Protective effect of trans-nerolidol on vascular endothelial cell injury induced by lipopolysaccharides / Efectos protectores del trans-nerolidol en lesiones inducidas por lipopolisacáridos en células endoteliales vasculares

This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated vimentin in human lung tissue.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500µm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.

Mexenone protects mice from LPS-induced sepsis by EC barrier stabilization.

Blood vessels permit the selective passage of molecules and immune cells between tissues and circulation. Uncontrolled inflammatory responses from an infection can increase vascular permeability and edema, which can occasionally lead to fatal organ failure. We identified mexenone as a vascular permeability blocker by testing 2,910 compounds in the Clinically Applied Compound Library using the lipopolysaccharide (LPS)-induced vascular permeability assay. Mexenone suppressed the LPS-induced downregulation of junctional proteins and phosphorylation of VE-cadherin in Bovine Aortic Endothelial Cells (BAECs). The injection of mexenone 1 hr before LPS administration completely blocked LPS-induced lung vascular permeability and acute lung injury in mice after 18hr. Our results suggest that mexenone-induced endothelial cell (EC) barrier stabilization could be effective in treating sepsis patients.

Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria.

BACKGROUND: It is generally accepted that endothelial cells (ECs), primarily rely on glycolysis for ATP production, despite having functional mitochondria. However, it is also known that ECs are heterogeneous, and their phenotypic features depend on the vascular bed. Emerging evidence suggests that liver sinusoidal ECs (LSECs), located in the metabolically rich environment of the liver, show high metabolic plasticity. However, the substrate preference for energy metabolism in LSECs remains unclear. METHODS: Investigations were conducted in primary murine LSECs in vitro using the Seahorse XF technique for functional bioenergetic assays, untargeted mass spectrometry-based proteomics to analyse the LSEC proteome involved in energy metabolism pathways, liquid chromatography-tandem mass spectrometry-based analysis of acyl-carnitine species and Raman spectroscopy imaging to track intracellular palmitic acid. RESULTS: This study comprehensively characterized the energy metabolism of LSECs, which were found to depend on oxidative phosphorylation, efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids and glutamine. Furthermore, despite its high availability, palmitic acid was not directly oxidized in LSEC mitochondria, as evidenced by the acylcarnitine profile and etomoxir's lack of effect on oxygen consumption. However, together with L-carnitine, palmitic acid supported mitochondrial respiration, which is compatible with the chain-shortening role of peroxisomal ß-oxidation of long-chain fatty acids before further degradation and energy generation in mitochondria. CONCLUSIONS: LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment. The functional reliance of LSECs on oxidative phosphorylation, which is not a typical feature of ECs, remains to be determined.

MAP4K4 exacerbates cardiac microvascular injury in diabetes by facilitating S-nitrosylation modification of Drp1.

Dynamin-related protein 1 (Drp1) is a crucial regulator of mitochondrial dynamics, the overactivation of which can lead to cardiovascular disease. Multiple distinct posttranscriptional modifications of Drp1 have been reported, among which S-nitrosylation was recently introduced. However, the detailed regulatory mechanism of S-nitrosylation of Drp1 (SNO-Drp1) in cardiac microvascular dysfunction in diabetes remains elusive. The present study revealed that mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) was consistently upregulated in diabetic cardiomyopathy (DCM) and promoted SNO-Drp1 in cardiac microvascular endothelial cells (CMECs), which in turn led to mitochondrial dysfunction and cardiac microvascular disorder. Further studies confirmed that MAP4K4 promoted SNO-Drp1 at human C644 (mouse C650) by inhibiting glutathione peroxidase 4 (GPX4) expression, through which MAP4K4 stimulated endothelial ferroptosis in diabetes. In contrast, inhibition of MAP4K4 via DMX-5804 significantly reduced endothelial ferroptosis, alleviated cardiac microvascular dysfunction and improved cardiac dysfunction in db/db mice by reducing SNO-Drp1. In parallel, the C650A mutation in mice abolished SNO-Drp1 and the role of Drp1 in promoting cardiac microvascular disorder and cardiac dysfunction. In conclusion, our findings demonstrate that MAP4K4 plays an important role in endothelial dysfunction in DCM and reveal that SNO-Drp1 and ferroptosis activation may act as downstream targets, representing potential therapeutic targets for DCM.

