Changcheng Zhou, Ph.D. - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Obesity is associated with several diseases. Inflammation is a common link between obesity and associated diseases. Two such diseases influencing the obese population are diabetes in the form of insulin resistance, and coronary artery disease in the form of atherosclerosis. Both of these obesity-induced complications are associated with inflammation in either the blood vessel wall (atherosclerosis) or in adipose tissue (insulin resistance). Many inflammatory pathways that contribute to atherosclerosis initiation and development are regulated by transcriptional factor nuclear factor-[unreadable]B (NF-[unreadable]B), a central coordinator of the innate and adaptive immune responses (8, 9). NF-[unreadable]B is rapidly activated in response to various stimuli, including cytokines, infectious agents and reactive oxygen species (10). I[unreadable]B kinase [unreadable] (IKK[unreadable]) is the predominant catalytic subunit of the IKK complex that is required for activation of NF-[unreadable]B by inflammatory mediators such as TNF[unreadable] and IL-1s in the canonical or classical activation pathway (11, 12). In the past decade, IKK[unreadable] has been established as a critical molecular link between inflammation and pathogenesis of several chronic diseases such as cancer. However, the role of IKK[unreadable] in atherosclerosis has not been thoroughly investigated. IKK[unreadable] activation has been detected in atherosclerosis and in vascular inflammatory reactions in animal models and humans (13-15). Despite the strong evidence suggesting the involvement of IKK[unreadable] activation in atherosclerosis progression, the role of IKK[unreadable]-mediated inflammatory functions by macrophages in atherosclerosis remain unclear. The previous studies using various mouse models have given inconsistent results and further research is urgently needed to define the role of macrophage IKK[unreadable] in atherosclerosis. Preliminary data demonstrate that deficiency of IKKbeta in macrophages promotes inflammation. In addition, pilot studies indicate that deficiency of IKKbeta in macrophages reduces diet-induced atherosclerosis. We hypothesize that IKKbeta plays an important role linking macrophage inflammation to obesity-induced atherosclerosis and insulin resistance. Studies will address the role of macrophage IKKbeta in experimental models of diet-induced atherosclerosis and insulin resistance.

DESCRIPTION (provided by applicant): The goal of this project is to investigate a novel mechanism linking exposure to endocrine disrupting chemicals (EDCs) with dyslipidemia and cardiovascular disease. The pregnane X receptor (PXR) is a nuclear receptor activated by numerous drugs, xenobiotic and dietary chemicals. Many EDCs activate PXR, including organochlorine and organophosphate pesticides, alkylphenols, phthalates, polychlorinated biphenyls, bisphenol A and its analogs. However, the role of PXR in mediating the pathophysiological effects of EDCs in humans and animals remains elusive. Mounting evidence implicates EDC exposure in the development of chronic human diseases but the contribution of these EDCs to the etiology of cardiovascular disease (CVD), obesity, and diabetes is poorly understood. We have recently revealed the pro-atherogenic effects of PXR in animal models and demonstrated that chronic PXR activation induces hyperlipidemia in wild-type mice and increases atherosclerosis in atherosclerosis-prone apolipoprotein E deficient (ApoE-/-) mice. Our central hypothesis is that EDCs which activate PXR will lead to hyperlipidemia and accelerated atherosclerosis in mice, thereby increasing the risk of CVD in exposed individuals. We propose the following specific aims to test this hypothesis: 1) What are the molecular mechanisms through which intestinal PXR activation induces hyperlipidemia? 2) How does exposure to FDA-approved phthalate substitute plasticizers elevate plasma lipid levels in mice? 3) Is EDC-mediated PXR activation necessary and sufficient to increase atherosclerosis development in ApoE-/- mice? Our research will establish the role of PXR in linking exposure to EDCs with hyperlipidemia and CVD, and will provide novel mechanistic links explaining how EDC exposure causes atherogenic effects. These studies are broadly translational and will provide strong evidence to inform future risk assessment for EDCs.

