University Of Missouri-Columbia

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Although there have been important advances in the detection and treatment of cancer, there remains an urgent need to develop new therapeutic strategies. Copper (Cu) is an essential micronutrient that is required in higher amounts by cancer cells relative to normal tissues. Several enzymes with key roles in cancer require Cu for their activity, including lysyl oxidases and several oncogenic kinases (e.g., MEK1; ULK1). In pre-clinical models of cancer, many studies have shown that tumor growth and metastasis is suppressed by Cu chelators added to the diet. In clinical trials, Cu depletion via an oral Cu chelator was found to significantly slow disease progression in patients with mesothelioma, and significantly extend survival in breast cancer patients. While it is clear that Cu depletion is a promising anticancer strategy, there is a need to develop therapies that specifically target pathways of Cu delivery to oncogenic enzymes. In the current proposal, we pursue a highly innovative approach by targeting the Cu transporter, ATP7A. Our extensive preliminary studies validate ATP7A as a therapeutic target including: 1) Intestine-specific deletion of murine ATP7A lowers systemic copper status to levels shown to be therapeutic in cancer patients; 2) ATP7A is required to deliver copper to the family of lysyl oxidases, which have well-documented roles in metastasis; 3) Targeted deletion of ATP7A in breast and lung cancer cell lines reduces primary tumor growth and metastasis in mice; 4) Elevated ATP7A expression is significantly correlated with lower survival in cancer patients. Based on these findings, we hypothesize that a small molecule inhibitor of ATP7A will be therapeutic in cancer. Using computer-aided drug design, we have identified a hit molecule called MKV3 that binds to ATP7A with nanomolar affinity and inhibits ATP7A activity in cancer cell lines. Mice treated with MKV3 showed reduced activity of the serum Cu biomarker, ceruloplasmin, and reduced tumor growth. In this proposal, we will conduct structure guided optimization to identify MKV3 analogs with improved potency and drug like properties (Aim 1); conduct pharmacokinetic studies to identify MKV3 analogs that are suitable for pharmacodynamic studies (Aim 2); and evaluate the most favorable MKV3 analog in mouse models of breast cancer (Aim 3).

NIH Research Projects · FY 2025 · 2021-08

Heart failure is a leading cause of morbidity and mortality worldwide. Cardiomyocyte survival and death play a crucial role in the pathogenesis of heart failure due to the limited capacity of cardiomyocytes to proliferate or repair. Recently, multiple clinical studies have identified TNF-related apoptosis inducing ligand (TRAIL) and its receptor, death receptor 5 (DR5), as being two of the most powerful predictive markers of heart failure development and severity. Additionally, whole transcriptome analysis from our laboratory identified TRAIL and DR5 alterations in a mouse model of heart failure and its involvement in cardioprotective, EGFR-dependent signaling. While there have been multiple studies demonstrating high expression of TRAIL and DR5 in the heart, their function has never been investigated. The role of TRAIL/DR5 in cancer has been extensively studied due to the ability of TRAIL to selective induce apoptosis in cancer cells, however, in non-transformed cell types, the function of TRAIL/DR5 is unclear. Due to the connection of TRAIL/ DR5 with heart failure and unidentified role of TRAIL/DR5 in the heart we have been exploring the impact of DR5 signaling in cardiomyocytes. Using pharmacological agonists of DR5, we observe that DR5 activation does not induce canonical death receptor signaling pathways in cardiomyocytes but activates the pro-growth and survival kinase ERK1/2. Using specific inhibitors for signal transduction pathways, we observe that ERK1/2 activation involves the transactivation of EGFR and results in cardiomyocyte hypertrophy. Therefore, we hypothesize that DR5 activation in cardiomyocytes plays a non-canonical, cardioprotective role through the activation of pro- growth and survival mechanisms. Completion of the following research proposal will contribute important information to this novel field of study through the identification of the function and signaling mechanisms initiated by DR5 activation in cardiomyocytes, the role of DR5 in the normal and failing heart and the determination of the potential of targeting TRAIL/DR5 as a therapeutic strategy in heart failure.

NIH Research Projects · FY 2024 · 2021-07

Project Summary Hypertension is a high-risk factor for stroke, cardiovascular diseases, and renal failure and it is one of the leading causes of death in the US and afflicts 75 million people. Primary hypertension, the most common form of hypertension, is associated with elevated sympathetic vasomotor tone and hyperactivity of the hypothalamic pituitary adrenal (HPA) axis. However, the role of hyperactivity of the HPA axis in elevated sympathetic outflow in primary hypertension remains largely unknown. The paraventricular nucleus (PVN) of the hypothalamus is a critical brain region that integrates neuroendocrine and cardiovascular functions. In primary hypertension, PVN presympathetic neuron activity is increased and provides excitatory drive to maintain heightened sympathetic vasomotor tone. Glutamatergic synaptic inputs to the PVN presympathetic neurons are enhanced in spontaneously hypertensive rats (SHRs). However, the cellular mechanisms underlying hyperactivity of PVN presympathetic neurons and enhanced excitatory synaptic inputs in SHRs remain unknown. The corticotrophin-releasing hormone (CRH)-containing neurons in the PVN (PVN-CRH neurons) are an essential component of the HPA axis. PVN-CRH neurons are activated in hypertension as indicated by increased expression levels of CRH protein and mRNA levels in the PVN in patients with primary hypertension. The objective of this project is to determine the role of PVN-CRH neurons in regulating blood pressure and sympathetic outflow in primary hypertension. Our pilot study found that PVN-CRH neuron activity was increased in SHRs, and selective inhibition or ablation of PVN-CRH neurons decreased arterial blood pressure in SHRs. In addition, selective inhibition of PVN-CRH neurons suppressed the activity of PVN presympathetic neurons in SHRs; the effect was eliminated by blocking CRH receptor 1. Thus, we will test our central hypothesis that increased activity of PVN-CRH neurons leads to hyperactivity of PVN presympathetic neurons and elevated sympathetic outflow in primary hypertension. We will first determine if hyperactivity of PVN-CRH neurons is required for high blood pressure and elevated sympathetic outflow in primary hypertension (Aim 1). We will also identify the synaptic mechanism underlying hyperactivity of PVN-CRH neurons in hypertension (Aim 2). Finally, we will determine the role of PVN-CRH neurons in the elevated activity of PVN presympathetic neurons and enhanced glutamatergic synaptic inputs in hypertension (Aim 3). Our proposed studies will greatly improve the understanding of the cellular and molecular mechanisms underlying primary hypertension. We expect our studies to provide novel information about the neuronal mechanisms responsible for primary hypertension and an important rationale for developing novel treatment strategies to reduce sympathetic vasomotor tone in primary hypertension.

