Search Results (198)

Vascular endothelial cells form a selective barrier that regulates the passage of substances and leukocytes between the bloodstream and surrounding tissues, thereby maintaining vascular homeostasis. Although endothelial barrier dysfunction is implicated in numerous diseases, the molecular mechanisms that protect against such dysfunction remain incompletely defined. Thrombin, an inflammatory mediator, increases endothelial permeability by inducing myosin light chain (MLC) phosphorylation through Rho/Rho-associated kinase (Rho-kinase)-mediated inhibition of myosin phosphatase. This process disrupts vascular endothelial cadherin (VE-cadherin)-based junctions and promotes radial stress fiber formation. Here, we demonstrate that the green tea catechin (–)-epigallocatechin-3-gallate (EGCG) reduces phosphorylation of the myosin phosphatase regulatory subunit MYPT1 at inhibitory sites and suppresses Rho-kinase signaling in endothelial cells. Together, these EGCG-mediated effects reduce MLC phosphorylation, inhibit radial stress fiber formation, and preserve VE-cadherin-mediated cell–cell adhesion, thereby maintaining endothelial barrier integrity. Full article

The colonic epithelial barrier is a multilayered defense system comprising the mucus layer, intestinal epithelial cells (IECs), and the underlying lamina propria. These components collectively maintain mucosal homeostasis and restrict microbial translocation. Disruption of this barrier is a hallmark of chronic intestinal inflammation particularly in IBDs, and is primarily driven by pro-inflammatory cytokines, such as TNF-α, IFN-γ, IL-1β, and IL-6. TNF-α and IFN-γ synergistically induce epithelial cell apoptosis and tight junction disassembly through mechanisms involving TNFR2 upregulation, myosin light chain kinase (MLCK) activation, and adherens junction destabilization. IL-1β amplifies paracellular permeability via NF-κB-dependent MLCK induction and OCLN downregulation, while IL-6 promotes barrier leakiness by upregulating CLDN-2 and sustaining self-reinforcing inflammatory loops that maintain chronic inflammation and impede epithelial repair. This leads to persistent immune-cell infiltration, chronic tight junction remodeling, and failure of barrier replenishment. Consequently, leaky colon facilitates microbial and antigen translocation into the lamina propria, further activating immune cells and perpetuating pro-inflammatory signaling. This review synthesizes current evidence and studies on the cooperative and self-reinforcing roles of pro-inflammatory cytokines, providing insight into the mechanisms underlying chronic intestinal barrier dysfunction and highlighting the need for therapeutic strategies that simultaneously target multiple inflammatory axes to restore barrier integrity in inflammatory bowel disorders. Full article

Background/Objectives: Infantile cardiomyopathy is a rare but often life-threatening condition in which monogenic causes are particularly relevant, especially when cardiac disease is preceded by hypotonia or multisystem involvement. Among sarcomeric genes, MYL2, encoding the ventricular regulatory myosin light chain, plays a critical role in myocardial contractility. However, biallelic MYL2-associated disease remains exceptionally rare, and its clinical spectrum is not fully defined. This study aims to describe a novel case and further delineate the phenotype of recessive MYL2-related cardiomyopathy. Methods: We report a male infant with congenital hypotonia and delayed motor development who underwent extensive metabolic, neuromuscular, and neuroimaging evaluation. Trio-based whole-exome sequencing was performed to identify a potential genetic etiology, followed by variant interpretation using standard bioinformatic and ACMG/AMP criteria. Results: The patient developed acute decompensated heart failure at approximately 10 months of age, with severe left ventricular systolic dysfunction and multiorgan failure, and died at 12 months despite maximal intensive care support. Whole-exome sequencing identified a homozygous MYL2 c.413T>A (p.Met138Lys) missense variant. The variant is absent or extremely rare in population databases, affects a highly conserved residue, is predicted to be deleterious by multiple in silico tools, and is compatible with autosomal recessive inheritance, with both parents confirmed as heterozygous carriers. In the context of a phenotype consistent with recessive MYL2-associated disease, these findings support a likely pathogenic interpretation. Conclusions: This case expands the allelic and phenotypic spectrum of recessive MYL2-associated cardiomyopathy and highlights the value of early genomic testing in infants with unexplained hypotonia and rapidly progressive cardiac dysfunction. Molecular diagnosis may aid in prognosis, clinical decision-making, and genetic counseling. Full article

