Junction Integrity Research Articles

Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles.

Lung bacterial infections, including hospital-acquired pneumonia, remain a serious problem for public health. Endothelial cell (EC) exposure to heat-killed Staphylococcus aureus (HKSA) represents a clinical scenario of high titers of killed bacterial particles present in the host after antibiotic therapy, which triggers inflammatory cascades, cytokine storms, and EC dysfunction leading to acute lung injury (ALI). GPR68 is a member of the proton-sensing G protein-coupled receptor family. Acting as a pH sensor, GPR68 becomes activated upon pH reduction and contributes to pathologic cell responses by activating ER stress and unfolded protein response. This study investigated the role of GPR68 in HKSA-induced EC dysfunction and HKSA-induced ALI. HKSA robustly increased GPR68 mRNA levels in human pulmonary EC and directly stimulated GPR68 activity. A selective GPR68 small molecule inhibitor, OGM-8345, attenuated HKSA-induced EC permeability and protected cell junction integrity. OGM-8345 inhibited HKSA-induced activation of inflammatory genes TNF-α, IL-6, IL-8, IL-1β, and CXCL5 and decreased cytokine secretion by HKSA-challenged EC. Co-treatment with the GPR68 activator Ogerin or medium acidification to pH 6.5 augmented HKSA-induced EC dysfunction, which was rescued by OGM-8345. Intratracheal HKSA injection increased vascular leak and lung inflammation in mice which were monitored by lung Evans blue extravasation, increased cell and protein count in bronchoalveolar lavage, and mRNA expression of inflammatory genes. ALI and barrier dysfunction was attenuated by OGM-8345. We show for the first time the role of GPR68 in mediating HKSA-induced lung injury and the strong potential for OGM-8345 as a therapeutic treatment of bacterial pathogen-induced ALI associated with tissue acidification.

SLAMF8 Disrupts Epithelial Barrier in Chronic Rhinosinusitis with Nasal Polyps via M1 Macrophage Polarization.

Recent studies show that M1 macrophages accumulate predominantly in non-eosinophilic chronic rhinosinusitis with nasal polyps (neCRSwNP). However, the precise mechanisms regulating M1 macrophages and their impact on the epithelial barrier remain unclear. We aim to investigate the expression and regulatory role of SLAMF8, a molecule exclusively expressed in myeloid cells, in M1 macrophage polarization and its potential contribution to neCRSwNP development. We evaluated SLAMF8 expression and its correlation with clinical variables using real-time quantitative polymerase chain reaction and Western blot in sinonasal mucosa samples from CRSwNP and control subjects. Immunofluorescence staining confirmed the co-expression of SLAMF8 with macrophages. After SLAMF8 knockdown, we explored the influence on macrophage M1 polarization and the effect on epithelial-mesenchymal transition (EMT) process and tight junction integrity in epithelial cells through an indirect co-culture system of M1 macrophages with human nasal epithelial cells. SLAMF8 was highly expressed on M1 macrophages in polyp tissues, notably in neCRSwNP, and correlated with disease severity indices only in neCRSwNP. SLAMF8 knockdown in THP-1 cells reduced M1 macrophage markers (CD86, iNOS, and NLRP3) and decreased secretion of inflammatory cytokines (IL-1β, IL-6, and TNF-α). Co-culture with M1 macrophage supernatant after SLAMF8 knockdown enhanced epithelial viability, reduced EMT and apoptosis, and upregulated tight junction markers Occludin and Claudin-4 in nasal epithelial cells. SLAMF8 elevation correlates with the EMT, epithelial tight junction and disease severity in neCRSwNP. The SLAMF8 upregulation promotes M1 macrophage polarization, by which facilitates EMT and impairs nasal epithelial barrier function. SLAMF8 may represent a novel therapeutic target for neCRSwNP.

GsMTx-4 combined with exercise improves skeletal muscle structure and motor function in rats with spinal cord injury.

