Mayo Clinic Rochester

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.

NIH Research Projects · FY 2025 · 2023-05

The overarching goal of this grant is to develop an optimal viro-immunotherapy approach for the treatment of relapsed refractory (R/R) T cell lymphoma (TCL). VSV-IFNβ-NIS is an oncolytic Vesicular Stomatitis Virus (VSV) engineered to selectively infect and kill tumor cells, while sparing normal cells. In the recently completed first-in-human dose escalation study utilizing a single intravenous (IV) infusion of VSV-IFNβ-NIS in patients with R/R multiple myeloma (MM) and TCL, we reported that the virus can be safely administered up to the highest dose level tested (1.7e11 TCID50) with no dose limiting toxicities. The most exciting and significant clinical activity was seen in patients with TCL who received one dose of 1.7e11 TCID50 VSV-IFNβ-NIS as a single agent, with 5 clinical responses (2 CR and 3 PR) in 10 heavily pretreated patients with multi-focal TCL. Pharmacokinetics (PK) and pharmacodynamics (PD) correlative analyses suggest that responses are due to a combination of direct oncolytic tumor destruction and immune-mediated tumor control. However, not all patients responded, and several patients had mixed tumor responses only. We recently showed in preclinical models that addition of anti-CTLA-4 (αCTLA4) and anti-PD1 (αPD1) antibodies given prior to VSV- IFNβ-NIS resulted in complete remission of established tumors in 100% of mice. Thus, the goal of this grant is to improve the 50% response rate and durability of response (DOR) in patients with TCL by maximizing the potency of VSV-IFNβ- NIS using immune checkpoint blockade (ICB) to amplify the antitumor activity of virotherapy-boosted tumor-reactive CTL. In parallel, we seek to identify biomarkers and immune correlates differentiating responders from non-responders in TCL through analysis of tumor biopsies and blood. We thus have the following specific aims: Specific Aim 1. Determine the safety, PK/PD and efficacy of one IV of VSV-IFNβ-NIS in combination with immune checkpoint antibodies for patients with R/R TCL. Hypothesis: Immune activation with αCTLA4 and αPD1 antibodies followed by destruction of TCL with VSV virotherapy will maximize the boosting of antitumor cytotoxic T cells to bring about long-term tumor control and remission. Specific Aim 2. Determine the baseline tumor gene expression profile (antiviral and immune) and tumor mutation burden (TMB) and evaluate their impact on clinical response. Hypothesis: A VSV permissive gene signature and high TMB are positive contributing factors to the depth and durability of response. Specific Aim 3. Determine the impact of VSV-IFNβ-NIS treatment with αCTLA4 and αPD1 immune boosting on the kinetics, magnitude and specificity of antitumor CTL and Treg immune responses. Hypothesis: Timely addition of the immune modulators with VSV virotherapy will result in enhanced frequency and duration of antigen reactive T cells. Upon completion of this study, we will achieve a deeper understanding of parameters that drive responses in viro- immunotherapy, and potentially derive a new treatment option worthy of further clinical development in patients with R/R TCL. Results from this study will also provide the foundation for building more effective dosing for other cancer indications.

NIH Research Projects · FY 2025 · 2023-05

PROJECT SUMMARY/ABSTRACT Adipose tissue phenotype and physiology are thought to be important contributors to overall metabolic health, and our preliminary data suggests that exercise training improves adipose tissue biology. The objectives of the proposed project are to elucidate how alterations in adipose tissue cellular composition and endocrine signaling may contribute to the beneficial adaptations to exercise and to generate new mechanistic insights into the role of adipose tissue in metabolic health. Aim 1 will study the effects of exercise training on adipose tissue macrophage populations and inflammatory profile and will examine the association of these changes with metabolic health. Older adults with prediabetes and obesity will participate in a 3-month exercise training intervention with metabolic health, adipose tissue macrophage populations, and adipose inflammatory molecular signatures measured before and after the training intervention. Aim 2 will determine how acute exercise triggers key signaling events (e.g., immune cell infiltration, release of adipokines, extracellular vesicles) in adipose tissue that are likely to have paracrine and endocrine effects. Leukocyte populations and inflammatory signatures will be assessed in adipose tissue biopsies collected from older adults before, immediately after, and 3 hours after a 30-minute bout of cycling exercise. The adipose tissue secretome will be assessed in plasma and in media collected from cultured human adipose tissue explants. Overall, this project will elucidate the effects of exercise on adipose tissue phenotype and provide mechanistic insight into the causes of metabolic dysfunction. The career development plan that accompanies the proposed project will facilitate my successful transition to independent research scientist. My career development plan will provide the foundation on which I can build an independent research program, comprising the following elements: didactic training in multidisciplinary fields; mentorship by experts in bioinformatics, clinical and translational research, diabetes, aging, and exercise; and hands-on experience in clinical research, bioinformatics, and scientific communication With the proposed career development plan, I will learn new statistical and bioinformatics approaches and laboratory techniques; gain basic knowledge in tangential fields of science to enable me to ask integrative questions and to seek out multidisciplinary collaborators; and lead an independent clinical trial, gaining vital experience in clinical research with guidance from experience mentors. The supportive environment fostered by my mentorship team and by Mayo Clinic will allow me to establish multidisciplinary collaborations and to become an independent investigator.

NIH Research Projects · FY 2026 · 2023-05

PROJECT SUMMARY Chronic myelomonocytic leukemia (CMML) is an aggressive hematological malignancy with dismal outcomes. There is an unmet need for CMML focused rationally derived therapies. CMML can be divided into “proliferative” (pCMML) and “dysplastic” subtypes, with pCMML having a high frequency of RAS pathway mutations and being associated with a median survival of <18 months. We have shown that in pCMML, mutant NRAS is a bona fide oncogenic driver and that RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic check point kinases such as PLK1. PLK1 was among the top protein coding genes upregulated in RAS mutant CMML and given that prior RNAi studies had shown increased sensitivity of RAS mutant cells to PLK1 inhibition and the fact that PLK1 physically interacts with RAF1 at the mitotic spindles, as well as the ongoing use of clinical grade PLK1 inhibitors in AML trials, we selected PLK1 as a therapeutic target in RAS mutant pCMML. We showed differential sensitivity of RAS mutant pCMML cells to PLK1 inhibition using in vitro (progenitor colony assays) and in vivo models (patient- derived xenografts) and demonstrated potential synergy with hypomethylating agents (HMA). We now propose a seminal Phase 1 clinical trial testing the safety and preliminary efficacy of Onvansertib, a novel, oral, PLK1 inhibitor in patients with relapsed/refractory pCMML, using an innovative BOIN (Bayesian Optimal Interval) design. Samples from trial patients will be used to assess pharmacokinetics, targeting efficacy and response correlations with PLK1 and KMT2A (regulator of RAS PLK1 axis) expression levels. We will also use our large CMML biorepository (n=177 RAS mutant samples) to develop in vitro and in vivo models to assess mutational, transcriptomic and epigenetic predictors of response and resistance; specifically the impact of individual RAS pathway mutations and cooccurring TET2 and ASXL1 mutations, the two most common response defining mutations in CMML, on Onvansertib responses. Based on our preliminary data, it is our central hypothesis that PLK1 inhibition is an effective treatment strategy in RAS mutant pCMML, with responses being influenced by defined mutational profiles and epigenetic features. We will address this hypothesis by first performing a Phase 1 clinical trial testing the safety and targeting efficacy of Onvansertib, an oral selective PLK1 inhibitor, in pCMML (AIM 1). Second, we will determine the in vitro impact of individual RAS pathway mutations and associated CMML transcriptomic and epigenetic profiles on Onvansertib responses (AIM 2). Finally, we will assess the impact of ASXL1/TET2 mutations on response to Onvansertib and Onvansertib plus HMA in RAS- pathway mutant CMML xenografts (AIM 3). If successful, this research will provide the first targeted therapy for RAS mutant myeloid neoplasms, setting the stage for personalized therapeutics in hematological malignancies.

