocols to the Human Subjects Protection Committee. The three major functions of the Administrative Core are: (1) Administrative: This includes functions which are not routine, but require organizational skills and initiative from our senior administrative personnel. These includes the daily supervision of clerical and accounting functions, but also they include translating into action the decisions made by the Principal Investigators, usually with the concurrence ofthe Internal Advisory Committee, without an undue expenditure ofthe investigators'time. This often requires considerable skills on the part of the administrative assistants to make the detailed changes necessary to redirect the flow of money or effort occasioned by the decision, and to assure that all ofthe Program Project personnel affected by the decision understand the administrative changes. (2) Clerical: Word processing and record keeping aspects of the Program Project must function routinely and accurately if the day-to-day operations ofthe Program Project are to proceed smoothly and well. It is important that competent administrative assistants oversee and participate in these routine operations, providing the essential discipline and continuity required for a successful operation. (3) Accounting: Accurate and prompt accounting is essential for the financial health of the Program Project. All personnel matters for the staff of the Program Project are coordinated by the administrative staff; including hiring, merit increases, and termination of Program Project employees. In addition, the Administrative Core is responsible for ordering and receiving all supplies purchased for the Cores and projects located at UCLA. Melenie Resales, Administrative Assistant to the Program Project, is located in an office adjacent to Dr. Lusis and is responsible for day-to-day administrative details and for expediting the decision made by the Principal Investigators. She will work closely with Ms. Freda Azbijari, an accountant, located and paid by the Dept. Of Medicine. Ms. Azbijari coordinates all UCLA and Cedars-Sinai;expenditures, and prepares each month an updated budget report showing how much has been spent or committed, allowing us to plan and project for the remainder of the budget year. In addition to day-to-day administration and accounting Ms. Resales and Ms. Azbijari will meet biweekly with Dr. Lusis, to discuss new decisions, new problems, and to follow-up on the progress made toward expediting decisions and solving problems. Ms. Resales will prepare an agenda and take notes of the discussion and decisions made at these meetings while Ms. Azbijari will report on the current budget status each month. Another administrative duty is arranging the meetings with External and Internal consultants. In general, these are attended by all Program Project investigators.

DESCRIPTION (provided by applicant): The pathogenesis of heart failure is complex, involving heterogeneous genetic and environmental factors. Genome-wide association studies (GWAS) have had limited success for heart failure and the genetic factors contributing to human heart failure remain poorly understood. A major challenge in the field is to fully understand the heterogeneity of the disease in order to develop more effective and personalized therapies. In this proposal, we have developed a novel approach by using GWAS across a hybrid mouse diversity panel (HMDP) under a well defined pathological stressor to systematically identify genetic factors and molecular networks implicated in heart failure. We believe this novel approach has several major advantages. First, the HMDP consists of ~100 common inbred and recombinant inbred (RI) strains which have been either entirely sequenced or densely genotyped [over 140,000 single nucleotide polymorphisms (SNPs)]. Second, the insights learnt from mouse models of heart failure should provide relevant guidance for future mechanistic, epidemiological and genetic studies in human. Lastly, Chronic excessive adrenergic overdrive is a well recognized major contributor to human heart failure and understanding the genetic modulators to betaAR signaling would have a major impact. With precisely administered chronic treatment of isoproterenol, a non-selective betaAR agonist, we will be able to quantitatively inflict a pathological insult in a relatively high-throughput manner. Accordingly, we propose to 1). Identify genetic loci in mouse contributing to cardiac responses to chronic beta-adrenergic stimulation by quantitative analysis of cardiac function and remodeling in the HMDP mice in response to chronic isoproterenol stimulation, and association analysis with an efficient mixed model algorithm to identify regions of the genome and potential candidate genes linking to the different features of cardiac response to chronic beta-adrenergic stimulation. 2). Model pathways contributing to regulation of heart function and hypertrophy by global expression array analyses of hearts before and after isoproterenol treatment to map loci contributing to differences in gene expression that are associated with chronic beta-adrenergic response, and construction co-expression networks to identify subnetworks associated with chronic beta-adrenergic response. In short, the systems approach designed in this proposal will bring novel insights to genes and their interacting networks implicated in betaAR signaling and heart failure.