Effects of Chronic Inflammatory Activation of Murine and Human Arterial Endothelial Cells at Normal Lipoprotein and Cholesterol Levels In Vivo and In Vitro.

The activation of endothelial cells is crucial for immune defense mechanisms but also plays a role in the development of atherosclerosis. We have previously shown that inflammatory stimulation of endothelial cells on top of elevated lipoprotein/cholesterol levels accelerates atherogenesis. The aim of the current study was to investigate how chronic endothelial inflammation changes the aortic transcriptome of mice at normal lipoprotein levels and to compare this to the inflammatory response of isolated endothelial cells in vitro. We applied a mouse model expressing constitutive active IκB kinase 2 (caIKK2)-the key activator of the inflammatory NF-κB pathway-specifically in arterial endothelial cells and analyzed transcriptomic changes in whole aortas, followed by pathway and network analyses. We found an upregulation of cell death and mitochondrial beta-oxidation pathways with a predicted increase in endothelial apoptosis and necrosis and a simultaneous reduction in protein synthesis genes. The highest upregulated gene was ACE2, the SARS-CoV-2 receptor, which is also an important regulator of blood pressure. Analysis of isolated human arterial and venous endothelial cells supported these findings and also revealed a reduction in DNA replication, as well as repair mechanisms, in line with the notion that chronic inflammation contributes to endothelial dysfunction.

TruD technology for the study of epi- and endothelial tubes in vitro.

Beyond the smallest organisms, animals rely on tubes to transport cells, oxygen, nutrients, waste products, and a great variety of secretions. The cardiovascular system, lungs, gastrointestinal and genitourinary tracts, as well as major exocrine glands, are all composed of tubes. Paradoxically, despite their ubiquitous importance, most existing devices designed to study tubes are relatively complex to manufacture and/or utilize. The present work describes a simple method for generating tubes in vitro using nothing more than a low-cost 3D printer along with general lab supplies. The technology is termed "TruD", an acronym for true dimensional. Using this technology, it is readily feasible to cast tubes embedded in ECM with easy access to the lumen. The design is modular to permit more complex tube arrangements and to sustain flow. Importantly, by virtue of its simplicity, TruD technology enables typical molecular cell biology experiments where multiple conditions are assayed in replicate.

Choline metabolism reprogramming mediates an immunosuppressive microenvironment in non-small cell lung cancer (NSCLC) by promoting tumor-associated macrophage functional polarization and endothelial cell proliferation.

INTRODUCTION: Lung cancer is a prevalent malignancy globally, and immunotherapy has revolutionized its treatment. However, resistance to immunotherapy remains a challenge. Abnormal cholinesterase (ChE) activity and choline metabolism are associated with tumor oncogenesis, progression, and poor prognosis in multiple cancers. Yet, the precise mechanism underlying the relationship between ChE, choline metabolism and tumor immune microenvironment in lung cancer, and the response and resistance of immunotherapy still unclear. METHODS: Firstly, 277 advanced non-small cell lung cancer (NSCLC) patients receiving first-line immunotherapy in Sun Yat-sen University Cancer Center were enrolled in the study. Pretreatment and the alteration of ChE after 2 courses of immunotherapy and survival outcomes were collected. Kaplan-Meier survival and cox regression analysis were performed, and nomogram was conducted to identify the prognostic and predicted values. Secondly, choline metabolism-related genes were screened using Cox regression, and a prognostic model was constructed. Functional enrichment analysis and immune microenvironment analysis were also conducted. Lastly, to gain further insights into potential mechanisms, single-cell analysis was performed. RESULTS: Firstly, baseline high level ChE and the elevation of ChE after immunotherapy were significantly associated with better survival outcomes for advanced NSCLC. Constructed nomogram based on the significant variables from the multivariate Cox analysis performed well in discrimination and calibration. Secondly, 4 choline metabolism-related genes (MTHFD1, PDGFB, PIK3R3, CHKB) were screened and developed a risk signature that was found to be related to a poorer prognosis. Further analysis revealed that the choline metabolism-related genes signature was associated with immunosuppressive tumor microenvironment, immune escape and metabolic reprogramming. scRNA-seq showed that MTHFD1 was specifically distributed in tumor-associated macrophages (TAMs), mediating the differentiation and immunosuppressive functions of macrophages, which may potentially impact endothelial cell proliferation and tumor angiogenesis. CONCLUSION: Our study highlights the discovery of ChE as a prognostic marker in advanced NSCLC, suggesting its potential for identifying patients who may benefit from immunotherapy. Additionally, we developed a prognostic signature based on choline metabolism-related genes, revealing the correlation with the immunosuppressive microenvironment and uncovering the role of MTHFD1 in macrophage differentiation and endothelial cell proliferation, providing insights into the intricate workings of choline metabolism in NSCLC pathogenesis.

