Thematic Research Projects
The thematic research areas of Wyoming INBRE-2 are structured around what are perceived as strengths and as having significant health relevance to make maximum use of existing research expertise. Cardiovascular diseases, diabetes and obesity are among the leading causes of morbidity and mortality in the United States, including rural areas like Wyoming. Effective management of cardiovascular diseases, especially cardiometabolic diseases, to improve the quality of life and reduce the overwhelming health care costs has become a burning issue for NIH and the American Heart and American Diabetes Associations. Eight research projects led by 8 junior investigators recruited to UW have been chosen. Four projects will be focused on cardiovascular diseases and four on diabetes and obesity. As diabetes and obesity are also newly classified under the cluster of cardiometabolic diseases, there are substantial interactions among these projects; especially between the basic science and clinical studies. Three of the projects are community-based research projects. Career development plans and discrete mentorships have been developed for all project investigators.
CURRENT THEMATIC INVESTIGATOR PROJECTS:
John Oakey Ph.D., Chemical Engineering, High Throughput Screening of β-Cell Response to Encapsulation
Pancreatic islet of Langerhans transplantation is an experimental, yet highly promising therapy for insulin-dependent diabetes mellitus (type I diabetes). However, a persistent obstacle to successful transplantation is the immunorejection of donor islets. One solution, islet encapsulation, protects islets from immunorejection by providing a physical barrier through which secreted insulin can diffuse while preventing contact with host cells and antibodies. This strategy promises to provide patients with functional, insulin-producing islets without need for immunosuppresants. Many biomaterials have been explored as candidate encapsulants, including a variety of natural and synthetic polymers. Photopolymerized poly(ethylene glycol) (PEG) hydrogels are particularly attractive cell encapsulants as a result of their excellent mechanical properties, diffusive properties, cytocompatibility and biopassivitiy within the body. The conditions that maximize β-cell efficacy and function, however, are a complex amalgam of PEG properties and the function of secondary extracellular matrix proteins that are formulated into the hydrogel. Given the number of components, formulation variables and degrees of freedom, a very small fraction of the overall composition space has been surveyed for the influence of cell-material interactions upon cell viability and function.
Our goal, therefore, is to create a platform that accelerates the process of design and discovery by screening candidate materials, conditions and cellular responses far more quickly and efficiently. This platform will utilize microfluidic cell encapsulation to rapidly generate micro-hydrogel particles containing individual cells and a broad range of material compositions. Hydrogel particles and indicators of cellular response will be evaluated by custom-built microfluidic flow cytometry. Success of this project will produce diabetes-specific outcomes in the form of high-throughput screening tools for guiding cellular therapies. More broadly, we will have created and demonstrated a platform by which we can screen any cell-matrix interaction, which represents a profound capability for the fields of tissue engineering regenerative medicine.
The initial phase of this project have been focused upon enabling technology and process development. We have successfully built and validated systems for producing hydrogel particles and screening particles. As a result, we have developed a hardware platform that is capable of producing large quantities of monodisperse polyethylene glycol (PEG)-based particles and a homebuilt flow cytometer that integrates with microfluidic flow devices. We have successfully made batches of particles with a broad range of PEG weight fraction and relative molecular weight that are barcoded to allow for the accurate optical discrimination of the particles’ polymer composition. We are currently producing batches of PEG particles that contain fluorescently-labeled proteins in order to study diffusion through hydrogel matrices in order to better understand the relationship of macromolecular composition to network structure and thereby molecular diffusivity. This study is important for three reasons: 1) It provides us with a simple, dynamic system with which we can validate our hardware, 2) it leverages the utility of high-throughput screening and multi-temporal screening to address an issue that has been data-limited and, 3) it leads us to the next phase of our project in which hydrogel particles and indicators of cellular viability and response will be evaluated in our flow cytometers. We are actively seeking funding for fundamental instrumentation development and disease-specific studies.
