C/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascu-lar disease. None of these snoRNAs has any known targets yet except for one,... Show moreC/D box small nucleolar RNAs (snoRNAs) of the DLK1-DIO3 locus are associated with vascular remodeling and cardiovascu-lar disease. None of these snoRNAs has any known targets yet except for one, AF357425/SNORD113-6. We previously showed that this snoRNA targets mRNAs of the integrin signaling pathway and affects arterial fibroblast function. Here, we aimed to identify whether AF357425/SNORD113-6 can also target small RNAs. We overexpressed or inhibited AF357425 in murine fibroblasts and performed small RNA sequencing. Expression of transfer (t)RNA fragments (tRFs) was predominantly regulated. Compared with overexpression, AF357425 knockdown led to an overall decrease in tRFs but with an enrichment in smaller tRFs (<30 nucleotides). We focused on tRNA leucine anti-codon TAA (tRNALeu(TAA)), which has a conserved predicted binding site for AF357425/ SNORD113-6. Adjacent to this site, the tRNA is cleaved to form tRFLeu 47-64 in both primary murine and human fibro-blasts and in intact human arteries. We show that AF357425/ SNORD113-6 methylates tRNALeu(TAA) and thereby prevents the formation of tRFLeu 47-64. Exposing fibroblasts to oxidative or hypoxic stress increased AF357425/SNORD113-6 and tRNALeu(TAA) expression, but AF357425/SNORD113-6 knockdown did not increase tRFLeu 47-64 formation under stress even further. Thus, independent of cellular stress, AF357425/SNORD113-6 protects against site-specific frag-mentation of tRNALeu(TAA) via 2'O-ribose-methylation. Show less
Jain, M.; Manjaly, G.; Maly, K.; Vries, M.R.D.; Janisiw, M.; Konig, L.; ... ; Jantsch, M.F. 2022
Adenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are in-terpreted as guanosines by the translational machinery. Conse-quently, A to I... Show moreAdenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are in-terpreted as guanosines by the translational machinery. Conse-quently, A to I editing in mRNAs can lead to their recoding and the formation of proteins not encoded in the genome. Filamin A is an actin-crosslinking protein. A to I editing in the filamin pre-mRNA leads to the exchange of a glutamine to an arginine in a highly interactive domain of the protein. However, the con-sequences of this editing event are still poorly understood. Here we show, using transgenic mice expressing either constitutively edited or constitutively uneditable filamin A that filamin A ed-iting critically controls angiogenesis in tumors but also in a mouse ischemia model. Hyper-editing reduces angiogenesis, while hypoediting leads to increased angiogenesis, possibly by altering vascular endothelial growth factor receptor 2 (VEGFR2) turnover. Further, FLNA editing of the tumor itself seemingly affects its metastatic potential by changing its inter-action with the extracellular matrix. We therefore identify fila-min A editing as a critical component for angiogenesis, tumor growth, and metastasis formation. Show less
Purpose of Review: Small non-coding RNAs regulate gene expression and are highly implicated in heart failure. Recently, an additional level of post-transcriptional regulation has been identified,... Show morePurpose of Review: Small non-coding RNAs regulate gene expression and are highly implicated in heart failure. Recently, an additional level of post-transcriptional regulation has been identified, referred to as the epitranscriptome, which encompasses the body of post-transcriptional modifications that are placed on RNA molecules. In this review, we summarize the current knowledge on the small non-coding RNA epitranscriptome in heart failure. Recent Findings: With the rise of new methods to study RNA modifications, epitranscriptome research has begun to take flight. Over the past 3 years, the number of publications on the epitranscriptome in heart failure has significantly increased, and we expect many more highly relevant publications to come out over the next few years. Currently, at least six modifications on small non-coding RNAs have been investigated in heart failure-relevant studies, namely N6-adenosine, N5-cytosine and N7-guanosine methylation, 2'-O-ribose-methylation, adenosine-to-inosine editing, and isomiRs. Their potential role in heart failure is discussed. Show less
