Muscle Cells Research Articles

Single‐nucleus RNA sequencing reveals midgut cellular heterogeneity and transcriptional profiles in Bombyx mori cytoplasmic polyhedrosis virus infection

AbstractThe gut is not only used by insects as an organ for the digestion of food and absorption of nutrients but also as an important barrier against the invasion and proliferation of pathogenic microorganisms. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), an insect‐specific virus, predominantly colonizes the midgut epithelial cells of the silkworm, thereby jeopardizing its normal growth. However, there is limited knowledge of the cellular immune responses to viral infection and whether the infection is promoted or inhibited by different types of cells in the silkworm midgut. In this study, we used single‐nucleus RNA sequencing to identify representative enteroendocrine cells, enterocytes, and muscle cell types in the silkworm midgut. In addition, by analyzing the transcriptional profiles of various subpopulations in the infected and uninfected groups, we found that BmCPV infection suppresses the response of the antiviral pathways and induces the expression of BmHSP70, which plays a role in promoting BmCPV replication. However, certain immune genes in the midgut of the silkworm, such as BmLebocin3, were induced upon viral infection, and downregulation of BmLEB3 using RNA interference promoted BmCPV replication in the midgut of B. mori. These results suggest that viral immune evasion and active host resistance coexist in BmCPV‐infected silkworms. We reveal the richness of cellular diversity in the midgut of B. mori larvae by single‐nucleus RNA sequencing analysis and provide new insights into the complex interactions between the host and the virus at the single‐cell level.

Vitamin D receptor and its antiproliferative effect in human pulmonary arterial hypertension

Vitamin D (vitD) deficiency is frequently observed in patients with pulmonary arterial hypertension (PAH) and, in these patients, low levels of vitD correlate with worse prognosis. The aim of this study was to examine the expression and the antiproliferative role of vitD receptor (VDR) and its signalling pathway in the human pulmonary vasculature. VDR presence and expression was analyzed in lungs, pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) from controls and PAH-patients. VDR expression and VDR-target genes were examined in PASMC treated with calcitriol. The antiproliferative effect of 48 h-calcitriol was studied in PASMC by MTT and BrdU assays. VDR is expressed in PASMC. It is downregulated in lungs and in PASMC, but not in PAEC, from PAH-patients compared to non-hypertensive controls. Calcitriol strongly upregulated VDR expression in PASMC and the VDR target genes KCNK3 (encoding TASK1), BIRC5 (encoding survivin) and BMP4. Calcitriol produced an antiproliferative effect which was diminished by silencing or by pharmacological inhibition of survivin or BMPR2, but not of TASK1. In conclusion, the expression of VDR is low in PAH-patients and can be rescued by calcitriol. VDR exerts an antiproliferative effect in PASMC by modulating survivin and the BMP signalling pathway.

AEBP1 is a negative regulator of skeletal muscle cell differentiation in oral squamous cell carcinoma

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.

Protein phosphatase-1 regulates the binding of filamin C to FILIP1 in cultured skeletal muscle cells under mechanical stress

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.

Dapagliflozin targets SGLT2/SIRT1 signaling to attenuate the osteogenic transdifferentiation of vascular smooth muscle cells

Vascular calcification is a complication that is frequently encountered in patients affected by atherosclerosis, diabetes, and chronic kidney disease (CKD), and that is characterized by the osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). At present, there remains a pressing lack of any effective therapies that can treat this condition. The sodium-glucose transporter 2 (SGLT2) inhibitor dapagliflozin (DAPA) has shown beneficial effects in cardiovascular disease. The role of this inhibitor in the context of vascular calcification, however, remains largely uncharacterized. Our findings revealed that DAPA treatment was sufficient to alleviate in vitro and in vivo osteogenic transdifferentiation and vascular calcification. Interestingly, our study demonstrated that DAPA exerts its anti-calcification effects on VSMCs by directly targeting SGLT2, with the overexpression of SGLT2 being sufficient to attenuate these beneficial effects. DAPA was also able to limit the glucose levels and NAD+/NADH ratio in calcified VSMCs, upregulating sirtuin 1 (SIRT1) in a caloric restriction (CR)-dependent manner. The SIRT1-specific siRNA and the SIRT1 inhibitor EX527 attenuated the anti-calcification effects of DAPA treatment. DAPA was also to drive SIRT1-mediated deacetylation and consequent degradation of hypoxia-inducible factor-1α (HIF-1α). The use of cobalt chloride and proteasome inhibitor MG132 to preserve HIF-1α stability mitigated the anti-calcification activity of DAPA. These analyses revealed that the DAPA/SGLT2/SIRT1 axis may therefore represent a viable novel approach to treating vascular calcification, offering new insights into how SGLT2 inhibitors may help prevent and treat vascular calcification.Graphical abstractDapagliflozin attenuated vascular smooth muscle cells osteogenic transdifferentiation and vascular calcification through the SIRT1-mediated deacetylation and degradation of HIF-1α in a manner dependent on caloric restriction.

