Porphyran Attenuates Neuronal Loss in the Hippocampal CA1 Subregion Induced by Ischemia and Reperfusion in Gerbils by Inhibiting NLRP3 Inflammasome-Mediated Neuroinflammation.

Porphyran, a sulfated polysaccharide found in various species of marine red algae, has been demonstrated to exhibit diverse bioactivities, including anti-inflammatory effects. However, the protective effects of porphyran against cerebral ischemia and reperfusion (IR) injury have not been investigated. The aim of this study was to examine the neuroprotective effects of porphyran against brain IR injury and its underlying mechanisms using a gerbil model of transient forebrain ischemia (IR in the forebrain), which results in pyramidal cell (principal neuron) loss in the cornu ammonis 1 (CA1) subregion of the hippocampus on day 4 after IR. Porphyran (25 and 50 mg/kg) was orally administered daily for one week prior to IR. Pretreatment with 50 mg/kg of porphyran, but not 25 mg/kg, significantly attenuated locomotor hyperactivity and protected pyramidal cells located in the CA1 area from IR injury. The pretreatment with 50 mg/kg of porphyran significantly suppressed the IR-induced activation and proliferation of microglia in the CA1 subregion. Additionally, the pretreatment significantly inhibited the overexpressions of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing protein-3 (NLRP3) inflammasome complex, and pro-inflammatory cytokines (interleukin 1 beta and interleukin 18) induced by IR in the CA1 subregion. Overall, our findings suggest that porphyran exerts neuroprotective effects against brain IR injury, potentially by reducing the reaction (activation) and proliferation of microglia and reducing NLRP3 inflammasome-mediated neuroinflammation.

Human Pluripotent Stem Cell-Derived Alveolar Organoids: Cellular Heterogeneity and Maturity.

Chronic respiratory diseases such as idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and respiratory infections injure the alveoli; the damage evoked is mostly irreversible and occasionally leads to death. Achieving a detailed understanding of the pathogenesis of these fatal respiratory diseases has been hampered by limited access to human alveolar tissue and the differences between mice and humans. Thus, the development of human alveolar organoid (AO) models that mimic in vivo physiology and pathophysiology has gained tremendous attention over the last decade. In recent years, human pluripotent stem cells (hPSCs) have been successfully employed to generate several types of organoids representing different respiratory compartments, including alveolar regions. However, despite continued advances in three-dimensional culture techniques and single-cell genomics, there is still a profound need to improve the cellular heterogeneity and maturity of AOs to recapitulate the key histological and functional features of in vivo alveolar tissue. In particular, the incorporation of immune cells such as macrophages into hPSC-AO systems is crucial for disease modeling and subsequent drug screening. In this review, we summarize current methods for differentiating alveolar epithelial cells from hPSCs followed by AO generation and their applications in disease modeling, drug testing, and toxicity evaluation. In addition, we review how current hPSC-AOs closely resemble in vivo alveoli in terms of phenotype, cellular heterogeneity, and maturity.

Acute Inhibition of the Human Kv1.5 Channel by H1 Receptor Antagonist Dimenhydrinate: Mode of Action.

Dimenhydrinate, an H1 receptor antagonist, is generally used for the prevention and treatment of nausea and vomiting. However, cardiac arrhythmias have been reported to be associated with the overdose of histamine H1 receptor antagonists, indicating the probable effect of antihistamines on ion channels. By using a two-microelectrode voltage clamp, we have herein studied the electrophysiological effects of dimenhydrinate on the human Kv1.5 channel in the Xenopus oocyte expression system. Dimenhydrinate acutely and reversibly suppressed the amplitudes of the peak and the steady-state current, within 6 min. The inhibitory effect of dimenhydrinate on the peak and the steady-state Kv1.5 currents increased progressively from -10 to +50 mV. At each test voltage, the drug suppressed both the peak and the steady-state currents to a similar extent. When the oocytes were stimulated at the rates of 5- and 30-s intervals, dimenhydrinate-induced a use-dependent blockade of the human Kv1.5 channel. Dimenhydrinate expedited the timecourse of the Kv1.5 channel activation more effectively than the timecourse of its inactivation. However, the activation and inactivation curves of the channel were not altered by the H1 receptor antagonist. In conclusion, we found that dimenhydrinate inhibits the human Kv1.5 channel by changing the channel's activation mode, thereby possibly increasing the possibility of triggering cardiac arrhythmias and affecting atrial fibrillation.

