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Cellular and Molecular Life Sciences :... Dec 2022The pathological proliferation of cells in vascular smooth muscle underlies neointimal hyperplasia (NIH) development during atherosclerosis. Circular RNAs (circRNAs),...
The pathological proliferation of cells in vascular smooth muscle underlies neointimal hyperplasia (NIH) development during atherosclerosis. Circular RNAs (circRNAs), which represent novel functional biomarkers and RNA-binding proteins, contribute to multiple cardiovascular diseases; however, their roles in regulating the vascular smooth muscle cell cycle remain unknown. Thus, we aimed to identify the roles of circRNAs in vascular smooth muscle during coronary heart disease (CHD). Through circRNA sequencing of CHD samples and human antigen R (ELAVL1) immunoprecipitation, we identified circRNAs that are associated with CHD and interact with ELAVL1. Our results suggested that the hsa_circ_0000280 associated with CHD inhibits cell proliferation and induces ELAVL1-dependent cell cycle arrest. Gain/loss-of-function experiments and assays in vivo indicated that hsa_circ_0000280 facilitates interactions between ELAVL1 and cyclin-dependent kinase suppressor 1 (CDKN1A) mRNA and stabilization of this complex and leads to cell cycle arrest at the G1/S checkpoint, inhibiting cell proliferation of vascular smooth muscle cells in vitro and NIH in vivo. Importantly, hsa_circ_0000280 reduced neointimal thickness and smooth muscle cell proliferation in vivo. Taken together, these findings reveal a novel pathway in which hsa_circ_0000280 facilitates the regulation of ELAVL1 on CDKN1A mRNA to inhibit NIH. Therefore, measuring and modulating their expression might represent a potential diagnostic or therapeutic strategy for CHD.
Topics: Humans; Hyperplasia; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; ELAV-Like Protein 1
PubMed: 36477660
DOI: 10.1007/s00018-022-04602-w -
Cells Apr 2022Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover... (Review)
Review
Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover relevant disease mechanisms and enhance the translation of therapeutic findings to strengthen neuromuscular disease precision medicine. By concentrating on idiopathic inflammatory muscle disorders, we summarize the recent evolution of the novel in vitro models to study disease mechanisms and therapeutic strategies. A particular focus is laid on the integration and simulation of multicellular interactions of muscle tissue in disease phenotypes in vitro. Finally, the requirements of a neuromuscular disease drug development workflow are discussed with a particular emphasis on cell sources, co-culture systems (including organoids), functionality, and throughput.
Topics: Coculture Techniques; Drug Development; Humans; Muscle Cells; Neuromuscular Diseases; Organoids
PubMed: 35406795
DOI: 10.3390/cells11071233 -
Analytica Chimica Acta Aug 2020Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of...
Restenosis, re-narrowing of arterial lumen following intervention for cardiovascular disease, remains a major issue limiting the long-term therapeutic efficacy of treatment. The signaling molecules, TGFβ (transforming growth factor-beta) and Smad3, play important roles in vascular restenosis, but very little is yet known about the down-stream dynamics in global protein expression and phosphorylation. Here, we develop a highly multiplexed quantitative proteomic and phosphoproteomic strategy employing 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags and The DiLeu Tool software to globally assess protein expression and phosphorylation changes in smooth muscle cells (SMCs) treated with TGFβ/Smad3 and/or SDF-1α (stromal cell-derived factor). A total of 4086 proteins were quantified in the combined dataset of proteome and phosphoproteome across 12-plex DiLeu-labeled SMC samples. 2317 localized phosphorylation sites were quantified, corresponding to 1193 phosphoproteins. TGFβ/Smad3 induced up-regulation of 40 phosphosites and down-regulation of 50 phosphosites, and TGFβ/Smad3-specific SDF-1α exclusively facilitated up-regulation of 27 phosphosites and down-regulation of 47 phosphosites. TGFβ/Smad3 inhibited the expression of contractile-associated proteins including smooth muscle myosin heavy chain, calponin, cardiac muscle alpha-actin, and smooth muscle protein 22α. Gene ontology and pathway enrichment analysis revealed that elevated TGFβ/Smad3 activated cell proliferation and TGFβ signaling pathway, sequentially stimulating phosphorylation of CXCR4 (C-X-C chemokine receptor 4). SDF-1α/CXCR4 activated extracellular signal-regulating kinase signaling pathway and facilitated the expression of synthetic marker, osteopontin, which was validated through targeted analysis. These findings provide new insights into the mechanisms of TGFβ regulated SMC dedifferentiation, as well as new avenues for designing effective therapeutics for vascular disease.
Topics: Cell Dedifferentiation; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Proteomics; Transforming Growth Factor beta
PubMed: 32800120
DOI: 10.1016/j.aca.2020.06.054 -
Cardiovascular Research Feb 2022The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation, and pro-inflammatory signalling. Therefore, we tested the hypothesis that...
AIMS
The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation, and pro-inflammatory signalling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis.
