UNASSIGNED: Tight control of cytoplasmic Ca2+ in endothelial cells is essential for the regulation of endothelial barrier function. Here, we investigated the role of Cavβ3, a subunit of voltage-gated Ca2+ (Cav) channels, in modulating Ca2+ signaling in brain microvascular endothelial cells (BMECs) and how this contributes to the integrity of the blood-brain barrier.
UNASSIGNED: We investigated the function of Cavβ3 in BMECs by Ca2+ imaging and Western blot, examined the endothelial barrier function in vitro and the integrity of the blood-brain barrier in vivo, and evaluated disease course after induction of experimental autoimmune encephalomyelitis in mice using Cavβ3-/- (Cav β3-deficient) mice as controls.
UNASSIGNED: We identified Cavβ3 protein in BMECs, but electrophysiological recordings did not reveal significant Cav channel activity. In vivo, blood-brain barrier integrity was reduced in the absence of Cavβ3. After induction of experimental autoimmune encephalomyelitis, Cavβ3-/- mice showed earlier disease onset with exacerbated clinical disability and increased T-cell infiltration. In vitro, the transendothelial resistance of Cavβ3-/- BMEC monolayers was lower than that of wild-type BMEC monolayers, and the organization of the junctional protein ZO-1 (zona occludens-1) was impaired. Thrombin stimulates inositol 1,4,5-trisphosphate-dependent Ca2+ release, which facilitates cell contraction and enhances endothelial barrier permeability via Ca2+-dependent phosphorylation of MLC (myosin light chain). These effects were more pronounced in Cavβ3-/- than in wild-type BMECs, whereas the differences were abolished in the presence of the MLCK (MLC kinase) inhibitor ML-7. Expression of Cacnb3 cDNA in Cavβ3-/- BMECs restored the wild-type phenotype. Coimmunoprecipitation and mass spectrometry demonstrated the association of Cavβ3 with inositol 1,4,5-trisphosphate receptor proteins.
UNASSIGNED: Independent of its function as a subunit of Cav channels, Cavβ3 interacts with the inositol 1,4,5-trisphosphate receptor and is involved in the tight control of cytoplasmic Ca2+ and Ca2+-dependent MLC phosphorylation in BMECs, and this role of Cavβ3 in BMECs contributes to blood-brain barrier integrity and attenuates the severity of experimental autoimmune encephalomyelitis disease.

The global incidence of cardiac diseases is increasing, imposing a substantial socioeconomic burden on healthcare systems. The pathogenesis of cardiovascular disease is complex and not fully understood, and the physiological function of the heart is inextricably linked to well-regulated cardiac muscle movement. Myosin light chain kinase (MLCK) is essential for myocardial contraction and diastole, cardiac electrophysiological homeostasis, vasoconstriction of vascular nerves and blood pressure regulation. In this sense, MLCK appears to be an attractive therapeutic target for cardiac diseases. MLCK participates in myocardial cell movement and migration through diverse pathways, including regulation of calcium homeostasis, activation of myosin light chain phosphorylation, and stimulation of vascular smooth muscle cell contraction or relaxation. Recently, phosphorylation of myosin light chains has been shown to be closely associated with the activation of myocardial exercise signaling, and MLCK mediates systolic and diastolic functions of the heart through the interaction of myosin thick filaments and actin thin filaments. It works by upholding the integrity of the cytoskeleton, modifying the conformation of the myosin head, and modulating innervation. MLCK governs vasoconstriction and diastolic function and is associated with the activation of adrenergic and sympathetic nervous systems, extracellular transport, endothelial permeability, and the regulation of nitric oxide and angiotensin II. Additionally, MLCK plays a crucial role in the process of cardiac aging. Multiple natural products/phytochemicals and chemical compounds, such as quercetin, cyclosporin, and ML-7 hydrochloride, have been shown to regulate cardiomyocyte MLCK. The MLCK-modifying capacity of these compounds should be considered in designing novel therapeutic agents. This review summarizes the mechanism of action of MLCK in the cardiovascular system and the therapeutic potential of reported chemical compounds in cardiac diseases by modifying MLCK processes.

UNASSIGNED: Pulmonary hypertension (PH) is a refractory disease characterized by elevated pulmonary artery pressure and resistance. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance by altering the blood dynamics and rheology. Our previous work indicated that polyethylene oxide (PEO) can significantly reduce the medial wall thickness and vascular resistance of the pulmonary arteries, but the specific mechanism is still unclear.
UNASSIGNED: This study was designed to investigate the role and mechanism of PEO on intracellular calcium [Ca2 +] i and cytoskeletal proteins of endothelial cells (ECs) induced by low shear stress (LSS) in PH. Primary Pulmonary Artery Endothelial Cells (PAECs) were subjected to steady LSS (1 dyn/cm2) or physiological shear stress (SS) (10 dyn/cm2) for 20 h in a BioFlux 200 flow system. Calcium influx assays were conducted to evaluate the mechanisms of PEO on [Ca2 +] i. Subsequently, taking the key protein that induces cytoskeletal remodeling, the regulatory light chain (RLC) phosphorylation, as the breakthrough point, this study focused on the two key pathways of PEO that regulate phosphorylation of RLC: Myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) pathways.
UNASSIGNED: Our current research revealed that PEO at LSS (1 dyn/cm2) significantly suppressed LSS-induced [Ca2 +] i and the expression level of transient receptor potential channel 1(TRPC1). In addition, ECs convert LSS stimuli into the upregulation of cytoskeletal proteins, including filamentous actin (F-actin), MLCK, ROCK, p-RLC, and pp-RLC. Further experiments using pharmacological inhibitors demonstrated that PEO at the LSS downregulated cytoskeleton-related proteins mainly through the ROCK and MLCK pathways.
UNASSIGNED: This study considered intracellular calcium and cytoskeleton rearrangement as entry points to study the application of PEO in the biomedical field, which has important theoretical significance and practical application value for the treatment of PH.

