Search Results (235)

Objectives: Saffron, a traditional Chinese medicine, is renowned for its pharmacological effects in promoting blood circulation, resolving blood stasis, regulating menstruation, detoxification, and alleviating mental disturbances. Trans-crocetin, its principal bioactive component, exhibits significant anti-hypoxic activity. The clinical development and therapeutic efficacy of trans-crocetin are limited by its instability, poor solubility, and low bioavailability. Conversion of trans-crocetin into trans-sodium crocetinate (TSC) enhances its solubility, stability, and bioavailability, thereby amplifying its anti-hypoxic potential. Methods: This study integrates network pharmacology with in vivo and in vitro validation to elucidate the molecular targets and mechanisms underlying TSC’s therapeutic effects against high-altitude acute lung injury (HALI), aiming to identify novel treatment strategies. Results: TSC effectively reversed hypoxia-induced biochemical abnormalities, ameliorated lung histopathological damage, and suppressed systemic inflammation and oxidative stress in HALI rats. In vitro, TSC mitigated CoCl₂-induced hypoxia injury in human pulmonary microvascular endothelial cells (HPMECs) by reducing inflammatory cytokines, oxidative stress, and ROS accumulation while restoring mitochondrial membrane potential. Network pharmacology and pathway analysis revealed that TSC primarily targets the EGFR/PI3K/AKT/NF-κB signaling axis. Molecular docking and dynamics simulations demonstrated stable binding interactions between TSC and key components of this pathway. ELISA and RT-qPCR confirmed that TSC significantly downregulated the expression of EGFR, PI3K, AKT, NF-κB, and their associated mRNAs. Conclusions: TSC alleviates high-altitude hypoxia-induced lung injury by inhibiting the EGFR/PI3K/AKT/NF-κB signaling pathway, thereby attenuating inflammatory responses, oxidative stress, and restoring mitochondrial function. These findings highlight TSC as a promising therapeutic agent for HALI. Full article

The endothelial glycocalyx (GCX) plays a crucial role in vascular health and integrity and influences many biochemical activities through mechanotransduction, in which heparan sulfate (HS) plays a major role. Endothelin-1 (ET-1) is a potent vasoregulator that binds to the endothelin B receptor (ETB) on endothelial cells (ECs), stimulating vasodilation, and to the endothelin A receptor on smooth muscle cells, stimulating vasoconstriction. While the shear stress (SS) dependence of ET-1 and HS is well documented, there is limited research documenting the SS dependence of the ETB. Understanding the SS dependence of the ETB is crucial for clarifying the role of hemodynamic forces in the endothelin system. We hypothesize that GCX HS regulates the expression of the ETB on the EC surface in an SS-dependent manner. Human lung microvascular ECs were exposed to SS in a parallel-plate flow chamber for 12 h. Damage to the GCX was simulated by treatment with 15 mU/mL heparinase-III during SS exposure. Immunostaining and qPCR were used to evaluate changes in ET-1, ETB, and HS expression. Results indicate that ETB expression is SS sensitive, with at least a 1.3-fold increase in ETB protein expression and a 0.6 to 0.4-fold-change decrease in ETB mRNA expression under SS. This discrepancy suggests post-translational regulation. In some cases, enzymatic degradation of HS attenuated the SS-induced increase in ETB protein, reducing the fold-change difference to 1.1 relative to static controls. This implies that ETB expression may be partially dependent on HS-mediated mechanotransduction, though inconclusively. Furthermore, ET-1 mRNA levels were elevated two-fold under SS without a corresponding rise in ET-1 protein expression or significant impact from HS degradation, implying that post-translational regulation of ET-1 occurs independently of HS. Full article