Regulatory Role of Nfix Gene in Sheep Skeletal Muscle Cell Development and Its Interaction Mechanism with MSTN

Skeletal muscle development is crucial for livestock production, and understanding the molecular mechanisms involved is essential for enhancing muscle growth in sheep. This study aimed to investigate the role of Nfix, a member of the nuclear factor I (NFI) family, in regulating muscle development in sheep, filling a significant gap in the current understanding of Nfix deficiency and its impact on skeletal muscle growth, as no similar studies have been reported in this species. Bioinformatic analysis, including temporal analysis of transcriptome data, identified Nfix as a potential target gene for muscle growth regulation. The effects of Nfix overexpression and knockout on the proliferation and differentiation of sheep skeletal muscle cells were investigated. Changes in the expression of associated marker genes were assessed to explore the regulatory link between Nfix and the myostatin (MSTN) gene. Additionally, target miRNAs for Nfix and MSTN were predicted using online databases such as miRWalk, resulting in the construction of an Nfix–miRNA–MSTN interactive regulatory network. The findings revealed that Nfix promotes the proliferation and differentiation of sheep skeletal muscle cells, with further analysis indicating that Nfix may regulate muscle cell development by modulating MSTN expression. This study provides preliminary insights into the function of Nfix in sheep skeletal muscle development and its regulatory interactions, addressing a critical knowledge gap regarding Nfix deficiency and its implications for muscle growth. These findings contribute to a better understanding of muscle biology in sheep and provide a theoretical foundation for future research into the regulatory mechanisms governing muscle development.

ANK Deficiency-Mediated Cytosolic Citrate Accumulation Promotes Aortic Aneurysm.

Disturbed metabolism and transport of citrate play significant roles in various pathologies. However, vascular citrate regulation and its potential role in aortic aneurysm (AA) development remain poorly understood. Untargeted metabolomics by mass spectrometry was applied to identify upregulated metabolites of the tricarboxylic acid cycle in AA tissues of mice. To investigate the role of citrate and its transporter ANK (progressive ankylosis protein) in AA development, vascular smooth muscle cell (VSMC)-specific Ank-knockout mice were used in both Ang II (angiotensin II)- and CaPO4-induced AA models. Citrate was abnormally increased in both human and murine aneurysmal tissues, which was associated with downregulation of ANK, a citrate membrane transporter, in VSMCs. The knockout of Ank in VSMCs promoted AA formation in both Ang II- and CaPO4-induced AA models, while its overexpression inhibited the development of aneurysms. Mechanistically, ANK deficiency in VSMCs caused abnormal cytosolic accumulation of citrate, which was cleaved into acetyl coenzyme A and thus intensified histone acetylation at H3K23, H3K27, and H4K5. Cleavage under target and tagmentation analysis further identified that ANK deficiency-induced histone acetylation activated the transcription of inflammatory genes in VSMCs and thus promoted a citrate-related proinflammatory VSMC phenotype during aneurysm diseases. Accordingly, suppressing citrate cleavage to acetyl coenzyme A downregulated inflammatory gene expression in VSMCs and restricted ANK deficiency-aggravated AA formation. Our studies define the pathogenic role of ANK deficiency-induced cytosolic citrate accumulation in AA pathogenesis and an undescribed citrate-related proinflammatory VSMC phenotype. Targeting ANK-mediated citrate transport may emerge as a novel diagnostic and therapeutic strategy in AA.

