NFKB1 Gene Mutant Was Associated with Prognosis of Coronary Artery Disease and Exacerbated Endothelial Mitochondrial Fission and Dysfunction

Abstract

Endothelial apoptosis is the core pathological change in atherosclerotic cardiovascular disease, including coronary artery disease (CAD). Determining the molecular mechanisms underlying endothelial apoptosis is important. Nuclear factor kappa B (NF-κB) is a crucial transcription factor for controlling apoptosis. Our previous study demonstrated that the -94 ATTG ins/del mutant in the promoter of NFKB1 gene (rs28362491) is a risk factor for CAD. In the present study, we found that NFKB1 rs28362491 polymorphism was positively associated with increased major adverse cardiac and cerebrovascular events (MACCEs) in CAD patients. After adjusting for confounding factors including age, smoking, hypertension, glucose, and low-density lipoprotein cholesterol, the mutant DD genotype was an independent predictor of MACCEs ( $\text{OR}=2.578$ , $95\%\text{CI}=1.64$ –4.05, $P=0.003$ ). The in vitro study showed that mutant human umbilical vein endothelial cells (DD-mutant HUVECs) were more susceptible to high-glucose/palmitate-induced apoptosis, which was accompanied by decreased p50 expression and increased expression of cleaved caspase-3, Cytochrome c, and phospho-p65 ( $P<0.05$ ). The mitochondrial membrane potential was significantly lower, while increasing levels of mtROS and more opening of the mPTP were observed in DD-mutant HUVECs ( $P<0.05$ ). Furthermore, the percentage of cells with fragmented or spherical mitochondria was significantly higher in DD-mutant HUVECs than in wild-type cells (genotype II HUVECs) ( $P<0.05$ ). In addition, after stimulation with high glucose/palmitate, the NFKB1 gene mutant significantly increased the expression of Drp1, which indicated that the NFKB1 gene mutant affected the expression of mitochondrial morphology-related proteins, leading to excessive mitochondrial fission. In conclusion, the mutant DD genotype of the NFKB1 gene was an independent predictor of worse long-term prognosis for CAD patients. DD-mutant HUVECs exhibited abnormal activation of the NF-κB pathway and increased Drp1 expression, which caused excessive mitochondrial fission and dysfunction, ultimately leading to increased apoptosis.