University Of Colorado Denver

NIH Research Projects · FY 2026 · 2022-01

ABSTRACT Recent reports from the American Journal of Public Health and the American Psychological Association identified a critical need to examine mechanisms by which exposure to psychosocial stress in childhood increases the risk for obesity and cardiovascular disease (CVD) in adulthood. Given there is a lag in time before the impact of psychosocial stress experienced in childhood is expressed as disease in adulthood, these calls-to-action urge researchers to investigate the role of modifiable factors over the course of childhood that may mitigate risk for later obesity and CVD. The proposed mixed-methods study is uniquely designed to answer these calls-to-action by examining how stress “gets under the skin” to put children at higher risk for later obesity and CVD, and ultimately health disparities by race/ethnicity. The main objectives of this study are to: (1) comprehensively examine the relationships between multi-level psychosocial stressors (i.e., individual, dyadic, household, societal) and their dimensions (i.e., severity, frequency, timing), biological factors (e.g., hypothalamic pituitary adrenal (HPA) axis activity), and child weight and emerging CVD risk over the course of childhood and (2) identify modifiable factors at the individual, parental, and familial level to interrupt these stress pathways. The proposed study will build on and expand a prior parent R01 study (HL126171). The parent R01 study is a two-phased, mixed-methods study including a prospective epidemiological cohort study with 1307 diverse parent/child (ages 5-16) dyads (≈200 each African American, Hispanic, Native American, Immigrant/Refugee, White) and an embedded ecological momentary assessment (EMA) sub-sample with 627 parent/child dyads (≈100 per each racial/ethnic group). Data was collected at two time points (baseline, 24-month follow-up). In the proposed study, online survey data and 7-day EMA data will be continued at 48 and 72 months, allowing for a total of four waves of data collection. Children, who are now ages 9-16 will be added to both the online survey and EMA data collection, in addition to participating in three 24 hr. dietary recalls and 7-day accelerometry. New biological measures (e.g., hair cortisol, body composition, arterial stiffness) with children and parents, neighborhood factors (e.g., child opportunity and disadvantage index) using geo-spatial measures, and societal-level factors (e.g., structural racism, sociopolitical shift, COVID-19) contributing to psychosocial stressors will also be added at both time points. Human-Centered Design multi-family focus groups will also be carried out to co-create intervention targets with families. This study will provide breadth and depth in understanding the pathways between multi-level psychosocial stressors and child weight and emerging CVD across important developmental milestones (e.g., puberty) and family life cycle stages (e.g., families with young children to families with adolescents). Importantly, this study will identify modifiable factors (e.g., family adaptability/resilience) that mitigate the negative impact of multi-level psychosocial stressors on child CVD that can be targeted in interventions.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Despite new treatment modalities, the incidence of breast cancer has remained steady in recent years with >250,000 new diagnoses and >40,000 deaths annually in the US. Concurrently, the proportion of US women with overweight or obesity continues to rise, and is approaching 70%. Obesity and metabolic disease (which occurs in lean and obese women) increase breast cancer incidence and worsen patient outcomes in women of all ages. Premenopausal women with obesity are at increased risk of triple negative (TN) breast cancer (lacking any targetable factors). Postmenopausal women with obesity incur more estrogen receptor (ER) positive breast cancer and are more likely to develop resistance to endocrine therapies. While estrogen is clearly an important part of this relationship, two key observations suggest that there may be estrogen-independent mechanisms at play: 1) obesity is accompanied by worse prognosis for estrogen-independent triple negative breast cancer; and 2) anti-estrogen therapies are less effective against ER+ breast tumors in women with obesity. Regardless of tumor subtype and menopausal status, excess weight is associated with poor outcomes for breast cancer patients. Weight loss is known to improve breast cancer outcomes, but most people cannot sustain the standard dietary weight loss strategies and weight regain is common. Intermittent energy restriction (IER) is a novel dietary weight loss strategy that may have more beneficial effects on metabolic health and on breast cancer risk and tumor progression. The work in this proposal will employ preclinical, clinical, and interventional studies, to examine a novel mechanism of obesity-associated tumor progression and the value and feasibility of innovative dietary interventions for eliminating obesity’s adverse effects on breast cancer. We have merged expertise in nutrition, obesity, and medical oncology to: 1) examine a novel role that cancer- associated fibroblasts (CAFs) and the tumor microenvironment (TME) may be playing in obesity-associated tumor progression; 2) investigate if the novel dietary weight loss strategy of IER can eliminate obesity-associated tumor progression; and 3) perform an ORBIT Phase IIa proof-of-concept study examining the ability of an IER- based weight loss intervention to reach meaningful clinical milestones in breast cancer patients afflicted with overweight and obesity and refine the intervention for delivery in a future randomized efficacy trial. If the objectives of this proposal are achieved, we will have: * Advanced our understanding of the obesity – breast cancer relationship, with evidence of a novel role for CAFs and the TME in obesity-associated tumor promotion for TN and ER+ breast cancer; * Established the foundation for IER weight loss trials in breast cancer survivors, with data that will help us adapt these strategies to the unique characteristics and needs of this patient population; and * Identified novel circulating biomarkers of a pro-metastatic TME, which may help identify patients most susceptible to metastatic disease.