Machender Kandadi, Ph.D. School of Pharmacy, Role of Reactive oxygen species (ROS) in anthrax lethal toxin associated cardiac dysfunction
Bacillus anthracis infection is a highly lethal disease and a major bioterrorism health threat today. In US, the 2001 outbreak of B. anthracis resulted 22 cases of anthrax, including 5 deaths. Anthrax infections are frequently associated with severe and often irreversible cardiovascular complications, suggesting that the toxins generated from Bacillus anthracis, namely lethal toxin and edema toxin may possess pernicious cardiovascular effects. Lethal toxin depresses left ventricular ejection fraction and edema toxin produces pronounced tachycardia. In addition, lethal toxin has also been shown to trigger diastolic dysfunction, and suppress contractility in the hearts in in-vivo models. However, the underlying mechanisms were not explored. We hypothesis that anthrax lethal toxin can directly affect cardiac contractility and we plan to test this with the following: Aim #1 will examine whether anthrax lethal toxin directly affects cardiomyocyte contractile function and also does anthrax lethal toxin induces excessive ROS production in myocardium; Aim #2 will examine whether ROS inhibition confer protection against anthrax lethal toxin induces cardiomyocyte contractile anomalies; Aim #3 will evaluate whether TLR4 receptor knockout ameliorate anthrax lethal toxin induced cardiac contractile dysfunction. Cardiac contractile properties of individual myocytes from cardiac catalase overexpressing mice and Toll like Receptor 4 (TLR4) knockout mice will be assessed following lethal toxin exposure. Various signaling downstream signaling events that are involved in anthrax toxicity would be studied using standard molecular biology techniques. Given that autophagy plays an extremely important role in maintenance of normal heart function and morphology in stress conditions. We would elucidate whether autophagy has any role in anthrax lethal toxin induced cardiac contractile dysfunction. Autophagy would be studied for all the aims described above. The outcome of this study will help in understanding the pathology and molecular signaling involved anthrax toxicity which in turn may help in designing treatment strategies in clinics.
Enette Larson-Meyer, Ph.D., R.D. Family and Consumer Sciences and Brenda Alexander, Ph.D. and Ann Marie Hart, APRN, BC (FNP), Role of Ghrelin and PYY in postpartum body weight regulation and presence in human milk.
Introduction & Background: Epidemiological studies suggest that childbearing is an important contributor to the development of obesity in many women, and that breastfeeding protects against overweight and obesity in the mother and infant. It is possible that concentrations of the orexogenic peptide ghrelin, and the satiety hormones polypeptide YY (PYY), glucagon-like peptide (GLP-1) and leptin are altered following child birth and during lactation, and are associated with body weight/body fat retention during the postpartum period. It is also plausible these hormones are present in human milk where they may regulate appetite and/or energy homeostasis in the breastfed infant. Research Problem & Purpose: The purpose if this project is to determine whether ghrelin, PYY, GLP-1 and leptin are altered during fasting and following meal ingestion in postpartum women relative to never-pregnant controls, and whether these same hormones are present in human fore-and hind milk. Specific Aims and Rational: The specific aims of this project three fold:
Aim 1. Determine how pregnancy and lactation affect the fasting and meal-induced concentrations of the appetite-related hormones.
Aim 2. Determine the role of the appetite-related hormones in predicting body weight-retention/loss in the year following childbirth.
Aim 3. Identify specific appetite-related hormones in breast milk and determine whether concentrations of these hormones differ in fore- compared to hind milk throughout lactation.
