Mayo Clinic Rochester

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Approximately 10% of neonates require assistance to begin breathing after birth. At-risk neonates, i.e., those requiring advanced resuscitation due to prematurity, brain injury, or other conditions, are at increased risk of mortality when born in a hospital without a neonatal intensive care unit (‘outborn’) compared to at-risk neonates born in a hospital with a NICU (‘inborn’). Furthermore, at-risk outborn neonates are more likely to suffer serious morbidity, e.g., pneumothorax, severe intraventricular hemorrhage (sIVH), and seizures, and receive cardiopulmonary resuscitation in the delivery room. We estimate that there are 10,000-15,000 at-risk outborn neonates each year who experience poorer health outcomes due to their birth location. However, there are limited strategies to optimize the resuscitation of at-risk neonates born in community hospitals that lack higher levels of neonatal care. There is a critical need to improve patient-important outcomes for this population. The overall objectives in this application are to (i) determine the impact of real-time, audio-video telemedicine consults provided by a neonatologist (termed teleneonatology) on the risk of early mortality and morbidity for at-risk outborn neonates, and (ii) evaluate the effect of teleneonatology on delivery room care provided to these neonates. The central hypothesis is that teleneonatology reduces early mortality and morbidity and improves delivery room care for at-risk outborn neonates. The rationale for this project is that teleneonatology brings resuscitation expertise to the bedside of at-risk neonates more effectively than a brief telephone consult, which is the current practice. Through teleneonatology, the neonatologist can visualize the neonate and provide step- by-step guidance to the community hospital care team. The central hypothesis will be tested by pursuing three specific aims: 1) Determine the impact of teleneonatology on the risk of early neonatal mortality (death within 7 days), 2) Identify the effect teleneonatology has on the risk of early morbidity (defined as pneumothorax, sIVH, or seizure during the first 7 days of life), and 3) Evaluate the impact of teleneonatology on delivery room care. The three aims are embedded into a single prospective, multi-center research trial. With sequential roll-out of the intervention using a cluster randomized stepped wedge design, neonatologists from four NICU ‘hub’ sites will provide teleneonatology consults to at-risk outborn neonates born at 27 community hospital ‘spoke’ sites. Completion of the proposed research will contribute the first-ever, high quality evidence on the impact of teleneonatology on neonatal health outcomes. The research is innovative because it uses video telemedicine to connect neonatologists exactly when needed to at-risk outborn neonates in a way that was not previously possible. This project is significant because it may identify an innovative care model that reduces the long- standing outcome disparities experienced by at-risk outborn neonates.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT The median survival in adults with repaired coarctation of aorta (COA) is ~55 years, and more that 50% of deaths are due to end-stage heart failure and sudden cardiac death from left ventricular (LV) systolic/diastolic dysfunction. LV dysfunction results from chronic pressure overload from hypertension, which in turn leads to LV remodeling (increased fibrosis and stiffness, and impaired relaxation). LV dysfunction has been observed in COA patients with borderline hypertension or stage 1 hypertension (B/S1) (blood pressure 120-139/80-89 mmHg), even though the severity of hypertension in these patients is considered significant enough to warrant antihypertensive therapy based on the current guidelines. Additionally, COA patients with B/S1 hypertension have impaired aerobic capacity and exhibit hypertensive response to exercise, both of which are associated with cardiovascular adverse events The pathophysiologic mechanisms responsible for LV remodeling and abnormal hemodynamic response to exercise in this subset of COA patients are not well understood but are postulated to be due to increased aortic stiffness. We recently demonstrated that a 2-week course of angiotensin-II receptor blocker (ARB) improved aortic stiffness, coronary flow reserve (CFR), cardiac output reserve and vasodilatory reserve (VDR) during exercise. However, it is unknown whether these hemodynamic changes will lead to LV reserve remodeling (decreased fibrosis and stiffness, and improved relaxation) and improved aerobic capacity during long-term therapy. Our long-term goal is to prevent early cardiovascular death in COA patients, by identifying and modifying the pathophysiologic mechanisms leading to LV dysfunction and vascular complications in this population. Our overall objective for this application is to determine whether ARB might promote LV reserve remodeling and improve aerobic capacity, and to delineate the mechanisms of response to ARB. Our central hypothesis is that ARB will promote LV reserve remodeling and improve aerobic capacity by improving CFR and VDR. This hypothesis will be tested by pursuing two specific aims: (1) Determine whether ARB promotes LV reverse remodeling in patients with repaired COA and B/S1 hypertension and delineate the mechanisms of response to ARB; (2) Determine whether ARB improves aerobic capacity and delineate the mechanisms of response to ARB. We will randomize 80 subjects 1:1 to ARB (losartan 50 mg) or placebo for 52 weeks. These subjects will undergo multi-domain assessment of cardiovascular structure and function at baseline and after 52 weeks of therapy. This proposal is innovative because it will novel magnetic resonance imaging techniques to assess cardiovascular response to ARB in patients with repaired COA. The results will be significant because it will enable the development of novel management paradigms for prevention of LV dysfunction and cardiovascular death in this population.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT Several forms of renal replacement therapy (RRT) exist to help patients with ESKD, but they are not equally desirable, with in-home dialysis and kidney transplantation being generally preferable to in-center hemodialysis. The best evidence available indicates that, compared to in-center dialysis, pre-emptive kidney transplantation and at-home dialysis modalities are associated with better quality of life, same or better survival, and are more cost-effective. Noting these benefits, the U.S. Department of Health and Human Services launched the Advancing American Kidney Health initiative in 2019. This program proposed a target of having 80% of ESKD patients on home dialysis or receiving a preemptive transplant by 2025. Yet, according to the U.S. Renal Data System, only 14% of patients diagnosed with ESKD in 2018 utilized a home dialysis modality or received preemptive transplant, despite the fact that the majority of patients are both medically and psychosocially eligible for these options. In part, this may be due to the work that RRT requires of patients to implement new healthcare “workload” into their lives. In the case of home dialysis, patients must shoulder these new tasks without the help of healthcare professionals traditionally found at in-center dialysis facilities. In the case of transplant, patients are often surprised by the lack of a return to “normal life” due challenges with immunosuppression, finances, and relationships that follow transplant. When treatment workload exceeds patient capacity, defined as patients’ abilities and resources to access and use healthcare services and enact self-care at home, patients are at risk for poorer outcomes. These outcomes are in part driven by unsustainable treatment burden, defined as the objective treatment work asked of patients and the subjective negative social and emotional consequences. Treatment burden is correlated with patient non-adherence and has been found to affect as many as 40% of all patients with chronic conditions. There is considerable evidence that patient capacity is a modifiable construct and relevant to CKD care. It is created through patient interaction with their biography (sense of self and life roles), resources, environment, experience of patient work, and social network. In the proposed study we will determine if patient capacity, among CKD patients who progress to kidney failure, correlates with the choice of RRT (Aim 1). We will also determine amongst ESKD patients on RRT whether higher capacity predicts the switch to a more desirable form of RRT and lower capacity predicts the switch to a less desirable form of RRT or withdrawing from RRT (Aim 2). Finally, this study will determine amongst CKD/ESKD patients medically eligible for transplant if patient capacity is prognostic for transplant referral, transplant approval, or death on the transplant wait list (Aim 3).

