University Of Washington

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Anorexia nervosa is the deadliest psychological disorder with an estimated 10% lifetime disease mortality rate, while over 1/3 of all cancer patients will die from disease-based anorexia, not the cancer itself. On the opposite end of the spectrum, over 2/3 of US adults are overweight or obese, and this number, as well as the rates of associated comorbidities such as heart disease, diabetes, and cancer, is only expected to increase in the coming years. Despite the opposite directionality of these eating disorders, dysfunctional eating in obesity and anorexia is mediated by common appetite circuitry in the central nervous system (CNS). Numerous studies have documented a coordinated and complex pattern of changes in multiple gene products in these appetite centers following periods of excessive or inadequate eating. These observations strongly suggest that the behavioral decision to eat excessively or inadequately is likely driven by a multitargeted, maladaptive genetic reprogramming process in CNS appetite centers. Thus, a core question is what global process could coordinate such changes in multiple gene products? My published studies have demonstrated that appetite changes align with changes in alternative polyadenylation (APA) in the hypothalamus. APA is a rapid, activity-dependent RNA processing mechanism that regulates mRNA transcript stability, maturation, and localization. I identified a significant APA pattern change on tissue inhibitor of metalloproteinases 2 (Timp2), a gene previously implicated in the development of an obese phenotype. Thus, I am exploring the hypothesis that Timp2 APA in the arcuate nucleus (ARC) of the hypothalamus meters the development of obesity. My proposed experiments in the F99 phase will show that 1) Timp2 mRNA is necessary for appetite control in the ARC, and that 2) APA regulation of ARC Timp2 is necessary to counteract hyperphagia and obesity. These studies will be the first to functionally link APA regulation to feeding behavior and will serve as the basis of further genome to behavioral phenome studies in my independent career. My Sponsor, Dr. Gary Wayman, and Co-Sponsors, Drs. Suzanne Appleyard and Emily Qualls-Creekmore, are established neuroscientists at Washington State University with expertise in molecular neuroscience (Wayman) and ingestive behavior (Appleyard and Qualls-Creekmore). My proposed Research and Training plan will strengthen my theoretical and technical understanding of neurogenetics. In the pre-doctoral F99 phase, I will learn shRNA and CRISPR/SaCas9 vector design and validation strategies, cell culture techniques, stereotaxic surgeries, and advanced metabolic analyses. In the postdoctoral K00 phase, I will build upon these skills and learn to use genetic mouse models, multi-omics, advanced bioinformatics, and AI computational models to map genome to phenome regulation. Overall, the proposed training will optimally position me to start an independent research career at a leading neuroscience research institute and advance our understanding of RNA regulation as a functional link between the genome and the behavioral phenome.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Cardiac fibrosis is the pathologic development of scar tissue around the muscle cells of the heart. It occurs with nearly every form of heart disease and rapidly progresses the heart to failure. A fundamental cellular determinant of fibrosis is the transition of fibroblasts into myofibroblasts, which are the primary producers of fibrotic matrix. Notably, myofibroblast revert to quiescence as organs including the heart recover from some injuries and fibrosis resolves. New evidence from liver and skin suggests these deactivated myofibroblasts are primed to reactivate with enhanced fibrotic responsiveness and wound healing capabilities as a protective adaptation to future injury. This has never been examined in the heart, but our preliminary data suggests cardiac fibrosis more than doubles when recovered hearts experience a second bout of pathologic stress. Given heart disease develops from cumulative rounds of stress and recovery, cardiac myofibroblasts likely undergo multiple cycles of deactivation and reactivation providing impetus for delineating these processes and determining how they are regulated. Our previous work suggests that p38 MAPK regulates the stability of the myofibroblast phenotype and the heart's fibrotic response. Moreover, recent findings suggest p38 inhibition appears to deactivate myofibroblasts reverting them to quiescence. This finding suggests that we can use experimental p38 perturbations of the myofibroblast state to study its impact on the heart's fibrotic response. Hence two comprehensive specific aims will be used to examine the overarching hypothesis that p38 regulation of myofibroblast state stability and the kinetics and fate trajectory of deactivation underlies long-term fibrotic responsiveness of the heart. Here single cell RNA sequencing in combination with myofibroblast lineage reporter mice engineered with targeted gain or loss of p38 function will be used to determine: (Aim 1) the role of p38 in regulating myofibroblast deactivation and resolving cardiac fibrosis, and (Aim 2) the effects of p38 activity on deactivated cardiac myofibroblast reactivation, fibrotic responsiveness, and fibrotic memory. Together these aims will attempt (A) to define the process and regulation of cardiac myofibroblast deactivation and reactivation, (B) to identify compensatory responses to perturbations in myofibroblast activity, and (C) to identify the function of deactivated myofibroblasts, their epigenetic memories of injury, and their role in enhancing the heart's fibrotic responsiveness.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY Cardiomyocytes sense macro scale mechanical cues to adapt their structure and function, and disturbances of these mechanical signals can cause a negative feedback loop that leads to changes in cardiomyocyte structure and ultimately decreased cardiac output. Important to this mechanical sensing are focal adhesions, the mechanosensitive protein complexes that attach the cell cytoskeleton to the underlying extracellular matrix (ECM). Although focal adhesions have been shown to be necessary for myofilament maturation and are sensitive to external substrate characteristics, little is known about the specific forces sensed at these complexes during the physiological contraction cycle or how this adhesive tension is regulated by cardiomyocyte contractility. Furthermore, alterations of cardiomyocyte contractile tension initiate maladaptive cell remodeling, but this mechanism and the involvement of focal adhesions are poorly understood. Given that focal adhesions of other cell types have been shown to distribute focal adhesion tension unevenly within individual cells and microdomains and that there are regional heterogeneities in cardiomyocyte strain, it is important to understand the spatial distribution of mechanosensing in cardiomyocytes as the myofibrils contract against the ECM. This proposal aims to fill the crucial gap in knowledge of the role of focal adhesions and their interactions with both the myofibril structures and the ECM for the mechanical homeostasis of contracting cardiomyocytes. Previous studies of focal adhesion sensation during contraction have been limited by the inability to measure exact force across the focal adhesions in a time and spatially resolved manner. A recently developed tool that has been used to study mechanically driven cell processes is the FRET (Förster Resonant Energy Transfer) tension sensor, which I have engineered to express endogenously in induced pluripotent stem cells within the focal adhesion gene vinculin. Preliminary data from stem cell derived cardiomyocytes that express this sensor show myofibril contraction confers an increase in global focal adhesion tension sensation in static cells. However, the spatial and temporal generation of force in cardiomyocytes during contraction is not yet known and will be examined in this project using cutting edge microscopy and image analysis techniques. Importantly, this model overcomes previous limitations of overexpression artifacts or inconsistent sensor expression. Thus, I propose to first quantify spatial and temporal physiological focal adhesion tension during static and dynamic cardiomyocyte contraction. Second, I will modulate both the inherent contractility of cardiomyocytes and their connection to the ECM to determine the internal and external regulation of focal adhesion tension in cardiomyocytes. These aims will provide a complete understanding of focal adhesion tension generation during CM contraction. This understanding may inform future studies to develop better targeted therapies to maintain mechanical homeostasis in heart disease.

