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Electroporation technique induced by high-voltage pulses has been successfully used to ablate tumor cells while preserving the function of normal blood vessels. Generally, the tumor blood vessels can provide a pathway to draw nutrients for tumor growth and contribute to invasion and metastasis, which is an obstacle to tumor treatment. The electroporation study of the endothelial cell, which is important in the vasculature microenvironment, is helpful to investigate the influence on both tumor and normal blood vessels. This study built a multicell-layer model of the vascular microenvironment to investigate the discriminative electroporation effect between normal and tumor blood vessels by high-frequency bipolar pulses (HFBPs) and monopolar pulses (MPs). The simulation results showed that both pore number and electroporation region in normal blood vessels are significantly lower than those in tumor blood vessels. The rich vascular smooth muscle cells existed in the normal blood vessels play a protective function for endothelial cells, compared with tumor blood vessels. However, the differences in pore number and electroporation region between normal and tumor blood vessels are gradually smaller with an increased electric field, which demonstrates that the electroporation pulse with higher intensity damages both normal and tumor blood vessels. HFBPs generate a weaker electroporation effect on both normal and tumor blood vessels than traditional MP. However, HFBPs are more suitable to electroporate tumor blood vessels, while preserving the normal blood vessels. Moreover, this study could also provide a multicell-layer model that can be used to analyze the cell electroporation effect in the vascular microenvironment. [ABSTRACT FROM AUTHOR]

Objective(s): Atrophy of the muscles following denervation can lead to the death of myofibers. This study evaluated the sciatic nerve and tibialis cranialis muscle (TCM) regeneration using scaffold and cells. Materials and Methods: Ninety adult male Wistar rats were divided into six main groups (n=15) and three subgroups (2, 4, and 8 weeks). Groups: control; without surgery, Tr; sciatic nerve transected in silicone tube, S; collagen gel put inside the silicone tube, MC; placed 3x104 mast cells mixed with scaffold, MSC; placed 3x104 mesenchymal stem cells mixed with scaffold, and MC+MSC; 3x104 of each of the mast cell and mesenchymal stem cells along with scaffold. Animals were euthanized and sampled for muscle and nerve histological and nerve immunohistochemical evaluations. Results: Diameter of muscle fibers, ratio of the muscle fiber's nuclei to the fibrocyte nuclei (mn/fn), ratio of the muscle fibers nuclei number to the muscle fiber's number (mn/mf), and ratio of the blood vessels number to the number of muscle fibers (v/mf) in all treatment groups, especially the MC + MSC group, increased compared to the Tr group but the number of mast cells, the percentage of sarcoplasmolysis, and necrosis fibers decreased. Histomorphometric results of the nerve in its various parts and immunohistochemistry results also showed improved nerve conduction, especially in the MC + MSC group. Conclusion: In this study, nerve improvement happened mainly for two reasons: cells and time. So, the most obvious improvement occurred in the group with mast and mesenchymal cells in the 8th week. [ABSTRACT FROM AUTHOR]

Key points The peristaltic reflex has been a central concept in gastrointestinal motility; however, evidence was published recently suggesting that post‐stimulus responses that follow inhibitory neural responses provide the main propulsive force in colonic motility. This new concept was based on experiments on proximal colon where enteric inhibitory neural inputs are mainly nitrergic. However, the nature of inhibitory neural inputs changes from proximal to distal colon where purinergic inhibitory regulation dominates. In spite of the transition from nitrergic to purinergic regulation, post‐stimulus responses and propulsive contractions were both blocked by antagonists of a conductance (ANO1) exclusive to interstitial cells of Cajal (ICC). How purinergic neurotransmission, transduced by PDGFRα+ cells, can influence ANO1 in ICC is unknown. We compared neural responses in proximal and distal colon. Post‐stimulus responses were blocked by inhibition of nitrergic neurotransmission in proximal colon, but P2Y1 receptor antagonists were more effective in distal colon. Ca2+ entry through voltage‐dependent channels (CaV3) enhances Ca2+ release in ICC. Thus, we reasoned that hyperpolarization caused by purinergic responses in PDGFRα+ cells, which are electrically coupled to ICC, might decrease inactivation of CaV3 channels and activate Ca2+ entry into ICC via anode‐break upon cessation of inhibitory responses. Post‐stimulus responses in distal colon were blocked by MRS2500 (P2Y1 receptor antagonist), apamin (SK channel antagonist) and NNC55‐0396 (CaV3 antagonist). These compounds also blocked propagating contractions in mid and distal colon. These data provide the first clear demonstration that integration of functions in the smooth muscle–ICC–PDGFRα+ cell (SIP) syncytium generates a major motility behaviour. Propagating propulsive contractions initiated by the enteric nervous system are a major motility behaviour in the colon. A major component of contractions, necessary for propulsive contractions, occurs at cessation of enteric inhibitory neurotransmission (post‐stimulus response) and is generated by interstitial cells of Cajal (ICC), which are electrically coupled to smooth muscle cells. The nature of enteric inhibitory neurotransmission shifts from proximal colon, where it is predominantly due to nitric oxide, to distal colon, where it is predominantly due to purine neurotransmitters. Different cells transduce nitric oxide and purines in the colon. ICC transduce nitric oxide, but another type of interstitial cell, PDGFRα+ cells, transduces input from purinergic neurons. However, the post‐stimulus responses in proximal and distal colon are still generated in ICC. This paper explores how integrated behaviours of ICC, PDGFRα+ cells and smooth muscle cells accomplish propulsive motility in the colon. [ABSTRACT FROM AUTHOR]

Abdominal aortic aneurysm (AAA) is a life-threatening condition characterized by localized dilation of the abdominal aorta, posing a significant risk of rupture and fatal hemorrhage. While surgical and endovascular repair techniques have advanced, the underlying mechanisms driving AAA development remain unclear, hindering the development of effective preventive and therapeutic strategies. Using bioinformatics analysis of publicly available data sets, the study identified a strong correlation between cell death (CD) score and different types of programmed cell death scores in AAA samples. WGCNA analysis revealed a module enriched in genes related to proteasome-mediated protein degradation, nuclear envelope, and endocytosis, significantly correlated with CD score. Further analysis identified ABI1 as a dominant feature gene, highlighting its potential role in AAA pathogenesis. In vitro validation using an Angiotensin II-induced AAA model in human aortic smooth muscle cells demonstrated that siRNA-mediated knockdown of ABI1 significantly reduced cell apoptosis, migration, and the expression of pro-apoptotic proteins, confirming ABI1's crucial role in promoting CD and AAA progression. The findings suggest that ABI1 may represent a promising therapeutic target for the prevention and treatment of AAA. Further research is warranted to fully understand the role of ABI1 in AAA and to develop targeted therapies based on this promising target. [ABSTRACT FROM AUTHOR]

The severe conditions at high altitudes, where yaks inhabit, contribute to delayed muscular growth and compromised tenderness of their muscle tissue. Myosatellite cells are responsible for the growth and regeneration of skeletal muscle after birth and have the potential to proliferate and differentiate, its development is closely related to meat quality, and the nuclear receptor gene NR1D1 is involved in muscle formation and skeletal muscle regulation. Therefore, in order to understand the effect of NR1D1 on muscle satellite cells, we identified the mRNA expression levels of marker genes specifically expressed in muscle satellite cells at different stages to determine the type of cells isolated. Eventually, we successfully constructed a primary cell line of yak muscle satellite cells. Then we constructed NR1D1 overexpression vector and interference RNA, and introduced them into isolated yak skeletal muscle satellite cells. We performed qPCR, CCK8, and fluorescence-specific to detect the expression of genes or abundance of proteins as markers of cell proliferation and differentiation. Compared with those in the control group, the expression levels of proliferation marker genes KI-67 , CYCLIND1 , and CYCLINA were significantly inhibited after NR1D1 overexpression, which was also supported by the CCK-8 test, whereas differentiation marker genes MYOD , MYOG , and MYF5 were significantly inhibited. Fluorescence-specific staining showed that KI-67 protein abundance and the number of microfilaments both decreased, while the opposite trend was observed after NR1D1 interference. In conclusion, we confirmed that NR1D1 inhibited the proliferation and differentiation of yak skeletal muscle satellite cells, which provides a theoretical basis for further research on the effect of NR1D1 on improving meat quality traits and meat production performance of yaks. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutations in the DMD gene. Muscle fibers rely on the coordination of multiple cell types for repair and regenerative capacity. To elucidate the cellular and molecular changes in these cell types under pathologic conditions, we generated a rhesus monkey model for DMD that displays progressive muscle deterioration and impaired motor function, mirroring human conditions. By leveraging these DMD monkeys, we analyzed freshly isolated muscle tissues using single-cell RNA sequencing (scRNA-seq). Our analysis revealed changes in immune cell landscape, a reversion of lineage progressing directions in fibrotic fibro-adipogenic progenitors (FAPs), and TGF-β resistance in FAPs and muscle stem cells (MuSCs). Furthermore, MuSCs displayed cell-intrinsic defects, leading to differentiation deficiencies. Our study provides important insights into the pathogenesis of DMD, offering a valuable model and dataset for further exploration of the underlying mechanisms, and serves as a suitable platform for developing and evaluating therapeutic interventions. [Display omitted] • Generation of DMD monkey model recapitulating the pathogenesis of human patients • The immune cell landscape undergoes highly dynamic changes in DMD monkeys • Abnormal differentiation of FAPs causes TGF-β-independent fibrosis • Profound muscle stem cell defects contribute to the muscle dysfunction of DMD A DMD rhesus monkey model exhibits profound changes in immune cells, fibro-adipogenic progenitors (FAPs), and muscle stem cells at the early stage of the disease, constituting a suitable model for investigating disease mechanisms. [ABSTRACT FROM AUTHOR]