Motor dysfunction and muscle atrophy are typical symptoms of patients with spinal cord injury (SCI). Exercise training is a conventional physical therapy after SCI, but exercise intervention alone may have limited efficacy in reducing secondary injury and promoting nerve regeneration and functional remodeling. Our previous research found that intramedullary pressure after SCI is one of the key factors affecting functional prognosis. It has been reported that GsMTx-4, a specific blocker of the mechanosensitive ion channels Piezo1, can protect the integrity of the neuromuscular junction and promote nerve regeneration, and thus has the potential as a therapeutic agent for SCI. In this study, we observed the combined and separate therapeutic effect of GsMTx-4 and exercise on the structure of the soleus muscle and motor function in rats with SCI. At 42 days post-injury, compared with SCI rats, the Basso-Beattie-Bresnahan score (P = 0.0007) and Gait Symmetry (P = 0.0002) were significantly improved after combination therapy. On histology of rat soleus muscle, compared with SCI rats, the combined treatment significantly increased the wet weight ratio, muscle fiber cross-sectional area and acetylcholinesterase (all P<0.0001). On histology of rat spinal tissue, compared with SCI rats, the combined treatment significantly increased neuron counts and BDNF levels, and significantly reduced the percentage of TUNEL-positive cells (all P<0.0001). On physiology of rat soleus muscle, compared with SCI rats, the combined treatment increased the succinate dehydrogenase expression (P<0.0001), while the expression of α-glycerophosphate dehydrogenase (P<0.0001) and GDF8 protein (P = 0.0008) decreased. Results indicate the combination therapy effectively improves histopathology of spinal cord and soleus muscle in SCI rats, enhancing motor function. This study was conducted on animal models, it offers insights for SCI treatment, advancing understanding of lower limb muscle pathology post-SCI. Further research is needed for clinical validation in the future.

P0055 Impact of Indole-3-Propionic Acid on Bacterial Invasion Potential of Klebsiella Strains Isolated from Pediatric Ulcerative Colitis Patients

Abstract Background Indole-3-propionic acid (IPA), a tryptophan metabolite produced by microbes, has demonstrated beneficial effects, including reversing dysbiosis, balancing bacterial populations, and reducing inflammation.1,2,3 However, the mechanisms behind these effects and their impact on pathogenic microbes remain unclear. This study aimed to evaluate the effects of IPA on bacterial invasion in vitro, using potential pathobionts (Klebsiella variicola and Klebsiella quasipneumoniae) isolated from non-inflamed sections of the colon in pediatric ulcerative colitis (UC) patients. We hypothesized that IPA could reduce virulence traits, promoting a more homeostatic environment, which could lower inflammation in pediatric UC patients. Methods Caco-2 cells (colonic epithelial cell line) were infected with Klebsiella strains, with or without 0.5 mM IPA. Bacterial invasion was assessed via gentamicin protection assay, and qPCR measured Interleukin-8, IL-10, and TNF-α expression in infected cells. Transepithelial electrical resistance (TEER) assay evaluated barrier integrity, and qPCR analyzed the expression of bacterial virulence genes, including entB (siderophores), manC (capsule), and hcp1 (type VI secretion) in IPA-treated and untreated cultures. Additionally, siderophore production was quantified using a Chrome-Azurol Sulfonate (CAS) assay, and Anthony’s stain visualized the bacterial capsule. Results IPA-treated Caco-2 cells showed significantly reduced bacterial invasion, with further reductions when bacteria were also exposed to IPA. qPCR revealed a significant decrease in IL-8 expression in IPA-treated cells during infection. IL-10 levels decreased non-significantly, and TNF-α expression showed variable results. A trend toward improved tight junction integrity was observed in the TEER assay with IPA treatment, although no significant differences were detected. IPA treatment increased the expression of entB, manC, and hcp1 genes, except for K. variicola, where manC expression remained unchanged. IPA significantly reduced the capsule thickness of K. variicola and resulted in a significant reduction in K. quasipneumoniae siderophore production. Conclusion IPA reduces bacterial invasion and modulates immune responses in Caco-2 cells infected with Klebsiella pathobiont strains. It reduces the virulence of K. quasipneumoniae by targeting siderophore production and diminishes the virulence of K. variicola by reducing capsule formation, suggesting its potential as an anti-virulence agent against these pathobionts. These findings highlight IPA’s potential as an immunomodulatory agent for managing IBD. However, further research is needed into other Klebsiella virulence pathways and host-microbiota interactions.

The Crucial Role of the Blood-Brain Barrier in Neurodegenerative Diseases: Mechanisms of Disruption and Therapeutic Implications.