NIH Research Projects · FY 2024 · 2023-08

PROJECT DESCRIPTION/ABSTRACT Development of effective therapies for glioblastoma (GBM) remains a major challenge despite decades of intensive research. Coupled with intra-tumoral heterogeneity and plasticity, the infiltration of normal brain tissue by GBM cells poses unique therapeutic challenges. Further, the specialized neurovascular unit that forms the blood-brain barrier (BBB) is partially intact in GBM and results in heterogeneous, sub-therapeutic delivery of most cytotoxic chemotherapies to regions of every GBM. We have previously shown that the efficacy of otherwise highly potent antibody-drug conjugates is specifically limited in GBM by poor delivery across the BBB. Like antibodies, single-strand DNA aptamers fold into unique 3-dimensional shapes with epitope binding affinities that rival those of antibodies, and some aptamers also efficiently traverse the BBB. In contrast to the laborious development of antibody-based therapeutics, the integration of solid-support synthesis, PCR amplification, and next-generation sequencing technologies enable massively parallel screening strategies, known as ‘systematic evolution of ligands by exponential enrichment (SELEX)’, to identify individual DNA aptamers with desired physical and biological features through successive rounds of negative and/or positive selection. Based on prior experience with this strategy, we hypothesize that tumor-specific DNA aptamer-drug conjugates (ApDCs) optimized for distribution across the BBB can be efficiently identified using in vivo SELEX with libraries of aptamer-drug conjugates and orthotopic GBM patient-derived xenografts. To address tumor heterogeneity, in vivo selection will be performed across multiple PDXs, and single cell sequencing technology will be leveraged to identify ApDCs that bind to diverse subsets of tumor cells and not normal cells within the brain. The goal of this application is to develop a robust platform for efficient screening of GBM-specific ApDCs. This will be accomplished by addressing three Aims. Aim 1 – R61: Optimize design and sequencing of aptamer-toxin libraries We will optimize strategies for toxin conjugation during library processing through multiple SELEX rounds. Extending our preliminary data demonstrating MMAE toxin stability to PCR thermal cycling, we will optimize toxin-conjugated PCR primers for library preparation and single-cell RNA/aptamer-seq. Aim 2 – R61: Determine optimum training round strategy to identify brain tumor-specific ApDCs We will optimize the time between DNA library injection and tissue collection. We also will evaluate a novel SELEX reward strategy based on selectively capturing aptamers only after cleavage of MMAE from an ApDC. Aim 3 – R33: Apply in vivo SELEX with orthotopic GBM PDXs to train ApDC libraries In vivo SELEX will be performed with an ApDC library across a heterogenous set of orthotopic GBM PDXs to understand the potential for targeting heterogeneous tumor and sparing normal cell populations.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Venous thromboembolism (VTE) continues to be a major health problem with over 500,000 VTE events in the US annually. Venous thromboembolism costs more than $8 billion per year to our health care system and causes more than 100,000 deaths per year in the US alone. Venous thromboembolism is particularly problematic in patients who experience trauma. Despite administration of chemoprophylaxis to trauma patients during hospitalization, about 5% of patients still develop symptomatic VTE prior to discharge, and, more alarmingly, 40 - 60% of patients who develop symptomatic VTE, do so after discharge. An accurate assessment of traumatic- injury based coagulopathies remains dependent on unavailable basic scientific knowledge needed to address the National Institutes of Health (NIH) initiative of defining the “role of laboratory monitoring…to help better define those at risk of bleeding and thrombosis.” To understand the etiologies of trauma-induced venous thromboembolism, a comprehensive approach that assesses plasma coagulation factor activities, platelet reactivity, and endothelial function within the context of pre-trauma patient characteristics (sex, BMI, smoking etc.) and “real-time” blood studies at the time of trauma is required”. Obtaining integrated lab and clinical data from a diverse trauma patient population is not readily feasible in a single investigator’s laboratory or site. The long-term goal is to develop novel predictive, diagnostic and treatment strategies that identify “at risk” individuals for VTE or bleeding soon after trauma. Our Central Hypothesis is that clinically significant thrombosis requires the integrated dysregulation of NETosis, thrombin generation, endothelial injury, and von Willebrand factor- platelet dysfunction. Our collaboration embodies all of the elements needed to improve the quality of care of trauma patients. The investigators are from multiple health-related professions and this leverages experiences and knowledge from different, but related disciplines. Our collaboration also encourages and fosters scientific inquiry with the scientific expertise that will create and disseminate new knowledge and applications. Finally, the team science approach that we have culminated is expected to provide for the highest quality of care and improvement of trauma health outcomes.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second deadliest cancer within the next 10 years. Investigations into novel mechanisms of PDAC transformation are urgently needed as no effective PDAC treatment currently exists. Transcriptional regulation during transformation is multifaceted with genetic mutations and epigenetic alterations playing vital interconnected roles. A key unelucidated facet of gene regulation in the context of transformation is the molecular mechanism which mediates changes in chromatin reorganization. ChIP-seq and ChIP-PCR studies in an inducible model of oncogenic KRAS, a key initiating factor in PDAC, demonstrate a relocation of heterochromatin to the nuclear periphery. These lamina associated domains (LADs) are enriched in H3K9me2 and positioned at the nuclear lamina. ChIP-seq and RNA-seq identified loss of active enhancer regions incorporated into LADs and downregulation of LAD associated genes, respectively. To investigate the mechanism regulating the assembly of LADs downstream of oncogenic KRAS we performed several molecular and biochemical studies. We conducted a screening BioID utilizing Lamin A, a core component of the nuclear lamina known to interact with LADs, as bait. Lamin A BioID identified a novel myosin, Myosin 18a (MYO18a), enriched at the nuclear lamina under oncogenic KRAS signaling. Immunocytochemistry revealed increased nuclear localization of MYO18a and enrichment at the lamina upon KRAS activation. Biochemical analysis validated MYO18a-Lamin A interaction and confirmed MYO18a interaction with chromatin. siRNA knockdown for MYO18a rescued expression of genes associated with oncogenic KRAS-mediated LADs. These data lead us to our central hypothesis that MYO18a acts as a downstream effector of KRAS to modulate chromatin positioning at the nuclear lamina to silence oncogenic gene expression. To test this hypothesis, we will conduct ChIP-seq and RNA-seq experiments in parallel to detect MYO18a associated LADs in in vitro models of PDAC. LAD assembly will then be investigated in the presence/absence of MYO18a. We will also develop MYO18a genetic engineered mouse models to explore the role of MYO18a and nuclear MYO18a in PDAC development and progression. Further study into this mechanism can provide valuable insight into gene regulation in the transformation process and will identify potentially druggable targets.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY / ABSTRACT Nonalcoholic fatty liver disease (NAFLD) is closely associated with the impairment of many metabolic pathways, including decreased hepatic insulin sensitivity and secretion, increased glucagon, and the risk of developing type 2 diabetes mellitus (T2DM). Conversely, patients with diabetes have a higher prevalence of steatohepatitis and end- stage liver disease. As an intermediate state of hyperglycemia easily detectable in clinical settings, prediabetes is the condition that precedes T2DM but the progression is variable, which could increase over time with associated risk factors. To date, the mechanisms underlying prediabetic progression are still not understood. We think simultaneous imaging of the abdominal organs’ abnormalities along the liver-pancreas axis and measures of insulin action, secretion, and hepatic extraction can provide insights into T2DM development, a better understanding of the relationship between NAFLD and diabetes, and an opportunity to intervene in prediabetic progression. The overall goal of this work is to develop an advanced multiparametric abdominal MRE method for characterizing pathophysiologic state of the liver-pancreas axis and prediabetic progression in NAFLD. • In Aim 1, A multifrequency, self-navigating, and hybrid radial-Cartesian 3D vector magnetic resonance elastography (MRE) technology will be developed and expanded on the liver, pancreas and fat data acquisition and image reconstruction. Ten healthy volunteers will be recruited during MRE driver, imaging sequence and reconstruction development and optimization. Benchmark for technical success will be evaluated with a test-retest repeatability and measurement agreement study on ten patients with diagnosed NAFLD. • In Aim 2, We will perform quantitative imaging assessments of the liver-pancreas axis in a cross-sectional study in 150 patients with diagnosed NAFLD with varying risk or progression to diabetes (i.e., 50 patients without diabetes, 50 patients with prediabetes, 50 patients with diabetes). Laboratory and clinical data, as well as insulin secretion and action measurements, will be collected within 30 days of MRI/MRE. Precursory tissue abnormalities and integrative metabolic-mechanical models will be trained and evaluated to assess the risk of developing T2DM. • In Aim 3, assuming 20% withdrawal rate, we plan to have one-year follow-up examinations in 120 patients out of those who have baseline examinations in Aim 2. The models trained in Aim 2 will be further tuned with longitudinal MRI/MRE changes for assessing bi-directional treatment effects on tissue composition, structural and metabolic features, bridging the cellular, gland, enzymes, and hormones to the liver-pancreas axis, thereby predicting progression or regression direction of diabetes development in NAFLD. We anticipate that this program will provide initial validation of multiparametric MRI/MRE and the derived surrogate to assess the liver-pancreas axis of diabetes progression in NAFLD. This project’s success will also provide a valuable noninvasive assessment tool for emerging therapeutic interventions.