Familial thoracic aortic aneurysm and dissection (FTAAD), caused by the pathogenic Myh11 K1256del variant, is characterized by impaired aortic contractility; however, how reduced contractility predisposes the aorta to dissection remains incompletely understood. In this study, we performed a data-driven trans-omic upstream analysis using Genome Enhancer to identify key regulatory mechanisms in aortas from Myh11 K1256del mice under baseline conditions, without exposure to exogenous pathological stimuli. Transcriptome analysis revealed enrichment of genes related to smooth muscle contraction and regulation of myosin light chain phosphatase activity. Upstream computational analysis of regulatory regions identified nuclear factor of activated T cells 1 and lymphoid enhancer-binding factor 1 as major transcription factors, and further highlighted Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) as a predicted central regulator of the dysregulated transcriptional network. Druggability analysis suggested ROCK1 and the JunB proto-oncogene AP-1 transcription factor subunit as potential therapeutic targets. Furthermore, it predicted 51 candidate therapeutants, including atorvastatin, GSK-269962A, and atovaquone. These findings indicate that even in the absence of overt pathological stimulation, aortic tissue carrying the Myh11 K1256del variant exhibits a transcriptional program centered on ROCK signaling, which may prime the aorta for maladaptive responses to additional stress and may enhance susceptibility to dissection. This computational analysis requires experimental validation, but may provide a hypothesis-generating framework for development of preventive pharmacological interventions against FTAAD. Full article

Protein inhibitor of neuronal NO synthase (PIN) or dynein light chain 8 (LC8) is a highly conserved protein interacting with multiple partners, like neuronal NO synthase (nNOS) or myosin Va to modulate a variety of cellular functions. As PIN is expressed in skeletal muscle, our aim was to investigate a possible role of PIN in glucose uptake in L6 and primary muscle cells. PIN overexpression resulted into a decrease in glucose uptake with reduced GLUT4 expression and translocation at the plasma membrane, similarly to the pharmacological blockade of nNOS with L-NAME. PIN effect is mediated by a reduction in nNOS protein level and a direct interaction with nNOS leading to a reduced NO production in L6 myocytes. Surprisingly, a siRNA targeting PIN decreased glucose uptake and GLUT4 translocation, suggesting the involvement of nNOS-independent effects. We therefore focused on myosin Va which interacts with PIN in L6 myocytes. Myosin Va silencing provoked a decrease in glucose uptake. As PIN siRNA also reduced myosin Va expression, this confirms the essential role of myosin Va in the observed effects of PIN silencing on glucose uptake. We conclude that PIN modulates glucose uptake and GLUT4 translocation in rat muscle cells, through NO-dependent and -independent mechanisms. Full article

Pyruvate kinase M2 (PKM2) influences meat quality through glycolysis and also exhibits its moonlighting function as a protein kinase that catalyzes protein phosphorylation. However, it remains unclear whether PKM2 phosphorylates myofibrillar proteins, thereby affecting postmortem myofibrillar protein stability. This study investigates PKM2’s non-canonical kinase function using quantitative phosphoproteomics and an in vitro myofibrillar protein incubation model to identify its phosphorylation substrates and functional impacts. The quantitative phosphoproteomics identified 441 phosphoproteins, 881 phosphopeptides, and 1040 phosphorylation sites. Notably, the myosin regulatory light chain (MRLC) was identified as a likely candidate phosphorylation substrate of PKM2 in vitro. The interaction between PKM2 and MRLC was confirmed using co-immunoprecipitation (Co-IP) and Western blotting. Furthermore, MRLC phosphorylation by PKM2 significantly inhibited its degradation and enhanced its stability. This work establishes an in vitro biochemical framework for the moonlighting role of glycolytic enzymes, suggesting a potential mechanistic pathway that might influence myofibrillar protein stability during meat aging. Full article

Tubule-derived pro-fibrotic mediators are central for the development of kidney fibrosis. We previously showed that fibrotic stimuli activate and elevate GEF-H1 (ARHGEF2) in tubular cells, leading to RhoA-dependent fibrotic reprogramming. In search of new mechanisms of GEF-H1 regulation, here we used immunoprecipitation and proximity ligation assay to show interaction between GEF-H1 and Myotonic Dystrophy Kinase-related Cdc42-binding kinase (MRCK)α in tubular cells. MRCKα silencing elevated GEF-H1 activity, and induced GEF-H1-dependent RhoA activation, stress fibre formation and myosin light chain phosphorylation. MRCKα depletion also elevated phospho-cofilin levels in a RhoA-dependent manner. The fibrogenic cytokine TGFβ1 rapidly increased binding between GEF-H1 and MRCKα, while MRCKα silencing augmented TGFβ1-induced GEF-H1 activation, suggesting a negative feedback loop. An mRNA array detecting fibrogenic genes revealed increase in a subset of basal and TGFβ1-induced genes following MRCKα depletion. MRCKα silencing promoted nuclear translocation of the profibrotic transcriptional co-activator Myocardin-Related Transcription Factor (MRTF), and MRTF-A+B depletion prevented increase in ACTA2 (α-smooth muscle actin), a key marker of fibrotic reprogramming. Finally, total MRCKα mRNA was reduced in a murine kidney fibrosis model, and immunohistochemistry revealed a decrease in tubular MRCKα. Taken together, we identified MRCKα as a new suppressor of GEF-H1/RhoA/MRTF signaling. Reduced MRCKα expression in kidney fibrosis may promote tubular fibrotic gene expression. Full article