Skeletal muscle formation involves tight interactions between muscle cells and associated connective tissue fibroblasts. Every muscle displays the same type of organisation, they are innervated in the middle and attached at both extremities to tendons. Myonuclei are heterogeneous along myotubes and regionalised according to these middle and tip domains. During development, as soon as myotubes are formed, myonuclei at muscle tips facing developing tendons display their own molecular program. In addition to molecular heterogeneity, a subset of tip myonuclei has a fibroblastic origin different to the classical somitic origin, highlighting a cellular heterogeneity of myonuclei in foetal myotubes. To gain insights on the functional relevance of myonucleus heterogeneity during limb development, we used 2D culture and co-culture systems to dissociate autonomous processes (occurring in 2D-cultures) from 3D-environment of tissue development. We also assessed the role of muscle contraction in myonucleus heterogeneity in paralysed limb muscles. The regionalisation of cellular heterogeneity was not observed in 2D cell culture systems and paralyzed muscles. The molecular signature of MTJ myonuclei was lost in a dish and paralysed muscles indicating a requirement of 3D-enviroment and muscle contraction for MTJ formation. Tip genes that maintain a regionalized expression at myotube tips in cultures are linked to sarcomeres. The behaviour of regionalized markers in cultured myotubes and paralyzed muscles allows us to speculate whether the genes intervene in myogenesis, myotube attachment or MTJ formation. Highlights: • The molecular signature of MTJ myonuclei is lost in cultured myotubes and paralysed muscles • Genes expressed in muscle tips that maintain their regionalised expression in cultured myotubes are linked to sarcomeric proteins • Cellular heterogeneity of myonuclei is observed in cultured myotubes but with no regionalisation • BMP signalling regulates fibroblast nucleus incorporation into cultured myotubes [ABSTRACT FROM AUTHOR]

The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1. [ABSTRACT FROM AUTHOR]

The actin-binding protein filamin c (FLNc) is a key mediator in the response of skeletal muscle cells to mechanical stress. In addition to its function as a structural scaffold, FLNc acts as a signaling adaptor which is phosphorylated at S2234 in its mechanosensitive domain 20 (d20) through AKT. Here, we discovered a strong dephosphorylation of FLNc-pS2234 in cultured skeletal myotubes under acute mechanical stress, despite high AKT activity. We found that all three protein phosphatase 1 (PP1) isoforms are part of the FLNc d18-21 interactome. Enzymatic assays demonstrate that PP1 efficiently dephosphorylates FLNc-pS2234 and in vitro and in cells upon PP1 activation using specific modulators. FLNc-pS2234 dephosphorylation promotes the interaction with FILIP1, a mediator for filamin degradation. Altogether, we present a model in which dephosphorylation of FLNc d20 by the dominant action of PP1c prevails over AKT activity to promote the binding of the filamin degradation-inducing factor FILIP1 during acute mechanical stress. [ABSTRACT FROM AUTHOR]

Excess cholesterol accumulation induces the accumulation of foam cells, eventually accelerating atherosclerosis progress. Historically, the mechanisms of macrophage-derived foam cells have attracted attention because of their central role in plaque development, which was challenged by lineage tracing in union with single-cell sequencing (sc-seq). Accumulated studies have uncovered how vascular smooth muscle cells (VSMCs) proliferate and migrate to the vascular intima and accumulate, then transform into foam cells induced by surplus lipids, finally accounting for 30% to 70% of the total foam cells within the plaque of both mice and humans. Therefore, the mechanisms of VSMC-derived foam cells have received increasing attention. The review intends to summarize the transformation mechanism of VSMCs into foam cells induced by oxidized low-density lipoproteins (ox-LDL) in atherosclerosis. [ABSTRACT FROM AUTHOR]

Cardiac action potential (AP) alternans have been linked to the development of arrhythmia. AP alternans may be driven by AP instabilities, Ca2+ transient (CaT) instabilities, or both. The mechanisms underlying CaT driven AP alternans is well-supported experimentally, but the ionic mechanism underlying alternans driven by AP instabilities remain incompletely understood. Here we used the Ca2+ buffer BAPTA to remove the CaT and generate a model of AP alternans driven primarily by AP instabilities. In isolated rabbit ventricle myocytes, AP alternans induced by rapid pacing were either critically damped and persisted over time, overdamped and ceased over seconds, or underdamped progressing to 2:1 capture. Control cells predominantly exhibited critically damped alternans. In contrast, removing CaT with BAPTA destabilized alternans formation in a concentration dependent manner. Importantly, alternans were easier to induce in CaT free cells as evidenced by a higher alternans threshold relative to control cells. While the L-type Ca2+ channel agonist Bay K 8644 had a minor effect on alternans formation in myocytes with conserved CaT, combining the agonist with BAPTA markedly promoted the formation of underdamped alternans and increased the alternans threshold more than four-fold as compared to controls. Our data support a mechanistic model in which AP alternans are a primary self-sustained event in which the CaT serves as a dampening cue that curbs alternans development, likely via a canonical negative feedback process involving Ca2+ induced inhibition of L-type Ca2+ current. [ABSTRACT FROM AUTHOR]

Background: Manual therapy (MT) has been widely used in China to treat local tissue inflammation for a long time. However, there is a lack of scientific evidence for using MT in anti‐inflammatory therapy, and its anti‐inflammatory mechanism needs further clarification. Methods: We utilized MT to treat cardiotoxin (CTX) injury‐induced skeletal muscle inflammation in C57BL6/J mice. We analyzed the underlying mechanism by integrating single‐cell RNA sequencing (scRNA‐seq) with molecular techniques. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were used to assess skeletal muscle inflammation and muscle fiber cross‐sectional area (CSA). scRNA‐seq, immunofluorescence, and western blot were performed to determine cellular and molecular outcome changes. Results: Compared with CTX injury‐induced skeletal muscle inflammatory mice, MT intervention significantly reduced proinflammatory cytokines interleukin (IL)‐1β, IL‐6, and tumor necrosis factor alpha (TNF‐α) expression levels; scRNA‐seq detected that neutrophil numbers and activity were maximum proportions increased in injured skeletal muscle among macrophage, T cells, B cells, endothelial cells, fast muscle cells, fibroblasts, and skeletal muscle satellite cells; and S100A9 gene expression was supreme in neutrophils. However, after treatment with MT, S100A9 protein expression and the numbers and activity of Ly6g+/Mpo+ neutrophils were significantly inhibited, thus reducing the inflammatory cytokine levels and exerting an anti‐inflammatory effect by early clearing neutrophils. Conclusion: MT can mitigate localized inflammation induced by injured skeletal muscle, achieved by decreasing S100A9 protein expression and clearing neutrophils in mice, which may help advance therapeutic strategies for skeletal muscle localized inflammation. [ABSTRACT FROM AUTHOR]

In severe skeletal muscle damage, muscle tissue regeneration process has to face the loss of resident muscle stem cells (MuSCs) and the lack of connective tissue necessary to guide the regeneration process. Biocompatible and standardized 3D structures that can be injected to the muscle injury site, conforming to the defect shape while actively guiding the repair process, holds great promise for skeletal muscle tissue regeneration. In this study, we explore the use of an injectable and porous lysine dendrimer/polyethylene glycol (DGL/PEG) hydrogel as an acellular support for skeletal muscle regeneration. We adjusted the DGL/PEG composition to achieve a stiffness conducive to the attachment and proliferation of murine immortalized myoblasts and human primary muscle stems cells, sustaining the formation and maturation of muscle fibers in vitro. We then evaluated the potential of one selected "myogenic-porous hydrogel" as a supportive structure for muscle repair in a large tibialis anterior muscle defect in rats. This injectable and porous formulation filled the defect, promoting rapid cellularization with the presence of endothelial cells, macrophages, and myoblasts, thereby supporting neo-myogenesis more specifically at the interface between the wound edges and the hydrogel. The selected porous DGL/PEG hydrogel acted as a guiding scaffold at the periphery of the defect, facilitating the formation and anchorage of aligned muscle fibers 21 days after injury. Overall, our results indicate DGL/PEG porous injectable hydrogel potential to create a pro-regenerative environment for muscle cells after large skeletal muscle injuries, paving the way for acellular treatment in regenerative muscle medicine. [ABSTRACT FROM AUTHOR]