DESCRIPTION (provided by applicant): Diabetic complications including heart disease, stroke, nephropathy, neuropathy, retinopathy, infectious diseases, sexual dysfunction, bone loss and dental diseases, are highly significant health problems and the underlying molecular pathways, likely modulated by hyperglycemia, remain poorly understood. In this study, we will use a combination of genetics and systems biology to identify molecular pathways that contribute to diabetic nephropathy and cardiovascular complications. We will use a novel mouse genome wide association strategy across a hybrid mouse diversity panel (HMDP). The HMDP consists of ~100 common inbred and recombinant inbred (RI) strains which have been either entirely sequenced or densely genotyped. We have already demonstrated the power of this approach to detect and finely map associations for complex traits, to suggest pathways associated with the traits and to rapidly identify the underlying gene variations. Because the HMDP is renewable (strains are commercially available), the genotyping does not need to be repeated and the panel can be assayed for multiple phenotypes providing cumulative biological insights. To identify the genetic factors contributing to diabetic complications, we will generate diabetic and euglycemic mice by crossing each strain of the HMDP to Akita/+ mice. The mouse Akita mutation (Ins-2 gene) induces the unfolded protein response and apoptosis in pancreatic islet beta cells, even in heterozygous F1 animals. Our early studies have shown that diabetic hyperglycemia severity is comparable across F1 animals with diverse genetic backgrounds. By contrast, different F1 strains exhibit varying responses to diabetes with respect to complications, including nephropathy, heart function, vascular calcification, and atherosclerosis. We will take advantage of current high-throughput transcriptomic and metabolomic assays, combined with dense genotype information and recent advances in computational methods, to identify candidate genes responsible for these diabetic complications. The candidate genes and pathways will be validated in genetically modified mice or in cell-based systems. We have recently shown that hyperglycemia induces bone morphogenetic protein (BMP) signaling in cultured human endothelial cells and in the blood vessels of Akita mice. This pathway clearly contributes to diabetes-mediated vascular calcification and we will test the hypothesis that BMP signaling is also involved in other macro- and micro-vascular complications of diabetes. The team of investigators has strong expertise in the characterization of multiple diabetic complications as well as a proven record in the analysis of complex traits in mice. To our knowledge, this novel project is the first large scale integrated genetics study to identify and finely map hyperglycemia modulated molecular pathways that determine susceptibility to the complications of diabetes. PUBLIC HEALTH RELEVANCE: Over 20 million people in the U.S. suffer from diabetes mellitus. And, while the risk for heart disease, stroke, nephropathy, neuropathy, retinopathy, infectious diseases, sexual dysfunction, and dental diseases are markedly increased in diabetic individuals, the underlying molecular pathways for these diabetic complications remain poorly understood. The goal of this project, identifying molecular mechanisms responsible for diabetes accelerated diseases, remains a critical goal in the drive to develop better treatments and preventative measures.

DESCRIPTION (provided by applicant): Unraveling the genetic basis of common polygenic diseases, such as hypertension, diabetes and heart failure, will require fresh approaches to view how genes work together in groups rather than singly. In this proposal, we investigate gene network analysis as a promising new approach. Our goal is to identify specific expression patterns of gene modules, rather than single genes, which predict susceptibility to heart failure (HF). A network analysis of DNA microarray data typically groups 20,000 genes into 20-30 modules, each containing 10's to 100's of gene, drastically reducing number of possible candidates required to perform a gene network- based Gene Module Association Study (GMAS), which will be complementary to GWAS. To test the GMAS concept, we will use a systems genetics approach integrating DNA microarray analysis with physiological studies and computational modeling, to examine whether gene module expression patterns predict susceptibility to heart failure (HF) induced by cardiac stress. For this purpose, we will utilize a novel resource developed at UCLA, the Hybrid Mouse Diversity Panel (HMDP), consisting of 102 strains of inbred mice from which a common mouse cardiac modular gene network comprised of 20 gene modules has been constructed. Our preliminary findings reveal that different HMDP strains show considerable variability in both gene module expression patterns and phenotypic response to chronic cardiac stress (isoproterenol). Using biological and computational experiments, we will test the hypothesis that gene module expression patterns among HMDP strains represent different good enough solutions, all of which are adequate for normal excitation-contraction- metabolism coupling, but have different abilities to adapt to chronic cardiac stress. Three Specific Aims integrating experimental and computational biology and combining discovery-driven, hypothesis-driven, and translational elements are proposed, towards the goal of relating HMDP results directly to human heart failure.