NIH Research Projects · FY 2024 · 2023-05

PROJECT SUMMARY / ABSTRACT The goals of this proposal are to: 1) obtain experimental skills and career training necessary to develop an independent research program investigating mechanisms of bone development and disease, and 2) characterize a novel target of bone remodeling called G protein-gated inwardly-rectifying K+ channel 3 (Girk3), a key regulator of potassium flux and physiological processes. Our data demonstrate that Girk3-/- mice develop high bone mass after skeletal maturity and have low interleukin-1 beta (IL-1β) and other circulating cytokines. The overall objective of this proposal is to test the hypothesis that Girk3 deletion enhances bone density in adult skeletons by altering the secretion of IL-1β and other monocyte-derived cytokines that modify bone resorption. During the mentored K99 phase, the specific aims are to identify how deletion of Girk3 in monocytes affects bone mass (Aim 1) and to determine how Girk3 regulates cytokine production by blood and bone marrow monocytes (Aim 2). Methods used to evaluate the role of Girk3 in bone remodeling will include dynamic and static histomorphometry, osteoblast and osteoclast differentiation assays, cytometry by time-of-flight (CyTOF), single cell RNA-sequencing (scRNA-seq), and bioinformatics. Completion of this aim will facilitate my transition into the independent R00 phase when the specific aim (Aim 3) will be to determine if Girk3 deletion can prevent bone loss in a murine model of osteoporosis via inhibition of IL-1β. The K99 phase will be conducted at Mayo Clinic and will focus on obtaining mentored training in professional development through meetings with a curated mentorship team, regular attendance and presentations at research seminars and national research conferences, participating in workshops on grantsmanship and responsible conduct in research, and seeking out networking opportunities, as well as conducting the experiments in Aims 1 and 2 and publishing the results. The R00 phase will be conducted in my independent laboratory and will focus on completion and publication of Aim 3 and developing an R01 application based on the results. The proposed plan synergizes new skills in osteoclast and ion channel biology, unbiased spectometry, single cell RNA sequencing and bioinformatics, and bone histomorphometry with prior expertise in endocrinology and osteoblast biology to create my own unique research trajectory. The career development plan and research strategy outlined in this application will produce a robust foundation for an independent research career in musculoskeletal biology.

NIH Research Projects · FY 2026 · 2023-05

Our preliminary data implicates inflamed adipose tissue as a factor that may lead to biochemical abnormalities in muscle and attenuate some of the adaptive responses to exercise in obese individuals. The objective of this project is to evaluate a hypothesis that adipose tissue inflammation activates inflammatory cascades in skeletal muscle that, in turn, attenuate molecular responses to exercise. Aim 1 will determine how adipose tissue phenotype influences skeletal muscle function and exercise response parameters in humans. Humans with obesity will complete studies to assess molecular response to acute exercise from protein synthesis rates, mRNA of exercise-responsive genes, and activation of signaling proteins in skeletal muscle. Subcutaneous adipose tissue (SAT) and intermuscular adipose tissue (IMAT) will be assessed using a combination of non-invasive imaging and biopsy-based molecular phenotyping. This aim will determine if acute exercise response is attenuated with increasing IMAT or in people with inflamed adipose tissue phenotype. Aim 2 will determine the mechanisms by which SAT and IMAT secretomes influence muscle phenotype and exercise response. Primary muscle cultures and adipose explants (SAT and IMAT) will be generated from participants in Aim 1. Myotubes will be exposed to conditioned media from IMAT, inflamed SAT, or non- inflamed SAT to evaluate their influence on molecular phenotype and exercise response pathways. Genetic and pharmacological approaches will be used to target key inflammatory pathways in skeletal muscle (TLR4, IKK, JAK/STAT) to assess their roles in exercise response in vitro. The contribution of the proposed research is expected to be a detailed understanding of the mechanistic links between adipose tissue inflammation, local inflammatory responses in skeletal muscle, and molecular response to acute exercise. The knowledge gained in the proposed study will have a positive impact because “exercise resistance” represents a significant barrier to the prevention and reversal of disease and disability in humans, and understanding the role of anatomically distinct adipose tissue pools in skeletal muscle physiology may lead to new approaches to enhance the beneficial adaptations to exercise in populations that stand to benefit most. Discovering new ways to enhance training responses in people, particularly those at risk for metabolic disorders, will have significant benefit since exercise non-responders have increased risk for metabolic disease.

NIH Research Projects · FY 2026 · 2023-05

Alcohol withdrawal is a critical component of development and persistence of addiction to alcohol and a cause for significant morbidity and mortality in patients with alcohol use disorders (AUD). Introduction of benzodiazepines resulted in reduced severity of alcohol withdrawal syndrome (AWS), and decreased mortality and frequency of complications. While research suggests involvement of certain neurotransmitter systems in the neurobiology of AWS, genetic markers associated with predisposition to AWS and its treatment response remain unknown. We therefore propose to perform comprehensive genetic analyses, including genome-wide association studies (GWASs), to identify genetic markers of AWS risk and response to benzodiazepine treatment of AWS. The proposed analyses will constitute the largest GWAS of AWS to date, with a sample size of AUD subjects 10 times larger than the only previously published AWS GWAS, and the first GWAS of benzodiazepine response. We will also investigate sex differences in genetic effects on AWS and response to its treatment. Our study will involve advanced analyses, including assessment of SNP-based heritability of AWS along with gene and pathway-level analyses (including drug-target enrichment) and fine-mapping to detect relevant genetic effects. We will also use polygenic risk scores to determine if genetic load for AUD-related traits is associated with AWS. The proposed study is aligned with NIAAA policy, which states that GWAS is “the preferred approach for the identification of and the confirmation of genes that harbor variants that contribute to AUD and related phenotypes, since results from these studies will likely provide potential insights into translational studies and new therapeutic targets”. The proposed research also supports NIH efforts to increase awareness and attention to sex in research. Our preliminary data and power calculations demonstrate that analysis of available data is expected to identify common genetic variants associated with AWS. Discovery of genetic variants that impact the risk of AWS will generate knowledge on its neurobiology and ultimately contribute to the development of tools for the identification of patients at risk and selection of treatment options based on an understanding of inter- individual differences in sensitivity to this central component of AUD. This line of research may also contribute to development of new AUD treatment approaches aimed to restore physiological dysfunction associated with those genetic variants.