Endothelial cell (EC) barrier integrity is tightly regulated by the activity of the non-muscle myosin light chain kinase (nmMLCK) under diverse pathological inflammatory conditions (pneumonia, sepsis) and exposure to mechanical stress. Inflammatory stimuli, including lipopolysaccharide (LPS), cytokines, and damage-associated molecular patterns (DAMPs), increase EC permeability through nmMLCK-dependent EC paracellular gap formation. However, the exact mechanisms by which nmMLCK regulates vascular barrier dysfunction in acute lung injury (ALI) remain incompletely understood. We hypothesized that inflammation-induced ROS results in the peroxynitrite-mediated nitration of nmMLCK that contributes to EC barrier disruption. Human lung EC exposure to either the peroxynitrite donor, SIN-1, or to LPS, triggered significant nmMLCK nitration, which was abolished by the oxidant scavenger, MnTMPyP. Mass spectrometry of SIN-1-treated nmMLCK identified multiple nitrated tyrosines. Nitration of Y1410 proved a critical PTM as site-directed substitution with alanine (Y1410A) abolished both SIN-1- and LPS-induced nmMLCK nitration. nmMLCK nitration disrupts wild-type nmMLCK interaction with Kindlin-2, a cytoskeletal regulator of vascular barrier stability, whereas EC transfected with the Y1410A nmMLCK mutant exhibited preserved Kindlin-2 binding, reflected by alterations in trans-EC electrical resistance (TEER). Consistent with these observations, LPS-challenged murine lungs displayed enhanced nmMLCK nitration and diminished nmMLCK-Kindlin-2 association. Functionally, SIN-1 markedly impaired EC barrier integrity (TEER), which was not observed in ECs expressing the Y1410A mutant. Together, these findings suggest that nmMLCK nitration at Y1410 is a critical molecular mechanism contributing to vascular leakage, highlighting this modification as a potential therapeutic target to reduce inflammation-induced vascular permeability. Given nmMLCK’s established role in barrier regulation, we hypothesized that LPS-induced peroxynitrite formation may promote the nitration of nmMLCK tyrosine residues: a PTM that potentially contribute to nmMLCK’s regulation of EC barrier integrity. Full article

Fucoidan improves host health by enriching beneficial taxa such as Bacteroides and Parabacteroides, yet the underlying mechanisms remain unclear. This study validated the association between these two genera and fucoidan-mediated mitigation of intestinal inflammation in mice. Subsequently, the effects of Parabacteroides goldsteinii and Bacteroides finegoldii were evaluated in colitis mice, and the contributions of microbiota-associated metabolites spermidine and betaine were investigated in vitro. Both strains reduced IL-6 (32–36%), TNF-α (30–37%), and IL-1β (40–45%) levels and increased levels of catalase (25–35%) and glutathione peroxidase (31–45%) in the colon. Mechanically, these strains suppressed activation of the NF-κB and MAPK pathways and preserved tight junction integrity by inhibiting myosin light chain kinase activation. These effects were associated with alterations of gut microbiota, characterized by decreased Proteobacteria and increased Bacteroidota, resulting in increased betaine (45–60%) and spermidine (90–112%). In vitro, betaine and spermidine alleviated LPS-induced inflammation and oxidative damage by regulating macrophage polarization. These results suggest that Bacteroides and Parabacteroides contribute to fucoidan-induced improvement of host health through betaine- and spermidine-related pathways. Future studies should clarify the origins of key metabolites and validate their causality and translational relevance using approaches such as fecal microbiota transplantation, metabolite tracing, and human-relevant systems. Full article