High-altitude pulmonary edema (HAPE) is a severe condition associated with high-altitude environments, and its molecular mechanism has not been fully elucidated. This study systematically analyzed the DNA methylation status of HAPE patients and healthy controls using reduced-representation bisulfite sequencing (RRBS) and 850K DNA methylation chips, identifying key differentially methylated regions (DMRs). Targeted bisulfite sequencing (TBS) revealed significant abnormalities in DMRs of five genes, azurocidin 1 (AZU1), growth factor receptor bound protein 7 (GRB7), mannose receptor C-type 2 (MRC2), RUNX family transcription factor 3 (RUNX3), and septin 9 (SEPT9). The abnormal expression of AZU1 was validated using peripheral blood leukocytes from HAPE patients and normal controls, as well as rat lung tissue, indicating its potential importance in the pathogenesis of HAPE. To further validate the function of AZU1, we conducted experimental studies using a hypobaric hypoxia injury model in Human Umbilical Vein Endothelial Cells (HUVEC). The results showed that AZU1 was significantly upregulated under hypobaric hypoxia. Knocking down AZU1 mitigates the reduction in HUVEC proliferation, angiogenesis, and oxidative stress damage induced by acute hypobaric hypoxia. AZU1 induces cellular oxidative stress via the p38/mitogen-activated protein kinase (p38/MAPK) signaling pathway. This study is the first to elucidate the mechanism of AZU1 in HAPE via the p38/MAPK pathway, offering novel insights into the molecular pathology of HAPE and laying a foundation for future diagnostic and therapeutic strategies. Full article

Orthoflavivirus omskense (Omsk hemorrhagic fever virus, OHFV) is a tick-borne flavivirus that causes Omsk hemorrhagic fever (OHF), a severe zoonotic disease endemic to Western Siberia. Despite the fact that the role of NS1 proteins of various mosquito-borne flaviviruses in pathogenesis was investigated and their ability to affect human endothelial permeability was shown, the role of the NS1 protein of OHFV in pathogenesis is unstudied. In this work, the ability of OHFV NS1 to induce human endothelial permeability was investigated for the first time. It was shown that recombinant OHFV NS1 produced in eucaryotic cells directly affects both human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC) in vitro. RNAseq of endothelial cells treated with OHFV NS1 indicated that OHFV NS1 enhances the expression of genes associated with cellular stress responses, vascular signaling, and cell–cell junction regulation, resulting in a nonspecific increase in the endothelial permeability of various vessels. These results suggest that the NS1 protein may contribute to OHFV pathogenesis by disrupting endothelial barrier function and promoting vascular leakage, potentially playing a role in the hemorrhagic manifestations of Omsk hemorrhagic fever. Full article

Viral lung infections are a never-ending threat to public health due to the emergence of new variants and their seasonal nature. While vaccines offer some protection, the need for effective antiviral drugs remains high. The existing research methods using 2D cell culture and animal models have their limitations. Human cell-based tissue engineering approaches hold great promise for bridging this gap. Here, we describe a microextrusion bioprinting approach to generate three-dimensional (3D) lung models composed of four cell types: endothelial cells, primary fibroblasts, macrophage cells, and epithelial cells. A549 and Calu-3 cells were selected as epithelial cells to simulate the cells of the lower and upper respiratory tract, respectively. Cells were bioprinted in a hydrogel consisting of alginate, gelatin, hyaluronic acid, collagen, and laminin-521. The models were cultured under air–liquid interface (ALI) conditions to further enhance their physiological relevance as lung cells. Their viability, metabolic activity, and expression of specific cell markers were analyzed during long-term culture for 21 days. The constructs were successfully infected with both a seasonal influenza A virus (IAV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant, demonstrating their potential for studying diverse viral infections. Full article

Orthoflavivirus encephalitidis (tick-borne encephalitis virus, TBEV) is of high concern due to its ability to cause severe neurological manifestations. Despite the fact that the role of NS1 proteins from various mosquito-borne flaviviruses in pathogenesis and their ability to affect human endothelial permeability have been investigated, TBEV NS1 has thus far been insufficiently studied. In this study, human endothelial permeability was assessed using TEER and transwell permeability assays. Signaling pathways were determined by RNAseq. The ability of the NS1 protein of TBEV to affect human endothelial permeability was investigated for the first time. It was shown that recombinant TBEV NS1 produced in eucaryotic cells directly affected human lung microvascular endothelial cells (HLMVECs) in vitro but not human umbilical vein endothelial cells (HUVECs). It was indicated that TBEV NS1 induced endothelial hyperpermeability of HLMVECs through activating TNF-α and other inflammatory signaling pathways. Full article

Current animal and in vitro cell culture models do not fully recapitulate the physiological and pathophysiological characteristics of the human lung. As a result, the translation of these models to clinical practice is very limited, and clinical trials initiated on the extrapolation of such data fail. Although current models are beneficial in fundamental research, there is a need to constantly improve models to more accurately predict outcomes in clinical trials and personalized medicine. Here, we report important strategies to develop a 3D lung model with human primary lung cells. Starting from the well-established air-liquid interface (ALI) culture system, we describe a gradual increase in the complexity of the system by co-culturing different primary cell types, by testing different coatings, and by adding a three-dimensional matrix. As a result, we have established a reproducible 3D in vitro model of the airway consisting of human primary cells representing a differentiated mucociliary airway epithelium, an underlying submucosa with fibroblasts, and an endothelial interface. Full article

