C2238/αANP modulates apolipoprotein E through Egr-1/miR199a in vascular smooth muscle cells in vitro

Abstract

Subjects carrying the T2238C ANP gene variant have a higher risk to suffer a stroke or myocardial infarction. The mechanisms through which T2238C/αANP exerts detrimental vascular effects need to be fully clarified. In the present work we aimed at exploring the impact of C2238/αANP (mutant form) on atherosclerosis-related pathways. As a first step, an atherosclerosis gene expression macroarray analysis was performed in vascular smooth muscle cells (VSMCs) exposed to either T2238/αANP (wild type) or C2238/αANP. The major finding was that apolipoprotein E (ApoE) gene expression was significantly downregulated by C2238/αANP and it was upregulated by T2238/αANP. We subsequently found that C2238/αANP induces ApoE downregulation through type C natriuretic peptide receptor (NPR-C)-dependent mechanisms involving the upregulation of miR199a-3p and miR199a-5p and the downregulation of DNAJA4. In fact, NPR-C knockdown rescued ApoE level. Upregulation of miR199a by NPR-C was mediated by a reactive oxygen species-dependent increase of the early growth response protein-1 (Egr-1) transcription factor. In fact, Egr-1 knockdown abolished the impact of C2238/αANP on ApoE and miR199a. Of note, downregulation of ApoE by C2238/αANP was associated with a significant increase in inflammation, apoptosis and necrosis that was completely rescued by the exogenous administration of recombinant ApoE. In conclusion, our study dissected a novel mechanism of vascular damage exerted by C2238/αANP that is mediated by ApoE downregulation. We provide the first demonstration that C2238/αANP downregulates ApoE in VSMCs through NPR-C-dependent activation of Egr-1 and the consequent upregulation of miR199a. Restoring ApoE levels could represent a potential therapeutic strategy to counteract the harmful effects of C2238/αANP.

Pathogenesis of Ischemic Stroke: Role of Epigenetic Mechanisms

“…Our group demonstrated that the T2238C variant of the atrial natriuretic peptide (ANP) gene, a nonmodifiable risk factor for cardiovascular diseases including stroke, modulates ApoE level through miR-199 in vitro. The miR-199 up-regulation induced by C2238/αANP, and not by the wild type ANP, produced a downregulation of ApoE associated with decreased cell viability and increased apoptosis, necrosis, and inflammation in human vascular smooth muscle cells [96]. These results support the growing importance of miRNAs in the pathogenesis of stroke.…”

Early Growth Response 1 (Egr-1) Is a Transcriptional Activator of β-Secretase 1 (BACE-1) in the Brain

“…Human subjects carrying the T2238C ANP gene variant have a higher risk to suffer a stroke or myocardial infarction. C2238/␣ANP modulates apoE through Egr-1/miR199a in vascular smooth muscle cells in vitro (52). It will be interesting to investigate whether Egr-1 regulates A␤ clearance via apoE in the brain.…”

Atrial Natriuretic Peptide ― Old But New Therapeutic in Cardiovascular Diseases ―

“…23 Interestingly, ANP-RR reduced apolipoprotein E (ApoE) gene expression via NPR-C in VSMCs, which suggests NPR-C stimulation by ANP-RR interferes with the vasoprotective actions of ApoE. 24 These findings suggest that the rs5065 minor allele may produce a "malignant" ANP for the endothelium leading to atherosclerotic diseases through a NPR-C pathway. Therefore NPR-C inhibition may be a seminal therapeutic target for subjects with rs5065 to attenuate the risk of atherosclerotic diseases.…”

“…Some reports suggest that NPR-C has roles in the L-type Ca 2+ current in myocytes and sino-arterial nodes together, 53 and to inhibit proliferation of cardiac fibroblasts with BNP. 54 On the other hand, it may be strategic to inhibit NPR-C because ANP-RR may mediate vascular injury via NPR-C; 24 as we stated before, because NPR-C seems to exhibit "pleiotropic" studies have been done because of the high cost and difficulties associated with large peptide synthesis for clinical use.…”