Light-sensitive Ca2+ signaling in the mammalian choroid

The choroid is the thin, vasculature-filled layer of the eye situated between the sclera and the retina, where it serves the metabolic needs of the light-sensing photoreceptors in the retina. Illumination of the interior surface of the back of the eye (fundus) is a critical regulator of subretinal fluid homeostasis, which determines the overall shape of the eye, but it is also important for choroidal perfusion. Noted for having some of the highest blood flow rates in the body, the choroidal vasculature has been reported to lack intrinsic, intravascular pressure-induced (myogenic) autoregulatory mechanisms. Here, we ask how light directly regulates choroid perfusion and ocular fluid homeostasis, testing the hypothesis that light facilitates ocular fluid absorption by directly increasing choroid endothelial permeability and decreasing choroid perfusion. Utilizing ex vivo pressurized whole-choroid and whole-eye preparations from mice expressing cell-specific Ca2+ indicators, we found that the choroidal vasculature has two intrinsically light-sensitive Ca2+-signaling mechanisms: One increases Ca2+-dependent production of nitric oxide in choroidal endothelial cells; the other promotes vasoconstriction through Ca2+ elevation in vascular smooth muscle cells. In addition, we found that choroidal flow, or pressure, modulates endothelial and smooth muscle photosensitivity and trans-retinal absorption of fluid into the choroid. These results collectively suggest that the choroid vasculature exhibits an inverted form of autoregulatory control, where pressure- and light-induced mechanisms work in opposition to regulate blood flow and maintain fluid balance in response to changes in light and dark, aligning with the metabolic needs of photoreceptors.

Hedgehog Signalling Pathway and Its Role in Shaping the Architecture of Intestinal Epithelium

The hedgehog (Hh) signalling pathway plays a key role in both embryonic and postnatal development of the intestine and is responsible for gut homeostasis. It regulates stem cell renewal, formation of the villous–crypt axis, differentiation of goblet and Paneth cells, the cell cycle, apoptosis, development of gut innervation, and lipid metabolism. Ligands of the Hh pathway, i.e., Indian hedgehog (Ihh) and Sonic hedgehog (Shh), are expressed by superficial enterocytes but act in the mesenchyme, where they are bound by a Patched receptor localised on myofibroblasts and smooth muscle cells. This activates a cascade leading to the transcription of target genes, including those encoding G1/S-specific cyclin-D2 and -E1, B-cell lymphoma 2, fibroblast growth factor 4, and bone morphogenetic protein 4. The Hh pathway is tightly connected to Wnt signalling. Ihh is the major ligand in the Hh pathway. Its activation inhibits proliferation, while its blocking induces hyperproliferation and triggers a wound-healing response. Thus, Ihh is a negative feedback regulator of cell proliferation. There are data indicating that diet composition may affect the expression of the Hh pathway genes and proteins, which in turn, induces changes in mucosal architecture. This was shown for fat, vitamin A, haem, berberine, and ovotransferrin. The Hh signalling is also affected by the intestinal microbiota, which affects the intestinal barrier integrity. This review highlights the critical importance of the Hh pathway in shaping the intestinal mucosa and summarises the results obtained so far in research on the effect of dietary constituents on the activity of this pathway.

Spatial transcriptomics reveals strong association between SFRP4 and extracellular matrix remodeling in prostate cancer

Prostate tumor heterogeneity is a major obstacle when studying the biological mechanisms of molecular markers. Increased gene expression levels of secreted frizzled-related protein 4 (SFRP4) is a biomarker in aggressive prostate cancer. To understand how SFRP4 relates to prostate cancer we performed comprehensive spatial and multiomics analysis of the same prostate cancer tissue samples. The experimental workflow included spatial transcriptomics, bulk transcriptomics, proteomics, DNA methylomics and tissue staining. SFRP4 mRNA was predominantly located in cancer stroma, produced by fibroblasts and smooth muscle cells, and co-expressed with extracellular matrix components. We also confirmed that higher SFRP4 gene expression is associated with cancer aggressiveness. Gene expression of SFRP4 was affected by gene promotor methylation. Surprisingly, the high mRNA levels did not reflect SFRP4 protein levels, which was much lower. This study contributes previously unknown insights of SFRP4 mRNA in the prostate tumor environment that potentially can improve diagnosis and treatment.

