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The Journal of Allergy and Clinical... Nov 2023The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could...
BACKGROUND
The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signaling in the regulation of airway smooth muscle (ASM) contraction, airway hyperresponsiveness, and airway remodeling, but evidence from human studies is lacking.
OBJECTIVE
We sought to compare the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo, and in murine airways dissected from healthy and house dust mite (HDM)-sensitized animals.
METHODS
Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilization assays were used to study human ASM cell responses.
RESULTS
S1P did not induce contraction of airways isolated from healthy and HDM-exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, a 30-minute exposure to S1P induced a significant concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways, respectively. HDM-sensitized mice demonstrated a significant increase in methacholine responsiveness, which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human ASM cells, an effect mediated through S1P receptor type 2, as shown by selective antagonism and S1P receptor type 2 small-interfering RNA knockdown.
CONCLUSIONS
Our data suggest that S1P released locally into the airways may be involved in the regulation of ASM hyperresponsiveness and hyperplasia, defining a novel target for future therapies.
Topics: Humans; Mice; Animals; Sphingosine-1-Phosphate Receptors; Methacholine Chloride; Asthma; Muscle, Smooth; Cell Proliferation
PubMed: 37474025
DOI: 10.1016/j.jaci.2023.05.028 -
PloS One 2023People experiencing asthma exacerbations are at increased risk of cardiovascular events. To better understand the relationship between asthma exacerbations and... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
People experiencing asthma exacerbations are at increased risk of cardiovascular events. To better understand the relationship between asthma exacerbations and cardiovascular risk, this randomized case-control, cross-over controlled trial assessed the immediate systemic inflammatory and vascular responses to acutely induced pulmonary inflammation and bronchoconstriction in people with asthma and controls.
METHODS
Twenty-six people with asthma and 25 controls underwent three airway challenges (placebo, mannitol, and methacholine) in random order. Markers of cardiovascular risk, including serum C-reactive protein, interleukin-6, and tumor necrosis factor, endothelial function (flow-mediated dilation), microvascular function (blood-flow following reactive hyperemia), and arterial stiffness (pulse wave velocity) were evaluated at baseline and within one hour following each challenge. The systemic responses in a) asthma/control and b) positive airway challenges were analyzed. (ClinicalTrials.gov reg# NCT02630511).
RESULTS
Both the mannitol and methacholine challenges resulted in clinically significant reductions in forced expiratory volume in 1 second (FEV1) in asthma (-7.6% and -17.9%, respectively). Following positive challenges, reduction in FEV1 was -27.6% for methacholine and -14.2% for mannitol. No meaningful differences in predictors of cardiovascular risk were observed between airway challenges regardless of bronchoconstrictor response.
CONCLUSION
Neither acutely induced bronchoconstriction nor pulmonary inflammation and bronchoconstriction resulted in meaningful changes in systemic inflammatory or vascular function. These findings question whether the increased cardiovascular risk associated with asthma exacerbations is secondary to acute bronchoconstriction or inflammation, and suggest that other factors need to be further evaluated such as the cardiovascular impacts of short-acting inhaled beta-agonists.
Topics: Humans; Methacholine Chloride; Cardiovascular Diseases; Pulse Wave Analysis; Risk Factors; Asthma; Bronchoconstriction; Bronchial Provocation Tests; Forced Expiratory Volume
PubMed: 37459335
DOI: 10.1371/journal.pone.0288623 -
Respiratory Research Oct 2023The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß-sympathomimetics) and, depending on the severity of disease, additional...
INTRODUCTION
The standard therapy for bronchial asthma consists of combinations of acute (short-acting ß-sympathomimetics) and, depending on the severity of disease, additional long-term treatment (including inhaled glucocorticoids, long-acting ß-sympathomimetics, anticholinergics, anti-IL-4R antibodies). The antidepressant amitriptyline has been identified as a relevant down-regulator of immunological T2-phenotype in asthma, acting-at least partially-through inhibition of acid sphingomyelinase (ASM), an enzyme involved in sphingolipid metabolism. Here, we investigated the non-immunological role of amitriptyline on acute bronchoconstriction, a main feature of airway hyperresponsiveness in asthmatic disease.
