6 research outputs found

    Role of MiR-148b in angiogenesis and endothelial cell plasticity

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    Endothelial cells (ECs) have a well-established role in the maintenance of vascular physiology. Moreover, ECs participate in many physiological processes such as angiogenesis, blood homeostasis, inflammatory response, lipid metabolism and many more. Angiogenesis is one of the most studied functions of endothelium, where ECs participate in the formation of the new blood vessels. Additionally, ECs can also exhibit a form of plasticity called endothelial-to-mesenchymal transition (EndMT), which is characterized by the loss of endothelial-specific morphology and markers and acquisition of mesenchymal-like phenotype. Both angiogenesis and EndMT can be regulated by physiological cues, such as inflammation and haemodynamic forces, as well as number of molecular stimuli, such as TGF-β. Interestingly, recent evidence suggests that angiogenesis and EndMT can be orchestrated by miRNAs. This thesis aims to address the hypothesis that miR-148b is a modulator of angiogenesis and endothelial cell plasticity in the form of EndMT. The preliminary high-throughput miRNA screen has identified miR-148b as a strong enhancer of HUVEC proliferation. Subsequent bioinformatic analysis and validation demonstrated that TGFB2 and SMAD2 are direct targets of miR-148b in ECs. Further experiments using gain- and loss-of-function approaches demonstrated that miR-148b regulates EC function. Specifically, overexpression of miR-148b enhanced EC migration, proliferation and in vitro angiogenesis, whereas its inhibition promoted EndMT, decreasing the expression of CD31 and VE-Cadherin and elevating collagen 1. Furthermore, inflammatory cytokine challenge decreased miR-148b levels in ECs, promoting EndMT, via upregulation of SMAD2, and enhancing reactive oxygen species production, all of which were abrogated by exogenous miR- 148b. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound angiogenesis and accelerated wound closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds impairing wound closure, which is reverted by SMAD2 silencing. Together, the data in this thesis supports the hypothesis that miR-148b regulates angiogenesis and endothelial cell plasticity, and provides the evidence that miR-148b upregulation enhances both in vitro and in vivo angiogenesis, while its knockdown promotes EndMT. This thesis demonstrates for the first time that miR-148b could be a key factor controlling EndMT and vascularization, thus opening a new avenue for therapeutic application of miR-148b in diseases that require vascular and tissue repair

    MicroRNA-148b Targets the TGF-β Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing

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    Transforming growth factor beta (TGF-β) is crucial for regulation of the endothelial cell (EC) homeostasis. Perturbation of TGF-β signaling leads to pathological conditions in the vasculature, causing cardiovascular disease and fibrotic disorders. The TGF-β pathway is critical in endothelial-to-mesenchymal transition (EndMT), but a gap remains in our understanding of the regulation of TGF-β and related signaling in the endothelium. This study applied a gain- and loss-of function approach and an in vivo model of skin wound healing to demonstrate that miR-148b regulates TGF-β signaling and has a key role in EndMT, targeting TGFB2 and SMAD2. Overexpression of miR-148b increased EC migration, proliferation, and angiogenesis, whereas its inhibition promoted EndMT. Cytokine challenge decreased miR-148b levels in ECs while promoting EndMT through the regulation of SMAD2. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound vascularization and accelerated closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds, resulting in impaired wound closure that was reversed by SMAD2 silencing. Together, these results demonstrate for the first time that miR-148b is a key factor controlling EndMT and vascularization. This opens new avenues for therapeutic application of miR-148b in vascular and tissue repair

    Targeting Non-coding RNA in Vascular Biology and Disease

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    Only recently have we begun to appreciate the importance and complexity of the non-coding genome, owing in some part to truly significant advances in genomic technology such as RNA sequencing and genome-wide profiling studies. Previously thought to be non-functional transcriptional “noise,” non-coding RNAs (ncRNAs) are now known to play important roles in many diverse biological pathways, not least in vascular disease. While microRNAs (miRNA) are known to regulate protein-coding gene expression principally through mRNA degradation, long non-coding RNAs (lncRNAs) can activate and repress genes by a variety of mechanisms at both transcriptional and translational levels. These versatile molecules, with complex secondary structures, may interact with chromatin, proteins, and other RNA to form complexes with an array of functional consequences. A body of emerging evidence indicates that both classes of ncRNAs regulate multiple physiological and pathological processes in vascular physiology and disease. While dozens of miRNAs are now implicated and described in relative mechanistic depth, relatively fewer lncRNAs are well described. However, notable examples include ANRIL, SMILR, and SENCR in vascular smooth muscle cells; MALAT1 and GATA-6S in endothelial cells; and mitochondrial lncRNA LIPCAR as a powerful biomarker. Due to such ubiquitous involvement in pathology and well-known biogenesis and functional genetics, novel miRNA-based therapies and delivery methods are now in development, including some early stage clinical trials. Although lncRNAs may hold similar potential, much more needs to be understood about their relatively complex molecular behaviours before realistic translation into novel therapies. Here, we review the current understanding of the mechanism and function of ncRNA, focusing on miRNAs and lncRNAs in vascular disease and atherosclerosis. We discuss existing therapies and current delivery methods, emphasising the importance of miRNAs and lncRNAs as effectors and biomarkers in vascular pathology