NIH Research Projects · FY 2025 · 2023-08

Gastrointestinal (GI) symptoms are significant non-motor manifestations of Parkinson disease (PD) that can precede PD motor symptoms by years; cognitive dysfunction is an important late, non-motor manifestation. Braak’s hypothesis proposes that α-synuclein (αS) aggregates propagate along vagal or olfactory afferents to the central nervous system (CNS), leading to motor and non-motor features of PD. However, αS aggregates are also identified in enteric neurons in elderly people without PD. Squalamine prevention of αS aggregates improved constipation in PD. Density of nigrostriatal dopamine transporters (as assessed by 123I-FP-CIT SPECT) are relevant to cognitive processing in PD. αS oligomers in CSF is a marker of early synucleinopathies and αS protein misfolding in PD implies propagation from the gut to brainstem via the vagus nerve. The relationship between density of nigrostriatal dopamine or aS misfolding in gastrointestinal mucosa and the GI pathophysiology in human PD is unknown. To date, prior studies have typically addressed one pathological mechanism and one GI manifestation. Our multidisciplinary characterization of GI and neural phenotype in PD is key for this first study in 3 groups of humans of the mechanistic roles of the central neural circuitry, central dopamine receptors’ density, autonomic pathways, as well as αS expression in the gastrointestinal and colonic mucosa and microbiome in gut pathophysiology in human PD. Our overall hypothesis is that reduced nigrostriatal dopamine transmitter, autonomic dysfunction and increased αS expression or misfolding in the gastric, duodenal or sigmoid mucosa are associated with abnormal GI motor and barrier functions and with submucosal neuronal dopamine and αS expression in patients with PD with GI symptoms. We propose a prospective cohort design study with two aims in 3 groups, that is, PD patients with Hoehn and Yahr motor stages 1-3 with/without GI symptoms, and age-matched controls with n=24 per group: Aim 1: To compare GI motor functions (gastric emptying and accommodation, colonic transit, defecatory function), small bowel permeability, duodenal and stool microbiome and putative mechanisms (GI and sigmoid mucosal transcriptome, including αS misfolding, tight junction protein, and dopamine receptor expression). Aim 2: To quantitate nigrostriatal dopamine transporter expression, autonomic symptoms, and vagal and sympathetic functions using validated 123I-FP-CIT SPECT, COMPASS-31 questionnaires, and CASS scores, respectively and compare the central dopamine transporter expression and autonomic functions In sub-aims, we compare central and autonomic functions, GI transit and permeability, and mucosal αS mucosal expression or mis-folding in the 3 groups. The significance of the proposal lies in identification of mechanisms, and potential targets for future interventions directed to vagal and autonomic dysfunction, aggregation or misfolding of αS in gut mucosa, as well as dopamine expression to treat GI manifestations of PD.

NIH Research Projects · FY 2025 · 2023-08

Heart transplantation is considered gold standard therapy for end-stage heart failure. However, demand currently far outstrips supply due to multiple challenges. An important limitation is the occurrence of primary graft dysfunction (PDG) in 10-20% of patients and contributes greatly to adverse clinical outcomes and resource utilization. PGD occurs when donor heart function and output is inadequate end organ perfusion. Risk for significant PDG increases when donor heart preservation time is greater than 4 hours. Valproic acid (VPA), a histone deacetylase inhibitor, is a “Food and Drug Administration (FDA)” approved drug traditionally used for the treatment of epilepsy. We now convincingly demonstrate that addition of VPA can dramatically improve donor heart function and improve ischemic tolerance compared to preservation using Histidine-Tryptophan- Ketoglutarate (HTK) preservation solution alone. This was seen in murine heart reperfusion models in the setting of ex-vivo perfusion and transplantation. Furthermore, we show evidence that VPA achieves this by upregulating tricarboxylic acid cycle enzyme Irg1 which produces the anti-inflammatory metabolite “itaconate”. Indeed, our cardiac reperfusion model confirms the impressive upregulation of Irg1 above baseline driven by VPA treatment, and this was accompanied by robust activation of antioxidant pathway mechanisms through Nrf2 transcription factor. Chromatin immunoprecipitation showed that VPA treatment increased Irg1 enhancer activity as indicated by increased occupancy by acetylated H3K27 histone. Importantly, VPA treatment of stored human donor hearts also upregulated Irg1 expression and decreased the expression of inflammatory mediators suggesting translational relevance for large animal and human clinical settings. For this proposal, we plan to: (1) Identify the cell type through which Irg1 acts and we hypothesize that it is most likely through cardiomyocytes (CM) and endothelial cells (EC). The is achieved using transgenic mice with conditional deficiency of Irg1 in these cell types using inducible Cre-Lox technology. We will also examine overexpression models using adeno-associated virus mediated expression Irg1 mRNA. (2) Using cell culture, we will determine whether Irg1/itaconate mediated alkylation modifications on Nrf2 pathway antioxidant proteins impacts their function. We will treat cells with itaconate and then identify as well as mutate relevant alkylation modifications sites at the cysteine residue of antioxidant proteins to determine their importance. (3) We will determine the efficacy of VPA for improving donor heart function and ischemic tolerance in pigs and humans. We will also corroborate mechanisms of VPA mediated cardioprotection identified in murine models. This project has critical clinical implications such as decreasing the PGD incidence, allow transport of donor hearts over longer distances to facilitate organ allocation, and improve clinical transplantation outcomes. Reduction in perioperative donor heart injury by harnessing the cardioprotective effect of VPA and Irg1/itaconate is novel and potentially relevant for preservation of other organs such as the livers and kidneys.