NIH Research Projects · FY 2026 · 2023-04

Abstract Numerous recent studies have consistently shown that likely no two cells in the human body have the same genomes, a phenomenon called somatic mosaicism. Mosaicism can be studied using various approaches, but the study of mutations directly in the cell promises a comprehensive characterization of mosaicism in any tissue. Analysis of single cell genome by cloning relies on natural DNA replication machinery in cells and, thus, minimizes errors in DNA during cloning; however, cloning is limited by the ability of cells to proliferate. Analysis by whole genome amplification (WGA) is hampered by introduced errors and non-uniformity of amplification. Here we propose to address the limitations of single cell cloning and single cell WGA by developing a hybrid approach that proceeds in two stages: 1) limited culturing of single cells to a micro-sized colony of 2-50 cells; and 2) WGA of the micro-size colonies to yield enough DNA material for sequencing. An optimized hybrid approach will enable rigorously and unbiasedly studying somatic mosaic at a single cell level throughout the human body without WGA artifacts. Finally, to preserve tissue cell heterogeneity and enable biobanking of tissues amenable to the developed hybrid approach, we will develop a storing protocol for tissues to preserve proliferative potential of cells in the stored tissues. Success of the project would enable comprehensive and accurate discovery of mutations in a single cell in a variety of tissues prioritized by SMaHT and beyond, deepening our understanding of the mosaicism of humans. 2

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT Dr. Yogesh Reddy, MBBS, MSc, is an Assistant Professor in the Department of Cardiovascular Medicine at Mayo Clinic. Dr. Reddy is seeking a Mentored Career Development Award to develop the training required to study the contribution of skeletal muscle abnormalities to exercise performance in patients with Pulmonary Arterial Hypertension (PAH). Dr. Reddy has received extensive training in pulmonary vascular hemodynamics in PAH during his previous training under the T32 grant, providing necessary skills and experience to study the central cardiovascular abnormalities that drive exercise intolerance in PAH. While therapies targeted to these central limitations have markedly improved clinical outcomes in PAH, a wide therapeutic gap remains, as nearly 90% of patients report significant residual symptoms even after treatment with pulmonary vasoactive therapy. Preliminary studies have suggested that peripheral abnormalities in skeletal muscle may contribute to the exercise intolerance of patients with PAH, but no study has yet used gold standard methods to measure skeletal muscle oxygen (O2) utilization during exertion in PAH. To fill this knowledge gap and build new skills in the characterization of peripheral deficits in PAH, Dr. Reddy proposes to: 1) determine the independent role of skeletal muscle and mitochondrial O2 transport abnormalities in exercise performance in PAH compared to controls, and 2) perform a randomized clinical trial of isolated quadriceps (knee-extension) based training to assess its effect on peripheral O2 transport and whole-body exercise performance in PAH. In this proposal, Dr. Reddy is seeking the primary mentorship of Dr. Barry Borlaug, an experienced, NIH-funded investigator with expertise in exercise physiology in pulmonary hypertension, who has a proven track record of successfully mentoring junior investigators. The mentorship team supervised by Dr. Borlaug includes multidisciplinary experts who will provide necessary advanced training in the fields of mitochondrial biology (Dr. Sreekumaran Nair), leg blood flow quantification and exercise training interventions (Dr. Thomas Olson), iterative mathematical modeling and biostatistical methods (Dr. Rickey Carter), patient reported outcomes (Dr. Shannon Dunlay) and clinical trials in PAH (Dr. Robert Frantz). As a result of the training proposed, Dr. Reddy will obtain the skills necessary to: 1) comprehensively evaluate mitochondrial and peripheral O2 transfer physiology during exertion, and 2) conduct randomized trials of an exercise-based intervention. The advanced training obtained during this award in skeletal muscle biology and exercise physiology will greatly complement the skills he has already obtained characterizing central hemodynamic abnormalities in PAH, uniquely positioning him to succeed as an independently-funded clinician scientist focused on the pathophysiology and treatment of exercise intolerance in PAH, understanding and investigating the problem “from all angles”. The data derived and expertise gained will form the basis for a competitive R01 application centered on novel treatments targeting peripheral deficits for patients with PAH.

NIH Research Projects · FY 2026 · 2023-04

Abstract/Project Summary Cholangiocarcinoma (CCA) is a highly lethal cancer that arises from the bile ducts and is often diagnosed at an advanced stage with very poor prognosis. Factors associated with development of CCA include inflammatory conditions of the bile ducts such as congenital cysts, gallstones, primary sclerosing cholangitis (PSC), chronic hepatitis from viral and other causes, and occupational exposure to toxins such as the organic solvents dichloromethane and 1,2-dichloropropane. In parts of Asia, liver fluke infestations of the bile ducts are a major risk factor. However, the majority of patients who develop CCA worldwide have no known major risk factor. Based on epidemiologic studies, it appears that CCA risk is due to a combination of genetic and environmental factors. Genome-wide association studies (GWAS) have identified genetic susceptibility loci for different cancer types, allowing development of risk models that can allow determination of an individual’s risk of cancer. GWAS have also provided valuable information on the biological and molecular pathways that contribute to risk of different cancers, allowing improved understanding of cancer development and progress in cancer prevention and treatment. Because CCA is a relatively less common cancer, it has been difficult to study large numbers of CCA patients and there have been no large CCA GWAS studies published, either for patients with de novo CCA or for CCA developing in patients with PSC. Although PSC patients are up to 150 times more likely to develop CCA than the general population, only 10-20% of PSC patients will progress to CCA. Variation in the risk of CCA among PSC patients may be caused by a complex interplay between genetic and environmental factors. Several studies indicate PSC has a strong genetic component; however, the impact of genetic factors in PSC-related CCA development is yet to be elucidated. We hypothesize that different host genetic variants are associated with CCA risk in de novo versus PSC-associated CCA cases. We have developed a large multi-institutional and multi-national collaboration to identify gene variants associated with CCA. The goal of this study is to use high-density single nucleotide polymorphism (SNP) analysis of genomic DNA from CCA patients and controls. In a first phase of the study, we have genotyped DNA from 2829 CCA patients, including 2412 of European and 417 of Asian descent. Of the European descent cases, 197 were PSC-associated. Comparing the results with controls from the PLCO cohort, we identified a variant in the HLA region on chromosome 6 that reached genome wide significance. A number of additional regions showed suggestive results. We now propose an expansion of this discovery phase to acquire, genotype and sequence DNA from an additional 7,267 CCA patients to confirm the validity of these suggestive results. We are also expanding recruitment efforts to enrich the cohort in samples from non-European patients. We will use sophisticated statistical genetic methods to analyze the results from the multi-ethnic cohort. Whole exome sequencing will also be used to identify rare variants associated with CCA.