Cigarette Smoke-Induced Respiratory Response: Insights into Cellular Processes and Biomarkers.

Cigarette smoke (CS) poses a significant risk factor for respiratory, vascular, and organ diseases owing to its high content of harmful chemicals and reactive oxygen species (ROS). These substances are known to induce oxidative stress, inflammation, apoptosis, and senescence due to their exposure to environmental pollutants and the presence of oxidative enzymes. The lung is particularly susceptible to oxidative stress. Persistent oxidative stress caused by chronic exposure to CS can lead to respiratory diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), and lung cancer. Avoiding exposure to environmental pollutants, like cigarette smoke and air pollution, can help mitigate oxidative stress. A comprehensive understanding of oxidative stress and its impact on the lungs requires future research. This includes identifying strategies for preventing and treating lung diseases as well as investigating the underlying mechanisms behind oxidative stress. Thus, this review aims to investigate the cellular processes induced by CS, specifically inflammation, apoptosis, senescence, and their associated biomarkers. Furthermore, this review will delve into the alveolar response provoked by CS, emphasizing the roles of potential therapeutic target markers and strategies in inflammation and oxidative stress.

Cyclic Phytosphingosine-1-Phosphate Primed Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice.

Background and Objectives: O-cyclic phytosphingosine-1-phosphate (cP1P) is a synthetic chemical and has a structure like sphingosine-1-phosphate (S1P). S1P is known to promote cell migration, invasion, proliferation, and anti-apoptosis through hippocampal signals. However, S1P mediated cellular-, molecular mechanism is still remained in the lung. Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are characterized by excessive immune response, increased vascular permeability, alveolar-peritoneal barrier collapse, and edema. In this study, we determined whether cP1P primed human dermal derived mesenchymal stem cells (hdMSCs) ameliorate lung injury and its therapeutic pathway in ALI mice. Methods and Results: cP1P treatment significantly stimulated MSC migration and invasion ability. In cytokine array, secretion of vascular-related factors was increased in cP1P primed hdMSCs (hdMSCcP1P), and cP1P treatment induced inhibition of Lats while increased phosphorylation of Yap. We next determined whether hdMSCcP1P reduce inflammatory response in LPS exposed mice. hdMSCcP1P further decreased infiltration of macrophage and neutrophil, and release of TNF-α, IL-1ß, and IL-6 were reduced rather than naïve hdMSC treatment. In addition, phosphorylation of STAT1 and expression of iNOS were significantly decreased in the lungs of MSCcP1P treated mice. Conclusions: Taken together, these data suggest that cP1P treatment enhances hdMSC migration in regulation of Hippo signaling and MSCcP1P provide a therapeutic potential for ALI/ARDS treatment.

PTD-FGF2 Attenuates Elastase Induced Emphysema in Mice and Alveolar Epithelial Cell Injury.

Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1-0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.

Establishment of a human induced pluripotent stem cell derived alveolar organoid for toxicity assessment.