Acod1/itaconate activates Nrf2 in pulmonary microvascular endothelial cells to protect against the obesity-induced pulmonary microvascular endotheliopathy.

BACKGROUND: Obesity is the main risk factor leading to the development of various respiratory diseases, such as asthma and pulmonary hypertension. Pulmonary microvascular endothelial cells (PMVECs) play a significant role in the development of lung diseases. Aconitate decarboxylase 1 (Acod1) mediates the production of itaconate, and Acod1/itaconate axis has been reported to play a protective role in multiple diseases. However, the roles of Acod1/itaconate axis in the PMVECs of obese mice are still unclear. METHODS: mRNA-seq was performed to identify the differentially expressed genes (DEGs) between high-fat diet (HFD)-induced PMVECs and chow-fed PMVECs in mice (|log2 fold change| ≥ 1, p ≤ 0.05). Free fatty acid (FFA) was used to induce cell injury, inflammation and mitochondrial oxidative stress in mouse PMVECs after transfection with the Acod1 overexpressed plasmid or 4-Octyl Itaconate (4-OI) administration. In addition, we investigated whether the nuclear factor erythroid 2-like 2 (Nrf2) pathway was involved in the effects of Acod1/itaconate in FFA-induced PMVECs. RESULTS: Down-regulated Acod1 was identified in HFD mouse PMVECs by mRNA-seq. Acod1 expression was also reduced in FFA-treated PMVECs. Acod1 overexpression inhibited cell injury, inflammation and mitochondrial oxidative stress induced by FFA in mouse PMVECs. 4-OI administration showed the consistent results in FFA-treated mouse PMVECs. Moreover, silencing Nrf2 reversed the effects of Acod1 overexpression and 4-OI administration in FFA-treated PMVECs, indicating that Nrf2 activation was required for the protective effects of Acod1/itaconate. CONCLUSION: Our results demonstrated that Acod1/Itaconate axis might protect mouse PMVECs from FFA-induced injury, inflammation and mitochondrial oxidative stress via activating Nrf2 pathway. It was meaningful for the treatment of obesity-caused pulmonary microvascular endotheliopathy.

Generation of Slco1a4-CreERT2-tdTomato Knock-in Mice for Specific Cerebrovascular Endothelial Cell Targeting.

The cerebrovascular endothelial cells with distinct characteristics line cerebrovascular blood vessels and are the fundamental structure of the blood-brain barrier, which is important for the development and homeostatic maintenance of the central nervous system. Cre-LoxP system-based spatial gene manipulation in mice is critical for investigating the physiological functions of key factors or signaling pathways in cerebrovascular endothelial cells. However, there is a lack of Cre recombinase mouse lines that specifically target cerebrovascular endothelial cells. Here, using a publicly available single-cell RNAseq database, we screened the solute carrier organic anion transporter family member 1a4 (Slco1a4) as a candidate marker of cerebrovascular endothelial cells. Then, we generated an inducible Cre mouse line in which a CreERT2-T2A-tdTomato cassette was placed after the initiation codon ATG of the Slco1a4 locus. We found that tdTomato, which can indicate the endogenous Slco1a4 expression, was expressed in almost all cerebrovascular endothelial cells but not in any other non-endothelial cell types in the brain, including neurons, astrocytes, oligodendrocytes, pericytes, smooth muscle cells, and microglial cells, as well as in other organs. Consistently, when crossing the ROSA26LSL-EYFP Cre reporter mouse, EYFP also specifically labeled almost all cerebrovascular endothelial cells upon tamoxifen induction. Overall, we generated a new inducible Cre line that specifically targets cerebrovascular endothelial cells.

