Plasma Levels of snoRNAs are Associated with Platelet Activation in Patients with Peripheral Artery Disease

In addition to supervised walking therapy, antithrombotic therapy and the management of risk factors, the treatment of peripheral artery disease (PAD) is limited to endovascular and surgical interventions, i.e., angioplasty with stent implantation and bypass surgery, respectively. Both are associated with a high restenosis rate. Furthermore, patients with PAD often suffer atherothrombotic events like myocardial infarction, transient ischemic attacks or stroke. Small ribonucleic acids (RNAs) have proven reliable biomarkers because of their remarkable stability. Small nucleolar RNAs (snoRNAs) guide modifications to small nuclear RNAs and ribosomal RNAs, enabling protein synthesis. In the current study, we measured four snoRNAs in 104 consecutive PAD patients who underwent elective infrainguinal angioplasty with stent implantation. We selected snoRNAs that showed significant overexpression in the plasma of end-stage PAD patients in a previous study. All four snoRNAs are transcribed from the 14q32 locus, which is strongly linked to human cardiovascular disease, including PAD and restenosis. We showed that the four selected 14q32 snoRNAs were abundantly expressed in the plasma of PAD patients. The plasma levels of these snoRNAs were not directly associated with target vessel restenosis, however, levels of SNORD113.2 and SNORD114.1 were strongly linked to platelet activation, which is an important determinant of long-term outcome, in PAD, and in cardiovascular disease in general

MEG8 regulates Tissue Factor Pathway Inhibitor 2 (TFPI2) expression in the endothelium

A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. The 14q32 locus contains many non-coding RNAs such as Maternally Expressed Gene 8 (MEG8). We observed an induction of this gene in ischemic heart disease. We investigated the role of MEG8 specifically in endothelial function as well as the underlying mechanism. We hypothesized that MEG8 plays an important role in cardiovascular disease via epigenetic regulation of gene expression. Experiments were performed in human umbilical vein endothelial cells (HUVECs). In vitro silencing of MEG8 resulted in impaired angiogenic sprouting. More specifically, total sprout length was reduced as was proliferation, while migration was unaffected. We performed RNA sequencing to assess changes in gene expression after loss of MEG8. The most profoundly regulated gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), was fivefold increased following MEG8 silencing. TFPI2 has previously been described as an inhibitor of angiogenesis. Mechanistically, MEG8 silencing resulted in a reduction of the inhibitory histone modification H3K27me3 at the TFPI2 promoter. Interestingly, additional silencing of TFPI2 partially restored angiogenic sprouting capacity but did not affect proliferation of MEG8 silenced cells. In conclusion, silencing of MEG8 impairs endothelial function, suggesting a potential beneficial role in maintaining cell viability. Our study highlights the MEG8/TFPI2 axis as potential therapeutic approach to improve angiogenesis following ischemia

MEG8 regulates Tissue Factor Pathway Inhibitor 2 (TFPI2) expression in the endothelium

A large portion of the genome is transcribed into non-coding RNA, which does not encode protein. Many long non-coding RNAs (lncRNAs) have been shown to be involved in important regulatory processes such as genomic imprinting and chromatin modification. The 14q32 locus contains many non-coding RNAs such as Maternally Expressed Gene 8 (MEG8). We observed an induction of this gene in ischemic heart disease. We investigated the role of MEG8 specifically in endothelial function as well as the underlying mechanism. We hypothesized that MEG8 plays an important role in cardiovascular disease via epigenetic regulation of gene expression. Experiments were performed in human umbilical vein endothelial cells (HUVECs). In vitro silencing of MEG8 resulted in impaired angiogenic sprouting. More specifically, total sprout length was reduced as was proliferation, while migration was unaffected. We performed RNA sequencing to assess changes in gene expression after loss of MEG8. The most profoundly regulated gene, Tissue Factor Pathway Inhibitor 2 (TFPI2), was fivefold increased following MEG8 silencing. TFPI2 has previously been described as an inhibitor of angiogenesis. Mechanistically, MEG8 silencing resulted in a reduction of the inhibitory histone modification H3K27me3 at the TFPI2 promoter. Interestingly, additional silencing of TFPI2 partially restored angiogenic sprouting capacity but did not affect proliferation of MEG8 silenced cells. In conclusion, silencing of MEG8 impairs endothelial function, suggesting a potential beneficial role in maintaining cell viability. Our study highlights the MEG8/TFPI2 axis as potential therapeutic approach to improve angiogenesis following ischemia

