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Objective To discuss the effect and mechanism of Rotigaptide on the autonomic contractile movement of gastric smooth muscle in diabetic rats. Methods The diabetic rat model (DM) was induced by streptozotocin (STZ). SD rats were divided into the control group and the DM group. In vitro muscle strip experiment was performed to observe changes of the autonomic contractile movement of the gastric smooth muscle before and after treatment with Rotigaptide in each group. Western blot assay was used to detect expression levels of Cx43 and PKCα in membrane protein and pulp protein, p-Cx43 Ser368 and p-PKCα Thr497 in gastric smooth muscle tissue of the control group, the DM group and the Rotigaptide group. Results Compared with the control group, the amplitude and frequency of autonomic contraction in gastric smooth muscle contraction were decreased in the DM group (P＜0.05). In the control group, there were no changes in amplitude and frequency of autonomic contraction in gastric smooth muscle after treatment with Rotigaptide. In the DM group, after treatment with Rotigaptide, the amplitude and frequency of autonomic contraction in gastric smooth muscle contraction were significantly reduced (P＜0.05). Compared with the control group, the Cx43 content of membrane protein was increased, the Cx43 content of pulp protein was decreased, the membrane Cx43/pulp Cx43 ratio increased, and the p-Cx43 Ser368 content of membrane protein was increased in the DM group (P＜0.05). Compared with the DM group, the Cx43 content of membrane protein was decreased (P＜0.05), the Cx43 content of pulp protein was increased and the membrane Cx43/pulp Cx43 ratio was decreased in the Rotigaptide group (P＜0.05). Compared with the control group, the content of membrane protein PKCα was decreased, the content of pulp protein PKCα was increased and the membrane PKCα/pulp PKCα ratio was decreased in the DM group (P＜0.05). Compared with the DM group, the content of membrane protein p-PKCα Thr497 was increased in the Rotigaptide group (P＜0.05). Conclusion Rotigaptide inhibits autonomic contraction of gastric smooth muscle in diabetic rats by down-regulating the number and the opening rate of gap junction by PKCα-Cx43 pathway activity. [ABSTRACT FROM AUTHOR]

Background: STIM1 (stromal interaction molecule 1) regulates store-operated calcium entry and is involved in pulmonary artery vasoconstriction and pulmonary artery smooth muscle cell proliferation, leading to pulmonary arterial hypertension (PAH)., Methods: Bioinformatics analysis and a 2-stage matched case-control study were conducted to screen for noncoding variants that may potentially affect STIM1 transcriptional regulation in 242 patients with idiopathic PAH and 414 healthy controls. Luciferase reporter assay, real-time quantitative polymerase chain reaction, western blot, 5-ethynyl-2'-deoxyuridine (EdU) assay, and intracellular Ca 2+ measurement were performed to study the mechanistic roles of those STIM1 noncoding variants in PAH., Results: Five noncoding variants (rs3794050, rs7934581, rs3750996, rs1561876, and rs3750994) were identified and genotyped using Sanger sequencing. Rs3794050, rs7934581, and rs1561876 were associated with idiopathic PAH (recessive model, all P <0.05). Bioinformatics analysis showed that these 3 noncoding variants possibly affect the enhancer function of STIM1 or the microRNA (miRNA) binding to STIM1 . Functional validation performed in HEK293 and pulmonary artery smooth muscle cells demonstrated that the noncoding variant rs1561876-G ( STIM1 mutant) had significantly stronger transcriptional activity than the wild-type counterpart, rs1561876-A, by affecting the transcriptional regulatory function of both hsa-miRNA-3140-5p and hsa-miRNA-4766-5p. rs1561876-G enhanced intracellular Ca 2+ signaling in human pulmonary artery smooth muscle cells secondary to calcium-sensing receptor activation and promoted proliferation of pulmonary artery smooth muscle cells under both normoxia and hypoxia conditions, suggesting a possible contribution to PAH development., Conclusions: The potential clinical implications of the 3 noncoding variants of STIM1 , rs3794050, rs7934581, and rs1561876, are 2-fold, as they may help predict the risk and prognosis of idiopathic PAH and guide investigations on novel therapeutic pathway(s)., Competing Interests: None.

Hirschsprung disease (HSCR) and its associated disorders (AD-HSCR) often result in severe hypoperistalsis caused by enteric neuropathy, mesenchymopathy, and myopathy. Notably, HSCR involving the small intestine, isolated hypoganglionosis, chronic idiopathic intestinal pseudo-obstruction, and megacystis-microcolon-intestinal hypoperistalsis syndrome carry a poor prognosis. Ultimately, small-bowel transplantation (SBTx) is necessary for refractory cases, but it is highly invasive and outcomes are less than optimal, despite advances in surgical techniques and management. Thus, regenerative therapy has come to light as a potential form of treatment involving regeneration of the enteric nervous system, mesenchyme, and smooth muscle in affected areas. We review the cutting-edge regenerative therapeutic approaches for managing HSCR and AD-HSCR, including the use of enteric nervous system progenitor cells, embryonic stem cells, induced pluripotent stem cells, and mesenchymal stem cells as cell sources, the recipient intestine's microenvironment, and transplantation methods. Perspectives on the future of these treatments are also discussed., (© 2023. The Author(s) under exclusive licence to Springer Nature Singapore Pte Ltd.)