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

Paricalcitol Has a Potent Anti-Inflammatory Effect in Rat Endothelial Denudation-Induced Intimal Hyperplasia.

Neointimal hyperplasia is the main cause of vascular graft failure in the medium term. Vitamin D receptor activation modulates the biology of vascular smooth muscle cells and has been reported to protect from neointimal hyperplasia following endothelial injury. However, the molecular mechanisms are poorly understood. We have now explored the impact of the selective vitamin D receptor activator, paricalcitol, on neointimal hyperplasia, following guidewire-induced endothelial cell injury in rats, and we have assessed the impact of paricalcitol or vehicle on the expression of key cell stress factors. Guidewire-induced endothelial cell injury caused neointimal hyperplasia and luminal stenosis and upregulated the expression of the growth factor growth/differentiation factor-15 (GDF-15), the cytokine receptor CD74, NFκB-inducing kinase (NIK, an upstream regulator of the proinflammatory transcription factor NFκB) and the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Immunohistochemistry confirmed the increased expression of the cellular proteins CD74 and NIK. Paricalcitol (administered in doses of 750 ng/kg of body weight, every other day) had a non-significant impact on neointimal hyperplasia and luminal stenosis. However, it significantly decreased GDF-15, CD74, NIK and MCP-1/CCL2 mRNA expression, which in paricalcitol-injured arteries remained within the levels found in control vehicle sham arteries. In conclusion, paricalcitol had a dramatic effect, suppressing the stress response to guidewire-induced endothelial cell injury, despite a limited impact on neointimal hyperplasia and luminal stenosis. This observation identifies novel molecular targets of paricalcitol in the vascular system, whose differential expression cannot be justified as a consequence of improved tissue injury.

Vascular development, remodeling and maturation.

The development of the vascular system is crucial in supporting the growth and health of all other organs in the body, and vascular system dysfunction is the major cause of human morbidity and mortality. This chapter discusses three successive processes that govern vascular system development, starting with the differentiation of the primitive vascular system in early embryonic development, followed by its remodeling into a functional circulatory system composed of arteries and veins, and its final maturation and acquisition of an organ specific semi-permeable barrier that controls nutrient uptake into tissues and hence controls organ physiology. Along these steps, endothelial cells forming the inner lining of all blood vessels acquire extensive heterogeneity in terms of gene expression patterns and function, that we are only beginning to understand. These advances contribute to overall knowledge of vascular biology and are predicted to unlock the unprecedented therapeutic potential of the endothelium as an avenue for treatment of diseases associated with dysfunctional vasculature.

Tracing embryonic hematopoiesis guides induction of pluripotent stem cells to hematopoietic progenitors.

In this issue of Developmental Cell, Fowler et al. applied genetic lineage-tracing mouse models to support the notion that artery endothelial cells are the predominant source of hematopoietic stem cells. They leveraged this and developed a method capable of efficiently differentiating human pluripotent stem cells into HLF+HOXA+ hematopoietic progenitors.

Doxycycline protects against sepsis-induced endothelial glycocalyx shedding.

Endothelial glycocalyx (eGC) covers the inner surface of the vessels and plays a role in vascular homeostasis. Syndecan is considered the "backbone" of this structure. Several studies have shown eGC shedding in sepsis and its involvement in organ dysfunction. Matrix metalloproteinases (MMP) contribute to eGC shedding through their ability for syndecan-1 cleavage. This study aimed to investigate if doxycycline, a potent MMP inhibitor, could protect against eGC shedding in lipopolysaccharide (LPS)-induced sepsis and if it could interrupt the vascular hyperpermeability, neutrophil transmigration, and microvascular impairment. Rats that received pretreatment with doxycycline before LPS displayed ultrastructural preservation of the eGC observed using transmission electronic microscopy of the lung and heart. In addition, these animals exhibited lower serum syndecan-1 levels, a biomarker of eGC injury, and lower perfused boundary region (PBR) in the mesenteric video capillaroscopy, which is inversely related to the eGC thickness compared with rats that only received LPS. Furthermore, this study revealed that doxycycline decreased sepsis-related vascular hyperpermeability in the lung and heart, reduced neutrophil transmigration in the peritoneal lavage and inside the lungs, and improved some microvascular parameters. These findings suggest that doxycycline protects against LPS-induced eGC shedding, and it could reduce vascular hyperpermeability, neutrophils transmigration, and microvascular impairment.