The 14q32 locus is an imprinted region in the human genome which contains multiple non-coding RNAs. We investigated the role of the long non-coding RNA maternally expressed gene 8 (MEG8) in... Show moreThe 14q32 locus is an imprinted region in the human genome which contains multiple non-coding RNAs. We investigated the role of the long non-coding RNA maternally expressed gene 8 (MEG8) in endothelial function and its underlying mechanism. A 5-fold increase in MEG8 was observed with increased passage number in human umbilical vein endothelial cells (HUVECs), suggesting MEG8 is induced during aging. MEG8 knockdown resulted in a 1.8-fold increase in senescence, suggesting MEG8 might be protective during aging. The endothelial barrier was also impaired after MEG8 silencing. MEG8 knockdown resulted in reduced expression of microRNA (miRNA)-370 and -494 but not -127, -487b and -410. Overexpression of miRNA-370 or -494 partially rescued the MEG8-silencing-induced barrier loss. Mechanistically, MEG8 regulates expression of miRNA-370 and -494 at the mature miRNA level through interaction with the RNA-binding proteins cold-inducible RNA-binding protein (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multi-enzyme complex subunit beta (HADHB). Mature miRNA-370 and miRNA-494 were found to interact with CIRBP, whereas precursor miRNA-370 and miRNA-494 were found to interact with HADHB. Individual CIRBP and 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. Show less
Homberg, D.A.L. van den; Kwast, R.V.C.T. van der; Quax, P.H.A.; Nossent, A.Y. 2022
N-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser... Show moreN-6-methyladenosine (m6A) is the most prevalent post-transcriptional RNA modification in eukaryotic cells. The modification is reversible and can be dynamically regulated by writer and eraser enzymes. Alteration in the levels of these enzymes can lead to changes in mRNA stability, alternative splicing or microRNA processing, depending on the m6A-binding proteins. Dynamic regulation of mRNA m6A methylation after ischemia and hypoxia influences mRNA stability, alternative splicing and translation, contributing to heart failure. In this study, we studied vasoactive microRNA m6A methylation in fibroblasts and examined the effect of hypoxia on microRNAs methylation using m6A immunoprecipitation. Of the 19 microRNAs investigated, at least 16 contained m6A in both primary human fibroblasts and a human fibroblast cell line, suggesting vasoactive microRNAs are commonly m6A methylated in fibroblasts. More importantly, we found that mature microRNA m6A levels increased upon subjecting cells to hypoxia. By silencing different m6A writer and eraser enzymes followed by m6A immunoprecipitation, we identified METTL4, an snRNA m6A methyltransferase, to be predominantly responsible for the increase in m6A modification. Moreover, by using m6A-methylated microRNA mimics, we found that microRNA m6A directly affects downstream target mRNA repression efficacy. Our findings highlight the regulatory potential of the emerging field of microRNA modifications. Show less
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... Show moreA 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. Show less
Ingen, E. van; Foks, A.C.: Woudenberg, T.; Bent, M.L. van der; Jong, A. de; Hohensinner, P.J.; Wojta, J.; ... ; Nossent, A.Y. 2021
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
Ingen, E. van; Foks, A.C.; Woudenberg, T.; Bent, M.L. van der; Jong, A. de; Hohensinner, P.J.; ... ; Nossent, A.Y. 2021
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macro-phage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened Ml polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
Inhibition of the 14q32 microRNAs, miR-329-3p and miR-495-3p, improves post-ischemic neovascularization. Cold-inducible RNA-binding protein (CIRBP) facilitates maturation of these microRNAs. We... Show moreInhibition of the 14q32 microRNAs, miR-329-3p and miR-495-3p, improves post-ischemic neovascularization. Cold-inducible RNA-binding protein (CIRBP) facilitates maturation of these microRNAs. We hypothesized that CIRBP deficiency improves post-ischemic angiogenesis via downregulation of 14q32 microRNA expression. We investigated these regulatory mechanisms both in vitro and in vivo. We induced hindlimb ischemia in Cirp(-/-) and C57Bl/6-J mice, monitored blood flow recovery with laser Doppler perfusion imaging, and assessed neovascularization via immunohistochemistry. Post-ischemic angiogenesis was enhanced in Cirp(-/-) mice by 34.3% with no effects on arteriogenesis. In vivo at day 7, miR-329-3p and miR-495-3p expression were downregulated in Cirp(-/-) mice by 40.6% and 36.2%. In HUVECs, CIRBP expression was upregulated under hypothermia, while miR-329-3p and miR-495-3p expression remained unaffected. siRNA-mediated CIRBP knockdown led to the downregulation of CIRBP-splice-variant-1 (CIRBP-SV1), CIRBP antisense long noncoding RNA (lncRNA-CIRBP-AS1), and miR-495-3p with no effects on the expression of CIRBP-SV2-4 or miR-329-3p. siRNA-mediated CIRBP knockdown improved HUVEC migration and tube formation. SiRNA-mediated lncRNA-CIRBP-AS1 knockdown had similar long-term effects. After short incubation times, however, only CIRBP knockdown affected angiogenesis, indicating that the effects of lncRNA-CIRBP-AS1 knockdown were secondary to CIRBP-SV1 downregulation. CIRBP is a negative regulator of angiogenesis in vitro and in vivo and acts, at least in part, through the regulation of miR-329-3p and miR-495-3p. Show less