Chronic hypoxia plays a central role in diverse pulmonary pathologies, but its effects on longitudinal changes in the biomechanical behavior of proximal pulmonary arteries remain poorly understood. Similarly, effects of normoxic recovery have not been well studied. Here, we report hypoxia-induced changes in composition, vasoactivity, and passive biaxial mechanics in the main branch pulmonary artery of male C57BL/6J mice exposed to 10% FiO2 for 1, 2, or 3 weeks. We observed significant changes in extracellular matrix, and consequently wall mechanics, as early as 1 week of hypoxia. While circumferential stress and stiffness returned toward normal values by 2-3 weeks of hypoxia, area fractions of cytoplasm and thin collagen fibers did not return toward normal until after 1 week of normoxic recovery. By contrast, elastic energy storage and overall distensibility remained reduced after 3 weeks of hypoxia as well as following 1 week of normoxic recovery. While smooth muscle and endothelial cell responses were attenuated under hypoxia, smooth muscle but not endothelial cell responses recovered following 1 week of subsequent normoxia. Collectively, these data suggest that homeostatic processes were unable to preserve or restore overall function, at least over a brief period of normoxic recovery. Longitudinal changes are critical in understanding large pulmonary artery remodeling under hypoxia, and its reversal, and will inform predictive models of vascular adaptation., (Copyright © 2024 by ASME.)

Background: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events., Methods: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells., Results: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P =0.0034) and adjusted multivariate model (hazard ratio, 1.56; P =0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P =0.018) and of plaque hemorrhage (hazard ratio, 1.7; P =0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFβ -triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells., Conclusions: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications., Competing Interests: Disclosures None.

Purpose: This study aimed to investigate the underlying factors driving the onset of myopia, specifically the role of the ciliary muscle's contraction in the elongation of the axial length of the eye., Methods: The retrospective study was conducted utilizing data from three ophthalmic centers in Shanghai and Beijing. Both Chinese and Caucasian children were involved. The axial length of the subjects' eyes was measured in both relaxed and contracted state of the ciliary muscle. A comprehensive mechanical model was also developed to observe the influence of ciliary muscle contraction on the axial length., Results: This study included a sample of 198 right eyes of 198 myopic children. Of these, 97 were male and 101 were female, 126 were of Chinese ethnicity and 72 were Caucasian. The age of onset for myopia ranged from 5.9 to 16.9 years old. The axial length of the eye decreased 0.028 ± 0.007mm following dilation, indicating relaxation of the ciliary muscle (t paired student = 15.16, p = 6.72 x 10-35). In contrast, ciliary muscle contraction resulted in an increase in axial length. Considering proportionality, a significant 90.4% (179 eyes) exhibited a reduced axial length, while a minor 9.6% (19 eyes) demonstrated an increase post-mydriasis. Finite element modeling demonstrated that muscle contraction caused a tension force that transmits towards the posterior pole of the eye, causing it to extend posteriorly., Conclusion: The contraction of the ciliary muscle leads to an extension of the axial length. This could potentially be the initiating factor for myopia., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Meng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Improvement in lower esophageal sphincter (LES) competency after laparoscopic Nissen fundoplication (LNF) is well established, yet esophageal body physiology data are limited. We aimed to describe the impact of LNF on whole esophagus physiology using standard and novel manometric characteristics., Methods: A cohort of patients with an intact fundoplication without herniation and no postoperative dysphagia were selected and underwent esophageal manometry at one-year after surgery. Pre- and post-operative manometry files were reanalyzed using standard and novel manometric characteristics and compared., Key Results: A total of 95 patients were included in this study. At 16.1 (8.7) months LNF increased LES overall and abdominal length and resting pressure (p < 0.0001). Outflow resistance (IRP) increased [5.8 (3-11) to 11.1 (9-15), p < 0.0001] with a 95th percentile of 20 mmHg in this cohort of dysphagia-free patients. Distal contractile integral (DCI) also increased [1177.0 (667-2139) to 1321.1 (783-2895), p = 0.002], yet contractile amplitude was unchanged (p = 0.158). There were direct correlations between pre- and post-operative DCI [R: 0.727 (0.62-0.81), p < 0.0001] and postoperative DCI and postoperative IRP [R: 0.347 (0.16-0.51), p = 0.0006]. Contractile front velocity [3.5 (3-4) to 3.2 (3-4), p = 0.0013] was slower, while distal latency [6.7 (6-8) to 7.4 (7-9), p < 0.0001], the interval from swallow onset to proximal smooth muscle initiation [4.0 (4-5) to 4.4 (4-5), p = 0.0002], and the interval from swallow onset to point when the peristaltic wave meets the LES [9.4 (8-10) to 10.3 (9-12), p < 0.0001] were longer. Esophageal length [21.9 (19-24) to 23.2 (21-25), p < 0.0001] and transition zone (TZ) length [2.2 (1-3) to 2.5 (1-4), p = 0.004] were longer. Bolus clearance was inversely correlated with TZ length (p = 0.0002) and time from swallow onset to proximal smooth muscle initiation (p < 0.0001). Bolus clearance and UES characteristics were unchanged (p > 0.05)., Conclusions & Inferences: Increased outflow resistance after LNF required an increased DCI. However, this increased contractile vigor was achieved through sustained, not stronger, peristaltic contractions. Increased esophageal length was associated with increased TZ and delayed initiation of smooth muscle contractions., (© 2024 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Major esophageal disorders involve obstructive transport of bolus to the stomach, causing symptoms of dysphagia and impaired clearing of the refluxed gastric contents. These may occur due to mechanical constriction of the esophageal lumen or loss of relaxation associated with deglutitive inhibition, as in achalasia-like disorders. Recently, immune inflammation has been identified as an important cause of esophageal strictures and the loss of inhibitory neurotransmission. These disorders are also associated with smooth muscle hypertrophy and hypercontractility, whose cause is unknown. This review investigated immune inflammation in the causation of smooth muscle changes in obstructive esophageal bolus transport. Findings suggest that smooth muscle hypertrophy occurs above the obstruction and is due to mechanical stress on the smooth muscles. The mechanostressed smooth muscles release cytokines and other molecules that may recruit and microlocalize mast cells to smooth muscle bundles, so that their products may have a close bidirectional effect on each other. Acting in a paracrine fashion, the inflammatory cytokines induce genetic and epigenetic changes in the smooth muscles, leading to smooth muscle hypercontractility, hypertrophy, and impaired relaxation. These changes may worsen difficulty in the esophageal transport. Immune processes differ in the first phase of obstructive bolus transport, and the second phase of muscle hypertrophy and hypercontractility. Moreover, changes in the type of mechanical stress may change immune response and effect on smooth muscles. Understanding immune signaling in causes of obstructive bolus transport, type of mechanical stress, and associated smooth muscle changes may help pathophysiology-based prevention and targeted treatment of esophageal motility disorders. NEW & NOTEWORTHY Esophageal disorders such as esophageal stricture or achalasia, and diffuse esophageal spasm are associated with smooth muscle hypertrophy and hypercontractility, above the obstruction, yet the cause of such changes is unknown. This review suggests that smooth muscle obstructive disorders may cause mechanical stress on smooth muscle, which then secretes chemicals that recruit, microlocalize, and activate mast cells to initiate immune inflammation, producing functional and structural changes in smooth muscles. Understanding the immune signaling in these changes may help pathophysiology-based prevention and targeted treatment of esophageal motility disorders.