Midkine promotes renal fibrosis by stabilizing C/EBPß to facilitate endothelial-mesenchymal transition.

Numerous myofibroblasts are arisen from endothelial cells (ECs) through endothelial to mesenchymal transition (EndMT) triggered by TGF-ß. However, the mechanism of ECs transforms to a different subtype, or whether there exists an intermediate state of ECs remains unclear. In present study, we demonstrate Midkine (MDK) mainly expressed by CD31 + ACTA2+ECs going through partial EndMT contribute greatly to myofibroblasts by spatial and single-cell transcriptomics. MDK is induced in TGF-ß treated ECs, which upregulates C/EBPß and increases EndMT genes, and these effects could be reversed by siMDK. Mechanistically, MDK promotes the binding ability of C/EBPß with ACTA2 promoter by stabilizing the C/EBPß protein. In vivo, knockout of Mdk or conditional knockout of Mdk in ECs reduces EndMT markers and significantly reverses fibrogenesis. In conclusion, our study provides a mechanistic link between the induction of EndMT by TGF-ß and MDK, which suggests that blocking MDK provides potential therapeutic strategies for renal fibrosis.

Aging-regulated PNUTS maintains endothelial barrier function via SEMA3B suppression.

Age-related diseases pose great challenges to health care systems worldwide. During aging, endothelial senescence increases the risk for cardiovascular disease. Recently, it was described that Phosphatase 1 Nuclear Targeting Subunit (PNUTS) has a central role in cardiomyocyte aging and homeostasis. Here, we determine the role of PNUTS in endothelial cell aging. We confirm that PNUTS is repressed in senescent endothelial cells (ECs). Moreover, PNUTS silencing elicits several of the hallmarks of endothelial aging: senescence, reduced angiogenesis and loss of barrier function. Findings are validate in vivo using endothelial-specific inducible PNUTS-deficient mice (Cdh5-CreERT2;PNUTSfl/fl), termed PNUTSEC-KO. Two weeks after PNUTS deletion, PNUTSEC-KO mice present severe multiorgan failure and vascular leakage. Transcriptomic analysis of PNUTS-silenced HUVECs and lungs of PNUTSEC-KO mice reveal that the PNUTS-PP1 axis tightly regulates the expression of semaphorin 3B (SEMA3B). Indeed, silencing of SEMA3B completely restores barrier function after PNUTS loss-of-function. These results reveal a pivotal role for PNUTS in endothelial homeostasis through a SEMA3B downstream pathway that provides a potential target against the effects of aging in ECs.

The stress-responsive cytotoxic effect of diesel exhaust particles on lymphatic endothelial cells.

Diesel exhaust particles (DEPs) are very small (typically < 0.2 µm) fragments that have become major air pollutants. DEPs are comprised of a carbonaceous core surrounded by organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs. Inhaled DEPs reach the deepest sites in the respiratory system where they could induce respiratory/cardiovascular dysfunction. Additionally, a previous study has revealed that a portion of inhaled DEPs often activate immune cells and subsequently induce somatic inflammation. Moreover, DEPs are known to localize in lymph nodes. Therefore, in this study we explored the effect of DEPs on the lymphatic endothelial cells (LECs) that are a constituent of the walls of lymph nodes. DEP exposure induced cell death in a reactive oxygen species (ROS)-dependent manner. Following exposure to DEPs, next-generation sequence (NGS) analysis identified an upregulation of the integrated stress response (ISR) pathway and cell death cascades. Both the soluble and insoluble components of DEPs generated intracellular ROS. Three-dimensional Raman imaging revealed that DEPs are taken up by LECs, which suggests internalized DEP cores produce ROS, as well as soluble DEP components. However, significant cell death pathways such as apoptosis, necroptosis, ferroptosis, pyroptosis, and parthanatos seem unlikely to be involved in DEP-induced cell death in LECs. This study clarifies how DEPs invading the body might affect the lymphatic system through the induction of cell death in LECs.