Ingen, E. van; Homberg, D.A.L. van den; Bent, M.L. van der; Mei, H.L.; Papac-Milicevic, N.; Kremer, V.; ... ; Nossent, A.Y. 2021
We have previously shown that C/D box small nucleolar RNAs (snoRNAs) transcribed from the DLK1-DIO3 locus on human chromosome 14 (14q32) are associated with cardiovascular disease. DLK1-DIO3... Show moreWe have previously shown that C/D box small nucleolar RNAs (snoRNAs) transcribed from the DLK1-DIO3 locus on human chromosome 14 (14q32) are associated with cardiovascular disease. DLK1-DIO3 snoRNAs are 'orphan snoRNAs' that have no known targets. We aimed to identify RNA targets and elucidate the mechanism-of-action of human SNORD113-6 (AF357425 in mice). As AF357425-knockout cells were non-viable, we induced overexpression or inhibition of AF357425 in primary murine fibroblasts and performed RNA-Seq. We identified several pre-mRNAs with conserved AF357425/SNORD113-6 D'-seed binding sites in the last exon/3' untranslated region (3'UTR), which directed pre-mRNA processing and splice-variant-specific protein expression. We also pulled down the snoRNA-associated methyltransferase fibrillarin from AF357425-High versus AF357425-Low fibroblast lysates, followed by RNA isolation, ribosomal RNA depletion and RNA-Seq. Identifying mostly mRNAs, we subjected these to PANTHER pathway analysis and observed enrichment for genes in the integrin pathway. We confirmed 2'O-ribose methylation in six integrin pathway mRNAs (MAP2K1, ITGB3, ITGA7, PARVB, NTN4 and FLNB). Methylation and mRNA expressions were decreased while mRNA degradation was increased under AF357425/SNORD113-6 inhibition in both murine and human primary fibroblasts, but effects on protein expression were more ambiguous. Integrin signalling is crucial for cell-cell and cell-matrix interactions, and correspondingly, we observed altered human primary arterial fibroblast function upon SNORD113-6 inhibition. Show less
Nguyen, T.T.M.; Bent, M.L. van der; Wermer, M.J.H.; Wijngaard, I.R. van den; Zwet, E.W. van; Groot, B. de; ... ; Nossent, A.Y. 2021
Early blood biomarkers to diagnose acute stroke could drastically reduce treatment delays. We investigated whether circulating small non-coding RNAs can serve as biomarkers to distinguish between... Show moreEarly blood biomarkers to diagnose acute stroke could drastically reduce treatment delays. We investigated whether circulating small non-coding RNAs can serve as biomarkers to distinguish between acute ischemic stroke (IS), intracerebral hemorrhage (ICH) and stroke mimics (SM). In an ongoing observational cohort study, we performed small RNA-sequencing in plasma obtained from a discovery cohort of 26 patients (9 IS, 8 ICH and 9 SM) presented to the emergency department within 6 h of symptom onset. We validated our results in an independent dataset of 20 IS patients and 20 healthy controls. ICH plasma had the highest abundance of ribosomal and tRNA-derived fragments, while microRNAs were most abundant in plasma of IS patients. Combinations of four to five tRNAs yielded diagnostic accuracies (areas under the receiver operating characteristics curve) up to 0.986 (ICH vs. IS and SM) in the discovery cohort. Validation of the IS and SM models in the independent dataset yielded diagnostic accuracies of 0.870 and 0.885 to distinguish IS from healthy controls. Thus, we identified tRNA-derived fragments as a promising novel class of biomarkers to distinguish between acute IS, ICH and SM, as well as healthy controls. Show less
Kwast, R.V.C.T. van der; Parma, L.; Bent, M.L. van der; Ingen, E. van; Baganha, F.; Peters, H.A.B.; ... ; Nossent, A.Y. 2020
