Smooth Contraction Research Articles

Aortic Remodeling Kinetics in Response to Coarctation-Induced Mechanical Perturbations.

Background: Coarctation of the aorta (CoA; constriction of the proximal descending thoracic aorta) is among the most common congenital cardiovascular defects. Coarctation-induced mechanical perturbations trigger a cycle of mechano-transduction events leading to irreversible precursors of hypertension including arterial thickening, stiffening, and vasoactive dysfunction in proximal conduit arteries. This study sought to identify kinetics of the stress-mediated compensatory response leading to these alterations using a preclinical rabbit model of CoA. Methods: A prior growth and remodeling (G&R) framework was reformulated and fit to empirical measurements from CoA rabbits classified into one control and nine CoA groups of various severities and durations (n = 63, 5-11/group). Empirical measurements included Doppler ultrasound imaging, uniaxial extension testing, catheter-based blood pressure, and wire myography, yielding the time evolution of arterial thickening, stiffening, and vasoactive dysfunction required to fit G&R constitutive parameters. Results: Excellent agreement was observed between model predictions and observed patterns of arterial thickening, stiffening, and dysfunction among all CoA groups. For example, predicted vascular impairment was not significantly different from empirical observations via wire myography (p-value > 0.13). Specifically, 48% and 45% impairment was observed in smooth muscle contraction and endothelial-dependent relaxation, respectively, which were accurately predicted using the G&R model. Conclusions: The resulting G&R model, for the first time, allows for prediction of hypertension precursors at neonatal ages that is currently challenging to examine in preclinical models. These findings provide a validated computational tool for prediction of persistent arterial dysfunction and identification of revised severity-duration thresholds that may ultimately avoid hypertension from CoA.

Unveiling Angiotensin II and Losartan-Induced Gene Regulatory Networks Using Human Urine-Derived Podocytes.

Podocytes are highly specialized cells that play a pivotal role in the blood filtration process in the glomeruli of the kidney, and their dysfunction leads to renal diseases. For this reason, the study and application of this cell type is of great importance in the field of regenerative medicine. Hypertension is mainly regulated by the renin-angiotensin-aldosterone system (RAAS), with its main mediator being angiotensin II (ANG II). Elevated ANG II levels lead to a pro-fibrotic, inflammatory, and hypertrophic milieu that induces apoptosis in podocytes. The activation of RAAS is critical for the pathogenesis of podocyte injury; as such, to prevent podocyte damage, patients with hypertension are administered drugs that modulate RAAS signaling. A prime example is the orally active, non-peptide, selective angiotensin-II-type I receptor (AGTR1) blocker losartan. Here, we demonstrate that SIX2-positive urine-derived renal progenitor cells (UdRPCs) and their immortalized counterpart (UM51-hTERT) can be directly differentiated into mature podocytes. These podocytes show activation of RAAS after stimulation with ANG II, resulting in ANG II-dependent upregulation of the expression of the angiotensin-II-type I receptor, AGTR1, and the downregulated expression of the angiotensin-II-type II receptor 2 (AGTR2). The stimulation of podocytes with losartan counteracts ANG II-dependent changes, resulting in a dependent favoring of the specific receptor from AGTR1 to AGTR2. Transcriptome analysis revealed 94 losartan-induced genes associated with diverse biological processes and pathways such as vascular smooth muscle contraction, the oxytocin signaling pathway, renin secretion, and ECM-receptor interaction. Co-stimulation with losartan and ANG II induced the exclusive expression of 106 genes associated with DNA methylation or demethylation, cell differentiation, the developmental process, response to muscle stretch, and calcium ion transmembrane transport. These findings highlight the usefulness of UdRPC-derived podocytes in studying the RAAS pathway and nephrotoxicity in various kidney diseases.