NIH Research Projects · FY 2025 · 2021-07

PROJECT SUMMARY Risky alcohol use and related behaviors among college students result in significant, adverse outcomes (e.g., injury, death). For example, an estimated 1,519 college student deaths each year are associated with alcohol. Accordingly, these behaviors constitute a serious public health concern that must be addressed across college campuses. NIAAA has prioritized the identification of interventions focused on reducing college drinking and these efforts have yielded a number of evidence-based strategies (EBSs), which were recently compiled in the College Alcohol Intervention Matrix (CollegeAIM; NIAAA, 2015a; 2019). CollegeAIM includes a list of evidence- based alcohol reduction strategies organized by approach (e.g., individual or environmental), cost, and level of efficacy. Colleges can use CollegeAIM to identify EBSs to implement on their campuses to combat risky drinking. Despite NIAAA prioritization and the availability of the CollegeAIM tool and existing EBSs to address risky drinking, relatively little is known about the adoption and implementation of EBSs for college drinking among stakeholders (individuals involved with EBS implementation) on college campuses, and further, to what extent CollegeAIM is used to identify EBSs for implementation. The proposed project will evaluate the use and effectiveness of CollegeAIM in the selection of EBSs for college student drinking across a statewide substance use coalition, with a long-term objective of supporting colleges’ use of CollegeAIM and thus, ultimately the implementation of EBSs to reduce risky drinking. The proposed project aims will be achieved with a mixed-methods design to assess stakeholders’ use and perspectives of the CollegeAIM tool, identify barriers and facilitators to using CollegeAIM to select appropriate interventions for their institutions, and identify institution characteristics that impact CollegeAIM use. This will be the first study to extensively evaluate CollegeAIM as a tool to identify EBSs on college campuses. The findings from the proposed project also will be instrumental in developing an organizational decision-support program, intended to be used in tandem with CollegeAIM to address college drinking, which will be evaluated in a follow-up R01 proposal. The proposed project and training of this K08 Mentored Clinical Scientist Research Career Development Award will be based at the University of Missouri, an ideal setting given the well-established training programs and group of experts across domains of alcohol research. The candidate will be mentored by Drs. Kenneth Sher and Kristin Hawley and an excellent team of collaborators who will support the candidate’s advancement through specialized training in addictions and implementation science. The candidate will gain the experience with mixed-methods design and analytic approaches and alcohol prevention research necessary to establish the candidate as an independent investigator who will be prepared to extend the findings and initiatives of the current study in future R-series proposals and well-positioned to form an implementation science research program in addictions.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Influenza, as a global threat to human health, continues to cause significant morbidity and high rates of mortality. To effectively control influenza, it is important to understand the interplay between the host and influenza by identifying host factors that regulate viral replication and defining the mechanisms by which influenza virus manipulates the cellular defense or signaling pathway. Sphingolipids are bioactive lipid mediators and include sphingosine 1-phosphate (S1P). Although S1P and its metabolizing enzymes, such as S1P lyase (SPL) and sphingosine kinase 2 (SK2), have been reported to regulate versatile cellular or disease processes, their roles in influenza virus infection are poorly understood. Preliminary data indicate that SPL promoted IKKε-mediated type I interferon (IFN) responses to display anti-influenza viral activity. However, influenza viruses effectively downregulated SPL, suggesting that influenza virus strives to evade the host defense mechanism. Furthermore, influenza virus increased the level of another S1P-metabolizing enzyme, SK2, which accelerated influenza virus replication. Inhibition of SK2 impaired influenza virus propagation in vitro and increased the viability of virus-infected mice, demonstrating the pro-influenza function of SK2. These findings heighten the need to further investigate the interplay between the S1P-metabolizing enzymes, host defense and signaling, and influenza virus. The research aims of this proposal include 1) determining the mechanisms by which influenza virus manipulates SPL and SK2 to enhance virus replication, 2) investigating the mechanisms of how these S1P-metabolizing enzymes display antiviral or pro-influenza viral activities, and 3) establishing the functions of the S1P-metabolizing enzymes during influenza virus infection in vivo. Collectively, these research results can define the regulatory functions of S1P-metabolizing enzymes that impact host defenses and influenza pathogenicity. Furthermore, the project could provide a foundation for designing new therapeutic interventions to cure influenza.

NIH Research Projects · FY 2025 · 2021-06

Project Summary/Abstract Chronic viral infections continue to cause significant morbidity and mortality in humans. Viruses often evade or suppress the host immunity to establish persistent infections. The clone 13 strain (Cl 13) of lymphocytic choriomeningitis virus (LCMV) induces a profound immune suppression and persists in the mouse. LCMV Cl 13 infection of mice has served as a valuable model system for the mechanistic study of viral regulation of host immunity and virus persistence. Sphingosine kinase (SK) 2 mediates the synthesis of sphingosine 1-phosphate (S1P) from sphingosine and controls diverse cellular conditions. However, the function of SK2 in host immune responses to virus infection remains poorly understood. The preliminary data demonstrate that SK2 deficiency in mice results in heightened T cell responses to LCMV Cl 13 infection, leading to lethal immunopathology associated with kidney disease. The data also indicate that LCMV Cl 13 increases the activation of SK2 in CD4+ T cells, which inhibits the expansion of virus-specific T cells. Importantly, the oral administration of the SK2-specific inhibitor into LCMV Cl 13-infected mice accelerates the clearance of the persistent infection. Therefore, the following research aims are developed to uncover the regulatory function of SK2 in virus- induced immune suppression, immune pathology, and virus persistence. First, the role of SK2 in CD4+ T cell suppression will be investigated during persistent LCMV Cl 13 infection as well as using human T cells from patients chronically infected with viruses. Second, the molecular mechanism by which SK2 suppresses virus- specific CD4+ T cell responses and restricts immune pathology will be determined upon LCMV infection. Lastly, the proposed study will further determine the therapeutic efficacy of the SK2-specific inhibitor during persistent LCMV infection and assess the features of the host immunity regulated by SK2 inhibition. Taken together, this research is expected to elucidate the mechanism by which SK2 regulates virus-specific T cell responses, and to define the function of SK2 in the imbalanced immune mechanism that can cause either immune pathologic kidney disease or persistent viral infection. The project could provide a framework for developing new immune therapeutic interventions for controlling chronic virus infections.

