RESUMEN

The Enhanced Permeability and Retention (EPR) effect, an elevated accumulation of drugs and nanoparticles in tumors versus in normal tissues, is a widely used concept in the field of cancer therapy. It assumes that the vasculature of solid tumors would possess abnormal, leaky endothelial cell barriers, allowing easy access of intravenous-delivered drugs and nanoparticles to tumor regions. However, the EPR effect is not always effective owing to the heterogeneity of tumor endothelium over time, location, and species. Herein, we introduce a unique nanoparticle-based approach, using MUC18-targeted gold nanorods coupled with mild hyperthermia, to specifically enhance tumor endothelial permeability. This improves the efficacy of traditional cancer therapy including photothermal therapy and anticancer drug delivery by increasing the transport of photo-absorbers and drugs across the tumor endothelium. Using single cell imaging tools and classic analytical approaches in molecular biology, we demonstrate that MUC18-targeted gold nanorods and mild hyperthermia enlarge the intercellular gaps of tumor endothelium by inducing circumferential actin remodeling, stress fiber formation, and cell contraction of adjacent endothelial cells. Considering MUC18 is overexpressed on a variety of tumor endothelium and cancer cells, this approach paves a new avenue to improve the efficacy of cancer therapy by actively enhancing the tumor endothelial permeability.

Asunto(s)

RESUMEN

PURPOSE OF REVIEW: Major Depressive Disorder (MDD) is characterized by persistent symptoms such as fatigue, loss of interest in activities, feelings of sadness and worthlessness. MDD often coexist with cardiovascular disease (CVD), yet the precise link between these conditions remains unclear. This review explores factors underlying the development of MDD and CVD, including genetic, epigenetic, platelet activation, inflammation, hypothalamic-pituitary-adrenal (HPA) axis activation, endothelial cell (EC) dysfunction, and blood-brain barrier (BBB) disruption. RECENT FINDINGS: Single nucleotide polymorphisms (SNPs) in the membrane-associated guanylate kinase WW and PDZ domain-containing protein 1 (MAGI-1) are associated with neuroticism and psychiatric disorders including MDD. SNPs in MAGI-1 are also linked to chronic inflammatory disorders such as spontaneous glomerulosclerosis, celiac disease, ulcerative colitis, and Crohn's disease. Increased MAGI-1 expression has been observed in colonic epithelial samples from Crohn's disease and ulcerative colitis patients. MAGI-1 also plays a role in regulating EC activation and atherogenesis in mice and is essential for Influenza A virus (IAV) infection, endoplasmic reticulum stress-induced EC apoptosis, and thrombin-induced EC permeability. Despite being understudied in human disease; evidence suggests that MAGI-1 may play a role in linking CVD and MDD. Therefore, further investigation of MAG-1 could be warranted to elucidate its potential involvement in these conditions.

Asunto(s)

RESUMEN

Despite of yet unknown mechanism, microvascular deposition of oligomeric Tau (oTau) has been implicated in alteration of the Blood-Brain Barrier (BBB) function in Alzheimer's disease (AD) brains. In this study, we employed an in vitro BBB model using primary mouse cerebral endothelial cells (CECs) to investigate the mechanism underlying the effects of oTau on BBB function. We found that exposing CECs to oTau induced oxidative stress through NADPH oxidase, increased oxidative damage to proteins, decreased proteasome activity, and expressions of tight junction (TJ) proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5. These effects were suppressed by the pretreatment with Fasudil, a RhoA/ROCK signaling inhibitor. Consistent with the biochemical alterations, we found that exposing the basolateral side of CECs to oTau in the BBB model disrupted the integrity of the BBB, as indicated by an increase in FITC-dextran transport across the model, and a decrease in trans endothelial electrical resistance (TEER). oTau also increased the transmigration of peripheral blood mononuclear cells (PBMCs) in the BBB model. These functional alterations in the BBB induced by oTau were also suppressed by Fasudil. Taken together, our findings suggest that targeting the RhoA/ROCK pathway can be a potential therapeutic strategy to maintain BBB function in AD.

Asunto(s)

RESUMEN

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.