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY Arboviruses maintained in a human-mosquito-human transmission cycle are responsible for fueling periodic outbreaks worldwide and are an increasing public health threat. A critical feature of arbovirus transmission cycles, and a major determinant of their geographic spread and pathogenesis, is the magnitude and duration of viremia in vertebrate hosts. However, few studies have investigated the molecular determinants of viremia. Recent studies published by the Morrison laboratory demonstrated that the murine scavenger receptor MARCO on liver macrophages removes chikungunya (CHIKV) particles and other arthritogenic alphaviruses, including Ross River (RRV) and o’nyong ‘nyong (ONNV) viruses, from murine circulation due to recognition of the lysine (K) residue at position 200 of CHIKV and ONNV E2 glycoprotein and 251 of RRV E2 glycoprotein. My preliminary studies further revealed that CHIKV clearance is also abrogated when mutations were introduced at glutamate (E)208 of E2 and K61 of E1 glycoproteins, and mass spectrometry analysis of the biochemical features important for viral clearance suggested that E1 K61 is methylated. Further analysis of position 208 of CHIKV E2 glycoprotein revealed the importance of a negative charge at this position for CHIKV removal from circulation. As a pattern recognition receptor, MARCO recognizes modified self and non-self molecules, and polymorphisms in human MARCO can predispose carriers to infectious diseases such as tuberculosis. Because the scavenger receptor cysteine-rich (SRCR) domain of MARCO is a binding site for endogenous ligands, such as modified low-density lipoprotein, I hypothesize that the SRCR domain of MARCO stably and noncovalently interacts with an exposed interface between the E1 and E2 glycoproteins of CHIKV, allowing for the removal of viral particles from circulation and a reduction in both the magnitude and duration of viremia. In Aim 1, I will define the residues and biochemical features of CHIKV important for MARCO-dependent clearance from circulation by manipulating surface features of virus particles, assessing how specific mutations impact viral dissemination, and identifying post-translational modifications at specific sites in the E1 and E2 glycoproteins. In Aim 2, I will elucidate the sites on MARCO responsible for binding arthritogenic alphaviruses with cell-based and biochemical approaches. In addition, I will determine the extent to which virus particles interact with human MARCO, and whether known polymorphisms in MARCO affect virus-MARCO interactions, viremia, or clinical outcomes. Taken together, by defining the molecular mechanism of interaction between MARCO and CHIKV, this proposal could provide insights into factors that influence alphaviral pathogenesis, elucidate the relationship between MARCO polymorphisms and viremia, and identify individuals or populations with an increased susceptibility to severe alphaviral infections and outbreaks, respectively.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY/ABSTRACT Immune checkpoint blocker therapy has recently greatly improved survival of patients with late- stage melanoma. However, about 2/3 of patients do not benefit from this therapy. One of main hurdles is that many melanoma tissues lack effector CD8+ T cells. The immature and dysfunctional blood vessels actively limit T cell infiltration. Recently vascular normalization has been demonstrated to be able to facilitate effector immune cell infiltration and to improve cancer immunotherapy. a novel pathway in the IGFBP7/CD93 interaction, both of which are selectively upregulated in tumor vasculature. Our preliminary study indicates that disrupting this interaction in vivo normalizes tumor vessels to reduce hypoxia and improve tumor perfusion in mouse melanoma models. Our examination of tumor tissues reveals that blockade of this pathway could reinvigorate tumor blood vessels to promote T cell infiltration while limit myeloid-derived suppressor cells in the tumor. Here we hypothesize that upregulation of this pathway contributes to the tumor vascular abnormality and targeting this pathway will offer a novel approach to facilitate melanoma immunotherapy. We will dissect how this pathway is induced in the tumor and consequently leads to tumor vascular dysfunction and then tumor outgrowth. The mechanisms by which CD93 regulates immune cell infiltration, as well as its expression causing resistance to anti-PD1 therapy, will be evaluated. By the completion of these studies, we will gain insight into the biological role of this pathway in the cancer microenvironment of melanoma and, more importantly, provide a new strategy of promoting immunotherapy in melanoma. Our studies uncovered

NIH Research Projects · FY 2025 · 2021-12

NIH Research Projects · FY 2026 · 2021-12

Project Summary Our application addresses a fundamental need of the scientific community focused on curing Multiple Sclerosis (MS). MS is an inflammatory disease of the central nervous system that affects more than 2.5 million people worldwide with an unknown etiology. Progress in the understanding of MS has been constrained by a lack of understanding of the reciprocal interactions between behaviorally-relevant neuronal activity and changes in myelination in health and disease. The link between behaviorally-relevant neuronal activity and circuit-specific changes in myelination remain unknown, as does the role of behavior interventions in enhancing remyelination. Recently, we have developed new approaches to visualize myelin, oligodendrocytes, and their precursors in the intact mouse brain, allowing an unprecedented view into their dynamics and behavior in health and disease. In this application, we propose to capitalize on the inter-individual, longitudinal, subcellular insights revealed by these techniques to discern the effects of changes in local circuit activity and myelination on motor behavior. The objectives of this proposal are: 1) evaluate how behaviorally-relevant neuronal circuit function regulates myelination during learning and myelin repair and 2) to elucidate how changes in myelin specificity modulate motor learning and behavior. This proposal represents a novel synthesis of approaches to study neural circuit function during behavior and oligodendrocyte biology, and breaks new ground in the understanding of the mechanisms underlying motor learning and dysfunction following demyelinating injuries.

NIH Research Projects · FY 2023 · 2021-09

Principles of presynaptic networks for single layer 2/3 neurons in ferret visual cortex Single neurons in neocortical circuits are driven by presynaptic networks composed of excitatory and inhibitory neurons. Each neuron’s population of presynaptic partners determines how incoming information is processed. A longstanding view of cortical circuits is that a majority of synaptic inputs originate from local networks through horizontal (recurrent) connections. However, the mechanisms by which recurrent networks shape the activity of cortical neurons is largely unknown. Additionally, synaptic and cellular mechanisms proposed by theoretical models rely on studies of the rodent visual cortex, which is increasingly shown to differ from that of carnivores and primates in organization and function. The proposed career development plan aims to address these problems by mapping presynaptic excitatory and inhibitory cells of single layer 2/3 neurons and dissecting how they act to selectively modulate neural activity in ferret V1 in vivo. This proposal uses a novel combination of advanced optical techniques and electrophysiology. The candidate has a deep background in in vivo physiology and optical imaging in a wide variety of mammalian species. The candidate proposes to receive training in state-of- the-art multiphoton holographic optogenetics and the use of novel molecular tools. The candidate will also receive guidance from mentors and advisors on professional development. This training will establish the necessary skills for a successful independent research career studying the role of presynaptic networks in fundamental cortical operations in a non-murine model system. The candidate will carry out the mentored phased under Dr. David Fitzpatrick, a world-prominent expert on the early visual-system and cortical processing of carnivores and primates. The candidate will be co-mentored by Dr. Hillel Adesnik, who is a pioneer in multiphoton holographic optogenetics and developed techniques the candidate proposes to use. Additional advising from Dr. Kristina Nielsen and Dr. Krishnan Padmanabhan will provide guidance in professional development and the transition to an academic position. MPFI will provide an excellent research environment, with abundant resources, technical support, and intellectual discussions with prominent scientists to help ensure successful completion of the proposed research and transition to independence. The candidate’s long-term aspirations are to build an innovative and multidisciplinary research program to establish fundamental principles of cortical circuits, ultimately providing a scaffold for understanding disorders, such as schizophrenia and autism, which show profound impairments in the processing of sensory signals.