Differential vegfc expression dictates lymphatic response during zebrafish heart development and regeneration.

Vascular endothelial growth factor C (Vegfc) is crucial for lymphatic and blood vessel development, yet its cellular sources and specific functions in heart development remain unclear. To address this, we created a vegfc reporter and an inducible overexpression line in zebrafish. We found vegfc expression in large coronary arteries, circulating thrombocytes, cardiac adipocytes, and outflow tract smooth muscle cells. Notably, while coronary lymphangiogenesis aligns with Vegfc-expressing arteries in juveniles, it occurs only after coronary artery formation. Vegfc overexpression induced ectopic lymphatics on the ventricular surface prior to arterial formation, indicating that Vegfc abundance, rather than arterial presence, drives lymphatic development. However, this overexpression did not affect coronary artery coverage, suggesting a specific role for Vegfc in lymphatic, rather than arterial, development. Thrombocytes emerged as the initial Vegfc source during inflammation following heart injuries, transitioning to endocardial and myocardial expression during regeneration. Lower Vegfc levels in the amputation model corresponded with a lack of lymphatic expansion. Importantly, Vegfc overexpression enhanced lymphatic expansion and promoted scar resolution without affecting cardiomyocyte proliferation, highlighting its role in regulating lymphangiogenesis and promoting heart regeneration.

Red Yeast Rice or Lovastatin? A Comparative Evaluation of Safety and Efficacy Through a Multifaceted Approach.

The increasing use of red yeast rice (RYR) as a natural supplement to manage blood cholesterol levels is driven by its active compound, monacolin K (MK), which is chemically identical to the statin drug lovastatin (LOV). Despite its growing popularity, concerns persists regarding the safety and efficacy of RYR compared to pure statins. This study aimed to evaluate the phytochemical composition, pharmacological effects, and safety profile of various RYR samples in comparison with LOV. RYR samples with different MK content were analyzed using HPLC-DAD to quantify monacolins and other bioactive compounds. The inhibitory activity on HMG-CoA reductase was assessed through an enzymatic assay, while pharmacokinetic properties were predicted using invitro simulated digestion and in silico models. Invitro cytotoxicity was evaluated in intestinal, hepatic, renal, and skeletal muscle cell models. Additionally, the transcriptional levels of muscle damage-related target genes were evaluated by qRT-PCR in skeletal muscle cells treated with a selection of RYR samples. Significant variability in the phytochemical composition of RYR sampleswas observed, particularly in the content of secondary monacolins, triterpenes, and polyphenols. The RYR phytocomplex exhibited superior inhibition of HMG-CoA reductase activity compared to isolated LOV, suggesting synergistic effects between secondary monacolins and other compounds. Molecular insights revealed that RYR samples had a lower impact on muscle cells than LOV, as reflected also by cell viability. These findings suggest that RYR could serve as a safe alternative to purified statins. However, further research is needed to fully elucidate the mechanisms behind the synergistic activity of the phytocomplex and to firmly establish the clinical efficacy of this natural product.

Localized release of muscle-generated BDNF regulates the initial formation of postsynaptic apparatus at neuromuscular synapses.

Growing evidence indicates that brain-derived neurotrophic factor (BDNF) is produced in contracting skeletal muscles and is secreted as a myokine that plays an important role in muscle metabolism. However, the involvement of muscle-generated BDNF and the regulation of its vesicular trafficking, localization, proteolytic processing, and spatially restricted release during the development of vertebrate neuromuscular junctions (NMJs) remain largely unknown. In this study, we first reported that BDNF is spatially associated with the actin-rich core domain of podosome-like structures (PLSs) at topologically complex acetylcholine receptor (AChR) clusters in cultured Xenopus muscle cells. The release of spatially localized BDNF is tightly controlled by activity-regulated mechanisms in a calcium-dependent manner. Live-cell time-lapse imaging further showed that BDNF-containing vesicles are transported to and captured at PLSs in both aneural and synaptic AChR clusters for spatially restricted release. Functionally, BDNF knockdown or furin-mediated endoproteolytic activity inhibition significantly suppresses aneural AChR cluster formation, which in turn affects synaptic AChR clustering induced by nerve innervation or agrin-coated beads. Lastly, skeletal muscle-specific BDNF knockout (MBKO) mice exhibit structural defects in the formation of aneural AChR clusters and their subsequent recruitment to nerve-induced synaptic AChR clusters during the initial stages of NMJ development in vivo. Together, this study demonstrated the regulatory roles of PLSs in the intracellular trafficking, spatial localization, and activity-dependent release of BDNF in muscle cells and revealed the involvement of muscle-generated BDNF and its proteolytic conversion in regulating the initial formation of aneural and synaptic AChR clusters during early NMJ development in vitro and in vivo.