The blood-brain barrier (BBB) is a crucial structure that maintains brain homeostasis by regulating the entry of molecules and cells from the bloodstream into the central nervous system (CNS). Neurodegenerative diseases such as Alzheimer's and Parkinson's disease, as well as ischemic stroke, compromise the integrity of the BBB. This leads to increased permeability and the infiltration of harmful substances, thereby accelerating neurodegeneration. In this review, we explore the mechanisms underlying BBB disruption, including oxidative stress, neuroinflammation, vascular dysfunction, and the loss of tight junction integrity, in patients with neurodegenerative diseases. We discuss how BBB breakdown contributes to neuroinflammation, neurotoxicity, and the abnormal accumulation of pathological proteins, all of which exacerbate neuronal damage and facilitate disease progression. Furthermore, we discuss potential therapeutic strategies aimed at preserving or restoring BBB function, such as anti-inflammatory treatments, antioxidant therapies, and approaches to enhance tight junction integrity. Given the central role of the BBB in neurodegeneration, maintaining its integrity represents a promising therapeutic approach to slow or prevent the progression of neurodegenerative diseases.

Sesamin Alleviates Allergen-Induced Diarrhea by Restoring Gut Microbiota Composition and Intestinal Barrier Function.

Food allergens are the key triggers of allergic diarrhea, causing damage to the immune-rich ileum. This weakens the mucosal barrier and tight junctions, increases intestinal permeability, and exacerbates allergen exposure, thereby worsening the condition. Sesamin, a natural lignan isolated from sesame seed, has shown potential in regulating immune responses, but its effects on intestinal health remain unclear. In this study, we constructed an ovalbumin (OVA)-induced allergic diarrhea mouse model, which demonstrated increased mast cell degranulation, reduced tight junction integrity, and impaired intestinal barrier function. Pro-inflammatory cytokines were significantly increased in the ileum, along with unbalanced cluster of differentiation 4 (CD4+) T-cell immunity, altered gut microbiota composition, and disrupted bacterial metabolism. Sesamin treatment significantly alleviated intestinal damage by modulating gut microbiota abundance, enhancing short-chain fatty acid (SCFA) production, and increasing SCFA receptor expression. This study suggests that sesamin may be a promising therapeutic candidate for allergic diarrhea and intestinal injury.

Nerve repair with polylactic acid and inosine treatment enhance regeneration and improve functional recovery after sciatic nerve transection.

Following transection, nerve repair using the polylactic acid (PLA) conduit is an effective option. In addition, inosine treatment has shown potential to promote nerve regeneration. Therefore, this study aimed to investigate the regenerative potential of inosine after nerve transection and polylactic acid conduit repair. C57/Black6 mice were subjected to sciatic nerve transection, repair with PLA conduit, and intraperitoneal injection of saline or inosine 1 h after injury and daily for 1 week. To assess motor and sensory recovery, functional tests were performed before and weekly up to 8 weeks after injury. Following, to evaluate the promotion of regeneration and myelination, electroneuromyography, morphometric analysis and immunohistochemistry were then performed. Our results showed that the inosine group had a greater number of myelinated nerve fibers (1,293 ± 85.49 vs. 817 ± 89.2), an increase in neurofilament high chain (NFH) and myelin basic protein (MBP) immunolabeling and a greater number of fibers within the ideal g-ratio (453.8 ± 45.24 vs. 336.6 ± 37.01). In addition, the inosine group presented a greater adenosine A2 receptor (A2AR) immunolabeling area. This resulted in greater compound muscle action potential amplitude and nerve conduction velocity, leading to preservation of muscle and neuromuscular junction integrity, and consequently, the recovery of motor and sensory function. Our findings suggest that inosine may enhance regeneration and improve both motor and sensory function recovery after nerve transection when repaired with a poly-lactic acid conduit. This advances the understanding of biomaterials and molecular treatments.

Using nutrition to help recovery from infections.

Antibiotics are a cornerstone of modern medicine, but antibiotic consumption can have depleting effects on the gut microbiota, potentially leading to gastrointestinal symptoms and other diseases, namely Clostridioides difficile infection. Because nutrition is a major driver of gut microbiota diversity and function, here we explore the current evidence on the potential of diets in alleviate the deleterious effects of antibiotics consumed during infections. Beneficial nutrients can enhance the symbiotic effect of the gut microbiota with the host, supporting anti-inflammatory responses and maintaining tight junction integrity. Short-chain fatty acids have been shown to positively affect the immune response, reducing the severity of C. difficile infection, whereas high-fibre diets have been shown to promote faster recovery of the gut microbiota after antibiotic therapy. The role of nutrition during infection is gaining momentum, with key findings exploring the effect of some nutrients in limiting the severity of infections and helping the microbiota recover from antibiotic-induced dysbiosis. Although this field is in its infancy, these findings open the possibility of personalised nutrition as a way of restoring microbiome diversity. But more work is needed to identify the most effective types and combinations of nutrients to achieve this.