NIH Research Projects · FY 2024 · 2022-07

Treatment dropout is a primary challenge for individuals suffering with posttraumatic stress disorder (PTSD) and alcohol use disorder (AUD) following trauma exposure. Treatments for PTSD+AUD are effective at reducing symptoms, yet treatment attrition is a critical issue. Dropout rates among individuals with PTSD+AUD are higher than for those with PTSD alone, and dropout is associated with less clinical improvement. A clearer understanding of the factors that contribute to dropout, including those who initiate services but do not start treatment (nonstarters) and those who start but do not complete treatment (noncompleters), will enable us to develop strategies that improve retention and treatment outcomes for this population. Examining modifiable and theoretically informed constructs, such as difficulty tolerating emotional distress and coping motives for alcohol use, may improve our understanding of treatment dropout. Additionally, observer ratings of in-session behavior, or “thin slice” ratings, is a method of measuring predictors more proximal to when dropout occurs and has the potential to be a robust predictor of dropout. Thin slice refers to short (e.g. 30 second) video clips of expressive behavior rated by trained observers on specified constructs or outcomes of interest. The proposed F32 will use mixed methods to explore reasons for treatment dropout using data collected from an existing clinical trial for women with PTSD and heavy drinking following sexual assault. Specific aims are: 1) To identify themes related to reasons for dropout for nonstarters and noncompleters by conducting qualitative interviews with providers of sexual assault services (N = 10), which will be used to develop the “thin slice” rating constructs of observed participant behavior for use in Aim 2. 2) To predict dropout and related treatment processes (N = 100) using thin slice ratings and advanced statistical methods. Treatment dropout will be examined within each treatment condition (imaginal exposure and alcohol skills training) and between treatment conditions to test whether certain thin slice constructs are better at predicting dropout for certain types of treatment. We will also examine trajectories of treatment processes related to dropout: difficulty tolerating emotions and alcohol craving. 3) To enrich and contextualize Aim 2 quantitative findings by concurrently conducting qualitative interviews exploring treatment initiation and dropout with women enrolled in a clinical trial for PTSD and heavy drinking following sexual assault (N = 25). The applicant will receive training in 1) PTSD and alcohol treatment process research, 2) conducting studies predicting dropout in clinical trials, 3) skill development for collecting and analyzing qualitative and quantitative data, and 4) manuscript and grant writing. The proposed study will make substantial contributions to our understanding of dropout, a necessary first step to the development of strategies to increase retention and improve treatment outcomes for this vulnerable population.

NIH Research Projects · FY 2024 · 2022-07

PROJECT SUMMARY/ABSTRACT Adolescent intimate partner violence (IPV) is a major public health problem in the United States. More than 60% of adolescents aged 12-18 in relationships report experiencing IPV (physical, sexual, and/or psychological abuse). Given the widespread prevalence and associated adverse outcomes, states are actively engaged in primary prevention strategies for reducing IPV, including the enactment and implementation of IPV laws. These state laws, which are rapidly expanding across the country, have the potential to alter the social context in which IPV occurs by encouraging or requiring school districts to adopt prevention education curricula and policies for addressing IPV. Such policies include definitions, training requirements for teachers and staff, and reporting procedures. Currently, 19 states have passed laws encouraging or mandating school IPV policies. Despite these laws gaining momentum in the past decade, there is a paucity of research examining the effectiveness of state laws and how schools implement policies to meet states’ legal mandates. The objectives of the proposed research in this Pathway to Independence Award are to determine if state IPV laws reduce the prevalence of adolescent IPV victimization and to assess if and how high schools implement these laws adequately. The specific aims are to: 1) estimate the effectiveness of state IPV laws on reducing the prevalence of adolescent IPV; 2) assess the implementation of IPV laws and the relationship between school IPV policies and IPV outcomes at the school-district level; 3) identify barriers and facilitators to implementing IPV policies in high schools; and 4) develop, refine, and test the feasibility and acceptability of a toolkit for implementation of IPV legislation in high schools. This study will provide the largest and most comprehensive evaluation of state IPV legislation to date. Findings will provide critical data on the effectiveness of school IPV policies in preventing adolescent IPV and actionable evidence for policy makers and agencies responsible for carrying out IPV policy implementation. To acquire the necessary skills to accomplish this project, I will receive training in three critical areas: 1) health policy and advanced econometrics; 2) implementation science; and 3) qualitative methods. I will also engage in professional development, extend my professional networks, and further develop my grant writing and oral presentation skills. I have assembled an interdisciplinary mentorship team with expertise in public health, medicine, public policy, law, and education to ensure completion of the proposed research and training plan and a successful transition to independence. This Pathway to Independence Award will enable me to develop into an interdisciplinary, independent investigator who examines the causes and consequences of IPV at the individual level and assesses how multilevel policies and interventions can prevent adolescent IPV.

NIH Research Projects · FY 2026 · 2022-07

Project Summary Prostate cancer is the second leading cause of cancer related death in men in the United States. Many of the newly diagnosed patients are indolent and would not die from prostate cancer even if they are left untreated. But lack of reliable markers to identify such indolent tumors has led to overtreatment of patients. To minimize overtreatment, patients with a lower Gleason Score are put on active surveillance and are not treated unless there is sign of disease progression. Although such active surveillance can minimize over-treatment, it may miss the opportunity for early intervention of aggressive tumors. Therefore, understanding molecular mechanisms underlying the aggressive nature of prostate cancer and identifying prognostic markers to distinguish indolent from aggressive prostate cancers are of great clinical significance. Human prostate cancers mostly originate in the peripheral zone (PZ). In addition, transition zone (TZ) tumors are often associated with favorable pathological features and better recurrence-free survival. These observations lead to the hypothesis that the differences in tissue microenvironment between the two zones influence the frequency and aggressiveness of the resulting tumors. We compared gene expression profiles between TZ and PZ stroma by an RNA-seq analysis and identify the transcription factor FOXF2 as one of the top genes expressed at a higher level in TZ stromal cells. We demonstrated that elevated Foxf2 expression in prostate stromal cells suppressed growth of prostate cancer xenografts in vivo. In Aim1, we will use genetically engineered mouse models to confirm that stromal Foxf2- mediated signaling suppresses tumor progression. In Aim 2, we will investigate the molecular mechanisms through which stromal Foxf2 suppresses tumor progression. In Aim 3, we will investigate how stromal FOXF2 expression is regulated.