The rational for this project is that when we understand how these hormones change following childbirth and during lactation and whether they are present in human milk (possibly in relation to maternal factors), we can then undertake targeted experimental and/or mechanistic studies to elucidate factors responsible for the obesity-protective effect of breastfeeding in mother and offspring. Biomedical Impact/Relevance to Field: Aberrations of ghrelin, PYY, GLP-1 and/or leptin may help explain why some women struggle to lose weight or are predisposed to body weight (or adiposity) gain following childbirth and during lactation. Concentrations of these hormones, if present in human milk, may also serve as short-term or long-term regulators of energy intake in the breastfed infant and offer a potential mechanism for the obesity-protective effects of breastfeeding. Current Project Status. As of March 3, 2012, we have enrolled and completed baseline data collection on 18 lactating postpartum women and 15 never-pregnant controls (for a total of 33 enrolled/baseline-completed volunteers) (Aim 1). We have enrolled 3 additional never-pregnant controls that are scheduled to complete baseline visits in April, and are waiting the delivery of several interested, currently pregnant participants. We have completed one- year follow-up visits on 11 postpartum lactating subjects and 8 never-pregnant controls (Aim 2). Additionally we submitted a manuscript on provocative findings from milk collected at baseline (4 weeks postpartum) in 13 postpartum women (Aim 3). In that publication we report the presence of PYY, GLP-1 and leptin in human milk, and that GLP-1 but not PYY or leptin concentrations change across a single feeding with concentrations of GLP-1 greater in hind- compared to fore-milk. Concentrations of milk leptin strongly correlated with maternal BMI. Action Plans for External Funding: Findings from our analysis of breast milk (Aim 3) will serve as preliminary data for a NIH grant application with a specific aim to determine whether the appetite regulation hormones in human milk—including ghrelin, PYY, GLP-1 and leptin-- are potential regulators of growth and body fat accretion in infants during the first year of life, and whether variations in these hormones among individual mothers are explained by differences in maternal nutrition and/or adiposity (October, 2012 submission).
Sreejayan Nair, Ph.D., School of Pharmacy,Cathepsin-K Mitigates Cardiac Dysfunction
Heart failure is the leading cause of morbidity and mortality in the United States. Cardiac hypertrophy, the adaptive response of the heart to pressure overload, has been recognized as a major independent risk factor for heart failure. Consequently, inhibition or regression of cardiac hypertrophy by pharmacologic interventions such as angiotensin converting enzyme inhibition has been shown to lower the risk of heart failure whereas persistence of hypertrophy has the opposite effect. However, despite the pivotal role of cardiac hypertrophy in heart failure, drugs that interrupt or reverse the progression of cardiac hypertrophy remain limited, warranting the development of novel therapeutic strategies to address this problem. Cathepsins are cysteine proteases that are ubiquitously expressed in various tissues and that play roles in cancer, autoimmune and cardiovascular diseases. Recent observations showing altered expression of cathepsins in the myocardium of humans with left-ventricular hypertrophy strongly suggest the involvement of cathepsins in heart failure. Our preliminary data show that cathepsin K (catK) the most potent proteolytic cathepsin is upregulated in the hypertrophic heart. However the causal role of catK in the development of cardiac hypertrophy and heart failure is largely unknown. The long-term goal of our research program is to understand how cathepsins can be manipulated for preventive and therapeutic purposes in heart failure. The objective of this research proposal, which is our next step in pursuit of that goal, is to determine the role of cat-K in the development of cardiac hypertrophy. Our central hypothesis is that cat-K is necessary for obesity- and pressure overload-mediated cardiac hypertrophic responses. Our hypothesis has been formulated on the basis of our preliminary data using cat-K-/- mice. The rationale for the proposed research is that, once it is known how cat-K regulates cardiac hypertrophy, pharmacological inhibitors of cat-K (which are currently used clinically to treat osteoporosis) can be assessed as new or innovative approaches for the prevention and treatment of heart failure. We plan to test our central hypothesis and, thereby, accomplish the objective of this application by pursuing the following two specific aims: 1. Determine the role of catK in the development of heart failure. Based on the preliminary data, the working hypothesis here is that catK knockout mitigates cardiac dysfunction and hypertrophy in a mouse model of pressure-overload-induced cardiac hypertrophy. 2. Identify intracellular signaling mechanisms modulated by cat-K leading to cardiac hypertrophy and dysfunction. We postulate, again on the basis of our preliminary data, that catK mediates cardiomyocyte apoptosis, autophagy and hypertrophy. The outcome of these studies will help identify molecular pathways that are mediated by cathepsins in the development of cardiac hypertrophy and dysfunctions. Such results are expected to have an important positive impact, because the identified mechanisms are highly likely to provide new targets for preventive and therapeutic interventions in addition to fundamentally advancing the growing knowledge of the mechanisms involved in the pathogenesis of heart failure. A NIHR15 grant proposal addressing the aforementioned aims was submitted in 2011, which received a priority score of 69. On resubmission the proposal receive a score of 26. The proposal will be submitted as a R01 for the June, 2012 deadline.