The goal of this project is to investigate the role of IKB kinase beta (IKKB), a central coordinator of innate immunity and inflammation through activation of NF-KB, in linking obesity to adipose tissue inflammation and adipogenesis. Obesity is associated with a state of chronic low-grade inflammation that has been considered to be a major contributor to diabetes and atherosclerosis. The transcriptional factor NF-KB is a primary mediator of inflammatory pathways that are linked to the development of obesity-associated insulin resistance and atherosclerosis. IKKB is the predominant catalytic subunit ofthe IKK complex that is required for canonical activation of NF-KB by inflammatory mediators. Given the defined role of IKKB in regulating NF-KB-mediated inflammation in several cell types, it is unclear whether IKKB contributes to obesity-induced inflammation in adipocytes. In the proposed studies, we will define the role of adipocyte-derived IKKB in high fat (HF) diet-induced obesity, adipose inflammation and insulin resistance. In preliminary studies during Phase I support of this project, we also defined the role of smooth muscle cell (SMC) IKKB in the development of obesity-associated atherosclerosis in LDL receptor (LDLR) deficient mice. To delete IKKB in SMCs, we used SM22-Cre transgenic male mice carrying floxed IKKB alleles that were mated to female LDLR-/- mice carrying floxed IKKB alleles. During the course of these studies, we made the novel finding that deficiency of IKKB in SMCs rendered mice resistant to the development of diet-induced obesity. Preliminary results demonstrate that SM22 is expressed in primary adipose stromal/vascular (SV) cells, consistent with recent reports that SM22-Cre is active in mesenchymal stem cells (MSCs) that give rise to adipose tissue. Notably, SV cells from SMC IKKB-deficient mice exhibited impaired adipogenic potential. These results implicate a previously unrecognized role of IKKB in the regulation of adipogenesis. The central hypothesis of this proposal is that HF-diet induced activation of IKKB promotes adipocyte differentiation, adipocyte inflammation, and insulin resistance. In the proposed studies, we will define mechanisms for IKKB-mediated regulation of adipogenesis. We will also define the effect of adipocyte IKKB deficiency on the development of obesity, adipose inflammation, and insulin resistance in response to a HF diet. The proposed studies will elucidate new aspects of adipocyte biology and metabolic control and may lead to novel therapeutic targets for obesity and diabetes.

Project Summary This is an R21 application intended to investigate the mechanisms of atherogenic effects of bisphenol A (BPA). BPA is a base chemical used extensively in polycarbonate plastics in many consumer products, and human exposure to BPA is ubiquitous. Higher BPA exposure has recently been associated with an increased risk of cardiovascular disease (CVD) in multiple human population-based studies. However, the mechanisms responsible for these associations remain unknown. Many environmental chemicals can activate the xenobiotic receptor pregnane X receptor (PXR) which, in turn, acts as a xenobiotic sensor to regulate xenobiotic metabolism and exhibits considerable differences in its pharmacology across species. Very recently, we revealed PXR's pro-atherogenic effects in animal models and found that chronic activation of mouse PXR increases atherosclerosis in atherosclerosis-prone apolipoprotein E deficient (ApoE-/-) mice. We also demonstrated that BPA is a potent activator of human but not mouse PXR, consequently, the choice of animal model is paramount in predicting the human risk assessment of BPA. Therefore, we generated novel PXR- humanized ApoE-/- mice (huPXR¿ApoE-/-, i.e., ApoE knockout mice with the human PXR transgene in place of mouse PXR) that can respond to human PXR ligands. Our central hypothesis is that chronic activation of PXR by exposure to BPA promotes foam cell formation and increases atherosclerotic lesion formation in PXR- humanized mice, thereby increasing the risk of CVD in exposed individuals. Two specific aims are proposed to test this hypothesis: 1) Does chronic exposure to BPA at doses relevant to human exposure increase atherosclerosis in PXR-humanized ApoE-/- mice? 2) What molecular pathway does BPA act through to influence atherogenesis? The proposed studies are the first to investigate the effects of BPA exposure on atherosclerosis in a suitable animal model, and to explore the precise molecular mechanisms by which BPA induces CVD at the molecular level.