RESUMEN

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Asunto(s)

RESUMEN

Introducción y objetivos La reperfusión coronaria produce un daño en la microcirculación y, en concreto, las células endoteliales. Este estudio evalúa el efecto del suero aislado tras la revascularización de pacientes con un infarto agudo de miocardio con elevación del segmento ST (IAMCEST) en la viabilidad celular, el grado de permeabilidad endotelial in vitro y la asociación de estos parámetros con una mayor extensión de los índices de resonancia magnética cardiaca (RMC) relacionados con el daño por reperfusión (edema, hemorragia y obstrucción microvascular). Métodos Se incubaron células endoteliales de arteria coronaria humana con suero aislado 24 h tras la revascularización de 43 pacientes con IAMCEST evaluados mediante RMC y 14 sujetos de control. Se testó el efecto del suero de pacientes con IAMCEST en la pérdida de viabilidad celular por activación de la apoptosis y la necrosis, así como en la permeabilidad y la estructura de la monocapa endotelial. Resultados El suero de pacientes con IAMCEST aumentó la apoptosis (p <0,01) y la necrosis (p <0,05) de células endoteliales de arteria coronaria humana y causó un incremento de la permeabilidad de la monocapa endotelial in vitro (p <0,01) debido a mayores espacios intercelulares (p <0,05 frente a los controles). Una mayor necrosis inducida por suero se asoció con más permeabilidad endotelial in vitro (p <0,05) y con una mayor extensión de los principales índices de daño tras reperfusión y mayor tamaño de infarto. Conclusiones El suero tras la reperfusión de pacientes con IAMCEST induce la apoptosis y la necrosis in vitro de las células endoteliales y la permeabilidad endotelial. Cuanto más potente sea el efecto inductor de necrosis, más deletéreas son las consecuencias en cuanto al daño estructural resultante. (AU)

Introduction and objectives Clinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction). Methods Human coronary artery endothelial cells were incubated with serum isolated 24hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer. Results Serum from STEMI patients increased apoptosis (P <.01) and necrosis (P <.05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P <.01), due to enlarged intercellular spaces (P <.05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P <.05) and with more extensive CMR-derived indices of reperfusion injury and infarct size. Conclusions Postreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure. (AU)

Asunto(s)

RESUMEN

Anomalous vascular endothelium significantly contributes to various cardiovascular diseases. VE-cadherin plays a vital role in governing the endothelial barrier. Krüppel-like factor 4(KLF4), as a transcription factor, which binds the VE-cadherin promoter and enhances its transcription. Tumor necrosis factor receptor-associated factor 7 (TRAF7) is an E3 ubiquitin ligase that has been shown to modulate the degradation of KLF4. H2S can covalently modify cysteine residues on proteins through S-sulfhydration, thereby influencing the structure and functionality of the target protein. However, the role of S-sulfhydration on endothelial barrier integrity remains to be comprehensively elucidated. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates endothelial integrity and its underlying mechanism. In this study, we observed that protein S-sulfhydration was reduced in the endothelium during diabetes and TRAF7 was the main target. Overexpression of TRAF7-Cys327 mutant could mitigate the endothelial barrier damage by weakening TRAF7 interaction with KLF4 and reducing ubiquitination degradation of KLF4. In conclusion, our research demonstrates that H2S plays a pivotal role in regulating S-sulfhydration of TRAF7 at Cys327. This regulation effectively inhibits the ubiquitin-mediated degradation of KLF4, resulting in an upregulation of VE-cadherin levels. This molecular mechanism contributes to the prevention of endothelial barrier damage.

Asunto(s)

RESUMEN

Endothelial dysfunction often accompanies sepsis. Sevoflurane (Sev) is a widely used inhaled anesthetic that has a protective effect on sepsis-associated damage. We aimed to elucidate the role of Sev in endothelial dysfunction by using a model of LPS induced HUVECs. Sev increased the viability and decreased the apoptosis of HUVECs exposed to LPS. Inflammation and endothelial cell adhesion were improved after Sev addition. Besides, Sev alleviated LPS-induced endothelial cell permeability damage in HUVECs. RORα served as a potential protein that bound to Sev. Importantly, Sev upregulated RORα expression and inhibited endoplasmic reticulum (ER) stress in LPS-treated HUVECs. RORα silencing reversed the impacts of Sev on ER stress. Moreover, RORα deficiency or tunicamycin (ER stress inducer) treatment restored the effects of Sev on the viability, apoptosis, inflammation and endothelial permeability damage of HUVECs exposed to LPS. Taken together, Sev ameliorated LPS-induced endothelial cell damage by targeting RORα to inhibit ER stress.

Asunto(s)