OBJECTIVE: Our previous studies have found that the composition ratio of Prevotella melaninogenica (Pm) on buccal mucosa surface of oral lichen planus (OLP) patients increased significantly compared with control. Furthermore, Pm could invade the epithelium of OLP patients. This study aimed to further explore the impact of Pm on oral keratinocytes.
METHODS: The Pm-human oral keratinocyte (HOK) co-culture model was established to detect monolayer permeability, zona occludens-1 (ZO-1) expression, and intracellular survival of Pm. We performed RNA-seq followed by identification of differentially expressed genes (DEGs) and Gene Ontology (GO) analysis, with a particular focus on myosin light chain kinase (MLCK). An MLCK inhibitor ML-7 was utilized in Pm-HOK co-culture model to assess its effects on monolayer permeability and ZO-1 expression.
RESULTS: HOK monolayer permeability was increased, and ZO-1 expression was decreased after co-culture (p < 0.05). Pm could survive in HOK cells. RNA-seq revealed MLCK was an upregulated common DEG. The expression of MLCK in the Pm-HOK co-culture model was upregulated. Inhibition of MLCK rescued the increased epithelial permeability, and ZO-1 expression was upregulated (p < 0.05).
CONCLUSIONS: MLCK may be involved in disrupting epithelial barrier function by Pm.

The main cause of inflammatory bowel disease (IBD) is abnormal intestinal permeability due to the disruption of the tight junction of the intestinal barrier through a pathogen-mediated inflammatory mechanism and an imbalance of the gut microbiota. This study aimed to evaluate whether 2-ketoglutaric acid alleviated permeability dysfunction with tight junction localization, activated the transforming growth factor beta-activated kinase 1 (TAK1) inflammation pathway, and regulated the homeostasis of the intestinal microbiome in vitro and in vivo IBD model. Our findings revealed that 2-ketoglutaric acid significantly suppressed abnormal intestinal permeability, delocalization of tight junction proteins from the intestinal cell, expression of inflammatory cytokines, such as TNF-α, both in vitro and in vivo. 2-Ketoglutaric acid was found to directly bind to TAK1 and inhibit the TNF receptor-associated factor 6 (TRAF6)-TAK1 interaction, which is related to the activation of nuclear factor kappa B (NF-κB) pathways, thereby regulating the expression of mitogen-activated protein kinase. Dietary 2-ketoglutaric acid also alleviated gut microbiota dysbiosis and IBD symptoms, as demonstrated by improvements in the intestine length and the abundance of Ligilactobacillus, Coriobacteriaceae_UCG_002, and Ruminococcaceae_unclassified in mice with colitis. This study indicated that 2-ketoglutaric acid binds to TAK1 for activity inhibition which is related to the NF-κB pathway and alleviates abnormal permeability by regulating tight junction localization and gut microbiome homeostasis. Therefore, 2-ketoglutaric acid is an effective nutraceutical agent and prebiotic for the treatment of IBD.

Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure.
We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154).
Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes.
cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

Fluoride is a common contaminant of groundwater and agricultural commodity, which poses challenges to animal and human health. A wealth of research has demonstrated its detrimental effects on intestinal mucosal integrity; however, the underlying mechanisms remain obscure. This study aimed to investigate the role of the cytoskeleton in fluoride-induced barrier dysfunction. After sodium fluoride (NaF) treatment of the cultured Caco-2 cells, both cytotoxicity and cytomorphological changes (internal vacuoles or massive ablation) were observed. NaF lowered transepithelial electrical resistance (TEER) and enhanced paracellular permeation of fluorescein isothiocyanate dextran 4 (FD-4), indicating Caco-2 monolayers hyperpermeability. In the meantime, NaF treatment altered both the expression and distribution of the tight junction protein ZO-1. Fluoride exposure increased myosin light chain II (MLC2) phosphorylation and triggered actin filament (F-actin) remodeling. While inhibition of myosin II by Blebbistatin blocked NaF-induced barrier failure and ZO-1 discontinuity, the corresponding agonist Ionomycin had effects comparable to those of fluoride, suggesting that MLC2 serves as an effector. Given the mechanisms upstream of p-MLC2 regulation, further studies demonstrated that NaF activated RhoA/ROCK signaling pathway and myosin light chain kinase (MLCK), strikingly increasing the expression of both. Pharmacological inhibitors (Rhosin, Y-27632 and ML-7) reversed NaF-induced barrier breakdown and stress fiber formation. The role of intracellular calcium ions ([Ca2+]i) in NaF effects on Rho/ROCK pathway and MLCK was investigated. We found that NaF elevated [Ca2+]i, whereas chelator BAPTA-AM attenuated increased RhoA and MLCK expression as well as ZO-1 rupture, thus, restoring barrier function. Collectively, abovementioned results suggest that NaF induces barrier impairment via Ca2+-dependent RhoA/ROCK pathway and MLCK, which in turn triggers MLC2 phosphorylation and rearrangement of ZO-1 and F-actin. These results provide potential therapeutic targets for fluoride-induced intestinal injury.