METHODS
After stimulation of precision cut lung slices (PCLS) from mice (wildtype and ASM-knockout), rats, guinea pigs and human lungs with mediators of bronchoconstriction (endogenous and exogenous acetylcholine, methacholine, serotonin, endothelin, histamine, thromboxane-receptor agonist U46619 and leukotriene LTD4, airway area was monitored in the absence of or with rising concentrations of amitriptyline. Airway dilatation was also investigated in rat PCLS by prior contraction induced by methacholine. As bronchodilators for maximal relaxation, we used IBMX (PDE inhibitor) and salbutamol (ß-adrenergic agonist) and compared these effects with the impact of amitriptyline treatment. Isolated perfused lungs (IPL) of wildtype mice were treated with amitriptyline, administered via the vascular system (perfusate) or intratracheally as an inhalation. To this end, amitriptyline was nebulized via pariboy in-vivo and mice were ventilated with the flexiVent setup immediately after inhalation of amitriptyline with monitoring of lung function.
RESULTS
Our results show amitriptyline to be a potential inhibitor of bronchoconstriction, induced by exogenous or endogenous (EFS) acetylcholine, serotonin and histamine, in PCLS from various species. The effects of endothelin, thromboxane and leukotrienes could not be blocked. In acute bronchoconstriction, amitriptyline seems to act ASM-independent, because ASM-deficiency (Smdp1) did not change the effect of acetylcholine on airway contraction. Systemic as well as inhaled amitriptyline ameliorated the resistance of IPL after acetylcholine provocation. With the flexiVent setup, we demonstrated that the acetylcholine-induced rise in central and tissue resistance was much more marked in untreated animals than in amitriptyline-treated ones. Additionally, we provide clear evidence that amitriptyline dilatates pre-contracted airways as effectively as a combination of typical bronchodilators such as IBMX and salbutamol.
CONCLUSION
Amitriptyline is a drug of high potential, which inhibits acute bronchoconstriction and induces bronchodilatation in pre-contracted airways. It could be one of the first therapeutic agents in asthmatic disease to have powerful effects on the T2-allergic phenotype and on acute airway hyperresponsiveness with bronchoconstriction, especially when inhaled.
Topics: Mice; Rats; Humans; Animals; Guinea Pigs; Bronchoconstriction; Methacholine Chloride; Amitriptyline; Histamine; Bronchodilator Agents; Serotonin; Acetylcholine; Sympathomimetics; 1-Methyl-3-isobutylxanthine; Dilatation; Lung; Asthma; Albuterol; Endothelins; Thromboxanes
PubMed: 37907918
DOI: 10.1186/s12931-023-02580-6 -
The Journal of Clinical Investigation Sep 2023Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote - with...
Asthma is a chronic inflammatory disease associated with episodic airway narrowing. Inhaled β2-adrenergic receptor (β2AR) agonists (β2-agonists) promote - with limited efficacy - bronchodilation in asthma. All β2-agonists are canonical orthosteric ligands that bind the same site as endogenous epinephrine. We recently isolated a β2AR-selective positive allosteric modulator (PAM), compound-6 (Cmpd-6), which binds outside of the orthosteric site and modulates orthosteric ligand functions. With the emerging therapeutic potential of G-protein coupled receptor allosteric ligands, we investigated the impact of Cmpd-6 on β2AR-mediated bronchoprotection. Consistent with our findings using human β2ARs, Cmpd-6 allosterically potentiated β2-agonist binding to guinea pig β2ARs and downstream signaling of β2ARs. In contrast, Cmpd-6 had no such effect on murine β2ARs, which lack a crucial amino acid in the Cmpd-6 allosteric binding site. Importantly, Cmpd-6 enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in guinea pig lung slices, but - in line with the binding studies - not in mice. Moreover, Cmpd-6 robustly potentiated β2 agonist-mediated bronchoprotection against allergen-induced airway constriction in lung slices obtained from a guinea pig model of allergic asthma. Cmpd-6 similarly enhanced β2 agonist-mediated bronchoprotection against methacholine-induced bronchoconstriction in human lung slices. Our results highlight the potential of β2AR-selective PAMs in the treatment of airway narrowing in asthma and other obstructive respiratory diseases.
