At the Program Project Grant at Atherosclerosis Research Unit, the focus is on the artery wall metabolism as it relates to inflammation and cardiovascular diseases.
In project 1, among other topics, the role of minimally oxidized LDL and phospholipid oxidation products; endothelial cell regulation; mechanisms of oxidation; inflammation; regulation of monocyte and neutrophil entry into the vessel wall and lipoprotein receptors are being studied. Oxidized phospholipids were shown in several studies to be found in the vasculature of animal models of atherosclerosis, in human atherosclerotic lesions, and in other inflammatory diseases.
In addition notch signaling in blood vessels; cellular and molecular mechanisms of vascular lumen formation; extracellular matrix, inflammation, and the angiogenic response and signaling circuitry in vascular morphogenesis are being investigated in Project 1.
The focus of Project 2 has been the cell biology of the artery wall and lipid metabolism; growth factors and endothelium. The antioxidant and anti-inflammatory properties of high density lipoprotein; the role of dysfunctional HDL in atherosclerosis; apolipoprotein A-I mimetic peptides and their role in atherosclerosis prevention and the effect of apolipoprotein mimetic peptides in inflammatory disorders other than atherosclerosis are among the areas being investigated. Here, oxidized phospholipids were demonstrated to cause vascular and nonvascular cells to initiate an inflammatory reaction. Determination of the mechanisms of action of apolipoprotein mimetic peptides in inflammation and atherosclerosis are among areas being actively pursued. To obtain apoA-I mimetic peptide at optimal cost the peptide has been expressed in tomatoes and it was shown that it reduces the systemic inflammation and atherosclerotic lesions in a mouse model. Studies with apolipoprotein A-I mimetic peptides suggest that the small intestine is a major tissue regulating systemic inflammation and may be important for determining the functionality of HDL.
The overall goal of Project 3 is to understand the role of bone morphogenetic proteins (BMPs), and their inhibitors and receptors in vascular disease such as atherosclerosis, vascular calcification, diabetic vascular disease and arteriovenous malformations (AVMs). A regulatory pathway was identified in which BMP-2/4 induces the expression of activin-like kinase receptor 1 (ALK1), a receptor for BMP-9. ALK1 is essential for angiogenesis and regulates expression of vascular endothelial growth factor (VEGF) and BMP-inhibitors including matrix Gla protein (MGP). It was found that this regulatory pathway is affected in vascular disease and appears to be essential for vascular homeostasis. A new model of adult stem cells, the so-called DFAT cells, for use in our studies has been established in Project 3.
Project 4 studies the nuclear receptors LXR and FXR and their roles in lipid homeostasis. Particular emphasis is placed on the LXR target gene ATP-binding cassette transporter G1 (ABCG1) and its role in sterol homeostasis and on the role of FXR in regulating metabolic pathways.
In Project 5 the overall goal is to understand the factors affecting susceptibility to cardiovascular and metabolic disorders. Common forms of these disorders involve many genetic and environmental contributions, greatly complicating both genetic and biochemical approaches. To simplify the analyses, Project 5 studies traits relevant to these disorders, such as atherosclerotic lesions, heart remodeling, obesity, and insulin resistance, among inbred strains of mice.
The power of mouse genetics. Over the last 100 years, a number of tools have been developed for studies with mice, such as the creation of inbred strains, the culture of embryonic stem cells and the engineering of genes. These make the mouse the most useful mammal for genetic studies. Among the hundreds of inbred strains of mice (each representing a unique gene pool in which natural variations have been fixed by inbreeding) are variations relevant to most human disorders. For example, when placed on a hyperlipidemic background, the size of atherosclerotic lesions varies more than 100-fold among the various strains. The challenge is to identify the underlying genetic differences and the pathways that they perturb.
Genetic dissection of complex traits in mice. In order to identify the genes underlying complex traits in mice, it is important to be able to map them with high precision. Traditional quantitative trait locus (QTL) analysis, involving genetic crosses between strains, has poor resolution, and has been only modestly successful. Together with the Eleazar Eskin group in Computer Sciences at UCLA, an association-based strategy was developed in Project 5 that has greatly improved resolution and made it possible to directly identify strong candidates. Second, to understand the pathways perturbed by the various genes, systems-based approaches along with recently developed high throughput technologies, such as expression arrays, next generation sequencing, and mass spectrometry was developed. Systems-based approaches, as contrasted with reductionistic approaches, attempt to move beyond the perspective of single genes to groups of genes. This is particularly important for studies of complex traits since they result from interactions between genes and between genes and the environment. Finally, classical gene engineering approaches can be used to test the resulting hypotheses.