NIH Research Projects · FY 2026 · 2023-08

Project Summary/Abstract Skeletal deterioration and related fracture risk is exacerbated in the elderly cancer survivors receiving radiation treatment (RTx), affecting independent living, and reducing quality of life. Cellular senescence, one of the major pathways induced following RTx-induced DNA damage, is characterized by a pro-inflammatory senescence associated secretory phenotype (SASP, consisting of chemokines, cytokines, growth factors, matrix degrading enzyme, etc), and is mainly regulated by cyclin dependent kinase inhibitors (CDKi’s) p16Ink4a and p21Cip1. Till recently, it was understood that p16Ink4a and p21Cip1 co-expressed in all senescent cells and regulated their function interchangeably. This theory however did not match with the expression pattern of p16Ink4a and p21Cip1 post-RTx or in aging. Using gene expression and RNA in situ hybridization studies, we have recently shown that cells express p21 or p16Ink4a in unique populations of bone marrow cells, osteoblasts, and osteocytes independent of the expression of either senescence marker. Only a small proportion of cells express both p16Ink4a and p21Cip1. Furthermore, we have compelling preliminary data using mass cytometry of bone cells, which allowed us to visualize these independent unique populations of p16Ink4a and p21Cip1 expressing cells without any coexpression of SASP, suggesting a physiological function of these CDKis. Interestingly, in a parallel analysis in radiated bones, we identified elevated levels of Cd11b+p21+ myeloid cell population, which was accompanied by expression of several SASP factors, thus allowing us to characterize these p21+ cells as p21+ senescent (p21+SEN) cells. In a recent seminal finding, using transgenic mice harboring transgenes that enable the selective elimination of either p16Ink4a or p21Cip1 expressing cells, the elimination of p21+SEN cells in the p21-ATTAC [apoptosis through targeted activation of caspase] mice, but not the elimination of p16+SEN cells in the p16-INK- ATTAC mice, could mitigate most of the RTx-related adverse events in bone in young mice. Whether this approach of clearance of p21+SEN cells will work to alleviate RTx-related bone deterioration in old mice, which have a pre-existing high burden of senescent cells, remains to be seen. Based on our compelling preliminary data, we will test our central hypothesis that: “Acute generation of p21+SEN cells mediate RTx-related skeletal deterioration and BMSC dysfunction, but targeted early clearance of p21+SEN cells can alleviate RTx-related chronic skeletal deterioration and promote fracture healing”. To test our central hypothesis, our aims are: (aim 1)To identify key mechanisms that are involved in RTx-related skeletal deterioration following early clearance of p21+SEN cells in young and aged mice, (aim 2): To assess bone architectural changes following cell specific clearance (using our novel Cre-LoxP mice, p21-LOX-ATTAC) of p21+Cd11b+SEN myeloid cells and (aim3): To assess if prior clearance of senescent cells pharmacologically or by genetic clearance of p21+SEN cells will promote fracture healing. The project will address questions related to basic biology of aging and role of p21 in skeletal cells and lay the groundwork to support the idea that an early intervention could prove effective to counter adverse changes from RTx, and to alleviate chronic skeletal deterioration and reduce the risk of potential fractures.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY/ABSTRACT Dr. Egan is a physician trained in clinical endocrinology and will undertake a four-year mentored research and career development program in the pathophysiology of gestational diabetes mellitus (GDM) at Mayo Clinic. She will complete a master’s degree in Clinical and Translational Science, gain experience in the conduct of in vivo studies in an obstetric population, acquire new skills in analysis and modeling of human physiological data, and develop scientific leadership skills and collaborative relationships. These activities will fill current gaps in knowledge and allow Dr. Egan to become an independent clinical investigator. Dr. Egan will be guided by a team of prominent researchers with relevant areas of expertise and an excellent track record in mentoring young faculty. This team will be led by her primary mentor, Dr Adrian Vella, whose research focuses on the pathogenesis of type 2 diabetes mellitus (T2DM). GDM is a common pregnancy complication and while the associated hyperglycemia resolves postpartum, it is a strong risk factor for T2DM. There is little understanding of maternal β-cell function in early pregnancy, how this may predict GDM in later pregnancy, and what changes occur postpartum. Dr. Egan’s preliminary data suggest that α-cell function also changes during pregnancy, but its role in GDM development and postpartum glucose tolerance is unknown. In the proposed in vivo studies, Dr. Egan will determine the degree of β-cell (Aim 1) and α-cell (Aim 2) dysfunction needed to produce GDM and examine the islet cell recovery (or lack thereof) that occurs postpartum. Dr. Egan will assess if GDM can be predicted earlier in pregnancy which would facilitate more timely intervention. She will also identify the residual defects in glucose metabolism that persist at one year postpartum. Such defects could contribute to the accelerated progression to T2DM observed in women with GDM (Aim 3). To accomplish these aims, Dr. Egan will study women during pregnancy and post-partum using state-of-the-art, non-invasive techniques to collect and examine detailed physiologic data. In keeping with the NIDDK mission, the proposed studies will address important aspects of diabetes development in women and provide the foundation for a future R01 application with the goal of developing novel diabetes prevention and treatment approaches.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Focal epilepsy is a network disease marked by focal areas of cortical hyperexcitability and interconnected brain regions that affect excitability. For many patients, it is challenging to accurately localize brain regions involved in seizure initiation and to determine nodes of the seizure network that affect excitability on an individual basis. We hypothesize that seizure-related brain tissue is chronically compromised and exhibits aberrant, interictal, hyperexcitability that can be interrogated dynamically using stimulation. We propose using novel stimulation- based biomarkers to develop reliable and precise estimates of seizure onset locations and related network nodes. Whereas stimulation-based biomarkers have typically utilized single pulses of electrical stimulation to map connectivity, we propose two diagnostic stimulation biomarkers which utilize multiple stimulation pulses, novel waveforms and time-varying amplitude envelopes to interrogate adaptation and inhibitory feedback. Leveraging high-channel count stimulation, we suggest a method to rapidly map modulatory network connections which could serve as implanted device targets. We utilize simultaneous single unit recordings to underpin our proposed invasive EEG biomarkers of hyperexcitability. Our aims are to: 1) Develop stimulation-based biomarkers of the seizure onset zone, 2) Identify patient- specific, modulatory network connections, and 3) Determine whether there are interictal single neuron signatures of hyperexcitability. To do this, we will use a newly developed external stimulator will allow for automated, efficient stimulation of 128-256 channels in epilepsy patients implanted with temporary invasive electrodes. Simultaneous recordings from microelectrodes will allow us to correlate single and multiunit activity with EEG activity recorded from macroelectrodes. We will examine these results within the novel mathematical framework of fractional dynamics that can link the timescales of responses to excitability. Grant outcomes will include a rapid protocol using stimulation-based interictal biomarkers to localize the seizure onset zone and identify relevant network nodes. Microelectrode recordings will provide a single/multiunit scale understanding of EEG excitability dynamics. This proposal explores the largely uncharted territory of stimulation-based biomarkers beyond single pulse electrical stimulation to improve treatment for drug-resistant epilepsy.

NIH Research Projects · FY 2025 · 2023-08

PROJECT DESCRIPTION/ABSTRACT Therapeutically resistant triple negative breast cancer (TNBC) is characterized by mesenchymal features that facilitate immune evasion and disease progression, resulting in high rates of death from the disease. New strategies targeting the mesenchymal phenotype to overcome therapeutic resistance are needed. Interactions between the cytoskeleton of cancer cells and the surrounding extracellular matrix play important roles in initiating local invasion and metastasis. The resulting mechanical stimuli activate signaling pathways which promote the transition of non-motile polarized epithelial cells to cells with mesenchymal properties that are able to invade surrounding tissues and evade immune surveillance, a process termed epithelial to mesenchymal transition (EMT). Fibrotic stiffening of the non-cellular stroma resulting from extracellular matrix deposition is a common feature of TNBC and other malignancies that is associated with therapeutic resistance and poor prognosis. How mechanical stress promotes EMT and cancer progression has not been elucidated. Ataxia Telangiectasia Mutated and Rad-3 Related (ATR) is best known as a regulator of the DNA damage response in replicating cells. In our preliminary data, we have found that high ATR protein expression is associated with reduced progression-free survival in TNBC. We discovered that ATR is post-translationally upregulated by a deubiquitinating enzyme, USP21, in response to mechanical stress, and that ATR regulates the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex to promote β-catenin nuclear translocation and mechanical stress-induced EMT. This new role for ATR in mechanical stress and EMT is independent of ATR’s established function in regulating the DNA damage response. In a clinical trial of anti-PD-1 immunotherapy for advanced TNBC, high ATR levels in biopsy specimens was correlated with EMT and reduced responsiveness. ATR inhibition could promote mesenchymal to epithelial transition and anti-tumor immunity in vivo. We hypothesize that ATR regulates the LINC complex following mechanical stress to promote β-catenin mediated EMT, immune evasion, and TNBC progression. We will test this hypothesis through three specific aims. Aim 1 will examine the impact of mechanical stress on ATR deubiquitination and the LINC complex; Aim 2 will evaluate how ATR influences β-catenin pathway activation and EMT; Aim 3 will investigate the role of ATR-SUN2 in stiffness and EMT associated immune evasion.