Alveolar epithelial cells (AECs) are vulnerable to injury, which can result in epithelial hyperplasia, apoptosis, and chronic inflammation. In this study, we developed human induced pluripotent stem cell (hiPS) cell-derived AECs (iAECs) and the iAECs based organoids (AOs) for testing AEC toxicity after chemical exposure. HiPS cells were cultured for 14 days with differentiation medium corresponding to each step, and the iAECs-based AOs were maintained for another 14 days. SFTPC and AQP5 were expressed in the AOs, and mRNA levels of SOX9, NKX2.1, GATA6, HOPX, and ID2 were increased. The AOs were exposed for 24 h to nine chemical substances, and IC50 values of the nine chemicals were determined using MTT assay. When the correlations between iAECs 2D culture and AOs 3D culture were calculated using Pearson's correlation coefficient r value, the nine chemicals that caused a significant decrease of cell viability in 3D culture were found to be highly correlated in 2D culture. The cytotoxicity and nitric oxide release in AO cultured with macrophages were then investigated. When AOs with macrophages were exposed to sodium chromate for 24 h, the IC50 value and nitric oxide production were higher than when the AOs were exposed alone. Taken together, the AO-based 3D culture system provides a useful platform for understanding biological characteristics of AECs and modeling chemical exposures.

Recombinant Human Bone Morphogenetic Protein-2 Priming of Mesenchymal Stem Cells Ameliorate Acute Lung Injury by Inducing Regulatory T Cells.

Mesenchymal stromal/stem cells (MSCs) possess immunoregulatory properties and their regulatory functions represent a potential therapy for acute lung injury (ALI). However, uncertainties remain with respect to defining MSCs-derived immunomodulatory pathways. Therefore, this study aimed to investigate the mechanism underlying the enhanced effect of human recombinant bone morphogenic protein-2 (rhBMP-2) primed ES-MSCs (MSCBMP2) in promoting Tregs in ALI mice. MSC were preconditioned with 100 ng/ml rhBMP-2 for 24 h, and then administrated to mice by intravenous injection after intratracheal injection of 1 mg/kg LPS. Treating MSCs with rhBMP-2 significantly increased cellular proliferation and migration, and cytokines array reveled that cytokines release by MSCBMP2 were associated with migration and growth. MSCBMP2 ameliorated LPS induced lung injury and reduced myeloperoxidase activity and permeability in mice exposed to LPS. Levels of inducible nitric oxide synthase were decreased while levels of total glutathione and superoxide dismutase activity were further increased via inhibition of phosphorylated STAT1 in ALI mice treated with MSCBMP2. MSCBMP2 treatment increased the protein level of IDO1, indicating an increase in Treg cells, and Foxp3+CD25+ Treg of CD4+ cells were further increased in ALI mice treated with MSCBMP2. In co-culture assays with MSCs and RAW264.7 cells, the protein level of IDO1 was further induced in MSCBMP2. Additionally, cytokine release of IL-10 was enhanced while both IL-6 and TNF-α were further inhibited. In conclusion, these findings suggest that MSCBMP2 has therapeutic potential to reduce massive inflammation of respiratory diseases by promoting Treg cells.

L-carnosine Attenuates Bleomycin-Induced Oxidative Stress via NFκB Pathway in the Pathogenesis of Pulmonary Fibrosis.

Idiopathic Pulmonary fibrosis (IPF), a chronic interstitial lung disease, has pulmonary manifestations clinically characterized by collagen deposition, epithelial cell injury, and a decline in lung function. L-carnosine, a dipeptide consisting of ß-alanine and L-histidine, has demonstrated a therapeutic effect on various diseases because of its pivotal function. Despite the effect of L-carnosine in experimental IPF mice, its anti-oxidative effect and associated intercellular pathway, particularly alveolar epithelial cells, remain unknown. Therefore, we demonstrated the anti-fibrotic and anti-inflammatory effects of L-carnosine via Reactive oxygen species (ROS) regulation in bleomycin (BLM)-induced IPF mice. The mice were intratracheally injected with BLM (3 mg/kg) and L-carnosine (150 mg/kg) was orally administrated for 2 weeks. BLM exposure increased the protein level of Nox2, Nox4, p53, and Caspase-3, whereas L-carnosine treatment suppressed the protein level of Nox2, Nox4, p53, and Caspase-3 cleavage in mice. In addition, the total SOD activity and mRNA level of Sod2, catalase, and Nqo1 increased in mice treated with L-carnosine. At the cellular level, a human fibroblast (MRC-5) and mouse alveolar epithelial cell (MLE-12) were exposed to TGFß1 following L-carnosine treatment to induce fibrogenesis. Moreover, MLE-12 cells were exposed to cigarette smoke extract (CSE). Consequently, L-carnosine treatment ameliorated fibrogenesis in fibroblasts and alveolar epithelial cells, and inflammation induced by ROS and CSE exposure was ameliorated. These results were associated with the inhibition of the NFκB pathway. Collectively, our data indicate that L-carnosine induces anti-inflammatory and anti-fibrotic effects on alveolar epithelial cells against the pathogenesis of IPF.