DESCRIPTION (provided by applicant): Congestive heart failure is a complex disease involving multiple genetic and environmental factors. Three years ago, the laboratories of Dr. Yibin Wang, a molecular biologist with expertise in heart failure, and Dr. Aldons Lusis, a geneticist working in the area of cardiovascular disease, joined forces to perform a genetic screen in a hybrid mouse diversity panel (HMDP) to identify genes contributing to common forms of heart failure. For the past two years, this work has been supported by a multi- PI R21. This support enabled us to complete the preliminary screen and identified over 30 genome-wide significant loci harboring genes contributing to different aspects of cardiac pathologies induced by chronic stimulation of the ?-adrenergic agonist isoproterenol (ISO), including hypertrophy, fibrosis, and cardiac dysfunction and remodeling. Moreover, gene expression profiles were obtained from all HMDP mouse hearts in control mice and following ISO treatment. These rich datasets containing genetic information detailed cardiac phenotype parameters and comprehensive cardiac transcriptome profiles from 107 inbred strains of mice will allow us to harness the power of genetics and systems approaches to identify novel molecular pathways contributing to the specific aspects of cardiac pathology during heart failure. Indeed, we observed a dramatic diversity of heart failure phenotypes among all HMDP strains following ISO stimulation and discovered a number of genetic loci and gene modules with significant association with cardiac hypertrophy and fibrosis. These data support the overall hypothesis that common genetic variants have a major contribution to the pathogenesis of heart failure. Uncovering the mechanistic basis of these newly discovered HF associated genes and their interactions via systems approach is the overarching goal of this proposal. Specifically, in Aim 1, we will extend our systems studies to discover genes and gene modules significantly associated with cardiac pathology induced by chronic angiotensin II treatment (AngII). We will identify unique and common genes involved in ?AR vs. ?AR-specific pathogenesis in heart. In Aim 2, we will investigate the molecular mechanisms underlying a candidate gene associated with heart failure, Miat, that encodes a long-non-coding (lnc)RNA with a previously unknown function in heart. In Aim 3, we will investigate the mechanism and functional role of Abcc6, a GWAS candidate gene, in stress induced cardiac fibrosis. These studies will reveal the underlying genetic contributions to specific features of heart failure, and the uncovered novel pathology associated genes and their interaction should provide new insights to the mechanism of the disease.

PROJECT SUMMARY Project 1 The nature of gene-by-diet interactions in obesity and other metabolic syndrome (MetSyn) traits is not well understood. Such interactions are likely to be key in understanding the worldwide ?epidemic of obesity?, particularly given the recent data implicating gut microbiota in cardio-metabolic traits. We propose a three- pronged approach to the problem. First, we will dissect gene-by-diet interactions affecting MetSyn traits (obesity, insulin resistance, fatty liver, plasma lipids) in a mouse population, the Hybrid Mouse Diversity Panel, that enables fine genetic mapping using association. Second, we will identify the underlying biochemical pathways using a systems genetics approach that allows us to follow the flow of information from DNA to transcriptome to proteome to metabolome to gut microbiome to MetSyn traits. Third, we will extend these findings to a human population, the METabolic Syndrome In Man (METSIM) study which consists of more than 10,000 men from Kuopio, Finland, that have been exquisitely characterized for MetSyn clinical traits, DNA variation and adipose tissue molecular phenotypes. The results will define mechanisms and genetic variations that underlie the striking divergent responses of individuals to unhealthy ?Western?-style diets rich in fat and sugar.