Muscle stem cells (MuSCs) enable muscle growth and regeneration after exercise or injury, but how metabolism controls their regenerative potential is poorly understood. We describe that primary metabolic changes can determine murine MuSC fate decisions. We found that glutamine anaplerosis into the tricarboxylic acid (TCA) cycle decreases during MuSC differentiation and coincides with decreased expression of the mitochondrial glutamate deaminase GLUD1. Deletion of Glud1 in proliferating MuSCs resulted in precocious differentiation and fusion, combined with loss of self-renewal in vitro and in vivo. Mechanistically, deleting Glud1 caused mitochondrial glutamate accumulation and inhibited the malate-aspartate shuttle (MAS). The resulting defect in transporting NADH-reducing equivalents into the mitochondria induced compartment-specific NAD+/NADH ratio shifts. MAS activity restoration or directly altering NAD+/NADH ratios normalized myogenesis. In conclusion, GLUD1 prevents deleterious mitochondrial glutamate accumulation and inactivation of the MAS in proliferating MuSCs. It thereby acts as a compartment-specific metabolic brake on MuSC differentiation. [Display omitted] • Glutamine is the main TCA cycle substrate in MuSCs, decreasing with differentiation • Glud1 loss impairs MuSC self-renewal and causes imbalanced differentiation and fusion • Glud1 loss traps mitochondrial glutamate and disrupts the malate-aspartate shuttle (MAS) • Restoring MAS in Glud1- deficient MuSCs blocks early differentiation and fusion imbalance Soro-Arnáiz and Fitzgerald et al. leverage ex vivo and in vivo models to identify how Glud1 determines muscle stem cell (MuSC) fate by controlling mitochondrial glutamate levels and, in turn, the activity of the malate aspartate shuttle. The resulting compartment-specific shift in NAD+/NADH ratios promotes precocious MuSC differentiation and fusion. [ABSTRACT FROM AUTHOR]

The endothelial cells of the blood circulation are exposed to hemodynamic forces, such as cyclic strain, hydrostatic forces, and shear stress caused by the blood fluid's frictional force. Endothelial cells perceive mechanical forces via mechanosensors and thus elicit physiological reactions such as alterations in vessel width. The mechanosensors considered comprise ion channels, structures linked to the plasma membrane, cytoskeletal spectrin scaffold, mechanoreceptors, and junctional proteins. This review focuses on endothelial mechanosensors and how they alter the vascular functions of endothelial cells. The current state of knowledge on the dysregulation of endothelial mechanosensitivity in disease is briefly presented. The interplay in mechanical perception between endothelial cells and vascular smooth muscle cells is briefly outlined. Finally, future research avenues are highlighted, which are necessary to overcome existing limitations. [ABSTRACT FROM AUTHOR]

Skeletal muscle constitutes the largest percentage of tissue in the animal body and plays a pivotal role in the development of normal life activities in the organism. However, the regulation mechanism of skeletal muscle growth and development remains largely unclear. This study investigated the effects of Ankrd1 on the proliferation and differentiation of C2C12 myoblasts. Here, we identified Ankrd1 as a potential regulator of muscle cell development, and found that Ankrd1 knockdown resulted in the proliferation ability decrease but the differentiation level increase of C2C12 cells. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyzes as well as RNA‐seq results showed that Ankrd1 knockdown activated focal adhesion kinase (FAK)/F‐actin signal pathway with most genes significantly enriched in this pathway upregulated. The integrin subunit Itga6 promoter activity is increased when Ankrd1 knockdown, as demonstrated by a dual‐luciferase reporter assay. This study revealed the molecular mechanism by which Ankrd1 knockdown enhanced FAK phosphorylation activity through the alteration of integrin subunit levels, thus activating FAK/Rho‐GTPase/F‐actin signal pathway, eventually promoting myoblast differentiation. Our data suggested that Ankrd1 might serve as a potential regulator of muscle cell development. Our findings provide new insights into skeletal muscle growth and development and valuable references for further study of human muscle‐related diseases. [ABSTRACT FROM AUTHOR]

Aging is associated with the steady decline of several cellular processes. The loss of skeletal muscle mass, termed sarcopenia, is one of the major hallmarks of aging. Aged skeletal muscle exhibits a robust reduction in its regenerative capacity due to dysfunction (i.e., senescence, lack of self‐renewal, and impaired differentiation) of resident muscle stem cells, called satellite cells. To replicate aging in vitro, immortalized skeletal muscle cells (myoblasts) can be treated with various agents to mimic age‐related dysfunction; however, these come with their own set of limitations. In the present study, we used sequential passaging of mouse myoblasts to mimic impaired differentiation that is observed in aged skeletal muscle. Further, we investigated mitochondrial apoptotic mechanisms to better understand the impaired differentiation in these "aged" cells. Our data shows that sequential passaging (>20 passages) of myoblasts is accompanied with significant reductions in differentiation and elevated cell death. Furthermore, high‐passage (HP) myoblasts exhibit greater mitochondrial‐mediated apoptotic signaling through mitochondrial BAX translocation, CYCS and AIFM1 release, and caspase‐9 activation. Finally, we show that inhibition of mitochondrial outer membrane permeability partly recovered differentiation in HP myoblasts. Together, our findings suggests that mitochondrial apoptotic signaling is a contributing factor to the diminished differentiation that is observed in aged myoblasts. [ABSTRACT FROM AUTHOR]

Lymphatic muscle cells (LMCs) are indispensable for proper functioning of the lymphatic system, as they provide the driving force for lymph transport. Recent studies have advanced our understanding of the molecular mechanisms that regulate LMCs, which control rhythmic contraction and vessel tone of lymphatic vessels—traits also found in cardiac and vascular smooth muscle. In this review, we discuss the molecular pathways that orchestrate LMC‐mediated contractility and summarize current knowledge about their developmental origin, which may shed light on the distinct contractile characteristics of LMCs. Further, we highlight the growing evidence implicating LMC dysregulation in the pathogenesis of lymphedema and other diseases related to lymphatic vessel dysfunction. Given the limited number and efficacy of existing therapies to treat lymphedema, LMCs present a promising focus for identifying novel therapeutic targets aimed at improving lymphatic vessel contractility. Here, we discuss LMCs in health and disease, as well as therapeutic strategies aimed at targeting them to improve lymphatic vessel function. [ABSTRACT FROM AUTHOR]

Phytoecdysteroids (PEs) are naturally occurring steroid compounds, that have recently gained significant attention, due to their diverse biological activities and high therapeutic potential. The aim of the present study was to quantify some PEs including 20-hydroxyecdysterone (20-HE), ponasterone A (PA), and turkesterone (TU) in selected plant foods and Rhaponticum carthamoides extract. Furthermore, the effects of 20-HE, TU, and R. carthamoides extract, were investigated with in vitro methods using isolated smooth muscle tissues. The levels of 20-HE in the analyzed samples exhibited significant differences, with kaniwa seed extract containing the highest amount, followed by spinach leaf extract, quinoa seed extract, and asparagus stem extract. The in vitro analyses suggested that R. carthamoides extract exhibits dose-dependent cytotoxic effects on smooth muscle cells, with low doses promoting contraction and higher doses inducing relaxation. Additionally, the extract demonstrated a significant inhibitory effect on ACh-induced contractions, while 20-HE enhanced the contractile response. The current findings highlighted phytoecdysteroids' potential for modifying gastrointestinal motility. [ABSTRACT FROM AUTHOR]

Sotatercept acts as a type IIA-Fc activin receptor, thereby scavenging free activin from its physiological membrane receptor. Through this type of action, sotaterpect leads to a rebalancing of the proliferation and antiproliferation pathways of pulmonary smooth muscle cells in response to bone morphogenic protein (BMP). Although sotatercept has been approved as the fourth pillar of therapy for group 1 pulmonary arterial hypertension (PAH) in the United States and Europe, several studies are ongoing to broaden the application of the drug to non-Group 1 PAH. We provide an overview of the clinical and preclinical evidence of targeting the activation pathway by sotatercept in the treatment of PAH. We also discuss other potential applications of sotatercept in the context of pulmonary hypertension other than PAH group 1. [ABSTRACT FROM AUTHOR]