NIH Research Projects · FY 2025 · 2021-09

Abstract There is an urgent need to implement effective tobacco cessation interventions in HIV care programs in low- and middle-income countries (LMICs) where most tobacco users and people with HIV live. India is an important setting in which to test tobacco cessation interventions with an estimated 275 million tobacco users and 2 million people living with HIV (PWH). Effective medications and behavioral interventions have not been well tested in HIV care settings in LMICs. In high income settings, Positively Smoke Free (PSF), a theory- based behavioral intervention, has demonstrated efficacy among PWH and been adapted for mobile phone delivery (PSF-M). Mobile health interventions offer scalability and may be more effective among PWH than face-to-face. Varenicline is the cessation medication with the strongest evidence of efficacy in PWH but achieving sufficient adherence to sustain long-term success is a challenge. We propose to combine the two most promising cessation interventions among PWH, varenicline and PSF-M, enhanced with varenicline adherence support. They will be tested at the Voluntary Health Services (VHS) Infectious Disease Medical Center, Chennai Antiviral Research and Treatment Clinical Research Site, where 24% of patients new to HIV care are current smokers or dual users of smoked and smokeless tobacco and 83% own mobile phones. In this setting we propose the following specific aims: (1) To demonstrate the effectiveness of an integrated intervention combining varenicline plus mobile behavioral treatment among tobacco users in HIV care on biochemically verified tobacco abstinence at 24 weeks compared to a standard care control; (2): To evaluate the implementation processes of the integrated intervention in an LMIC HIV practice; and (3): To measure the costs and cost-effectiveness of an integrated intervention with varenicline and PSF-M. To accomplish these aims, PSF-M will be adapted to the VHS context and novel content about smokeless tobacco and medication adherence self-efficacy will be added. A randomized two-arm trial will compare the combination of varenicline and PSF-M to a standard care control. We will evaluate implementation processes within HIV care workflows including acceptability, appropriateness, feasibility and costs and conduct cost-effectiveness analysis to assess the clinical impact and value of the integrated intervention if scaled-up. The work proposed is responsive to the specific interests in RFA-CA-20-037 by testing the effectiveness of tobacco cessation interventions with demonstrated efficacy in other settings and adapted for an LMIC context, evaluating the implementation process from multiple stakeholder perspectives in an HIV care practice, and assessing the clinical impact and value of the integrated intervention if implemented at scale. The successful completion of this work will move the field forward by advancing our understanding of the effectiveness of an integrated tobacco cessation intervention in HIV care settings and projecting the population level impacts of implementing the integrated intervention for PWH in India or other LMICs.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT/SUMMARY Autosomal dominant polycystic kidney (ADPKD) affects an estimated 600,000 individuals in the US and accounts for 5% of patients with end-stage kidney disease (ESKD). At the current time there is only one approved therapy available for patients diagnosed with rapidly progressing disease. This underlines the need for identification of additional measures to slow kidney disease progression in ADPKD patients. Kidney stone disease is highly prevalent, increasingly common, and associated with considerable comorbidity among patients affected with ADPKD. In this proposal, we build on our preliminary data which demonstrates that patients with ADPKD and kidney stone disease have a more rapid loss of kidney function compared to ADPKD patients without kidney stone disease. We will leverage an interdisciplinary team that is uniquely poised to define kidney stone disease in ADPKD by combining expertise in large data analytics and high-dimensional microbiome and metabolomic data. The overall goal of this application is to demonstrate that kidney stone disease in patients with ADPKD represents a significant risk factor for faster kidney disease progression and that antibiotic exposure increases stone risk in this population. A secondary goal is to determine how dysbiosis of the gut microbiome contributes to kidney stone disease. We hypothesize that kidney stone disease contributes to more rapid decline in kidney function in ADPKD through worsening inflammation and that antibiotics contribute to kidney stone disease by perturbing the gut-PKD axis through alterations of the gut microbiome. We test this hypothesis in 3 specific aims. In aim 1, we seek to describe the distribution and composition of kidney stones by sex and age and to determine the link between kidney stone disease and decline in kidney function independent of markers of inflammation and other known risk factors for kidney disease progression. In Aim 2 we investigate the link between antibiotic exposure and kidney stone disease while in Aim 3 we will define the link between oral antibiotic exposure and change in urinary kidney stone risk factors resultant from changes in the microbiome. We will leverage the Intermountain Healthcare System database with medical and pharmacy coverage, and clinical data including stone analysis and the longitudinal data and samples from the NIDDK sponsored HALT-PKD clinical trials. These resources will provide access to 1,823 patients with ADPKD including 296 with kidney stones. We will recruit 100 patients with ADPKD with or without antibiotic exposure to facilitate microbiome and stone risk analysis in aim 3. A barrier to developing new therapies for stone prevention is a lack of understanding of how perturbations of the gut microbiome and downstream metabolite changes in the intestinal and urinary tracts contributes to kidney stone disease. Understanding the gut-PKD axis from the proposed studies will introduce a new paradigm for kidney stone prevention in ADPKD and will provide key insights into novel therapeutic targets for kidney stone disease in ADPKD.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY The objective of this combined UH2/UH3 application is to develop a non-invasive, accurate, and cost-effective saliva-based test for early detection of squamous cell carcinoma of the head and neck (SCCHN) recurrence. SCCHN includes squamous cell carcinomas derived from oral cavity, tongue, pharynx and larynx. It is the 7th most common cancer in US and worldwide by incidence, and the 3rd in US and 4th worldwide by 5-year prevalence. SCCHN is a biologically aggressive cancer in which high rates of recurrence (local, locoregional or distant) contribute significantly to poor patient survival. Current clinical practice methods for detection of SCCHN recurrence are either subjective, invasive, hard to access, not able to detect recurrence in a timely manner or expensive. There is an unmet medical need for an objective, non-invasive, easy to access test that is able to detect SCCHN recurrence in a timely and a cost-effective way. Our solution is “HNKlear”, a non- invasive, saliva-based candidate test for early detection of SCCHN recurrence. HNKlear incorporates a 7- methylated microRNA biomarker panel. The real-time nature of our test will provide more timely and precise detection of SCCHN recurrence. In our published proof of concept studies, HNKlear demonstrated 92% sensitivity and 98% specificity in ~300 SCCHN and control tissue samples and 85% sensitivity and 95% in ~200 SCCHN and control saliva samples using continuous variables, which supports the clinical translation of the test. We propose a combined UH2/UH3 application for the clinical translation of HNKlear. In the UH2 phase, we will analytically validate the test and algorithm in a pre-clinical study and in a new patient cohort. Success of this phase will let us lock down the test and cut-off when the milestone is met. We will then conduct a clinical study in the UH3 phase to demonstrate the clinical validity of HNKlear for SCCHN recurrence in an observational clinical follow-up study incorporating HNKlear into current clinical practice. Success of this phase will generate robust evidence of the clinical validity of HNKlear for early detection of SCCHN recurrence, and will further enable clinical application of HNKlear as a Laboratory Developed Test.