Mineralocorticoid receptor antagonism attenuates arteriovenous fistula stenosis by modulating the phenotype of vascular smooth muscle cells.

Fistula stenosis is a primary contributor to arteriovenous fistula (AVF) failure in maintenance hemodialysis patients. Emerging data indicated excessive fibrotic remodeling was the primarily contributor to fistula stenosis during AVF remodeling. The mineralocorticoid receptor (MR) has been implicated in vascular remodeling across various cardiovascular pathologies. However, its role in AVF remodeling, particularly concerning fibrotic remodeling, remains elusive. MR expression and the phenotypes of vascular smooth muscle cells (VSMCs) were assessed in dysfunctional AVF. The effects of MR on VSMC phenotypic switching were examined in vitro, and the protective effects of MR antagonists on AVF outcome were evaluated in a rat AVF model. Dysfunctional fistula exhibited significant medial fibrosis and extracellular matrix deposition, alongside markedly increased MR activity. In the dysfunctional fistula vessels, VSMC displayed reduced expression of the contractile marker SMMHC and featured characteristic of a synthetic phenotype, including increased osteopontin expression and heightened proliferation. In vitro studies with cultured VSMC revealed that MR overactivity induced by aldosterone led to phenotypic switching from contractile to synthetic state, concomitant with EGFR-ERK1/2 pathway overactivation. These effects were largely abolished by the MR antagonist fineronone. Knockdown of EGFR expression abrogated ERK1/2 phosphorylation and inhibited the VSMC phenotypic switching. Conversely, ectopic overexpression of EGFR in VSMC diminished the protective effect of finerenone. In rat AVF models, pharmacologic targeting of MR with finerenone significantly improved AVF outcomes, characterized by increased luminal diameters and flow volume, reduced medial fibrosis, and inhibited VSMC phenotypic switching. These beneficial outcomes were likely attributable to a restrained activity of EGFR-ERK1/2 pathway in VSMC and elevated NO production in endothelial cells. Our study demonstrated that therapeutic targeting of MR may improve AVF outcome by modulating VSMC phenotypic switching. These findings offer promising avenues for further clinical investigations aimed at optimizing AVF outcomes in the hemodialysis population.

ScRNA-seq and bulk RNA-seq identified NUPR1 as novel biomarkers related to CD4 + T cells infiltration for abdominal aortic aneurysm.

Developing a molecular signature associated with CD4 + T cell infiltration is essential for identifying biomarkers in abdominal aortic aneurysms (AAA). Establishing such a signature is vital for improving diagnostic accuracy and therapeutic strategies for AAA. This study focuses on CD4 + T cells, which are pivotal in the immune microenvironment of AAA, to pinpoint key targets. We identified CD4 + T cell-related biomarkers in AAA using bulk and single-cell RNA sequencing data from the GEO database. We employed CIBERSORT to assess immune cell infiltration and applied weighted gene co-expression network analysis and differential expression analyses to pinpoint key genes. A nomogram and related score were developed based on these genes. Single-cell RNA sequencing further analyzed their expression across cell types, and KEGG/GO analyses were conducted for candidate genes. Four genes (NUPR1, CCL4L2, CCL3L3, MMP9) were identified finally. Validation via qPCR and immunohistochemistry showed NUPR1 downregulation in aneurysms and an inverse relationship with CD4 + T cells infiltration. Immunofluorescence results indicated NUPR1 was mainly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs). After VSMCs-specific overexpression of NUPR1 via adeno-associated virus, the AAA diameter decreased, while treatment with the NUPR1 nuclear translocation inhibitor ZZW-115 hydrochloride had no effect on AAA size. Overexpression of NUPR1 in VSMCs suppresses the migration of CD4 + T cells. The four-gene signature accurately predicts CD4 + T cell infiltration in AAA patients and may serve as a clinical index. NUPR1 could be a therapeutic target for the interaction between CD4 + T cells and AAA.