NIH Research Projects · FY 2026 · 2023-04

Chronic liver disease (CLD) is a highly common cause of death due to the development of cirrhosis and consequential life-threatening portal hypertension (PHTN). Hepatic venous pressure gradient (HVPG) measurement is the gold standard to evaluate PHTN but not readily available in routine clinical settings due to its invasiveness and requirement of adequate operator skill and experience. Thus, an urgent need for a safe, reliable, operator-independent noninvasive method exists for diagnosing and monitoring PHTN to allow early targeted interventions. Biological soft tissues usually have a biphasic architecture, including a solid matrix (cells, extracellular matrix) saturated with fluid (interstitial fluid and blood). MR elastography (MRE) can quantitatively assess multiple tissue mechanical properties. Within them, liver stiffness, which comprises solid stress and fluid pressure, is widely used clinically to assess hepatic fibrosis as a liver biopsy alternative. Our prior studies show that MRE-assessed viscoelasticity can distinguish solid-related fibrosis and fluid-related inflammation in early-stage CLD. Other hepatosplenic MRE parameters (compressibility and nonlinearity) are promising biomarkers for hemodynamics-associated fluid pressure but require systematic evaluation. The overall goal of this work is to develop an advanced multiparametric 3D vector MRE (3DV MRE) technique of the liver and spleen for fully characterizing PHTN in advanced CLD. • In Aim 1, a dual-frequency, self-navigating, and hybrid radial-Cartesian 3DV MRE method will be developed for characterizing multiple mechanical properties in both small animals and human subjects. We will develop a model-based iterative reconstruction and a 3D neural network inversion to calculate viscoelasticity, compressibility, and nonlinearity in the liver and spleen from the hybrid 3DV MRE data. • In Aim 2, we will use 3DV MRE imaging on three PHTN rat models (diet-induced nonalcoholic steatohepatitis (N=22), cirrhosis-induced PHTN after bile duct ligation surgery (N=22), and congestion-induced PHTN after partial inferior vena cava ligation surgery (N=22)). Technical integrity, scientific rigor, and diagnostic accuracy will be assessed by comparing multiple imaging biomarkers with in vivo interstitial fluid and portal pressure measurements, ex vivo dynamic mechanical analysis testing results, and histologic features. • Before the clinical translation, we will evaluate the repeatability of MRE biomarkers in 5 controls and 5 clinical patients using a test-retest strategy. Finally, a pilot clinical study in 10 controls and 50 clinical patients with endoscopy or HVPG will rigorously cross-validate the diagnostic accuracy of the PHTN predictors. Advanced 3DV MRE development enables other investigators and us to explore this promising technology for many other etiologies related to fluid pressure. This project’s success will also provide a valuable noninvasive assessment tool for emerging therapeutic interventions.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY We have repeatedly observed that cells acquire the ability to escape from viral replication/lysis upon long-term low-level exposure to Vesicular Stomatitis Virus (VSV). Whilst investigating the mechanisms associated with escape, we showed that APOBEC3B is a major effector of mutational plasticity in cells which evade viral replication/therapies including VSV. RNAseq of cells which escaped VSV identified ~300 coding mutations with APOBEC3B signatures, some of which we hypothesized would directly affect the ability of the virus to replicate within target cells. Of these mutations, a single C-T point mutation in the Cold Shock Domain Containing E1 (CSDE1) gene (CSDE1C-T), which generates a mutant CSDE1 protein CSDE15P-S, was expressed at high clonality in both human and murine cells, of different histological types, which became resistant to VSV. These data show, for the first time to our knowledge, that CSDE1 plays a highly significant role in replication of, and oncolysis by, VSV. We also observed that VSV can co-evolve to complement cellular mutations such as CSDE1C-T. Thus, forced evolution of wild type VSV through CSDE15P-S cells allowed us to track the emergence of a mutant virus VSV-IFNß-IGR P/MC-U. This virus contained a highly specific mutation in the Intergenic region between the P and M genes of VSV (IGR P/MC-U) (located in the only perfect consensus binding site for CSDE1 in the VSV genome) and completely rescued high level replication of virus in cells expressing mutant CSDE15P-S. On the basis of these data, here we will test our overarching hypothesis that CSDE1 is a critical mediator of the replication of VSV; that mutation in CSDE1, such as at CSDE1C-T, allows for target cell escape from replication of VSV; and that VSV can evolve compensatory mutations to recover replication fitness in CSDE1(C-T)-mutated cells. To test this hypothesis, we have formulated four Specific Aims in which we will: (Aim 1) define how CSDE1 mediates replication of VSV, and how mutations in CSDE1 are critically associated with escape from VSV; (Aim 2) understand how the IGR P/MC-U mutation in VSV complements CSDE15P-S; (Aim 3) track the induction of the IGR P/MC-U mutation as the virus undergoes strong selective pressures against its replication and identify the cellular anti-viral mutational pathways which imprint mutational signatures onto VSV genomes; and (Aim 4) exploit our discovery of CSDE1 as a critical co- factor for VSV replication to generate CSDE1-expressing VSV with enhanced efficacy as both vaccination and oncolytic platforms. Overall, these studies will have significant impact in understanding VSV/host cell interactions, in defining pathways by which viruses can usurp anti-viral mutagenic pathways to seed escape- competent quasi species and will inform development of novel, improved VSV for vaccination and oncolysis.

NIH Research Projects · FY 2026 · 2023-03

PROJECT SUMMARY Senescent cells (SCs) cause senescence, a dominant risk factor for acute kidney injury (AKI). SCs are cell cycle-arrested (due to upregulated cell cycle inhibitors p16Ink4a and p21Cip1) and display a senescence- associated secretory phenotype (SASP) which is proinflammatory and proapoptotic. Senolytics, agents that kill SCs, are now in clinical trials. We demonstrate senescence in the heme protein-mediated AKI model (HP-AKI) as indicated by multiple indices. The significance of such changes – injurious or protective – is unknown as regards AKI. The AKI field recognizes that mitochondrial injury drives AKI, while the senescence field recognizes that mitochondrial injury elicits senescence; this application uniquely unites these two concepts. Early in HP-AKI, we demonstrate that mitochondria are injured and their heme content increased; normal mitochondria, exposed to such heme content, cease functioning. Heme, a prooxidant tetrapyrrole, present in the ubiquitous family of heme proteins, is freed when heme proteins are destabilized because of cellular stress. We also demonstrate that heme induces p16Ink4a/p21Cip1 and a SASP, and suppresses PGC-1α. In exploring heme-induced mitochondrial injury and induction of p16Ink4a/p21Cip1, we focused on two transcription factors both upregulated by mitochondrial injury, one, ETS1, being an inducer of p16Ink4a, the other, ATF4, an inducer of p21Cip1. Our preliminary data demonstrate that these 4 principal molecules (ETS1, ATF4, p16Ink4a, p21Cip1) are all induced in HP-AKI; in heme-exposed renal proximal tubular epithelial cells in vitro; and in renal ischemia-reperfusion injury (IRI). Our hypothesis is that AKI results from heme-mediated mitochondrial injury and ensuing senescence, a thesis to be tested in three aims. Aim I: Define the role of heme-mediated mitochondrial injury in ETS1 and ATF4 expression, senescence, and AKI. Using complementary in vivo and in vitro approaches, we will sequentially examine the role of heme-mediated mitochondrial injury; the contribution of ETS1, ATF4, and ETS1/ATF4-independent pathways; and the involvement of senescence in AKI. Aim II: Define the roles of p21Cip1 and p16Ink4a in AKI: Genetic strategies. The role of p21Cip1 in AKI will be examined by inducible deletion of high p21Cip1-expressing cells and with proximal tubule-specific p21 KO mice. The role of p16Ink4a will be examined by inducible deletion of high p16Ink4a-expressing cells and by inducible proximal tubule-specific deletion of high p16Ink4a-expressing cells. Aim III: Define the effect of senolytics in AKI. SCs survive because of upregulated anti-apoptotic pathways, while senolytics kill SCs but not non-SCs. This aim examines the efficacy of senolytics in AKI, and in reducing the sensitivity of aged mice to AKI. In sum, this resubmitted R01 examines senescence in AKI, linking it sequentially to heme-mediated mitochondrial injury, the transcription factors ETS1 and ATF4, and p16Ink4a and p21Cip1. This R01 offers novel insights regarding the role of senescence in the pathogenesis of AKI and the therapeutic utility of senolytics.