Developing a polymer-based photocrosslinked 3D printable scaffolds comprised of gelatin methacryloyl (G) and hyaluronic acid methacryloyl (H) incorporated with two molecular weights of polyethylene glycol diacrylate (P) of various concentrations that enables rabbit adipose-derived stem cells (rADSCs) to survive, grow, and differentiate into smooth muscle cells (SMCs). Then, the chemical modification and physicochemical properties of the PGH bioinks were evaluated. The cell viability was assessed via MTT, CCK-8 assay and visualized employing Live/Dead assay. In addition, the morphology and nucleus count of differentiated SMCs were investigated by adopting TRAP (tartrate-resistant acid phosphatase) staining, and quantitative RT-PCR analysis was applied to detect gene expression using two different SMC-specific gene markers α-SMA and SM-MHC. The SMC-specific protein markers namely α-SMA and SM-MHC were applied to investigate SMC differentiation ability by implementing Immunocytofluorescence staining (ICC) and western blotting. Moreover, the disk, square, and tubular cellular models of PGH7 (GelMA/HAMA=2/1) + PEGDA-8000 Da, 3% w/v) hybrid bioink were printed using an extrusion bioprinting and cell viability of rADSCs was also analysed within 3D printed square construct practising Live/Dead assay. The results elicited the overall viability of SMCs, conserving its phenotype in biocompatible PGH7 hybrid bioink revealing its great potential to regenerate SMCs associated organs repair., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Stem cells respond and remember mechanical cues from the microenvironment, which modulates their therapeutic effects. Chromatin organization and energy metabolism regulate the stem cell fate induced by mechanical cues. However, the mechanism of mechanical memory is still unclear. This study aimed to investigate the effects of mechanical amplitude, frequency, duration, and stretch cycle on mechanical memory in mesenchymal stem cells. It showed that the amplitude was the dominant parameter to the persistence of cell alignment. F-actin, paxillin, and nuclear deformation are more prone to be remolded than cell alignment. Stretching induces transcriptional memory, resulting in greater transcription upon subsequent reloading. Cell metabolism displays mechanical memory with sustained mitochondrial fusion and increased ATP production. The mechanical memory of chromatin condensation is mediated by histone H3 lysine 27 trimethylation, leading to much higher smooth muscle differentiation efficiency. Interestingly, mechanical memory can be transmitted based on direct cell-cell interaction, and stretched cells can remodel the metabolic homeostasis of static cells. Our results provide insight into the underlying mechanism of mechanical memory and its potential benefits for stem cell therapy., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The mechanism of action of bronchial thermoplasty (BT) treatment for patients with severe asthma is incompletely understood. This study investigated the 2.5-year impact of BT on airway smooth muscle (ASM) mass and clinical parameters by paired data analysis in 22 patients. Our findings demonstrate the persistence of ASM mass reduction of >50% after 2.5 years. Furthermore, sustained improvement in asthma control, quality of life and exacerbation rates was found, which is in line with previous reports. An association was found between the remaining ASM and both the exacerbation rate (r=0.61, p=0.04 for desmin, r=0.85, p<0.01 for alpha smooth muscle actin (SMA)) and post-bronchodilator forced expiratory volume in 1 s predicted percentage (r=-0.69, p=0.03 for desmin, r=-0.58, p=0.08 for alpha SMA). This study provides new insight into the long-term impact of BT., Competing Interests: Competing interests: No conflicts of interest exist for the following authors—PW, AWMG, JNSd'H, NHTtH, JJTHR, TM, EJMW and PS. JTA reports research grants from Boston Scientific during the conduct of the study. PB reports research grants from Boston Scientific, non-financial support from Boston Scientific related to investigator-initiated research grant, research grants from ZonMw and research grants from Dutch Lung Foundation, during the conduct of the study., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Many vascular disorders arise as a result of dysfunctional smooth muscle cells. Tissue engineering strategies have evolved as key approaches to generate functional vascular smooth muscle cells for use in cell-based precision and personalized regenerative medicine approaches. This article highlights some of the challenges that exist in the field and presents some of the prospects for translating research advancements into therapeutic modalities. The article emphasizes the need for better developing synergetic intracellular and extracellular cues in the processes to generate functional vascular smooth muscle cells from different stem cell sources for use in tissue engineering strategies.