? DESCRIPTION (provided by applicant): The overall objective of the Center Pharmacogenomics of Statin Therapy (POST) is to apply genomic, transcriptomic, and metabolomic analyses, together with studies in cellular and animal models, and innovative informatic tools, to identify and validate biomarkers for efficacy of statin drugs in reducing riskof cardiovascular disease (CVD), and for adverse effects of statins, specifically myopathy and type 2 diabetes. This multidisciplinary approach is enabled by a team of investigators with expertise in genomics (human, mouse, and molecular), statistics and informatics, and clinical medicine and pharmacology. The Center is comprised of three Projects, two Research Cores, and an Administrative Core. A major aim of Project 1 is the identification of cellular transcriptomic and metabolomic markers for clinical efficacy and adverse effects of statins. This will be accomplished by analyses in statin-exposed lymphoblast cell lines derived from patients with major adverse coronary events, or onset of myopathy or type 2 diabetes on statin treatment, compared with unaffected statin- treated controls. In addition, using genome wide genotypes from these patients, DNA variants will be identified that are associated with statin-induced changes in the transcripts and metabolites that most strongly discriminate affected patients and controls. Project 2 will use a unique, well-characterized panel of 100 inbred mouse strains to discover genetic variation associated with statin-induced myopathy and dysglycemia. Mechanisms underlying these effects will be investigated, with emphasis on the role of dysregulation of autophagy by statin treatment. Projects 1 and 2 will also use relevant cellular and mouse models, respectively, to perform functional studies to validate effects of genes identified in all POST projects as strong candidates for modulating statin efficacy or adverse effects. In Project 3, information derived from genome-wide genotypes, electronic health records, and pharmacy data in a very large and diverse population-based patient cohort will be leveraged to identify and replicate genetic associations with statin efficacy (lipid lowering and CVD event reduction) and adverse effects (myopathy and type 2 diabetes), as well as to assess the overall heritability of these responses. The Clinical Core, based in Kaiser Permanente of Northern California, will provide the clinical information and biologic materials for both Projects1 and 3. Investigators in the Informatics Core will optimize data analysis and integration of results across all projects. The Administrative Core will provide scientific leadership and management of the Center, and foster scientific interactions and training opportunities. Overall, the research program of this Center provides an innovative model for a systems approach to pharmacogenomics that incorporates complementary investigative tools to discover and validate genetically influenced determinants of drug response. Moreover, the findings have the potential for guiding more effective use of statins for reducing CVD risk and minimizing adverse effects, and identifying biomarkers of pathways that modulate the multiple actions of this widely used class of drugs.

PROJECT SUMMARY/ABSTRACT In the present cycle, we developed a renewable mouse resource for genetic analysis of complex cardiovascular traits. The resource, which we term the Hybrid Mouse Diversity Panel (HMDP), consists of 100 selected inbred strains that have been largely sequenced and that exhibit great diversity in their responses to atherosclerosis. This resource can be used to map complex traits with excellent resolution and to perform pathway analysis using systems genetics . In Aim 1, we propose to exploit this resource for the analysis of arterial inflammation and intestinal lipid metabolism, the themes of this Program. We will examine higher order genetic interactions using pathway analysis and network modeling to identify novel mechanisms for atherosclerosis in collaboration with the projects in this PPG. We will also create and maintain a systems genetics database for the larger cardiovascular research community. In Aim 2, we will study in detail a novel regulatory pathway, involving the transcription factor Zhx2, that we identified in the present cycle using a systems genetics approach. Mice carrying a naturally occurring deficiency of Zhx2 expression have a 10-fold to 20-fold reduction in lesion size, and bone marrow transplantation studies indicate that this effect is mediated largely by hematopoietic cells. The mutation does not affect levels of monocytes, but rather promotes macrophage apoptosis, likely involving the NF-?B pathway. We will identify the pathways perturbed by Zhx2 deficiency and examine macrophage survival and growth using in vivo labeling and parabiosis experiments.