NIH Research Projects · FY 2024 · 2021-09

This Project Summary was originally submitted with R01GM143774 and is included here unchanged to satisfy submission system requirements. Project Summary During mitosis, the mother cell divides by the formation of a cleavage furrow, leaving two daughter cells connected by a thin intercellular bridge. The resolution of this bridge, abscission, leads to the separation of the two daughter cells. During ingression of the cleavage furrow, the central spindle microtubules are compacted to form a structure known as the midbody (MB). It is now well established that MB regulates cytokinesis by recruiting abscission-mediating proteins, such as ESCRT complex, as well as several regulators of abscission checkpoint. Until recently, the MB was thought to be discarded after division by releasing it into extracellular space. However, recently it was shown that MBs accumulate in stem and cancer cells after mitosis has been completed (called MBsomes) and it has been proposed that MBsomes function as novel signalling platforms that regulate cell differentiation and proliferation. Recently we developed a protocol for purification of post- mitotic MBs and completed their proteomic and RNAseq analyses that led to identification of several mRNAs and mRNA-binding proteins that accumulate at the MB. Importantly, MB-enriched mRNAs encode several ESCRT complex subunits, as well as proteins that stimulate cell proliferation. Furthermore, we show that these MB-enriched mRNAs can be transferred to the neighboring cells via post-mitotic MB internalization. Based on all of these findings, we hypothesize that targeting of selected mRNAs to the MB during cytokinesis play a key role in regulating cell abcission and post-mitotic MBsome signaling. Here we propose three specific aims to test this hypothesis. First, we will map and characterize the domain(s) within 3'-UTR that are needed for mRNA targeting during cytokinesis. We will then use candidate approach, as well as proteomic and CRISPR screens, to identify RNA-binding proteins that interact with these 3”-UTR domains and regulate mRNA targeting and localized translation at the MB. Second, we will test the possibility that MB accumulation/translation of ESCRT mRNAs mediates ESCRP complex targeting to the MB. Third, we will test whether MBsome-dependent transfer of specific mRNAs, such as mRNA encoding proliferation regulator CENP-E contribute to MBsome-induced cell proliferation.

NIH Research Projects · FY 2025 · 2021-09

Abstract: IgE-mediated food allergy to peanuts (PN) and/or tree nuts (TN), is a major health problem in the United States, affecting approximately 4% of children and up to 2% of adults. Co-allergy among these foods is relatively common and is difficult to identify given the more common finding of co-sensitization. Recent progress with early administration of these foods and oral immunotherapy, especially in conjunction with anti-IgE have merit. Unfortunately, these approaches are not successful for all patients and, even when successful, have limitations regarding compliance and unpredictable breakthrough. There are significant, unmet needs to 1) understand the immunologic details of IgE mediated activation of mast cells by allergens from PN and TN, 2) understand the molecular basis for co-allergy among TN and between PN and TN, 3) develop improved diagnostics to identify clinically relevant peanut and tree nut allergy and 4) design new approaches to interfere with allergic reactions caused by peanuts. The overarching concept of this proposal is that the 2S albumins are the most important allergens of peanuts and tree nuts and are the key to understanding PN and TN allergy and cross-reactivity and to developing potent diagnostic and potentially therapeutic reagents. Preliminary data show that we have 1) developed a sensitive ELISA assay, 2) identified the critical amino acids within IgE-binding peptides, 3) demonstrated that conformationally constrained (3D) peptides bind IgE strongly and 4) shown that patients with PN allergy alone and PN + TN allergy identify different patterns of peptides in a microarray assay. We hypothesize that 1) we can optimize IgE binding to existing peptides and discover novel peptides with enhanced binding, 2) there are cross- reacting epitopes of PN and selected TN and 3) IgE binding to existing and novel peptides will have potential predictive value for important clinical outcomes. We propose to 1) perform positional amino acid (aa) screening to optimize binding of IgE to peptides, 2) utilize click chemistry and stapling technology to enhance IgE binding and resistance to proteases and 3) use microarray technology to assess IgE binding with well-defined samples from patients with PN, WN, PecN, CN and PisN allergy and from those undergoing clinical trials. Success in this project will establish a new intellectual framework regarding allergen/IgE interactions, describe, at least in part, the molecular basis for these co-allergies, design new diagnostics and move us along the path toward development of an oral, peptide based, treatment for peanut allergy.