NIH Research Projects · FY 2026 · 2023-08

ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in the United States. NAFLD is a conglomerate of the relatively nonprogressive non-alcoholic fatty liver (NAFL) and progressive non-alcoholic steatohepatitis (NASH). While hepatic steatosis is a conserved feature of both NAFL and NASH, the latter is characterized by liver injury, inflammation, fibrosis, and the risk of liver cancer and cirrhosis. At the present time, there are no effective noninvasive and scalable screening strategies to distinguish between NAFL and NASH nor monitor NASH progression. Thus, there is a significant unmet need for biomarkers which are relatively easy to obtain, can be tested repeatedly over time, can distinguish NAFL from NASH, are pathophysiologically in- formed, and help risk stratify patients with NASH. Extracellular vesicles (EVs) carry signals from diseased cells and organs and therefore represent ideal biomarkers for NAFL and NASH. However, the development of EV based biomarkers has been confounded by several challenges. First challenge relates to the large sample volume requirements of mass spectrometry approaches traditionally used for EV analysis (e.g. LC-MS/MS). This limits their utility for the analysis of scarce patient samples. Second challenge lies in the fact that EVs released from the diseased liver become diluted by circulation and represent only a small subset (~1%) of total EVs present in peripheral blood. This makes it difficult to identify biomarkers of disease progression or therapy re- sponse. In this project, we will address the aforementioned challenges by developing novel technologies: 1) nanoprojectile (NP) secondary ion mass spectrometry (SIMS) that will enable multiplexed analysis of EVs using microliters of sample and 2) microfluidic organotypic liver cultures that will allow us to harvest undiluted liver EVs from healthy or diseased tissue. Upon completion of this project, we will have in hand a powerful mass spectrometry technology for proteomic and lipidomic analysis of EVs. The identification of EV biomarkers in this project will, in the future, be parlayed into a blood-based test that will allow to diagnose NAFLD, distinguish NAFL from NASH and help risk stratify patients. Such a test will replace highly invasive liver biopsy currently used for diagnosis and will revolutionize the care of patients with NAFLD. It will also allow early diagnosis of NASH in at risk populations.

NIH Research Projects · FY 2025 · 2023-07

Abstract Dysregulation of myelopoiesis can be either cell-intrinsic or cell-extrinsic. Cell-intrinsic mutations can result in myelodysplastic syndromes (MDSs) or myeloproliferative disorders (MPNs), while cell-extrinsic systemic inflammation promotes emergency myelopoiesis. However, the cause of the enhanced myelopoiesis is not always clear, and these two mechanisms are not necessarily mutually exclusive. Patients with chronic inflammation or autoimmune disease are at an increased risk for MDS, and chronic inflammation is often observed in MDS patients. Provocatively, our preliminary data demonstrates that a cell-extrinsic emergency myelopoiesis progressively develops when the inner mitochondrial membrane transporter ABCB7 is conditionally deleted in differentiating B cells in the bone marrow. ABCB7 is intrinsically required for B cell development beyond the pro-B cell stage. In 4-5 month old mice engineered to lack ABCB7 in B-lineage cells, bone marrow hematopoiesis is disrupted. Bone marrow erythropoiesis is dramatically reduced, and bone marrow myelopoiesis is greatly enhanced. To compensate for the loss of bone marrow marrow erythropoiesis, stress erythropoiesis is initiated in the spleen, however, the mice remain anemic. Importantly, the enhanced numbers of myeloid cells being produced in the bone marrow were not labelled with a cre-dependent reporter, establishing that the myeloid expansion is cell-extrinsic. ABCB7-deficient pro-B cells exhibit iron overload and DNA damage, which could provide the stimuli for initiation of chronic inflammation leading to progressive cell- extrinsic emergency myelopoiesis. Intriguingly, ABCB7 expression is decreased in myelodysplastic disorders in which patients have a mutation in the RNA splicing gene SF3B1. ABCB7 RNA levels are decreased by 60- 70% in SF3B1-mutated CD34+ cells from MDS-RS patients due to aberrant splicing and nonsense-mediated decay. We hypothesize that myelodysplasia in patients with SF3B1-mutated myelodysplastic disorders is not strictly cell-intrinsic, but instead there is a cell-extrinsic contribution mediated by decreased expression of ABCB7 in B-lineage precursor cells. To test this hypothesis, we have created a novel dox-regulated ABCB7 mouse model that allows us to address the hematopoietic consequences of decreased ABCB7 expression. These studies will establish that reduced expression of ABCB7, in both mouse and human hematopoiesis, leads to dysfunctional myelopoiesis due to B-lineage cells. Our studies will have important therapeutic implications for treating SF3B1-mutated myeloid disorders, as elimination of the B-lineage cells could contribute to amelioration of disease.

NIH Research Projects · FY 2025 · 2023-07

Mayo Clinic, which serves ~1.3M patients annually, has a history of excellence in clinical research and clinical trials. Its state-of-the-art research facilities in Rochester, Minnesota, Jacksonville, Florida, and Scottsdale, Arizona enable coordinated clinical trials in the upper Midwest, Southeast, and Southwest United States. The Department of Neurology, which has over 200 child and adult neurologists, has an enterprise-wide leadership structure that supports its integrated practice, research, and education mission. Mayo Clinic Neurology cares for >90,000 adult and pediatric patients annually and has a robust clinical research enterprise. Departmental faculty have clinical expertise and clinical trial experience across the full spectrum of neurological diseases, including rare and ultra-rare conditions. Mayo Clinic is ideally positioned to provide NeuroNEXT the diverse patients with unmet needs and the clinical trialists with expertise in these diseases. Investigator-initiated clinical trials by Mayo neurology faculty will be directed towards NeuroNEXT funding mechanisms. This grant application aims to support, expand, and innovate neuroscience clinical trial activities at all Mayo Clinic sites by providing the infrastructure to ensure robust integration within NeuroNEXT, while leveraging our institutional strengths. Furthermore, the grant will support an innovative program to train the next generation of clinical trialists to thrive in an evolving landscape of innovative trial design and modern regulatory science.