Empagliflozin Attenuates Neointima Formation After Arterial Injury and Inhibits Smooth Muscle Cell Proliferation and Migration by Suppressing Platelet-Derived Growth Factor-Related Signaling.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events. However, the precise mechanisms beyond glycemic control are not fully understood. The objective of this study was to determine the role of PDGF (platelet-derived growth factor)-related signaling in empagliflozin-mediated inhibition of neointima formation. Adult male nondiabetic Wistar rats were subjected to carotid artery balloon injury. Empagliflozin (30 mg/kg per day) was administered by oral gavage for 18 days beginning 4 days before surgery. The invitro effects of empagliflozin on rat aortic vascular smooth muscle cell (VSMC) proliferation and migration were also determined. Empagliflozin attenuated balloon injury-induced neointima formation in carotid arteries. In VSMCs, empagliflozin attenuated PDGF-BB-induced proliferation and migration. Moreover, empagliflozin-treated VSMCs did not undergo apoptosis or cytotoxic death. Empagliflozin suppressed PDGF-related signaling, including phosphorylation of PDGF receptor β, Akt, and STAT3 (signal transducer and activator of transcription 3). Overactivation of PDGF signaling attenuated empagliflozin-mediated inhibition of VSMC function. SGLT2 mRNA levels in rat VSMCs were undetectable, and SGLT2 silencing did not alter the empagliflozin-mediated effects, supporting the SGLT2-independent effects of empagliflozin on VSMC. This study highlights the crucial role of suppressing PDGF-related signaling in mediating the beneficial effects of empagliflozin on neointima formation and VSMC function, which are independent of SGLT2 and glycemic control. Our study provides a novel mechanistic aspect of empagliflozin for the prevention of vascular stenosis disorders.

Deciphering the inhibitory effects of trimetazidine on pulmonary hypertension development via decreasing fatty acid oxidation and promoting glucose oxidation

Pulmonary hypertension (PH) is a devastating disease characterized by vascular remodeling, resulting in right ventricular failure and death. Dysregulation of energy metabolism is linked to PH pathogenesis. Trimetazidine (TMZ), a selective long-chain 3-ketoacyl coenzyme A thiolase inhibitor, is critical in maintaining energy metabolism. Despite the indicated TMZ’s inhibitory effect on pulmonary vascular remodeling in PH development, the integrated evaluation of the changes in biomolecules, such as metabolites and transcripts, that TMZ induces in the lung and heart tissues is largely unknown in vivo. For an improved understanding of the molecular mechanism involving the effects of TMZ on PH development, we performed a comprehensive analysis of the changes in cardiac metabolites and pulmonary transcripts of SU5416-Hypoxia (Su/Hx) rats treated with TMZ. Metabolomic analysis of the Su/Hx-induced PH hearts demonstrated that TMZ reduced the long-chain fatty acid concentration. Additionally, TMZ alleviated PH degree and excessive strain on the right heart functions in rats with Su/Hx-induced PH. We identified the candidate target genes for TMZ treatment during PH development. Interestingly, the mRNA levels of the fatty acid transporters were substantially downregulated by TMZ administration in the lungs with Su/Hx-induced PH. Notably, TMZ suppressed excessive proliferation of human pulmonary artery smooth muscle cells under hypoxic conditions. Our study suggests that TMZ ameliorates PH development by involving energy metabolism in the lungs and heart.

M6A Modification of Profilin-1 in Vascular Smooth Muscle Cells Drives Phenotype Switching and Neointimal Hyperplasia via Activation of the p-ANXA2/STAT3 Pathway.

