Identification of Epigenetic Regulators of Vascular Calcification with a CRISPR-Based Screen.

Abstract

BACKGROUND Vascular calcification, mainly driven by osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs), is a common pathological condition in patients with chronic kidney disease. However, the roles of other epigenetic regulators in this process remain largely unexplored. CRISPR screen is a highly efficient strategy widely used in identifying genes related to various biological processes. However, the lack of suitable cell sorting strategies combined with CRISPR screen meant this technology had not been applied to gene screening in vascular calcification. METHODS We performed an epigenetic-focused CRISPR screen in primary human VSMCs and identified key genes and pathways underlying osteogenic transdifferentiation, based on sgRNA enrichment in RANKL + (calcified) and RANKL - (noncalcified) VSMCs isolated by magnetic activated cell sorting. To validate the screen results, potential genes with different rankings were validated by siRNA intervention and Alizarin Red S staining. Integrating CRISPR results with transcriptome data revealed 17 critical regulators. We further investigated the top hit, Anthrax Toxin Receptor 1 (ANTXR1), in vascular calcification by examining clinical human samples and intervention in mice model. RESULTS Through CRISPR screen, we identified 122 potential genes positive-regulating vascular calcification and 116 negative-regulating genes. Phenotypic experimental results further verified the roles of genes with different rankings in osteogenic transdifferentiation of VSMCs, reinforcing the validity of our CRISPR screen system. Notably, integrative analysis of CRISPR screen with transcriptome data revealed ANTXR1 as a critical factor regulating vascular calcification. Furthermore, detection of clinical human samples confirmed the upregulation of ANTXR1 during calcification. Knockdown of ANTXR1 suppressed vascular calcification in mice model; likewise, overexpression promoted vascular calcification and the osteogenic transdifferentiation of VSMCs. CONCLUSIONS Our epigenetic-focused CRISPR screen and transcriptome analysis identified critical epigenetic genes involved in vascular calcification.