Human pluripotent stem cell-derived macrophages and macrophage-derived exosomes: therapeutic potential in pulmonary fibrosis.

Pulmonary fibrosis (PF) is a fatal chronic disease characterized by accumulation of extracellular matrix and thickening of the alveolar wall, ultimately leading to respiratory failure. PF is thought to be initiated by the dysfunction and aberrant activation of a variety of cell types in the lung. In particular, several studies have demonstrated that macrophages play a pivotal role in the development and progression of PF through secretion of inflammatory cytokines, growth factors, and chemokines, suggesting that they could be an alternative therapeutic source as well as therapeutic target for PF. In this review, we describe the characteristics, functions, and origins of subsets of macrophages involved in PF and summarize current data on the generation and therapeutic application of macrophages derived from pluripotent stem cells for the treatment of fibrotic diseases. Additionally, we discuss the use of macrophage-derived exosomes to repair fibrotic lung tissue.

Reduced receptor for advanced glycation end products is associated with α-SMA expression in patients with idiopathic pulmonary fibrosis and mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease. Despite alveolar epithelial cells is crucial role in lung, its contribution and the associated biomarker remain unknown in the pathogenesis of IPF. Recently, environmental factors including stone dust, silica and cigarette smoking were found as risk factors involved in IPF. Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super family of cell surface receptors. It has been shown that interaction between RAGE and its ligands on immune cells mediates cellular migration and regulation of pro-inflammation. RAGE is highly expressed in the lung, in particular, alveolar epithelial cells. Therefore, we determined whether RAGE expression is associated with fibrosis-associated genes in patients with IPF and mice. RESULTS: When bleomycin (BLM) was intratracheally administered to C57BL/6 mice for 1, 2 weeks, macrophage and neutrophils were significantly increased. The fibrotic nodule formed and accumulation of collagen was determined after BLM injection in H&E- and Masson's trichrome staining. Levels of elastin, Col1a1 and fibronectin were increased in quantitative real-time PCR and protein levels of α-SMA was increased in western blot analysis. In the lung tissues of 1 mg/kg BLM-induced mice, RAGE expression was gradually decreased in 1- and 2 weeks in immunohistochemistry and western blot analysis, and 3 mg/kg of BLM-induced mice exhibited decreased RAGE levels while α-SMA expression was increased. We next determined RAGE expression in the lungs of IPF patients using immunohistochemistry. As a result, RAGE expression was decreased, while α-SMA expression was increased compared with non-IPF subjects. CONCLUSIONS: Our findings suggest that reduced RAGE was associated with increased fibrotic genes in BLM-induced mice and patients with IPF. Therefore, RAGE could be applied with a biomarker for prognosis and diagnosis in the pathogenesis of IPF.

An Update on the Role of Nrf2 in Respiratory Disease: Molecular Mechanisms and Therapeutic Approaches.