Isolated airway smooth muscle has been extensively investigated since 1840 to understand the pharmacology of airway diseases. There has often been poor predictability from murine experiments to drugs evaluated in patients with asthma or chronic obstructive pulmonary disease (COPD). However, the use of isolated human airways represents a sensible strategy to optimise the development of innovative molecules for the treatment of respiratory diseases. This review aims to provide updated evidence on the current uses of isolated human airways in validated in vitro methods to investigate drugs in development for the treatment of chronic obstructive respiratory disorders. This review also provides historical notes on the pioneering pharmacological research on isolated human airway tissues, the key differences between human and animal airways, as well as the pivotal differences between human medium bronchi and small airways. Experiments carried out with isolated human bronchial tissues in vitro and ex vivo replicate many of the main anatomical, pathophysiological, mechanical and immunological characteristics of patients with asthma or COPD. In vitro models of asthma and COPD using isolated human airways can provide information that is directly translatable into humans with obstructive lung diseases. Regardless of the technique used to investigate drugs for the treatment of chronic obstructive respiratory disorders (i.e., isolated organ bath systems, videomicroscopy and wire myography), the most limiting factors to produce high-quality and repeatable data remain closely tied to the manual skills of the researcher conducting experiments and the availability of suitable tissue., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Objective: To compare the collagen, elastic fibers, and smooth muscle content of the clitoris and the glans penis in young adults., Materials and Methods: The clitoris and the glans penis of six women and six men (mean age 25±3) who died as a result of accidents were excised. The samples were placed under a formaldehyde solution and histologically processed. Masson's trichrome and Weigert's resorcin-fuchsin stain was used to highlight the elastic fibers, smooth muscle, and collagen. Stereological analysis was conducted in 5 random fields of 5 slides for each sample. For statistical analysis, the unpaired t-test was used to compare values between groups, and a value of P<0.05 was considered as significant for all analyses., Results: Stereology revealed a mean smooth muscle content of 35.84±6.46% and 31.64±4.74% for the clitoris and glans penis, respectively, while it also revealed collagen content of 26.11±7.41% and 28.44±3.55% and elastic fibers content of 24.12±4.34% and 30.97±6.13% for the clitoris and glans penis, respectively. The statistical analysis showed no significant differences between them., Conclusion: Regardless of anatomical differences, the volumetric density of collagen, elastic fibers, and smooth muscle were similar for the clitoris and glans penis in young adults, a feature possibly explained by their embryology., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