Why not just study human populations? Over the last several years, human genetic studies, particularly genome-wide association studies (GWAS), have taught us a great deal about many different common disorders. But these have important limitations, such as the inability to identify interactions and to analyze molecular mechanisms. Notably, GWAS studies of most straits have succeeded in explaining a very small fraction of the genetic component. For example, GWAS of about a quarter million individuals for body mass index explained only about 1.5% of the trait variation, despite the fact that the trait has more than 50% heritability. Clearly, studies in animal models, where the genetics and environment can be controlled, will be essential for a full understanding of complex diseases. http://labs.genetics.ucla.edu/lusis/
Among the areas that the Project 6 concentrates on are: nuclear receptors in lipid metabolism; endocrine functions of adipose tissue; the diverse biology of PPARgamma and integration of metabolism and inflammation by lipid-activated nuclear receptors..
The nuclear hormone receptors are a family of ligand-activated transcription factors that play diverse roles in mammalian physiology. While it has long been recognized that these proteins are central to development and homeostasis in vertebrate organisms, recent work has begun to define an unexpected role for members of this superfamily in human disease. Obesity, diabetes and cardiovascular disease are the leading causes of morbidity and mortality in industrialized societies. The common thread that links these disorders is a dysregulation of lipid metabolism. Recent years have seen a new paradigm emerge for the transcriptional regulation of metabolic pathways with the discovery of nuclear receptors that are activated by lipids. Included in this group are PPARgamma, which is activated by fatty acids, and LXR, which is activated by cholesterol metabolites. These receptors modulate differentiation and lipid homeostasis in multiple cell types, and the pathways they control have critical links to metabolic disease. The present focus is on defining the role of the PPAR and LXR signaling pathways in macrophages and adipocytes. In addition coordination of inflammation and metabolism by PPAR and LXR nuclear receptors; adipocyte progenitors; GPIHBP1, an endothelial cell transporter for lipoprotein lipase; LXR signaling pathways and atherosclerosis; transcriptional and posttranscriptional control of cholesterol homeostasis by LXRs; transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR and feedback regulation of cholesterol uptake by the LXR-IDOL-LDLR axis are being studied. A combination of molecular biology, mouse genetics and gene array technology is used in the studies and the goal is to understand the importance of these receptors in both normal physiology and in disease. As ligand-activated transcription factors, nuclear receptors make ideal drug targets. Recent work suggests that both PPARg and LXR may represent targets for therapuetic intervention in atherosclerosis.
Core A, Essential Laboratory Services
Core Leader: Jesus Araujo, M.D., Ph.D. – Associate Professor
Co-Core Leader: Diana Shih, Ph.D. – Associate Professor
Core A has been a critical element in this Program Project since its inception. There are two components to this core. The CHS portion of Core A provides human and mouse cells (monocytes and endothelial cells) to the Component Projects as well as general services such laboratory maintenance and oversight of environmental safety compliance. The MRL portion of Core A provides analytical chemistry assays for the Component Projects and other Cores. Core A continues to be a cost-effective way of providing these essential services to the Component Projects and other Cores.
Core B, Morphology Core
Core Leader: Thomas A. Drake, M.D. – Professor
Collaborators: Michael C. Fishbein, M.D. - Professor
The Morphology Core provides comprehensive morphologic procedures and analyses for the Component Projects. All Component Projects will use genetically defined and/or engineered mice that will require morphologic analysis. Thus, a Core is required to provide the highest quality, most efficient, and cost- effective morphologic services. The most heavily used service of this Core has been and will continue to be quantitative analyses of atherosclerotic lesions. The Core will provide quantitative and detailed cellular analysis of atherosclerotic lesions in the aortic root and innominate arteries of mice and will provide quantitative analysis of the percent of aorta with atherosclerotic lesions using en face analysis. Additionally, a broad range of morphologic procedures are required by all the Projects that includes analysis of various of tissues for specific cells, and labeling and analysis of cultured cells and isolated cells with fluorescent probes for confocal microscopy. This Core is a cost-effective, efficient and high quality means of providing these services.