PARENT ABSTRACT The overall objective of the Center Pharmacogenomics of Statin Therapy (POST) is to apply genomic, transcriptomic, and metabolomic analyses, together with studies in cellular and animal models, and innovative informatic tools, to identify and validate biomarkers for efficacy of statin drugs in reducing risk of cardiovascular disease (CVD), and for adverse effects of statins, specifically myopathy and type 2 diabetes. This multidisciplinary approach is enabled by a team of investigators with expertise in genomics (human, mouse, and molecular), statistics and informatics, and clinical medicine and pharmacology. The Center is comprised of three Projects, two Research Cores, and an Administrative Core. A major aim of Project 1 is the identification of cellular transcriptomic and metabolomic markers for clinical efficacy and adverse effects of statins. This will be accomplished by analyses in statin-exposed lymphoblast cell lines derived from patients with major adverse coronary events, or onset of myopathy or type 2 diabetes on statin treatment, compared with unaffected statin-treated controls. In addition, using genome wide genotypes from these patients, DNA variants will be identified that are associated with statin-induced changes in the transcripts and metabolites that most strongly discriminate affected patients and controls. Project 2 will use a unique, well- characterized panel of 100 inbred mouse strains to discover genetic variation associated with statin- induced myopathy and dysglycemia. Mechanisms underlying these effects will be investigated, with emphasis on the role of dysregulation of autophagy by statin treatment. Projects 1 and 2 will also use relevant cellular and mouse models, respectively, to perform functional studies to validate effects of genes identified in all POST projects as strong candidates for modulating statin efficacy or adverse effects. In Project 3, information derived from genome-wide genotypes, electronic health records, and pharmacy data in a very large and diverse population-based patient cohort will be leveraged to identify and replicate genetic associations with statin efficacy (lipid lowering and CVD event reduction) and adverse effects (myopathy and type 2 diabetes), as well as to assess the overall heritability of these responses. The Clinical Core, based in Kaiser Permanente of Northern California, will provide the clinical information and biologic materials for both Projects 1 and 3. Investigators in the Informatics Core will optimize data analysis and integration of results across all projects. The Administrative Core will provide scientific leadership and management of the Center, and foster scientific interactions and training opportunities. Overall, the research program of this Center provides an innovative model for a systems approach to pharmacogenomics that incorporates complementary investigative tools to discover and validate genetically influenced determinants of drug response. Moreover, the findings have the potential for guiding more effective use of statins for reducing CVD risk and minimizing adverse effects, and identifying biomarkers of pathways that modulate the multiple actions of this widely used class of drugs. ADMINISTRATIVE SUPPLEMENT ABSTRACT In response to NOT-AG-18-008, our goal is to extend the validation and application of our data integration methodologies into Alzheimer?s disease research. This administrative supplement is designed to extend the work of the existing subaward to the University of Pennsylvania subcontract for the POST Informatics Core. The PGRN POST Informatics Core serves as the central hub for data sharing and coordination across the three POST projects in the PGRN P50 award. One of our jobs is annotating the extensive information that will be collected and providing analysis expertise to the projects as needed. However, to make great strides in scientific progress and ensure that the collective whole of the Center is greater than the sum of the parts, a key function of the Informatics Core is to serve as ?The Integrator? to combine these data and information. We and others have shown that integration of complementary omics-based data can provide emergent insights into biological processes compared to what can be learned through any single approach alone. The methods that we are developing to integrate data for statin pharmacogenomic phenotypes will be equally applicable in the area of Alzheimer?s disease. Additionally, recent emphasis on open data science by Alzheimer?s disease researchers provides ample data for us to interrogate our method. We have developed novel statistical analysis tools such as the Analysis Tool for Heritable and Environmental Network Associations (ATHENA), and data visualization tools, such as PhenoGram, both of which are designed to collect and combine information from diverse data sources. With these tools, we will leverage publicly available Alzheimer?s disease datasets to maximize the knowledge gleaned about disease risk for Alzheimer?s diseases. The methodologies that we have been developing as part of the PGRN POST award for the past 2.5 years are clearly applicable to the study of Alzheimer?s disease risk. An important validation step of the application of our methodologies is to apply them to different types of datasets and in different phenotypic areas. This administrative supplement focused on extended research into having an Alzheimer?s disease focus is a great mechanism to simultaneously allow us to validate our methodologies with different types of data and potentially identify important risk factors and pathways toward a better understanding of the etiology of Alzheimer?s disease. Finally, we may have the opportunity to identify cross biological implications due to the known pleiotropic relationships between Alzheimer?s disease and cardiovascular disease.

Project Description Gut microbiota have been associated with many different disorders, including cardiovascular disease. One common mechanism involves the production, from dietary components, of metabolites that enter the circulation and affect physiologic functions such as inflammation. We propose to perform a comprehensive screen of gut microbiota-derived metabolites that contribute to cardio-metabolic disorders. Using a panel of genetically diverse inbred strains of mice, we will identify microbes and microbiota-derived metabolites that associate with atherosclerosis, followed by validation in human cohorts and mechanistic studies in germ-free mice. The work will be done in three laboratories with complimentary skills: A. Lusis (genetics), F. Rey (microbiology), and Z. Wang (metabolomics). All of the investigators have worked together for several years. The proposal represents an extension of a screen we previously performed using a panel of 100 inbred strains of mice for atherosclerosis (900 mice total). In that screen, we observed over a 200-fold range of lesion development. We now propose to analyze the microbiomes (Aim 1) and plasma metabolomes (Aim 2) of the mice and to relate these to atherosclerosis traits. We will then prioritize the significant associations by studying these in an atherosclerosis case-control human population (Aim 3). Finally, we will study the mechanisms by which the metabolites affect disease using germ-free mouse models (Aim 4). In preliminary studies, the levels of trimethylamine-N-oxide, another microbe-derived molecule shown to contribute to human atherosclerosis, were significantly correlated with lesion development. And, using a subset of the panel, we identified two microbes (A. muciniphila and R. intestinalis) associated with cardiometabolic traits and showed that these exhibited the predicted effects when used to colonize mice. These preliminary studies provide strong validation for the overall approach. We anticipate identifying several novel metabolites associated with atherosclerosis and related traits, and exploring the underlying mechanisms. This should pave the way for novel therapies that target the microbiome.