A human pluripotent stem cell-derived in vitro model of the blood-brain barrier in cerebral malaria.

BACKGROUND: Blood-brain barrier (BBB) disruption is a central feature of cerebral malaria (CM), a severe complication of Plasmodium falciparum (Pf) infections. In CM, sequestration of Pf-infected red blood cells (Pf-iRBCs) to brain endothelial cells combined with inflammation, hemolysis, microvasculature obstruction and endothelial dysfunction mediates BBB disruption, resulting in severe neurologic symptoms including coma and seizures, potentially leading to death or long-term sequelae. In vitro models have advanced our knowledge of CM-mediated BBB disruption, but their physiological relevance remains uncertain. Using human induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs), we aimed to develop a novel in vitro model of the BBB in CM, exhibiting enhanced barrier properties. METHODS: hiPSC-BMECs were co-cultured with HB3var03 strain Pf-iRBCs up to 9 h. Barrier integrity was measured using transendothelial electrical resistance (TEER) and sodium fluorescein permeability assays. Localization and expression of tight junction (TJ) proteins (occludin, zonula occludens-1, claudin-5), cellular adhesion molecules (ICAM-1, VCAM-1), and endothelial surface markers (EPCR) were determined using immunofluorescence imaging (IF) and western blotting (WB). Expression of angiogenic and cell stress markers were measured using multiplex proteome profiler arrays. RESULTS: After 6-h of co-culture with Pf-iRBCs, hiPSC-BMECs showed reduced TEER and increased sodium fluorescein permeability compared to co-culture with uninfected RBCs, indicative of a leaky barrier. We observed disruptions in localization of occludin, zonula occludens-1, and claudin-5 by IF, but no change in protein expression by WB in Pf-iRBC co-cultures. Expression of ICAM-1 and VCAM-1 but not EPCR was elevated in hiPSC-BMECs with Pf-iRBC co-culture compared to uninfected RBC co-culture. In addition, there was an increase in expression of angiogenin, platelet factor-4, and phospho-heat shock protein-27 in the Pf-iRBCs co-culture compared to uninfected RBC co-culture. CONCLUSION: These findings demonstrate the validity of our hiPSC-BMECs based model of the BBB, that displays enhanced barrier integrity and appropriate TJ protein localization. In the hiPSC-BMEC co-culture with Pf-iRBCs, reduced TEER, increased paracellular permeability, changes in TJ protein localization, increase in expression of adhesion molecules, and markers of angiogenesis and cellular stress all point towards a novel model with enhanced barrier properties, suitable for investigating pathogenic mechanisms underlying BBB disruption in CM.

Melatonin alleviates myocardial dysfunction through inhibition of endothelial-to-mesenchymal transition via the NF-κB pathway.

Endothelial-to-mesenchymal transition (EndMT) is a complex biological process of cellular transdifferentiation by which endothelial cells (ECs) lose their characteristics and acquire mesenchymal properties, leading to cardiovascular remodeling and complications in the adult cardiovascular diseases environment. Melatonin is involved in numerous physiological and pathological processes, including aging, and has anti-inflammatory and antioxidant activities. This molecule is an effective therapeutic candidate for preventing oxidative stress, regulating endothelial function, and maintaining the EndMT balance to provide cardiovascular protection. Although recent studies have documented improved cardiac function by melatonin, the mechanism of action of melatonin on EndMT remains unclear. The present study investigated the effects of melatonin on induced EndMT by transforming growth factor-ß2/interleukin-1ß in both in vivo and in vitro models. The results revealed that melatonin reduced the migratory ability and reactive oxygen species levels of the cells and ameliorated mitochondrial dysfunction in vitro. Our findings indicate that melatonin prevents endothelial dysfunction and inhibits EndMT by activating related pathways, including nuclear factor kappa B and Smad. We also demonstrated that this molecule plays a crucial role in restoring cardiac function by regulating the EndMT process in the ischemic myocardial condition, both in vessel organoids and myocardial infarction (MI) animal models. In conclusion, melatonin is a promising agent that attenuates EC dysfunction and ameliorates cardiac damage compromising the EndMT process after MI.