Core C, Transgenic and Gene Targeting Core
Core Leader: Diana M. Shih, Ph.D. – Associate Professor
Collaborator: A. Francis Stewart – Professor
Core C will assist the component projects in the construction of novel, genetically modified animal models by gene targeting and transgenic mouse techniques. This core will also help in the construction of recombinant adenoviruses. Most component Projects in the current grant cycle have used the services of this Core and in the next grant cycle all 6 component Projects are expected to use the services of this core.
Lusis Lab: Director: Aldons J. Lusis, Ph.D.
For the past 30 years, our lab has been interested in common, complex forms of cardiovascular and metabolic disorders. In an attempt to simplify the analysis of such multifactorial traits, we helped develop mouse models of lipid metabolism and atherosclerosis in the early 1980s. Once the models were established, we spent much of the 1980s cloning candidate genes involved in lipoprotein metabolism and mapping them in mice and humans. In the early 1990s, with the development of more sophisticated methods for mapping complex traits, we were among the first groups to utilize quantitative trait locus (QTL) analysis. Using this approach, we mapped numerous loci for obesity, lipoprotein metabolism, and atherosclerosis. But, like other researchers, we found it difficult to find the underlying genes because of the poor resolution of QTL mapping.
With the completion of the Genome Project in the early 1990s, it became possible to examine gene expression on a global level. In the early 1990s, we integrated transcriptome microarray analysis with common genetic variation in mice to help develop an approach known as “systems genetics”. The idea is very simple: genetic variation contributes to complex traits such as atherosclerosis in part by perturbing patterns of gene expression and, by monitoring such patterns globally, we hope to understand some of the underlying disease pathways. We found that it was possible to map, either in mice or in humans, thousands of loci contributing to variations in gene expression, and these were termed expression quantitative trait loci, or eQTL. We also developed statistical methods to connect such loci to complex clinical traits.
Recently, together with the laboratory of Eleazar Eskin in Computer Sciences, we developed a reference resource for systems genetics called the Hybrid Mouse Diversity Panel (HMDP). The HMDP consists of about 100 common and recombinant inbred strains which have been entirely sequenced or densely genotyped. The HMDP strains are commercially available and, thus, can be assayed for multiple phenotypes by different laboratories, providing cumulative biological insights. This, of course, is ideal for a systems genetics approach, since multiple “intermediate phenotypes” such as transcript levels, protein levels, and metabolite levels, can be assayed in genetically identical animals and then integrated. The HMDP also has two additional very important advantages. First, the mapping resolution in the HMDP, which involves association, is much better, by at least an order of magnitude, than classical linkage analysis. Second, the HMDP is ideal for examining gene-by-environment interactions, since genetically identical animals can be examined before and after a particular environmental perturbation.
Over the past several years, genome-wide association studies in large human populations have revealed numerous loci contributing to common diseases. However, it has proved difficult to understand the pathways by which the genetic variations contribute to disease, and such studies are poorly powered to examine environmental interactions. A major problem in human studies, of course, is the difficulty of obtaining appropriate tissues which are required for analysis of molecular phenotypes such as transcript levels or protein levels. Our current studies are focused on aspects of common metabolic and cardiovascular disorders that are difficult to address directly in humans. In particular, we are utilizing systems genetics along with the HMDP (and, in some cases, primary human cell lines) to help dissect the genetics of obesity, atherosclerosis, and heart failure.
Biomineralization Research Laboratory.
Co-Principal Investigators: Linda L. Demer, MD, PhD and Yin Tintut, PhD.
Our laboratory pioneered studies of the mechanisms of artery wall calcification and its paradoxical relationship with osteoporosis. We currently focus our investigations on the mechanisms of inflammatory factors on vascular and bone cell calcification as well as on bone anabolic effects of intermittent parathyroid hormone treatment.
Our current funding is from the NIH [National Heart Lung and Blood Institute (NHLBI) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)].