Small extracellular vesicles derived from miRNA-486 overexpressed dental pulp stem cells mitigate high altitude pulmonary edema through PTEN/PI3K/AKT/eNOS pathway.

High altitude pulmonary edema (HAPE) is a life-threatening, non-cardiogenic pulmonary edema characterized by rapid onset and high mortality. Extracellular vesicles of mesenchymal stem cells are used in the treatment of a variety of lung diseases, but their use in HAPE remains underreported. This study explores the therapeutic potential of miRNA-486 modified extracellular vesicles from dental pulp stem cells (sEVmiR-486) against HAPE, aiming to decipher the associated molecular mechanisms. The rat HAPE model was established by exposing subjects to a simulated high-altitude, low-oxygen environment within a specialized chamber. The HAPE-afflicted rats received sEVNull and sEVmiR-486 intravenously, and the therapeutic effect was assessed through histopathological analysis, pulmonary artery pressure, lung water content, as well as markers of oxidative stress and inflammation. To supplement in vivo findings, pulmonary microvascular endothelial cells (PMVEC) were stressed with cobalt chloride to emulate hypoxic damage, and then treated with sEVNull and sEVmiR-486 to unravel the mechanism of action. The sEVNull mitigated pathological changes in the lungs, reduced pulmonary artery pressure and lung water content, and alleviated oxidative stress and inflammatory responses in cases of HAPE. Moreover, sEVNull enhanced vascular reactivity and restored pulmonary permeability and tight junction integrity, these effects were intensified by miRNA-486 overexpression. Notably, sEVmiR-486 attenuated oxidative damage in hypoxic PMVEC cells by modulating the PTEN/PI3K/Akt/eNOS signaling pathway. miRNA-486 fortified DPSC-sEVs intervention as a novel and potent treatment strategy for HAPE.

Designing and evaluating a novel blood-brain barrier in vitro model of teleost for reproducing alterations in brain infecting

A new blood-brain barrier (BBB) in vitro model of tilapia (Oreochromis niloticus) was successfully established by co-culture with brain microvascular endothelial cell line TVEC-01 and brain astrocyte cell line TA-02 of tilapia. Experiments with the expression levels of BBB-related genes, TEER value detection and four-hour water-leaking test have shown that the BBB in vitro model has an excellent barrier effect. In bacterial penetration experiments, the pathogenic strain of Streptococcus agalactiae was able to pass through the BBB in vitro model and initiate a series of immune responses, among which TA-02 contributed to the expression of pro-inflammatory cytokines and TVEC-01 contributed to the expression of anti-inflammatory cytokines, providing an excellent research tool and theoretical basis for further study of meningitis. Moreover, the qRT-PCR and transmission electron microscopy revealed that the pathogenic strain of S. agalactiae effectively penetrated the BBB in vitro model of tilapia during early-stage infection without destroying tight junction integrity. This suggested that, in the initial phases of infection, the pathogenic strain of S. agalactiae may breach the BBB via a transcellular pathway rather than a paracellular pathway. Summarily, a novel BBB in vitro model of tilapia was successfully designed and evaluated for reproducing alterations in brain infecting.

Cost-Effective Bioimpedance Spectroscopy System for Monitoring Syncytialization In Vitro: Experimental and Numerical Validation of BeWo Cell Fusion.

The placenta plays a critical role in nutrient and oxygen exchange during pregnancy, yet the effects of medicinal drugs on this selective barrier remain poorly understood. To overcome this, this study presents a cost-effective bioimpedance spectroscopy (BIS) system to assess tight junction integrity and monolayer formation in BeWo b30 cells, a widely used model of the multinucleated maternal-fetal exchange surface of the placental barrier. Cells were cultured on collagen-coated porous membranes and treated with forskolin to induce controlled syncytialization. Electrical impedance was measured using an entry level impedance analyzer, while immunofluorescence staining was used to confirm monolayer formation and syncytialization. The measurements and staining confirmed the formation of a confluent monolayer on day 4. In fact, the electrical resistance tripled for treated samples indicating a more electrically restrictive barrier. This resistance remained constant for treated samples reflecting the intact barrier's integrity over the next 3 days. The measurements show that, on day 4, the electrical capacitance of the cells decreased for the treated samples as opposed to the untreated samples. This reflects that the surface area of the BeWo b30 cells decreased when the samples were treated with forskolin. Finally, a COMSOL model was developed to explore the effects of electrode positioning, depth, and distance on TEER measurements, explaining discrepancies in the literature. In fact, there was a substantial 97% and 39.4% difference in the obtained TEER values. This study demonstrates the AD2 device's feasibility for monitoring placental barrier integrity and emphasizes the need for standardized setups for comparable results. The system can hence be used to analyze drug effects and nutrient transfer across the placental barrier.