The role of mechano-regulated YAP/TAZ in erectile dysfunction

Phosphodiesterase type 5 inhibitors (PDE5is) constitute the primary therapeutic option for treating erectile dysfunction (ED). Nevertheless, a substantial proportion of patients, approximately 30%, do not respond to PDE5i treatment. Therefore, new treatment methods are needed. In this study, we identified a pathway that contributes to male erectile function. We show that mechano-regulated YAP/TAZ signaling in smooth muscle cells (SMCs) upregulates adrenomedullin transcription, which relaxed the SMCs to maintain erection. Using single-nucleus RNA sequencing, we investigated how penile erection stretches the SMCs, inducing YAP/TAZ activity. Subsequently, we demonstrate that YAP/TAZ plays a role in erectile function and penile rehabilitation, using genetic lesions and various animal models. This mechanism relies on direct transcriptional regulation of adrenomedullin by YAP/TAZ, which in turn modulates penile smooth muscle contraction. Importantly, conventional PDE5i, which targets NO-cGMP signaling, does not promote erectile function in YAP/TAZ-deficient ED model mice. In contrast, by activating the YAP/TAZ-adrenomedullin cascade, mechanostimulation improves erectile function in PDE5i nonrespondent ED model rats and mice. Furthermore, using clinical retrospective observational data, we found that mechanostimulation significantly promotes erectile function in patients irrespective of PDE5i use. Our studies lay the groundwork for exploring the mechano-YAP/TAZ-adrenomedullin axis as a potential target in the treatment of ED.

Structural basis of α1A-adrenergic receptor activation and recognition by an extracellular nanobody

The α1A-adrenergic receptor (α1AAR) belongs to the family of G protein-coupled receptors that respond to adrenaline and noradrenaline. α1AAR is involved in smooth muscle contraction and cognitive function. Here, we present three cryo-electron microscopy structures of human α1AAR bound to the endogenous agonist noradrenaline, its selective agonist oxymetazoline, and the antagonist tamsulosin, with resolutions range from 2.9 Å to 3.5 Å. Our active and inactive α1AAR structures reveal the activation mechanism and distinct ligand binding modes for noradrenaline compared with other adrenergic receptor subtypes. In addition, we identified a nanobody that preferentially binds to the extracellular vestibule of α1AAR when bound to the selective agonist oxymetazoline. These results should facilitate the design of more selective therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

The intermediate filament protein nestin serves as a molecular hub for smooth muscle cytoskeletal signaling

BackgroundThe recruitment of the actin-regulatory proteins cortactin and profilin-1 (Pfn-1) to the membrane is important for the regulation of actin cytoskeletal reorganization and smooth muscle contraction. Polo-like kinase 1 (Plk1) and the type III intermediate filament protein vimentin are involved in smooth muscle contraction. Regulation of complex cytoskeletal signaling is not entirely elucidated. The aim of this study was to evaluate the role of nestin (a type VI intermediate filament protein) in cytoskeletal signaling in airway smooth muscle.MethodsNestin expression in human airway smooth muscle (HASM) was knocked down by specific shRNA or siRNA. The effects of nestin knockdown (KD) on the recruitment of cortactin and Pfn-1, actin polymerization, myosin light chain (MLC) phosphorylation, and contraction were evaluated by cellular and physiological approaches. Moreover, we assessed the effects of non-phosphorylatable nestin mutant on these biological processes.ResultsNestin KD reduced the recruitment of cortactin and Pfn-1, actin polymerization, and HASM contraction without affecting MLC phosphorylation. Moreover, contractile stimulation enhanced nestin phosphorylation at Thr-315 and the interaction of nestin with Plk1. Nestin KD also diminished phosphorylation of Plk1 and vimentin. The expression of T315A nestin mutant (alanine substitution at Thr-315) reduced the recruitment of cortactin and Pfn-1, actin polymerization, and HASM contraction without affecting MLC phosphorylation. Furthermore, Plk1 KD diminished nestin phosphorylation at this residue.ConclusionsNestin is an essential macromolecule that regulates actin cytoskeletal signaling via Plk1 in smooth muscle. Plk1 and nestin form an activation loop during contractile stimulation.

