The broad objectives of the proposed project are to: (1) test the hypothesis that alternative splicing events and alternative splicing regulators modulate human macrophage activation within a cardiometabolic disease context, (2) determine whether genetic variants associated with cardiometabolic disease traits alter the splicing code in modulating macrophage activation, (3) train the PI further in translational functional genomics research, and (4) provide the PI with individualized mentoring to ensure her transition to the role of an independent investigator. Cardiometabolic diseases, which continue to contribute to significant morbidity and mortality worldwide, are associated with chronic systemic inflammation, which can activate macrophages to shift toward the causally implicated pro-inflammatory M1 phenotype seen in cardiometabolic disease-associated tissues. The mechanisms underlying this process remain incompletely understood despite previous characterization of transcript-level changes, and a missing link is whether post-transcriptional regulation is involved.! With the advent of RNA sequencing (RNA-seq), alternative splicing (AS)?the generation of multiple different mRNA isoforms from a single gene?is emerging as a pervasive cell-specific mechanism that can lead to human diseases if dysregulated. Because AS plays an impactful role in cellular differentiation, it may contribute to the phenotypic plasticity of activated macrophages, critical players in both tissue homeostasis and the pathogenesis of cardiometabolic diseases. Although few studies describe isoform-level differences in the extremes of macrophage phenotypes, our preliminary RNA-seq studies of M1-activated and resting human monocyte-derived macrophages (HMDMs) have identified more than 200 AS events for genes associated with inflammation and cell survival. This proposal outlines a plan to study how regulation of AS events and the shift in isoform abundance from these events underlie cardiometabolic disease-promoting macrophage phenotypes, as well as a plan to study how genetic variants associated with cardiometabolic disease traits affect macrophage activation through the splicing code. The PI will engage in a rigorous training program of didactic coursework and mentoring by senior RNA and splicing biologists, macrophage experts, and computational biologists to gain additional skills relevant to conduct of the proposed aims and to functional genomics investigation. In undertaking the proposed studies and training plan, the PI will make significant contributions to the understanding of macrophage activation in the cardiometabolic context, mature as a translational investigator in functional genomics, and prepare to compete successfully for R01 funding for future projects aimed at precision medicine approaches toward complex human diseases.
Systemic chronic inflammation seen in cardiometabolic diseases, which confer significant morbidity and mortality worldwide, can induce macrophages to shift toward a pro-inflammatory phenotype in disease relevant tissues, e.g., the arterial wall in atherosclerosis and adipose in obesity. The mechanistic underpinnings of how macrophages shift phenotypes, an area for possible therapeutic targeting, remain incompletely understood; and a missing link is whether alternative splicing, which generates multiple protein isoforms, also contributes to the function of key proteins directly relevant to macrophage phenotypes and disease-relevant functions. The research outlined in this proposal will shed light on the possibility that alternative splicing modulates macrophage activation, providing a translational framework for future therapeutic targeting of the cardiometabolic disease-promoting, pro-inflammatory macrophage.
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