Coronary artery calcification (CAC) is a hallmark of atherosclerosis and a critical factor in the development and progression of coronary artery disease (CAD). This review aims to address the complex pathophysiological mechanisms underlying CAC and its relationship with CAD. We examine the cellular and molecular processes that drive the formation of calcified plaques, highlighting the roles of inflammation, lipid accumulation, and smooth muscle cell proliferation. Additionally, we explore the genetic and environmental factors that contribute to the heterogeneity in CAC and CAD presentation among individuals. Understanding these intricate mechanisms is essential for developing targeted therapeutic strategies and improving diagnostic accuracy. By integrating current research findings, this review provides a comprehensive overview of the pathways linking CAC to CAD, offering insights into potential interventions to mitigate the burden of these interrelated conditions. [ABSTRACT FROM AUTHOR]

Atherosclerosis (AS) is an inflammatory disease characterized by arterial inflammation. One important trigger for AS development is the excessive migration of vascular smooth muscle cells (VSMCs); however, the mechanism underlying this phenomenon remains unclear. Therefore, we investigated the role of DNA polymerase β (Pol β), a crucial enzyme involved in base excision repair, VSMC migration, and subsequent AS development. In this study, we revealed a significant increase in Pol β content within AS plaques in ApoE−/−Pol β+/+ mice. In vitro experiments demonstrated a significant decrease in hCASMC viability and migration ability upon Pol β knockdown, whereas the subsequent recovery of Pol β expression reversed this effect. Moreover, our investigations revealed that Pol β knockdown leads to the inhibition of the POSTN gene transcription by suppressing the YY1/TGF-β1 pathway, resulting in the decreased expression of the protein periostin during VSMC migration. Collectively, our findings provide insights into the role of Pol β in AS development, offering a novel approach for the clinical treatment of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Cell death is a critical biological process necessary for development, tissue maintenance, and defense against diseases. To date, more than 20 forms of cell death have been identified, each defined by unique molecular pathways. Understanding these different forms of cell death is essential for investigating the pathogenesis of diseases such as cancer, neurodegenerative disorders, and autoimmune conditions and developing appropriate therapies. Paraptosis is a distinct form of regulated cell death characterized by cytoplasmic vacuolation and dilatation of cellular organelles like the mitochondria and endoplasmic reticulum (ER). It is regulated by several signaling pathways, for instance, those associated with ER stress, calcium overload, oxidative stress, and specific cascades such as insulin-like growth factor I receptor (IGF-IR) and its downstream signaling pathways comprising mitogen-activated protein kinases (MAPKs) and Jun N-terminal kinase (JNK). Paraptosis has been observed in diverse biological contexts, including development and cellular stress responses in neuronal, retinal, endothelial, and muscle cells. The induction of paraptosis is increasingly important in anticancer therapy, as it targets non-apoptotic stress responses in tumor cells, which can be utilized to induce cell death. This approach enhances treatment efficacy and addresses drug resistance, particularly in cases where cancer cells are resistant to apoptosis. Combining paraptosis-inducing agents with traditional therapies holds promise for enhancing treatment efficacy and overcoming drug resistance, suggesting a valuable strategy in anticancer therapy. [ABSTRACT FROM AUTHOR]

From quiescence to activation and myogenic differentiation, muscle stem cells (MuSCs) experience drastic alterations in their signaling activity and metabolism. Through balanced cycles of fission and fusion, mitochondria alter their morphology and metabolism, allowing them to affect their decisive role in modulating MuSC activity and fate decisions. This tightly regulated process contributes to MuSC regulation by mediating changes in redox signaling pathways, cell cycle progression, and cell fate decisions. In this review, we discuss the role of mitochondrial dynamics as an integral modulator of MuSC activity, fate, and maintenance. Understanding the influence of mitochondrial dynamics in MuSCs in health and disease will further the development of therapeutics that support MuSC integrity and thus may aid in restoring the regenerative capacity of skeletal muscle. [ABSTRACT FROM AUTHOR]

Simple Summary: In pancreatic ductal adenocarcinoma (PDAC), the interaction between tumor cells and the tumor microenvironment is critical in regulating cancer progression. In particular, cancer-associated fibroblasts (CAFs) affect the invasive activity and resistance to chemotherapy of tumor cells by means of contact-mediated and paracrine signals, the latter including transforming growth factor beta (TGF-β). By using advanced transwell and spheroid co-culture models as exploratory tools, we showed while that TGF-β is involved in the interplay between PDAC cells and fibroblasts and promotes the acquisition of aggressive phenotypes, additional as-yet-unknown factors play a role. We propose that these advanced cell culture models provide a pathologically relevant tractable system to investigate the role of the tumor microenvironment in PDAC progression and develop novel treatment strategies. Background: The interaction between cancer cells and cancer-associated fibroblasts (CAFs) is a key determinant of the rapid progression, high invasiveness, and chemoresistance of aggressive desmoplastic cancers such as pancreatic ductal adenocarcinoma (PDAC). Tumor cells are known to reprogram fibroblasts into CAFs by secreting transforming growth factor beta (TGF-β), amongst other cytokines. In turn, CAFs produce soluble factors that promote tumor-cell invasiveness and chemoresistance, including TGF-β itself, which has a major role in myofibroblastic CAFs. Such a high level of complexity has hampered progress toward a clear view of the TGFβ signaling loop between stromal fibroblasts and PDAC cells. Methods: Here, we tackled this issue by using co-culture settings that allow paracrine signaling alone (transwell systems) or paracrine and contact-mediated signaling (3D spheroids). Results: We found that TGF-β is critically involved in the activation of normal human fibroblasts into alpha-smooth muscle actin (α-SMA)-positive CAFs. The TGF-β released by CAFs accounted for the enhanced proliferation and resistance to gemcitabine of PDAC cells. This was accompanied by a partial epithelial-to-mesenchymal transition in PDAC cells, with no increase in their migratory abilities. Nevertheless, 3D heterospheroids comprising PDAC cells and fibroblasts allowed monitoring the pro-invasive effects of CAFs on cancer cells, possibly due to combined paracrine and physical contact-mediated signals. Conclusions: We conclude that TGF-β is only one of the players that mediates the communication between PDAC cells and fibroblasts and controls the acquisition of aggressive phenotypes. Hence, these advanced in vitro models may be exploited to further investigate these events and to design innovative anti-PDAC therapies. [ABSTRACT FROM AUTHOR]

Ganglioneuroblastoma is a rare peripheral neuroblastic tumor located anywhere in the sympathetic nervous system but rarely in the retropharyngeal space. Diagnosis can often be difficult based on imaging alone. We describe one case of a child presenting with snoring. The lesion was located in the rare retropharyngeal space, and its histology finally revealed ganglioneuroblastoma. Therefore, clinicians should be aware of pediatric patients with respiratory symptoms indicating cervical ganglioneuroblastoma. To make a definite diagnosis as soon as possible, a core needle biopsy or even immunohistochemistry may need to be performed before surgery. [ABSTRACT FROM AUTHOR]

Objective: In our study, we aim to explore the structural differences between the aortic root and the pulmonary artery to better understand the process of pulmonary autograft dilatation during the Ross procedure. Materials and methods: We studied twenty human fetuses (aged 14–36 weeks of gestation) and four adults (one female and three males, aged 30–45 years, mean age = 37 ± 16 years). Samples of aortic root and pulmonary artery were obtained through dissection. Histological examinations, including hematoxylin–eosin, Masson's trichrome, and orcein staining, as well as immunohistochemical technique with caldesmon staining, were performed. Microscopic counting was conducted to assess the number of elastic laminae and smooth muscle cells in each arterial wall. Statistical analyses were performed using R software. Means and standard deviations were used to present central tendencies and data dispersion for elastic laminae and smooth muscle. Results: Significant histological differences were observed between the aortic root and pulmonary artery in both adults and fetuses. In fetuses, no difference was found between the two vessels in terms of elastic laminae (p = 0.26) and smooth muscle cells (p = 0.69). However, in adults, significant differences were found for elastic laminae (p < 0.001) and smooth muscle cells (p < 0.001) between the aorta and pulmonary artery. Conclusions: The microscopic vascular structure impacts the mechanical properties of the pulmonary autograft wall and explains its observed dilatation remote from the Ross procedure due to wall stresses related to systemic pressure. [ABSTRACT FROM AUTHOR]