Nuclear factor erythroid 2-related factor (Nrf2) is a transcriptional activator of the cell protection gene that binds to the antioxidant response element (ARE). Therefore, Nrf2 protects cells and tissues from oxidative stress. Normally, Kelch-like ECH-associated protein 1 (Keap1) inhibits the activation of Nrf2 by binding to Nrf2 and contributes to Nrf2 break down by ubiquitin proteasomes. In moderate oxidative stress, Keap1 is inhibited, allowing Nrf2 to be translocated to the nucleus, which acts as an antioxidant. However, under unusually severe oxidative stress, the Keap1-Nrf2 mechanism becomes disrupted and results in cell and tissue damage. Oxide-containing atmospheric environment generally contributes to the development of respiratory diseases, possibly leading to the failure of the Keap1-Nrf2 pathway. Until now, several studies have identified changes in Keap1-Nrf2 signaling in models of respiratory diseases, such as acute respiratory distress syndrome (ARDS)/acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma. These studies have confirmed that several Nrf2 activators can alleviate symptoms of respiratory diseases. Thus, this review describes how the expression of Keap1-Nrf2 functions in different respiratory diseases and explains the protective effects of reversing this expression.

Human pluripotent stem-cell-derived alveolar organoids for modeling pulmonary fibrosis and drug testing.

Detailed understanding of the pathogenesis and development of effective therapies for pulmonary fibrosis (PF) have been hampered by lack of in vitro human models that recapitulate disease pathophysiology. In this study, we generated alveolar organoids (AOs) derived from human pluripotent stem cells (hPSCs) for use as an PF model and for drug efficacy evaluation. Stepwise direct differentiation of hPSCs into alveolar epithelial cells by mimicking developmental cues in a temporally controlled manner was used to generate multicellular AOs. Derived AOs contained the expected spectrum of differentiated cells, including alveolar progenitors, type 1 and 2 alveolar epithelial cells and mesenchymal cells. Treatment with transforming growth factor (TGF-ß1) induced fibrotic changes in AOs, offering a PF model for therapeutic evaluation of a structurally truncated form (NP-011) of milk fat globule-EGF factor 8 (MFG-E8) protein. The significant fibrogenic responses and collagen accumulation that were induced by treatment with TGF-ß1 in these AOs were effectively ameliorated by treatment with NP-011 via suppression of extracellular signal-regulated kinase (ERK) signaling. Furthermore, administration of NP-011 reversed bleomycin-induced lung fibrosis in mice also via ERK signaling suppression and collagen reduction. This anti-fibrotic effect mirrored that following Pirfenidone and Nintedanib administration. Furthermore, NP-011 interacted with macrophages, which accelerated the collagen uptake for eliminating accumulated collagen in fibrotic lung tissues. This study provides a robust in vitro human organoid system for modeling PF and assessing anti-fibrotic mechanisms of potential drugs and suggests that modified MGF-E8 protein has therapeutic potential for treating PF.

Organoid Model in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive- fibrosing disease characterized by extensive deposition of extracellular matrix (ECM), scarring of the lung parenchyma. Despite increased awareness of IPF, etiology and physiological mechanism of IPF are unclear. Therefore, preclinical model will require relevant and recapitulative features of IPF. Recently, pluripotent stem cells (PSC)-based organoid studies are emerging as an alternative approach able to recapitulate tissue architecture with remarkable fidelity. Moreover, these biomimetic tissue models can be served to investigate the mechanisms of diverse disease progression. In this review, we will overview the current organoids technology for human disease modeling including lung organoids for IPF.

Suppression of voltage-gated K+ channels by darifenacin in coronary arterial smooth muscle cells.