Isaridin E, a cyclodepsipeptide derived from the marine fungus Beauveria felina (SYSU-MS7908), has been demonstrated to possess multiple biological properties. In this study, we employed both lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) and a LPS-induced murine endotoxemia model to investigate its anti-inflammatory effects. Our results revealed that isaridin E suppressed the expression of pro-inflammatory cytokines and adhesion molecules in a concentration dependent manner, while also reducing monocyte adhesion to endothelial cells. Furthermore, this compound attenuated vascular hyperpermeability and inflammatory cell infiltration in the lungs, as well as preserving the integrity of the aortic and pulmonary tissues. At the molecular level, isaridin E was found to downregulate TLR4 expression, increase IκBα levels, and inhibit the LPS-induced phosphorylation and nuclear translocation of NF-κB p65. In conclusion, our findings indicate that isaridin E exerts robust anti-inflammatory effects in LPS-induced endotoxemia through the suppression of the TLR4/NF-κB signaling axis, positioning it as a promising therapeutic candidate for vascular inflammatory disorders. Full article

Integrin β4 (ITGB4) mediates lung endothelial cell (EC) inflammation attenuated by simvastatin, an HMG CoA-reductase inhibitor. The cytoplasmic domain of ITGB4 is predicted to bind kindlin-2. Kindlin-2 expression is mediated by SMURF1, an E3 ubiquitin ligase that promotes kindlin-2 ubiquitination and degradation. We hypothesized that increased kindlin-2 expression via the inhibition of SMURF1 mediates EC inflammatory responses relevant to acute lung injury (ALI). To investigate this, human lung ECs were treated with simvastatin (5 µM, 16 h) prior to the immunoprecipitation of kindlin-2 and Western blotting for ITGB4. Next, ECs were treated with a SMURF1 inhibitor, A01, and increased kindlin-2 expression was confirmed. In assays of barrier function, kindlin-2 was silenced (siRNA) in ECs prior to thrombin and measurements of transendothelial resistance (TER) and FITC-dextran transwell flux. Repeat assessments of barrier function were performed in A01-treated ECs. Finally, mice were pretreated with A01 prior to LPS; bronchoalveolar lavage (BAL) fluid was collected, and their lungs were used for histology. Simvastatin increased ITGB4:kindlin-2 association, while A01 increased kindlin-2 expression. Thrombin-induced EC barrier disruption was both increased after kindlin-2 silencing and decreased by A01. Finally, murine ALI was significantly attenuated by A01. Our findings suggest that the augmentation of kindlin-2 may serve as a novel ALI therapeutic strategy. Full article

Drug development for human disease relies on preclinical model systems such as human cell cultures and animal experiments before therapeutic treatments can ultimately be tested on humans in clinical studies. We here describe the generation of a novel human cell line (HLMVEC/SVTERT289) that we generated by transfection of microvascular endothelial cells from healthy donor lung tissue with the catalytic domain of telomerase and the SV40 large T/small t-antigen. These cells exhibited satisfactory growth characteristics and largely maintained their native characteristics, including morphology, cell surface marker expression, angiogenic potential and the protein composition of secreted extracellular vesicles. In order to test their suitability as a disease model, we simulated mechanical stress induced by cyclic stretch as encountered in ventilator-induced lung injury using the FlexCell^® system and compared their performance to primary lung endothelial cells. In this setting, HLMVEC/SVTERT289 cells exhibited significantly higher neprilysin activity on the cell surface and extracellular vesicles secreted from the cell line exhibited higher Tissue Factor and ACE2 expression but lower ACE expression and ACE activity than vesicles released from the primary cells. This study provides an unprecedented and detailed characterization of the HLMVEC/SVTERT289 cell line, which should help to appraise its suitability in different molecular studies. Full article