Objective: Protein kinase C (PKC) has been implicated in the increased contraction of human airway smooth muscle cells (HASMCs) in asthma. Using the three-dimensional collagen gel contraction system, the study aimed to determine the effects of LY333531, a specific inhibitor of the PKC-β isoform, on the contraction of tumor necrosis factor (TNF)-α-sensitized HASMCs. Methods: Cultured HASMCs were divided into five groups: the control group received no treatment, and the cells in the TNF-α group were sensitized with 10 ng/mL TNF-α for 48 h, while TNF-α was administered to sensitize HASMCs in the presence of 0.1, 0.2, and 0.5 μM LY333531 for 48 h in the 0.1LY, 0.2LY, and 0.5LY groups, respectively. Following this, HASMCs contraction was stimulated with 1 mM acetylcholine (ACh) for up to 24 h in each group and assessed using a three-dimensional collagen gel contraction assay. Furthermore, western blot and immunofluorescence analysis were performed. Results: The collagen gel contraction assay revealed that TNF-α increased the protein expression of phosphorylated PKC-β2, CPI-17, and MLC while exacerbating ACh-induced HASMCs contraction. LY333531 significantly attenuated HASMCs contraction and downregulated the protein expression of both p-CPI-17 and p-MLC. Conclusions: At least in part by regulating CPI-17 and MLC phosphorylation, LY333531 attenuates augmented contraction of TNF-α-sensitized HASMCs in a collagen gel contraction system. [ABSTRACT FROM AUTHOR]

Aim: Vascular smooth muscle cell (VSMC) phenotypic switching has been reported to regulate vascular function and thoracic aortic aneurysm and dissection (TAAD) progression. Early growth response 1 (Egr1) is associated with the differentiation of VSMCs. However, the mechanisms through which Egr1 participates in the regulation of VSMCs and progression of TAAD remain unknown. This study aimed to investigate the role of Egr1 in the phenotypic switching of VSMCs and the development of TAAD. Methods: Wild‐type C57BL/6 and SMC‐specific Egr1‐knockout mice were used as experimental subjects and fed β‐aminopropionitrile for 4 weeks to construct the TAAD model. Ultrasound and aortic staining were performed to examine the pathological features in thoracic aortic tissues. Transwell, wound healing, CCK8, and immunofluorescence assays detected the migration and proliferation of synthetic VSMCs. Egr1 was directly bound to the promoter of Krüppel‐like factor 5 (KLF5) and promoted the expression of KLF5, which was validated by JASPAR database and dual‐luciferase reporter assay. Results: Egr1 expression increased and was partially co‐located with VSMCs in aortic tissues of mice with TAAD. SMC‐specific Egr1 deficiency alleviated TAAD and inhibited the phenotypic switching of VSMC. Egr1 knockdown prevented the phenotypic switching of VSMCs and subsequently suppressed the migration and proliferation of synthetic VSMCs. The inhibitory effects of Egr1 deficiency on VSMCs were blunted once KLF5 was overexpressed. Conclusion: Egr1 aggravated the development of TAAD by promoting the phenotypic switching of VSMCs via enhancing the transcriptional activation of KLF5. These results suggest that inhibition of SMC‐specific Egr1 expression is a promising therapy for TAAD. [ABSTRACT FROM AUTHOR]

BACKGROUND: The formation of ice crystals will have adverse effects on aquatic products, and the key to ensure the long‐term preservation and better quality preservations of the product is to evaluate the intercellular ice crystal formation to find suitable refrigeration conditions and cryoprotectants. RESULTS: The ice crystal formation was successfully captured by using an inverted microscope cryomicroscopic system equipped with a low‐temperature stage, the ice crystals formed under different freezing methods between tuna muscle cells were observed directly, the deformation degree of muscle tissue pores during crystallization was evaluated, and the effect of freeze–thaw times on tuna samples was analyzed. The effects of the use of cryoprotectant such as cellobiose and carboxylated cellulose nanofibers on ice‐growth inhibition were investigated, and the reliability of the ice crystal observation results was further verified by the determination of physical properties. The results showed that carboxylated cellulose nanofibers had the best ice‐growth inhibition effect, they prevented about 50% cell deformation compared with the control group, and also reduced the minimum size of ice crystal formation. In addition, the addition of cellobiose and sodium tripolyphosphate gave the ice crystals a more uniform size and roundness. CONCLUSION: The experiment proposed a stable and clear observation method for the process of intercellular ice crystal formation, and the accuracy of the observation method was further verified by some physical indicators. This may help in the selection of suitable measurement methods to directly observe ice crystal formation behavior and screen cryoprotectants. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Single‐molecule localisation microscopy (SMLM) has the potential to reveal the underlying organisation of specific molecules within supramolecular complexes and their conformations, which is not possible with conventional microscope resolution. However, the detection efficiency for fluorescent molecules in cells can be limited in SMLM, even to below 1% in thick and dense samples. Segmentation of individual complexes can also be challenging. To overcome these problems, we have developed a software package termed PERPL: Pattern Extraction from Relative Positions of Localisations. This software assesses the relative likelihoods of models for underlying patterns behind incomplete SMLM data, based on the relative positions of pairs of localisations. We review its principles and demonstrate its use on the 3D lattice of Z‐disk proteins in mammalian cardiomyocytes. We find known and novel features at ~20 nm with localisations of less than 1% of the target proteins, using mEos fluorescent protein constructs. LAY DESCRIPTION: Single‐molecule localisation microscopy (SMLM) finds the positions of fluorescently labelled molecules in cells with a precision of between 1 and 10 nm. Interpreting this data helps us to uncover how these molecules are organised within cells and within specific structures important for their cellular roles. However, imperfect fluorescent labelling and low signal‐to‐noise ratios in experimental SMLM data limit its ability to detect all of the fluorescent molecules. Sometimes less than 1% of these molecules are located with high precision, which makes determining their molecular arrangement very challenging. To overcome this challenge, we have developed a new method called PERPL: Pattern Extraction from Relative Positions of Localisations (https://github.com/AlistairCurd/PERPL‐Python3, https://www.youtube.com/watch?v=uB1aU8QC%5fZ4) to analyse this type of data. In this method, we determine the relative positions of pairs of precisely localised molecules in 3D, and determine characteristic relative positions from collecting data over multiple instances of the molecular complex in a single cell, or over multiple cells. The combination of the most common relative positions of pairs of molecules gives us information about their 3D arrangement and the structure of the complex they are found in. We then build mathematical models for the distribution of relative positions in different candidate structures, which represent hypotheses about the underlying molecular organisation. Fitting these models to the experimental data allows us to select the model most likely to represent the best hypothesis. We demonstrate this method on an eightfold symmetric protein distribution in the nuclear pore complex and on a repeating pattern in muscle cell protein arrangements found from approx. 1% and 0.1% of these molecules in different SMLM techniques. We also explore the patterns of other muscle cell proteins that are as yet less well understood. PERPL can also analyse the arrangement of more than one molecule of interest in a complex and features within standard pixelated image data that can be described by coordinates. Finally, PERPL can also be used to test systematic variability due to SMLM data acquisition techniques, when a well‐characterised sample is used, or to test the variability of a sample. [ABSTRACT FROM AUTHOR]

In the fields of cultured meat, biopharmaceuticals, cell therapy, and tissue engineering, large numbers of mammalian cells are required; thus, highly-concentrated cell cultures are widely adopted. In general, such cultures can lead to cell damage caused by waste product accumulation and nutritional inadequacy. In this study, a novel co-culture system where the recombinant lactate-assimilating cyanobacterial strain, KC0110, derived from euryhaline Picosynechococcus sp. PCC 7002, and mammalian muscle cells cultured across porous membranes been developed. By using the KC0110 strain, the amount of ammonium and lactate excreted from C2C12 mouse muscle cells into the culture significantly decreased. Importantly, pyruvate and some amino acids, including pyruvate-derived amino acids, also increased significantly compared to those in monoculture of C2C12 cells. It is believed that the organic acids secreted by the KC0110 strain enhance the growth of mammalian cells, leading to a reduction in high-concentration culture-induced mammalian cell damage [lactate dehydrogenase (LDH) release] through cyanobacterial co-culture. These results show that, through co-cultivation with cyanobacteria, it is possible to culture mammalian cells, alleviating cell damage, even in highly-concentrated cultures. This study demonstrated an in vitro "symbiotic circular system" that can interchange metabolites produced by phototrophs and mammalian cells. [ABSTRACT FROM AUTHOR]