Darifenacin, an anticholinergic agent, has been used to treat overactive bladder syndrome. Despite its extensive clinical use, there is little information about the effect of darifenacin on vascular ion channels, specifically K+ channels. This study aimed to investigate the effect of the anti-muscarinic drug darifenacin on voltage-gated K+ (Kv) channels, vascular contractility, and coronary blood flow in rabbit coronary arteries. We used the whole-cell patch-clamp technique to evaluate the effect of darifenacin on Kv channels. Darifenacin inhibited the Kv current in a concentration-dependent manner. Applying 1 µM darifenacin shifted the activation and inactivation curves toward a more positive and negative potential, respectively. Darifenacin slowed the time constants of recovery from inactivation. Furthermore, blockade of the Kv current with darifenacin was increased gradually by applying a train of pulses, indicating that darifenacin inhibited Kv currents in a use- (state)-dependent manner. The darifenacin-mediated inhibition of Kv currents was associated with the Kv1.5 subtype, not the Kv2.1 or Kv7 subtype. Applying another anti-muscarinic drug atropine or ipratropium did not affect the Kv current or change the inhibitory effect of darifenacin. Isometric organ bath experiments using isolated coronary arteries were applied to evaluate whether darifenacin-induced inhibition of the Kv channel causes vasocontraction. Darifenacin substantially induced vasocontraction. Furthermore, darifenacin caused membrane depolarization and decreased coronary blood flow. From these results, we concluded that darifenacin inhibits the Kv currents in concentration- and use- (state)-dependent manners. Inhibition of the Kv current with darifenacin occurred by shifting the steady-state activation and inactivation curves regardless of its anti-muscarinic effect.

Diesel Particulate Matter 2.5 Induces Epithelial-to-Mesenchymal Transition and Upregulation of SARS-CoV-2 Receptor during Human Pluripotent Stem Cell-Derived Alveolar Organoid Development.

Growing evidence links prenatal exposure to particulate matter (PM2.5) with reduced lung function and incidence of pulmonary diseases in infancy and childhood. However, the underlying biological mechanisms of how prenatal PM2.5 exposure affects the lungs are incompletely understood, which explains the lack of an ideal in vitro lung development model. Human pluripotent stem cells (hPSCs) have been successfully employed for in vitro developmental toxicity evaluations due to their unique ability to differentiate into any type of cell in the body. In this study, we investigated the developmental toxicity of diesel fine PM (dPM2.5) exposure during hPSC-derived alveolar epithelial cell (AEC) differentiation and three-dimensional (3D) multicellular alveolar organoid (AO) development. We found that dPM2.5 (50 and 100 µg/mL) treatment disturbed the AEC differentiation, accompanied by upregulation of nicotinamide adenine dinucleotide phosphate oxidases and inflammation. Exposure to dPM2.5 also promoted epithelial-to-mesenchymal transition during AEC and AO development via activation of extracellular signal-regulated kinase signaling, while dPM2.5 had no effect on surfactant protein C expression in hPSC-derived AECs. Notably, we provided evidence, for the first time, that angiotensin-converting enzyme 2, a receptor to mediate the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) entry into target cells, and the cofactor transmembrane protease serine 2 were significantly upregulated in both hPSC-AECs and AOs treated with dPM2.5. In conclusion, we demonstrated the potential alveolar development toxicity and the increase of SARS-Cov-2 susceptibility of PM2.5. Our findings suggest that an hPSC-based 2D and 3D alveolar induction system could be a useful in vitro platform for evaluating the adverse effects of environmental toxins and for virus research.

WKYMVm ameliorates acute lung injury via neutrophil antimicrobial peptide derived STAT1/IRF1 pathway.

Formyl peptide receptors (FPRs) are mainly expressed on leucocytes and sense microbe-associated molecular pattern (MAMP) molecules, thereby regulating leukocyte chemotaxis and activation. The formyl peptide receptor 2 (FPR2) selective agonist WKYMVm (Trp-Lys-Met-Val-D-Met) has shown potent pro-angiogenic, anti-inflammatory, and anti-apoptotic properties. In this study, we investigated whether WKYMVm exhibits bactericidal activity during neutrophil accumulation in acute lung injury (ALI) in mice and determined its cellular signaling pathways in HL-60 neutrophil-like cells. A daily intraperitoneal treatment of ALI mice with WKYMVm (2.5- and 5 mg/kg/d) daily over four days decreased the levels of proinflammatory cytokines TNF-α, IL-6, and IL-1ß, while it increased the MPO and NO release by differentiated HL-60 neutrophil-like cells. The IRF1 level and STAT1 phosphorylation at S727 were increased in the lungs of mice with ALI treated with WKYMVm. Lung histology induced by ALI was unaffected by treatment with WKYMVm. In vitro, WKYMVm increased MPO, NO, and SOD activity, as well as IRF1 and STAT1 phosphorylation at Ser727. Taken together, our data suggest therapeutic potential of WKYMVm, via FPR2-dependent regulation of STAT1/IRF1, in ALI.