NIH Research Projects · FY 2025 · 2021-09

PROPOSAL SUMMARY Diffuse intrinsic pontine gliomas (DIPGs) are aggressive brainstem tumors in children with no curative therapies available. DIPGs are canonically driven by recurrent mutations in the histone 3 gene (H3K27M). This substitution imparts broad dysregulation of the histone post-translational modifications (PTMs) that regulate the recruitment and initiation of transcriptional machinery. Processive transcription, or the mechanics of RNA Pol II as it actively transcribes across chromatin, is both dependent on and actively propagates chromatin states such as dynamic accessibility and transcription-associated PTMs (tPTMs). Disorders of transcription dynamics have demonstrated pathogenic roles in cancer development, and inhibition of transcription machinery is an effective therapy in these models. We have recently shown that the H3K27M mutation activates regulators of transcriptional elongation, including CDK9. We have demonstrated that inhibition of CDK9-dependent transcriptional elongation is an effective therapy in DIPG, but the contribution of processive transcription to DIPG oncogenic transformation is unknown. The overall hypothesis of this proposal is that the H3K27M mutation promotes CDK9-dependent nascent transcription, which in turn contributes to both the establishment of an oncogenic chromatin state as well as the adaptive response to standard-of-care radiation therapy. Using a combination of CRISPR-edited model systems, patient derived cultures, and both patient-derived xenograft and syngeneic engineered mouse models, we will test this hypothesis by 1) defining the role of processive transcription in H3K27M-mediated oncogenesis, 2) determining the impact of CDK9 inhibition on processive transcription, and 3) characterizing the role of transcriptional induction in response to ionizing radiation. Successful completion of the proposal will allow us to comprehensively map the impact of the H3K27M mutation on the nascent transcriptional landscape. This data will enable us to define a novel transcriptional framework for understanding DIPG’s chromatin-mediated oncogenesis, and it will demonstrate how exploiting this transcriptional dependence may be leveraged to improve the patient benefit derived from radiation therapy. The proposed career development plan leverages these studies to provide advanced training in the conduct of rigorous hypothesis-driven research, the molecular study of transcriptional regulation, and representative pre- clinical cancer modeling. The mentorship team reflects nationally-recognized senior scientists who possess both focused expertise in these areas of study as well as a strong commitment to my career development. The training plan outlines how I will refine my expertise through a combination of didactic course work, focused workshops, national meetings, and mentorship guidance. Collectively, this training platform will facilitate my transition to independence as a basic-translational researcher with a long-term goal of applying novel chromatin- and transcription-based strategies to improve patient outcomes in DIPG.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY We have discovered that a single hydride induces global changes to both structure and dynamics within multiple members of an enzyme family, providing a fundamental link between enzyme structure, dynamics, and allostery that has implications to the entire oxidoreductase superfamily. Specifically, the BLVRB family are NADPH-dependent reductases present in multiple organisms where they regulate cellular redox through the reduction of biliverdin-to-bilirubin and a wide array of flavin substrates. While our recent publications have revealed that coenzyme binding is coupled to global conformational and dynamic changes, we have now discovered that there are largescale changes coupled to the oxidation state of the coenzyme as far as 23 Å away. Thus, structural catalytic the central premise of this application is that a coenzyme's hydride is globally coupled to both and dynamic changes within an enzyme family and that such global coupling is integrally related to function. The novelty here is that we will explicitly determine how a single hydride, i.e., the difference between NADPH/NADP+, is globally linked (Aim 1) and how this global coupling controls enzyme function (Aim 2). Further innovation includes the following. First, we have discovered that hydride-coupled networks can be modulated by mutations directly to the enzyme/coenzyme interface but also to distally coupled sites, which gives us the unique opportunity to determine the role of these networks in function. Second, we have discovered that evolutionarily changing residues modulate hydride coupled networks and function, providing remarkable insight into the evolutionary role of hydride-mediated coupling and function. Evolutionary differences will therefore be exploited to identify allosteric networks coupled to the oxidative state of the coenzyme and simultaneously reveal their evolutionary roles in function. Based on our preliminary data that includes NMR, X-ray crystallographic, and biochemical studies, we hypothesize that the coenzyme oxidation induces its own conformational change that is further propagated globally through the enzyme in multiple BLVRB family members (referred to as “insideout” coupling) and that networks coupled to these changes modulate function (referred to as “outsidein” coupling). We will address this hypothesis through the following: Aim 1) Determine how a single hydride modulates the global dynamics and structure within the BLVRB family of enzymes. NMR solution studies using CSPs, relaxation studies, and ensembles methods will be used to determine how a single hydride imparts its global regulation to dynamics and structure using three distinct BLVRB family members with both active site and distal differences (human, hyrax, and mosquito). Aim 2) Determine the functional role of networks coupled to the oxidative state of the coenzyme. Biochemical and biophysical methods will be used to determine the functional role of hydride-mediated global regulation, which include both the role of direct interactions with the coenzyme's hydride as well as the role of networks of communication coupled to the coenzyme (allostery).

NIH Research Projects · FY 2025 · 2021-09

Emerging research is providing compelling evidence that the vestibular system contributes not only to basic reflexes (e.g. vestibulo-ocular, postural) but also to complex cognitive processes including spatial memory and navigation; self-motion perception and motor planning; and executive function. Vestibular function declines with healthy aging, and studies from our group and others have shown that vestibular loss in aging adults is associated with known age-related reductions in cognitive skills including spatial memory and navigation ability, and self-motion perception and motor planning. In a pilot study supported by the NIDCD, we found that older adults with vestibular loss had reduced hippocampal volumes, as well as atrophy of subfields of the thalamus and the basal ganglia. During this pilot study, our group built a pipeline for analysis of neuroimaging data from the Baltimore Longitudinal Study of Aging (BLSA) at the Center for Imaging Science (CIS) in the Johns Hopkins Department of Biomedical Engineering. We established the computational infrastructure for both volumetric analysis and shape analysis. In this proposal, we seek to leverage this infrastructure to more comprehensively investigate the impact of aging on three core central vestibular pathways, and examine the link between structural changes in these pathways and functional changes in clinical skills mediated by these pathways. Specifically, we aim to: Aim 1 Investigate the relationship between peripheral vestibular sensory loss associated with aging and the structure of central vestibular pathways, specifically considering 3 primary central vestibular pathways: 1) spatial cognitive; 2) sensorimotor; 3) prefrontal cortex (executive function). Aim 2 Examine the longitudinal relationships between peripheral vestibular loss and structural changes in central vestibular pathways using longitudinal models and also the novel change-point model in aging adults. Aim 3 Explore the association between structural changes in central vestibular pathways and clinical functional changes cross-sectionally and longitudinally. This proposal represents a unique opportunity to leverage strengths in vestibular physiology and computational neuroimaging to increase our fundamental understanding of the impact of healthy aging on central vestibular networks, and associated clinical consequences. Findings from this study will be used to drive further critical research questions, including 1) Does vestibular loss contribute to the accelerated decline in cognitive ability and brain structure that occurs in individuals with cognitive impairment and Alzheimer’s disease; and 2) Can vestibular interventions for vestibular loss prevent/mitigate changes in central vestibular pathways?

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN) channels are critical determinants of membrane potential and excitability in many types of cells throughout the body, including cardiac pacemaker cells, central and peripheral neurons, many types of sensory cells, and interstitial cells of Cajal in the colon and bladder. Consistent with this widespread distribution, HCN channels have been identified as potential drug targets for treatment of a long list of conditions including angina, heart failure, epilepsy, neuropathic pain, depression, gastrointestinal dysmotility, and neurogenic bladder. However, the single FDA-approved HCN channel drug (ivabradine) is limited, owing in part to its non-selective block of all four mammalian HCN channels isoforms and its off-target block of Kv11.1 (hERG), Nav1.5, and Cav1.2 channels. The need for new, isoform-specific HCN channel activators and inhibitors has been widely recognized but the lack of information about allosteric and isoform-specific regulation of HCN channels is a roadblock to the development of novel therapeutics. The long-term goals of this project are to identify naturally-occurring, allosteric regulators of HCN channels and to understand their mechanisms of action. Achieving these goals will advance understanding of the physiological and molecular functions of HCN channels and aid in the development of new HCN channel drugs. The current proposal focuses on our exciting discovery of LRMP and IRAG as two novel, isoform- specific protein interaction partners of HCN4 channels. LRMP and IRAG are homologous ER transmembrane proteins that have large cytoplasmic domains. Importantly, LRMP and IRAG only modulate the HCN4 isoform. Moreover, the two proteins have opposing effects on HCN4: LRMP causes a loss-of-function (LOF) by decreasing the canonical depolarizing shift in voltage-dependence induced by cAMP while IRAG causes gain- of-function (GOF) by shifting the basal voltage dependence of HCN4 to more positive potentials. Preliminary data establish that IRAG is co-expressed with HCN4 in cardiac pacemaker cells. Proposed aims will identify interaction sites on the three proteins, determine the molecular mechanisms for the distinct and isoform- specific effects, and evaluate their role in pacemaker cells.