The small GTPase RhoA and its downstream effector Rho-kinase (ROCK) have emerged as pivotal regulators of vascular smooth muscle cell (VSMC) contraction, endothelial function, and vascular remodeling. Activation of the RhoA/ROCK pathway enhances calcium (Ca²⁺) sensitivity by inhibiting myosin light chain phosphatase (MLCP), thereby promoting sustained vascular tone independent of intracellular Ca²⁺ levels. In endothelial cells (ECs), RhoA/ROCK signaling contributes to nitric oxide (NO) dysregulation, oxidative stress, cytoskeletal reorganization, and inflammatory activation. Cumulative evidence implicates this pathway in the development and progression of hypertension and other cardiovascular diseases, where maladaptive vascular remodeling, VSMC proliferation, and endothelial dysfunction drive increased vascular resistance. Translational studies have identified ROCK inhibitors and indirect modulators such as statins as promising therapeutic strategies. This review integrates recent mechanistic insights into RhoA/ROCK regulation of vascular function with clinical and translational perspectives on targeting this pathway in hypertension. Full article

Background/Objective: Larazotide acetate (LA) is a synthetic octapeptide under development as a therapeutic candidate for celiac disease, acting to reduce intestinal permeability and regulate tight junctions (TJs). Although several studies have shown barrier-protective effects, the cellular mechanisms underlying LA’s actions in the intestinal epithelium remain unclear. This study aimed to elucidate the mechanistic roles of LA in maintaining intestinal epithelial integrity during cellular injury. Methods: C2BBe1 and leaky IPEC-J2 cell monolayers were pretreated with 10 mM LA and subjected to anoxia/reoxygenation (A/R) injury. Transepithelial electrical resistance (TEER), TJ protein localization, and phosphorylation of myosin light chain-2 (MLC-2) were analyzed. In addition, RNA sequencing was conducted to identify differentially expressed genes and signaling pathways affected by LA treatment. Results: LA pretreatment significantly increased TEER and preserved TJ protein organization during A/R injury. Transcriptomic analysis revealed enrichment of genes related to barrier regulation, small GTPase signaling, protein phosphorylation, proliferation, and migration. LA pretreatment markedly reduced MLC-2 phosphorylation, likely through modulation of the ROCK pathway, consistent with RNA-seq findings. Moreover, LA enhanced cellular proliferation, validating transcriptomic predictions. Conclusions: LA exerts a protective effect on intestinal epithelial integrity by stabilizing tight junctions, reducing MLC-2 phosphorylation, and promoting epithelial proliferation. These findings highlight a novel mechanism for LA and support its therapeutic potential in treating gastrointestinal disorders associated with “leaky gut” and mucosal injury. Full article

In this study, we aimed to characterize intramuscular fat (IMF) tissue in fattening steers through a comparison with subcutaneous fat (SCF) tissue. The IMF of the longissimus thoracis et lumborum and the SCF of the back fat from three fattening steers (mean body weight of 703.50 ± 11.45 kg) were collected, and the muscle tissue, connective tissue, and fascia were carefully removed. Gene and protein expressions and the lipid contents were assessed via transcriptomic, proteomic, and lipidomic analyses, respectively. Subsequently, tissue-specific factors were identified using integrated analysis. The results revealed that the expressions of sarcoplasmic/endoplasmic reticulum Ca²⁺ transporting 2 (ATP2A2), enolase 3 (ENO3), fructose-bisphosphatase 2 (FBP2), myosin heavy chain 7 (MYH7), myosin light chain 3 (MYL3), myosin light chain kinase (MYLK), glycogen phosphorylase (PYGM), troponin C1 (TNNC1), and tropomyosin 2 (TPM2) significantly increased in IMF at both the mRNA and protein levels, whereas those of fatty acid-binding protein 4 (FABP4), stearoyl-CoA desaturase (SCD), and apolipoprotein E (APOE) were reduced. The abundances of both phosphatidylinositol (PI) (18:1/20:4) and phosphatidylcholine (PC) (15:0/18:2) were positively correlated with APOE. Conversely, that of PI (18:1/20:4) was negatively correlated with ENO3 and PYGM, whereas PC (15:0/18:2) was negatively correlated with TNNC1 and MYLK. In conclusion, we identified calcium signaling and glycolysis as key IMF-regulating pathways. ATP2A2, ENO3, FBP2, MYH7, MYL3, MYLK, PYGM, TNNC1, TPM2, and LPE 18:0 were negatively associated with IMF deposition, whereas FABP4, SCD, APOE, PI (18:1/20:4), and PC (15:0/18:2) were positively associated with it. These findings offer underlying IMF-related targets to promote IMF deposition in cattle. Full article