#2696 IN SILICO ELECTROPHYSIOLOGICAL STUDY REVEALS TAMSULOSIN MEDIATE URETER SMOOTH MUSCLE CONTRACTION BY INCREASING POTASSIUM CURRENT

Abstract Background and Aims Tamsulosin is a common α1-AR antagonist that is prescribed to patients with acute kidney stone episodes [Scotland et al., 2021]. According to accumulated experimental data, abnormal peristaltic contraction of the ureter smooth muscle (USM) causes acute kidney stone episodes [Lang et al., 2006]. Intracellular electrical activities like membrane depolarization and action potentials play important roles in modulating the USM contraction by releasing intracellular calcium from the sarcoplasmic reticulum. Therefore, an electrophysiological study will be helpful to assess the USM cell's electrical activities and in diagnosing abnormal USM contraction. Transient potassium channels (KA) are prevalent in different systems and hold immense importance for maintaining/performing selective electrophysiological functions. The present study investigates the altered electrophysiological characteristics of the USM cell because of the induction of Tamsulosin doses. Method The single isolated USM cell comprises several voltage-gated ion channels, such as two voltage-gated calcium (T-type, and L-type) channels, one voltage-gated fast potassium (KA) channel, one calcium-dependent large conductance potassium channel, and an HCN channel. The individual ion channel currents and the membrane potential are recorded by utilizing both the voltage clamp and current clamp protocols. A Tamsulosin drug (0.5 mg) model for the KA channel is introduced by multiplying the maximal conductance of the KA channel with a scaling factor between 0 and 1 to mimic the drug concentration. Statistical analysis protocols are evaluated for analytical purposes. Results In this in silico electrophysiological model, the resting membrane potential is maintained at −75 mV, as this value is cited by various experimental studies. The ionic concentrations and biophysical parameters for all ion channels are varied in the physiological ranges. Under the voltage clamp protocol, the voltage steps are increased from a holding potential of −80 mV to −10 mV with a 10 mV of step voltage. In Figure 1, the red and blue solid lines represent recorded outward KA current from the isolated USM cell under both control and applied Tamsulosin dose conditions. Figure 1 reveals that the KA current is elevated after the application of the Tamsulosin drug regarding all step voltages. As a result, the total outward current is elevated and the USM action potential duration is shortened. It is also observed that the steady-state activation curve of the KA channel is shifted to more negative after applying Tamsulosin. The USM cell is electrically less excitable with the Tamsulosin. Conclusion This first in-silico study reveals the KA ionic current, which plays a major role in coordinating ureteral contractions, is altered due to Tamsulosin. It implies that the pharmacological maneuver of Tamsulosin is substantially beneficial to treat dysfunctional peristalsis by reducing the symptoms and installation complexities from the ureteral stents procedure.

#3357 MORPHOMETRIC AND FUNCTIONAL EVALUATION OF A NON-RENAL VASCULATURE IN FEMALE AND MALE PKD/MHM (CY/+) RATS

Abstract Background and Aims Polycystic kidney disease (PKD) is a cystic genetic disorder of the kidneys. Vascular abnormalities are the most important non-cystic complications in PKD. In the present study, we evaluated aortic morphometry and vascular function in female and male PKD/Mhm (Cy/+) rats, an animal model of autosomal dominant PKD. Method Thoracic aortic rings from six month-old heterozygous PKD/Mhm (Cy/+) female (n = 10; 307±4g) and male (n = 10; 470±24g) and age-matched non-affected homozygous (+/+) Control-female (n = 9; 312±6g) and Control-male (n = 13; 460±14g) rats were used a) to evaluate both contractile/relaxation responses ex vivo using organ bath experiments and b) to study aortic morphometry assessed by hematoxylin&eosin staining. Furthermore, immunohistochemistry for cleaved poly (ADP-ribose) polymerase (PARP)-1 was performed. Results Although there was no significant difference in body weight, serum creatinine levels were increased in the PKD-female and PKD-male rats compared with the respective controls (PKD-female 50.0±1.7 vs. Control-female 43.9±2.1 µmol/L; PKD-male 178.7±21.0 vs. Control-male 40.4± 1.3 µmol/L, p<0.05) and were found to be higher in PKD-male rats than in the PKD-females (p<0.0001). Compared to Control-male, PKD-male rats showed decreased maximum relaxation to acetylcholine (55±2% vs. 77±1%, p<0.05), indicating impaired endothelial function. Contractile responses to phenylephrine (3.0±0.1g vs. 3.4±0.1g, p<0.05) and high potassium (3.3±0.1g vs. 3.7±0.1g, p<0.05) were reduced in the PKD-male compared to Control-male. Additionally, in PKD-male aortas, the concentration-response curve to sodium nitroprusside, an endothelium-independent vasodilator, was shifted to the right compared to Control-male. Morphometrical analysis revealed that wall thickness and wall cross-sectional area normalized to body weight, and the wall:lumen area ratio were significantly higher in PKD-male aortas compared to Control-male. Additionally, PKD-male showed increased cleaved PARP-1 immunoreactivity confined to endothelial level compared to Control-male. All these parameters were unchanged in PKD-female rats compared to Control-female. Conclusion Six month-old PKD-male, not female, rats show endothelial dysfunction, impaired smooth muscle contraction and relaxation, pathological changes in aortic morphometry, and apoptosis in a non-renal vasculature. The rat model of autosomal dominant PKD in males can be used to identify novel targets for the treatment of this disease.