The high prevalence and burden of uterine fibroids in women raises questions about the origin of these benign growths. Here, we propose that fibroids should be understood in the context of human evolution, specifically the advent of bipedal locomotion in the hominin lineage. Over the ≥7 million years since our arboreal ancestors left their trees, skeletal adaptations ensued, affecting the pelvis, limbs, hands, and feet. By 3.2 million years ago, our ancestors were fully bipedal. A key evolutionary advantage of bipedalism was the freedom to use hands to carry and prepare food and create and use tools which, in turn, led to further evolutionary changes such as brain enlargement (encephalization), including a dramatic increase in the size of the neocortex. Pelvic realignment resulted in narrowing and transformation of the birth canal from a simple cylinder to a convoluted structure with misaligned pelvic inlet, mid-pelvis, and pelvic outlet planes. Neonatal head circumference has increased, greatly complicating parturition in early and modern humans, up to and including our own species. To overcome the so-called obstetric dilemma provoked by bipedal locomotion and encephalization, various compensatory adaptations have occurred affecting human neonatal development. These include adaptations limiting neonatal size, namely altricial birth (delivery of infants at an early neurodevelopmental stage, relative to other primates) and mid-gestation skeletal growth deceleration. Another key adaptation was hyperplasia of the myometrium, specifically the neomyometrium (the outer two-thirds of the myometrium, corresponding to 90% of the uterine musculature), allowing the uterus to more forcefully push the baby through the pelvis during a lengthy parturition. We propose that this hyperplasia of smooth muscle tissue set the stage for highly prevalent uterine fibroids. These fibroids are therefore a consequence of the obstetric dilemma and, ultimately, of the evolution of bipedalism in our hominin ancestors., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Nanofibrous scaffolds have attracted much attention in bladder reconstruction approaches due to their excellent mechanical properties. In addition, their biological properties can be improved by combination with biological materials. Taking into account the advantages of nanofibrous scaffolds and decellularized extracellular matrix (dECM) in tissue engineering, scaffolds of poly-L-lactic acid (PLLA) coated with decellularized human amnion membrane (hAM) or sheep bladder (SB)-derived ECM proteins are developed (amECM-coated PLLA and sbECM-coated PLLA, respectively). The bladder regenerative potential of modified electrospun PLLA scaffolds is investigated in rabbits. The presence of ECM proteins is confirmed on the nanofibers' surface. Coating the surface of the PLLA nanofibers improves cell adhesion and proliferation. Histological and immunohistochemical evaluations show that rabbits subjected to cystoplasty with a multilayered PLLA scaffold show de novo formation and maturation of the multilayered urothelial layer. However, smooth muscle bundles (myosin heavy chain [MHC] and α-smooth muscle actin [α-SMA] positive) are detected only in ECM-coated PLLA groups. All groups show no evidence of a diverticulumor fistula in the urinary bladder. These results suggest that the biofunctionalization of electrospun PLLA nanofibers with ECM proteins can be a promising option for bladder tissue engineering. Furthermore, hAM can also replace animal-sourced ECM proteins in bladder tissue regeneration approaches., (© 2023 Wiley-VCH GmbH.)

The tunica muscularis of mammalian esophagi is composed of striated muscle and smooth muscle. Contraction of the esophageal striated muscle portion is mainly controlled by cholinergic neurons. On the other hand, smooth muscle contraction and relaxation are controlled not only by cholinergic components but also by non-cholinergic components in the esophagus. Adenosine triphosphate (ATP) is known to regulate smooth muscle contraction and relaxation in the gastrointestinal tract via purinergic receptors. However, the precise mechanism of purinergic regulation in the esophagus is still unclear. Therefore, the aim of the present study was to clarify the effects of ATP on the mechanical responses of the esophageal muscle in mice. An isolated segment of the mouse esophagus was placed in a Magnus's tube and longitudinal mechanical responses were recorded. Exogenous application of ATP induced contractile responses in the esophageal preparations. Tetrodotoxin, a blocker of voltage-dependent sodium channels in neurons and striated muscle, did not affect the ATP-induced contraction. The ATP-evoked contraction was blocked by pretreatment with suramin, a purinergic receptor antagonist. RT-PCR revealed the expression of mRNA of purinergic receptor genes in the mouse esophageal tissue. The findings suggest that purinergic signaling might regulate the motor activity of mouse esophageal smooth muscle.

Background: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall., Methods: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1β activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs., Results: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept., Conclusions: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated., Competing Interests: Disclosures N.J. Galant is a cofounder and CEO of Paradox Immunotherapeutics. The other authors report no conflicts.

Advanced maternal age during pregnancy is associated with increased risk of vaginal tearing during delivery and maladaptive postpartum healing. Although the underlying mechanisms of age-related vaginal injuries are not fully elucidated, changes in vaginal microstructure may contribute. Smooth muscle cells promote the contractile nature of the vagina and contribute to pelvic floor stability. While menopause is associated with decreased vaginal smooth muscle content, whether contractile changes occur before the onset of menopause remains unknown. Therefore, the first objective of this study was to quantify the active mechanical behavior of the murine vagina with age. Further, aging is associated with decreased vaginal elastin content. As such, the second objective was to determine if elastic fiber disruption alters vaginal contractility. Vaginal samples from mice aged 2-14 months were used in maximum contractility experiments and biaxial extension-inflation protocols. To evaluate the role of elastic fibers with age, half of the vaginal samples were randomly allocated to enzymatic elastic fiber disruption. Contractile potential decreased and vaginal material stiffness increased with age. These age-related changes in smooth muscle function may be due, in part, to changes in microstructural composition or contractile gene expression. Furthermore, elastic fiber disruption had a diminished effect on smooth muscle contractility in older mice. This suggests a decreased functional role of elastic fibers with age. Quantifying the age-dependent mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties provides a first step towards better understanding how age-related changes in vaginal structure may contribute to tissue integrity and healing. STATEMENT OF SIGNIFICANCE: Advanced maternal age at the time of pregnancy is linked to increased risks of vaginal tearing during delivery, postpartum hemorrhaging, and the development of pelvic floor disorders. While the underlying causes of increased vaginal injuries with age and associated pathologies remain unclear, changes in vaginal microstructure, such as elastic fibers and smooth muscle cells, may contribute. Menopause is associated with fragmented elastic fibers and decreased smooth muscle content; however, how reproductive aging affects changes in the vaginal composition and the mechanical properties remains unknown. Quantifying the mechanical contribution of smooth muscle cells and elastic fibers to vaginal properties with age will advance understanding of the potential structural causes of age-related changes to tissue integrity and healing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2023. Published by Elsevier Ltd.)