NIH Research Projects · FY 2025 · 2022-07

ABSTRACT The Bioengineering Cardiovascular Training Grant (BCTG) program provides Bioengineering training for six NIH supported and one Bioengineering department supported PhD students committed to careers in cardiovascular research. Cardiovascular (CV) diseases are the leading cause of mortality in the US and progress in this area will require training outstanding scientists with a variety of backgrounds in bioengineering to develop new and innovative approaches for study, diagnosis and treatment of CV disease. The UW and Department of Bioengineering have been leaders in CV research and technology development for over 50 years. BCTG faculty in multiple departments in the College of Engineering and School of Medicine have strong collaborative programs in this area. Our program benefits the public by training professionals in basic science and translational research that integrates engineering, mathematics, chemistry, physiology, physics and computational skills to promote fundamental discoveries and develop new technologies. Students gain interdisciplinary skills and the value of collaboration. The program emphasizes new technology development in diagnostics and therapeutics to study and treat CV pathologies, improving early detection and disease management, to improve quality of life for heart failure patients. We believe that the success of translational research requires highly trained scientists to be placed in both academia and industry, with skills to successfully transition research from universities to commercialization and the clinic. For this renewal proposal we have added several novel components to provide training in research and professional skills that spans the biomedical pipeline from discovery & mechanistic studies to translational research to commercialization or clinical development. The 2-year program involves 1) research in the laboratory of a chosen mentor, 2) a unique curriculum with opportunities for training in clinical translation and commercialization, and 3) clinical preceptorships that are unique to our program. Trainees attend a weekly seminar series focused on CV research, with an associated journal club and special sessions with speakers that are limited to trainees. Trainees also attend lectures and discussion groups in the Biomedical Research Integrity summer program, sponsored by the School of Medicine. Additional new components include outreach and research mentoring to under-represented groups to increase the diversity of the workforce in STEM fields, and a course that provides resources and seminars for professional skills development and training in research reproducibility, rigor and transparency. Trainees are selected based on strong quantitative and engineering backgrounds, training environment of faculty mentors, appropriateness of the research to our programmatic focus and the potential for innovative, collaborative and translational research. Trainees present at yearly symposiums and national meetings, and write a yearly progress report that includes feedback questions to continually improve our program. Trainees are required to apply for fellowships at the end of the two-year training period to begin establishing a record of successful competition in gaining research support.

NIH Research Projects · FY 2025 · 2022-07

Abstract/Project Summary: The purpose of this study is to determine whether breast MRI radiomic features can be utilized to optimize treatment of ductal carcinoma in situ (DCIS), the earliest form of breast cancer diagnosed. Although DCIS survival rates approach 100%, there is concern that its management generally results in overtreatment, exposing many of the 50,000 U.S. women diagnosed each year to unnecessary anxiety and morbidity. The vast majority of DCIS is detected in asymptomatic women in whom suspicious calcifications are identified on mammography and characterized using limited tissue histopathology. Unfortunately, conventional imaging and pathology have not proven reliable for distinguishing low vs. high-risk DCIS. Specifically, it is unclear at diagnosis which forms of DCIS will upstage to invasive disease or have an ipsilateral breast recurrence (IBR) after treatment. This limited risk-stratification is due in part to inadequate sampling of the entire DCIS lesion and an inability to account for peritumoral microenvironment features. This results in unnecessary surgery, radiation therapy, and medical therapy for as many as half of women diagnosed with DCIS. Breast MRI is commonly and easily performed, able to best depict DCIS span, and can assess tumor and peritumoral heterogeneity rooted in biological features such as angiogenesis, making it an appealing choice for a radiomics assay to improve DCIS risk assessments. The Quantitative Breast Imaging Lab at the University of Washington has shown that quantitative MRI features are associated with DCIS grade, a molecular marker of recurrence (Oncotype DX DCIS Score), and IBR. The Computational Biomarker Imaging Group at the University of Pennsylvania has pioneered breast MRI radiomic phenotyping and shown radiomic measures of breast cancers correlate with genomic features and recurrence. The Center for Statistical Sciences at Brown University has expertise with radiomics, machine learning, and statistical analyses for imaging trials from ECOG-ACRIN. In this collaborative application, we hypothesize that breast MRI radiomic signatures of DCIS will result in distinct phenotypes that are prognostic and can be integrated with clinical, molecular, and pathologic markers to optimize DCIS treatment. To test this hypothesis, we will create a multi-institutional database of over 1400 MRIs, including exams from the ECOG-ACRIN E4112 trial, with curated outcomes (e.g., upstage to invasion, DCIS Score, and IBR). Leveraging a novel approach to harmonize multicenter data (nested-Combat radiomic feature standardization), we will discover and validate MRI radiomic phenotypes and assess those phenotypes’ associations with invasive upstaging, Oncotype DX DCIS Score, and 5- and 10-year IBR. Finally, we will determine whether integration of these phenotypes into existing clinical prognostic indices (e.g., Van Nuys Prognostic Index) can provide more precise estimates of IBR. If successful, this study will help clinicians de-escalate DCIS therapy in low-risk patients and address an important public health goal: decreasing breast cancer overtreatment.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Over 1.8 million Americans have rheumatoid arthritis (RA). Compared to the general population these patients are at an increased risk of developing cancer, especially lymphoma and lung cancer. There has been a theoretical concern that use of immunosuppressive agents may impair immune responses to tumors placing patients at risk for developing new cancers and for recurrence of previously treated cancers. These concerns have made clinicians reluctant to use standard therapies for RA in patients with an active or recently diagnosed cancer. Although only a limited number of studies have evaluated their safety, they have in fact not shown any signal of harm from disease modifying anti-rheumatic drugs (DMARDs) in patients with RA and cancer. However, these studies have numerous limitations including focus on mostly patients with remote (> 5 years) history of cancer, small sample sizes, focus on only certain DMARDs, and/or limited information on cancer- specific mortality and other outcomes that might matter to patients. Also, nothing is known about the perspectives and experiences of patients with RA who are being treated for a cancer and how this experience might shape their willingness to use DMARDs. Treatment guidelines for the management of RA support a shared decision-making (SDM) approach that attempts to leverage the professional expertise of the rheumatologist to uphold what is most important to each patient. We herein propose a mixed methods approach to identify the optimal ways to enhance delivery of care for these patients and to provide a foundation for shared decision making to ensure alignment between treatment decisions and the values, goals, and preferences of individual patients. The studies proposed here will build an evidence base to identify the safest DMARD options for patients with RA who develop cancer (Aim 1); elicit the experiences, perceptions, and most valued outcomes of patients with RA and an active or recently diagnosed (<5 years) cancer, their family members and clinicians who care for these patients (Aim 2). The overall goal of this K23 mentored career development proposal is to support Dr. Namrata Singh’s development as an independent, patient-oriented researcher whose long-term goal is to develop a research agenda focused on the identification of factors that contribute to suboptimal treatment and outcomes for patients with rheumatic diseases, especially those with cancer. The training and research plans proposed here will allow her to develop critical skills in advanced pharmacoepidemiology and in qualitative research methods. Combined with an outstanding mentorship team, the University of Washington’s world-class facilities, and a rigorous training plan, this project will prepare Dr. Singh to successfully obtain future funding to adapt and test a decision aid for SDM implementation among patients with RA and an active or recent cancer.