Project Summary The rise in long-term HIV survivorship as a result of the introduction of effective antiretroviral therapy (ART) over the past decades has generated new awareness of the sequelae of both HIV infection and of the therapeutics used to treat HIV patients. Accumulating evidence of comorbidities associated with HIV infection and treatment with antiretroviral (ARV) drugs includes significant dyslipidemia and elevated risk of developing atherosclerotic cardiovascular disease (CVD). The goal of this project is to investigate a novel mechanism of ART-associated dyslipidemia and cardiovascular disease (CVD). Several ARV drugs have been identified as potent agonists for the pregnane X receptor (PXR), a nuclear receptor activated by numerous drugs, xenobiotic and dietary chemicals. PXR functions as a xenobiotic sensor that induces expression of genes required for xenobiotic metabolism in the liver and intestine. In the past decade, the function of PXR in the regulation of drug and xenobiotic metabolism has been extensively studied by many laboratories and the role of PXR as a xenobiotic sensor has been well established. We have recently revealed the pro-atherogenic effects of PXR in animal models and demonstrated that chronic PXR activation induces hyperlipidemia in wild-type mice and increases atherosclerosis in atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice. Preliminary studies demonstrated that HIV protease inhibitor amprenavir activates PXR and induces hyperlipidemia in wild- type mice but not in PXR-deficient mice; furthermore, follow-up study of currently used first-line ARV drugs subsequently found that these drugs activated PXR and induced PXR-mediated gene expression. The goal of this project is to generate novel mechanistic insights into ART-associated dyslipidemia and risk of CVD. Our central hypothesis is that long-term treatment with ARV drugs that activate PXR will lead to aberrant lipid homeostasis and accelerated atherosclerosis. We propose the following specific aims to test this hypothesis: 1) Determine the contribution of PXR towards the adverse effects of currently recommended ARV drugs on lipid homeostasis; 2) Define the molecular mechanisms through which PXR agonistic ARV drugs induce hyperlipidemia; and 3) Determine the impact of ARV drug-mediated PXR activation on macrophage functions and atherosclerosis development. These studies will provide critical mechanistic insights and new understandings of ART-associated dyslipidemia and CVD risk and will have direct clinical consequences for patients undergoing long-term treatment with PXR agonistic drugs.

? DESCRIPTION (provided by applicant): Obesity is associated with a state of chronic inflammation that is thought to be a major contributor to diabetes and atherosclerosis. Many inflammatory pathways that contribute to the development of insulin resistance and atherosclerosis are regulated by IKK?/NF-?B signaling. However, the role of IKK? in metabolic disorders and atherosclerosis remains elusive. Our initial studies showed that IKK? deficiency (driven by a SM22Cre-IKK?- flox system) in smooth muscle cells (SMCs) rendered mice resistant to the development of diet-induced obesity, metabolic disorders, and atherosclerosis. We made the novel finding that SM22 is expressed in primary adipose stromal vascular (SV) cells and IKK? positively regulates adipogenesis, indicating that chronic inflammation might be an important initial trigger for the adipocyte differentiation in response to high-fat diet consumption. Our central hypothesis is that IKK? is essential for adipogenesis and atherogenesis, and that high-fat diet-induced IKK? activation promotes adipocyte differentiation and adipose inflammation, leading to increased obesity, insulin resistance, and atherosclerosis. Three specific aims are proposed: (1) Define the role of IKK? in the regulation of murine and human adipocyte differentiation in response to overnutrition; (2) Determine the contribution of adipocyte IKK? to the development of obesity-associated insulin resistance and atherosclerosis; and (3) Determine the impact of pharmacological inhibition of IKK? by antisense oligonucleotides on diet-induced obesity and atherosclerosis. The proposed studies will define the role of IKK? in adipogenesis and atherogenesis and will reveal a critical link between overnutrition, inflammation, and cardiometabolic disease. Our studies will also provide strong evidence for use of appropriate IKK? inhibitors as an innovative therapeutic strategy to treat obesity and cardiometabolic disease.

The goal of this project is to investigate the role of IKB kinase beta (IKKß), a central coordinator of innate immunity and inflammation through activation of NF-?B, in linking obesity to adipose tissue inflammation and adipogenesis. Obesity is associated with a state of chronic low-grade inflammation that has been considered to be a major contributor to diabetes and atherosclerosis. The transcriptional factor NF-?B is a primary mediator of inflammatory pathways that are linked to the development of obesity-associated insulin resistance and atherosclerosis. IKKß is the predominant catalytic subunit of the IKK complex that is required for canonical activation of NF-?B by inflammatory mediators. Given the defined role of IKKß in regulating NF-?B-mediated inflammation in several cell types, it is unclear whether IKKß contributes to obesity-induced inflammation in adipocytes. In the proposed studies, we will define the role of adipocyte-derived IKKß in high fat (HF) diet-induced obesity, adipose inflammation and insulin resistance. In preliminary studies during Phase I support of this project, we also defined the role of smooth muscle cell (SMC) IKKß in the development of obesity-associated atherosclerosis in LDL receptor (LDLR) deficient mice. To delete IKKß in SMCs, we used SM22-Cre transgenic male mice carrying floxed IKKß alleles that were mated to female LDLR-/- mice carrying floxed IKKß alleles. During the course of these studies, we made the novel finding that deficiency of IKKß in SMCs rendered mice resistant to the development of diet-induced obesity. Preliminary results demonstrate that SM22 is expressed in primary adipose stromal/vascular (SV) cells, consistent with recent reports that SM22-Cre is active in mesenchymal stem cells (MSCs) that give rise to adipose tissue. Notably, SV cells from SMC IKKß-deficient mice exhibited impaired adipogenic potential. These results implicate a previously unrecognized role of IKKß in the regulation of adipogenesis. The central hypothesis of this proposal is that HF-diet induced activation of IKKß promotes adipocyte differentiation, adipocyte inflammation, and insulin resistance. In the proposed studies, we will define mechanisms for IKKß-mediated regulation of adipogenesis. We will also define the effect of adipocyte IKKß deficiency on the development of obesity, adipose inflammation, and insulin resistance in response to a HF diet. The proposed studies will elucidate new aspects of adipocyte biology and metabolic control and may lead to novel therapeutic targets for obesity and diabetes.