NIH Research Projects · FY 2026 · 2023-07

PROJECT ABSTRACT The intestinal lumen contains a plethora of proteins from the diet and microbiota that require tolerogenic responses. If tolerance is not properly mounted against these innocuous proteins the mucosal immune system constantly encounters, individuals become progressively at-risk for inflammatory disorders including food allergies or inflammatory bowel diseases. As these disorders increase in incidence, particularly within the pediatric population, understanding how the immune system encounters luminal antigens during early life must be thoroughly explored for the prevention and treatment of these disorders. Currently, exclusive breastfeeding is the recommended dietary practice for infants through the first three months, followed by complementary breastfeeding with introduction of solid foods. Yet the world health organization estimates only 30% of infants globally are exclusively breastfed in the first three months, and alternative diets ranging from infant formula to goat’s milk are used for a variety of reasons. Breastfeeding is significantly associated with decreased risk of food allergy and IBD, and a number of beneficial components of breast milk have been identified. We have previously shown epidermal growth factor (EGF) is highly concentrated in breastmilk, particularly early in lactation. Immediately following delivery, EGF inhibits antigen delivery within the neonates intestine, and a lack of dietary EGF is associated with increased intestinal permeability. As the infant ages, EGF in breastmilk decreases allowing antigen delivery to occur and FoxP3+ regulatory T cells develop in response to orally derived antigens during this time. Thus, maternal EGF regulates antigen delivery until a time when the infant is prepared to develop tolerogenic responses to encountered antigen. Our preliminary data shows decreased dietary EGF or disrupting the Epidermal Growth Factor Receptor within intestinal cells of the neonate resulted in early antigen delivery, decreased FoxP3+ regulatory T cells at the time of weaning, and an increased predisposition to intestinal inflammation in a model of colitis. Interestingly, while FoxP3+ regulatory T cell differentiation was initiated in response to early antigen delivery, these cells eventually lost FoxP3 expression but remained in the intestine, becoming effector cells. Antigen delivery was also associated with an increase in CX3CR1+ F4/80+ antigen presenting cells, however the role neonatal antigen presenting cells downstream of antigen delivery remains unknown. These data suggest early antigen delivery in the absence of maternal EGF regulation disrupts oral tolerance during early life. Here we will 1) determine the effect of neonatal antigen delivery on antigen presenting cells in the colon and 2) determine the mechanism through which neonatal antigen delivery abrogates regulatory T cells. This work has important implication in why antigen delivery during early life is regulated by breast milk, and the consequences of early antigen delivery in the absence of maternal regulation.

NIH Research Projects · FY 2026 · 2023-07

Project Summary/Abstract Synthetic opioid-involved overdose deaths have increased sharply. Fentanyl is driving many of those overdose deaths. However, oxycodone is one of the most prescribed opioid medications in the US. Current in vitro assays and in vivo models designed to study the pathophysiology of opioid use disorder (OUD) and to discover potential therapeutic targets are useful, but there is a need for additional model systems. Our preliminary data and serval preclinical study using single-cell sequencing have revealed that each opioid agent might have unique molecular profiles and mechanisms of action. Those findings highlight the need for additional models to evaluate drug action in the brain at the single-cell level. Our research team combines expertise in addiction medicine, pharmacogenomics, and bioinformatics, expertise required to develop a computational and experimental framework to integrate gene expression and chromatin accessibility in induced pluripotent stem cell (iPSC)- derived brain organoids. The goal of the proposed study is to provide novel mechanistic insight into drug action at single-cell resolution. Our research strategy involves the use of single-cell sequencing technology and iPSC-derived 3D brain organoids to identify molecular signatures for OUD using two commonly prescribed synthetic opioids: oxycodone and fentanyl as molecular probes. Aim 1, we will define molecular characteristics of response to synthetic opioids: oxycodone and fentanyl exposure of iPSC-derived forebrain organoids from both OUD patients and healthy controls at the single-cell level. Aim 2, we propose to reconstruct transcriptional regulons in different cell types in the brain organoids by applying novel network biology approaches to prioritize potential candidates, to detect meaningful biological information embedded in the sea of Big Data and to uncover novel regulatory mechanisms that explain the properties of biological phenotypes. These approaches could help to develop mechanistic hypothesis for experimental validation. Aim 3, we will study genes and pathways identified from Aim 1 and Aim 2 with regard to their potential use as novel drug targets for OUD treatment or prevention, by pursuing functional genomic studies using appropriate iPS-derived CNS cell types and brain organoids Our findings will enhance the general understanding of drug mechanism(s) of action and the underlying pathophysiology responsible for opioid addiction in a drug-dependent fashion, thus opening new avenues to discover novel therapeutic targets for the treatment of OUD. In summary, this proposal is based on extensive preliminary data, and decades of experience in using drugs as “molecular probes” for underlying genomic and other omic mechanisms. As a result, the proposed studies have significant implications for molecular mechanisms leading to understanding of the pathophysiology of OUD as well as the discovery of novel therapeutic agents for OUD treatment and/or prevention. If successful, our research approaches could be generalizable to other opioids and to other substance use disorders.