NIH Research Projects · FY 2025 · 2021-09

A substantial cadre of investigators at the University of Colorado are focused on characterizing the molecular origins and causal mechanisms that drive the initiation and preclinical phases of rheumatic and autoimmune diseases. One of the most important outcomes of those efforts has been the demonstration in populations at- risk for future disease that dysbiosis and chronic inflammation at mucosal sites can promote the initial local development of disease-specific loss of tolerance to autoantigens. Ultimately, this process leads to systemic autoimmunity and the development of diseases including rheumatoid arthritis (RA) and spondyloarthroarthritis (SpA). Building on that concept with unique at-risk populations and an extensive programmatic infrastructure, a P30 Rheumatic Disease Research Resource Center (RDRRC) entitled “Center for Mucosal Immunobiology and Rheumatic Disease Pathogenesis” is proposed. Included in the Center are an Administrative Core, a Population and Data Sciences Core, and a Mucosal Immunobiology Core. This Center is specifically designed to facilitate studies of human disease pathogenesis that incorporate the broad range of mucosal and systemic immunologic techniques, as well as develop new approaches, to study these integrated mechanisms in already recruited cohorts of individuals throughout the preclinical and then clinically active phases of disease. The Center includes 43 internal and external members who have in aggregate $77M in related annual direct cost grant funding. In addition to providing access to stored biospecimens and data from ongoing population studies, the Center will provide consultative and member discount mechanisms for all of the critical aspects of cohort development, epidemiologic assessment, data management and data analyses. In addition, the Center will provide members access to technologies that focus on analyses of the microbiome and microbial:cell interactions, as well as informative mucosal and peripheral immune biomarkers. The Center will build on a recent $80M investment from the Dean to fund four other relevant programs with which this Center will interact. Dr. Michael Holers, Professor of Medicine and Immunology, will serve as the Center Director, and Dr. Kristi Kuhn, Associate Professor of Medicine, will serve as the Associate Director. Both will interact with Advisory and Executive Committees, and in the latter will be joined by Core Directors and Co-Directors with domain expertise. As a key component of the mission of the Center, a Pilot & Feasibility Grants Program will be developed with substantial institutional support. Additional Enrichment Program activities will include a seminar series, technology assessment series, and an annual symposium. A major focus will be on early career investigator development through enhanced Core access as well as Grants Review and a Scholars Programs. Social media, web-based outreach and newsletters will provide comprehensive communication. Finally, a Patient Impact Program will be utilized for the assessment and enhanced translation of novel biomarkers and therapeutically relevant RDRRC discoveries into direct patient care with the goal to improve outcomes.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Sensorineural hearing loss (SNHL) is a leading cause of disability and affects ~1.4 billion people globally, including different age groups and ethnicities. Although around 150 genes have been identified for SNHL, the Hispanic population remains understudied for SNHL, with most Hispanic studies focused on a single gene GJB2. A few countries studied – Chile, Mexico/Hispanic-American, Nicaragua, the Philippines – have a low prevalence of GJB2 variants, suggesting that SNHL cohorts from these countries have novel genes or variants for discovery. Additionally, Hispanic children are at risk for environmental exposures to chemicals that may lead to epigenetic modifications and cause SNHL. Our overarching hypothesis is that SNHL has a unique, population-specific allelic and epigenetic spectrum in Hispanic-descent populations. We assembled an international group of researchers with complementary expertise in otology, genetics, epigenomics and functional genomics, with previous collaborative experience that signals this project will be highly productive. In our previous studies, we identified novel variants in Hispanic-American and Filipino patients with SNHL, including genetic variants that were associated with temporal bone anomalies and predictive of cochlear implant outcomes. Nicaraguan families were submitted for exome sequencing and were negative for variants; these families likely harbor non-coding variants or have epigenetic mechanisms of SNHL. We have in place efficient pipelines for the identification of novel SNHL genes in families and differentially methylated regions (DMRs) in case-control cohorts, as well as validation methods in animal models and epithelial cells. Our goal is to determine genetic and epigenetic risk factors in Hispanic children with SNHL. For Aim 1, we will identify SNHL variants from next-generation sequence data using a tiered approach, which includes Sanger sequencing, filtering, homozygosity mapping, linkage analysis and transmission disequilibrium tests. We will recruit 500 Hispanic families, including large families sufficient for genome-wide significant linkage, and submit DNA samples to sequencing and analyses. Novel SNHL genes and variants identified in these families will be followed up with protein localization and hearing studies in mouse and zebrafish models and mutation constructs in epithelial cells. For Aim 2, we will perform an epigenome-wide association study by profiling the methylome of a well-powered cohort of 500 SNHL probands and 500 hearing children matched by age, sex and population, in order to identify DMRs that are associated with SNHL. For the top identified DMRs, we will utilize CRISPR-dCas9 technology on epithelial cells to determine if targeting the methylation site will affect gene expression. Integration of methylation profiles and genetic data using methylation quantitative trait locus analysis will aid in understanding genetic vs. environmental contributions to SNHL. Overall this project will impact genetic screening protocols and genetic counseling particularly in Hispanic populations, as well as improve understanding of the hearing mechanism and lead to new targets for the development of treatment of SNHL.