NIH Research Projects · FY 2026 · 2022-07

Project Summary In support of the NHGRI Strategic Plan to Diversify the Genomics Workforce, we propose the University of Washington Genomics Training for Data Scientists program (UW-GRTDS). This interdisciplinary program provides masters students in data science from underrepresented minority (URM) backgrounds with paid research experiences in genomics research laboratories at UW. During their research experience, students will receive mentorship from their genomics research faculty mentor, a data science faculty mentor, the UW-GRTDS Program Manager, and the Genome Sciences trainee community. UW-GRTDS students will be welcomed into the genomics training environment by participating in career development and networking events with other graduate students from URM backgrounds. The long-term objective of this program is to create a more inclusive environment for URM data scientists in order to encourage them to pursue careers in genomics research. Throughout this program we will identify and share strategies to enhance and support similar programs in order to serve the formation of a more diverse genomics workforce.

NIH Research Projects · FY 2024 · 2022-07

Familial hypertrophic cardiomyopathy (HCM) is a genetic cardiomyopathy affecting 1 in 500 US adults.1,2 Mutation in myosin heavy chain seven (MYH7), a sarcomeric thick filament protein, accounts for 20-40% of HCM cases.3,4 However, current understanding lacks a generalizable mechanism through which specific MYH7 variants result in HCM, and no specific disease-modifying therapy exists.5 The MYH7 S2 domain, host to numerous identified pathogenic variants, interacts with the C1C2 domain of cardiac myosin binding protein C (cMyBP-C).6 We have generated hiPSC-derived cardiomyocytes (hiPSC-CMs) from an HCM patient with MYH7 variant E848G. Our preliminary data suggest E848G disrupts the S2/C1C2 interaction and reduces MYH7 abundance. Thus, my central hypothesis is pathogenic MYH7 S2 variants that disrupt S2/C1C2 interaction and reduce MYH7 protein abundance result in contractile function; restoration of S2/C1C2 interaction (Aim 1) or MYH7 abundance (Aim 2) may improve contractile function. My specific aims are to: (1) demonstrate disruption of MYH7 S2/C1C2 interaction results in contractile dysfunction; and (2) elucidate the mechanism of E848G-induced loss of MYH7 protein abundance and test whether restoring normal MYH7 protein abundance can rescue contractile function. I will generate pathogenic MYH7 S2 variants with CRISPR/Cas9 for use in a mammalian two-hybrid approach to quantify dysfunction of the S2/C1C2 interaction. I will restore S2/C1C2 interaction disrupted by pathogenic MYH7 S2 variant by targeted mutation of the complementary amino acid in C1C2, ideally recovering contractile function in corresponding engineered heart tissues (EHTs). These findings will be applied to MYH7 S2 variants of unknown significance to test for S2/C1C2 integrity and verify in hiPSC-CMs. This aim will establish a novel rapid method to functionally reclassify MYH7 S2 variants of unknown significance. In the second aim, I will characterize hypocontractility in EHTs derived from MYH7-E848G hiPSC- CMs and correlate with observed loss in MYH7 protein abundance. I will use fluorescent recovery after photobleaching (FRAP) to capture the dynamics of MYH7 cycling in sarcomeres with and without the E848G variant. Overexpression of MYH7 and silencing of the mutant allele will test the relationship between MYH7 abundance and contractile function. These findings will then be corroborated in other MYH7 pathogenic variants. This aim will establish a generalizable mechanism through which MYH7 variants lose MYH7 protein abundance and consequently lose contractile function. In sum, these experiments will enable diagnostic tools for predicting pathogenicity of MYH7 S2 variants and therapeutic approaches to address contractile function. This project will take place in the highly supportive and collaborative environment of the University of Washington Department of Medicine. With the mentorship of my Sponsor and Co-Sponsor (Dr. Daniel Yang and Dr. Charles E. Murry, respectively), this project will provide the training required for me to realize my goal of establishing an independent research laboratory at the crossroads of cellular biology, tissue engineering, and clinical application.

NIH Research Projects · FY 2026 · 2022-07

Nonmedical (recreational) cannabis use is now legal in many states. Additional states are considering legalization, despite the weakness of the current evidence base regarding potential impacts on public health. This R01 proposal seeks 5 years of funding to understand: Aim 1) changes in cannabis use across the first decade following cannabis legalization; Aim 2) changes in alcohol use, nicotine use, and their co-use with cannabis following cannabis legalization; and Aim 3) whether psychosocial consequences of cannabis use change in a legal cannabis context. This knowledge is critical to inform policy, support efforts to maintain hard- won reductions in teen cannabis use since the 2000s, and promote responsible use by adults. The proposed project is grounded in life course theories of development and substance use, and uniquely suited to address stated goals. It continues and expands upon the Seattle Social Development Project-The Intergenerational Project (SSDP-TIP), which aims to understand the impact of cannabis legalization on youth and parent cannabis and other drug use (n = 426 families, 80% living in Washington State). SSDP-TIP includes a parent drawn from another ongoing longitudinal panel study that began in 1985, their oldest biological child, and a second caregiver (usually the spouse) when available. Seven waves of pre-legalization data (2002-2011; mean child age in 2011 = 12) and three waves of short-term post-legalization data (2015-2017; mean child age in 2017 = 18) from offspring and parents are available. The proposed study will add three additional annual data collections post-legalization in 2022, 2023, and 2024 with parents and offspring (mean offspring age in 2024 = 25; range: 11-36 years). For adolescent offspring, the accelerated longitudinal design allows us to compare offspring measured before legalization to offspring from later birth cohorts measured at the same ages, but after legalization. This permits the disentangling of child age and policy effects, and facilitates understanding of the implications of cannabis legalization for prevention in ways that other studies cannot. The inclusion of young adult offspring allows examination of the impact of cannabis legalization on patterns of use and consequences across the 20s and into the early 30s. The inclusion of parents permits examination of post- legalization changes in cannabis and other drug use and consequences across the 40s. SSDP-TIP is the only study in Washington State with longitudinal data from youth and their parents beginning 10 years before legalization. With the proposed data collections, it will also stretch to 12 years post-legalization. This prospective, longitudinal design provides important advantages over large, repeated cross sectional studies by enabling causal ordering of legalization and changes in behavior, disentanglement of change due to legalization versus time or age, and study of both within- and between-person change. Findings will inform the timing of efforts to prevent underage and problematic cannabis, tobacco, and alcohol use and help to clarify public health impacts in the context of cannabis legalization.