NIH Research Projects · FY 2025 · 2021-05

ABSTRACT The overall objective of the proposed K23 is to support Dr. Donte Bernard in acquiring the skills necessary to become an independent health disparities investigator with a program of research focused on the explication and reduction of the mental health sequelae associated with identity-based trauma among Black youth. Trauma exposure represents a significant public health concern that is nearly ubiquitous in the lives of Black youth. Yet, there is a lack of research that has disentangled the unique effects of identity-based trauma relative to other traumatic experiences. Moreover, a gap remains in the identification of culturally relevant constructs that may underlie the link between trauma and negative mental health outcomes and may serve as malleable targets for interventions to promote resiliency in the aftermath of trauma exposure. The need for this high impact research—and, as such, highly trained clinical scientists to lead this research—is significant and consistent with NIMHD priorities to scientifically understand the causes of health disparity. The proposed K23 directly addresses these limitations through a promising candidate, a comprehensive Training Plan that is supported by a team of highly successful mentors and renowned research environment, and novel, mixed methods research, leveraging an active NIMH R01-funded longitudinal study on child victimization and mental health, to better understand (a) experiences and responses to identity-based trauma (via qualitative methods); (b) the unique effect of identity-based trauma—above and beyond other psychosocial traumatic events—on internalizing and externalizing mental health sequalae; and (c) how identity-related vigilance and specific dimensions of one’s sociocultural identity influence the relationship between identity-based trauma and mental health outcomes cross sectionally and over time. Findings from these primary study aims will inform a preliminary culturally informed outline for a treatment to promote mental health resiliency in the aftermath of trauma (Exploratory Aim). On-site mentors (Drs. Danielson, Hughes-Halbert, Moreland, and Mueller) have extensive knowledge in child traumatic stress research, including trauma, longitudinal and mixed-methods approaches, and intervention development and evaluation. An off-site mentor (Dr. Joe at Washington University in St. Louis) will provide additional guidance in mechanisms and examinations of externalizing mental health in relation to traumatic experiences. The mentorship, coursework, seminars, workshops, and conference attendance afforded by the K23 will ensure that the candidate achieves numerous training goals, such as enhancing knowledge of trauma; developing expertise in mechanisms that may undergird the relationship between trauma and mental health; and developing competence in qualitative and mixed methods. The K23 activities will prepare Dr. Bernard to lead a program of high impact, rigorously designed mental health disparities research focusing on trauma among Black youth.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY The uterus in women is a unique organ in its ability to undergo repeated physiological damage and repair during the monthly menstrual cycle. The endometrium, in particular, is extensively regenerated following menstrual shedding. Our long-term research goal is to understand the normal mechanisms of endometrial regeneration and repair and how these processes, when mis-regulated, contribute to diseases/dysfunction such as endometrial cancer, endometriosis, thin endometrium, Asherman’s Syndrome and infertility. In this project, experiments are designed to investigate mesenchymal-epithelial transition (MET) as a mechanism of endometrial epithelial regeneration. Research shows that MET is one mechanism by which the endometrial epithelium is regenerated postpartum and in a menses-like model in mice and has been proposed as a mechanism in women. During MET, which is a type of cellular transdifferentiation, a mesenchymal cell is reprogrammed and converted into an epithelial cell. To our knowledge, the endometrium is the only tissue that uses cellular transdifferentiation under normal physiological conditions (e.g. postpartum and menses-like repair) in the adult. Unfortunately, our understanding of this unique repair mechanism is very incomplete. Two specific aims will further investigate MET in epithelial regeneration: (1) Test the function of MET-derived endometrial epithelial cells; and (2) Compare MET by endometrial-derived and bone marrow (BM)-derived mesenchymal cells. A combination of mouse models including lineage tracing, menses-like endometrial breakdown and repair and a novel orthotopic transplantation technique along with scRNA-seq will be employed to address fundamental questions about the function, characteristics, and origin of MET-derived epithelial cells. Particularly, whether they are bona fide endometrial epithelial cells and whether they originate from endometrial stromal cells and/or bone marrow cells, will be investigated. Importantly, orthotopic transplantation will be used to assess MET by human stromal cells as in vivo studies cannot be performed in women. Proper endometrial regeneration, including replacement of lost or damaged epithelial cells, is necessary for preparation of the uterus for subsequent reproductive cycles and pregnancy. No other organ is subject to such extreme tissue regeneration as that seen in the uterus during the menstrual cycle. It is perhaps because of the extent of damage and repair that the uterus undergoes that it is subject to development of diseases. Increased understanding of endometrial repair mechanisms will provide greater insight into how these processes, when gone awry, contribute to endometrial diseases and impact fertility ultimately leading to better therapeutics.

NIH Research Projects · FY 2026 · 2021-04

Abstract A remarkable trait of the healthy brain is that it can generate stable behaviors that last for decades. When this fails to occur, a range of neurological disorders follow. An emerging view is that neurons can sense disturbances in their activity and then make compensatory adjustments to stabilize their function, a process referred to broadly as “homeostatic plasticity.” Insights into homeostatic plasticity have improved our understanding of how neurons may remain stable in an ever-changing environment. Despite much progress, how these mechanisms work in the intact brain to produce behaviors across the lifespan remains largely unknown, and therefore, represents a major gap in basic neurobiology. We address this issue using an innovative model where there exists a direct relationship between homeostatic compensation in neurons and regulation of a tractable behavior during adult life: the respiratory motor system in frogs. For long periods each year, motor circuits that control breathing in these animals are inactive because they hibernate in water and do not breathe air. Our group recently discovered this environment leads to compensatory changes in motoneurons that allow the circuit to work appropriately when animals must breathe again after months of inactivity, thereby linking plasticity that stabilizes neuronal function to a vital and tractable behavior. Here, we exploit this system to test three hypotheses that address the central question of how homeostatic mechanisms arise in vivo to support adaptive behavior. Based on our preliminary data, we hypothesize that (1) this network relies on multiple forms of intrinsic and synaptic motor plasticity to generate appropriate output, (2) intrinsic and synaptic compensation follow unique time courses during inactivity due to distinct gene regulatory networks, and (3) activity and environmental stimuli interact to differentially regulate intrinsic and synaptic compensation. These hypotheses will be tested with an integrative approach that blends patch clamp electrophysiology to measure plasticity at the cellular level, single-cell RNA sequencing and quantitative PCR to link gene expression to physiology, electromyography to measure neuromuscular function in vivo, and extracellular recording to assess function of intact circuits. Overall, this work will inform how neurons integrate multiple types of plasticity to produce essential behaviors, a goal that must be achieved to understand how circuit function remains healthy throughout life in many individuals but fails in others to cause disease.

NIH Research Projects · FY 2025 · 2021-04

Project Summary and Abstract Inflammatory Bowel Disease (IBD) is an increasingly prevalent chronic disease marked by aberrant immune responses and intestinal and extra-intestinal inflammation. IBD is comorbid with cardiovascular disease and associated with decreased blood flow to the intestines, with a critical but poorly-studied role for the mesenteric (MAs) arteries that regulate intestinal perfusion. Perivascular sensory nerves (PSNs) continuously innervate the MA adventitia and perivascular adipose tissue (PVAT), regulating vasomotor function and facilitating blood flow by dilating MAs and inhibiting sympathetic vasoconstriction. With IBD, these PSN functions are severely impaired, and the PVAT that is normally anticontractile becomes procontractile through an unknown mechanism, further impairing MA dilation. The sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP) may also be important in these IBD-related vascular dysfunctions, as they are linked to disease severity and can mediate perivascular neuro-immune and neuro-adipose signaling through activation of local immune cells. Previous work demonstrates that macrophages accumulate in both the MA adventitia and PVAT during IBD, and macrophage depletion restores the ability of PSNs to dilate MAs and inhibit sympathetic constriction. This suggests that perivascular macrophages can modulate arterial function with IBD, likely through a mechanism involving sensory neurotransmitters in the adventitia and PVAT. What remains unclear is how macrophages participate in PSN, PVAT, and blood flow dysfunction and when these changes occur in IBD development. This project will test the overall hypothesis that PSN neurotransmitter released in adventitia and PVAT of MAs promotes macrophage activation, accumulation, and inflammatory mediator release, leading to vasomotor defects and impaired blood flow early in IBD pathogenesis. To investigate these relationships, the immune-driven, Helicobacter hepaticus-induced IL10-/- mouse model of IBD will be used to address 3 research Aims. Aim 1 will use confocal imaging, flow cytometry, and in vivo blood flow measurements at timepoints throughout IBD development to determine when macrophage infiltration causes PSN and PVAT dysfunction and impairs blood flow compared to the development of colon inflammation. Aim 2 will use sensory denervation and transgenic mice lacking PVAT in conjunction with isolated artery preparations to determine whether the presence and activity of PSNs and/or PVAT drive macrophage infiltration around MAs with IBD. Aim 3 will use advanced imaging, primary adventitial and PVAT macrophages, and biochemical assays to test whether sensory neuropeptides can activate macrophages from the MA adventitia and PVAT to release inflammatory mediators. This project will uniquely define the role of PSNs and their signaling pathways in neuro-immune-adipose interactions mediating vasomotor function, and it will determine how these pathways are affected during the pathogenesis of IBD. Results will provide new insight towards developing selective therapeutic strategies for treating vascular dysfunction and impaired intestinal blood flow to improve quality of life for IBD patients.

