Long noncoding RNA ANRIL regulates endothelial cell activities associated with coronary artery disease by up-regulating CLIP1, EZR, and LYVE1 genes.

Coronary artery disease (CAD) is the leading cause of death worldwide. Long noncoding RNAs (lncRNAs) are a class of noncoding transcripts of > 200 nucleotides and are increasingly recognized as playing functional roles in physiology and disease. ANRIL is an lncRNA gene mapped to the chromosome 9p21 genetic locus for CAD identified by the first series of genome-wide association studies (GWAS). However, ANRIL's role in CAD and the underlying molecular mechanism are unknown. Here, we show that the major ANRIL transcript in endothelial cells (ECs) is DQ485454 with a much higher expression level in ECs than in THP-1 monocytes. Of note, DQ485454 expression was down-regulated in CAD coronary arteries compared with non-CAD arteries. DQ485454 overexpression significantly reduced monocyte adhesion to ECs, transendothelial monocyte migration (TEM), and EC migration, which are critical cellular processes involved in CAD initiation, whereas siRNA-mediated ANRIL knockdown (KD) had the opposite effect. Microarray and follow-up quantitative RT-PCR analyses revealed that the ANRIL KD down-regulated expression of AHNAK2, CLIP1, CXCL11, ENC1, EZR, LYVE1, WASL, and TNFSF10 genes and up-regulated TMEM100 and TMEM106B genes. Mechanistic studies disclosed that overexpression of CLIP1, EZR, and LYVE1 reversed the effects of ANRIL KD on monocyte adhesion to ECs, TEM, and EC migration. These findings indicate that ANRIL regulates EC functions directly related to CAD, supporting the hypothesis that ANRIL is involved in CAD pathogenesis at the 9p21 genetic locus and identifying a molecular mechanism underlying lncRNA-mediated regulation of EC function and CAD development.

Splice variants of lncRNA RNA ANRIL exert opposing effects on endothelial cell activities associated with coronary artery disease.

Each gene typically has multiple alternatively spliced transcripts. Different transcripts are assumed to play a similar biological role; however, some transcripts may simply lose their function due to loss of important functional domains. Here, we show that two different transcripts of lncRNA gene ANRIL associated with coronary artery disease (CAD) play antagonizing roles against each other. We previously reported that DQ485454, the short transcript, is downregulated in coronary arteries from CAD patients, and reduces monocyte adhesion to endothelial cells (ECs) and transendothelial monocyte migration (TEM). Interestingly, the longest transcript NR_003529 is significantly upregulated in coronary arteries from CAD patients. Overexpression of ANRIL transcript NR_003529 increases monocyte adhesion to ECs and TEM, whereas knockdown of NR_003529 expression reduces monocyte adhesion to ECs and TEM. Much more dramatic effects were observed for the combination of overexpression of NR_003529 and knockdown of DQ485454 or the combination of knockdown of NR_003529 and overexpression of DQ485454. The antagonizing effects of ANRIL transcripts NR_003529 and DQ485454 were associated with their opposite effects on expression of downstream target genes EZR, CXCL11 or TMEM106B. Our results demonstrate that different transcripts of lncRNA can exert antagonizing effects on biological functions, thereby providing important insights into the biology of lncRNA. The data further support the hypothesis that ANRIL is the causative gene at the 9p21 CAD susceptibility locus.

Androgen inhibits key atherosclerotic processes by directly activating ADTRP transcription.

Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. In this study, we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription in a dose- and time-dependent manner, and the effect was abolished by three different androgen inhibitors, including pyrvinium pamoate, bicalutamide, and cyproterone acetate. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE, TGTTCT) located at +324bp from the ADTRP transcription start site. The ARE is required for concentration-dependent transcriptional activation of ADTRP. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP and its downstream genes PIK3R3 and MIA3. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.

Feedback regulation of coronary artery disease susceptibility gene ADTRP and LDL receptors LDLR/CD36/LOX-1 in endothelia cell functions involved in atherosclerosis.

A high level of low-density lipoprotein cholesterol (LDL) is one of the most important risk factors for coronary artery disease (CAD), the leading cause of death worldwide. However, a low concentration of LDL may be protective. Genome-wide association studies revealed that variation in ADTRP gene increased the risk of CAD. In this study, we found that a low concentration of oxidized-LDL induced the expression of ADTRP. Further analyses showed that knockdown of the expression of LDL receptor genes LDLR, CD36, or LOX-1 significantly downregulated ADTRP expression, whereas overexpression of LDLR/CD36/LOX-1 markedly increased ADTRP expression through the NF-κB pathway. Like ADTRP, LDLR, CD36 and LOX-1 were all involved in endothelial cell (EC) functions relevant to the initiation of atherosclerosis. Downregulation of LDLR/CD36/LOX-1 promoted monocyte adhesion to ECs and transendothelial migration of monocytes by increasing expression of ICAM-1, VCAM-1, E-selectin and P-selectin, decreased EC proliferation and migration, and increased EC apoptosis, thereby promoting the initiation of atherosclerosis. Opposite effects were observed with the overexpression of ADTRP and LDLR/CD36/LOX-1 in ECs. Interestingly, through the NF-κB and AKT pathways, overexpression of ADTRP significantly upregulated the expression of LDLR, CD36, and LOX-1, and knockdown of ADTRP expression significantly downregulated the expression of LDLR, CD36, and LOX-1. These data suggest that ADTRP and LDL receptors LDLR/CD36/LOX-1 positively regulate each other, and form a positive regulatory loop that regulates endothelial cell functions, thereby providing a potential protective mechanism against atherosclerosis. Our findings provide a new molecular mechanism by which deregulation of ADTRP and LDLR/CD36/LOX-1 promote the development of atherosclerosis and CAD.