Rickettsia disrupts and reduces endothelial tight junction protein zonula occludens-1 in association with inflammasome activation.

Rickettsia spp. cause life-threatening diseases in humans. The fundamental pathophysiological changes in fatal rickettsial diseases are disrupted endothelial barrier and increased microvascular permeability. However, it remains largely unclear how rickettsiae induce microvascular endothelial injury. In the present study, we demonstrated that Rickettsia conorii infection disrupts the continuous immunofluorescence expression of the interendothelial tight junction protein, zonula occludens-1 (ZO-1), in infected monolayers of microvascular endothelial cells (MVECs), accompanied by significantly diminished total expression levels of ZO-1. Interestingly, R. conorii activated inflammasome in MVECs, as evidenced by cleaved caspase-1 and IL-1β in the cell lysates in association with significantly elevated expression levels of nucleotide binding and oligomerization domain, leucine-rich repeat, and pyrin containing protein 3 (NLRP3). Furthermore, selective inhibition of NLRP3 by MCC950 significantly suppressed the activation and cleavage of caspase-1 induced by R. conorii in endothelial cells, which further prevented the disruption of interendothelial junctions and reduction of ZO-1 expression. Of note, pharmaceutical inhibition of NLRP3 mitigated the disrupted endothelial integrity caused by R. conorii, measured by fluorescein isothiocyanate-dextran passage in a Transwell assay, independent of bacterial growth and cellular cytotoxicity. Taken together, our results suggest that R. conorii affected microvascular endothelial junction integrity likely via diminishing and interrupting the junctional protein ZO-1 in association with activating NLRP3 inflammasome. These data not only highlight the potential of ZO-1 as a biomarker for Rickettsia-induced microvascular injury but also provide insight into targeting NLRP3 inflammasome/ZO-1 signaling as a potentially adjunctive therapeutic approach for severe rickettsioses.

Vitexin mitigates oxidative stress, mitochondrial damage, pyroptosis and regulates small nucleolar RNA host gene 1/DNA methyltransferase 1/microRNA-495 axis in sepsis-associated acute lung injury.

This study examinedvitexin's effect on sepsis-induced acute lung injury. We used network pharmacology and in vivo and in vitro experiments were performed to elucidate vitexin's role in preventing pyroptosis and regulating small nucleolar RNA host gene 1 (SNHG1)/DNA methyltransferase 1 (DNMT1)/microRNA-495 (miR-495 axis. We developed an acute lung injury model using C57BL/6 mice and MLE-12 cells. Through a combination of network pharmacology and in vitro screening, vitexin was identified as the most promising anti-inflammatory compound. Multiple techniques such as western blotting, real-time PCR, Hematoxylin and eosin staining, immunohistochemistry, andTUNEL assay were used. Additionally, immunofluorescence, DCFDA and TMRE staining, flow cytometry, methylation-specific PCR, and gene transfection techniques were performed to elucidate vitexin's potential targets and underlying mechanisms. Vitexin treatment significantly reduced lung damage, neutrophil infiltration, and inflammation while improving tight junction integrity. In LPS-treated RAW264.7 macrophages and a septic mouse BALF-induced MLE-12 cell injury model, vitexin demonstrated anti-inflammatory effects, promoted M2 macrophage polarization, and enhanced regenerative markers. It also decreased oxidative stress, mitigated apoptosis and pyroptosis, and improved mitochondrial function. Our research uncovered a novel epigenetic regulatory mechanism involving lncRNA SNHG1, DNMT1, and miR-495. Vitexin's ability to reduce inflammation, counteract oxidative stress, and modulate epigenetic processes. These findings underscore the promising role of vitexin as a treatment for ALI generated by sepsis. The SNHG1/miR-495 axis, which has been identified, represents a new target for future therapies in acute lung injury.