NIH Research Projects · FY 2024 · 2023-07

Abstract T cell anergy is a cell-intrinsic program of non-responsiveness to self-antigens, and is an important component of peripheral tolerance. As the induction and maintenance of anergy is critical to preventing breakdown of tolerance and initiation of autoimmunity, it is important to understand the mechanisms and molecules that regulate anergy. Initially, T cell anergy was induced by TCR stimulation in the absence of costimulation and a variety of stimuli have been used in vitro to induce anergy. This early work demonstrated the critical importance of activation of the Ras/Map kinase pathway upon TCR/CD28 stimulation for T cell activation, IL-2 production and proliferation. However, the mechanism is not clear as to how the Ras/Map kinase pathway is downregulated in anergic T cells. Recently, endogenously anergic T cells in WT mice have been identified and are characterized by high expression of CD73 and FR4. We have found that these naturally anergic FR4+CD73+ CD4+ T cells in vivo from WT mice express lower levels of Runx1 protein as compared to other CD4+ T cell subsets. TCR/CD28-induced activation of CD4+ T cells with conditional deletion of Runx1 resulted in lower IL-2 expression and reduced proliferation as compared to activation of WT CD4+ T cells. In addition, there was a higher frequency of naturally anergic FR4+CD73+ CD4+ T cells in CD4- cre Runx1 conditional knockout (cKO) mice or tamoxifen-treated ER-cre Runx1 cKO mice in mixed bone marrow chimeras. Thus, deletion of Runx1 in mature CD4+ T cells predisposes them to the development of T cell anergy. To understand the mechanism(s) responsible for hypo-responsiveness of Runx1-deficient T cells upon TCR/CD28 stimulation, RNAseq analysis was performed on WT and Runx1-deficient CD4+ T cells. Runx1-deficient CD4+ T cells upregulated expression of Dab2IP, an adaptor molecule that has been shown in non-hematopoietic cells to be a negative regulator of Ras signaling. Dab2IP contains a Ras-GAP domain, which leads to the inactivation of Ras through accelerating hydrolysis of GTP to GDP. To determine whether increased Dab2IP expression could inhibit T cell activation and promote anergy induction, we generated a novel cre-dependent, dox-regulatable Dab2IP transgenic mouse. Our initial results demonstrate that similar to CD4+ T cells conditionally deleted for Runx1, Dab2IP Tg CD4+ T cells have increased frequencies of naturally anergic FR4+CD73+ CD4+ T cells in their memory cell pool. Our hypothesis is that increased expression of Dab2IP interferes with normal TCR/CD28 signaling, preventing T cell activation, and predisposing T cells to the development of anergy.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY / ABSTRACT Wrist osteoarthritis (OA) is a prevalent, debilitating condition; however, limited attention has focused on the scaphotrapeziotrapezoid (STT) joint. The STT is positioned on the radial side of the wrist, spanning both rows of carpal bones and bridging the thumb and wrist joints. STT OA impacts between 15% and 24% of adults over 45 years of age, with increased prevalence and incidence given advancing age and female sex. However, compared to hand joints, the STT has received limited diagnostic and therapeutic attention. The wrist is anatomically complex: the number, unique geometries, small size, and close proximities of carpal bones pose challenges for diagnostic imaging and guiding evidence-based surgical interventions. Due to intricate carpal interrelationships, a strong biomechanical foundation of normal STT function during motion and under loading is critical for tailoring interventions that remedy symptomatic STT OA without compromising the remainder of the carpus or thumb. Thus, there is a need to understand the complex interplay between carpal bones during motion in unaffected participants. Specifically, there is a need to measure bone motion dynamically to understand the relationships between carpal bones during various motions of the wrist and hand under different loading conditions. 4DCT (3DCT over time) yields a time series of image volumes captured during motion with high spatial and temporal resolution, offering the exciting capability to capture bones dynamically. Interosseous proximity distributions, a proxy for pressure, and centers of closest proximity will be used to describe the joint relationships as a function of motion and loading, conferring an in-depth understanding of the STT joint in both unaffected and pathological states. Further, the magnitude and variability of joint space and shape at each STT articulation have not been rigorously quantified. Statistical shape modeling (SSM) and machine learning of STT carpal bones collected from unaffected and pathological STT joints will allow us to elucidate the interactions between interacting geometries of articulating surfaces during motion and bone morphology. Our study aims to understand STT arthrokinematics in normal and pathological conditions in the following aims. Aim 1: Quantify STT arthrokinematics during unresisted and resisted wrist and thumb motions, separately, in participants without wrist OA and quantify normal variations in STT morphology. Aim 2: Quantify STT arthrokinematics during unresisted versus resisted activities in patients with early-stage STT OA using 4DCT and compare 3D morphologies of unaffected and pathologic STT joints. 4DCT data elucidate motions and loads that stress the joint, while morphologic data indicate sites of structural change. A parallel analysis will allow us to define structure-function interactions at the STT joint. The Aims will culminate in a comprehensive view of how STT loading impacts arthrokinematics in participants with and without OA. An integrative understanding of morphology and the role of load in provocative maneuvers will enhance our understanding of this complex joint, promoting biomechanically motivated diagnostic and treatment strategies.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY / ABSTRACT Cirrhotic stage liver disease is the 11th leading cause of mortality in the US, and no treatment exists for late- stage disease other than liver transplantation. Thus, the overall objective of this proposal is to elucidate novel mechanisms that drive the release of fibrogenic signals leading to liver fibrosis progression and to guide the development of potential treatment strategies. Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Our preliminary data in primary human and mouse HSCs as well as in vivo demonstrate that 1. platelet-derived growth factor B (PDGF) induces metabolic reprogramming by increasing glycolysis; 2. PDGF- mediated glycolysis increase the transcriptional activation mark, histone 3 lysine 9 acetylation (H3K9ac) on the promoter region of vesicle trafficking-related Ras-related protein Rab (RAB) genes; 3. glycolysis promotes EV release and enrichment with fibrogenic proteins; and 4. in vivo glycolysis inhibition by HSC-selective hexokinase 2 (HK2) deletion abrogates liver fibrosis. We have utilized our novel findings to generate the CENTRAL HYPOTHESIS of the current proposal that PDGF-mediated glycolysis in HSCs induces fibrogenic EV release through H3K9ac-dependent transcriptional upregulation of RABs to amplify liver fibrosis. We will employ sophisticated cellular and animal models, including in vitro and in vivo utilization of dCas-KRAB model, in vivo HSC-specific HK2 deletion model as well as acetyl-coA-deficient HSCs, to investigate the following integrated, yet independent aims. In Aim 1, we will test the hypothesis that PDGF increases glycolysis through lysine-deficient kinase 1 (WNK1) phosphorylation to mediate glucose transporter 1 (GLUT1) translocation to the plasma membrane. We will uncover the kinase signaling leading to glycolysis in HSCs by: a. studying how PDGF increases glycolysis through phosphorylation of WNK1, a novel PDGF downstream lysine-deficient kinase; and b. investigating how WNK1 phosphorylation promotes GLUT1 translocation to the plasma membrane to increase glycolysis, which represents a new mechanism in HSCs. In Aim 2, we will test the hypothesis that glycolysis leads to EV release by upregulating the transcription of RABs through acetyl coenzyme A (acetyl-CoA)-mediated H3K9ac. We will dissect how glycolysis drives epigenetic regulation of vesicle trafficking gene program to control EV release by: a. studying how PDGF promotes the accumulation of the metabolite acetyl-CoA to increase H3K9ac; and b. examining how H3K9ac promotes RAB transcription to induces EV release. In Aim 3, we will test the hypothesis that HSC-specific glycolysis and subsequent epigenetic regulation of fibrogenic EV release amplifies in vivo liver fibrosis. We will investigate the mechanism of in vivo liver fibrosis amplification by: a. studying how glycolysis-mediated EVs amplify liver fibrosis by targeting HSCs; and b. investigating how the disruption of epigenetic regulation of RABs ameliorates liver fibrosis. This novel and innovative line of inquiry will define an HSC-specific glycolysis-dependent model of liver fibrosis amplification and set a trajectory towards new and significant advances to treat liver fibrosis and cirrhosis in humans.

NIH Research Projects · FY 2025 · 2023-07

Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. The mission of the Mayo Clinic Medical Scientist Training Program (MSTP) is to train clinician-scientist leaders of the future. Our program is designed to mentor trainees whom we individually guide on their journeys to develop technical, operational, and professional skills to support a productive career in which they integrate research and clinical activities to advance biomedical science. In order to fulfill our mission of developing future clinician-scientist leaders, we propose four measurable objectives that will serve as the foundation of our program: 1) To provide a fully integrated scientific and clinical training program that will help trainees develop the skills needed to recognize and address important clinical problems while simultaneously enabling our graduates to move easily between research and clinical environments; 2) To develop future leaders who possess the self-confidence and communication skills to share scientific concepts with colleagues and individuals at all scientific levels; 3) To encourage trainees to develop self-reflective skills that will lead to rigor in their own science and 4) To provide trainees with experiences that reflect their own long-term scientific goals in academia or other careers. This MSTP admits nine MD-PhD trainees per year from a pool of over three hundred applicants. Ninety-six carefully vetted and trained laboratory mentors will provide a safe learning environment and guide rigorous research. The co-Directors, Drs. Schimmenti and Kaufmann, have over thirty years of combined experience in MD-PhD leadership and are committed to the success and well-being of every trainee. Mayo Clinic’s three-part mission comprised of patient care, research, and education, provides a rich environment for training. The average time to degree for the last ten years of graduates is 8.18 years, with a mean of 4.3 first author papers per trainee and 8.7 total papers per trainee. Trainees are required to submit an F award or equivalent; and the success rate over the last ten years has been 58%. Trainees match well, with three of five trainees matching in physician scientist training programs in 2021. A self-reflective iterative evaluation process has been developed and implemented to support cycles of program improvement with goals to support rigor and reproducibility training, trainee retention, decreased time to degree, scholarship, and career satisfaction. The long-term goal of the program is to provide each dual degree graduate a firm foundation for a lifetime of career success in research and research related fields.