The Energy Balance and Body Composition Core (Core D) serves investigators across the institution to quantify energy balance and body composition in mice. Accurate quantification of these parameters is essential for studies focused on obesity, with wide-spread capabilities for metabolism-related studies. The core houses 24 LabMaster TSE® chambers that quantify food and water intake, physical activity and indirect calorimetry in mice, a common model for studies focused on the effect of diet or genetics on the development of obesity. The Core also houses an EchoMRI® to quantify lean and fat mass and total body water in conscious mice. Both systems allow for non-invasive longitudinal assessment of energy balance and body composition. Competent core staff maintain and oversee the use of the LabMaster TSE® system and train personnel in use of the EchoMRI®. Core Directors assist in study design and data handling and analysis. Over 50 Principal Investigators use the LabMaster TSE® system annually for studies on >700 mice/year, while the EchoMRI® is used by more than 25 laboratories annually. These systems are not duplicated at the institution or within the Commonwealth, and are vital to our ability to reduce the epidemic of obesity in Kentucky. In Phase III of the program, we will broaden our focus to include obesity as a risk factor for cardiovascular diseases, diabetes, cancer and neurodegenerative diseases. This will increase use of the system to support an anticipated larger user base with targeted growth within these areas across the institution. We will also purchase a new system, from institutional support, that will markedly expand capabilities of this core in quantifying energy metabolism in mice. Also, during Phase III of the program the core for this system, including all equipment, will be relocated to a modern animal care facility within a new research building that is dedicated to the study of these health disparities. We anticipate that this core will transition to self-sustaining capacity over the course of Phase III support of the COCVD.

PROJECT SUMMARY Despite major advances in developing diagnostic techniques and effective treatments, atherosclerotic cardiovascular disease (CVD) is still the leading cause of mortality and morbidity worldwide. Recent large- scale human studies have implicated a novel link between exposure to endocrine disrupting chemicals (EDCs) and CVD. However, how exposure to EDCs influences CVD risk is still poorly understood, and continues to hamper rational assessment of the health risks of EDC exposure. We have previously identified many plastic- associated EDCs as potent agonists of the xenobiotic sensor pregnane X receptor (PXR), which has provided an important tool for the study of new mechanisms through which EDC exposure impacts diseases. Our laboratory was the first to reveal the novel function of PXR in the regulation of atherosclerosis development, and has also demonstrated that several widely-used EDCs increase atherosclerosis and dyslipidemia through PXR signaling in mouse models. To understand the detailed mechanisms underlying EDC-induced dyslipidemia and atherosclerosis, novel tissue-specific PXR knockout mice have been generated, and preliminary studies demonstrated that exposure to a newly identified PXR agonistic EDC increased intestinal lipid absorption, hyperlipidemia, and hepatic steatosis in a PXR-dependent manner. Further, EDC-mediated PXR activation led to elevated circulating levels of ceramides, a class of bioactive sphingolipids that has been independently associated with increased CVD risk in humans. Our exciting preliminary findings support a central hypothesis that plastic-associated EDCs that activate PXR stimulate intestinal lipid absorption and ceramide production, leading to increased dyslipidemia, hepatic steatosis, and atherosclerosis. We propose three specific aims to test this hypothesis: 1) Determine the tissue-specific contribution of PXR signaling towards EDC-induced dyslipidemia and ceramide production using novel conditional knockout mice; 2) Define the enterohepatic signaling through which PXR agonistic EDCs regulate lipid and ceramide homeostasis; and 3) Determine the impact of EDC-mediated PXR activation on atherosclerosis development. Influences of the chemical environment on human health have become the subject of intense interest but very few studies in the EDC research field have focused on the impact of EDCs on atherosclerosis development. This renewal application will expand our initial research scope, pursue new research directions, utilize newly developed animal models, and combine in vitro, ex vivo, and in vivo approaches to investigate EDCs? atherogenic effects. The proposed studies will contribute to our understanding of ?gene-EDC interactions? in predisposing individuals to atherosclerosis and other chronic diseases.