American Society for Gastrointestinal Endoscopy guideline on the diagnosis and management of GERD: summary and recommendations.

This clinical practice guideline from the American Society for Gastrointestinal Endoscopy (ASGE) provides an evidence-based approach for strategies to diagnose and manage GERD. This document was developed using the Grading of Recommendations Assessment, Development, and Evaluation framework and serves as an update to the 2014 ASGE guideline on the role of endoscopy in the management of GERD. This updated guideline addresses the indications for endoscopy in patients with GERD as well as in the emerging population of patients who develop GERD after sleeve gastrectomy or peroral endoscopic myotomy. It also discusses how to endoscopically evaluate gastroesophageal junctional integrity in a comprehensive and uniform manner. Importantly, this guideline also discusses management strategies for GERD including the role of lifestyle interventions, proton pump inhibitors (PPIs), and endoscopic antireflux therapy (including transoral incisionless fundoplication [TIF], radiofrequency energy, and combined hiatal hernia repair and TIF [cTIF]) in the management of GERD. The ASGE suggests upper endoscopy for the evaluation of GERD in patients with alarm symptoms, with multiple risk factors for Barrett's esophagus, and with a history of sleeve gastrectomy. The ASGE recommends careful endoscopic evaluation, reporting, and photo-documentation of objective GERD findings with attention to gastroesophageal junction landmarks and integrity in patients who undergo upper endoscopy to improve care. In patients with GERD symptoms, the ASGE recommends lifestyle modifications. In patients with symptomatic and confirmed GERD with predominant heartburn symptoms, the ASGE recommends medical management including PPIs at the lowest dose for the shortest duration possible while initiating discussion about long-term management options. In patients with confirmed GERD with small hiatal hernias (≤2 cm) and Hill grade I or II who meet specific criteria, the ASGE suggests evaluation for TIF as an alternative to chronic medical management. In patients with persistent GERD with large hiatal hernias (> 2cm) and Hill grade III or IV, the ASGE suggests either cTIF or surgical therapy based on multidisciplinary review. This document summarizes the methods, analyses, and decision processes used to reach the final recommendations and represents the official ASGE recommendations on the above topics.

1,8-Cineole reduces pulmonary vascular remodelling in pulmonary arterial hypertension by restoring intercellular communication and inhibiting angiogenesis.

Pulmonary Arterial Hypertension (PAH) is characterized by pulmonary vascular remodelling, often associated with disruption of BMPR2/Smad1/5 and BMPR2/PPAR-γ signalling pathways that ultimately lead to right ventricle failure. Disruption of intercellular junctions and communication and a pro-angiogenic environment are also characteristic features of PAH. Although, current therapies improve pulmonary vascular tone, they fail to tackle other key pathological features that could prevent disease progression. In this scenario, aromatic plants emerge as promising sources of bioactive compounds, with 1,8-cineole standing out due to its hypotensive properties and cardioprotective effect in PAH. The present study aims to explore for the first time the effect of 1,8-cineole in pulmonary vascular remodelling associated with PAH. Resorting to the monocrotaline (MCT)-induced PAH animal model, the effect of 1,8-cineole on vascular remodelling including interstitial collagen accumulation, smooth muscle cell proliferation and protein levels of BMPR2 pathway-related proteins, was assessed by microscopy and western blot (WB) analysis. The integrity of gap junctions, pulmonary surfactant, mitochondrial structure and endothelial cell barrier were evaluated by transmission electron microscopy, confocal microscopy and WB analysis. Furthermore, the effect of 1,8-cineole on angiogenesis was determined on pulmonary artery endothelial cells (PAEC) submitted to hypoxia using the scratch wound and Matrigel angiogenesis assays, and the number of sprouts on isolated healthy and diseased pulmonary artery rings, treated with the compound, enabled the validation of these effects. 1,8-Cineole mitigated PAH-associated derailment of both BMPR2/Smad1/5 and BMPR2/PPAR-γ pathways and concomitantly reduced interstitial fibrosis and the arterial medial layer thickness in pulmonary arteries. The compound restored gap junction, lung surfactant and mitochondrial integrity and preserved endothelial barrier integrity. Furthermore, 1,8-cineole exerted an anti-angiogenic effect, by impairing the formation of vessel-like structures in PAEC and sprouting formation in isolated pulmonary arteries. The present study brings new insights about the mechanisms whereby 1,8-cineole impacts pulmonary vascular remodelling and demonstrates the potential of 1,8-cineole as a therapeutic strategy to hamper PAH progression.