NIH Research Projects · FY 2025 · 2021-03

Summary Sjögren’s syndrome (SS), an autoimmune exocrinopathy of the salivary and lacrimal glands, affects ~ 4 million Americans, 90% of whom are women. SS is characterized by sialadenitis and dacryoadenitis, decreased saliva (i.e., xerostomia) and tear production (i.e., xerophthalmia) and the presence in blood serum of autoantibodies against Ro/SSA and La/SSB. Xerostomia and xerophthalmia in SS patients can lead to periodontitis, yeast and bacterial infections, digestive disorders and vision deterioration that severely reduce the quality of life for patients. Ultimately, chronic inflammation in SS leads to secondary autoimmune diseases, tissue fibrosis and lymphoma. Therapy for SS is limited to symptom management through external hydration, artificial saliva and tears and muscarinic receptor agonists that induce fluid secretion from residual exocrine acinar cells. Such remedies are universally judged to be inadequate and thus, development of more effective SS treatments is essential. Our research focuses on cell surface P2X7 and P2Y2 receptors for extracellular ATP, the intracellular chemical form of energy that when released from damaged salivary glands initiate inflammatory responses. Our studies show that P2X7R and P2Y2R antagonists enhance saliva secretion and reduce lymphocytic foci in salivary glands of two different mouse models of SS. Antagonism of the P2X7R also reduces lymphocytic accumulation in the lacrimal glands and increases tear secretion. These antagonists have not been used to treat human SS, although P2X7R is upregulated in salivary glands of SS patients compared to non-SS controls. P2X7R activation in salivary glands also induces maturation and release of IL-1β, an SS- related cytokine that upregulates P2Y2R in immune and epithelial cells, suggesting that P2X7R and P2Y2R contribute together to SS development. This project will investigate the ability of P2X7R and/or P2Y2R antagonists to increase saliva and/or tear secretion and reduce sialadenitis and/or dacryoadenitis in mouse models of SS. These findings will be validated by assessing P2X7R and P2Y2R expression in archived human SS and control minor salivary gland biopsies and evaluating effects of P2X7R and/or P2Y2R antagonism in freshly isolated human salivary and lacrimal gland cells. Specific Aim 1 will investigate the hypothesis that P2X7R and P2Y2R play sequential roles in chronic sialadenitis and glandular dysfunction in SS mouse models and can be antagonized to treat SS in vivo. Specific Aim 2 will investigate the hypothesis that P2X7R and P2Y2R activation in lacrimal gland epithelial cells promotes dry eye disease in mouse models of SS. Specific Aim 3 will investigate P2X7R and P2Y2R-mediated proinflammatory responses in human primary salivary and lacrimal gland cells and human SS minor salivary gland biopsies. Successful completion of this proposal will represent a critical step towards realization of the ultimate goal of targeting the P2X7R and/or P2Y2R to treat SS in humans.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT Vascular insulin resistance is a hallmark of type 2 diabetes (T2D) and dampening of insulin-induced vasodilation is its primary consequence. Notably, in T2D, reduced insulin-stimulated vasodilation and blood flow to tissues such as skeletal muscle significantly limits glucose uptake and contributes to impaired glucose control. A detailed understanding of the precipitating factors and mechanisms underlying the defects in vasodilator actions of insulin is critical for the development of therapeutic strategies aimed at improving glycemic control and protecting against cardiovascular disease. Based on our prior work and most recent and exciting preliminary data, we propose the novel hypothesis that ADAM17-mediated shedding of the insulin receptor alpha (IRα) from endothelial cells impairs insulin-stimulated vasodilation in T2D. We further propose that the increased activity of endothelial ADAM17 is attributed to protein kinase-C (PKC) activation and subsequent externalization of phosphatidylserine (PS) to the outer leaflet of the cell membrane, which serves to guide ADAM17 to its targeted substrates. As exogenous PS is a competitive inhibitor of ADAM17 sheddase activity, we will also determine the efficacy of oral administration of PS for restoring vascular insulin sensitivity in T2D patients. We will test our innovative hypotheses with gain- and loss-of-function genetic-manipulation experiments in human cultured endothelial cells, in isolated resistance arteries harvested from patients undergoing abdominal surgery, and in patients with T2D. Experimental results will determine the role of PS externalization-ADAM17 activation-IRα shedding as a mechanism impairing the vasodilatory actions of insulin in T2D. Specifically, in Aim 1, we will determine the mechanism by which PKC causes the externalization of PS and whether PS externalization is needed for PKC-dependent activation of ADAM17 in endothelial cells. Next, in Aim 2, we will determine the role of ADAM17 activity in IRα shedding and subsequent impairment of insulin-stimulated vasodilation in T2D. Finally, in Aim 3, we will perform a randomized double-blind clinical trial to determine the therapeutic efficacy of oral administration of the competitive inhibitor of ADAM17 sheddase activity, PS, on insulin-stimulated leg blood flow in patients with T2D. Our team is poised to move cardiovascular and diabetes research forward with a project that will exert a sustained, powerful impact across a number of levels of inquiry that are novel conceptually, mechanistically, methodologically, and therapeutically. Indeed, this proposal represents a paradigm shift from our current mechanistic understanding of vascular insulin resistance. Targeting ADAM17 activation holds extraordinary promise for correcting vascular insulin resistance and ultimately preventing/treating T2D-associated metabolic and cardiovascular diseases.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY/ABSTRACT This project supports the career development of Dr. Katherene Anguah in her path to become an independent translational researcher focusing on how dietary components may benefit cardiometabolic health through the control of appetite. The mentored plan builds on previous training by providing additional technical, academic, and professional development skills to facilitate research independence. An interdisciplinary team of mentors includes experts in human clinical feeding studies, functional magnetic resonance imaging, microbiome analysis, and stable isotope labeled, targeted metabolomics. The 5-year plan includes the opportunity to advance through both didactic instruction and experiences aimed at expanding skills in research management and scientific writing. Rationale: Strong evidence supports the association between high fiber (HiFi) diets (e.g. legumes, nuts, vegetables) and a reduced risk for chronic conditions such as cardiovascular disease (CVD), type 2 diabetes and some forms of cancer. However, the current U.S. average consumption of dietary fiber of 17g/day is significantly below the recommendation level of 25g/d for women and 38g/d for men. Furthermore, fiber fermentation to produce short chain fatty acid (SCFA) products and alterations in microbial composition and activity may be mechanisms linking a HiFi diet to improved health. Importantly, much of the data, including findings supporting a beneficial role of SCFA have been derived from animal studies. Human studies are now needed to advance the understanding of the translational significance of rodent studies and the potential benefit of fiber on microbial metabolites and cardiometabolic health, glucose regulation, appetite and satiety. The central hypothesis is that that the mechanisms by which dietary fiber provides metabolic benefit include direct physical effects in the upper gastrointestinal tract to slow nutrient absorption, and indirect effects to reduce food intake mediated by SCFA-induced secretion of intestinal hormones resulting in increased satiety. Design: Using fiber derived from peas, Aim 1 will test the effect of a HiFi diet on appetite, satiety, and cardiometabolic health and whether elevated SCFA concentration mediates improved satiety. Aim 2 will quantitate the changes in microbial composition and colonic SCFA production rate during HiFi feeding and whether any changes are potential mediators of observed benefits on satiety and cardiometabolic risk factors. Relevance: These studies will significantly expand the understanding of mechanisms by which dietary fiber improves satiety and cardiometabolic health in humans.