Involvement of Rictor/mTORC2/Akt/GLUT4 pathway in the regulation of energy metabolism in the gastric smooth muscle of diabetic rats.

Diabetes mellitus can be accompanied by a variety of complications. The purpose of the present study was to characterize the Rictor/mTOR complex 2 (mTORC2)/Akt/glucose transporter 4 (GLUT4) pathway and its effects on energy metabolism in the gastric smooth muscle of diabetic rats. Diabetes was induced in rats using streptozotocin and their phenotype was compared with untreated rats. The relationship between gastric motility and energy metabolism was analyzed by comparing the contraction and ATP metabolism of muscle strips. Western blotting was used to detect the expression of key proteins in the pathway. The diabetic rats demonstrated less frequent and less powerful gastric smooth muscle contractions. The concentrations of ADP, AMP, and ATP, and the energy charge in gastric smooth muscle changed in different periods of diabetes, and these changes were consistent with changes in mechanistic target of rapamycin (mTOR) protein content. The expression of the key intermediates in signal transduction in the Rictor/mTORC2/Akt/GLUT4 pathway also underwent significant changes. Rictor protein expression increased during the development of diabetes, but the activation of mTORC2 did not increase with the increase in Rictor expression. GLUT4 translocation is regulated by Akt and its expression change during the development of diabetes. These findings suggest that altered energy metabolism is present in gastric smooth muscle that is associated with changes in the Rictor/mTORC2/Akt/GLUT4 pathway. Rictor/mTORC2/Akt/GLUT4 pathway may be involved in the regulation of energy metabolism in the gastric smooth muscle of diabetic rats and the development of diabetic gastroparesis.

Evolution and function of calponin and transgelin.

Calponin and transgelin (originally named SM22) are homologous cytoskeleton proteins that regulate actin-activated myosin motor functions in smooth muscle contraction and non-muscle cell motility during adhesion, migration, proliferation, phagocytosis, wound healing, and inflammatory responses. They are abundant cytoskeleton proteins present in multiple cell types whereas their physiological functions remain to be fully established. This focused review summarizes the evolution of genes encoding calponin and transgelin and their isoforms and discusses the structural similarity and divergence in vertebrate and invertebrate species in the context of functions in regulating cell motility. As the first literature review focusing on the evolution of the calponin-transgelin family of proteins in relevance to their structure-function relationship, the goal is to outline a foundation of current knowledge for continued investigations to understand the biological functions of calponin and transgelin in various cell types during physiological and pathological processes.

A dynamical model of TGF-β activation in asthmatic airways.

Excessive activation of the regulatory cytokine transforming growth factor $\beta $ (TGF-$\beta $) via contraction of airway smooth muscle (ASM) is associated with the development of asthma. In this study, we develop an ordinary differential equation model that describes the change in density of the key airway wall constituents, ASM and extracellular matrix (ECM), and their interplay with subcellular signalling pathways leading to the activation of TGF-$\beta $. We identify bistable parameter regimes where there are two positive steady states, corresponding to either reduced or elevated TGF-$\beta $ concentration, with the latter leading additionally to increased ASM and ECM density. We associate the former with a healthy homeostatic state and the latter with a diseased (asthmatic) state. We demonstrate that external stimuli, inducing TGF-$\beta $ activation via ASM contraction (mimicking an asthmatic exacerbation), can perturb the system irreversibly from the healthy state to the diseased one. We show that the properties of the stimuli, such as their frequency or strength, and the clearance of surplus active TGF-$\beta $, are important in determining the long-term dynamics and the development of disease. Finally, we demonstrate the utility of this model in investigating temporal responses to bronchial thermoplasty, a therapeutic intervention in which ASM is ablated by applying thermal energy to the airway wall. The model predicts the parameter-dependent threshold damage required to obtain irreversible reduction in ASM content, suggesting that certain asthma phenotypes are more likely to benefit from this intervention.

