PROJECT 4 SUMMARY Early life stress (ELS), defined as adverse experiences occurring before age 18, is a highly prevalent risk factor for cardiovascular disease (CVD). However, the development of effective strategies to prevent CVD in individuals exposed to ELS is hindered by lack of knowledge about physiological pathways underlying ELS effects on CVD, as well as malleable lifestyle factors that may mitigate these effects. Cross-species animal studies guiding Project 1 and Project 2 implicate specific mechanistic pathways that link ELS with CVD, including upregulation of histone deacetylase 9 (HDAC9), NADPH oxidase 2 (NOX2), and cytokines; reduced diversity in the gut microbiome; and reduction of short-chain fatty acids (SCFA). Together, these pathways induce CVD through reprogramming of the immune and vascular system in rodent models of ELS. However, we do not know whether these pathways lead to CVD in people who experienced ELS and whether they can be modified by lifestyle factors, such as diet, physical activity, and supportive parenting, during development. Project 4 will leverage an existing, ethnically diverse cohort of 1,000 adults (age 29) from Birmingham, Alabama, who have been characterized for ELS and lifestyle factors at ages 11, 13, 16, and 19, to test the hypotheses that 1) ELS induces vascular dysfunction and hypertension through sustained pro-inflammatory reprogramming of the gut microbiome and peripheral blood mononuclear cell transcriptome, and that 2) these effects may be modified by protective lifestyle factors during development. The proposed assessment at age 29 will include a comprehensive evaluation of stress and lifestyle factors in adulthood; vascular function; gut microbiome; SCFAs; and peripheral blood mononuclear cell (PBMC) transcriptome. Data from all five time points spanning ages 11 to 29 will be integrated to test 1) the relationship between prospectively measured ELS and immune and vascular function in adulthood; 2) whether these relationships are mediated by pro-inflammatory profiles of the gut microbiome and PBMC transcriptome in adulthood; and 3) whether diet quality, physical activity, and supportive parenting during development modify the relationships between ELS and adult gut microbiome, PBMC transcriptome, and immune and vascular function. Project 4 is conceptually and translationally innovative by testing specific gut microbiome and transcriptome pathways discovered in rodents in humans. Together with Project 3 which tests complementary metabolome and methylome pathways in adolescence, the two human projects will guide future mechanistic studies by identifying new multi-omic pathways that link ELS with vascular dysfunction during critical developmental periods spanning adolescence to young adulthood. The integrated findings from this PPG will elucidate causal pathways and protective factors related to ELS-induced CVD risk, directly informing the development of novel evidence-based interventions.