NIH Research Projects · FY 2025 · 2021-01

Project Summary The recalcitrant and harmful chemicals such as PFASs (per- and polyfluoroalkyl substances), endangers the environmental, wild-life, and human health profoundly. Among different strategies, bioremediation was established as an effective and reliable solution for remediating persistent environmental contaminants like PFASs. Fungi, such as basidiomycetes (i.e. white rot fungi) are used in bioremediation of PFAS for their strong extracellular biocatalytic capacity with great promise. However, several factors limit commercial applications: 1) need for nutrient addition as the carbon source for the microbe; 2) need to immobilize fungus biomass as pellets to prevent fungus dispersion onto reactor wall; 3) bacterial competition; 4) low efficiency due to the low chemical availability to the fungal mycelium and slow fungus growth. The proposed research will address the imminent challenges of remediating persistent and toxic environmental contaminants using the uniquely designed Nanomaterial-Fungus Framework (NFF). The NFF is a system that novel nano-materials create a biomimic scaffold where fungus can grow, and the scaffold enriches trace level contaminants that fungus can degrade. We aim to unveil the fundamental biodegradation mechanisms of the NFF system, which provides future guidance to modify and improve the system. The engineered NFF system will offer a novel strategy that applies toward a broad range of environmental pollutant bioremediation practices.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY The goal of this study is to examine the functions of the zinc finger protein ZPR1 in R-loops metabolism and neurodegeneration. R-loops are formed during transcription and consist of RNA-DNA hybridized strands and a complementary DNA strand. R-loop accumulation results in DNA damage leading to neurodegeneration associated with genetic neurodegenerative diseases, including spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). ZPR1 is evolutionary conserved in eukaryotes and is essential for cell viability. However, very little is known about the ZPR1 biological functions that may contribute to cell viability and human disease etiology. ZPR1 interacts directly with translation elongation factor 1A (EF1A), RNA Polymerase II and Senataxin (SETX), which are also conserved in eukaryotes. SETX is an RNA-DNA helicase required for resolution of R- loops. Mutations in SETX are associated with a group of untreatable neurodegenerative disorders, including ataxia oculomotor apraxia type 2, autosomal dominant SMA and ALS4, characterized by defects in R-loop metabolism. The molecular mechanisms of R-loop resolution are largely unknown. Our preliminary data show that ZPR1 deficiency causes R-loop accumulation and neurodegeneration. ZPR1 overexpression reduces R- loops and rescues DNA damage in neurons and patient cells, and prevents neurodegeneration in SMA mice. ZPR1 binds to RNA-DNA hybrids and associates with R-loops in vivo. ZPR1 interacts with SETX and ZPR1 is required for the formation of SETX complexes with R-loops suggesting that ZPR1 may help recruit SETX to R- loops. We have created novel Zpr1 mutant mice, with double and quadruple point mutations in the Zpr1 locus to selectively disrupt ZPR1-EF1A complexes. ZPR1 mutant mice show accumulation of R-loops and develop neurodegenerative disease-like phenotypes similar to reported for patients with SETX mutations. Together, these findings raise a hypothesis that ZPR1 complexes with EF1A and SETX may play distinct and critical roles in R-loop resolution and provide a foundation for investigating the function of ZPR1-EF1A and ZPR1-SETX complexes in R-loop metabolism. The specific aims are to examine: (Aim 1) the molecular basis of ZPR1- dependent accumulation of co-transcriptional R-loops and neurodegeneration using Zpr1 conditional mice; (Aim 2) the function of ZPR1-EF1A complexes in R-loop resolution using novel mouse models to disrupt ZPR1-EF1A complexes in vivo in motor neurons that we have generated, and the mechanism of GTP/GDP-dependent resolution of RNA and DNA strands by ZPR1-EF1A complexes; (Aim 3) the function of ZPR1-SETX complexes in R-loop metabolism, genome integrity and ALS4 pathogenesis. ZPR1-SETX complexes are disrupted in ALS4 patients with SETX mutation. The effect of disruption of ZPR1-SETX complexes on R-loop metabolism, DNA replication fork and genome integrity using cell-based models, including patient cells. This study will provide comprehensive insight into the molecular basis of pathogenesis caused by defects in R-loop metabolism that would be a breakthrough towards developing targeted therapeutic strategies for a group of incurable diseases.

NIH Research Projects · FY 2025 · 2020-12

Project Summary/Abstract Excess lipids increase the total intramyocellular lipid content and the ectopic fat storage resulting in lipotoxicity and insulin resistance in skeletal muscles, which is one of the main targets of insulin and its action is central for the maintenance of glucose homeostasis. Consumption of a diet high in fat and refined sugars, a Western Diet (WD), activates mineralocorticoid receptors (MRs) to induce muscle lipid metabolic disorders and insulin resistance. Recent data further indicate that cell specific endothelial cell (EC) MR (ECMR) activation mediates WD-induced muscle lipid metabolism disorders, impaired insulin metabolic signaling, and tissue insulin resistance. In this regard, ECMR activation increase CD36 expression in skeletal muscle arterioles and tissues, which promotes excessive free fatty acid trafficking across the muscle vasculature, leading to skeletal muscle lipid accumulation, CD36 palmitoylation and insulin resistance. There is a relationship between microvascular endothelial dysfunction and muscle metabolic disorders and insulin resistance through exosomes. Recent data suggest that EC derived exosomal proteins, such as exosomal CD36, can promote lipid accumulation and metabolic disorders. Upon being released from ECs, exosomal CD36 can be up-taken by the neighboring skeletal muscle and thus promote muscle lipid accumulation. The hypothesis of this application is that activation of ECMRs induces EC CD36 expression and release of EC-derived exosomal CD36 which increases free fatty acid uptake in ECs and translocation to skeletal muscle cells, leading to skeletal muscle intramyocellular lipid deposits and insulin resistance. Objective 1 of this application is to understand the role and mechanisms of ECMR signaling on CD36 expression, free fatty acid uptake in ECs and skeletal muscle cells, and related muscle lipid deposition and insulin resistance. Objective 2 of this application is to investigate the role and mechanisms of enhanced ECMR signaling on the EC-derived exosomal CD36 release and its role in facilitating increased skeletal muscle cell CD36 to further promote muscle fatty acid uptake, IMC lipid deposition and insulin resistance. Accordingly, in vitro cell treated with free fatty acid and in vivo mice fed a WD will be used to set up a model of ECMR/ECCD36 activation, obesity and insulin resistance. The proposed work should provide a better understanding the role of ECMRs and EC exosomal CD36 in the development of skeletal muscle insulin resistance and provide an important biomarker for the early diagnosis and prevention of this increasing cause of diabetes.