Long non-coding RNA MEG8 induces endothelial barrier through regulation of microRNA-370 and -494 processing

The 14q32 locus is an imprinted region in the human genome which contains multiple noncoding RNAs. We investigated the role of Maternally Expressed Gene 8 (MEG8) in endothelial function and the underlying mechanism. A 5-fold increase in MEG8 was observed with increased passage number in Human Umbilical Vein Endothelial Cells, suggesting MEG8 is induced during aging. MEG8 knockdown resulted in a 1.8-fold increase in senescence, suggesting MEG8 might be protective during aging. Endothelial barrier was impaired after MEG8 silencing. MEG8 knockdown resulted in reduced expression of miRNA-370 and -494 but not -127, -487b and -410. Overexpression of miRNA-370/-494 partially rescued MEG8-silencing-induced barrier loss. Mechanistically, MEG8 regulates expression of miRNA-370 and -494 at the mature miRNA level through interaction with RNA binding proteins Cold Inducible RNA Binding Protein (CIRBP) and Hydroxyacyl-CoA Dehydrogenase Trifunctional Multi-enzyme Complex Subunit Beta (HADHB). Precursor and mature miRNA-370/-494 were shown to interact with HADHB and CIRBP respectively. CIRBP/HADHB silencing resulted in downregulation of miRNA-370 and induction of miRNA-494. These results suggest MEG8 interacts with CIRBP and HADHB and contributes to miRNA processing at the post-transcriptional level

Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

BACKGROUND AND PURPOSE: The angiotensin II type 1 receptor (AT(1)R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT(1)R signals in a Gαq/11-dependent or -independent manner. EXPERIMENTAL APPROACH: We performed a global miRNA array analysis of angiotensin II (Ang II)-mediated miRNA regulation in HEK293N cells overexpressing the AT(1)R and focused on separating the role of Gαq/11-dependent and -independent pathways. MiRNA regulation was verified with quantitative PCR in both HEK293N cells and primary cardiac myocytes and fibroblasts. KEY RESULTS: Our studies revealed five miRNAs (miR-29b, -129-3p, -132, -132* and -212) that were up-regulated by Ang II in HEK293N cells. In contrast, the biased Ang II analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gαq/11-independent signalling, failed to regulate miRNAs in HEK293N cells. Furthermore, Ang II-induced miRNA regulation was blocked following Gαq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly down-regulated miR-129-3p. CONCLUSIONS AND IMPLICATIONS: Five miRNAs were regulated by Ang II through mechanisms depending on Gαq/11 and Erk1/2 activation. These miRNAs may be involved in Ang II-mediated cardiac biology and disease, as several of these miRNAs have previously been associated with cardiovascular disease and were found to be regulated in cardiac cells

Cardiovascular complications of diabetes: role of non-coding RNAs in the crosstalk between immune and cardiovascular systems

Abstract Diabetes mellitus, a group of metabolic disorders characterized by high levels of blood glucose caused by insulin defect or impairment, is a major risk factor for cardiovascular diseases and related mortality. Patients with diabetes experience a state of chronic or intermittent hyperglycemia resulting in damage to the vasculature, leading to micro- and macro-vascular diseases. These conditions are associated with low-grade chronic inflammation and accelerated atherosclerosis. Several classes of leukocytes have been implicated in diabetic cardiovascular impairment. Although the molecular pathways through which diabetes elicits an inflammatory response have attracted significant attention, how they contribute to altering cardiovascular homeostasis is still incompletely understood. In this respect, non-coding RNAs (ncRNAs) are a still largely under-investigated class of transcripts that may play a fundamental role. This review article gathers the current knowledge on the function of ncRNAs in the crosstalk between immune and cardiovascular cells in the context of diabetic complications, highlighting the influence of biological sex in such mechanisms and exploring the potential role of ncRNAs as biomarkers and targets for treatments. The discussion closes by offering an overview of the ncRNAs involved in the increased cardiovascular risk suffered by patients with diabetes facing Sars-CoV-2 infection. Graphical Abstrac

Role of LCN2 in a murine model of hindlimb ischemia and in peripheral artery disease patients, and its potential regulation by miR-138-5P

Background and aims: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD. Methods and results: WT and Lcn2-/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2-/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2-/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA+, p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54). Conclusions: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2