Motility of detrusor smooth muscle includes adrenergic relaxation and cholinergic contraction. Since the latter may be deregulated in overactive bladder (OAB) pathophysiology, anticholinergics are the standard therapy but occasionally less tolerated due to side effects such as dry mouth and constipation. β 3 adrenoceptor agonists also alleviate OAB symptoms by relaxing the detrusor muscle. Their age dependence, however, is far from understood. To address this issue, we induced contractions with KCl (60 mM) and carbachol (from 10 nM to 100 μM) in the presence of the β 3 adrenoceptor agonist CL316,243 (from 0.1 to 10 μM) in both human and rat muscle strips. Our results confirmed that both contractions were attenuated by β 3 adrenoceptor activation in both species, but with differing age dependence. In humans, specimens from mid-life subjects showed a significantly more pronounced effect of CL316,243 in attenuating carbachol-induced contractions than those from aged subjects (Cohen's d of maximal attenuation: 1.82 in mid-life versus 0.13 in aged) without altering EC 50 . Conversely, attenuation of KCl responses by CL316,243 increased during ageing (Spearman correlation coefficient = -0.584, P<0.01). In rats, both KCl- and carbachol-induced contractions were significantly more attenuated by CL316,243 in samples from adolescent as compared to aged samples. Immunohistochemistry in human detrusor sections proved β 3 adrenoreceptor abundance to remain unaltered during ageing. In conclusion, our findings suggest differential age-dependent changes in human β 3 adrenoceptor-dependent attenuation of detrusor contraction in terms of electromechanical versus pharmacomechanical coupling; they may help understand the differential responsiveness of OAB patients to β 3 agents., (© 2023. The Author(s).)

Presbyopia is an age-related ocular disorder where accommodative ability declines so that an individual's focusing range is insufficient to provide visual clarity for near and distance vision tasks without corrective measures. With age, the eye exhibits changes in biomechanical properties of many components involved in accommodation, including the lens, sclera, and ciliary muscle. Changes occur at different rates, affecting accommodative biomechanics differently, but individual contributions to presbyopia are unknown. We used a finite element model (FEM) of the accommodative mechanism to simulate age-related changes in lens stiffness, scleral stiffness, and ciliary contraction to predict differences in accommodative function. The FEM predicts how ciliary muscle action leads to lens displacement by initializing a tensioned unaccommodated lens (Phase 0) then simulating ciliary muscle contraction in accommodation (Phase 1). Model inputs were calibrated to replicate experimentally measured lens and ciliary muscle in 30-year-old eyes. Predictions of accommodative lens deformation were verified with additional imaging studies. Model variations were created with altered lens component stiffnesses, scleral stiffness, or ciliary muscle section activations, representing fifteen-year incremental age-related changes. Model variations predict significant changes in accommodative function with age-related biomechanical property changes. Lens changes only significantly altered lens thickening with advanced age (46% decrease at 75 years old) while sclera changes produced progressive dysfunction with increasing age (23%, 36%, 49% decrease at 45, 60, and 75 years old). Ciliary muscle changes effected lens position modulation. Model predictions identified potential mechanisms of presbyopia that likely work in combination to reduce accommodative function and could indicate effectiveness of treatment strategies and their dependency on patient age or relative ocular mechanical properties., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Age-associated myometrial dysfunction can prompt complications during pregnancy and labor, which is one of the factors contributing to the 7.8-fold increase in maternal mortality in women over 40. Using single-cell/single-nucleus RNA sequencing and spatial transcriptomics, we have constructed a cellular atlas of the aging myometrium from 186,120 cells across twenty perimenopausal and postmenopausal women. We identify 23 myometrial cell subpopulations, including contractile and venous capillary cells as well as immune-modulated fibroblasts. Myometrial aging leads to fewer contractile capillary cells, a reduced level of ion channel expression in smooth muscle cells, and impaired gene expression in endothelial, smooth muscle, fibroblast, perivascular, and immune cells. We observe altered myometrial cell-to-cell communication as an aging hallmark, which associated with the loss of 25 signaling pathways, including those related to angiogenesis, tissue repair, contractility, immunity, and nervous system regulation. These insights may contribute to a better understanding of the complications faced by older individuals during pregnancy and labor., (© 2024. The Author(s).)

Airway smooth muscle (ASM) remodeling in asthmatic airways may contribute to persistent airflow limitation and airway hyperresponsiveness. CD4 + T cells infiltrate the ASM layer where they may induce a proliferative and secretory ASM cell phenotype. We studied the interaction between activated CD4 + T cells and ASM cells in co-culture in vitro and investigated the effects of CD4 + T cells on chemokine production by ASM cells. CD4 + T cells induced marked upregulation of C-X-C motif chemokine ligands (CXCL) 9, 10, and 11 in ASM cells. Blockade of the IFN-γ receptor on ASM cells prevented this upregulation. Furthermore, T cell-derived IFN-γ and LIGHT (lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes) synergize in a dose-dependent manner to coordinately enhance CXCL9, 10, and 11 expression. The synergistic property of LIGHT was mediated exclusively through the lymphotoxin-β receptor (LTBR), but not herpes virus entry mediator (HVEM). Disruption of LTBR signaling in ASM cells reduced CXCL9, 10, and 11 production and ASM cell-mediated CD4 + T cell chemotaxis. We conclude that the LIGHT-LTBR signaling axis acts together with IFN-γ to regulate chemokines that mediate lymphocyte infiltration in asthmatics., (© 2024 Federation of American Societies for Experimental Biology.)