NIH Research Projects · FY 2025 · 2022-07

Project abstract BRAIN pioneers are people who take on significant risk as participants in first-in-human or early neurotechnology studies for the sake of helping to further science. Their willingness to try invasive and non- medically necessary devices, their investments of significant time and energy, and their close relationships with study teams, typically built over 3-5 years of intensive experiments, make them relatively unique as research participants. Their well-being is integral to the continued development of the field of neural technology, yet relatively little neuroethical attention has been focused on the structures of support, from researchers and from family members, that enable BRAIN pioneers to successfully participate in neural device trials. In this study, we: 1) Utilize an embedded ethicist to observe interactions between participants in long-term, implantable neural device trials, their family partners, and the research team to identify modes of care and support provided in the experimental setting; 2) Use qualitative interviewing to explore how participants, family members and researchers articulate the role, benefits, and limitations of family and researcher care and support before, during, and after a trial; and 3) Develop recommendations for best practices for a more inclusive support structure in research design and practice.

NIH Research Projects · FY 2024 · 2022-06

Abstract Worldwide, five million adolescents and young adults (AYA) are living with HIV. More than 90% of global HIV- related mortality among AYA occurs in sub-Saharan Africa. A limited but growing evidence base of interventions available for adolescents in sub-Saharan Africa highlight the importance of retention in care for improving treatment outcomes. To meet AYA where they are in their engagement with the healthcare system, interventions need to be feasible, acceptable, and appropriate for local healthcare settings and practice. This F31 project leverages a cluster randomized controlled trial, Data-informed Stepped Care to Improve Adolescent HIV Outcomes (UG3/UH3 HD096906; PIs: Kohler, John-Stewart) that is testing a Stepped Care intervention to improve retention among adolescents and young adults living with HIV in western Kenya. This F31 project aims to evaluate early phase implementation outcomes of the Stepped Care intervention as it is delivered across 10 healthcare facilities. Aim 1 will identify adaptations made to the intervention (materials, procedures, or delivery) that influence acceptability, feasibility, and appropriateness. The Framework for Reporting Adaptations and Modifications to Evidence-based interventions (FRAME) will be used to characterize adaptations and modifications necessary for facility-specific optimization of the intervention. Aim 2 will identify the barriers and facilitators that impact penetration, coverage, and fidelity of the Stepped Care intervention. We will apply the Consolidated Framework for Implementation Research (CFIR) to examine barriers and facilitators of implementation to understand the context and intervention characteristics influencing implementation of the Stepped Care intervention. Finally, Aim 3 will match barriers to successful implementation to targeted strategies to overcome them, producing a package of intervention implementation options that promote long-term intervention adoption and sustainability. Findings from this project will contribute to understanding optimization of Stepped Care and provide valuable information about the context necessary for successful scale-up of Stepped Care interventions for adolescents and young adults within Kenya or scale out to other global settings. The research plan will provide the F31 candidate, an implementation science pre-doctoral student, with rigorous training in (1) implementation science conceptual frameworks, models, design and methodology; (2) training in qualitative methods; and (3) content-area expertise regarding evidence-based interventions for adolescents and young adults living with HIV.

NIH Research Projects · FY 2026 · 2022-06

Skin and soft tissue infections (SSTIs) are among the most common infections encountered in ambulatory and inpatient settings with cellulitis and abscess being most common. Effective treatments are needed as SSTIs are a high-volume condition, and the overall healthcare burden remains high. SSTIs are one of the top causes for hospital readmissions. Furthermore, the standard of care, incision and drainage (I&D), is one of the most painful procedures in the emergency department. In addition to pain and the potential for unsightly scarring, other complications from I&D include damage to surrounding structures, an extension of the infection into adjacent tissues, bacteremic complications and reoccurrence. Furthermore, patient populations with psychosocial risk factors (e.g., homelessness, IV drug use) are prone to secondary infections and wound care remains problematic in these populations. Although bacteria can develop drug resistance, they remain susceptible to mechanical damage. In particular, short intense pulses of Focused Ultrasound (FUS), histotripsy, non-invasively generates localized cavitation that mechanically disrupts the bacteria and represents a new paradigm for non- invasive abscess treatment. We propose that histotripsy can non-invasively disinfect and liquefy many superficial and intramuscular abscesses allowing for complete fine needle aspiration. Our Aims are to expand our animal model to include MRSA pathogens and perform animal studies to advance this technology toward first in human studies. If successful, this approach could decrease patient morbidity, eliminate hospital stays, and lower healthcare costs.

NIH Research Projects · FY 2025 · 2022-06

Globally, 15 million preterm births occur annually, with the majority born in low- and middle-income countries. Prematurity is a leading cause of both under-5 mortality and future neurodevelopmental delay. A long- established link between periodontal disease and preterm birth exists, yet common dental interventions such as scaling and planing fail to prevent preterm birth. Xylitol, a sugar substitute commonly found in gum products, prevents caries and periodontitis with presumed dampening of local and systemic inflammation, a risk factor for neurodevelopmental delay in offspring. We recently completed a cluster-randomized trial in Malawi designed to test the efficacy of xylitol gums to prevent preterm birth and immediate neonatal outcomes. This follow-up study proposal seeks to meaningfully advance our findings by evaluating if exposure to xylitol gum administered preconception or during early pregnancy improves neurodevelopmental outcomes in offspring (up to 6 years of age). Specifically, we will test the hypothesis by formally evaluating whether chewing xylitol gum prevents adverse neurodevelopmental outcomes by leveraging subjects from the Prevention of Prematurity and Xylitol (PPaX) Trial that enrolled 10,404 pregnant women in Malawi from 2015-2019. The Specific Aims of the research are to: 1) Evaluate the sensitivity and specificity of the MDAT as compared to the KABC-II neurodevelopment diagnostic tool in assessing neurodevelopmental delay of formerly term children aged 4-6 years of age; 2) Evaluate the sensitivity and specificity of the MDAT as compared to the KABC- II in assessing neurodevelopmental delay of formerly preterm children aged 4-6 years of age; 3) Determine the impact of xylitol gum exposure during pregnancy on the neurodevelopmental outcomes of 200 sex-matched term born children (100 controls, 100 xylitol) and 200 preterm born children (100 controls, 100 xylitol). Validating the MDAT by comparing it to the KABC-II neurodevelopmental assessment in former preterm and term neonates in Malawi will allow for more efficient, cost-effective studies in the future and build capacity in low-resourced settings to identify children with neurodevelopmental delay more easily and provide more timely intervention. If xylitol use during pregnancy is found to prevent neurodevelopmental delay in offspring, this low-cost solution will prove to be a game-changer in preventing neurodevelopmental delay worldwide. Proposed Career and Learning Objectives are to: 1) Attain advanced skills in epidemiology, study design, data management, and analytic methods to correctly implement research focusing on evaluating neonatal outcomes in settings of health disparities, and 2) Enhance skills in database development, scientific writing, presentations, and grant preparation, and 3) Attain advanced skills, including neurodevelopmental test selection, examiner training and oversight, test interpretation and knowledge needed to conduct rigorous neurodevelopmental outcomes research. These skills will facilitate the applicant’s cutting-edge research on perinatal interventions to improve neonatal health and future neurodevelopmental outcomes in the most vulnerable populations.