35kDa SPECIFIC-SIZED HYALURONAN AMELIORATES HIGH-FAT DIET-INDUCED LIVER INJURY IN MURINE MODEL OF MODERATE OBESITY.

Obesity is a growing concern in the US and world-wide, associated with an increased risk for several cardiometabolic diseases, including metabolic associated steatotic liver disease (MASLD). Currently, therapeutic interventions to prevent and/or treat MASLD are limited, and research is needed to identify new therapeutic targets. The specific-sized 35kDa fragment of hyaluronan (HA35), has gut protective and anti-inflammatory properties and a previous pilot clinical study reported it is well tolerated in healthy individuals. Here we tested the hypothesis that HA35 treatment ameliorates high fat diet-induced liver injury. Five-week-old male C57BL/6J mice were allowed ad lib access to control chow or high fat fructose and cholesterol (FFC) diet over a period of 12 weeks. HA35 was administered at 15mg/kg via oral gavage on the last 6 days of the study as a therapeutic intervention. Mice on FFC diet-gained more body weight compared to those on chow diet, with final body weights ranging from 30.8-45.6 g. FFC diet caused hepatocyte injury, increased expression of inflammatory cytokine/chemokine mRNA, as well as indicators of liver fibrosis. When mice were stratified based on their final body weight, only mice <40g were protected by treatment with HA35. In this group, treatment with HA35 also restored tight junction integrity in the colon and increased expression of α -defensins in the small intestine. Taken together the data suggests that HA35 is an effective therapeutic in ameliorating high fat diet-induced liver inflammation and fibrosis in moderately obese, but not severe, conditions.

Correlation of the Endoscopic Esophagogastric Junction Integrity with Symptomatic GERD in Patients Undergoing Work-Up for Metabolic and Bariatric Surgery.

Gastroesophageal reflux disease (GERD) is a common adverse effect after metabolic and bariatric surgery (MBS). Identifying patients with preexisting GERD is critical for preoperative planning. The American Foregut Society (AFS) recently proposed a new endoscopic classification system for objective assessment of the esophagogastric junction (EGJ) integrity, building upon the Hill classification. Grade 1 represents an intact EGJ, while grades 2, 3, and 4 represent partial, moderate, and complete disruption of the ARB. Unlike Hill classification, the AFS classification includes objective measurement of hiatal axial length and aperture diameter. The study aimed to evaluate the ability of the AFS hiatus classification to predict GERD severity using symptom questionnaires. We performed a prospective study of obese patients who underwent endoscopy as work-up for MBS. The endoscopy was evaluated for esophagitis, Barrett's esophagus, and AFS grade. All patients were also surveyed preoperatively with the GERD-HRQL, GerdQ, and RSI. The correlation between AFS grades and questionnaire scores was analyzed using Spearman's test. A total of 393 patients were included in the study. There were 81% female, with a mean age of 36 ± 10.7years and a mean BMI of 41.7 ± 7.2kg/m2. The AFS grades were distributed as follows: 11 (2.8%) patients had grade 1; 137 (34.9%) had grade 2; 162 (41.2%) had grade 3; and 83 (21.1%) had grade 4. There was a positive but weak correlation between AFS grades and all scoring systems, including GERD-HRQL (r = 0.201), heartburn (r = 0.203), regurgitation (r = 0.212), RSI (r = 0.110), and GerdQ scores (r = 0.202). However, the proportion of patients with esophagitis increased progressively with increasing grades (0% in grade 1, 2.2% in grade 2, 9.9% in grade 3, and 32.5% in grade 4, p = 0.01). The AFS hiatus classification can stratify the population with obesity based on rate of esophagitis and symptom scores. This study supports the practical utility of the AFS classification as an adjunct in the detection of patients who are at risk for GERD after MBS. Further validation studies with pH testing are needed.

Targeting Inflammation and Skin Aging via the Gut-Skin Axis: The Role of Lactiplantibacillus plantarum HY7714-Derived Extracellular Vesicles.