NIH Research Projects · FY 2024 · 2020-09

High levels of alcohol consumption clearly place individuals at great risk and present a significant health and economic burden to society. Emerging evidence indicates that many young adults engage in what can be called extreme drinking (i.e., drinking at levels likely to lead to BACs > .16). Despite recent attention to extreme drinking5,6, we know surprisingly little about this behavior beyond associations revealed by cross‐sectional studies that rely exclusively on retrospective self‐report. The proposed study is designed to provide some of the first comprehensive data about influences on the extreme drinking phenotype, and to compare these with those identified for the typical binge drinking phenotype. Whether there are unique causes and correlates of extreme drinking (compared to binge drinking) is an empirical question that has not been tested. There are challenges to investigating extreme drinking, including 1) overcoming the limitations of retrospective self‐report, 2) adequately measuring personological and environmental influences, and 3) capturing the temporal associations of these diverse influences and their impact on extreme drinking occasions. The proposed project is designed to meet these challenges using a combination of laboratory, genetic, and ecological momentary assessment (EMA) methods. Our multi‐method approach will combine laboratory alcohol administration, EMA, and real‐time BAC assessment to capture the interplay between a broad range of potential influences on extreme drinking, extend our investigation outside the lab and into the natural drinking environment, and explore the temporal associations of influences on extreme drinking. We focus on four core constructs central to current theoretical models of addiction that are hypothesized to influence substance use through in‐the‐moment processes: reward sensitivity (RS), incentive salience (IS), impulsivity/loss of control (Imp), and negative affectivity (NA). We will recruit a sample of 400 young adults (ages 21‐29), ascertained from a statewide DMV database, who have a recent legal action with a recorded BAC consistent with extreme drinking (≥ .12). Using a longitudinal burst design, we will follow participants over a 12‐month period, with five self‐report assessments and four, two‐week EMA bursts. A baseline laboratory session will assess behavioral, trait, and electrophysiological markers of core study constructs. We aim to (1) Evaluate the validity and utility of real‐time assessments for identifying extreme drinking and alcohol‐related behavior. This aim could inform estimation methods for characterizing extreme drinking and guide refinement of definitions of problematic drinking profiles. (2) Characterize the structural influence of stable individual differences, transient intra‐individual factors, and environmental variables on risky, binge, and extreme drinking occasions and alcohol‐related negative consequences. This aim will reveal the incremental validity of state (EMA) and trait (lab; baseline questionnaires; polygenic risk scores [PRSs] when applicable) indices of core study constructs in predicting extreme drinking occasions within and between individuals; as well as test their interaction with specific contextual factors to predict extreme drinking behavior. (3) Identify multidimensional profiles associated with stable or highly variable binge and extreme drinking behavior. Our longitudinal burst design allows us to test hypotheses about the stability of drinking behavior over time.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) form a metabolic relationship known as the “proline cycle”, a novel pathway that impacts cellular growth and death pathways. The proline cycle is central to the metabolic shift that enables tumorigenesis and supports the metastatic cascade of cancer cells. PRODH is upregulated in metastasizing breast cancer cells, and PYCR1 is one of the most consistently upregulated enzymes across multiple cancer cell types. Thus, proline cycle enzymes have been proposed as potential cancer drug targets. A major goal of this project is to gain insight into whether these enzymes have binding sites that can be exploited for small molecule binding and establish target tractability, i.e., the likelihood of identifying modulators that interact effectively with these targets. Initial results are encouraging, as the chemical probes developed in the first phase of this project show activity in cellular and animal models of cancer. These results motivate our proposal to (1) develop new and more potent reversible inhibitors of PRODH and PYCR1, (2) explore the mechanism-based covalent inactivation of PRODH, and (3) define the mechanistic roles of the proline cycle using chemical probes in cancer cells. With these studies we will mechanistically dissect the role of proline metabolism in cancer progression. We expect this knowledge will in the long-term aid the development of new therapeutic strategies against cancer.

NIH Research Projects · FY 2025 · 2020-09

PROJECT SUMMARY/ABSTRACT Acute myeloid leukemia (AML) accounts for more than 40% of leukemia mortality in the United States. Each year around ten thousand people die from the disease, most within a few years of diagnosis. Despite advances in our understanding of the disease, few improvements in the therapy of AML have been made. Specific targets and novel strategies to eliminate AML stem cells are required for AML treatment. In preliminary studies, the PI’s lab has observed the following: 1) The expression of DPP4 negatively correlates with AML patient overall survival in 3 different databases. 2) DPP4 inhibitors can prevent AML development in vivo. 3) DPP4 inhibitors can prevent both human and mouse AML cells growth in vitro. 4) DPP4 knock out (KO) AML cells transplanted mice exhibit delay and reversal of AML development in two retroviral-induced AML mouse models. 5) DPP4 KO AML stem cells and progenitors (AML-SCP) are restrained in the bone marrow, with increased apoptosis, and reduced self-renewal ability. 6) DPP4 knockdown prevents the growth of human AML cells. 7) The activation of Src, IkB, and p65 is reduced in DPP4 KO AML cells. Based on these preliminary data, the central hypothesis is: DPP4 regulated trafficking, activation and apoptosis of AML-SCPs are critical for human AML development, which will be addressed in three specific aims. Aim 1: Determine the role of DPP4 in human AML development. The localization, apoptosis, and self-renewal of DPP4 deficient human AML-SCP will be investigated by colony forming unit assay and human AML cells xenografted mouse model. Using the xenograft model, the effect of DPP4 inhibitors treatment alone or in combination with standard chemotherapy to prevent AML development will be evaluated. Aim 2: Examine regulation of AML- SCP engraftment to the BM by DPP4. This will include identification of the critical cytokines regulated by DPP4 for AML-SCP trafficking. To test this, the PI will use migration and engraftment assays. In addition, the PI will investigate localization and niche cells interaction of DPP4 KO AML-SCPs in the BM microenvironment by imaging studies. Aim3: Investigate the role of DPP4 in AML-SCP survival. We will explore the critical domain of DPP4, if DPP4-Src interaction is essential for AML-SCP survival, how DPP4 regulates the activity and protein level of Src in AML-SCP and determine if dual therapy with DPP4 and Src inhibitors has greater benefits against human AML-SCP survival. Collectively, the proposed research will broadly impact the field by identification of a novel treatment for AML, via the strategy of confinement of AML-SCP to bone marrow, and improving the understanding of the role of microenvironment in the development of AML-SCPs.