Inherent differences of small airway contraction and Ca2+ oscillations in airway smooth muscle cells between BALB/c and C57BL/6 mouse strains.

BALB/c and C57BL/6 mouse strains are widely used as animal model in studies of respiratory diseases, such as asthma. Asthma is characterized by airway hyperresponsiveness, which is eventually resulted from the excessive airway smooth muscle (ASM) contraction mediated by Ca2+ oscillations in ASM cells. It is reported that BALB/c mice have inherently higher airway responsiveness, but show no different contractive response of tracheal ring as compared to C57BL/6 mice. However, whether the different airway responsiveness is due to the different extents of small airway contraction, and what's underlying mechanism remains unknown. Here, we assess agonist-induced small airway contraction and Ca2+ oscillations in ASM cells between BALB/c and C57BL/6 mice by using precision-cut lung slices (PCLS). We found that BALB/c mice showed an intrinsically stronger extent of small airway narrowing and faster Ca2+ oscillations in ASM cells in response to agonists. These differences were associated with a higher magnitude of Ca2+ influx via store-operated Ca2+ entry (SOCE), as a result of increased expression of SOCE components (STIM1, Orai1) in the ASM cells of small airway of BALB/c mice. An established mathematical model and experimental results suggested that the increased SOC current could result in increased agonist-induced Ca2+ oscillations. Therefore, the inherently higher SOC underlies the increased Ca2+ oscillation frequency in ASM cells and stronger small airway contraction in BALB/c mice, thus higher airway responsiveness in BALB/c than C57BL/6 mouse strain.

Hypoxic response patterns in lung tissue: An integrated analysis of comparative physiological and transcriptomic studies from Neodon fuscus and Lasiopodomys brandtii

Oxygen (O2) is essential for the survival and reproduction of most species. However, in recent years, with global climate change and the increasing impact of human activities on the ecosystem, the frequency of extreme environmental events, such as heat waves, cold waves, droughts, and floods, has increased, resulting in dramatic changes in environmental oxygen concentration (hypoxic environment, etc.), threatening the survival of animals and pushing them toward extreme adaptation. In this context, how organisms, especially those with differences in original habitats, adapt to low oxygen environment is particularly important. In this study, we systematically analyzed hypoxic response patterns in lung tissues of small rodents Neodon fuscus, Lasiopodomys brandtii, and Mus musculus with different experiences of natural hypoxia tolerance through laboratory simulation of hypoxia environment, combined with hematological, histological, and transcriptomic analysis. Our results show that all three species exhibit increased antioxidant defense and damage repair ability to a certain extent under hypoxia, although the specific molecular mechanisms are not the same. L. brandtii showed better damage repair ability than the others, which is likely to be closely related to the intermittent hypoxia environment experienced in the natural environment, and genes such as Glrx5, Prdx2, Col1a1, Lama1, and Eln may play a crucial role in this process. In addition, we found that both N. fuscus and L. brandtii appropriately enhanced oxygen transport in tissues under hypoxic conditions, with a series of functional genes related to hemoglobin synthesis and vascular smooth muscle contraction were significantly up-regulated in both species of voles, such as Actg2, Ptgir, Alas2, Hba, Hbb and Bpgm. Our results, to a certain extent, reveal the differences and similarities of hypoxic response patterns in lung tissues of small rodents with different hypoxic life histories and provide a relatively perfect analytical paradigm for related studies.

Quantitative Proteomic and Phosphoproteomic Profiling of Lung Tissues from Pulmonary Arterial Hypertension Rat Model.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH.

A 19-protein signature to predict outcomes in a cohort of stage II and III CRC.