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Vestibular dysfunction becomes more prevalent with age and it is estmated that more than 80% of people over 80 years old experience dysfunction5. Furthermore, approximately 8 million adults in the US suffer from balance impairment due to damage to the peripheral vestibular system, but effective treatments for balance dysfunction are virtually non-existent. Vestibular hair cells within vestibular canal and otolith organs convert hair bundle motion into receptor potentials and sensory information is relayed to the brain by action potentials in vestibular afferent nerves. Afferents in central zones of vestibular neuroepithelia exhibit different responses to vestibular stimuli than afferents in peripheral zones. There are three types of vestibular afferents: calyx-only afferents innervate one or more type I hair cells, bouton dendrites innervate type II hair cells and dimorphic afferents contact both hair cell types. Calyx-only afferents are present only in central zones and have irregular firing patterns, whereas dimorphic afferents exist in both zones and have regular firing patterns. We will study age-related dysfunction in calyx-bearing afferents in gerbil vestibular organs using novel preparations developed in the laboratories of the principal investigators. We will use electrophysiological, hair bundle stimulation, immunohistochemical and behavioral approaches to address age-related changes in mature and aged vestibular epithelia. In Aim 1 we will determine if functional changes in vestibular hair cell mechanotransduction and/or basolateral currents occur with age. Aim 2 will test the hypotheses that synaptic degeneration of calyx terminals will manifest as morphological uncoupling of type I hair cells from their associated calyces and deficits in vestibular evoked responses and behaviors. In Aim 3 we will directly investigate changes at the type I hair cell/calyx synapse by recording spontaneous activity and responses to hair bundle stimulation in mature and aged calyx afferents. Our investigative team is uniquely positioned to carry out the proposed studies. Results from this work will provide new information on how the aging process impacts peripheral vestibular signals and may inform development of vestibular neurotherapeutics targeting afferent nerves in order to restore normal vestibular function.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Pediatric sepsis is a major global public health problem associated with millions of deaths every year. In the United States, 1 in every 10-15 children with sepsis or septic shock will die during their hospitalization, and many survivors will have a lower health-related quality of life. We collaborated with an international task force to develop and validate the novel Phoenix criteria for sepsis and septic shock in children, which were published in two high-impact articles in JAMA and are now the reference standard for diagnosing pediatric sepsis around the world.8,9 The criteria are based on the Phoenix Sepsis Score (PSS), which we developed using a data- driven approach and >3.6 million patient records and include information for four organ dysfunctions. In order to maximize the impact of the Phoenix criteria for pediatric sepsis, several knowledge gaps must be addressed. In this study we will: evaluate the prognostic accuracy of the Phoenix sepsis criteria and the PSS before and after the COVID-19 pandemic and in the setting of specific organisms and infectious syndromes as well as varying vaccination status of at-risk children (SA 1); derive and validate sepsis trajectories based on the changes in the PSS over time that can help inform clinical decision-making (SA 2); prospectively validate the Phoenix sepsis criteria and the PSS overall and in high-risk patient subgroups (SA 3); and design and implement generalizable clinical workflows that maximize the impact of the Phoenix sepsis criteria and PSS trends (SA 4). We will leverage and expand our existing dataset by adding 2 new sites, 5 additional years of data from each site, and new data elements including vaccination records. We will also conduct a prospective study at four sites to validate the performance of the Phoenix criteria as CDS tools. We have enhanced our accomplished investigative team by adding new sites and expertise in human-computer interaction, CDS tool design, and pediatric hospital medicine. We expect the results of this proposal to have a powerful and sustained impact on the science of pediatric sepsis and ultimately improve sepsis recognition, accelerate appropriate effective treatment, decrease unnecessary treatment, and improve the outcomes of children with sepsis around the world.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY To protect the host and perform their symbiotic role, intestinal epithelial cells secrete large amounts of highly glycosylated mucin to physically distance yet provide bacteria habitat and fuel, while establishing apical junction complexes that regulate nutrient and waste flux. In return, a healthy microbiota functions to provide metabolites the epithelium relies on for function. We recently published that the purine hypoxanthine (Hpx) is a microbiota-derived metabolite that the mucosa depends upon for energy balance, barrier function, and wound healing, suggesting Hpx as a limiting substrate for homeostatic mucosal metabolism and function. In unbiased extensions of that work, we demonstrated that Hpx induces TP53-inducible glycolysis and apoptosis regulator (TIGAR) expression and activates AMP-activated kinase (AMPK) in vitro and in vivo. TIGAR increases metabolite flux through the pentose phosphate pathway while AMPK is a master regulator of metabolism that promotes energy balance. We hypothesize that Hpx fundamentally molds epithelial metabolism through TIGAR and AMPK as mechanisms that support intestinal homeostasis and wound healing. In this, purine depletion and reconstitution by colonization experiments with a developed mutant E. coli enriched in Hpx production will be employed to determine the influence of microbiota-derived Hpx on mucosal energy metabolism, barrier function, and wound healing. The applicant, Dr. Lee, has established a scientific niche in which to build a foundation for independent research in the role of microbiota-derived purines in gut mucosal energy metabolism and function. Dr. Lee and his mentor, Dr. Colgan, assembled an advisory committee to regularly meet as a group with Dr. Lee throughout the duration of the award to provide feedback on the research, critique the research plans, monitor publications, and provide career advice. Drs. Colgan and Lee also identified microbiology and immunology courses to facilitate expansion of Dr. Lee's foundation of knowledge in fundamental intestinal processes, and established tutelage in colonic enteroid harvesting/culturing for study and in histopathological analyses. Dr. Lee will submit his work to present at conferences specific to his research to share his research and network with colleagues and potential collaborators, and get valuable feedback from the scientific community. Dr. Lee's development will benefit from continued participation in the Mucosal Inflammation Program (MIP), a multi- disciplinary, multi-departmental program initiated to study mechanisms of mucosal inflammation and resolution. The MIP fosters a unique lab environment for collaboration between physician scientists, clinicians, and research scientists, and works to establish an environment for young investigators to flourish and develop. The facilities and resources available to and development plan built for Dr. Lee provide an ideal environment and path for his successful transition to independence.