Thoracic aortic dissection (TAD) is a severe disease, characterized by numerous apoptotic vascular smooth muscle cells (VSMCs). EDIL3/Del-1 is a secreted protein involved in macrophage efferocytosis in acute inflammation. Here, we aimed to investigate whether EDIL3 promoted the internalization and degradation of apoptotic VSMCs during TAD. The levels of EDIL3 were decreased in the serum and aortic tissue from TAD mice. Global edil3 knockout (edil3−/−) mice and edil3−/− bone marrow chimeric mice exhibited a considerable exacerbation in β-aminopropionitrile monofumarate (BAPN)-induced TAD, accompanied with increased apoptotic VSMCs accumulating in the damaged aortic tissue. Two types of phagocytes, RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were used for in vitro efferocytosis assay. edil3-deficient phagocytes exhibited inefficient internalization and degradation of apoptotic VSMCs. Instead, EDIL3 promoted the internalization phase through interacting with phosphatidylserine (PtdSer) on apoptotic VSMCs and binding to the macrophage ITGAV/αv-ITGB3/β3 integrin. In addition, EDIL3 accelerated the degradation phase through activating LC3-associated phagocytosis (LAP). Mechanically, following the engulfment, EDIL3 enhanced the activity of SMPD1/acid sphingomyelinase in the phagosome through blocking ITGAV-ITGB3 integrin, which facilitates phagosomal reactive oxygen species (ROS) production by NAPDH oxidase CYBB/NOX2. Furthermore, exogenous EDIL3 supplementation alleviated BAPN-induced TAD and promoted apoptotic cell clearance. EDIL3 may be a novel factor for the prevention and treatment of TAD.Abbreviations: BAPN: β-aminopropionitrile monofumarate; BMDM: bone marrow-derived macrophage; C12FDG: 5-dodecanoylaminofluorescein-di-β-D-galactopyranoside; CTRL: control; CYBB/NOX2: cytochrome b-245, beta polypeptide; DCFH-DA: 2',7'-dichlorofluorescin diacetate; EDIL3/Del-1: EGF-like repeats and discoidin I-like domains 3; EdU: 5-ethynyl-2'-deoxyuridine; EVG: elastic van Gieson; H&E: hematoxylin and eosin; IL: interleukin; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAC: N-acetylcysteine; PtdSer: phosphatidylserine; rEDIL3: recombinant EDIL3; ROS: reactive oxygen species; SMPD1: sphingomyelin phosphodiesterase 1; TAD: thoracic aortic dissection; TEM: transmission electron microscopy; VSMC: vascular smooth muscle cell; WT: wild-type [ABSTRACT FROM AUTHOR]

Aims: This study aimed to determine the efficacy and safety of iltamiocel investigational autologous muscle cell therapy in females with stress urinary incontinence (SUI). Methods: Adult females were randomized 2:1 to iltamiocel (150 × 106 cells) or placebo and stratified by severity and prior SUI surgery. The primary objective was efficacy based on the frequency of stress incontinence episodes (SIE) recorded in a 3‐day diary at 12 months posttreatment. After 12 months, placebo participants could elect to receive open‐label iltamiocel. Efficacy and safety analyses were performed using all patients as treated populations. Results: The study enrolled 311 patients, 297 were randomized to either iltamiocel (n = 199) or placebo (n = 98). Of the 295 participants that completed 12 months blinded follow‐up, the proportion achieving the primary endpoint of ≥ 50% SIE reduction was not statistically different between treatment groups (52% vs. 53.6%; p = 0.798). A significantly greater proportion of iltamiocel participants in the prior SUI surgery stratum group achieved ≥ 75% SIE reduction compared with placebo, (40% vs. 16%; p = 0.037). Treatment response was maintained at 24 months in 78.4% and 64.9% of iltamiocel participants who achieved ≥ 50% and ≥ 75% SIE reduction, respectively, at Month 12. Adverse events related to the treatment were reported in 19 (9.5%) iltamiocel participants and 6 (6.1%) placebo participants. Conclusion: The study did not meet its primary endpoint however, iltamiocel cell therapy is safe and may be ideally suited to female patients who have undergone prior surgery for SUI. Additional study in this group of patients with high unmet medical needs is warranted. Trial Registration: ClinicalTrials.gov identifier: NCT01893138; EudraCT number: 2014‐002919‐41. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) play an important role in the progression of atherosclerosis (AS). This study aimed to explore the exact role and mechanism of circ_0002984 in oxidized low-density lipoprotein (ox-LDL)-mediated human vascular smooth muscle cells (HVSMCs). The model of smooth muscle cell phenotype switching was constructed by treating HVSMCs with ox-LDL. The levels of circ_0002984, let-7a-5p, and kruppel-like factor 5 (KLF5) were measured by quantitative real-time PCR or western blot assay. Cell proliferation, migration, and apoptosis were detected by Cell Counting Kit-8 (CCK-8), EdU staining, wound healing assay, transwell assay, and flow cytometry. The expression of cleaved-caspase-3 and KLF5 was examined by western blot. The relationship between let-7a-5p and circ_0002984 or KLF5 was verified by dual-luciferase reporter assay or RIP assay. The results showed that circ_0002984 and KLF5 were up-regulated, while let-7a-5p was down-regulated in AS patients and ox-LDL-disposed HVSMCs. Silence of circ_0002984 suppressed proliferation and migration, and promoted apoptosis in ox-LDL-stimulated HVSMCs. Moreover, circ_0002984 sponged let-7a-5p to regulate the proliferation, migration, and apoptosis in ox-LDL-resulted HVSMCs. In addition, KLF5 was a target of let-7a-5p and its overexpression reversed the effect of let-7a-5p on the proliferation, migration, and apoptosis in ox-LDL-treated HVSMCs. Also, circ_0002984 positively regulated KLF5 expression by absorbing let-7a-5p. The promotion effect of circ_0002984 on the proliferation and migration of ox-LDL-treated HVSMCs was reversed by KLF5 silencing. Taken together, depletion of circ_0002984 inhibited the proliferation and migration of ox-LDL-stimulated HVSMCs, which might be achieved by modulating the let-7a-5p/KLF5 axis. [ABSTRACT FROM AUTHOR]

The stability of wave conduction in the heart is strongly related to the proper interplay between the electrophysiological activation and mechanical contraction of myocytes and extracellular matrix (ECM) properties. In this study, we statistically compare bioengineered cardiac tissues cultured on soft hydrogels (E ≃ 12 kPa) and rigid glass substrates by focusing on the critical threshold of alternans, network-physiological tissue properties, and the formation of stable spiral waves that manifest after wave breakups. For the classification of wave dynamics, we use an improved signal oversampling technique and introduce simple probability maps to identify and visualize spatially concordant and discordant alternans as V- and X-shaped probability distributions. We found that cardiac tissues cultured on ECM-mimicking soft hydrogels show a lower variability of the calcium transient durations among cells in the tissue. This lowers the likelihood of forming stable spiral waves because of the larger dynamical range that tissues can be stably entrained with to form alternans and larger spatial spiral tip movement that increases the chance of selftermination on the tissue boundary. Conclusively, we show that a dysfunction in the excitation-contraction coupling dynamics facilitates life-threatening arrhythmic states such as spiral waves and, thus, highlights the importance of the network-physiological interplay between contractile myocytes and the ECM. [ABSTRACT FROM AUTHOR]

Background: Hemorrhoids are very common in patients with human immunodeficiency virus (HIV) infection. The risk of postoperative infection is significantly greater in HIV-positive patients than in HIV-negative individuals, and the wound healing time is significantly prolonged. This study aimed to investigate the role of HIV-associated hemorrhoids from the perspective of vascular smooth muscle cell (VSMC) function. Methods: A total of 24 hemorrhoid tissue samples (note: grade IV hemorrhoids were absence) were collected and subjected to Masson staining to evaluate fibrosis in this study. mRNA and protein levels were monitored by qPCR and WB analysis, respectively. Immunofluorescence was conducted to evaluate PKG1 and α-SMA expression. To establish a cell model in vitro, VSMCs were stimulated with envelope glycoprotein (gp) 120, which is a type of HIV envelope protein. Cell proliferation was assessed via a CCK-8 assay and EdU staining. Moreover, a wound healing assay was performed to assess cell migration. Results: Our data confirmed that fibrosis was present in hemorrhoid tissues from HIV-infected patients and that PKG1 expression was upregulated. Moreover, the administration of HIV gp120 promoted the proliferation and migration of VSMCs. Similarly, fibrosis-related markers (α-SMA, MMP2, MMP3, and TIMP1) were markedly upregulated. However, silencing PKG1 inhibited the proliferation, migration, and expression of fibrosis-related markers in gp120-challenged VSMCs. Conclusion: The present research revealed that PKG1 regulated the proliferation, migration, and fibrosis of VSMCs, thereby exerting detrimental effects on HIV-associated hemorrhoids. [ABSTRACT FROM AUTHOR]