Objectives: Angiogenic T cells (Tang) are crucial in promoting angiogenesis, with the loss of CD28 serving as a marker for highly differentiated and senescent T cells. This study aims to investigate the characteristics and potential roles of CD8⁺CD28^null Tang in patients with ANCA-associated vasculitis (AAV). Methods: A cohort of AAV patients and matched healthy controls (HCs) were analyzed. Flow cytometry was used to assess the profiles of circulating CD8⁺CD28^null Tang. In vitro functional assays were performed to evaluate the pathogenic properties of CD8⁺CD28^null Tang. Results: CD8⁺CD28^null Tang levels were significantly higher in the peripheral blood of AAV patients compared to HCs, and their levels were further increased in AAV patients with MPO⁺, p-ANCA⁺, or interstitial lung disease compared to their respective control groups. Additionally, there was a positive correlation between both the percentage and absolute count of CD8⁺CD28^null Tang and the Birmingham Vasculitis Activity Score (BVAS). In patients with a good treatment response, both the percentage and absolute count of CD8⁺CD28^null Tang were significantly reduced, and this reduction was positively correlated with the decrease in BVAS scores. In vitro studies revealed that CD8⁺CD28^null Tang displayed enhanced chemotactic properties, induced apoptosis in human umbilical vein endothelial cells (HUVECs), and inhibited their proliferation, migration, and tube formation. Conclusions: AAV patients exhibit increased levels of circulating CD8⁺CD28^null Tang, which can be reduced following effective treatment. Furthermore, CD8⁺CD28^null Tang may contribute to the pathogenesis of AAV by promoting apoptosis and inhibiting the proliferation, migration, and tube formation of HUVECs. Full article

Tissue acidification resulting from dysregulated cellular bioenergetics accompanies various inflammatory states. GPR68, along with other members of proton-sensing G protein-coupled receptors, responds to extracellular acidification and has been implicated in chronic inflammation-related diseases such as ischemia, cancer, and colitis. The present study examined the role of extracellular acidification on human pulmonary endothelial cell (EC) permeability and inflammatory status per se and investigated potential synergistic effects of acidosis on endothelial dysfunction caused by bacterial lipopolysaccharide (LPS, Klebsiella pneumoniae). Results showed that medium acidification to pH 6.5 caused a delayed increase in EC permeability illustrated by a decrease in transendothelial electrical resistance and loss of continuous VE-cadherin immunostaining at cell junctions. Likewise, acidic pH induced endothelial inflammation reflected by increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1, upregulated mRNA transcripts of tumor necrosis factor-α, IL-6, IL-8, IL-1β, and CXCL5, and increased secretion of ICAM-1, IL-6, and IL-8 in culture medium monitored by ELISA. Among the GPCRs tested, acidic pH selectively increased mRNA and protein expression of GPR68, and only the GPR68-specific small molecule inhibitor OGM-8345 rescued acidosis-induced endothelial permeability and inflammation. Furthermore, acidic pH exacerbated LPS-induced endothelial permeability and inflammatory response in cultured lung macrovascular as well as microvascular endothelial cells. These effects were suppressed by OGM-8345 in both EC types. Altogether, these results suggest that GPR68 is a critical mediator of acidic pH-induced dysfunction of human pulmonary vascular endothelial cells and mediates the augmenting effect of tissue acidification on LPS-induced endothelial cell injury. Full article

Objectives: This research aims to investigate the mechanisms of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC), particularly focusing on the role of the epithelial–mesenchymal transition (EMT) within the tumor microenvironment (TME). Materials and Methods: We employed an in vitro three-dimensional organoid model that mirrors the physiology of human lung cancer. These organoids consist of lung cancer cells harboring specific EGFR mutations, human mesenchymal stem cells, and human umbilical vein endothelial cells. We analyzed EMT and drug resistance markers, and evaluated the effects of the anti-angiogenic agent Bevacizumab and micro-RNA miR200c. Results: The study identified a significant link between EMT and EGFR-TKI resistance. Notable findings included a decrease in E-cadherin and an increase in Zinc Finger E-Box Binding Homeobox 1 (ZEB1), both of which influenced EMT and resistance to treatment. Bevacizumab showed promise in improving drug resistance and mitigating EMT, suggesting an involvement of the Vascular Endothelial Growth Factor (VEGF) cascade. Transfection with miR200c was associated with improved EMT and drug resistance, further highlighting the role of EMT in TKI resistance. Conclusions: Our research provides significant insights into the EMT-driven EGFR-TKI resistance in NSCLC and offers potential strategies to overcome resistance, including the use of Bevacizumab and miR200c. However, due to the limitations in organoid models in replicating precise human cancer TME and the potential influence of specific EGFR mutations, further in vivo studies and clinical trials are necessary for validation. Full article