Hughes-Stovin syndrome is a rare disease characterized by thrombophlebitis and multiple pulmonary and/or bronchial aneurysms. The etiology and pathogenesis of HSS are incompletely known. The current consensus is that vasculitis underlies the pathogenic process, and pulmonary thrombosis follows arterial wall inflammation. As such, Hughes-Stovin syndrome may belong to the vascular cluster with lung involvement of Behçet syndrome, although oral aphtae, arthritis, and uveitis are rarely found. Behçet syndrome is a multifactorial polygenic disease with genetic, epigenetic, environmental, and mostly immunological contributors. The different Behçet syndrome phenotypes are presumably based upon different genetic determinants involving more than one pathogenic pathway. Hughes-Stovin syndrome may have common pathways with fibromuscular dysplasias and other diseases evolving with vascular aneurysms. We describe a Hughes-Stovin syndrome case fulfilling the Behçet syndrome criteria. A MYLK variant of unknown significance was detected, along with other heterozygous mutations in genes that may impact angiogenesis pathways. We discuss the possible involvement of these genetic findings, as well as other potential common determinants of Behçet/Hughes-Stovin syndrome and aneurysms in vascular Behçet syndrome. Recent advances in diagnostic techniques, including genetic testing, could help diagnose a specific Behçet syndrome subtype and other associated conditions to personalize the disease management.

In severe acute pancreatitis (SAP), intestinal mucosal barrier damage can cause intestinal bacterial translocation and induce or aggravate systemic infections. Heme oxygenase-1 (HO-1) is a validated antioxidant and cytoprotective agent. This research aimed to investigate the effect and mechanism of HO-1 on SAP-induced intestinal barrier damage in SAP rats. Healthy adult male Sprague-Dawley rats were randomly separated into the sham-operated group, SAP group, SAP + Hemin group, and SAP + Znpp group. The rat model of SAP was established by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Hemin (a potent HO-1 activator) and Znpp (a competitive inhibitor of HO-1) were injected intraperitoneally in the selected groups 24 h before SAP. Serum and intestinal tissue samples were collected for analysis after 24 h in each group. Hemin pretreatment significantly reduced systemic inflammation, intestinal oxidative stress, and intestinal epithelial apoptosis in SAP by increasing HO-1 expression. Meanwhile, pretreatment with Hemin abolished the inhibitory effect on the expression of the tight junction proteins and significantly inhibited the activation of the MLCK/P-MLC signaling pathway. Conversely, ZnPP completely reversed these effects. Our study indicates that upregulation of HO-1 expression attenuates the intestinal mucosal barrier damage in SAP. The protective effect of HO-1 on the intestine is attributed to MLCK/p-MLC signaling pathway inhibition.

Aflatoxin B1 (AFB1) is a widespread mycotoxin in food and feed. Although the liver is the main target organ of AFB1, the intestine is the first exposure organ to AFB1. However, the mechanism by which AFB1 induced intestinal barrier dysfunction via regulating the farnesoid X receptor (FXR)-mediated myosin light chain kinase (MLCK) signaling pathway has rarely been studied. In vivo, AFB1 exposure significantly decreased the small intestine length and increased the intestinal permeability. Meanwhile, AFB1 exposure markedly suppressed the protein expressions of FXR, ZO-1, occludin, and claudin-1 and enhanced the protein expression of MLCK. In vitro, AFB1 exposure induced intestinal barrier dysfunction by the elevation in the FITC-Dextran 4 kDa flux and inhibition in the transepithelial electrical resistance in a dose-dependent manner. In addition, AFB1 exposure downregulated the mRNA and protein expressions of FXR, ZO-1, occludin, and claudin-1, redistributed the ZO-1 protein, and enhanced the protein expressions of MLCK and p-MLC. However, fexaramine (Fex, FXR agonist) pretreatment markedly reversed the AFB1-induced FXR activity reduction, MLCK protein activation, and intestinal barrier impairment in vitro and in vivo. Moreover, pretreatment with the inhibition of MLCK with ML-7 significantly alleviated the AFB1-induced intestinal barrier dysfunction and tight junction disruption in vitro. In conclusion, AFB1 induced intestinal barrier impairment via regulating the FXR-mediated MLCK signaling pathway in vitro and in vivo and provided novel insights to prevent mycotoxin poisoning in the intestine.