Topics: Humans; Mice; Animals; Guinea Pigs; Methacholine Chloride; Ligands; Asthma; Lung; Binding Sites; Receptors, Adrenergic, beta-2
PubMed: 37432742
DOI: 10.1172/JCI167337 -
Brain and Nerve = Shinkei Kenkyu No... Dec 2023Eserine, well-known as physostigmine, is classified as an alkaloid. It is a cholinesterase inhibitor and appears in Agatha Christie's novel entitled, Crooked House and...
Eserine, well-known as physostigmine, is classified as an alkaloid. It is a cholinesterase inhibitor and appears in Agatha Christie's novel entitled, Crooked House and Curtain: Poirot's Last Case. In clinical medicine, eserine was used as an ophthalmic treatment for glaucoma and considered as a treatment for myasthenia gravis, Alzheimer's disease, and hereditary cerebellar ataxias. Currently, it is used as a treatment for anticholinergic poisoning.
Topics: Humans; Physostigmine; Cholinesterase Inhibitors; Myasthenia Gravis
PubMed: 38097226
DOI: 10.11477/mf.1416202536 -
Journal of Dental Research Feb 2024Tight junction proteins play a crucial role in paracellular transport in salivary gland epithelia. It is clear that severe xerostomia in patients with HELIX syndrome is...
Tight junction proteins play a crucial role in paracellular transport in salivary gland epithelia. It is clear that severe xerostomia in patients with HELIX syndrome is caused by mutations in the claudin-10 gene. However, little is known about the expression pattern and role of claudin-10 in saliva secretion in physical and disease conditions. In the present study, we found that only claudin-10b transcript was expressed in human and mouse submandibular gland (SMG) tissues, and claudin-10 protein was dominantly distributed at the apicolateral membranes of acini in human, rat, and mouse SMGs. Overexpression of claudin-10 significantly reduced transepithelial electrical resistance and increased paracellular transport of dextran and Na in SMG-C6 cells. In C57BL/6 mice, pilocarpine stimulation promoted secretion and cation concentration in saliva in a dose-dependent increase. Assembly of claudin-10 to the most apicolateral portions in acini of SMGs was observed in the lower pilocarpine (1 mg/kg)-treated group, and this phenomenon was much obvious in the higher pilocarpine (10 mg/kg)-treated group. Furthermore, 7-, 14-, and 21-wk-old nonobese diabetic (NOD) and BALB/c mice were used to mimic the progression of hyposalivation in Sjögren syndrome. Intensity of claudin-10 protein was obviously lower in SMGs of 14- and 21-wk-old NOD mice compared with that of age-matched BALB/c mice. In the cultured mouse SMG tissues, interferon-γ (IFN-γ) downregulated claudin-10 expression. In claudin-10-overexpressed SMG-C6 cells, paracellular permeability was decreased. Furthermore, IFN-γ stimulation increased p-STAT1 level, whereas pretreatment with JAK/STAT1 antagonist significantly alleviated the IFN-γ-induced claudin-10 downregulation. These results indicate that claudin-10 functions as a pore-forming component in acinar epithelia of SMGs, assembly of claudin-10 is required for saliva secretion, and downregulation of claudin-10 induces hyposecretion. These findings may provide new clues to novel therapeutic targets on hyposalivation.
Topics: Humans; Mice; Rats; Animals; Submandibular Gland; Pilocarpine; Mice, Inbred C57BL; Claudins; Sjogren's Syndrome; Tight Junctions; Xerostomia; Claudin-4
PubMed: 38058154
DOI: 10.1177/00220345231210547 -
JCI Insight Jan 2024Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of...