NIH Research Projects · FY 2025 · 2020-08

Abstract The heart’s pumping capacity is determined by myofilament loaded shortening and power output since the ventricles always work against an afterload to eject blood. The molecular mechanism to explain afterload dependence of ventricular function has eluded cardiac muscle physiologists and cardiologists for decades, and this mechanism could be pivotal for novel small molecule therapies aimed to help heart failure patients. In the previous cycle of the project, the MPI team (a) collectively published 33 peer-reviewed papers, (b) discovered a thin filament mechanism to enhance power reserve capacity in myofilaments from human failing hearts, (c) demonstrated phosphorylation-dependent MyBP-C induced interfilamentary drag, (d) integrated our FiberSim and FiberVent computational models, and (e) adapted cMyBP-C’s putative regulatory functions to our FiberSim model to derive new hypotheses for the current proposal. The new objectives are to (i) use biochemical, biophysical, and transgenic tools to discern cMyBP-C’S role in regulating myofilament power and afterloaded cardiac contractions and (ii) integrate these control mechanisms into our multi-scale computational model that can predict how sarcomere-level modifications impact hemodynamics. The experimental approach is strategically designed to address three independent yet complementary aims. Aim 1 will test the novel hypotheses that cMyBP-C is the load sensor, phosphorylation of cMyBP-C enhances the load-dependence, and these effects translate to ventricular function. Experiments will manipulate cMyBP-C’s phosphorylation state to optimize the load dependence of myofilament power and the afterload dependence of ventricular performance. Aim 2 tests the hypothesis that cMyBP-C tunes load dependence by its N-terminal position, which is modulated by its phosphorylation state using innovative FRET and fluorescent polarization methodologies. In Aim 3, computer models of sarcomere and organ-level function will be deployed to test molecular mechanisms of load-dependent contraction and hemodynamics. This work extends beyond the sarcomere and has the potential to identify high-value therapeutic targets to optimize ventricular performance in heart failure patients.

NIH Research Projects · FY 2024 · 2020-08

ABSTRACT Sulfur mustard gas (SM), a vesicating and warfare agent, has been used in many wars since World War I; most recently in Syria. SM rapidly penetrates the eye on contact and causes blindness by injuring corneal tissue- organization and function. Clinically, patients show a pathology termed as Mustard Gas Keratopathy that involves severe ocular inflammation, recurrent epithelial-erosions, epithelial-stromal separation limbal stem cell deficiency, corneal ulceration, haze and neovascularization. MGK pathophysiology is biphasic including acute and delayed-onset, and involves multiple mechanisms. We developed a novel, multimodal, non-steroidal topical ophthalmic drops, Turbo Eye Drop (TED), containing 4 FDA-approved generic drugs with differing mode of action, and stable at ambient temperature. Our pilot studies found that topical TED efficaciously treats acute and delayed-onset MGK in rabbits in vivo and human cornea ex vivo without significant side effects. Our central hypothesis is that topical TED treats acute and delayed-onset MGK in vivo by curbing SM-induced early inflammatory responses, extracellular matrix degradation, and production of excessive pro-fibrotic and pro- angiogenic factors without significant side effects. This project tests two novel hypotheses to establish an efficacious and safe topical therapy for acute and delayed-onset MGK in vivo, using four specific aims: Aim-1 defines TED treatment for acute MGK in vivo by testing the hypothesis that increasing frequency and duration of TED application will potently treat acute MGK and blindness without significant side effects. Aim-2 establishes TED treatment for delayed-onset MGK in vivo by testing the hypothesis that low TED topical dosing for longer duration will effectively cure delayed-onset MGK without issues in rabbits. Aim-3 uncovers mechanisms used by TED in mitigating acute and delayed-onset MGK in vivo and in vitro. Aim-4 secures intellectual property rights, develops regulatory strategies, and advances TED topical ophthalmic drops as an antidote for SM-induced ocular injury towards human application. This will be accomplished using an established SM-vapor rabbit in vivo and human cornea organ culture ex vivo models, GMP-grade TED eye drops, and monitoring eyes in live rabbits in a time-dependent manner with clinical eye exams and diagnostic imaging. The characterization of mechanisms used by TED in mitigating MGK will be studied using corneal tissues collected after euthanasia by measuring integrity of corneal epithelial basement membrane, epithelial-stromal organization, and collagen fibril arrangement using qPCR, ELISAs, immunofluorescence, H&E, and transmission electron microscopy techniques utilizing our published methods. Successful completion of the project will lead to the development of an effective and safe therapy for acute and delayed MGK and medical countermeasure to minimize ocular obliteration caused by the accidental or intentional use of SM in humans, and therefore will have very high impact in field and public safety.

NIH Research Projects · FY 2026 · 2020-08

Summary Cardiopulmonary toxicities following thoracic radiotherapy and PD-1 blocking immunotherapy have a major impact on quality of life and survival. Therefore, there is an unmet need to have early diagnostic test and intervention for fatal toxicities. Our proposal aims to understand immune mechanisms that modulate potentially life-threatening cardiopulmonary toxicities. We propose to study the toxicities in both mouse models and patients by analyzing blood samples. To achieve our goals, we have assembled a collaborative team across three institutions with a group of expert consultants in order to identify biological correlates and therapeutic targets to ameliorate these autoimmune toxicities. Our prior studies showed excessive mortality in mice simultaneously exposed to radiotherapy and PD-1 inhibition, which we show is dependent on both the cytokine IL-17A and the B-cell. Since both Th17/IL-17A and humoral immunity are implicated in autoimmune diseases, we hypothesize that toxicities result from the unchecked adaptive Th17 response due to PD-1 blockade, combined with autoantibodies against heart and lung tissues generated by the pro-inflammatory B- lymphocytes. We will employ mouse models and pharmacological inhibitors to dissect the underlying autoimmune mechanisms and measure key components of Th17 and B-cell response in prospectively collected blood samples from lung cancer patients undergoing combine radiotherapy and immunotherapy. Aim 1: To determine whether IL-17A/Th17 responses mediate the toxicities. We hypothesize that both innate and adaptive immunity contributes to the toxicities through the link of IL-17A. We will generate KO mice unable to produce IL-17A through either neutrophils or CD4 T cells. We expect that the toxicities are attenuated when Th17/IL-17A are blunted in these mouse models. To determine whether IL-17A/Th17 can be used as predictive biomarkers for the toxicities, we will examine dynamic changes of Th17/IL-17A in serum samples from our patients. Aim 2: To determine the role of humoral response in mediating the toxicities. We hypothesize that pro-inflammatory Tbet+ B-lymphocytes drive autoantibody production which results in the toxicities. We will use Tbetflox/flox CD19cre mice as our model, in which mature Tbet+ B cells are absent. This approach will be complemented by pharmacological depletion of B cells using anti-CD20 or by neutralizing autoantibodies with IVIg in wild-type mice. We expect that the toxicities are attenuated in these models. Furthermore, we will test whether pharmacological inhibitors of Th17/IL-17A reduce Tbet+ B cells and autoantibodies. Finally, the rise of autoantibodies in blood will be captured in mice and patients as a surrogate for the toxicities. Our study of basic mechanisms in preclinical models, combined with analysis of patient samples, will lead to novel diagnostics for early detection and improved therapies for severe cardiopulmonary toxicities.