Baskaran Thyagarajan Ph.D., Analysis of Role of TRPV1 channels in the regulation of Metabolic Diseases.
Obesity is the hallmark of cardiovascular complications and a threat for current and future healthcare demands. Although life-style modification presents an effective way to manage obesity, the coexistence of comorbid conditions like hypertension, diabetes, or atherosclerosis often necessitates pharmacotherapy for management.
Recent research suggests the role of transient receptor potential vanilloid receptor 1 (TRPV1) in the pathophysiology of obesity, diabetes and cardiovascular complications. However, the precise role of TRPV1 in the regulation of these diseases remains unknown. This proposal stems from our hypothesis that TRPV1 is a potential target against adipogenesis and obesity. Our primary focus is to investigate how the molecular mechanisms of adipogenesis are regulated by TRPV1 via Ca2+ dependent protein kinases, phospholipids and phosphatases as these critically regulate the functions of TRPV1. Our work emphasizes the critical role of regulation of adipogenesis by TRPV1 agonists, which modulate lipid homeostasis in animals and humans. We propose to investigate the regulatory role of TRPV1 activation by capsaicin and SA13353 (a specific TRPV1 agonist) in weight gain leading to obesity in mouse model. We hypothesize that cellular mechanisms that facilitate TRPV1 desensitization inhibit adipogenesis.
Figure 1. Hypothetical scheme represents the catalysis of differentiation of preadipocytes to adipocytes (adipogenesis) by peroxisome proliferation activating receptor gamma (PPARγ). The effects of capsaicin, protein kinase C (PKC), Ca2+/calmodulin dependent protein kinase (CaM Kinase II), calcineurin, phospholipase C (PLC) and PPARγ agonists on adipogenesis and TRPV1 ion channel functions are described. +, –, and ? - denote activation, inhibition and undetermined effect, respectively. TM, transmembrane domain; N and C, N and C termini; [Ca2+]e, extracellular Ca2+.
To investigate this overarching hypothesis, we plan to study the modulations of TRPV1 activity by capsaicin/SA13353 using cultured preadipocyte 3T3-L1 and primary adipose cells and animal models (wild type and transgenic, TRPV1-/-) to analyze
1. The effects of TRPV1 agonists on
i. Ca2+ influx induced calcineurin activation
ii. Ca2+ influx induced desensitization of TRPV1 (via calcineurin activation)
(i and ii down-regulate PPARγ)
2. The effects of long-term desensitization of TRPV1 via PLC pathway or loss of TRPV1 (TRPV1 knockout, TRPV1-/-) inhibits adipogenesis; and
3. The effects of TRPV1 sensitization by
i. PKC activation
ii. CaM Kinase II activation
(i and ii promote adipogenesis)
The outcome of this proposal will provide new insight into the role of TRPV1 channels in the regulation of adipogenesis. Our experimental plans will investigate potentials of TRPV1 channel agonists as novel therapeutic agents to treat obesity and the related cardiovascular complications. The data obtained from this proposal will form the foundation for our future research work and for the submission of a compelling and fundable R01 grant application to NIH within 2 years. Besides serving our immediate goals, the data obtained will help developing new pharmacotherapy for the treatment of metabolic syndrome.
Meijun Zhu Ph.D., Animal Sciences, Maternal Obesity, Stem Cell Factor/c-Kit Signaling and Mast Cells in the Development of Intestine and Incidence of Inflammatory Bowel Diseases in Progeny.