METHODS AND RESULTS
SM22-Cre+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/flox/Ldlr-/-, and Ldlr-/-) were fed a western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn -/-/Ldlr-/- mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 weeks, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn-/-/Ldlr-/- mice. SMC-Dbn-/-/Ldlr-/- lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn-/- arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn-/- SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbnflox/flox SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. During early atherogenesis, SMC-Dbn-/-/Ldlr-/- aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn-/- SMCs were reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs.
CONCLUSION
We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells.
Topics: Animals; Atherosclerosis; Cell Transdifferentiation; Cells, Cultured; Cross-Sectional Studies; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidase 1; Neuropeptides
PubMed: 33914863
DOI: 10.1093/cvr/cvab156 -
Journal of the American Heart... Apr 2020Background Vascular smooth muscle cell phenotypic change and consequential intimal hyperplasia (IH) cause arterial stenosis and posttreatment restenosis. Smad3 is a...
Background Vascular smooth muscle cell phenotypic change and consequential intimal hyperplasia (IH) cause arterial stenosis and posttreatment restenosis. Smad3 is a master transcription factor, yet its underlying functional mechanisms in this disease context are not well defined. Methods and Results In cultured smooth muscle cells, Smad3 silencing and overexpression respectively reduced and increased the mRNA and protein of NRP2 (neuropilin 2), a recently reported pro-IH signaling factor. Smad3 silencing attenuated pro-IH smooth muscle cell phenotypes including proliferation, migration, and dedifferentiation (reduced smooth muscle α-actin). While increased Smad3 enhanced these phenotypes, NRP2 silencing abolished this enhancement. Interestingly, the 5' untranslated region but not the promoter of NRP2 was indispensable for Smad3-enhanced transcriptional activity (luciferase assay); both chromatin immunoprecipitation and electrophoretic mobility shift assay showed predominant Smad3 binding in the +51 to +78 bp region of NRP2's 5' untranslated region. In vivo, Smad3 haploinsufficiency reduced NRP2 (immunostaining) and IH (by 47%) in wire-injured mouse femoral arteries. Conclusions Smad3 controls NRP2 expression by occupying its 5' untranslated region in promoting smooth muscle cell phenotypic change in vitro. This and in vivo results shed new light on the long-debated role of Smad3 in IH.
Topics: 5' Untranslated Regions; Animals; Binding Sites; Cell Movement; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Humans; Male; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neointima; Neuropilin-2; Protein Binding; Smad3 Protein; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta1; Vascular System Injuries
PubMed: 32306814
DOI: 10.1161/JAHA.119.015487 -
Cells Sep 2023Olive flounder () muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached...
Olive flounder () muscle satellite cells (OFMCs) were obtained by enzymatic primary cell isolation and the explant method. Enzymatic isolation yielded cells that reached 80% confluence within 8 days, compared to 15 days for the explant method. Optimal OFMC growth was observed in 20% fetal bovine serum at 28 °C with 0.8 mM CaCl and the basic fibroblast growth factor (BFGF) to enhance cell growth. OFMCs have become permanent cell lines through the spontaneous immortalization crisis at the 20th passage. Olive flounder skeletal muscle myoblasts were induced into a mitogen-poor medium containing 2% horse serum for differentiation; they fused to form multinucleate myotubes. The results indicated complete differentiation of myoblasts into myotubes; we also detected the expression of the myogenic regulatory factors myoD, myogenin, and desmin. Upregulation (Myogenin, desmin) and downregulation (MyoD) of muscle regulation factors confirmed the differentiation in OFMCs.
Topics: Animals; Satellite Cells, Skeletal Muscle; Flounder; Myogenin; Desmin; Muscle Fibers, Skeletal; Muscle, Skeletal
PubMed: 37759547
DOI: 10.3390/cells12182325 -
International Journal of Molecular... Aug 2020Cellular stress has been considered a relevant pathogenetic factor in a variety of human diseases. Due to its primary functions by means of contractility, metabolism,... (Review)
Review
Cellular stress has been considered a relevant pathogenetic factor in a variety of human diseases. Due to its primary functions by means of contractility, metabolism, and protein synthesis, the muscle cell is faced with continuous changes of cellular homeostasis that require rapid and coordinated adaptive mechanisms. Hence, a prone susceptibility to cellular stress in muscle is immanent. However, studies focusing on the cellular stress response in muscular disorders are limited. While in recent years there have been emerging indications regarding a relevant role of cellular stress in the pathophysiology of several muscular disorders, the underlying mechanisms are to a great extent incompletely understood. This review aimed to summarize the available evidence regarding a deregulation of the cellular stress response in individual muscle diseases. Potential mechanisms, as well as involved pathways are critically discussed, and respective disease models are addressed. Furthermore, relevant therapeutic approaches that aim to abrogate defects of cellular stress response in muscular disorders are outlined.