3129 Background: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide and remains an area of unmet clinical need. Despite advances in treating stage II and III disease, 30-50% of patients relapse. Additionally, variable survival outcomes are seen in patients with similar disease stages, making it difficult to predict relapses. Here we outline some of the proteomic pathways of early-stage CRC along with the identification of a proteomic-based prognostic signature that predicts relapse in stage II/III CRC. Methods: Archival formalin fixed paraffin embedded tumour (TUM) specimens and matched normal adjacent tissues (NAT) from a cohort of 495 CRC patients (with upfront resected stage II and III CRC) were prepared for proteomic analyses using data-independent acquisition mass spectrometry (DIA-MS). TUM samples with &lt; 20% tumour were excluded from the analysis. Differential expression analyses were performed to identify the top differentially expressed proteins (DEP) within TUM. DEP were subjected to crosstalk and pathway enrichment analysis (PEA). Survival analysis based on initial univariate and subsequent 100 runs of multivariate Cox regression with Least Absolute Shrinkage and Selection Operator (LASSO) was performed to obtain a reduced list of candidate proteins associated with recurrence-free survival (RFS). The proteins that appeared in greater than 95% of the LASSO runs were used in a multivariate Cox model with recursive feature selection to yield the final list of 19 proteins. A risk score was calculated as the sum of the intensities of each of the 19 proteins multiplied by the corresponding regression coefficients. The median of the risk scores was used as the threshold to dichotomise patients into either a low- or a high-risk group. Results: 399 TUM and 381 NAT were analysed, showing 799 DEP, with 529 upregulated and 270 downregulated in TUM vs NAT, respectively. PEA of the upregulated proteins showed enrichment of pathways related to extracellular matrix organisation, tRNA processing and collagen formation. PEA of the downregulated DEPs revealed pathways related to smooth muscle contraction, focal adhesion and oxidation. The 19-protein signature dichotomised patients into low- and high-risk groups with a C-index of 0.829. The signature was prognostic for RFS (hazard ratio (HR) 2.7, 95% confidence interval (CI): [2.24 – 3.18]) and for overall survival (OS) (HR 1.6, 95%CI [1.40, 1.76]). The prognostic significance of the signature was maintained after adjusting for other clinical variables for both RFS and OS (both p&lt;0.001) using multivariate Cox regression. Conclusions: Our findings show that CRC disrupts the extracellular matrix and upregulates collagen formation, making these potential targets for novel therapies. We have also identified a 19-protein signature that is associated with survival in our cohort independent of other clinical variables.

Single-cell analysis of adult human heart across healthy and cardiovascular disease patients reveals the cellular landscape underlying SARS-CoV-2 invasion of myocardial tissue through ACE2

BackgroundThe distribution of ACE2 and accessory proteases (ANAD17 and CTSL) in cardiovascular tissue and the host cell receptor binding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are crucial to understanding the virus’s cell invasion, which may play a significant role in determining the viral tropism and its clinical manifestations.MethodsWe conducted a comprehensive analysis of the cell type-specific expression of ACE2, ADAM17, and CTSL in myocardial tissue from 10 patients using RNA sequencing. Our study included a meta-analysis of 2 heart single-cell RNA-sequencing studies with a total of 90,024 cells from 250 heart samples of 10 individuals. We used co-expression analysis to locate specific cell types that SARS-CoV-2 may invade.ResultsOur results revealed cell-type specific associations between male gender and the expression levels of ACE2, ADAM17, and CTSL, including pericytes and fibroblasts. AGT, CALM3, PCSK5, NRP1, and LMAN were identified as potential accessory proteases that might facilitate viral invasion. Enrichment analysis highlighted the extracellular matrix interaction pathway, adherent plaque pathway, vascular smooth muscle contraction inflammatory response, and oxidative stress as potential immune pathways involved in viral infection, providing potential molecular targets for therapeutic intervention. We also found specific high expression of IFITM3 and AGT in pericytes and differences in the IFN-II signaling pathway and PAR signaling pathway in fibroblasts from different cardiovascular comorbidities.ConclusionsOur data indicated possible high-risk groups for COVID-19 and provided emerging avenues for future investigations of its pathogenesis.Trial registration(Not applicable).