Fine particulate matter (PM) 2.5 is the main component of air pollution causing pathological responses primarily in the respiratory and cardiovascular systems. Therefore, it is urgent to explore valid strategies to inhibit the adverse reactions induced by PM2.5. In our previous studies, we have revealed that intratracheal instillation of PM2.5 evoked airway remodeling, pulmonary inflammatory, and oxidative stress responses in rat lungs by upregulating VEGFA levels in bronchial epithelial cells and by activating ANGII/AT1R axis activation in vascular endothelial cells. The same results were obtained when human bronchial epithelial cells (Beas−2B) and human umbilical vein endothelial cells (HUVECs) cells were exposed to PM2.5 in vitro. Curcumin is a dietary polyphenol with protective properties, including anti−inflammatory and antioxidant effects. This study aims to determine the potential role of curcumin in protecting against PM2.5−induced adverse responses in the bronchial epithelium and vascular endothelium and the mechanism involved. To this end, we pretreated cells with curcumin (diluted 1000 times in sterile saline) for 2 h and then exposed them to PM2.5. Our results from RT−PCR, a luciferase reporter assay, and ELISA indicated that curcumin pretreatment effectively inhibited PM2.5−induced VEGFA elevation in Beas−2B cells by over 60% via blocking HIF1α accumulation and HIF1 transactivity, Moreover, curcumin also exerted a protective role in suppressing PM2.5−induced ANGII/AT1R axis components expression in HUVEC by over 90% via targeting the transcriptional factors, AP−1 and HIF1. Under the same conditions, curcumin pretreatment also blocked the downstream signaling events following ANGII/AT1R pathway activation, the increase in chemokines and cell adhesion molecules (sICAM−1, VCAM−1, E−Selectin, P−Selectin, IL−8, MCP−1) that drive monocyte−endothelial cell adhesion, as well as the elevated production of oxidative stress mediators (ROS and MDA) in HUVECs according to the data from immunofluorescence and flow cytometric assays. Most importantly, administration of curcumin resulted in an 80% reduction of the HIF1− and AP−1−dependent upregulation of VEGFA and AGT/AT1R axis components and impeding the resultant pro−inflammatory and oxidative responses in the lung of the rats exposed to PM2.5. Taking these data together, we disclosed the important role and mechanism of curcumin in protecting against PM2.5−induced adverse reactions in the bronchial epithelium and vascular endothelium. Curcumin might be used as a feasible and safe dietary agent to reduce the health risk of PM2.5. Full article

Background/Objectives: Endothelial hyperpermeability is the hallmark of severe disease, including sepsis and acute respiratory syndrome (ARDS). The development of medical countermeasures to treat the corresponding illness is of utmost importance. Synthetic somatostatin analogs (SSA) are FDA-approved drugs prescribed in patients with neuroendocrine tumors, and they act via growth hormone (GH) suppression. Preclinical investigations suggest that Octreotide (OCT) alleviates Lipopolysaccharide (LPS)-induced injury. The aim of the study is to investigate the involvement of activating transcription factor 6 (ATF6) in the protective effects of OCT in endothelial dysfunction. To the best of our knowledge, the available information on that topic is limited. Methods: Human lung microvascular endothelial cells (HULEC-5a) and bovine pulmonary artery endothelial cells (BPAEC) which expressed elevated levels of ATF6 due to AA147 were exposed to OCT or vehicle. Protein expression, endothelial permeability, and reactive oxygen species (ROS) generation were assessed utilizing Western blot analysis, Fluorescein isothiocyanate (FITC)-Dextran assay, and Dichlorofluorescein diacetate measurements, respectively. Results: Our observations suggest that ATF6 activation significantly improves OCT-induced endothelial barrier enhancement. This combination led to increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (Grp94), which are downstream unfolded protein response (UPR) targets. Moreover, ATF6 activation prior to OCT treatment resulted in decreased activation of myosin light chain 2 (MLC2) and cofilin; and reduced reactive oxygen species (ROS) generation. ATF6 activation enhanced the anti-inflammatory effects of OCT, as reflected in the suppression of transducer and activator of transcription (STAT) 1, STAT3, and P38 phosphorylation. Conclusions: Our findings suggest that ATF6 activation prior to OCT treatment enhances the beneficial effects of OCT in the endothelium. Full article