Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis

Smooth muscle cell (SMC) proliferation is a hallmark of vascular injury and disease. Global hypomethylation occurs during SMC proliferation in culture and in vivo during neointimal formation. Regardless of the programmed or stochastic nature of hypomethylation, identifying these changes is important in understanding vascular disease, as maintenance of a cells' epigenetic profile is essential for maintaining cellular phenotype. Global hypomethylation of proliferating aortic SMCs and concomitant decrease of DNMT1 expression were identified in culture during passage. An epigenome screen identified regions of the genome that were hypomethylated during proliferation and a region containing Collagen, type XV, alpha 1 (COL15A1) was selected by ‘genomic convergence' for characterization. COL15A1 transcript and protein levels increased with passage-dependent decreases in DNA methylation and the transcript was sensitive to treatment with 5-Aza-2′-deoxycytidine, suggesting DNA methylation-mediated gene expression. Phenotypically, knockdown of COL15A1 increased SMC migration and decreased proliferation and Col15a1 expression was induced in an atherosclerotic lesion and localized to the atherosclerotic cap. A sequence variant in COL15A1 that is significantly associated with atherosclerosis (rs4142986, P = 0.017, OR = 1.434) was methylated and methylation of the risk allele correlated with decreased gene expression and increased atherosclerosis in human aorta. In summary, hypomethylation of COL15A1 occurs during SMC proliferation and the consequent increased gene expression may impact SMC phenotype and atherosclerosis formation. Hypomethylated genes, such as COL15A1, provide evidence for concomitant epigenetic regulation and genetic susceptibility, and define a class of causal targets that sit at the intersection of genetic and epigenetic predisposition in the etiology of complex diseas

Iron-Responsive miR-485-3p Regulates Cellular Iron Homeostasis by Targeting Ferroportin

<div><p>Ferroportin (FPN) is the only known cellular iron exporter in mammalian cells and plays a critical role in the maintenance of both cellular and systemic iron balance. During iron deprivation, the translation of FPN is repressed by iron regulatory proteins (IRPs), which bind to the 5′ untranslated region (UTR), to reduce iron export and preserve cellular iron. Here, we report a novel iron-responsive mechanism for the post-transcriptional regulation of FPN, mediated by miR-485-3p, which is induced during iron deficiency and represses FPN expression by directly targeting the FPN 3′UTR. The overexpression of miR-485-3p represses FPN expression and leads to increased cellular ferritin levels, consistent with increased cellular iron. Conversely, both inhibition of miR-485-3p activity and mutation of the miR-485-3p target sites on the FPN 3′UTR are able to relieve FPN repression and lead to decreased cellular iron levels. Together, these findings support a model that includes both IRPs and microRNAs as iron-responsive post-transcriptional regulators of FPN. The involvement of microRNA in the iron-responsive regulation of FPN offers additional stability and fine-tuning of iron homeostasis within different cellular contexts. MiR-485-3p-mediated repression of FPN may also offer a novel potential therapeutic mechanism for circumventing hepcidin-resistant mechanisms responsible for some iron overload diseases.</p> </div

MiR-485-3p-mediated FPN repression is sufficient to alter endogenous cellular iron status.

<p>(A) Western blot analysis of FPN and α-tubulin in HepG2 cells transfected with miR-485 (pc-miR-485) or vector control (pc-vector), with densitometric analysis of FPN normalized to tubulin levels. (B) Corresponding ferritin protein levels from samples shown in (3A) as measured by ferritin ELISA. Data are the mean ± SEM (n = 3). (C) QRT-PCR analysis of miR-485-3p expression in HepG2 cells transfected with indicated concentrations of pc-miR-485. Data expressed as fold change in expression relative to RNU48 control (n = 3). (D) QRT-PCR analysis of TFRC mRNA expression in HepG2 cells expressing increasing concentrations of miR-485, relative to tubulin control (n = 3). (E) Western blot analysis of FPN, IRP2 and α-tubulin protein levels in HepG2 cells treated with control (AMO-CNTL) or miR-485-3p-blocking (AMO-485-3p) antisense mediated oligonucleotides and subjected to iron supplementation (FAC) or iron depletion (DFE). Densitometric analysis shows indicated protein expression normalized to tubulin levels, relative to the iron-replete (FAC) condition. (F to G) Corresponding TFRC (F) and FTL (G) mRNA expression, relative to tubulin control, in HepG2 treated with either AMO-CNTL and AMO-485-3p and exposed to iron-rich or iron-deficient condition (n = 3). * Significantly different by Student's t-test: **p<0.01.</p

MiR-485-3p directly targets FPN.

