Abstract Body: Background: Vascular diseases, including atherosclerosis and aneurysms, are the leading causes of death in the US and world-wide. Spatial transcriptomics alongside lineage-tracing and lineage-ablation data implicates critical roles for adventitial fibroblasts (AdvFibs) in both atherosclerosis and aneurysm, but no therapy to date targets this population of cells. In response to vascular stressors, AdvFibs are activated, expand rapidly, and undergo global transcriptomic alterations. However, it is currently unknown which genes regulate this activation and proliferation, nor whether this process is implicated in human vascular diseases.

Methods: To identify novel AdvFib regulators, we performed a novel genome wide CRSIPRi screen of AdvFib activation, followed by siRNA-mediated in vitro validation. The results of the screen were then functionally annotated against human genetic signals and scRNAseq data to predict the role of AdvFib activation in human vascular diseases.

Results: Our CRISPRi screen identified 768 inhibitors of fibroblast activation and 818 genes that promote AdvFib activation (p-value < 0.05). Beyond the expected regulators of the cell cycle and other essential genes, we found disproportionate enrichment for genes involved in TGF-beta and innate immune response pathways, revealing their importance in AdvFib activation. Human disease association analysis using PhenGenI revealed a significant association between identified regulators of AdvFib activation and genes implicated in hypertension, stroke, and venous thromboembolism, suggesting roles for AdvFib activation in these diseases. Additionally, identified regulators of AdvFib activation contain key GWAS signals for atherosclerosis and aneurysm, including FHL3, SKI, ZEB2, CDKN1A, and CDKN2A which, when integrated with summary-level Mendelian randomization, suggest a probable protective role of AdvFib activation in aneurysm and a detrimental one in atherosclerosis.

Conclusion: Our genome wide CRISPRi screen identified numerous potential regulators of AdvFib activation, many of which have known genetic and observational associations with human vascular diseases. This study affirms the potentially important role AdvFib plays in human vascular disease and may pave the way to future therapeutics targeting this often-neglected cell population.