Topics: Animals; Endoplasmic Reticulum Stress; Humans; Muscle Cells; Muscular Diseases; Oxidative Stress; Stress, Physiological; Unfolded Protein Response
PubMed: 32823799
DOI: 10.3390/ijms21165830 -
Proceedings of the National Academy of... Jan 2024Fatty expansion is one of the features of muscle degeneration due to muscle injuries, and its presence interferes with muscle regeneration. Specifically, poor clinical...
Fatty expansion is one of the features of muscle degeneration due to muscle injuries, and its presence interferes with muscle regeneration. Specifically, poor clinical outcomes have been linked to fatty expansion in rotator cuff tears and repairs. Our group recently found that fibroblast growth factor 8b (FGF-8b) inhibits adipogenic differentiation and promotes myofiber formation of mesenchymal stem cells in vitro. This led us to hypothesize that FGF-8b could similarly control the fate of muscle-specific cell populations derived from rotator cuff muscle involved in muscle repair following rotator cuff injury. In this study, we isolate fibro-adipogenic progenitor cells (FAPs) and satellite stem cells (SCs) from rat rotator cuff muscle tissue and analyzed the effects of FGF-8b supplementation. Utilizing a cell plating protocol, we successfully isolate FAPs-rich fibroblasts (FIBs) and SCs-rich muscle progenitor cells (MPCs). Subsequently, we demonstrate that FIB adipogenic differentiation can be inhibited by FGF-8b, while MPC myogenic differentiation can be enhanced by FGF-8b. We further demonstrate that phosphorylated ERK due to FGF-8b leads to the inhibition of adipogenesis in FIBs and SCs maintenance and myofiber formation in MPCs. Together, these findings demonstrate the powerful potential of FGF-8b for rotator cuff repair by altering the fate of muscle undergoing degeneration.
Topics: Rats; Animals; Rotator Cuff; Adipogenesis; Fibroblast Growth Factor 8; Rotator Cuff Injuries; Muscle Cells; Muscle Development
PubMed: 38147545
DOI: 10.1073/pnas.2314585121 -
The Journal of Physiology Jul 2015Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various... (Review)
Review
Vascular smooth muscle cells (SMCs), a major structural component of the vessel wall, not only play a key role in maintaining vascular structure but also perform various functions. During embryogenesis, SMC recruitment from their progenitors is an important step in the formation of the embryonic vascular system. SMCs in the arterial wall are mostly quiescent but can display a contractile phenotype in adults. Under pathophysiological conditions, i.e. vascular remodelling after endothelial dysfunction or damage, contractile SMCs found in the media switch to a secretory type, which will facilitate their ability to migrate to the intima and proliferate to contribute to neointimal lesions. However, recent evidence suggests that the mobilization and recruitment of abundant stem/progenitor cells present in the vessel wall are largely responsible for SMC accumulation in the intima during vascular remodelling such as neointimal hyperplasia and arteriosclerosis. Therefore, understanding the regulatory mechanisms that control SMC differentiation from vascular progenitors is essential for exploring therapeutic targets for potential clinical applications. In this article, we review the origin and differentiation of SMCs from stem/progenitor cells during cardiovascular development and in the adult, highlighting the environmental cues and signalling pathways that control phenotypic modulation within the vasculature.
Topics: Animals; Cell Differentiation; Humans; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Stem Cells
PubMed: 25952975
DOI: 10.1113/JP270033 -
Scientific Reports Jul 2021Caenorhabditis elegans (C. elegans) can produce various motion patterns despite having only 69 motor neurons and 95 muscle cells. Previous studies successfully elucidate...
Caenorhabditis elegans (C. elegans) can produce various motion patterns despite having only 69 motor neurons and 95 muscle cells. Previous studies successfully elucidate the connectome and role of the respective motor neuron classes related to movement. However, these models have not analyzed the distribution of the synaptic and gap connection weights. In this study, we examined whether a motor neuron and muscle network can generate oscillations for both forward and backward movement and analyzed the distribution of the trained synaptic and gap connection weights through a machine learning approach. This paper presents a connectome-based neural network model consisting of motor neurons of classes A, B, D, AS, and muscle, considering both synaptic and gap connections. A supervised learning method called backpropagation through time was adapted to train the connection parameters by feeding teacher data composed of the command neuron input and muscle cell activation. Simulation results confirmed that the motor neuron circuit could generate oscillations with different phase patterns corresponding to forward and backward movement, and could be switched at arbitrary times according to the binary inputs simulating the output of command neurons. Subsequently, we confirmed that the trained synaptic and gap connection weights followed a Boltzmann-type distribution. It should be noted that the proposed model can be trained to reproduce the activity patterns measured for an animal (HRB4 strain). Therefore, the supervised learning approach adopted in this study may allow further analysis of complex activity patterns associated with movements.
Topics: Animals; Caenorhabditis elegans; Computer Simulation; Connectome; Locomotion; Models, Neurological; Motor Neurons; Muscle Cells; Nerve Net
PubMed: 34215774
DOI: 10.1038/s41598-021-92690-2