Mesothelial cells, in the outermost layer of internal organs, are essential for both organ development and homeostasis. Although the parietal mesothelial cell is the primary origin of mesothelioma that may highjack developmental signaling, the signaling pathways that orchestrate developing parietal mesothelial progenitor cell (MPC) behaviors, such as MPC pool expansion, maturation, and differentiation, are poorly understood. To address it, we established a robust protocol for culturing WT1+ MPCs isolated from developing pig and mouse parietal thorax. Quantitative qPCR and immunostaining analyses revealed that BMP4 facilitated MPC differentiation into smooth muscle cells (SMCs). In contrast, FGF2 significantly promoted MPC progenitor pool expansion but blocked the SMC differentiation. BMP4 and FGF2 counterbalanced these effects, but FGF2 had the dominant impact in the long-term culture. A Wnt activator, CHIR99021, was pivotal in MPC maturation to CALB2+ mesothelial cells, while BMP4 or FGF2 was limited. Our results demonstrated central pathways critical for mesothelial cell behaviors. [ABSTRACT FROM AUTHOR]

The interactions between myogenic cells and adipocytes play an important role in improving carcass traits and the efficiency of energy utilization. However, there are few reports about the interaction between them mediated by small extracellular vesicles (sEV). In this study, sEV derived from porcine primary skeletal muscle stem cells (MuSCs) was found to be involved in the inhibition of porcine primary adipocyte viability, triglyceride content, Oil Red O enrichment and the expression of adipogenic genes. When the MuSCs were treated with insulin (INS) and oleic acid (OA), the effects of their secreted sEVs on adipose precursor cells were reversed, suggesting that the signaling effects of sEV are related to their own heterogeneity. Further by component heterogeneity analysis, miR-146a-5p was found to be enriched in sEVs of MuSCs and to regulate and suppress adipogenesis through its heterogeneity. This study provides an important mechanism and molecular target for small extracellular vesicles to regulate the interaction between muscle and adipose tissue and improve carcass traits at the intercellular level. [ABSTRACT FROM AUTHOR]

Immune system and inflammation had a great influence on the progression of muscle atrophy. However, the causal relationship with specific immune cell traits remained uncertain. The aim of this study was to elucidate the genetic influences on these associations, providing insights into the underlying mechanisms of muscle atrophy. A bidirectional two-sample Mendelian randomization (MR) analysis was conducted to investigate the causal relationship between immune cell phenotypes and muscle atrophy. Data on immune cell phenotypes were obtained from a research cohort containing data on 731 immune cell phenotypes and data on muscle atrophy were sourced from a Finnish database. MR analysis was performed using the MR-Egger method, weighted median, inverse variance weighting, heterogeneity testing, sensitivity analysis, and multiplicity analysis, with results subjected to false discovery rate(FDR) correction. Additionally, the UK Biobank cohort was utilized as an external validation. A total of 31 immune phenotypes with causal relationships with muscle atrophy were identified, including various phenotypes of conventional dendritic cells, myeloid cells, T cells/B cells/natural killer cells, regulatory cells, and T cell maturation stages. Among them, 12 immune phenotypes were identified as exhibiting a positive causal relationship with muscle atrophy, while 19 immune phenotypes were demonstrated to have a negative causal association, highlighting the complex interactions between immune cells and muscle health. The results of the reverse MR analysis indicated that a negative correlation between muscle atrophy and CD28 on secreting Treg (OR = 0.9038, 95%CI:0.8308 ~ 0.9832, P = 0.0186). A significant positive correlation was revealed by external datasets between the CD25 on IgD + CD38- immune phenotype and the risk of muscle atrophy, which was consistent with the trend observed in the training group (OR = 1.1041, 95% CI: 1.1005–1.1076, P = 0.0263). No evidence of pleiotropy was observed, and the reliability of these findings was demonstrated by the leave-one-out analysis. The findings highlight significant correlations between certain immune cell features and muscle atrophy, providing potential targets for further investigation of immunological mechanisms and therapeutic interventions for this condition. [ABSTRACT FROM AUTHOR]

Infection is a known cause of abdominal aortic aneurysm (AAA), and matrix metalloproteases‐2 (MMP‐2) secreted by vascular smooth muscle cells (SMCs) plays a key role in the structural disruption of the middle layer of the arteries during AAA progression. The periodontal pathogen Porphyromonas gingivalis is highly associated with the progression of periodontitis. GroEL protein of periodontal pathogens is an important virulence factor that can invade the body through either the bloodstream or digestive tract and is associated with numerous systemic diseases. Although P. gingivalis aggravates AAA by increasing the expression of MMP‐2 in animal studies, the molecular mechanism through which P. gingivalis regulates the expression of MMP‐2 is still unknown and requires further investigation.In this study, we first confirmed through animal experiments that P. gingivalis GroEL promotes MMP‐2 secretion from vascular SMCs, thereby aggravating Ang II‐induced aortic remodeling and AAA formation. In addition, rat vascular SMCs and A7r5 cells were used to investigate the underlying mechanisms in vitro. The results demonstrated that GroEL can promote the interaction between the K639 site of MMP‐2 and SUMO‐1, leading to MMP‐2 SUMOylation, which inhibits the reoccurrence of non–K639‐mediated monoubiquitylation. Hence, the monoubiquitylation‐mediated lysosomal degradation of MMP‐2 is inhibited, consequently promoting MMP‐2 stability and production. SUMOylation may facilitate intra‐endoplasmic reticulum (ER) and Golgi trafficking of MMP‐2, thereby enhancing its transport capacity. In conclusion, this is the first report demonstrating the presence of a novel posttranslational modification, SUMOylation, in the MMP family, suggesting that P. gingivalis GroEL may exacerbate AAA formation by increasing MMP‐2 production through SUMOylation in vascular SMCs. This study also provides a novel perspective on the role of SUMOylation in MMP‐2–induced systemic diseases. [ABSTRACT FROM AUTHOR]

Multinucleated skeletal muscle cells need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in cells that already harbor hundreds of nuclei. Here we utilize nuclear RNA-sequencing approaches and develop a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth. Skeletal muscle cells contain hundreds of nuclei, but can also add new nuclei in response to various stimuli. Here, the authors perform lineage tracing on the newly fused nuclei showing that these exhibit unique transcriptional states depending on the stimulus. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscles possess unique abilities known as adaptation or plasticity. When exposed to external stimuli, such as mechanical loading, both myofiber size and myonuclear number increase. Muscle stem cells, also known as muscle satellite cells (MuSCs), play vital roles in these changes. HeyL, a direct target of Notch signaling, is crucial for efficient muscle hypertrophy because it ensures MuSC proliferation in surgically overloaded muscles by inhibiting the premature differentiation. However, it remains unclear whether HeyL is essential for MuSC expansion in physiologically exercised muscles. Additionally, the influence of myofiber type on the requirement for HeyL in MuSCs within exercised muscles remains unclear. Methods: We used a voluntary wheel running model and HeyL-knockout mice to investigate the impact of HeyL deficiency on MuSC-derived myonuclei, MuSC behavior, muscle weight, myofiber size, and myofiber type in the running mice. Results: The number of new MuSC-derived myonuclei was significantly lower in both slow-twitch soleus and fast-twitch plantaris muscles from exercised HeyL-knockout mice than in control mice. However, expect for the frequency of Type IIb myofiber in plantaris muscle, exercised HeyL-knockout mice exhibited similar responses to control mice regarding myofiber size and type. Conclusions: HeyL expression is crucial for MuSC expansion during physiological exercise in both slow and fast muscles. The frequency of Type IIb myofiber in plantaris muscle of HeyL-knockout mice was not significantly reduced compared to that of control mice. However, the absence of HeyL did not affect the increased size and frequency of Type IIa myofiber in plantaris muscles. In this model, no detectable changes in myofiber size or type were observed in the soleus muscles of either control or HeyL-knockout mice. These findings imply that the requirement for MuSCs in the wheel-running model is difficult to observe due to the relatively low degree of hypertrophy compared to surgically overloaded models. [ABSTRACT FROM AUTHOR]