PROJECT SUMMARY This proposal is a continuation of work currently funded as a project in a PPG headed by Dr. Alan Fogelman. The PPG must be discontinued because of a limit on the number of cycles allowed, a recent NIH rule. During the last cycle of the grant, we studied 100 diverse strains of mice on a ?humanized? hAPOE- Leiden, hCETP background for atherosclerosis traits and for global transcriptomics and metabolomics. We now propose to analyze the data using novel computational approaches, including integration with human data from Genome-Wide Association Studies (GWAS) and expression datasets such as STARNET (Aim 1). We will also continue to make our ?systems genetics? data available to all interested investigators, noting that they have now proved useful to many laboratories (Aim 1). Such data generate hypotheses which must be experimentally tested, and we have chosen two genes/pathways based on our long-term interest in inflammation. We will study macrophage colony stimulating factor (M-CSF) as a key regulator of macrophage proliferation (Aim 2). We originally identified M-CSF and other CSFs as the first molecular markers of inflammation in atherosclerosis several decades ago and have continued to study them. Our preliminary data indicate that local M-CSF regulation is key in atherogenesis, and we will test the hypothesis and explore the roles of the three major isoforms. We will also study the transcription factor Zhx2, which we very recently showed to be a key driver of macrophage apoptosis in lesions (Aim 3). Our preliminary data suggest that it interacts with cholesterol loading and other stresses which will be tested. We note that genetic ablation of these two genes has some of the largest effects on lesion size observed. We anticipate that our studies will provide a more comprehensive view of the pathways underlying CVD, a better integration of human and mouse data and an improved understanding of macrophage growth and turnover in lesions. We are hopeful that the studies will lead to new therapeutic or diagnostic advances.

PROJECT SUMMARY Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder which comprises of a spectrum of hepatic abnormalities ranging from simple steatosis to steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Despite significant research efforts, the etiology of this disease is poorly understood; in particular, factors associated with progression from steatosis to NASH are unknown. We have developed mouse models from the Hybrid Mouse Diversity Panel (HMDP) that exhibit the spectrum of NAFLD observed in humans. The overall goal of our proposal is to use population-based approaches in mice to identify pathways and higher order biological networks that contribute to the development and progression of NAFLD. Using Mergeomics, an association-based modeling method we developed, we previously identified and validated several genes associated with steatosis from a cohort of HMDP mice fed a high fat, high sucrose diet. Applying the same strategy to a novel transgenic HMDP mice model of NASH, we have now identified several high confidence NASH candidate genes. In Aim 1, we will perform transcriptomic and metabolomics profiling on resistant and susceptible strains to examine the progression of NASH. We will identify and validate candidate genes for NASH progression using multi-omics approaches and Adeno-Associated Virus (AAV) vectors for rapid screening in mice. We will also identify cell-specific changes in gene expression and cell composition related to liver fibrosis and other NAFLD features. This will allow us to follow functional changes in the major hepatic cell types as well as populations of stellate cells and infiltrated inflammatory cells during NASH progression. In Aim 2, we will examine five prioritized genes contributing to hepatic fibrosis, including one gene, Mgp, that we recently validated using knockout mice. Mechanistic studies will be performed to investigate how these genes affect fibrosis. Additional candidate genes identified in Aim 1 will be examined with a similar strategy. Results from these studies will reveal the underlying genetic mechanisms contributing to NAFLD and may identify potential therapeutic targets.

