Abstract 23: Identification of NCF4 as a Novel Regulator in Arterial Remodeling and Advanced Atherosclerosis

Arterial wall remodeling is a central multifactorial process in the development and progression of cardiovascular diseases. We employed an approach aimed at observing genetic variants associated with the progression of carotid intima-media thickness (cIMT) in order to identify novel pathways effecting vessel remodeling. This was achieved by conducting gene-centric analysis of 400,000 variants in 3,042 subjects with repeated cIMT measurements. Rs16997464 on chr22 intergenic between neutrophil cytosolic factor-4 ( NCF4) and colony stimulating factor 2 receptor beta ( CSF2RB ) was associated with cIMT progression at array-wide significance (p -7 ). The potential causative genes within this locus were investigated using a human vascular and non-vascular tissue biobank. Expression of 9 genes near rs16997464, were analyzed with the most significant association being with NCF4 in aortic adventitia. The effect of the variant on the function of the NCF4 gene product was further analyzed by comparing the oxidative burst capacity of neutrophils from subjects with different rs16997464 genotypes. We observed that neutrophils homozygous for the minor T allele, associated with slower cIMT progression, produced more extracellular ROS than neutrophils homozygous for the G allele, indicating a functional effect of rs16997464 on the NCF4 gene product p40 phox , a component of the NADPH oxidase 2 complex (NOX2). In parallel, we investigated if the chr22 locus also influenced the cellular composition of the atherosclerotic plaque, by utilizing data from the Athero-Express Biobank. Here we found that the minor T allele associated with a higher smooth muscle cell (SMC) content in the plaque. Finally, using a partial ligation model in mice where ncf4 is mutated, resulting in a reduced but not absent NOX2-associated ROS formation, we observed a reduced neointima formation in the ncf4 -mutated strain compared with wild-type littermates. Thus, this study identified rs16997464 in the NCF4-CSF2RB locus as a novel genetic determinant of cIMT progression, and provides evidence suggesting that NCF4 is involved in SMC proliferation and alteration of vessel wall pathophysiology.