NIH Research Projects · FY 2025 · 2022-06

Modified Project Summary/Abstract Section The broad goal of this project is to understand how force generation by kinesin-related motors and dynamic microtubules controls the fidelity of chromosome segregation. Both microtubules and motors represent excellent targets for anti-cancer drugs but to make wise choices for developing therapeutics it is necessary to understand their contribution to cell division in detail. MCAK/Kif2C is a MT depolymerizing kinesin that controls MT length within the spindle and supresses chromosome instability (CIN). Using CRISPR/Cas9 engineered cells, rapamycin-dependent relocalization and long-term live imaging we have pinpointed the precise contribution that this protein makes to enhance the fidelity of chromosome segregation which will enable us to understand why this protein rescues CIN in tumorigenic cells and its future potential as a therapeutic target. MCAK/Kif2C is also strongly associated with centrosomes where it has the potential to suppress MT outgrowth and influence centrosome separation. We have identified two other poorly studied centrosome-associated kinesins, Kif25 and Kif9, that function in centrosome cohesion and centrosome satellite positioning respectively and that also impact centrosome separation and positioning. Altered centrosome positioning in cell culture has limited effects on cell viability. However, Both Kif9 and Kif25 are widely expressed in vertebrates. Because Kif25, in particular, is most highly expressed in brain it affords an opportunity to investigate the role of centrosome cohesion in vertebrate development and neural stem cell division using morpholinos directed against Kif25 introduced into early zebrafish embryos. We aim to use live and fixed-cell imaging tools and transgenic cell lines to investigate, with precision, the contribution that these kinesins make to preserving the fidelity of the genome and to long-term cell fate. Ultimately this knowledge base can be leveraged to develop new therapies that target kinesin enzymology or MT dynamics.

NIH Research Projects · FY 2026 · 2022-06

Empirical data indicate American Indian youth are vulnerable to ill health due to adverse socio-demographic indices and little physical activity, sedentary behaviors and inadequate nutrition. These factors, which form a toxic triangle of risk, are compounded by behavioral risks factors such as alcohol, tobacco, and other drug use (ATOD). If current patterns persist, by 2050 one out of three youth from the Choctaw Nation of Oklahoma, the third largest AI tribe, will be living with Type 2 diabetes and 70% of the Nation will be obese. Wakaya: Rising Up for Choctaw Youth Health, is an experiential, outdoor, nature-based program grounded in Choctaw values. It is a multi-level intervention that integrates components of motivational interviewing, the information- motivation-behavioral skills model, the Positive Youth Leadership Development framework, and the NIMHD Multilevel Socio-Ecological Research Framework. With a prospective two-group randomized waitlist-control trial design among 176 at-risk Choctaw youth (ages 14-18 years), we will assess the culturally grounded intervention's impact on the primary outcomes of physical activity, sedentary behavior, and healthful food habits; as well as the secondary outcomes of weight/BMI; and ATOD. Additionally, guided by the NIMHD Multilevel Socioecological Framework, we will examine the potential mechanisms of change by analyzing how pathway variables (e.g., nature connectedness) mediate intervention effects. Finally, through Topic Modeling of qualitative interview data and Ripple Effect Mapping via youth participant and tribal leader focus groups (n=64), we will explore the community-level impact of Wakaya and create a visual map representing the interconnected ripple effects of the intervention into the community. The program, supported by promising preliminary data on this approach among adults and acceptability data from youth, involves a 3-month outdoor intervention (with 4 individual sessions, weekly group sessions, 2 overnight culture immersion camps (7 days total), and a 4-day Choctaw Trail of Tears walk. Additionally, the intervention builds youth health leadership and community organizing skills. The research team comprises experts in American Indian health from academe as well as Choctaw Nation and collaborative efforts among two tribal Departments- Historic Preservation and Behavioral Health. Findings will be disseminated scientifically as well as shared with the tribe for programmatic implementation. If efficacious, this intervention will provide evidence for interventions that promote physical activity and nutrition programs that target green spaces, community gardens, natural/nature trails, and built environments. The proposed study provides significant and practical information in several areas, including the feasibility of delivering interventions in nature-based settings across multilevel stakeholders. Should it be efficacious, the program has potential for widespread adaptation and dissemination to other tribal communities and could be generalizable to other chronic co-occurring physical activity and food habit-related conditions.

NIH Research Projects · FY 2025 · 2022-06

Natural Language Processing (NLP) methods have been broadly applied to clinical problems, from recognition of clinical findings in physician notes to identification of transcribed speech samples indicating changes in cognitive status. Deep transformer networks (DTNs) have dramatically advanced NLP accuracy. These deep learning models have multiple hidden layers that may correspond to billions of trainable parameters, allowing them to apply information learned from training on large unlabeled corpora to a specific task of interest. However, their size leaves them especially vulnerable to confounding bias, induced by variables that can influence both the predictor (text) and the outcome (e.g. an associated diagnosis) of a predictive model. Such systematic biases are a recognized danger in the application of artificial intelligence methods to clinical problems, and are the focus of NLM NOT-LM-19-003 which invites applications proposing methods to identify and address them. Deep learning models in general require large amounts of training data, spurring initiatives to aggregate medical data from across institutional siloes. This can increase data set size and enhance model portability, but leaves the resulting models vulnerable to confounding by provenance, where models learn to recognize the origin of dataset components and make biased predictions based on site-specific class distributions (e.g. COVID prevalence). Such models will assign classes based on indicators of dataset provenance, rather than diagnostically meaningful linguistic differences, and make erroneous predictions when the provenance-specific distributions at the point of deployment differ from those in the training set. Confounding of this nature is a pervasive problem that presents a fundamental barrier to the portability of trained models, and threatens the utility of datasets assembled from across institutions and services. Unlike traditional statistical and machine learning models, with deep transformer networks feature representations are distributed across parameters spread throughout the entire network. New methods are needed to meet the challenge of identifying and mitigating the influence of confounding variables in such models. In the proposed research we will develop a systematic approach to Deconfounding Deep Transformer Networks (DeconDTN), embodied in an eponymous and publicly available set of open source tools for (1) identification of provenance-related biases, (2) mitigation of these biases using a novel set of validated methods, and (3) systematic evaluation of the resulting effects on model performance. While DeconDTN will be generally applicable, development and evaluation will occur in the context of three use cases involving data sets drawn from different sources: classification of speech transcripts from participants with dementia drawn from two locations, identification of goals-of-care discussions in clinical notes drawn from multiple studies involving a range of clinical services, and prediction of COVID-19 status in notes drawn from different clinical units. Our driving hypothesis is that the resulting models will make more accurate predictions in these heterogenous datasets than corresponding models without correction for confounding by provenance.