Empagliflozin dilates the rabbit aorta by activating PKG and voltage-dependent K+ channels.

We investigated the vasodilatory effects of empagliflozin (a sodium-glucose co-transporter 2 inhibitor) and the underlying mechanisms using rabbit aorta. Empagliflozin induced vasodilation in a concentration-dependent manner independently of the endothelium. Likewise, pretreatment with the nitric oxide synthase inhibitor L-NAME or the SKca inhibitor apamin together with the IKca inhibitor TRAM-34 did not impact the vasodilatory effects of empagliflozin. Pretreatment with the adenylyl cyclase inhibitor SQ22536 or a guanylyl cyclase inhibitor ODQ or a protein kinase A (PKA) inhibitor KT5720 also did not alter the vasodilatory response of empagliflozin. However, the vasodilatory effects of empagliflozin were significantly reduced by pretreatment with the protein kinase G (PKG) inhibitor KT5823. Although application of the ATP-sensitive K+ (KATP) channel inhibitor glibenclamide, large-conductance Ca2+-activated K+ (BKCa) channel inhibitor paxilline, or inwardly rectifying K+ (Kir) channel inhibitor Ba2+ did not impact the vasodilatory effects of empagliflozin, pretreatment with the voltage-dependent K+ (Kv) channel inhibitor 4-AP reduced the vasodilatory effects of empagliflozin. Pretreatment with DPO-1 (Kv1.5 channel inhibitor), guangxitoxin (Kv2.1 channel inhibitor), or linopirdine (Kv7 channel inhibitor) had little effect on empagliflozin-induced vasodilation. Application of nifedipine (L-type Ca2+ channel inhibitor) or thapsigargin (sarco-endoplasmic reticulum Ca2+-ATPase pump inhibitor) did not impact empagliflozin-induced vasodilation. Therefore, empagliflozin induces vasodilation by activating PKG and Kv channels.

Inhibition of voltage-dependent K+ channels by iloperidone in coronary arterial smooth muscle cells.

Iloperidone, a second-generation atypical antipsychotic drug, is widely used in the treatment of schizophrenia. However, the side-effects of iloperidone on vascular K+ channels remain to be determined. Therefore, we explored the effect of iloperidone on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch-clamp technique. Iloperidone inhibited vascular Kv channels in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of 2.11 ± 0.5 µM and a Hill coefficient of 0.68 ± 0.03. Iloperidone had no effect on the steady-state inactivation kinetics. However, it shifted the steady-state activation curve to the right, indicating that iloperidone inhibited Kv channels by influencing the voltage sensors. Application of 20 repetitive depolarizing pulses (1 and 2 Hz) progressively increased the inhibition of the Kv current in the presence of iloperidone. Furthermore, iloperidone increased the recovery time constant from Kv channel inactivation, suggesting that iloperidone-induced inhibition of Kv channels is use (state)-dependent. Pretreatment with a Kv1.5 inhibitor (diphenyl phosphine oxide 1 [DPO-1]) inhibited the Kv current to a level similar to that with iloperidone alone. However, pretreatment with a Kv2.1 or Kv7.X inhibitor (guangxitoxin or linopirdine) did not affect the inhibitory effect of iloperidone on Kv channels. Therefore, iloperidone directly inhibits Kv channels in a concentration- and use (state)-dependent manner independently of its antagonism of serotonin and dopamine receptors. Furthermore, the primary target of iloperidone is the Kv1.5 subtype.