NIH Research Projects · FY 2026 · 2023-03

Breast screening has rapidly transitioned in the US to digital breast tomosynthesis (DBT), an x-ray technology in which 3-D images are reconstructed from a limited number of low-dose x-ray source projections. DBT offers superior tissue visualization allowing for the direct measurement of the actual volume of dense tissue, rather than an estimated percent or volume (from a 2-D mammogram). Since breast density is a strong predictor of masking and risk, DBT volumetric density measures, including our recently developed and first of its kind, fully automated 3-D measure, have the potential to improve individualized breast cancer (BC) risk prediction. No studies to date have evaluated DBT volumetric density measures in large, diverse cohorts or subpopulations to understand the impact of these measures to improve prediction of masking and risk in order to tailor prevention and screening approaches. Our goal is to comprehensively examine DBT volumetric density measures as risk factors for invasive, interval and advanced BC, and evaluate their impact on clinically relevant BC risk models and artificial intelligence (AI) algorithms across multiple racial groups. We propose this research in three large breast screening cohorts that perform routine DBT, each with comprehensive clinical risk factors, multiple DBT per woman, follow-up and BC outcomes. Specifically, we will establish a nested case-control study of over 3,000 invasive BC cases and 9,000 controls matched on facility, age, race, ethnicity, date of enrollment DBT and follow-up time and estimate novel research and commercial DBT volumetric measures as well as ascertain clinical BI-RADS density from DBT screening exams from enrollment up to 6 months prior to diagnosis (or corresponding follow-up for controls). In Aim 1, we will evaluate DBT volumetric density measures and their combinations as risk factors for invasive, interval and advanced BC, at enrollment DBT exam, as well as DBT exams within five years of the cancer (or follow-up for controls), using state of the art commercial and research algorithms. We will also assess differences in associations by time of DBT exam, age, race, menopausal status and body mass index. In Aim 2, we will evaluate the contribution of DBT volumetric density measures to clinical BC risk models, including the BCSC 5-year risk model, the novel BCSC 6-year cumulative risk model for advanced cancer, and secondarily, the Tyrer-Cuzick model for both 5 and 10- year risk. Using these results, we will determine the impact of DBT density measures on high-risk thresholds for preventative therapy and tailored imaging. Finally, in Aim 3, we will evaluate the contribution of DBT volumetric density measures to three novel AI algorithms developed for BC risk and detection, with risk of invasive, advanced and interval BC in the short- and longer-term. Our innovative study will be the largest to inform how novel DBT volumetric density measures can augment BC risk-stratification and prediction across multiple races and build a diverse resource to evaluate new DBT measures and risk models as they evolve. These findings will build an evidence base to inform personalized prevention approaches.

NIH Research Projects · FY 2026 · 2023-03

Glioma intelligence from behind enemy lines Molecularly diverse gliomas may leverage convergent metabolic survival pathways that can be therapeutically targetable. Microdialysis enables sampling of the extracellular microenvironment and represents a previously underutilized opportunity to characterize and pharmacodynamically monitor living human gliomas, in situ. Our preliminary data from intraoperatively acquired glioma microdialysate reveal strong enrichment for methionine-associated pathways of cancer resiliency, including polyamine synthesis. Specifically, results to date have identified guanidinoacetate (GAA) as the most highly upregulated metabolite in glioma microdialysate, which we hypothesize results from upregulated polyamine synthesis within the tumor. This study will determine the reproducibility and potential therapeutic implications of our findings across a larger cohort of gliomas, asking if microdialysis could be leveraged to obtain mechanistic feedback during early phase clinical evaluation of candidate therapies. To interrogate methionine metabolism human gliomas in situ, we will perform intra-operative microdialysis and methionine tracing, comparing the metabolome of microdialysate and tissue from tumor and adjacent brain. Resected tissue will be used to determine the cellular source of methionine-associated metabolites. Recent studies have demonstrated that diverse tumors can escape DMFO-mediated polyamine metabolism by upregulation of polyamine transporters. Dual blockade of polyamine synthesis and polyamine transports with  DMFO+AMXT 1501 has been shown to improve outcomes in preclinical models. To mechanistically interrogate polyamine metabolism we will perform a combination of preclinical and clinical studies leveraging microdialysis. In a phase 0 study, patients will be randomized to vehicle, DMFO, or DMFO+AMXT 1501, prior to dual administration of DMFO+AMXT to determine the extracellular pharmacodynamic changes induced by early therapeutic stress. Collectively, these studies will test how microdialysis can be used to perform biochemical reconnaissance within the live human glioma, with and without therapeutic challenge, to gain “glioma intelligence from behind enemy lines.”