Background: Cellular and cytoskeletal events of overactive bladder (OAB) have not been sufficiently explored in human bladder due to different limitations. Bladder outlet obstruction (BOO) had been induced in different animal models with different methods to induce (OAB). Similarity of the animal models of BOO to the human OAB is postulated but has not been confirmed. The interstitial cells of Cajal (ICCs), and telocytes (TCs) are an important players in smooth muscles conductivity, they had not been well investigated in the previous BOO models. Objectives are to investigate the morphological pattern of cellular, cytoskeleton and telocytes distribution in BOO rat model and to match the events in two time periods and compare it to the findings in real-world human OAB., Methods: Female Sprague-Dawley rats (Rattus norvegicus) were randomly divided into: sham (n = 10), BOO 6 W (n = 10), BOO 8 W (n = 10). Operative procedure to Induce BOO was done under anesthesia with intraperitoneal Ketamine administration. The Effect of induction of BOO was evaluated after 6 and 8 weeks. The rats were anesthetized, and the urinary bladder was removed, while the rat was unconscious under anaesthesia it was transferred to the inhalation anaesthesia cage for euthanasia, rats were sacrificed under light anesthesia using isoflurane. Care of animals, surgical procedure, and euthanasia adhered to Guide for the Care and Use of Laboratory Animals, and AVMA Guidelines for the Euthanasia of Animals. The retrieved bladder was processed for examination with histopathology, immunohistochemistry (IHC), and transmission electron microscopy (EM)., Results: Histological examination of the bladder shows thinner urothelium, condensation of collagen between muscle bundles. IHC with c-kit shows the excess distribution of ICCs between smooth muscle bundles. EM shows frequent distribution of TCs that were situated between collagen fibers. Finings in BOO 6 W group and BOO 8 W group were comparable., Conclusion: The animal model study demonstrated increased collagen/ smooth muscle ratio, high intensity of ICCs and presence of TCs. Findings show that a minimally invasive procedure to induce BOO in rats had resulted in an OAB that has morphological changes that were stable in 6 & 8 weeks. We demonstrated the distribution of TCs and ICCs in the rat animal model and defined them. The population of TCs in the BOO rat model is described for the first time, suggests that the TCs and ICCs may contribute to the pathophysiology of OAB. Similarity of animal model to human events OAB was demonstrated. These findings warrant further study to define the role of TCs in OAB., Clinical Trial Registry: The study does not require a clinical trial registration; it is an experimental animal study in basic science and does not include human subjects., (© 2024. The Author(s).)

Prune belly syndrome (PBS), also known as Eagle-Barret syndrome, is a rare, multi-system congenital myopathy primarily affecting males. Phenotypically, PBS cases manifest three cardinal pathological features: urinary tract dilation with poorly contractile smooth muscle, wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, and intra-abdominal undescended testes. Genetically, PBS is poorly understood. After performing whole exome sequencing in PBS patients, we identify one compound heterozygous variant in the PIEZO1 gene. PIEZO1 is a cation-selective channel activated by various mechanical forces and widely expressed throughout the lower urinary tract. Here we conduct an extensive functional analysis of the PIEZO1 PBS variants that reveal loss-of-function characteristics in the pressure-induced normalized open probability (NPo) of the channel, while no change is observed in single-channel currents. Furthermore, Yoda1, a PIEZO1 activator, can rescue the NPo defect of the PBS mutant channels. Thus, PIEZO1 mutations may be causal for PBS and the in vitro cellular pathophysiological phenotype could be rescued by the small molecule, Yoda1. Activation of PIEZO1 might provide a promising means of treating PBS and other related bladder dysfunctional states., (© 2024. The Author(s).)