Recurrent implantation failure versus recurrent implantation success: a preliminary study at proteomic level

Objective: This study aimed to find proteomic differences in endometrial receptivity between recurrent implantation failure (RIF) and recurrent implantation success (RIS). Methods This study was a basic proteomic analysis of endometrial biopsies from RIF and RIS women. Differentially expressed proteins between RIF and RIS were screened via isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Furthermore, bioinformatic analysis was launched to analyze the difference in the endometrial receptivity between RIF and RIS women. Quantitative P CR and Western blotting were used to validated the study. Results This study identified 625 differentially expressed proteins in the RIF women compared with RIS women. Bioinformatics analysis demonstrated that these differentially expressed proteins were enriched in ‘Wnt signaling,’ ‘Vascular smooth muscle contraction,’ ‘Ubiquitin mediated proteolysis,’ ‘Tight junction,’ or ‘TGF-beta signaling’ related processes or pathways. Quantitative PCR and Western blotting validation experiments revealed that Claudin-4, p38, PRKAB, IKB-b were unregulated, myosin-2, PKG2 were downregulated significantly in RIF. Conclusion This study indicated that the alternations of key signal pathways play important roles in the failure of embryo implantation, further studies are warranted to verify if targeting these molecules might be useful to improve the embryo implantation or not.

A pharmacologically pre-contracted smooth muscle bowel model for the study of highly-potent opioid receptor agonists and antagonists

Isolated organ models are a versatile tool for pharmacological and toxicological research. Small bowel has been used to assess the inhibition of smooth muscle contraction by opioids. In the present study, we set out to establish a pharmacologically stimulated rat bowel model. The effects of carfentanil, remifentanil and the new synthetic opioid U-48800 and their respective antagonists naloxone, nalmefene and naltrexone were studied in a small bowel model in rats. The IC50 values of the tested opioids were as follows: carfentanil (IC50 = 0.02 µmol/L, CI 0.02–0.03 µmol/L) ≫ remifentanil (IC50 = 0.51 µmol/L, CI 0.40–0.66 µmol/L) ≫ U-48800 (IC50 = 1.36 µmol/L, CI 1.20–1.54 µmol/L). The administration of the opioid receptor antagonists naloxone, naltrexone and nalmefene led to progressive, parallel rightward shifts of the dose-response curves. Naltrexone was most potent in antagonizing the effects of U-48800, whereas naltrexone and nalmefene were most effective in antagonizing the effects of carfentanil. In summary, the current model seems to be a robust tool to study opioid effects in a small bowel model without the necessity of using electrical stimulation.

Focal adhesion kinase activation is involved in contractile stimulation-induced detrusor muscle contraction in mice

Recent studies suggested smooth muscle contraction may involve mechanisms besides the myosin regulatory light chain (MLC) phosphorylation-induced actomyosin crossbridge cycling. This study aims to determine if focal adhesion kinase (FAK) activation is involved in mouse detrusor muscle contraction. The mouse detrusor muscle strips were preincubated with PF-573228 (2 μM), latrunculin B (1 μM), or the same volume of vehicle (DMSO) for 30 min. The contractile responses to KCl (90 mM), electrical field stimulation (EFS, 2–32 Hz), or carbachol (CCh, 10−7.5-10−4.5 M) were measured. In a separate experiment, the phosphorylated FAK (p-FAK) and MLC (p-MLC) levels were measured in the detrusor strips stimulated with CCh (10 μM) after incubation with PF-573228 or vehicle (DMSO) compared to those with vehicle incubation but without CCh stimulation. KCl-induced contractile responses decreased significantly after incubation with PF-573228 or latrunculin B compared to the corresponding vehicle-treated strips (p < 0.0001). The contractile responses induced by EFS were markedly inhibited by preincubation with PF-573228 at 8, 16, and 32 Hz (p < 0.05) or latrunculin B at 16 and 32 Hz (p < 0.01). Following the application of PF-573228 or Latrunculin B, CCh-induced dose-response contractions were lower than the corresponding vehicle group (p = 0.0021 and 0.0003, respectively). Western blot examination showed that CCh stimulation enhanced the expression of p-FAK and p-MLC, while preincubation with PF-573228 prevented the increase of p-FAK but not p-MLC. In conclusion, FAK activation involves tension development induced by contractile stimulation in the mouse detrusor muscle. This effect is likely caused by promoting actin polymerization rather than elevating MLC phosphorylation.