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Central venous catheters are indispensable healthcare devices used for a range of applications from hemodialysis to critically ill patients. Unfortunately, central venous catheters provide a route for entry of pathogens into the bloodstream, resulting in central line-associated bloodstream infection (CLABSI). The pathophysiology of CLABSI comprises two main routes of infection: the extraluminal route for short-term central venous catheters, where microorganisms enter from the insertion site and colonize the catheter tip; and the intraluminal route for long-term central venous catheters, where frequent line manipulation introduces microorganisms into the lumen. While many approaches for CLABSI prevention focus on aseptic techniques to mitigate extraluminal and, to some extent, intraluminal infections, intraluminal infections remain a major source of CLABSI. Here, we propose to develop a non-antibiotic approach to prevent CLABSI using controlled electrochemical reactions occurring in the catheter lumen to generate the biocide hypochlorous acid (HOCl). In preliminary work, we showed antimicrobial activity of electrochemical HOCl generation on catheter surfaces, and in a preliminary in vitro catheter model, demonstrated that this strategy may prevent CLABSI. We further showed that HOCl concentrations and delivery rates are controllable by tuning electrochemical parameters. We term the devices we propose to develop that will generate intraluminal HOCl as a CLABSI prevention strategy, electrochemical intravascular catheters (e-catheters). eCatheters will use a novel intraluminal electrochemical system designed to deliver HOCl at concentrations ‘tuned” to prevent microbial cell growth and biofilm formation without causing host toxicity. The e-catheters will be controlled by custom-designed micropotentiostats for use in animals (and, eventually, in humans). The developed devices will be tested against 12 species of bacteria and yeast in vitro and evaluated in a rabbit model of intravascular catheter-associated infection to assess prevention of Staphylococcus aureus and Klebsiella pneumoniae CLABSI. The innovative e-catheter strategy provides an original way to address CLABSI prevention, avoiding conventional antibiotics and therefore selective pressure on commensal microbiota and emergence of antibiotic resistance.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY/ABSTRACT Insufficient T cell infiltration is a major challenge in adoptive transfer therapies like CAR-T. Therefore, one strategy to improve therapy is to enhance T cell trafficking into tumors. However, current therapies targeting T cell activities largely consist of immune checkpoint modulators, and very little innovation has occurred in therapeutic design targeting T cell intrinsic regulators of intratumoral accumulation. This is due, in part, to an incomplete understanding of the regulatory pathways involved in T cell trafficking. We recently identified Adapter protein 2 associated kinase 1 (Aak1) as an important regulator of T cell chemotaxis into tumors in an in vivo forward genetic screen. The primary objective of this project is to measure the translational potential of AAK1 as a therapeutic target in cancer to augment adoptive transfer therapies, with the additional goal of better understanding molecular functions of Aak1 as a regulator of chemokine receptor Cxcr3 internalization. These goals will be accomplished in three aims. Aim 1 will quantify the impact of genetic modification of Aak1 on tumor infiltration of adoptively transferred T cells in a preclinical solid tumor model. Aim 2 will determine whether Aak1 kinase activity is required for chemokine-induced internalization of Cxcr3 in primary T cells. Aim 3 will measure the degree to which Aak1 modification impacts therapeutic efficacy in adoptive transfer therapies. This proposal has several innovative aspects, including characterization of a novel, T cell specific Aak1 knockout mouse, functional and mechanistic testing of a novel Aak1 mutant construct, and evaluation of Aak1 as a novel therapeutic target to limit T cell chemotaxis into inflamed tissue. Successful completion of this project will benefit development of novel treatment strategies for solid tumors, and findings can broadly be applied to any T cell adoptive transfer approach and is not limited to individual CAR or TCR engineered platforms.

NIH Research Projects · FY 2026 · 2023-02

PROJECT SUMMARY Chronic kidney disease (CKD) is estimated to affect 30 million Americans and incur healthcare cost of $30.9 billion every year. There is an urgent need of a safe and noninvasive clinical tool for accurate staging of CKD, which is essential for its management. The importance of renal parenchyma perfusion and vasculature morphology for CKD characterization has been documented by many studies, but is not used clinically due to lack of translatable imaging solutions. In this project, we will use a novel super-resolution ultrasound imaging (SRUI) technology, which can resolve 50-micron renal cortex microvessels and measure their blood flow speed in human, to quantify cortex microvessel morphology and perfusion for accurate CKD characterization. Aim 1: Technical development. New acquisition and processing methods will be developed to enhance performance of SRUI through phantom and patient experiments. Novel quantitative parameters of SRUI for renal cortex will be developed, which includes vessel density, diameter, and tortuosity, as well as mean blood flow velocity, micro- Resistive Index of arterioles and venules, and perfusion index. Aim 2: Animal validations. We will study 7 normal pigs and 14 CKD pigs with renal artery stenosis (RAS) to validate SRUI measurements using independent measurements obtained through contrast enhanced CT, micro-CT, and histology. Aim 3: Clinical study. We will study 50 healthy volunteers to establish the normal range of SRUI parameters and study 116 CKD patients with clinically indicated renal biopsy to investigate the efficacy of SRUI for CKD staging, using biopsy histology as the reference standard. Statistical difference between CKD patients and healthy controls and differences across CKD stages defined by histology will be evaluated. The association of each ultrasound parameter with histology CKD score will be assessed. Univariate and multivariate logistic regression and ROC analyses (receiver operating characteristic) analyses will be performed to assess the performance of SRUI, conventional ultrasound (renal length, cortex thickness, Doppler renal resistive index, and shear wave elastography), and clinical parameters (eGFR and proteinuria) for distinguishing histology CKD stages. A subset (N=45) of patients will be scanned by two sonographers randomly selected from a pool of five sonographers. Intraclass correlation coefficients will be used to evaluate the inter-sonographer agreement. The inter-sonographer variance will be calculated to estimate the minimum detectable difference for longitudinal follow-ups. Successful completion of this project will result in a safe, noninvasive, cost-effective, and accessible ultrasound technology for accurate characterization of chronic kidney disease to guide treatment decision making.

NIH Research Projects · FY 2025 · 2023-02

ABSTRACT In the United States, half a million adolescents suffer from an eating disorder. With only 66 certified providers nationally, Family-Based Treatment (FBT), a first-line evidence-based treatment for adolescent eating disorders, is not readily available to most families. This provider shortage leaves most young patients without care and undoubtedly contributes to the chronicity and lethality of these conditions. Patients with eating disorders generally make their first contact with the healthcare system in primary care. As such, equipping primary care providers (PCPs) with effective means to treat these patients has potential to democratize care, improve rates of early intervention, and enhance patient outcomes. Family-Based Treatment for Primary Care (FBT-PC) is a novel intervention for delivery by a PCP in primary care that uses FBT strategies. Data support proof-of-concept for this adaptation. We have several study aims. (1) We will finalize the FB-PC intervention through an open case series. (2) We will establish the feasibility and acceptability of FBT-PC for caregivers, patients, and PCPs in a pilot randomized controlled trial. Finally, (3) we will test preliminary target engagement of FBT-PC and determine whether it is associated with improved caregiver self-efficacy and, through this mechanism, symptom remission. Remission will be defined as weight restoration to 95% of expected body weight and a score within 1 SD of community norms on the Eating Disorder Examination-Questionnaire. We also propose (4) an exploratory aim to evaluate baseline characteristics of our sample to determine for whom the FBT-PC intervention is most beneficial. To accomplish all aims we will complete an open case series (n = 6), followed by a pilot trial in which we will randomly assign 40 patients (ages 7-18 years) with restrictive eating disorders and their caregiver(s) to FBT-PC or a control condition of standard FBT. Families will attend up to 18 sessions over 6 months. Goals from the open case series (Aim 1) will include the development of tools for FBT-PC training and implementation, including treatment and training protocols and fidelity measures. Feasibility (Aim 2) will be assessed through an evaluation of recruitment and retention. Acceptability (Aim 2) will be evaluated using mixed methods surveys and interviews of caregivers, patients and PCPs on the topics of tolerability, fit, and burden. We will also assess the degree to which FBT-PC engages our proposed mechanism of change, caregiver self-efficacy, to facilitate symptom remission (Aim 3). Effect sizes will be calculated for FBT-PC with a goal of ≥ 0.5, comparable with those found in FBT trials. Finally, baseline sample characteristics (Exploratory Aim) will be assessed including caregiver perceptions about their child’s illness, referral method, length of illness, and symptom severity. Once we have established feasibility, acceptability, and target engagement of the FBT-PC intervention, we intend to use these findings in support of a large pragmatic clinical trial to evaluate the noninferiority of effectiveness of FBT-PC versus standard FBT.