NIH Research Projects · FY 2026 · 2023-07

PROJECT SUMMARY/ABSTRACT In 2017, ~750K Americans were diagnosed with end-stage kidney disease (ESKD), which rises 3% yearly. 87% will have renal replacement therapy with hemodialysis (HD), with preferred vascular access through arteriovenous fistula (AVF). AVFs have ~62% year patency due to venous stenosis (VS) and neointimal hyperplasia (VNH) causing reduced blood flow and suboptimal HD which is treated with percutaneous transluminal angioplasty (PTA) at >$3B/yr. Monocyte and macrophage recruitment occurs to the injured vessel wall after PTA of stenotic arteriovenous fistulas (AVF) through increased expression of MCP-1 leading to VS/VNH. Bindarit is an oral selective inhibitor of MCP-1, - 2, and -3 and we encapsulated it in polylactic-co-glycolic acid (PLGA) nanoparticles embedded in a thermosensitive Pluronic F127 hydrogel (BN NP) for periadventitial delivery to the outflow vein to test in this proposal. Scanning electron microscope and dynamic light scattering were used to characterize the BN NP and control nanoparticles (NP C). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to study drug release kinetics. Immediately after PTA, in a murine model of AVF stenosis, BN NP or NP C was administrated to the periadventitia of outflow veins. Animals were sacrificed 3 and 21 days later for gene expression, histomorphometric, and immunohistochemical analyses. Doppler ultrasound was performed weekly. There was no difference in the size and storage modulus of BN NP compared controls. Pharmacokinetic analysis demonstrated increased drug release from BN NP when compared to controls. BN NP treated vessels had reduced MCP-1, MCP-2 and MCP-3 gene and protein levels, reduced CCR2, increased FABP4/IL8, macrophage/monocyte abundance, proinflammatory cytokines, reduced CD4 (+) cells, reduced endothelial inflammation, and venous fibrosis resulting in positive vascular remodeling and improved patency with reduced VS/VNH. There was increased peak velocity 21 days after PTA in the BN NP group. Periadventitial administration of BN NP to the outflow vein after PTA results in decreased VS/VNH. Central Hypothesis. Periadventitial delivery of Bindarit NPs to AVF outflow vein after PTA decreases Mcp-1, -2, -3, CCR2 expression with increased FABP4/IL8 leading to less immune, macrophage cell infiltration with reduction in smooth muscle cells, fibrosis, and VS/VNH. We propose three specific aims: Aim 1: Determine how Bindarit NPs reduce MCP-1, -2, and -3 leading to decreased monocyte to macrophage differentiation, migration, proliferation, leukocyte chemoattraction by endothelial cells and inflammatory cytokine expression. Aim 2: Assess the role(s) of Bindarit NPs on CCR2 and FABP4/IL8 on reducing VS/VNH after PTA of stenotic AVFs. Aim 3: Ascertain the safety and efficacy of Bindarit NPs on reducing VS/VNH after PTA in pigs with CKD.

NIH Research Projects · FY 2025 · 2023-07

Project Summary Bone lesions from solid tumors such as breast, prostate, or kidney cancers, tumors originating in the bone marrow such as multiple myeloma (MM), or other non-malignant musculoskeletal pathologies can occur anywhere in the skeleton. These bone lesions cause pain and spinal cord compressions, leading to pathologic fractures and paralysis, thereby diminishing the patient's quality of life. Current therapies rely on diagnosing these bone lesions by whole-body X-ray or bone scans, which only identify them at advanced stages. While whole-body magnetic resonance imaging (WBMRI) is recommended for pretreatment assessment (e.g., in MM), MRI is often limited to spine and pelvic regions in practice to minimize patient discomfort, compromised image quality from geometric distortion, and high costs due to prolonged acquisition times. To address this unmet clinical need, we developed a novel WBMRI technique: `Dual-Echo T2-weighted acquisition for Enhanced Conspicuity of Tumors' (DETECT), for improved lesion visualization by simultaneously separating the confounding signals of fat and fluid. Compared to WBMRI with diffusion-weighted imaging (DWI), single- shot DETECT increased lesion detection (>40%) in considerably shorter scan times (<10 min) and without image distortions. This method also improved robustness to motion in the thoracic and abdomen regions, however, it suffers from image blurring due to T2-decay particularly in spine and extremities, limiting the diagnostic performance. In the current proposal, we will address these limitations by developing the next- generation WBMRI-DETECT using an efficient combination of single-shot and multi-shot acquisitions. DETECT also generates fat signal for quantitative fat fraction (FF) maps that can be used as a prognostic biomarker in MM, since tumor cells replace fat, a major constituent of bone marrow. This method also led us to develop a DETECT-based DWI technique for accurate measurement of apparent diffusion coefficient (ADC). The specific aims are: 1) To develop an integrated WBMRI using single-shot and multi-shot DETECT, along with quantitative FF maps; 2) To develop a DETECT-based DWI with accurate ADC measurements; and 3) To evaluate the integrated WBMRI, including DETECT-DWI and contrast-enhanced perfusion, for efficient bone lesion detection and therapy response assessment. We will use bone lesions in MM as the proof-of-concept disease to achieve these project goals. The successful outcome of this project will be an efficient WBMRI protocol with accurate FF and ADC measures as imaging biomarkers, validated in detection and measuring therapy response in MM patients. This WBMRI in combination with contrast-enhanced MRI including perfusion, will be an excellent cost-effective and practical approach (<45 minutes of table time) for widespread use in clinical practices across the world. This will benefit MM patients and patients suffering from other bone lesions, including pediatric patients during long follow-ups, without the drawbacks of PET/CT. This will provide relevant clinical information for treatment decisions to positively impact patients' quality of life and overall survival.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY / ABSTRACT Left heart disease (LHD) leads to pulmonary hypertension (PH-LHD, aka Group 2 PH), right ventricular (RV) failure, and increased mortality and morbidity. Advances in pulmonary vascular biology gleaned from study of the pulmonary arterial (PA) circulation in Group 1 PH and relevant animal models have led to effective therapies for Group 1 PH. Trials of Group 1 PH therapies in PH-LHD have shown highly variable (favorable, neutral or harmful) effects. We propose that two critical knowledge gaps contribute to variability in therapeutic response and impede progress in treating PH-LHD: (1) the lack of a mechanistically informative hemodynamic classification system defining the nature (vasoconstriction vs remodeling) and location (PA vs pulmonary venous (PV)) of pulmonary vascular disease in LHD, and (2) lack of understanding of vessel specific (PV vs PA) biological pathways mediating pulmonary vascular disease in PH-LHD. The objective of this proposal is to address these knowledge gaps and enable therapeutic innovation in PH-LHD. Based on extensive preliminary studies in human and experimental (Exp) PH-LHD, our central hypothesis is that PH-LHD is a phenotypically diverse entity whose ultimate therapeutic approach will be defined by unique hemodynamic phenogroups and vessel specific (PA vs PV) pathophysiological perturbations. In human and Exp PH-LHD, we will use novel hemodynamic assessments to phenotype PH-LHD according to pulmonary vascular resistance (PVR), vasoreactivity, and the longitudinal distribution of PVR (Aim 1). Findings will be validated in human PH-LHD by assessing phenogroup-specific differences in aerobic capacity, RV reserve function and exertional lung congestion. Findings in Exp PH-LHD will be validated by defining PA and PV remodeling (quantitative histomorphometry). Our broad hypothesis is that both the primary mechanism and location of the elevated PVR in PH-LHD have clinical implications and anatomical underpinnings. In human and Exp PH-LHD, we will then (Aim 2) use histochemical, proteomic, and transcriptomic based techniques and bioinformatic analyses to define vessel specific mechanisms across PH-LHD phenogroups. These studies will couple the Aim 1 hemodynamic phenotyping approach to vessel specific vascular biology. In Aim 3, we will determine if therapeutic agents based on our omics studies in human and Exp PH-LHD will ameliorate PV or PA remodeling and delay the progression of PH in Early or Late Exp PH-LHD phenogroups. The research outcome from this work will be a new hemodynamic classification of PH-LHD linked to specific pathophysiology and therapeutic targets, thus enabling individualized medicine approaches to PH-LHD.