NIH Research Projects · FY 2026 · 2022-05

Abstract This proposal seeks to identify potential allosteric properties in adhesins of human enterobacterial pathogens - Escherichia coli, Klebsiella pneumoniae/oxytoca, Enterobacter spp, Proteus mirabilis, and Salmonella – that are assembled via a chaperone-usher pathway (CUP). To date, only the mannose-specific, type 1 fimbrial adhesin of E. coli, FimH, has been demonstrated to be an allosteric protein that can exist in alternative functional (active/inactive) conformations. This property allows bacteria that contain FimH as part of hair-like surface appendages, fimbriae or pili, to bind ligand presented on host cells rapidly from an inactive conformation and to remain bound for very long lifetimes under shear force by transiting to an active conformation. The long-lived (slow dissociation) binding involves formation of so-called `catch-bonds' that can be activated and become stronger under tensile mechanical force and involve an allosteric switch. To date no other bacterial adhesin has been demonstrated to be allosteric and to exist in alternative functional (active/inactive) conformations. To identify other adhesins that work via similar mechanisms, we will focus on adhesins that are part of fimbriae or pili and belong to the same CUP structural class as FimH. We recently identified a set of aliphatic or aromatic residues that act as molecular toggles that control the allosteric switch between active and inactive conformations by switching their orientation between the protein core and surface. It is possible to stabilize either active or inactive conformation of the adhesin by “surface locking” such toggles through substitution to hydrophilic charged residues. We will use putative analogs of the FimH toggles to identify the existence of allosteric states in other CUP adhesins that are homologous or non-homologous to FimH, using mutagenesis, various functional assays, and three types of structural analysis – NMR, X-ray crystallography, and cryo-EM. Success of our studies will contribute to understanding of general mechanisms of bacterial adhesion to host cells and, ultimately, to the design of optimized vaccines and small molecule inhibitors. If certain adhesins are found to be allosteric, in-depth analysis of their physiologically-relevant structure/functional properties and significance for pathogenesis as well as practical implementation of the findings will be the focus of future studies.

NIH Research Projects · FY 2026 · 2022-05

Project Summary/Abstract This project will build a new kidney biopsy cohort to characterize the molecular, morphometric, and metabolic features of diabetic kidney disease (DKD) over the modern clinical course of type 1 diabetes (T1D). Landmark kidney biopsy studies have enhanced our understanding of DKD pathogenesis. However, advances in continuous glucose monitoring and automated insulin delivery have changed diabetes management and the clinical course of DKD in T1D. Moreover, innovation in molecular methods to interrogate kidney tissue, such as single-cell RNA sequencing (scRNA-seq), allows characterization of DKD at a resolution not previously possible. Based on published work and our preliminary data, we hypothesize that perturbed kidney energetics and hypoxia are central metabolic pathways in the development of DKD in T1D. We will test this hypothesis by creating a unique new longitudinal kidney biopsy cohort (N=100) spanning the critical duration of T1D over which DKD initiates and progresses (5-30 years) and leveraging our existing vanguard biopsy cohort (N=30). Normative kidney biopsy data will be provided from our existing cohort of healthy controls (N=20), the Kidney Precision Medicine Project (KPMP), and additional living kidney donor biopsies. We will implement state-of- the-art molecular (scRNA-seq) and morphometric interrogation of kidney tissue and rigorous metabolic phenotyping. Specifically, we aim to: (1) define differences in kidney energetics and hypoxia over the course of T1D; (2) test associations of the transcriptomic signatures of hypoxia with the structural lesions and clinical manifestations of progressive DKD; and (3) explore the mechanistic correlates of perturbed kidney energetics and hypoxia within a subset of participants with T1D with repeat kidney biopsies. This work will help define the role of perturbed energetics and hypoxia in DKD as well as risk factors for and consequences of kidney hypoxia in T1D. This study will also generate a valuable repository of data, biosamples, and kidney tissue for further analysis of DKD in T1D, made publicly available through the KPMP platform.

NIH Research Projects · FY 2026 · 2022-05

Project Summary/Abstract The peptide neurotensin (NTS) is known to be a potent modulator of dopamine neuron activity, and NTS signaling has been linked to various modalities of behavioral reinforcement and reward, as well as to the behavioral response to drugs of abuse and the motivation for drug taking. Pharmacological studies have found that in the ventral tegmental area (VTA), NTS binding to its receptors depolarizes dopamine neurons through a variety of second messenger cascades, resulting in increased dopamine neuron firing and downstream dopamine release. However, much less is known about the physiological release of NTS from endogenous brain circuits and what role these circuits may play in regulating motivated behavior. Using retrograde mapping we identified 23 brain regions that send NTS input to the VTA; however, few of these inputs are well-studied for their role in NTS signaling, and little is known about what stimuli or behavioral actions may activate NTS neurons. Furthermore, though peptidergic neurons typically co-release a fast neurotransmitter, such as glutamate or GABA, current methods for circuit-specific activation fail to distinguish between downstream effects triggered by peptides versus those evoked by fast transmitters. We hypothesize that different NTS projections to the VTA may play distinct roles in modulating motivated behavior, and that NTS acts synergistically with co-released fast transmitters to govern the dynamics of downstream dopamine neuron activation. Here we propose to determine the neurotransmitter co-expression of select NTS inputs to the VTA and map their synaptic connectivity to dopamine and non-dopamine neurons. We also propose to use viral CRISPR gene mutagenesis techniques in combination with optogenetics to isolate the peptide and fast transmitter components of NTS inputs to the VTA. We will dissect the role of these components in regulating different modalities of behavioral reinforcement and will measure the response of dopamine neurons in vivo to stimulation of specific inputs. Finally, we will use fiber photometry to measure the activity profiles of NTS neurons during learning and performance of a cued reinstatement food reward task. Together, these experiments will add unprecedented circuit-specific precision to our understanding of how these critical peptidergic inputs influence the activity state of dopamine neurons and govern reward and reinforcement learning.

NIH Research Projects · FY 2026 · 2022-05

PROJECT SUMMARY/ABSTRACT Unplanned ICU admissions from the acute care floor are common and most often related to the development of respiratory failure or cardiac dysfunction, with respiratory or circulatory impairment the reason for ICU admission in over 60% of cases. These admissions are associated with high morbidity and mortality and are accompanied by other important repercussions for patients and their family members, including inadequate communication and lower family ratings of care. Unplanned ICU admissions are strong indicators of adverse events, yet little is known about mechanisms underlying these admissions. Prior work has focused on patient characteristics associated with unplanned ICU admissions, including age and severity of illness, but there is limited evidence examining non-patient factors that also contribute, such as human (e.g., clinician), organizational, or technical failures. It is critical to understand how these non-patient factors affect unplanned ICU admissions, because admissions resulting from these factors may be preventable. It is also imperative to differentiate between non-patient factors (human vs organizational vs technical) and their associated mechanisms because they call for unique interventions. To fill this key knowledge gap, we propose to identify contributing factors and mechanisms for unplanned ICU admissions and connect these factors and mechanisms to patient- and family-centered outcomes. This objective will be met by achievement of three specific aims involving a cohort of patients transferred from acute care to the ICU at an academic medical center, a safety-net hospital, and a community hospital. The first aim will use root cause analysis to adapt and refine an existing framework for classification of adverse events, the PRISMA (Prevention and Recovery Information System for Monitoring and Analysis) model. The adapted PRISMA model will allow us to identify multiple factors – patient, human (e.g., clinician), organizational, and technical — and associated mechanisms contributing to unplanned ICU admissions. The second aim will examine associations between factors and mechanisms contributing to unplanned ICU admissions and family member symptoms of psychological distress, including symptoms of depression, anxiety, and post-traumatic stress. The final aim will compare ICU-free days and costs of care across factors and mechanisms contributing to unplanned ICU admissions. Our team has extensive research experience with seriously ill patients and their family members, with expertise in development of interventions to improve patient- and family-centered outcomes, medical decision making, healthcare systems, and quality and safety. We are well-positioned to identify factors and mechanisms contributing to unplanned ICU admissions and generate the knowledge needed to develop interventions with the greatest potential to improve outcomes for patients, family members, and the healthcare system.