NIH Research Projects · FY 2026 · 2023-02

ABSTRACT Clear cell renal cell carcinoma (ccRCC) accounts for ~75% of kidney cancers and is the 8th leading cause of cancer death in the United States. After completion of The Cancer Genome Atlas (TCGA) Project, clinically actionable mutations were identified in virtually every solid tumor. One major exception, however, is RCC, where the current standard of care, checkpoint inhibitor and anti-VEGF therapy, does not take into account that ~50% of RCCs have mutations in chromatin regulators. After first-line therapy, response rates are 20% and there are no FDA-approved therapies that target chromatin regulators, highlighting the need to identify how loss-of-function genotypes can be therapeutically targeted. The epigenome is profoundly disrupted in cancers including ccRCC. Aside from the near ubiquitous loss of VHL, the mutational landscape of ccRCC is dominated by loss-of-function mutations in epigenetic regulators, including SETD2, BAP1, and PBRM1. SETD2 loss has now been firmly linked to poor outcome and metastasis. The molecular phenotype of H3K36me3 deregulation in SETD2-mutant ccRCC makes this an ideal scenario to study from the angle of synthetic lethality because it induces global epigenetic changes that must be compensated for, creating unique vulnerabilities. Targeting factors that exhibit genetic epistasis with known cancer mutations to drive a synthetic lethal phenotype is a proven therapeutic approach. We performed an unbiased CRISPR/Cas9 screen to identify factors that exhibit synthetic lethality with SETD2 loss-of-function. The epigenetic factor NSD1, a writer of H3K36me1/2 acting through the H3K36 pathway, was identified. Based on these findings, we hypothesize that suppression of the H3K36 axis in the form of its epigenetic writers (NSD1 in a SETD2 loss context) drives a synthetic lethal phenotype mediated by distinct enhancer remodeling accompanied by expression defects incompatible with cell viability. Identification of NSD1, which is part of a larger family of three related proteins (NSD1/2/3) within the H3K36 signaling axis in turn, will lead to novel approaches for individualized therapeutics to target SETD2 loss-of-function, classically defined as ‘undruggable’. We will address this hypothesis with three aims. In aim 1 we functionally characterize the synthetic lethal phenotypes associated with H3K36 writers NSD1, NSD2, and NSD3 in isogenic SETD2 ccRCC cell lines. In aim 2 we demonstrate the utility of pharmacologic inhibitors of H3K36 signaling in driving synthetic lethality in SETD2-mutant cells and elucidate their biological underpinnings. Finally, in aim 3 we validate the efficacy and specificity of genetic and pharmacologic targeting of the H3K36 signaling axis to induce SETD2-mutant synthetic lethality in vivo using mouse models. Our studies will shed new light on how epigenetic regulators and the H3K36 axis specifically, drive cancer and metastasis when deregulated. This is expected to positively affect human health by generating preclinical evidence for new ways to treat SETD2-mutant ccRCC that will minimize off-target side effects, and enhance survival for patients with ccRCC, particularly those with more aggressive/metastatic disease.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY Intestinal health relies on the homeostatic function of intestinal macrophages in controlling gut inflammation. Defects in establishing this macrophage function can lead to unresolved inflammation as seen in inflammatory bowel disease (IBD). Recent studies have highlighted the impact of tissue microenvironments on establishing macrophage tissue-specific functions. Of specific interest, metabolites produced by gut bacteria, such as short-chain fatty acids and secondary bile acids, exert profound immunomodulatory effects on macrophage functional polarization. They suppress the expression of pro-inflammatory cytokines and transform macrophages to anti-inflammatory phenotype. What remains lacking, however, is the knowledge of how macrophages sense bacterial metabolites and mediate their immunomodulatory effects, especially in the gut microenvironment. Cellular metabolism regulates macrophage functions. We have previously demonstrated that macrophage pro-inflammatory response can be regulated by controlling metabolic substrate uptake. We therefore propose that metabolite sensing in macrophages is mediated via coordinated expression of transport proteins, which transport specific metabolites across plasma membranes and allow them to integrate into intracellular metabolism or to be directly sensed by intracellular receptors. Our long-term goal is to identify transporter targets that promote bacterial metabolite sensing and macrophage homeostatic function in order to control intestinal inflammation. Our preliminary data have indicated that macrophages exhibit distinct transporter reprogramming during functional polarization. There, we identified that SLCO3A1, an organic anion transporter and a recently discovered IBD-associated gene, is specifically upregulated during macrophage pro-inflammatory activation. Overexpression of SLCO3A1 enhances bile acid uptake. Also, the expression of SLCO3A1 is specific for tissue-resident macrophages from both small and large intestines as compared to other tissue-resident macrophages. Based on these observations, the overall goal of this project is to define the role of macrophage SLCO3A1 in metabolite sensing and intestinal tissue homeostasis. Our central hypothesis is that the expression of SLCO3A1 in macrophages facilitates the sensing of bacterial metabolites that promotes macrophage homeostatic function and the prevention of IBD. This project will focus on the following specific aims: (1) Define the mechanism by which SLCO3A1 regulates bile acid sensing and its immunosuppressive effect in macrophages. (2) Determine the role of SLCO3A1 in intestinal macrophages and the development of IBD. Altogether, this project will elucidate the mechanisms by which SLCO3A1 regulates the sensing of bacterial metabolites and promotes the homeostatic function of macrophages in controlling intestinal inflammation. We believe that completion of this project will provide mechanistic insights into important principles that govern the macrophage metabolite sensing in the intestinal microenvironment and the prevention of IBD.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY/ABSTRACT While cardiovascular disease (CVD) is the leading cause of death in men and women, substantial sex differences exist in disease prevalence and prognosis. A key impact of menopause is risk of future CVD. Post-menopausal women are twice as likely to have heart failure with preserved ejection fraction (HFpEF) and heart failure hospitalization rates disproportionately increase in women as they age. We hypothesize that a key contributor to this trend are hormonal changes during menopausal transition which accelerate myocardial stiffening. The impact of menopausal transition on myocardial stiffness is unknown. If understood, new disease prevention and therapy monitory strategies with significant potential for long-term benefits could be investigated. Elevated myocardial stiffness, a precursor of several CVDs, can go undetected prior to heart failure symptoms from diastolic dysfunction because left ventricular (LV) ejection fraction is preserved, and absolute LV chamber stiffness is not routinely measured. Current quantitative stiffness measurements require ex vivo mechanical testing or invasive pressure-volume measurements, hampering their adoption as practical clinical biomarkers. This proposal’s objective are to 1) establish a baseline for normal stiffness values during natural aging through menopausal transition and 2) identify sex as a biological variable. To accomplish these goals, we will develop a free breathing 3D non-invasive cardiac magnetic resonance elastography (cMRE) technique to measure myocardial stiffness. The technological advances in this study will make myocardial stiffness imaging in heart failure patients with dyspnea more reliably, regional, and enable quantitative cross-sectional and longitudinal monitoring of myocardial stiffness. Our preliminary data shows that LV myocardial stiffness increases significantly in healthy women, but not men, after the age of 50, which corresponds to the average age of menopausal transition. If cMRE can be used to detect elevated myocardial stiffening during menopausal transition and prior to the development of diastolic disfunction, the monitoring of current and new preventive interventions prior to heart failure symptoms will be made possible. To accomplish these objectives, we will: • Develop a 3D, free-breathing cMRE application. • Retrospectively bin cMRE data for multiple 3D MRE volumes that vary during the cardiac cycle. • Validate cMRE in static and pulsatile realistic cardiac phantoms. • Identify a clinical and technically feasible acquisition durations in a normal volunteer pilot study. • Evaluate the influence of natural aging, sex, and menopausal transition on myocardial stiffness. The natural evolution of myocardial stiffness in natural aging, in relation to sex-biases, and throughout menopausal transition will be evaluated using a newly developed and implemented free breathing non-invasive quantitative cMRE approach, which may have significant implications for the prevention of heart failure.