NIH Research Projects · FY 2024 · 2020-07

SUMMARY The conversion of proliferating skeletal muscle precursors (myoblasts) to terminally-differentiated myocytes is a critical step in skeletal muscle development and repair; control of this process is therefore of fundamental importance in both muscle development and muscle regeneration after injury. The tendency for myogenic cells cultured densely to differentiate and, conversely, the resistance to differentiation of cells at low density has been called the 'Community Effect'; understanding this phenomenon represents a basic question in muscle biology. Based on our initial observation that EphA7, a juxtacrine signaling molecule, is expressed on myocytes during embryonic and fetal myogenesis and on nascent myofibers during muscle regeneration in vivo we examined the muscle phenotype of EphA7-/- mice. We found that their hindlimb muscles possess fewer myofibers at birth, and those myofibers are reduced in size and have fewer myonuclei and reduced overall numbers of precursor cells throughout postnatal life. Adult EphA7-/- mice have reduced numbers of satellite cells and exhibit delayed and protracted muscle regeneration, and satellite cell-derived myogenic cells from EphA7-/- mice are delayed in their expression of differentiation markers in vitro. Exposure to exogenous EphA7 extracellular domain will rescue the null phenotype, and will also accelerate commitment to differentiation in WT cells, which led us to propose a model in which EphA7 expression on differentiated myocytes promotes commitment of adjacent myoblasts to terminal differentiation via reverse signaling. The experiments we propose in Aims 1 and 2 will address the role of EphA7 in myogenic commitment in both the myocyte ("How does commitment to differentiation lead to expression of EphA7?") and the myoblast ("How does receiving an EphA7 signal lead to commitment to differentiation?"). Once they have differentiated, myocytes must fuse with each other or with a growing myotube in order to generate a functional muscle cell (the contractile myocyte fiber), thus this also represents a critical process in both development and regeneration. However, the molecular requirements for fusion in mammalian muscle cells have been elusive. Our data suggest EphA7 also promotes myogenic fusion, possibly via different molecular mechanisms/interactions from its role in promoting myogenic differentiation. The experiments we propose in Aim 3 will test the ability of EphA7 to promote fusion in myogenic and nonmyogenic cells, determine whether it associates with the cell-surface fusogen myomaker, and identify other protein-protein interactions it is participating in at the interface of myocytes and growing myotubes in cis (on the same cell membrane) or in trans (on opposing cell membranes). Collectively, these studies will address the molecular mechanisms regulating two fundamental cellular processes during myogenic differentiation; they also have the potential to provide insight into potential innovations in muscle stem cell expansion in vitro and thus applications in tissue engineering and regenerative medicine.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Strong evidence implicates the sympathetic nervous system as a key regulator of peripheral vascular tone and blood pressure during hypoxia. Herein, we present striking sex-differences in the neurovascular response to hypoxia that challenge current dogma. Our results are corroborated by epidemiological data showing sex disparities in the prevalence of hypertension and progression of cardiovascular disease in conditions of hypoxemia (i.e., sleep apnea). However, contributing mechanisms remain a critically unanswered question. The present study will fill this gap in knowledge while also determining whether these mechanisms are impaired with obesity. Nearly 70% of the US population is overweight or obese, with the prevalence of obesity even greater in patients with sleep apnea. Obese adults exhibit greater sympathetic nervous system activity and higher risk for hypertension than normal weight adults. Emerging data indicate the impact of obesity on cardiovascular health is disproportionate in women versus men and it is reasonable to propose this is exaggerated with the addition of hypoxic stress. The purpose of this application is to examine key mechanisms contributing to sex-differences in hypoxic vasodilation and the impact of obesity, with particular emphasis on the sympathetic nervous system. Our central hypothesis is that young premenopausal, normal weight women are protected from the sympathetic vasoconstrictor effects of hypoxia, and the “beneficial” effect of female sex is lost with obesity. Based on strong preliminary data, we anticipate α-adrenergic mediated vasoconstriction is exaggerated and β-adrenergic and downstream nitric oxide-mediated vasodilation are attenuated during hypoxia in obese women. We will test our central hypothesis via the following specific aims: The first aim of this project will determine sex differences in α-adrenergic receptor mediated vasoconstriction during acute hypoxia as well as the impact of obesity. We propose a comprehensive approach of intra-arterial drug infusions of α-adrenergic agonists and antagonists, combined with direct measures of muscle sympathetic nerve activity in normal weight men, normal weight women, and obese women. The second aim of this project will determine the direct and modulatory effect of the β-adrenergic receptors on hypoxic vasodilation as well as the impact of obesity. We will collect human arterial endothelial cells and measure the peripheral vascular response to hypoxia prior to and following intra-arterial infusion of select β-adrenergic agonists and antagonists. This experimental approach will allow us to strategically assess β-adrenergic receptor activity, sensitivity, and expression in the context of hypoxia as well as down- stream mechanisms. Our proposed findings will advance the fundamental, mechanistic understanding of hypoxic vascular control in women, and results will ultimately guide the development of new strategies to treat and prevent vascular pathophysiology in sleep apnea and other conditions of hypoxia.

NIH Research Projects · FY 2024 · 2020-06

Project Summary Patients with severe, refractory asthma constitute approximately 5% of all asthma patients but are responsible for over 50% of all asthma-related healthcare costs. Biologic treatments for asthma, drugs which target specific etiological pathways in particular asthma cases, have begun to emerge in the market as the first individualized treatments for severe asthma. Cost of these biologics currently ranges from $10-$40k per year and does not always improve asthma symptoms in some patients. The clinical decision to keep a patient on a particular biologic is based on exacerbation count and asthma questionnaire responses at 12 weeks following treatment initiation, by which time the patient has had 3 doses (at 0 weeks, 4 weeks, and 8 weeks). Exacerbation count and patient responses to questionnaires were the primary endpoints of the clinical trials which resulted in approval and are the primary biomarkers used in clinical evaluation of response, but patient- specific biomarkers of lung function would be most beneficial for evaluation of response to these patient- specific drugs. The lack of biomarkers which can predict response to biologic treatment is an unmet clinical need. This study will address this unmet need by evaluating the potential of hyperpolarized 129Xe MRI (HPG) to detect and predict biologic response in individual patients. Hyperpolarization is the process by which the nuclear magnetism of 129Xe is greatly enhanced allowing it to be directly imaged by MRI. Subjects inhale the xenon gas, and MRI are collected yielding high-resolution maps of regional lung ventilation. The overarching hypothesis is that HPG MR can be performed in a routine clinical setting and will predict which patients respond to treatment with mepolizumab, an anti-IL-5 biologic, 8 to 12 weeks sooner than standard clinical assessment. HPG MRI will be performed in asthma patients slated to receive treatment by mepolizumab at baseline, 4-week, and 12-week time points in order to assess its potential to assess, detect, and predict patient response to mepolizumab. This may dramatically reduce costs and improve outcomes for these patients.