<p>(A) Quantitative real-time PCR (QRT-PCR) analysis of miR-485-3p (left) and miR-194 (right) expression in K562, HEL, HEK-293, and HepG2 cells after treatment with 100 µM DFE, relative to baseline control. Data expressed as fold change in expression relative to RNU48 control (n = 4). (B) Schematic of the FPN 3′UTR with sequence alignments of predicted miR-194 and miR-485-3p target binding sites. (C) Fold change in luminescence of FPN 3′UTR luciferase reporter in HepG2 cells co-transfected with miR-485 expression construct (pc-miR-485), relative to vector control (pc-vector) (n = 3). (D) Fold change in luminescence of FPN 3′UTR luciferase reporter co-transfected with antisense-mediated oligonucleotides (AMOs) against miR-485-3p (AMO-485-3p), expressed as fold change ± SEM relative to non-targeting control AMO (AMO-CNTL (n = 4). (E) Fold change in luminescence of mutant MT-448+618 FPN 3′UTR, MT-618 FPN 3′UTR, MT-448 FPN 3′UTR, WT 3′UTR and empty luciferase reporter, expressed as fold change ± SEM relative to wild type FPN 3′UTR reporter (n = 4). (F) Activity of FPN 3′UTR, mutant MT-618 FPN 3′UTR, and empty control luciferase reporters following treatment with indicated concentrations of DFE in HepG2 cells. Data is expressed as fold change in luminescence ± SEM relative to baseline condition (0 µM DFE) (n = 3). * Significantly different by Student's t-test: *p<0.05, **p<0.01, ***p<0.0001.</p

Phase 1 Study of a Sulforaphane-Containing Broccoli Sprout Homogenate for Sickle Cell Disease

<div><p>Sickle cell disease (SCD) is the most common inherited hemoglobinopathy worldwide. Our previous results indicate that the reduced oxidative stress capacity of sickle erythrocytes may be caused by decreased expression of NRF2 (Nuclear factor (erythroid-derived 2)-like 2), an oxidative stress regulator. We found that activation of NRF2 with sulforaphane (SFN) in erythroid progenitors significantly increased the expression of NRF2 targets <i>HMOX1</i>, <i>NQO1</i>, and <i>HBG1</i> (subunit of fetal hemoglobin) in a dose-dependent manner. Therefore, we hypothesized that NRF2 activation with SFN may offer therapeutic benefits for SCD patients by restoring oxidative capacity and increasing fetal hemoglobin concentration. To test this hypothesis, we performed a Phase 1, open-label, dose-escalation study of SFN, contained in a broccoli sprout homogenate (BSH) that naturally contains SFN, in adults with SCD. The primary and secondary study endpoints were safety and physiological response to NRF2 activation, respectively. We found that BSH was well tolerated, and the few adverse events that occurred during the trial were not likely related to BSH consumption. We observed an increase in the mean relative whole blood mRNA levels for the NRF2 target <i>HMOX1</i> (p = 0.02) on the last day of BSH treatment, compared to pre-treatment. We also observed a trend toward increased mean relative mRNA levels of the NRF2 target <i>HBG1</i> (p = 0.10) from baseline to end of treatment, but without significant changes in HbF protein. We conclude that BSH, in the provided doses, is safe in stable SCD patients and may induce changes in gene expression levels. We therefore propose investigation of more potent NRF2 inducers, which may elicit more robust physiological changes and offer clinical benefits to SCD patients.</p><p><b><i>Trial Registration</i>:</b> ClinicalTrials.gov <a href="http://www.clinicaltrials.gov/ct2/show/NCT01715480" target="_blank">NCT01715480</a></p></div

Adverse Events.

<p>Adverse Events.</p

Effect of BSH ingestion on whole blood mRNA in SCD subjects.

<p>The composite of the mean for relative mRNA expression of A) NQO1, B) HMOX1, and C) HBG1/(HBB+HBG1) for all patients during the study period. All expression was set relative to pre-treatment values. Expression was normalized with GAPDH, and all statistical analyses were performed using Friedman’s test (* = p<0.05).</p

Clinical Parameters.

<p>Clinical Parameters.</p