Background and Aims

Background: Modulation of vessel growth holds great promise for the treatment of peripheral artery disease (PAD) in patients with type 2 diabetes (T2D). However, the prognosis of T2D patients with PAD remains poor as breakthrough therapies remain to be developed. Epigenetic modifications, namely DNA methylation and histone posttranslational modifications, are emerging as key players in cardiovascular disease. Aim: To investigate whether epigenetic changes modulate post-ischemic vascularization in experimental diabetes.

Methods

Experiments were performed in primary human aortic endothelial cells (HAECs) exposed to hyperglycemia as well as in Lep^db/db mice carrying the genetic deletion of the methyltransferase SETD7 (Setd7^-/--Lep^db/db). Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings.

Results

RNA sequencing (RNA-seq) in glucose-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). Both SETD7 gene silencing and pharmacological inhibition by (R)PFI-2 rescued hyperglycemia-induced impairment of HAECs migration and tube formation, while SETD7 overexpression blunted the angiogenic response. RNA-seq, ingenuity pathway analysis (IPA) and ChIP assays showed that SETD7-dependent H3K4me1 regulates the transcription of the angiogenesis inhibitor semaphoring-3G (SEMA-3G). Indeed, SEMA-3G overexpression impaired angiogenic properties in SETD7-depleted HAECs. Endothelial sprouting was defective in aortas from Lep^db/db as compared to WT mice, whereas angiogenic response was preserved in Setd7^-/--Lep^db/db mice. Finally, SETD7/SEMA-3G axis was upregulated in muscle specimens from T2D patients.

Conclusions

Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in T2D patients with PAD.

Background and Aims

The nuclear receptor corepressor 1 (NCOR1) plays an important role in the regulation of gene expression under physiological and pathological conditions by connecting chromatin-modifying enzymes, coregulators and transcription factors. Recently we demonstrated that the deletion of macrophage NCOR1 aggravates the disease development by promoting CD36-triggered foam cell formation via PPARγ derepression. Since NCOR1 acts as a central regulator of hepatic lipid homeostasis, we speculated that its deletion in the liver will also affect atherosclerosis development.

Methods

To verify our hypothesis, we generated hepatocyte-specific Ncor1 knockout mice on a Ldlr-/- background. Besides assessing the progression of the disease in thoraco-abdominal aortas en face, we analyzed cholesterol and bile acid metabolism at expression and functional level.

Results

Our data demonstrate that liver-specific Ncor1 knockouts develop less atherosclerotic lesions than control mice on an atherosclerosis-prone background. Interestingly, plasma cholesterol levels of liver-specific Ncor1 knockout mice were higher compared to controls under chow diet, but lower after challenging mice with an atherogenic high-cholesterol diet for 12 weeks. Moreover, the content of hepatic cholesterol and triglycerides was decreased in liver-specific Ncor1 knockout compared to control mice. This improved lipid profile is secondary to changes in liver bile acid species and an improved fecal excretion of sterols.

Conclusions

Our experiments suggest that NCOR1 exerts central functions in the regulation of cholesterol and/or bile acid synthesis, transport and excretion under different dietary conditions. Liver NCOR1 could therefore be a novel target for the development of anti-atherosclerotic drugs.

Background and Aims

Atherosclerosis is inflammatory. The resolution of inflammation is an emerging area. Genome wide association studies (GWAS) have revealed a that vascular risk loci include SMARCA4, a SWI/SNF chromatin remodelling gene important for gene activation. We tested whether SMARCA4 is has a patho-mechanism relevant to atherosclerosis.

Methods

We studied cultured human blood-derived macrophages, stimulated with heme or cyclic-AMP. Gene expression was by RT-qPCR by -2ΔΔCt, and chromatin immunoprecipitation (ChIP) by our published methods.