The obesity rate increased more than 2 fold in recent decades. According to the latest NHANES survey (1999-2002), 29% of non-pregnant women 20-39 years of age are obese. At the same time, inflammatory bowel diseases (IBD) and related allergic diseases are also increasing. Is there a link between the increase in maternal obesity (MO) and the increase in IBD and related allergic diseases in offspring? Gut is the largest immune organ, and the majority of key developmental milestones of gut accomplishes during the fetal stage. Recent studies clearly show that gut epithelial integrity and barrier function is the central predisposing factor to IBD, food allergy as well as autoimmune diseases. The objective of this project is to explore the impact of MO on fetal and offspring gut development, inflammation, and barrier function, providing an explanation for the surge of IBD and related diseases in recent decades. We are employing the unique strength of the availability of transgenic mice including TLR4 knockout (TLR4-/-) mice, mast cell deficient (c-Kit-/-) mice and IBD-susceptible (IL10-/-) mice to exploring 1) role TLR4 signaling in maternal obesity and its associated inflammation in offspring gut development; and 2) role of mast cells in pre-disposing OB offspring mice to IBD.
Mice study is ongoing as planned. Currently, we have gotten sufficient TLR4 (-/+) mice which were treated with either control (Con) or obesogenic (OB) diet and given birth. The offspring born to both Con and OB mothers were fed with the same control diet after weaning till 2-month and 4-month of age, when offspring mice will be sacrificed and gut will be collected for analyzing the role of TLR4 signaling in maternal obesity and its associated inflammation in offspring gut through biochemical and histological analysis. Currently, we have collected 2-m-old TLR4 offspring mice and will collect samples from 4-month-old TLR4 knockout offspring mice in June.
Meanwhile, we are cross-breeding IL10 (-/-) mice with mast cell deficient mice (c-Kit-/-) to get mast cell heterozygous IL10 knockout mice (c-Kit-/+, IL10-/-). Currently, the colonies of c-Kit-/+, IL10-/- mice are increasing as planned and we have the first group of c-Kit-/+, IL10-/- mice under maternal obesity treatment at the end of 2011. We are expecting to have our first set of gut samples from offspring (c-Kit-/-, IL10-/- and c-Kit+/+, IL10-/-) mice born to C and OB mothers (c-Kit-/+, IL10-/-) collected in the following months.
In addition, we are also looking into the role of mast cells in IBD onset and incidence by cross-breeding c-Kit (-/-) and IL10 (-/-) mice that can spontaneously develop IBD. Currently, gut tissues from c-kit(-/-), IL10(-/-) and c-Kit (+/+), IL10 (-/-) have been harvested and we are undertaking related biochemical and histological analysis. Recently, we also looked into the protective roles of bioactive food components, especially grape seed extract on IBD onset and disease index.
Besides in vivo mice experiment, we have been conducting in vitro cell culture studies using murine mastocytoma cell line P815 and primary bone marrow derive mast cells derived from wild type and TLR4 knockout mice to test our hypothesis: palmitic acid (PA) induces mast cell activation and proliferation through TLR4 and SCF/c-Kit signaling. One abstract was submitted to 2012 Experimental Biology meeting and one manuscript is in preparation. Three papers in related topics were published in 2011.
We have been actively acquiring for external funds. Please see the following for details.
Mei-Jun Zhu (PI), Stephen P. Ford, and Claudio Fiocchi. NIH R01. Improving gastrointestinal immune response through fetal programming. $1,571,125 Not funded
Mei-Jun Zhu (PI), NIH R15. Maternal Obesity, AMPK and Neonatal Gut Development. $424,500 Impact score: 21
Mei-Jun Zhu (PI). NIH R01. Obesity, AMP-activated -protein kinase, and gut epithelial renewal and barrier function $1,809,875 Pending
Mei-Jun Zhu (PI). DOD. Grape seed extract, gut epithelium barrier function and inflammatory bowel diseases $750,000 Preparing