The development of the metanephros in one-humped camels involves a complex series of interactions between epithelial and mesenchymal cells. As a result, there is a synchronized differentiation process of stromal, vascular and epithelial cell types during glomerulogenesis, angiogenesis and tubulogenesis. In the current work, the metanephros of camel foetuses were divided into four stages where kidneys from each stage were processed and immunoassayed, followed by quantitative analysis to determine target protein intensities throughout metanephrogenesis in the camel. This study demonstrated robust expression of α-smooth muscle actin (α-SMA) in the glomerular mesangium, as well as in interlobular and glomerular arterioles during the earlier stages of development. However, in the late stages, α-SMA expression became more localized around the blood capillaries in both the cortex and medulla. Strong expression of CD34 was observed in the immature glomerular and peritubular endothelial cells within the subcapsular zone, as well as in the glomerular, proximal tubular and distal tubular epithelium of stage one foetuses, although its expression gradually diminished with foetal maturation. The expression pattern of osteopontin was prominently observed in the distal convoluted tubules throughout all stages, however, no expression was detected in the proximal tubules, glomeruli and arterioles. E-cadherin was detected in the developing renal tubular epithelial cells but not in the glomeruli. In conclusion, this study reveals the spatiotemporal distribution of key proteins, including α-SMA, CD34, Osteopontin and E-cadherin, which play a crucial role in metanephrogenesis in camel foetuses., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Acetylcholine is the primary excitatory neurotransmitter in visceral smooth muscles, wherein it binds to and activates two muscarinic receptors subtypes, M 2 and M 3 , thus causing smooth muscle excitation and contraction. The first part of this review focuses on the types of cells involved in cholinergic neurotransmission and on the molecular mechanisms underlying acetylcholine-induced membrane depolarisation, which is the central event of excitation-contraction coupling causing Ca 2+ entry via L-type Ca 2+ channels and smooth muscle contraction. Studies of the muscarinic cation current in intestinal myocytes (mI CAT ) revealed its main molecular counterpart, receptor-operated TRPC4 channel, which is activated in synergy by both M 2 and M 3 receptors. M 3 receptors activation is of permissive nature, while activation of M 2 receptors via G i/o proteins that are coupled to them plays a direct role in TRPC4 opening. Our understanding of signalling pathways underlying mI CAT generation has vastly expanded in recent years through studies of TRPC4 gating in native cells and its regulation in heterologous cells. Recent studies using muscarinic receptor knockout have established that at low agonist concentration activation of both M 2 receptor and the M 2 /M 3 receptor complex elicits smooth muscle contraction, while at high agonist concentration M 3 receptor function becomes dominant. Based on this knowledge, in the second part of this review we discuss the cellular and molecular mechanisms underlying the numerous anticholinergic effects on neuroactive drugs, in particular general anaesthetics and anxiolytics, which can significantly impair gastrointestinal motility. This article is part of the Special Issue on "Ukrainian Neuroscience"., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Salivary gland myoepithelial cells regulate salivary secretion and have been implicated in the histological diversity of salivary gland tumors. However, isolation of myoepithelial cells has been difficult owing to a lack of detailed functional analysis and cell surface markers. Therefore, we aimed to isolate myoepithelial cells from adult mouse submandibular glands using the epithelial marker EpCAM and cell adhesion factor CD49f as indicators and characterize them via sphere-forming culture. Functional analysis of specific gene expression in myoepithelial cells is possible via cell transfection experiments using the piggyBac transposon vector system. Here, we describe detailed methods and tips for the isolation and functional analysis of myoepithelial cells., (© 2023. Springer Science+Business Media, LLC.)

There are few scientific reports on the histology of the phallus of ratite birds. The aim of this study was to conduct a histochemical analysis to determine the distribution of smooth muscle cells and the volumetric density (Vv) of the fibres of the elastic system in the ostrich phallus. Adult ostriches, 14 months old, were used. The phalluses were fixed in Bouin's solution and then transferred to a buffered formalin solution. They were then processed using standard histological stains for paraffin and slices were obtained. The following techniques were performed: HE, Picrosirius red, Alcian Blue at pH 1.0 and 2.5. The Periodic acid-Schiff reagent and Weigert's Resorcin-Fuchsin with previous oxidation were performed. The M42 test system was used to quantify the elastic system fibres. For immunohistochemical analysis, an anti alpha smooth muscle actin monoclonal antibody was used. The surface of the phallus is covered by a non-keratinized stratified squamous epithelium, which becomes stratified cylindrical in the region of the spermatic sulcus. No glands associated with the connective tissue were observed. The Vv of the elastic system fibres was 4.75%. Smooth muscle cells were visualized only in the walls of blood vessels through immunostaining, with an absence in the lymphatic sinuses. Despite similarities with other birds, such as the presence of a fibrous external axis, a lymphatic core, and a spermatic groove, the ostrich phallus shows marked differences, including the absence of an elastic core, a non-keratinized lining epithelium, and the absence of glands throughout its extension., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Competing Interests: The authors declare no conflicts of interest.

Background: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype.Methods: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin.Results: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype.Conclusions: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization. [ABSTRACT FROM AUTHOR]

The BBSome-a complex consisting of 8 Bardet-Biedl syndrome proteins-is involved in the regulation of various cellular processes. Recently, the BBSome complex has emerged as an important regulator of cardiovascular function with implications for disease. In this study, we examined the role of the BBSome in vascular smooth muscle and its effects on the regulation of cardiovascular function. Smooth muscle-specific disruption of the BBSome through tamoxifen-inducible deletion of Bbs1 gene-a critical component of the BBSome complex-reduces relaxation and enhances contractility of vascular rings and increases aortic stiffness independent of changes in arterial blood pressure. Mechanistically, we demonstrate that smooth muscle Bbs1 gene deletion increases vascular angiotensinogen gene expression implicating the renin-angiotensin system in these altered cardiovascular responses. Additionally, we report that smooth muscle-specific Bbs1 knockout mice demonstrate enhanced ET-1 (endothelin-1)-induced contractility of mesenteric arteries-an effect reversed by blockade of the AT1 (angiotensin type 1 receptor) with losartan. These findings highlight the importance of the smooth muscle BBSome in the control of vascular function and arterial stiffness through modulation of renin-angiotensin system signaling. [ABSTRACT FROM AUTHOR]