NIH Research Projects · FY 2026 · 2023-01

ABSTRACT Moderate (<60%) O2 (hyperoxia) in premature infants promotes bronchial airway hyperresponsiveness (AHR) via effects on airway smooth muscle (ASM), a cell type that also contributes to impaired bronchodilation, and remodeling (proliferation, altered extracellular matrix (ECM)). Thus understanding mechanisms by which O2 affects bronchial airways is critical for therapeutic strategies in a vulnerable population. We propose a protective role for hydrogen sulfide (H2S) in developing airways that can be leveraged in prematurity, thus providing clinical significance to our proposal. We hypothesize that the protective endogenous H2S system is detrimentally influenced by O2, but that exogenous H2S donors can be used to counteract detrimental effects of O2 on contractility and remodeling. Little is known regarding regulation of endogenous H2S in developing bronchial airways, and mechanisms by which moderate O2 reduces H2S. Conversely, the mechanisms by which H2S impacts on developing airways to alleviate O2 effects are unknown. We propose 3 Aims using human fetal lung and in vivo neonatal mouse models of O2 to explore these concepts. Aim 1: In developing human ASM, determine influence of O2 on endogenous H2S; Aim 2: In developing human ASM, determine mechanisms by which H2S alleviates O2-enhanced airway contractility and remodeling; Aim 3: In a newborn mouse model of hyperoxia, determine effects of H2S on airway contractility and remodeling. In Aim 1, we will use 18-22 wk gestation human fetal ASM (fASM) to examine mechanisms by which O2 decreases H2S, focusing on ROS, mitochondria, and alterations in the methionine-transsulfuration balance that can drive changes in the H2S synthesis enzyme CBS. Aim 2 explores downstream effects of H2S (via donors NaHS and GYY4137, and enhancement of endogenous H2S) in the context of contractility and remodeling following 40% O2. Here, the focus is on three key mechanisms: suppression of HIF1α, activation of Nrf2, and enhancement of cAMP. In Aim 3, in vitro studies are integrated using a newborn mouse model of hyperoxia where early exposure to moderate oxygen levels results in sustained AHR and remodeling. The efficacy of H2S donors in alleviating AHR and remodeling is assessed. Clinical significance lies in establishing the importance of H2S in O2 effects on developing airway towards future therapeutic targeting for neonatal asthma.

NIH Research Projects · FY 2026 · 2023-01

Renal artery stenosis (RAS) remains a common cause of hypertension and end-stage renal disease in the elderly population, associated with increased morbidity and mortality. Recent data suggest that renal ischemia in RAS interferes with endogenous kidney repair mechanisms, such as CD133+/CD24+ scattered tubular-like cells (STCs), which can proliferate and their progeny re-differentiate into tubular epithelial cells to replace lost neighboring injured tubular cells. Our previous studies have shown that experimental RAS impairs the reparative capacity of swine STCs by inducing structural and functional abnormalities in their mitochondria. However, the processes underpinning RAS-induced STC mitochondrial damage remain unclear. Micro-RNAs (miRNAs) function as post-transcriptional regulators of gene expression. MiRNA genes are transcribed in the nucleus, which results in the production of pri- and pre-miRNA precursors, and subsequently mature miRNAs. Although most mature miRNAs are present in the cytosol, few miRNAs, known as ‘mitomiRs’, translocate to the mitochondrion to silence gene expression related to mitochondrial functions. Our preliminary data show that RAS increases expression of the mitomiR-181c in swine STCs associated with decreased expression of its mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) encoded mitochondrial gene targets, and in turn mitochondrial structural abnormalities and dysfunction. In addition, we found that the promoters and enhancers of the miR-181c gene (MIR181C) exhibit hyper 5-hydroxymethylation of cytosine (5hmC), an epigenetic mark generated by the oxidation of 5mC by the ten-eleven translocation methylcytosine dioxygenase (TET) enzyme. Excitingly, in our pilot studies anti-miR-181c, the TET inhibitor Bobcat339, and inhibitors of the mitomiR import proteins ameliorate mitochondrial damage in swine-STCs. Our central hypothesis is thus that altered miR-181c expression in STCs underlies RAS-induced STC mitochondrial damage, blunting the paracrine function and capacity of STCs to preserve the post- stenotic kidney. Three specific aims will be pursued: Aim 1: will test whether increased miR-181c expression in RAS-STCs induces mitochondrial structural damage and dysfunction in STCs. Aim 2: will test whether RAS imposes epigenetic changes that increase miR-181c expression in STCs. Aim 3: will test whether aberrant miR-181c mitochondrial import contributes to STC dysfunction. Successful studies will provide novel insight into the vulnerability of this repair system and may contribute towards development of feasible clinically relevant tools for improving the utility and efficacy of kidney repair in renal disease.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY We propose to develop and optimize an advanced neurochemical recording technique that would be able to measure relatively rapid physiologically representative second-to-second changes in tonic concentrations of specific neurochemicals, such as serotonin, in the brains of awake behaving animals. Microdialysis, a commonly used in vivo sampling technique, is able to measure changes that occur in tonic levels. However, in practice the sampling timescale is significantly limited to minute-to-minute changes and it suffers from poor spatial resolution and induces significant tissue damage. Present voltammetry technique can provide tonic measurement capability. However, it raises concerns for accurate quantification due to the limited approach to eliminate background capacitive current. Furthermore, limited biofouling has been shown, limiting its use for long-term tonic serotonin measurements in awake behaving animals. The proposed electrochemical technique we call N-shaped Multiple Cyclic Square Wave Voltammetry (N-MCSWV) will enable second-to-second measurements of tonic extracellular levels of serotonin with exceptional spatial resolution, sensitivity, specificity, selectivity, and diminished biofouling. This proposal leverages our unique expertise in neuroscience, electrochemistry, software development, and engineering to develop and validate this novel neurochemical recording technology for broad use in basic neuroscience research, clinical brain neuromodulation, and a variety of electrochemical applications. Our initial animal studies will guide and inform the application of our investigational technique for use by the general neuroscience and medical community. Our proposal seeks to (1) establish N-MCSWV as a reliable research tool that is capable of identifying and quantifying tonic serotonin extracellular levels in vivo with unsurpassed sensitivity, selectivity, and minimize biofouling, (2) apply Fourier transform electrochemical impedance spectroscopy to N-MCSWV to monitor the degree of electrode biofouling in vivo, and (3) validate the use of N-MCSWV for in vivo, acutely and chronically, selective measurement of tonic serotonin concentrations.