Results

We describe an pathway that modifies chromatin in response to heme. This pathway includes early clearance of BACH1 repressor from the HMOX1 enhancer, associated with the histone acetylation of the same site (H3K9Ac). This contrasted with gene-selective H3S10 phosphorylation of genes that respond to cyclic-AMP but not hemin (FOS and NR4A2). Interestingly, at the HMOX1 cis-regulatory sequence, chromatin looping and recruitment of SMARCA4 occurred prior to recruitment of p-ATF1, which we have shown is reuired for activation by hemin. Knockdown of SMARCA4 suppressed p-ATF1 binding to HMOX1 but increased its binding to the cyclic-AMP response elements of the enhancers for FOS and NR4A2. This resulted in suppressed HMOX1 mRNA levels, but increased mRNA levels for FOS and NR4A2. Downstream, si-SMARCA4 allowed heme to induce PLA2G7, which encodes platelet-activating-factor acetyl hydrolase (PAF-AH).

Conclusions

These data point to a role for SMARCA4 in chromatin remodelling in advance of ATF1, with the ability to alter its preferred target genes. Taken together, these data indicate that SMARCA4, an atherosclerosis risk gene, is associated with a novel mechanism key to switching between leukocyte-resolution and erythrocyte-resolution.

Background and Aims

Macrophages produce exosomes that can travel in the circulation and be detected in atherosclerotic lesions. But whether they impact lesion dynamics is currently unknown. We investigated the therapeutic value of exosomes produced by M2-like macrophages in controlling inflammation and atherosclerosis in mice.

Methods

Using cushioned-density gradient ultracentrifugation, exosomes were isolated from conditioned medium of cultured naïve bone marrow derived macrophages (BMDM-exo) and BMDM exposed to interleukin 4 (IL-4) to polarize them into an M2 phenotype (BMDM-IL-4-exo).

Results

Our results show that BMDM-IL-4-exo reduced Tnf and Il1b mRNA expression in recipient cultured BMDM and enhanced mitochondrial oxidative metabolism that fostered their M2 differentiation. Infusions of BMDM-IL-4-exo into Apoe^–/– mice fed a western diet reduced myeloid cells in the circulation by controlling hematopoiesis in the bone marrow. This profoundly altered the inflammatory profile of Ly-6C^hi monocytes and increased M2 marker expression in peritoneal macrophages. BMDM-IL-4-exo also reduced the necrotic core and numbers of macrophages in aortic root lesions. To identify molecular mechanisms responsible for these protective effects, we performed unbiased RNA sequencing of BMDM exosomes which revealed that BMDM-IL-4-exo are enriched in microRNA-99a/146b/378a. Gene editing to selectively knock down these microRNA in macrophages resulted in exosomes that were less effective in reducing NF-ĸB signaling and levels of TNFa in recipient macrophages.

Conclusions

Our findings reveal that M2-like macrophage exosomes are potent anti-inflammatory mediators through the delivery of select microRNA to recipient cells. Therefore, further engineering macrophage exosomes with microRNA targeting inflammatory pathways could provide a therapeutic approach to control atherosclerosis.

Background and Aims

Inflammatory processes are essential mediators of cardiovascular disease and its attenuation or modulation may reduce the burden of atherosclerosis, myocardial infarction and heart failure. A better understanding of the mechanisms controlling the recruitment of leukocytes to the lesion, a limiting step of inflammation, is paramount for the development of better therapies.

Aim: Identify endothelial miRNAs that modulated recruitment of leukocytes to inflammatory regions to gain a better understanding of cell adhesion modulatory mechanisms.

Methods

A functional high-throughput screening (HTS) using a robotic platform was performed to identify miRNAs modulating monocyte adhesion using a library of human miRNA mimics. Additional analysis were performed in order to validate identified targets.

Results

Functional HTS and validation screening identified 38 miRNAs decreasing monocyte adhesion. We observed that miRNA-induced reduction of monocyte adhesion was partially mediated by downregulation of inflammatory genes such as VCAM1 or ICAM-1 in response to TNF-α or IL-1β stimulation. Computational analysis identified potential pathways mediating thess effects for a reduced number of miRNA candidates. These pathways included actin cytoskeleton modulators (Ras small GTPases) and important mediators of inflammatory signaling transduction pathways. Lastly, we confirmed miRNA-mediated gene downregulation of several of these potential mediators which could explain the reduced inflammatory response of endothelial cells under these conditions.

Conclusions

We identified miRNAs able to modulate monocyte cell adhesion under inflammatory conditions. A better understanding of the role of specific microRNAs regulating the immune-endothelial cell interaction may lead to novel therapeutic strategies.