Intestinal mucosal tissues are prone to infections, often leading to inflammation. Lactic acid bacteria in the gut can modulate these inflammatory responses, but the interaction between host cells and lactic acid bacteria remains unclear. This study examines how Lactiplantibacillus plantarum HY7714 alleviates intestinal inflammation using gut-on-a-chip technology and in vitro models. Inflammation was induced using a gut-on-a-chip, and changes in cell morphology and barrier function were analyzed. Extracellular vesicles (EVs) derived from HY7714-improved intestinal cell structure repaired damage and restored tight junction integrity. Additionally, they attenuated inflammatory cytokines by regulating the MyD88/mTOR/NF-κB signaling pathway. RNA sequencing revealed downregulation of vicinal oxygen chelate (VOC) family proteins and proline aminopeptidase, both linked to inflammation and extracellular matrix interactions in skin health. Therefore, we explored the effects of HY7714 EVs on skin cells. The findings showed that HY7714 EVs reduced cytotoxicity and downregulated metalloproteinase expression in skin cells exposed to UVB radiation, indicating their potential anti-aging and anti-photoaging properties. These findings suggest that HY7714-derived EVs enhance both intestinal and skin health by reducing inflammation and improving barrier function, with potential benefits for the gut-skin axis.

Enhanced anti-inflammatory effects and metabolic profiles of cyanidin-3-glucoside via nanoliposomal encapsulation in Caco-2 and RAW 264.7 co-culture system

Cyanidin-3-glucoside (C3G) has been found to prevent/treat intestinal disease and maintain gut homeostasis. Numerous studies have proved that nanoliposomes can improve the absorption and utilization of food nutrients. However, the impacts of C3G nanoliposomes (C3G-NL) are still unkown. This study investigated the effects of cyanidin-3-glucoside nanoliposomes (C3G-NL) on intestinal inflammation and permeability using a Caco-2/RAW264.7 co-culture model. ELISA and qPCR results showed that C3G-NL reduced pro-inflammatory markers, including TNF-α, IL-6, IL-8, cyclooxygenase-2, and inducible nitric oxide synthase, while inhibiting the NF-κB/MAPK pathway. C3G-NL also improved intestinal barrier integrity by increasing transepithelial electrical resistance and upregulating tight junction proteins (zonula occludens-1, occludin, claudin-1). Additionally, C3G-NL enhanced C3G metabolism, leading to the formation of intermediary metabolites like chalcone and dihydroflavonols, which contributed to the synthesis of other anthocyanins (delphinidin, peonidin, petunidin, malvidin, and pelargonidin). Overall, C3G-NL helps mitigate inflammation and restore tight junction integrity via metabolic regulation.

Endothelial Rho kinase controls blood vessel integrity and angiogenesis.

The Rho kinases 1 and 2 (ROCK1/2) are serine-threonine specific protein kinases that control actin cytoskeleton dynamics. They are expressed in all cells throughout the body, including cardiomyocytes, smooth muscle cells and endothelial cells, and intimately involved in cardiovascular health and disease. Pharmacological ROCK inhibition is beneficial in mouse models of hypertension, atherosclerosis, and neointimal thickening that display overactivated ROCK. However, the consequences of endothelial ROCK signaling deficiency in vivo remain unknown. To address this issue, we analyzed endothelial cell (EC) specific ROCK1 and 2 deletions. We generated Cdh5-CreERT2 driven, tamoxifen inducible loss of function alleles of ROCK1 and ROCK2 and analyzed mouse survival and vascular defects through cellular, biochemical, and molecular biology approaches. We observed that postnatal or adult loss of endothelial ROCK1 and 2 was lethal within a week. Mice succumbed to multi-organ hemorrhage that occurred because of loss of vascular integrity. ECs displayed deficient cytoskeletal actin polymerization that prevented focal adhesion formation and disrupted junctional integrity. Retinal sprouting angiogenesis was also perturbed, as sprouting vessels exhibited lack of polymerized actin and defective lumen formation. In a three-dimensional endothelial sprouting assay, combined knockdown of ROCK1/2 or knockdown or ROCK2 but not ROCK1 led to reduced sprouting, lumenization and cell polarization defects caused by defective actin and altered VE-cadherin dynamics. The isoform specific role of endothelial ROCK2 correlated with ROCK2 substrate specificity for FAK and LIMK. By analyzing single and three allele mutants we show that one intact allele of ROCK2 is sufficient to maintain vascular integrity in vivo. Endothelial ROCK1 and 2 maintain junctional integrity and ensure proper angiogenesis and lumen formation. The presence of one allele of ROCK2 is sufficient to maintain vascular growth and integrity. These data indicate the need of careful consideration for the use of ROCK inhibitors in disease settings.