    The Human-Specific and Smooth Muscle Cell-Enriched LncRNA SMILR Promotes Proliferation by Regulating Mitotic CENPF mRNA and Drives Cell-Cycle Progression Which Can Be Targeted to Limit Vascular Remodeling.

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    RATIONALE: In response to blood vessel wall injury, aberrant proliferation of vascular smooth muscle cells (SMCs) causes pathological remodeling. However, the controlling mechanisms are not completely understood. OBJECTIVE: We recently showed that the human long noncoding RNA, SMILR, promotes vascular SMCs proliferation by a hitherto unknown mechanism. Here, we assess the therapeutic potential of SMILR inhibition and detail the molecular mechanism of action. METHODS AND RESULTS: We used deep RNA-sequencing of human saphenous vein SMCs stimulated with IL (interleukin)-1α and PDGF (platelet-derived growth factor)-BB with SMILR knockdown (siRNA) or overexpression (lentivirus), to identify SMILR-regulated genes. This revealed a SMILR-dependent network essential for cell cycle progression. In particular, we found using the fluorescent ubiquitination-based cell cycle indicator viral system that SMILR regulates the late mitotic phase of the cell cycle and cytokinesis with SMILR knockdown resulting in ≈10% increase in binucleated cells. SMILR pulldowns further revealed its potential molecular mechanism, which involves an interaction with the mRNA of the late mitotic protein CENPF (centromere protein F) and the regulatory Staufen1 RNA-binding protein. SMILR and this downstream axis were also found to be activated in the human ex vivo vein graft pathological model and in primary human coronary artery SMCs and atherosclerotic plaques obtained at carotid endarterectomy. Finally, to assess the therapeutic potential of SMILR, we used a novel siRNA approach in the ex vivo vein graft model (within the 30 minutes clinical time frame that would occur between harvest and implant) to assess the reduction of proliferation by EdU incorporation. SMILR knockdown led to a marked decrease in proliferation from ≈29% in controls to ≈5% with SMILR depletion. CONCLUSIONS: Collectively, we demonstrate that SMILR is a critical mediator of vascular SMC proliferation via direct regulation of mitotic progression. Our data further reveal a potential SMILR-targeting intervention to limit atherogenesis and adverse vascular remodeling

    Novel transcript discovery expands the repertoire of pathologically-associated, long non-coding RNAs in vascular smooth muscle cells

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    Vascular smooth muscle cells (VSMCs) provide vital contractile force within blood vessel walls, yet can also propagate cardiovascular pathologies through proliferative and pro-inflammatory activities. Such phenotypes are driven, in part, by the diverse effects of long non-coding RNAs (lncRNAs) on gene expression. However, lncRNA characterisation in VSMCs in pathological states is hampered by incomplete lncRNA representation in reference annotation. We aimed to improve lncRNA representation in such contexts by assembling non-reference transcripts in RNA sequencing datasets describing VSMCs stimulated in vitro with cytokines, growth factors, or mechanical stress, as well as those isolated from atherosclerotic plaques. All transcripts were then subjected to a rigorous lncRNA prediction pipeline. We substantially improved coverage of lncRNAs responding to pro-mitogenic stimuli, with non-reference lncRNAs contributing 21–32% for each dataset. We also demonstrate non-reference lncRNAs were biased towards enriched expression within VSMCs, and transcription from enhancer sites, suggesting particular relevance to VSMC processes, and the regulation of neighbouring protein-coding genes. Both VSMC-enriched and enhancer-transcribed lncRNAs were large components of lncRNAs responding to pathological stimuli, yet without novel transcript discovery 33–46% of these lncRNAs would remain hidden. Our comprehensive VSMC lncRNA repertoire allows proper prioritisation of candidates for characterisation and exemplifies a strategy to broaden our knowledge of lncRNA across a range of disease states.British Heart Foundation, European Research Council Advanced Grant VASCMIR and British/Israeli Collaborative grant BIRAX
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