Prolonged seizures can disrupt stem cell behavior in the adult hippocampus, an important brain structure for spatial memory. Here, using a mouse model of pilocarpine-induced status epilepticus (SE), we characterized spatiotemporal expression of Lin28a mRNA and proteins after SE. Unlike Lin28a transcripts, induction of LIN28A protein after SE was detected mainly in the subgranular zone, where immunoreactivity was found in progenitors, neuroblasts, and immature and mature granule neurons. To investigate roles of LIN28A in epilepsy, we generated Nestin-Cre:Lin28aloxP/loxP (conditional KO [cKO]) and Nestin-Cre:Lin28a+/+ (WT) mice to block LIN28A upregulation in all neuronal lineages after acute seizure. Adult-generated neuron- and hippocampus-associated cognitive impairments were absent in epileptic LIN28A-cKO mice, as evaluated by pattern separation and contextual fear conditioning tests, respectively, while sham-manipulated WT and cKO animals showed comparable memory function. Moreover, numbers of hilar PROX1-expressing ectopic granule cells (EGCs), together with PROX1+/NEUN+ mature EGCs, were significantly reduced in epileptic cKO mice. Transcriptomics analysis and IHC validation at 3 days after pilocarpine administration provided potential LIN28A downstream targets such as serotonin receptor 4. Collectively, our findings indicate that LIN28A is a potentially novel target for regulation of newborn neuron-associated memory dysfunction in epilepsy by modulating seizure-induced aberrant neurogenesis.
Topics: Animals; Nestin; Pilocarpine; Seizures; Status Epilepticus; Hippocampus; Neurogenesis; Epilepsy
PubMed: 38193536
DOI: 10.1172/jci.insight.175627 -
Scientific Reports Dec 2023Transient brain insults including status epilepticus (SE) can initiate a process termed 'epileptogenesis' that results in chronic temporal lobe epilepsy. As a...
Transient brain insults including status epilepticus (SE) can initiate a process termed 'epileptogenesis' that results in chronic temporal lobe epilepsy. As a consequence, the entire tri-synaptic circuit of the hippocampus is fundamentally impaired. A key role in epileptogenesis has been attributed to the CA1 region as the last relay station in the hippocampal circuit and as site of aberrant plasticity, e.g. mediated by acquired channelopathies. The transcriptional profiles of the distinct hippocampal neurons are highly dynamic during epileptogenesis. Here, we aimed to elucidate the early SE-elicited mRNA signature changes and the respective upstream regulatory cascades in CA1. RNA sequencing of CA1 was performed in the mouse pilocarpine-induced SE model at multiple time points ranging from 6 to 72 h after the initial insult. Bioinformatics was used to decipher altered gene expression, signalling cascades and their corresponding cell type profiles. Robust transcriptomic changes were detected at 6 h after SE and at subsequent time points during early epileptogenesis. Major differentially expressed mRNAs encoded primarily immediate early and excitability-related gene products, as well as genes encoding immune signalling factors. Binding sites for the transcription factors Nfkb1, Spi1, Irf8, and two Runx family members, were enriched within promoters of differentially expressed genes related to major inflammatory processes, whereas the transcriptional repressors Suz12, Nfe2l2 and Rest were associated with hyperexcitability and GABA / glutamate receptor activity. CA1 quickly responds to SE by inducing transcription of genes linked to inflammation and excitation stress. Transcription factors mediating this transcriptomic switch represent targets for new highly selected, cell type and time window-specific anti-epileptogenic strategies.
Topics: Mice; Animals; Hippocampus; Status Epilepticus; Epilepsy, Temporal Lobe; Neurons; Pilocarpine; Transcription Factors; Disease Models, Animal
PubMed: 38092829
DOI: 10.1038/s41598-023-49310-y -
Stem Cell Research & Therapy Jul 2023Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably,...
BACKGROUND
Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably, many therapeutics shown to be efficacious in animal models have not proved effective in humans. Thus, there is a need for a drug screening platform based on human tissue. The aim of this study was to develop a robust protocol for generating atrial cardiomyocytes from human-induced pluripotent stem cells.