NIH Research Projects · FY 2026 · 2022-05

The main goals of this project are to identify mechanisms underlying thrombogenesis in patients with left atrial (LA) fibrosis and to validate this new knowledge via a prospective proof-of-concept clinical study. Atrial fibrillation (AFib) affects millions of Americans and carries a five-fold increased risk of stroke, a leading cause of mortality and morbidity. Around 30% of all ischemic strokes are caused by thromboembolism in AFib patients. In patients without AFib, embolic strokes of undetermined source (ESUS) account for an additional 30% of ischemic strokes. Current stroke risk stratification tools in AFib and ESUS (e.g., CHA2DS2-VASc) are deficient in predictive accuracy, leaving many patients either under-treated for stroke prevention or over- treated and subjected to unnecessary bleeding risk. The growing evidence that LA fibrosis serves as a mechanistic nexus between AFib and ESUS is a very promising advance that could open new avenues for stroke prevention. However, taking advantage of this opportunity requires detailed knowledge of the mechanism(s) by which fibrotic atria are prone to thrombosis, with or without AFib. Fibrosis has complex structural, electrical, and contractile effects in the LA. These phenomena may independently or synergistically influence thrombosis risk by altering LA hemodynamics, but prior work has not systematically assessed inter-dependencies or clarified each factor’s relative importance. This is due to difficulties associated with experimental manipulation and acquisition of clinical measurements. Advances in computational modeling offer an unprecedented opportunity to address this critical knowledge gap. Specifically, the stage is set to create a multi-scale, multi- physics framework that can comprehensively simulate the pro-thrombotic potential of each unique patient-specific LA fibrosis pattern. Our central hypothesis is that LA fibrosis is a key mechanistic factor in determining each individual’s risk of thromboembolic stroke due to structural, electrical, and contractile factors. Our approach consists of three specific aims. Aim 1 will develop and calibrate a computational framework that integrates electrophysiological, biomechanical, and mechano- fluidic modeling in patient-specific LA models, paying special attention to resolving the effects of fibrosis. We will parameterize the framework using multi-modality magnetic resonance imaging acquisitions in AFib patients with prior stroke and non-AFib, non-stroke controls. Aim 2 will use the new computational framework to systematically characterize mechanistic connections between LA fibrosis and thrombogenesis. We will examine how each individual’s mix of fibrosis extent/pattern, LA anatomy, and susceptibility to emergent electromechanical phenomena combine (with or without simulated AFib) to create a thrombogenic milieu that can be characterized by computational modeling. Aim 3 will validate the mechanistic connections between fibrosis and risk of recurrent stroke/brain microinfarction in a proof-of-concept prospective clinical study. We will examine a high-risk cohort of ESUS patients, but notably without a current indication for oral anticoagulation. We will test if model-predicted thrombogenic combinations of LA shape, fibrosis pattern, deranged electromechanics, and disrupted blood flow exist in patients who experience more adverse outcomes. Our validated multi-physics modeling framework will, for the first time, yield new insight on fibrosis-mediated stroke mechanisms, and pave the way for new treatments for millions of patients who are borderline candidates for anticoagulation (e.g., individuals with ESUS or AFib with intermediate risk scores).

NIH Research Projects · FY 2025 · 2022-05

PROJECT SUMMARY Atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death in the US. Despite guidelines promoting aggressive anti-atherosclerotic therapies, there is »5%/year residual ASCVD risk in patients who achieve profound LDL-C lowering (median 30 mg/dL) with combined statin and PCSK9 inhibitor therapy. New treatment strategies are needed to target the mechanisms beyond LDL to reduce this residual risk. Histologic studies have demonstrated that plaque neovasculature constitutes the main entrance for inflammatory cells into plaques and provides a major source for the formation and progression of intraplaque hemorrhage (IPH). IPH, mainly resulting from plaque neovascularization, is a common feature of advanced atherosclerotic lesions and a critical element leading to accelerated plaque progression, plaque instability and ischemic vascular events in humans. We found that plaque neovessel permeability (measured as Ktrans, using dynamic contrast enhanced magnetic resonance imaging) is strongly correlated with macrophage content and that greater adventitial Ktrans is associated with IPH. Recent studies have identified that CD163+ macrophages are associated with IPH and can further promote neovascularization leading to IPH progression. On the other hand, B1 cell-derived IgM can inhibit inflammation and reduce atherosclerosis, and circulating human B1 cells are inversely associated with coronary plaque volume and instability features. Our preliminary data showed that plasma IgM levels are reduced in patients with carotid IPH and B1 cells are inversely associated with IPH progression. We, therefore, propose to study the role of B1 cell-derived IgM and B1 cells in IPH pathology. To test a novel hypothesis that B1 cell-derived IgM levels are reduced in patients with IPH and that the reduction in protective IgMs results in unobstructed IPH-promoted inflammation and neovessel permeability, thereby exacerbating plaque progression, we propose to conduct comprehensive studies including: (1) histological examination of CEA specimens to determine whether plaques with increased Ktrans and/or IPH have a lower density of IgM and a higher density of CD163+ macrophages; (2) a longitudinal clinical follow-up study in 250 patients to determine whether lower IgM levels and B1 cells predict progression of Ktrans and IPH and whether IgM production and effect on macrophages are different in B1 cells in patients with and without IPH; (3) in vitro mechanistic studies of endothelial sprouting and leakiness using 3D microvessels to determine the effects of IgM and B1 cells on RBC-induced changes in macrophages and vascular permeability. This proposal utilizes state-of-the-art imaging technique for quantification of vascular permeability and IPH and 3D microvessels for study how hemoglobin-stimulated macrophages and B1 cells and/or IgM influence endothelial function related vascular permeability. Our study will gain new knowledge to uncover inflammatory mechanisms in IPH pathogenesis and to discover potential therapeutic targets with a goal of reduced vascular permeability to prevent IPH and its progression, which will ultimately reduce residual ASCVD risk.