In-stent restenosis is characterized by a significant reduction in lumen diameter within the stented segment, primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMCs) and neointimal hyperplasia. PFN1 (profilin-1), an actin-sequestering protein extensively studied in amyotrophic lateral sclerosis, remains less explored in neointimal hyperplasia. Utilizing single-cell RNA sequencing alongside data from in-stent restenosis patients and various experimental in-stent restenosis models (swine, rats, and mice), we investigated the role of PFN1 in promoting VSMC phenotype switching and neointimal hyperplasia. Single-cell RNA sequencing of stenotic rat carotid arteries revealed a critical role for PFN1 in neointimal hyperplasia, a finding corroborated in stented swine coronary arteries, in-stent restenosis patients, PFN1SMC-IKO (SMC-specific PFN1 knockout) mice, and PFN1 overexpressed mice. PFN1 deletion was shown to suppress VSMC phenotype switching and neointimal hyperplasia in PFN1SMC-IKO mice subjected to a wire-injured model. To elucidate the observed discordance in PFN1 mRNA and protein levels, we identified that METTL3 (N6-methyladenosine methyltransferase) and YTHDF3 (N6-methyladenosine-specific reader) enhance PFN1 translation efficiency in an N6-methyladenosine-dependent manner, confirmed through experiments involving METTL3 knockout and YTHDF3 knockout mice. Furthermore, PFN1 was mechanistically found to interact with the phosphorylation of ANXA2 (annexin A2) by recruiting Src, promoting the phosphorylation of STAT3, a typical transcription factor known to induce VSMC phenotype switching. This study unveils the significance of PFN1 N6-methyladenosine modification in VSMCs, demonstrating its role in promoting phenotype switching and neointimal hyperplasia through the activation of the p-ANXA2 (phospho-ANXA2)/STAT3 pathway.

Morphological Evidence for a Unique Neuromuscular Functional Unit of the Human Vocalis Muscle

Human vocalization is a complex process that is still only partially understood. Previous studies have suggested the possibility of a localized neuromuscular network of the larynx. Here we investigate this structure in human dissection specimens using multiple immunofluorescence and transmission electron microscopy (TEM). In the area of the pars interna of the thyroarytenoid muscle, muscle fibers are present that are clearly differentiated from skeletal or cardiac muscle cells and show an intermediate ultrastructure. In addition, intramuscular neurons are present that are detectable by both electron and fluorescence microscopy and may have a sensory function in a local neuronal network. Also, several types of sensory and motor synapses are detectable and distributed throughout the pars interna of the thyroarytenoid muscle, with multisynaptic muscle fibers being a common feature. These findings suggest the existence of a previously unrecognized type of muscle fiber coupled to an intramuscular neuronal network, the presence of which could explain functional peculiarities at the laryngeal level.

Rhythmic Contractions of Lymph Vessels and Lymph Flow Are Disrupted in Hypertensive Rats.

Hypertension increases the risk of lymphedema in patients with comorbidities, but whether hypertension directly compromises lymph vessel (LV) function and lymph flow is unclear. We compared the contractions of mesenteric LVs ex vivo and lymph flow in vivo between normotensive and Ang II (angiotensin II)-induced hypertensive rats and explored the ionic basis of contractile patterns. Key studies were recapitulated in spontaneously hypertensive rats and control Wistar-Kyoto rats. Video microscopy continuously recorded the diameters of cannulated rat mesenteric LVs, and high-speed optical imaging estimated mesenteric lymph flow in vivo. Jess capillary Western electrophoresis evaluated expression levels of ion channel proteins. Isolated LVs from Ang II-induced hypertensive rats exhibited dysrhythmic contractions, whereas LVs from both Ang II-induced hypertensive rats and spontaneously hypertensive rats exhibited reduced diastolic diameters and cross-sectional flow. Mesenteric lymph flow in vivo was 2.9-fold lower in Ang II-induced hypertensive rats compared with normotensive rats. Surprisingly, the LVs from Ang II-induced hypertensive rats expressed fewer intact L-type Ca2+ channel pore proteins and more modulatory cleaved C-terminal fragments. However, pharmacological block of voltage-gated K+ channels but not other K+ channel types in control LVs established the pattern of contractile dysfunction observed in hypertension. Jess capillary Western electrophoresis analysis confirmed a loss of Shaker-type KV1.2 channels in LVs from hypertensive rats. We provide initial evidence of lymphatic contractile dysfunction and compromised lymph flow in hypertensive rats, which may be caused by a loss of KV1.2 channels in the lymphatic muscle cells.