Background: Anti-amyloid-β (Aβ) immunotherapy trials have revealed amyloid-related imaging abnormalities (ARIA) as the most prevalent and serious adverse events linked to pathological changes in cerebral vasculature. Recent studies underscore the critical involvement of perivascular macrophages and the infiltration of peripheral immune cells in regulating cerebrovascular damage. Specifically, Aβ antibodies engaged at cerebral amyloid angiopathy (CAA) deposits trigger perivascular macrophage activation and the upregulation of genes associated with vascular permeability. Nevertheless, further research is needed to understand the immediate downstream consequences of macrophage activation, potentially exacerbating CAA-related vascular permeability and microhemorrhages linked to Aβ immunotherapy. Methods: This study investigates immune responses induced by amyloid-targeting antibodies and CAA-induced microhemorrhages using RNA in situ hybridization, histology and digital spatial profiling in an Alzheimer's disease (AD) mouse model of microhemorrhage. Results: In the present study, we have demonstrated that bapineuzumab murine surrogate (3D6) induces profound vascular damage, leading to smooth muscle cell loss and blood–brain barrier (BBB) breakdown. In addition, digital spatial profiling (DSP) reveals that distinct immune responses contribute to vascular damage with peripheral immune responses and perivascular macrophage activation linked to smooth muscle cell loss and vascular fibrosis, respectively. Finally, RNA in situ hybridization identifies two distinct subsets of Trem2+ macrophages representing tissue-resident and monocyte-derived macrophages around vascular amyloid deposits. Overall, these findings highlight multifaceted roles of immune activation and vascular damage in driving the development of microhemorrhage. Conclusions: In summary, our study has established a significant link between CAA-Aβ antibody immune complex formation, immune activation and vascular damage leading to smooth muscle cell loss. However, the full implications of this cascade on the development of microhemorrhages requires further exploration. Additional investigations are warranted to unravel the precise molecular mechanisms leading to microhemorrhage, the interplay of diverse immune populations and the functional roles played by various Trem2+ macrophage populations in response to Aβ immunotherapy. [ABSTRACT FROM AUTHOR]

Rotator cuff tear (RCT) is the primary cause of shoulder pain and disability and frequently trigger muscle degeneration characterised by muscle atrophy, fatty infiltration and fibrosis. Single‐nucleus RNA sequencing (snRNA‐seq) was used to reveal the transcriptional changes in the supraspinatus muscle after RCT. Supraspinatus muscles were obtained from patients with habitual shoulder dislocation (n = 3) and RCT (n = 3). In response to the RCT, trajectory analysis showed progression from normal myonuclei to ANKRD1+ myonuclei, which captured atrophy‐and fatty infiltration‐related regulons (KLF5, KLF10, FOSL1 and BHLHE40). Transcriptomic alterations in fibro/adipogenic progenitors (FAPs) and muscle satellite cells (MuSCs) have also been studied. By predicting cell–cell interactions, we observed communication alterations between myofibers and muscle‐resident cells following RCT. Our findings reveal the plasticity of muscle cells in response to RCT and offer valuable insights into the molecular mechanisms and potential therapeutic targets of RCT. [ABSTRACT FROM AUTHOR]

Neurons typically release both a neurotransmitter and one or more neuropeptides, but how these signals are integrated within neural circuits to generate and tune behaviors remains poorly understood. We studied how the two hermaphrodite-specific neurons (HSNs) activate the egg-laying circuit of Caenorhabditis elegans by releasing both the neurotransmitter serotonin and NLP-3 neuropeptides. Egg laying occurs in a temporal pattern with approximately 2-min active phases, during which eggs are laid, separated by approximately 20-min inactive phases, during which no eggs are laid. To understand how serotonin and NLP-3 neuropeptides together help produce this behavior pattern, we identified the G-protein-coupled receptor neuropeptide receptor 36 (NPR-36) as an NLP-3 neuropeptide receptor using genetic and molecular experiments. We found that NPR-36 is expressed in, and promotes egg laying within, the egg-laying muscle cells, the same cells where two serotonin receptors also promote egg laying. During the active phase, when HSN activity is high, we found that serotonin and NLP-3 neuropeptides each have a different effect on the timing of egg laying. During the inactive phase, HSN activity is low, which may result in release of only serotonin, yet mutants lacking either serotonin or nlp-3 signaling have longer inactive phases. This suggests that NLP-3 peptide signaling may persist through the inactive phase to help serotonin signaling terminate the inactive phase. We propose a model for neural circuit function in which multiple signals with short- and long-lasting effects compete to generate and terminate persistent internal states, thus patterning a behavior over tens of minutes. • C. elegans HSNs release NLP-3 peptides and serotonin to activate egg laying • These signals act via different receptors on the same muscles to pattern behavior • The two signals together initiate egg laying after ∼20-min inactive phases • Each signal then causes egg-laying events at different frequencies over ∼2 min Individual neurons typically release both a neurotransmitter and neuropeptides, but how these signals function together remains largely unclear. Butt et al. determine how serotonin and neuropeptides released by a pair of C. elegans neurons combine their effects on the same target cells to generate a complex temporal pattern of egg-laying behavior. [ABSTRACT FROM AUTHOR]

Severe asthma is characterized by increased cell volume (hypertrophy) and enhanced contractile function (hyperresponsiveness) of the airway smooth muscle cells (ASMCs). The causative relationship and underlying regulatory mechanisms between them, however, have remained unclear. Here, we manipulated the single-cell volume of in vitro cultured human ASMCs to increase from 2.7 to 5.2 and 8.2 × 103 μm3 as a simulated ASMC hypertrophy by culturing the cells on micropatterned rectangular substrates with a width of 25 μm and length from 50 to 100 and 200 μm, respectively. We found that as the cell volume increased, ASMCs exhibited a pro-contractile function with increased mRNA expression of contractile proteins, increased cell stiffness and traction force, and enhanced response to contractile stimulation. We also uncovered a concomitant increase in membrane tension and Piezo1 mRNA expression with increasing cell volume. Perhaps more importantly, we found that the enhanced contractile function due to cell volume increase was largely attenuated when membrane tension and Piezo1 mRNA expression were downregulated, and an auto-regulatory loop between Piezo1 and YAP mRNA expression was also involved in perpetuating the contractile function. These findings, thus, provide convincing evidence of a direct link between hypertrophy and enhanced contractile function of ASMCs that was mediated via Piezo1 mRNA expression, which may be specifically targeted as a novel therapeutic strategy to treat pulmonary diseases associated with ASMC hypertrophy such as severe asthma. [ABSTRACT FROM AUTHOR]

Endothelial dysfunction (ED) is closely associated with most cardiovascular diseases. Experimental models are needed to analyze the potential impact of ED on cardioprotection in constant pressure Langendorff systems (CPLS). One cardioprotective strategy against ischemia/reperfusion injury (I/RI) is conditioning with the lipid emulsion Intralipid (IL). Whether ED modulates the cardioprotective effect of IL remains unknown. The aim of the study was to transfer a protocol using a constant flow Langendorff system for the induction of ED into a CPLS, without the loss of smooth muscle cell functionality, and to analyze the cardioprotective effect of IL against I/RI under ED. In isolated hearts of male Wistar rats, ED was induced by 10 min perfusion of a Krebs–Henseleit buffer containing 60 mM KCl (K+), and the vasodilatory response to the vasodilators histamine (endothelial-dependent) and sodium–nitroprusside (SNP, endothelial-independent) was measured. A CPLS was employed to determine cardioprotection of pre- or postconditioning with 1% IL against I/RI. The constant flow perfusion of K+ reduced endothelial response to histamine but not to SNP, indicating reduced vasodilatory functionality of endothelial cells but not smooth muscle cells. Preconditioning with IL reduced infarct size and improved cardiac function while postconditioning with IL had no effect. The induction of ED neither influenced infarct size nor affected the cardioprotective effect by preconditioning with IL. This protocol allows for studies of cardioprotective strategies under ED in CLPS. The protection by preconditioning with IL seems to be mediated independently of a functional endothelium. [ABSTRACT FROM AUTHOR]

Diabetes (DM) patients have an increased risk (~50%) for sudden cardiac death (SCD), mostly as a result of ventricular arrhythmias. The molecular mechanisms involved remain partially defined. The potent proinflammatory lipid mediator leukotriene (LT) B4, is pathologically elevated in DM compared to nondiabetic patients, resulting in increased LTB4 accumulation in heart, leading to an increased risk for life‐threatening proarrhythmic signatures. We used electrophysiology, immunofluorescence, and confocal microscopy approaches to evaluate LTB4 cellular effects in guinea pig heart and ventricular myocytes. We have observed that LTB4 is increased in multiple mouse models (C57BL/6 J/Lepob/ob and PANIC‐ATTAC) of DM, promotes profound cellular arrhythmogenesis (spontaneous beats and early after depolarizations, EADs), and severely depresses the rapidly activating delayed rectifier K current (hERG1/IKr) density in HEK293 cells and guinea pig ventricular myocytes. We have further found that guinea pigs challenged with LTB4 displayed a significantly prolonged QT interval, and that this can be prevented with LTB4R inhibition, suggesting that preventing such LTB4R effects may be therapeutically beneficial in DM. Our data suggests that a further elucidation of LTB4 vulnerable substrates, and how this leads to ventricular arrhythmias, is likely to lead to continued improvements in management options, and the development of new therapies for prevention of SCD in DM patients. [ABSTRACT FROM AUTHOR]

Background: The efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model. Methods: We developed an IAS-targeted in vivo model via rapid freezing (at − 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking. Results: In vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc20, Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups. Conclusions: These findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone. [ABSTRACT FROM AUTHOR]