Adenosine-to-inosine (A-to-I) editing in the seed sequence of microRNAs can shift the microRNAs' targetomes and thus their function. Using public RNA-sequencing data, we identified 35 vasoactive... Show moreAdenosine-to-inosine (A-to-I) editing in the seed sequence of microRNAs can shift the microRNAs' targetomes and thus their function. Using public RNA-sequencing data, we identified 35 vasoactive microRNAs that are A-to-I edited. We quantified A-to-I editing of the primary (pri-)microRNAs in vascular fibroblasts and endothelial cells. Nine pri-microRNAs were indeed edited, and editing consistently increased under ischemia. We determined mature microRNA editing for the highest expressed microRNAs, i.e., miR-376a-3p, miR-376c3p, miR-381-3p, and miR-411-5p. All four mature microRNAs were edited in their seed sequence. We show that both ADAR1 and ADAR2 (adenosine deaminase acting on RNA 1 and RNA 2) can edit pri-microRNAs in a microRNA-specific manner. MicroRNA editing also increased under ischemia in vivo in a murine hindlimb ischemia model and ex vivo in human veins. For each edited microRNA, we confirmed a shift in targetome. Expression of the edited microRNA targetomes, not the wild type targetomes, was downregulated under ischemia in vivo. Furthermore, microRNA editing enhanced angiogenesis in vitro and ex vivo. In conclusion, we show that microRNA A-to-I editing is a widespread phenomenon, induced by ischemia. Each editing event results in a novel microRNA with a unique targetome, leading to increased angiogenesis. Show less
Myostatin is a negative regulator of muscle cell growth and proliferation. Furthermore, myostatin directly affects the expression of 14q32 microRNAs by binding the 14q32 locus. Direct inhibition of... Show moreMyostatin is a negative regulator of muscle cell growth and proliferation. Furthermore, myostatin directly affects the expression of 14q32 microRNAs by binding the 14q32 locus. Direct inhibition of 14q32 microRNA miR-495-3p decreased postinterventional restenosis via inhibition of both vascular smooth muscle cell (VSMC) proliferation and local inflammation. Here, we aimed to investigate the effects of myostatin in a mouse model for postinterventional restenosis. In VSMCs in vitro, myostatin led to the dose-specific downregulation of 14q32 microRNAs miR-433-3p, miR-494-3p, and miR-495-3p. VSMC proliferation was inhibited, where cell migration and viability remained unaffected. In a murine postinterventional restenosis model, myostatin infusion did not decrease restenosis, neointimal area, or lumen stenosis. Myostatin inhibited expression of both proliferation marker PCNA and of 14q32 microRNAs miR-433-3p, miR-494-3p, and miR-495-3p dose-specifically in cuffed femoral arteries. However, 14q32 microRNA expression remained unaffected in macrophages and macrophage activation as well as macrophage influx into lesions were not decreased. In conclusion, myostatin did not affect postinterventional restenosis. Although myostatin inhibits 14q32 microRNA expression and proliferation in VSMCs, myostatin had no effect on macrophage activation and infiltration. Our findings underline that restenosis is driven by both VSMC proliferation and local inflammation. Targeting only one of these components is insufficient to prevent restenosis. Show less
MicroRNAs are posttranscriptional regulators of gene expression. As microRNAs can target many genes simultaneously, microRNAs can regulate complex multifactorial processes, including post-ischemic... Show moreMicroRNAs are posttranscriptional regulators of gene expression. As microRNAs can target many genes simultaneously, microRNAs can regulate complex multifactorial processes, including post-ischemic neovascularization, a major recovery pathway in cardiovascular disease. MicroRNAs select their target mRNAs via full complementary binding with their seed sequence, i.e., nucleotides 2-8 from the 5' end of a microRNA. The exact sequence of a mature microRNA, and thus of its 5' and 3' ends, is determined by two sequential cleavage steps of microRNA precursors, Drosha/DGCR8 and Dicer. When these cleavage steps result in nucleotide switches at the 5' end, forming a so-called 5'-isomiR, this results in a shift in the mature microRNAs seed sequence. The role of 5'-isomiRs in cardiovascular diseases is still unknown. Here, we characterize the expression and function of the 5'-isomiR of miR-411 (ISO-miR-411). ISO-miR-411 is abundantly expressed in human primary vascular cells. ISO-miR-411 has a different "targetome" from WT-miR-411, with only minor overlap. The ISO-miR-411/WT-miR-411 ratio is downregulated under acute ischemia, both in cells and a murine ischemia model, but is upregulated instead in chronically ischemic human blood vessels. ISO-miR-411 negatively influences vascular cell migration, whereas WT-miR-411 does not. Our data demonstrate that isomiR formation is a functional pathway that is actively regulated during ischemia. Show less
Kwast, R.V.C.T. van der; Quax, P.H.A.; Nossent, A.Y. 2020
Therapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both... Show moreTherapeutic neovascularization can facilitate blood flow recovery in patients with ischemic cardiovascular disease, the leading cause of death worldwide. Neovascularization encompasses both angiogenesis, the sprouting of new capillaries from existing vessels, and arteriogenesis, the maturation of preexisting collateral arterioles into fully functional arteries. Both angiogenesis and arteriogenesis are highly multifactorial processes that require a multifactorial regulator to be stimulated simultaneously. MicroRNAs can regulate both angiogenesis and arteriogenesis due to their ability to modulate expression of many genes simultaneously. Recent studies have revealed that many microRNAs have variants with altered terminal sequences, known as isomiRs. Additionally, endogenous microRNAs have been identified that carry biochemically modified nucleotides, revealing a dynamic microRNA epitranscriptome. Both types of microRNA alterations were shown to be dynamically regulated in response to ischemia and are able to influence neovascularization by affecting the microRNA's biogenesis, or even its silencing activity. Therefore, these novel regulatory layers influence microRNA functioning and could provide new opportunities to stimulate neovascularization. In this review we will highlight the formation and function of isomiRs and various forms of microRNA modifications, and discuss recent findings that demonstrate that both isomiRs and microRNA modifications directly affect neovascularization and vascular remodeling. Show less
Devaux, Y.; Nossent, A.Y.; EU CardioRNA COST Action 2019
We have previously shown that third-generation antisense (3GA) inhibition of 14q32 microRNA (miRNA)-494 reduced early development of atherosclerosis. However, patients at risk of atherosclerotic... Show moreWe have previously shown that third-generation antisense (3GA) inhibition of 14q32 microRNA (miRNA)-494 reduced early development of atherosclerosis. However, patients at risk of atherosclerotic complications generally present with advanced and unstable lesions. Here, we administered 3GAs against 14q32 miRNA-494 (3GA-494), miRNA-329 (3GA-329), or a control (3GA-ctrl) to mice with advanced atherosclerosis. Atherosclerotic plaque formation in LDLr-/- mice was induced by a 10-week high-fat diet and simultaneous carotid artery collar placement. Parallel to 3GA-treatment, hyperlipidemia was normalized by a diet switch to regular chow for an additional 5 weeks. We show that, even though plasma cholesterol levels were normalized after diet switch, carotid artery plaque progression continued in 3GA-ctrl mice. However, treatment with 3GA-494 and, in part, 3GA-329 halted plaque progression. Furthermore, in the aortic root, intra-plaque collagen content was increased in 3GA-494 mice, accompanied by a reduction in the intra-plaque macrophage content. Proatherogenic cells in the circulation, including inflammatory Ly6C(hi) monocytes, neutrophils, and blood platelets, were decreased upon miRNA-329 and miRNA-494 inhibition. Taken together, treatment with 3GA-494, and in part with 3GA-329, halts atherosclerotic plaque progression and promotes stabilization of advanced lesions, which is highly relevant for human atherosclerosis. Show less
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... Show moreIn 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. Show less
We have previously shown that third-generation antisense (3GA) inhibition of 14q32 microRNA (miRNA)-494 reduced early development of atherosclerosis. However, patients at risk of atherosclerotic... Show moreWe have previously shown that third-generation antisense (3GA) inhibition of 14q32 microRNA (miRNA)-494 reduced early development of atherosclerosis. However, patients at risk of atherosclerotic complications generally present with advanced and unstable lesions. Here, we administered 3GAs against 14q32 miRNA-494 (3GA-494), miRNA-329 (3GA-329), or a control (3GA-ctrl) to mice with advanced atherosclerosis. Atherosclerotic plaque formation in LDLr−/− mice was induced by a 10-week high-fat diet and simultaneous carotid artery collar placement. Parallel to 3GA-treatment, hyperlipidemia was normalized by a diet switch to regular chow for an additional 5 weeks. We show that, even though plasma cholesterol levels were normalized after diet switch, carotid artery plaque progression continued in 3GA-ctrl mice. However, treatment with 3GA-494 and, in part, 3GA-329 halted plaque progression. Furthermore, in the aortic root, intra-plaque collagen content was increased in 3GA-494 mice, accompanied by a reduction in the intra-plaque macrophage content. Pro-atherogenic cells in the circulation, including inflammatory Ly6Chi monocytes, neutrophils, and blood platelets, were decreased upon miRNA-329 and miRNA-494 inhibition. Taken together, treatment with 3GA-494, and in part with 3GA-329, halts atherosclerotic plaque progression and promotes stabilization of advanced lesions, which is highly relevant for human atherosclerosis. Show less