METHODS
A novel protocol for atrial differentiation, with optimized timing of retinoic acid during mesoderm formation, was compared to two previously published methods. Each differentiation method was assessed for successful formation of a contractile syncytium, electrical properties assayed by optical action potential recordings and multi-electrode array electrophysiology, and response to the G-protein-gated potassium channel activator, carbamylcholine. Atrial myocyte monolayers, derived using the new differentiation protocol, were further assessed for cardiomyocyte purity, gene expression, and the ability to form arrhythmic rotors in response to burst pacing.
RESULTS
Application of retinoic acid at day 1 of mesoderm formation resulted in a robust differentiation of atrial myocytes with contractile syncytium forming in 16/18 differentiations across two cell lines. Atrial-like myocytes produced have shortened action potentials and field potentials, when compared to standard application of retinoic acid at the cardiac mesoderm stage. Day 1 retinoic acid produced atrial cardiomyocytes are also carbamylcholine sensitive, indicative of active I currents, which was distinct from ventricular myocytes and standard retinoic addition in matched differentiations. A current protocol utilizing reduced Activin A and BMP4 can produce atrial cardiomyocytes with equivalent functionality but with reduced robustness of differentiation; only 8/17 differentiations produced a contractile syncytium. The day 1 retinoic acid protocol was successfully applied to 6 iPSC lines (3 male and 3 female) without additional optimization or modification. Atrial myocytes produced could also generate syncytia with rapid conduction velocities, > 40 cm s, and form rotor style arrhythmia in response to burst pacing.
CONCLUSIONS
This method combines an enhanced atrial-like phenotype with robustness of differentiation, which will facilitate further research in human atrial arrhythmia and myopathies, while being economically viable for larger anti-arrhythmic drug screens.
Topics: Animals; Female; Male; Humans; Induced Pluripotent Stem Cells; Atrial Fibrillation; Myocytes, Cardiac; Carbachol; Cell Differentiation; Action Potentials; Tretinoin
PubMed: 37501071
DOI: 10.1186/s13287-023-03405-5 -
International Journal of Molecular... Jan 2024In humans and animal models, temporal lobe epilepsy (TLE) is associated with reorganization of hippocampal neuronal networks, gliosis, neuroinflammation, and loss of...
In the Rat Hippocampus, Pilocarpine-Induced Status Epilepticus Is Associated with Reactive Glia and Concomitant Increased Expression of CD31, PDGFRβ, and Collagen IV in Endothelial Cells and Pericytes of the Blood-Brain Barrier.
In humans and animal models, temporal lobe epilepsy (TLE) is associated with reorganization of hippocampal neuronal networks, gliosis, neuroinflammation, and loss of integrity of the blood-brain barrier (BBB). More than 30% of epilepsies remain intractable, and characterization of the molecular mechanisms involved in BBB dysfunction is essential to the identification of new therapeutic strategies. In this work, we induced status epilepticus in rats through injection of the proconvulsant drug pilocarpine, which leads to TLE. Using RT-qPCR, double immunohistochemistry, and confocal imaging, we studied the regulation of reactive glia and vascular markers at different time points of epileptogenesis (latent phase-3, 7, and 14 days; chronic phase-1 and 3 months). In the hippocampus, increased expression of mRNA encoding the glial proteins GFAP and Iba1 confirmed neuroinflammatory status. We report for the first time the concomitant induction of the specific proteins CD31, PDGFRβ, and ColIV-which peak at the same time points as inflammation-in the endothelial cells, pericytes, and basement membrane of the BBB. The altered expression of these proteins occurs early in TLE, during the latent phase, suggesting that they could be associated with the early rupture and pathogenicity of the BBB that will contribute to the chronic phase of epilepsy.
Topics: Animals; Humans; Rats; Blood-Brain Barrier; Collagen; Disease Models, Animal; Endothelial Cells; Epilepsy; Epilepsy, Temporal Lobe; Hippocampus; Neuroglia; Pericytes; Pilocarpine; Rats, Sprague-Dawley; Status Epilepticus; Platelet Endothelial Cell Adhesion Molecule-1; Receptors, Platelet-Derived Growth Factor; Receptor, Platelet-Derived Growth Factor beta
PubMed: 38338969
DOI: 10.3390/ijms25031693