It has previously been reported that the RhoA/Rho-associated kinase (ROCK) pathway is involved in depolarization-induced contraction triggered by high [K⁺] stimulation in rat caudal arterial smooth muscle. Furthermore, we reported that activation of the upstream Ca²⁺-dependent proline-rich tyrosine kinase 2 (Pyk2) leads to phosphorylation of myosin targeting subunit of myosin light chain phosphatase (MYPT1) and 20 kDa myosin light chain (LC₂₀). These findings suggest that Rho guanine nucleotide exchange factors (RhoGEFs) or Rho GTPase-activating proteins (RhoGAPs) may mediate RhoA activation downstream of Pyk2, thereby contributing to depolarization-induced contraction. However, it remains unclear whether Pyk2 directly interacts with RhoGEFs or RhoGAPs. In this study, we investigated the interaction between Pyk2 and RhoGEFs or RhoGAPs during depolarization stimulation of rat caudal arterial smooth muscle. We examined the interaction between Pyk2 and RhoGEFs or RhoGAPs, which previously were identified in smooth muscle, specifically in rat caudal arterial smooth muscle, in response to 60 mM K⁺ stimulation by immunoprecipitation analysis. ArhGEF11, ArhGEF12, phosphorylated ArhGAP42 at Tyr792 (pTyr792-ArhGAP42) and phosphorylated ArhGAP42 at Tyr376 (pTyr376-ArhGAP42) co-immunoprecipitated with Pyk2. The co-immunoprecipitation of pTyr792-ArhGAP42, but not pTyr376-ArhGAP42, with Pyk2 was inhibited by a Pyk2 inhibitor, sodium salicylate. Furthermore, 60 mM K⁺ stimulation increased ArhGAP42 phosphorylation at Tyr792, which was also suppressed by sodium salicylate. These findings indicate that Pyk2-mediated phosphorylation of ArhGAP42 at Tyr792 may play a role in depolarization-induced contraction of rat caudal arterial smooth muscle. Full article

This study investigated the effects of genotype and sex on carcass traits, myosin heavy chain (MyHC) isoforms, and meat quality in pigs raised under Thai commercial conditions. Fifty pigs (25 barrows and 25 gilts) from five genotypes—purebred Duroc (D), LWLR1 (Large White_old × Landrace_old), LWLR2 (Large White_new × Landrace_new), DLWLR1 (Duroc × LWLR1), and DLWLR2 (Duroc × LWLR2)—were slaughtered at ~110 kg body weight. LWLR1 is traditional maternal line, whereas LWLR2 is a newly selected line for higher intramuscular fat (IMF). Significant genotype × sex interactions were observed for dressing percentage, carcass length, bone percentage, purge loss, pH₂₄, and L* (lightness) (p < 0.05). The Longissimus lumborum (LL) muscle of Duroc pigs showed the lowest pH₄₅ and shear force but highest IMF, whereas LWLR1 pigs exhibited higher crude protein, polyunsaturated fatty acids, and flavor-enhancing nucleotides. The LL muscle of LWLR2 and DLWLR2 pigs showed greater expression of MyHC I and IIA, as well as calpain 1 (CAPN1), calpain 2 (CAPN2), and calpastatin (CAST) genes. In contrast, Duroc pigs had the highest MyHC IIX and lowest IIB expression, opposite to patterns in LWLR1 and DLWLR1. Principal component analysis revealed distinct genotype-based clustering, emphasizing the influence of maternal lineage on pork quality and offering valuable insights for genetic selection strategies. Full article

Sodium hypochlorite solution has a good bacterial reduction effect. Thus, in order to understand the effect of sodium hypochlorite on tilapia storage, in this study, tilapia fillets were treated with sodium hypochlorite and Proclin 300 rinsing, the activity of histatinase protease was measured, and the muscle quality was assessed by the texture, colour, pH, myofibrillar fibril fragmentation index (MFI) and its structural changes. The results showed that sodium hypochlorite rinsing could significantly reduce the activities of histatinase enzymes B, L and D. Meanwhile, the sodium hypochlorite-treated group showed less degradation of myosin heavy chain, pro-myosin and myosin light chain, less degradation of texture and colour, better integrity in the fish myocytes, and a lower myofibrillar fragmentation index (MFI). In conclusion, sodium hypochlorite improved the storage quality of tilapia in several ways. Firstly, it inhibited protease activity, thereby maintaining the structural integrity of fish muscle fibres. Secondly, it reduced the rate of deterioration during storage. Full article