Project Summary Human and mouse studies have identified changes in the gut microbiome associated with progression of atherosclerosis. While gut microbes provide many benefits to the host (e.g. provide metabolic capabilities not represented in our human genome), they also can be detrimental. Coexistence with our gut microbes is largely enabled by the intestinal barrier?composed of a luminal mucus layer, epithelial cells and an inner functional immunological barrier? which limits the entry of toxins and microbial pro-inflammatory molecules. Previous studies have shown that diet and microbial metabolism modulate intestinal barrier function. Recent work from our team and others has linked changes in the gut microbiome with alterations in intestinal barrier function and cardiometabolic disease. However, the role of intestinal barrier function on atherosclerosis development and the microbial, dietary and host factors that control this process remain largely unexplored. Defining these will open new avenues for disease prevention and treatment, as both diet and the gut microbiome can be modified. We have identified both microbial and host targets associated with intestinal homeostasis, inflammation, and atherosclerosis. Briefly, we examined a panel of over 100 different genetically diverse inbred strains of mice (known as the Hybrid Mouse Diversity Panel, HMDP) for both atherosclerosis susceptibility and gut microbiota composition. In this screen, we identified several bacterial taxa associated with atherosclerosis protection and experimentally validated one predicted protective microbe, Roseburia intestinalis, whose effect depends on the availability of dietary substrates (i.e., fiber) that promote its growth and butyrate production. Moreover, we showed that the beneficial effects of R. intestinalis are associated with improved intestinal barrier function and lower plasma levels of LPS. Furthermore, these effects are mimicked by delivering butyrate to the distal gut. Our HMDP studies also revealed a poorly understood protein expressed primarily in intestinal dendritic cells and macrophages, ADAM-like Decysin-1 (Adamdec1), as a protein responsive to microbiome composition and contributing to intestinal homeostasis, glucose homeostasis and systemic inflammation. In this application, we propose to follow-up on these exciting observations to gain novel mechanistic insights into how modulation of intestinal homeostasis via diet-butyrate-producing bacteria interactions and Adamdec1 affect progression of atherosclerosis. We provide a strong validation for the overall approach, and the work will be done in two laboratories with complementary skills: Dr. Rey (microbiology, gnotobiotic mouse models) and Dr. Lusis (genetics, atherosclerosis). The investigators have worked together for several years. We anticipate discovery of novel mechanisms by which gut bacteria modulate development of atherosclerosis, which should pave the way for novel cardiovascular disease therapies that target the gut microbiome.

ABSTRACT Daily physical activity is a known intervention to prevent or ameliorate complications associated with metabolic-related diseases. However, the mechanisms underlying metabolic adaptations to chronic exercise training remain inadequately understood. Moreover, human studies show a broad range of exercise adaptation with some individuals refractory to specific metabolic improvements associated with training. Since conventional animal studies have focused primarily on few rodent strains, in the current application we will interrogate exercise training adaptations in ~100 diverse male and female strains of inbred mice known as the UCLA Hybrid Mouse Diversity Panel (HMDP), and integrate these data with existing MoTrPAC data repositories at the Bioinformatics Center (BIC), Stanford University. In Aim 1 we will determine the regulatory loci controlling exercise metabolism and integrate several ?omics? platforms (transcriptomics, proteomics, metabolomics) using Bayesian analyses and Mergeomics to identify regulatory networks and key driver nodes underlying exercise training in mice and validate these findings with publicly available data from the MoTrPAC consortium studies of exercise training in rats and humans. In Aim 2 we will use Quantitative Endocrine Network Interaction Estimation (QENIE) to functionally annotate novel inter-tissue communication circuits (between cardiac and skeletal muscle, and liver, and fat) that are critical for endurance exercise adaptation. Our findings provided herein show remarkable, sex-specific tissue crosstalk that occurs to maintain metabolic homeostasis and in response to exercise. We will construct communication networks between the four tissues in mouse and validate these against the plasma proteome of human and rat (provided by the MoTrPAC consortium BIC) similar to multi-species validation studies published previously by our group (PMCID6399495, 5935137). Moreover, we will also perform molecular validation studies confirming exercise-stimulated changes in novel circulating factors, and determine functionality of these communication networks using conventional loss and gain of expression approaches. Our findings will be of strong scientific impact as we will identify novel exercise-induced regulatory nodes and key driver pathways underlying improvements in metabolism, determine novel exercise driven endocrine interactions, and generate a mouse sample biobank and data repository that will be integrated into the MoTrPAC data hub for novel hypothesis generation by the entire research community.