NIH Research Projects · FY 2025 · 2021-09

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Changes in the legal landscape of cannabis have occurred simultaneously with increases in use among adults. Research efforts have focused on examining consequences of use, however, characterizing the impact of cannabis abstinence on health among regular cannabis users is also a relevant public health concern. Few research studies have compared cognition or psychiatric disorder symptoms between abstinent cannabis users, using contingency management (CM), to individuals in a control condition (monitoring), which does not require changes in cannabis use. The current study aims to extend the findings to an older population, for a longer period of cannabis abstinence, and to examine all cognitive domains. Thus, the first aim is to test the impact of cannabis abstinence for 6 weeks (42 days) on memory, attention, processing speed, language, and executive function (EFs) on adults, who are between 31 to 47 years old. The second aim is to explore how cannabis abstinence effects psychiatric disorder symptoms. The third aim (exploratory) is to examine how genetic risk for various psychiatric disorder symptoms by environment (cannabis abstinence versus continued use) interactions impact cognition. The proposed study is novel in that we will use experimental manipulation to create discordance in cannabis use among monozygotic (MZ) twin pairs. This research design will require significantly fewer resources than large-scale randomized controlled trials because the use of MZ twin pairs requires a smaller sample size and is still sufficiently powered to detect small effects. Fifty identical twin pairs (N=100), who are concordant on cannabis use, will be recruited from the Colorado Twin Registry, which have all been genotyped. Each twin, within a twin pair, will be randomly assigned to either CM, incentive-based protocol to promote abstinence, or control, no changes in cannabis use requested, for 42 days. Participants will be assessed across 42 days on measures of cognition and psychiatric disorder symptoms (baseline, day 14, day 28, and day 42). The method proposed in this project could be used to understand other outcomes from cannabis abstinence (e.g., physical health) or abstinence from other substances. The aims of the research project map on to the training goals: 1) clinical trial methodology, 2) longitudinal statistical analysis, and 3) behavioral genetics. Experts in clinical trial methodology, longitudinal statistical analysis, and behavioral genetics, will guide the research and training program. The team of researchers consists of Drs. John Hewitt and Christian Hopfer (primary mentors, training aim 3), Drs. Susan Mikulich-Gilbertson (mentor, training aim 2) and Naomi Friedman (consultant, training aim 2), Drs. Randi Schuster (co-mentor, training aim 1), Paula Riggs, and Kent Hutchison (consultants, training aim 1). Each training aim consists of one-on-one meetings with mentors or consultants, workshops, courses, seminars, specific analytic projects, responsible conduct of research training and preparation and submission of an R01 application in the final years. Completion of these research and training aims will provide me with the necessary skills to conduct recovery studies and provide preliminary data for an R01 application.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY, Overall The main goal of the Colorado (CO) Head and Neck Cancer (HNC) SPORE is to advance translational research to improve survival and quality of life for HNC patients. Optimal treatment for HNC patients is critically important because the head and neck organs support critical functions such as breathing, nourishing and communicating, and thus HNC can lead to significant morbidity and mortality. The CO HNC SPORE takes advantage of our expertise in basic and clinical sciences, and uses unique model systems to identify novel molecular and cellular mechanisms of HNC pathogenesis targetable by therapeutic interventions to treat all cancer types arising from head and neck anatomic sites. Three projects cover the treatment spectrum of head and neck squamous cell carcinoma (HNSCC) and include both tobacco-related and human papillomavirus (HPV)-related HNSCC. Project 1 studies novel immunotherapy mechanisms by inhibiting EphB4-EFNB2 interactions between immune cells and the endothelium. It will test if blockade of EphB4-EFNB2 signaling at the tumor endothelial barrier hinders Tregs' and TAMs' ability to infiltrate and promote cancer survival or suppress Teff function. The applicability of pre-clinical data to clinic will be assessed in samples from HNSCC patients treated with an EphB4- EFNB2 inhibitor during a window trial. Project 2 investigates if dual inhibition of TGFβ/PD-L1 combined with radiation therapy (RT) induces in situ vaccination, reverses immune suppression, and overcomes RT resistance. It will translate its findings with a trial of the TGFβ/PD-L1 dual inhibitor M7824 combined with RT in locally recurrent and oligometastatic HNSCC patients. Project 3 will study mechanisms of protein elongation inhibition in HNSCC, identifying key proteins targeted by the novel inhibitor SVC112 (a drug discovered in Colorado that is nearing clinical testing), and translating our findings by testing the distribution and prognostic significance of its target (eEF2) in patient samples. It will use immune relevant models including syngeneic and humanized mice to study immune-dependent and –independent mechanisms of SVC112 and study if protein elongation inhibition impacts the tumor microenvironment and enhances RT in HNSCC. The developmental research program (DRP) is designed to attract current HNC researchers and researchers from other fields to conduct innovative research in all types of cancers arising from head and neck tissues. The career enhancement program (CEP) is designed to solicit junior researchers to develop research projects to transition into independent HNC researchers. We encourage underrepresented minority (URM) and people with disabilities to apply for DRP and CEP projects. The CO HNC SPORE also includes Biospecimen/Pathology, Data Science, and Administrative Cores. In sum, the CO HNC SPORE will solidify in-depth HNC translational research and expand our team of dedicated HNC researchers. These activities will improve the care spanning the entire spectrum of HNC treatment from improving cures to developing innovative palliation strategies for HNC patients.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY Chronic kidney disease (CKD) is a major public health concern that has reached epidemic proportions. Hypertension is a leading modifiable risk factor for cardiovascular disease (CVD) and end-stage kidney disease, yet 50-70% of adults with CKD fail to achieve blood pressure (BP) control to <130/80 mmHg. A key process linking high systolic BP (SBP) to CVD is vascular endothelial dysfunction, mediated by excessive reactive oxygen species (ROS)-induced oxidative stress and reductions in nitric oxide (NO) bioavailability. NO is also critical in the regulation of renal blood flow (RBF), which is intimately related to BP and vascular function. Guidelines recommend a stepwise combination of lifestyle modifications and drug therapy to lower BP, yet adherence to lifestyle modifications such as aerobic exercise is poor in patients with CKD. Drug regimens often involve multiple medications, as hypertension is challenging to control in CKD. High-resistance inspiratory muscle strength training (IMST) is a novel lifestyle intervention involving repeated inhalations against a resistive load using a hand-held device. In a randomized, double-blind, sham controlled, parallel group design, R21-funded pilot study in 36 midlife/older men and women with baseline SBP ≥120 mmHg, we showed that IMST (30 breaths [5 minutes]/day at 75% of maximal inspiratory pressure, 6 days [30 minutes]/week for 6 weeks) had excellent adherence (95% of prescribed sessions completed) and lowered casual (resting) SBP by 9±2 mmHg. IMST improved endothelial function (brachial artery flow-mediated dilation, FMDBA) by 40%, linked to increased endothelial NO synthase (eNOS) activation and NO bioavailability, reduced ROS production and oxidative stress, and changes in circulating factors. Importantly, the effects of IMST on SBP and FMDBA were even greater in individuals with an estimated glomerular filtration rate (eGFR) <75 mL/min/1.73m2. To establish the efficacy of high-resistance IMST in midlife/older adults (≥50 years) with moderate-to-severe CKD (eGFR 20-59 mL/min/1.73m2) and inadequately controlled hypertension (SBP 130-159 mm Hg), we propose a randomized, parallel group, sham-controlled, double-blind, clinical trial to evaluate the effects of a clinically relevant treatment duration of IMST (3 months) on SBP, FMDBA, NO bioavailability, eNOS activation, ROS/oxidative stress, circulating factors, and RBF. Aim 1: To measure casual SBP (primary outcome) and 24-hour (ambulatory) SBP (secondary outcome) before (baseline) and after 3 months of IMST or Sham training. Aim 2: To measure FMDBA (secondary outcome) before and after IMST or Sham training. Aim 3: To determine: a) endothelial cell culture eNOS, NO and ROS production pre-post IMST or Sham serum exposure; b) markers of oxidative stress and antioxidant status in biopsied endothelial cells; c) the identity of the plasma metabolites involved; d) RBF by functional magnetic resonance imaging. Aim 4: To assess adherence (completed:prescribed sessions), safety, and tolerability of IMST vs. Sham.