A Biomimetic Approach Utilizing Pulsatile Perfusion Generates Contractile Vascular Grafts.

Surgical implantation of decellularized cadaveric arteries is routinely used to treat right-sided congenital cardiac lesions. These acellular conduits lack the capacity for somatic growth and are prone to stenosis and calcification, necessitating multiple operations throughout childhood. Islet-1+ cardiovascular progenitor cells (CPCs) have demonstrated the capacity for differentiation into all cell types of the heart and outflow tracts. We hypothesize that CPC seeding of decellularized pulmonary arteries and bioreactor culture under physiologic flow conditions will drive vascular differentiation of CPCs and result in a conduit more suitable for implantation and long-term growth. We began by decellularizing ovine pulmonary arteries and characterizing the composition of the extracellular matrix (ECM). Hemodynamic testing of decellularized vessels in a custom bioreactor was used to define the scaffold mechanical properties over a range of pressures and flow rates. Next, our expanded ovine CPCs were suspended in growth media and injected intramurally into decellularized pulmonary arteries that were subsequently cultured in either static or pulsatile cultures. A combination of immunohistochemistry, real-time polymerase chain reaction (PCR), and tissue bath contraction studies were used to evaluate the bioengineered arteries before transplantation. Pulmonary artery patches from the most favorable culture conditions were then implanted into juvenile sheep to provide proof of concept. Hematoxylin and eosin staining indicated complete removal of cell nuclei (n = 9), whereas double-stranded DNA isolation from tissue homogenates showed 99.1% DNA removal (p < 0.01, n = 4). Furthermore, trichrome and elastin staining verified maintenance of collagen and elastin. Immunohistochemistry and PCR analyses (n = 4 per group) confirmed contractile smooth muscle presence on only our 3-week pulsatile scaffolds via presence of calponin 1 and myosin heavy chain 11. Tissue bath studies demonstrated that smooth muscle contraction generated by our 3-week pulsatile scaffolds (2.23 ± 0.19 g, n = 4) is comparable with native tissue contraction strength (2.78 ± 0.06 g, n = 4). Ovine transplantation confirmed that our graft can be safely implanted, retains contractile smooth muscle cells, and recruits native endothelium. Longer duration of physiologic pulsatile culture drives differentiation of CPCs seeded on ECM conduits toward a mature, contractile phenotype that is maintained for several weeks in vivo. Longer term studies to assess somatic growth potential are needed. Impact statement The current field of vascular transplantation relies on cadaveric and synthetic grafts to treat right-sided congenital cardiac lesions. These grafts do not grow somatically with our patients. This results in multiple reoperations throughout childhood to increase the size of the graft. Our bioengineered alternative demonstrates successful implantation, contractile smooth muscle cells, and a native endothelial layer. This research demonstrates a pilot study confirming the viability of a bioengineered alternative to the current standard of care in the field of vascular transplantation.

The mast cell stimulator compound 48/80 causes urothelium-dependent increases in murine urinary bladder contractility.

Mast cells and degranulation of preformed inflammatory mediators contribute to lower urinary tract symptoms. This study investigated pathways by which the mast cell stimulator compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that 1) mast cell degranulation causes spontaneous urinary bladder smooth muscle contractions and 2) these contractions are caused by urothelium-derived PGE2. Urothelium-intact and -denuded urinary bladder strips were collected from mast cell-sufficient (C57Bl/6) and mast cell-deficient (B6.Cg-Kitw-sh) mice to determine if compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were used to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell-sufficient and -deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blockade of P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished compound 48/80 responses. However, only combined blockade of PGE2 (EP1), PGF2α (FP), and thromboxane A2 (TP) receptors inhibited compound 48/80-induced responses. Thus, the effects of compound 48/80 are urothelium dependent but independent of mast cells. Furthermore, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory nonneurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using compound 48/80 to determine mast cell-dependent responses in the urinary bladder.NEW & NOTEWORTHY Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.