RESUMEN

Increased vascular permeability is a prevalent feature in a wide spectrum of clinical conditions, but no effective treatments to restore the endothelial barrier are available. Idiopathic systemic capillary leak syndrome (ISCLS) is a life-threatening Paroxysmal Permeability Disorder characterized by abrupt, massive plasma extravasation. This condition serves as a robust model for investigating therapeutic approaches targeting interendothelial junctions. We conducted a single-center, interventional in vitro study at the Referral Center for ISCLS in Italy, involving four diagnosed ISCLS patients, aiming at investigating the effects of FX06, a Bß15-42 fibrin-derived peptide binding to VE-Cadherin, on endothelial barrier exposed to intercritical and acute ISCLS sera. The Transwell Permeability Assay was used to assess the permeability of human umbilical vein endothelial cells (HUVECs) exposed to ISCLS sera with or without FX06 (50 µg/ml). Acute ISCLS serum was also tested in a three-dimensional microfluidic device. Nitric oxide (NO), VE-Cadherin localization, and cytoskeletal organization were also assessed. In two and three-dimensional systems, ISCLS sera increased endothelial permeability, with a more pronounced effect for acute sera. Furthermore, acute sera altered VE-Cadherin localization and cytoskeletal organization. NO levels remained unchanged. FX06 restored the endothelial barrier function by influencing cellular localization rather than VE-Cadherin levels. In conclusion, FX06 prevents and reverts the hyperpermeability induced by ISCLS sera. These preliminary yet promising results provide initial evidence of the in vitro efficacy of a drug targeting the underlying pathophysiological mechanisms of ISCLS. Moreover, this approach may hold potential for addressing hyperpermeability in a spectrum of clinical conditions beyond ISCLS.

Asunto(s)

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: As a classic traditional Chinese medicine, Magnolia officinalis (M. officinalis) is widely used in digestive diseases. It has rich gastrointestinal activity including inflammatory bowel disease (IBD) treatment, but the mechanism is not clear. AIM OF THE STUDY: In recent years, there has been a growing interest in investigating the regulatory effects of herbal compounds on transient receptor potential (TRP) channel proteins. Transient receptor potential vanilloid 4 (TRPV4), a subtype involved in endothelial permeability regulation, was discussed as the target of M. officinalis in the treatment of IBD in the study. Based on the targeting effect of TRPV4, this study investigated the active ingredients and mechanism of M. officinalis extract in treating IBD. MATERIALS AND METHODS: To reveal the connection between the active ingredients in M. officinalis and TRPV4, a bioactivity-guided high performance liquid chromatography system coupled with mass spectrometry identification was utilized to screen for TRPV4 antagonists. TRPV4 siRNA knockdown experiment was employed to validate the significance of TRPV4 as a crucial target in regulating endothelial permeability by honokiol (HON). The interaction of the active ingredient representing HON with TRPV4 was confirmed by molecular docking, fluorescence-based thermal shift and live cell calcium imaging experiments. The potential binding sites and inhibitory mechanisms of HON in TRPV4 were analyzed by molecular dynamics simulation and microscale thermophoresis. The therapeutic effect of HON based on TRPV4 was discussed in DSS-IBD mice. RESULTS: Our finding elucidated that the inhibitory activity of M. officinalis against TRPV4 is primarily attributed to HON analogues. The knockdown of TRPV4 expression significantly impaired the calcium regulation and permeability protection in endothelial cells. The mechanism study revealed that HON specifically targets the Q239 residue located in the ankyrin repeat domain of TRPV4, and competitively inhibits channel opening with adenosine triphosphate (ATP) binding. The immunofluorescence assay demonstrated that the administration of HON enhances the expression and location of VE-Cadherin to protect the endothelial barrier and attenuates immune cell infiltration. CONCLUSIONS: The finding suggested that HON alleviates IBD by improving endothelial permeability through TRPV4. The discovery provides valuable insights into the potential therapeutic strategy of active natural products for alleviating IBD.

Asunto(s)

RESUMEN

INTRODUCTION AND OBJECTIVES: Clinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction). METHODS: Human coronary artery endothelial cells were incubated with serum isolated 24hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer. RESULTS: Serum from STEMI patients increased apoptosis (P <.01) and necrosis (P <.05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P <.01), due to enlarged intercellular spaces (P <.05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P <.05) and with more extensive CMR-derived indices of reperfusion injury and infarct size. CONCLUSIONS: Postreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure.

Asunto(s)

RESUMEN

INTRODUCTION: Major traumatic injury is associated with early hemorrhage-related and late-stage deaths due to multiple organ failure (MOF). While improvements to hemostatic resuscitation have significantly reduced hemorrhage-related deaths, the incidence of MOF among trauma patients remains high. Dysregulation of vascular endothelial cell (EC) barrier function is a central mechanism in the development of MOF; however, the mechanistic triggers remain unknown. Accelerated fibrinolysis occurs in a majority of trauma patients, resulting in high circulating levels of fibrin(ogen) degradation products, such as fragment X. To date, the relationship between fragment X and EC dysregulation and barrier disruption is unknown. The goal of this study was to determine the effects of fragment X on EC barrier integrity and expression of paracellular junctional proteins that regulate barrier function. METHODS: Human lung microvascular endothelial cells (HLMVECs) were treated with increasing concentrations of fragment X (1, 10, and 100 µg/mL), and barrier function was monitored using the xCELLigence live-cell monitoring system. Quantitative PCR (qPCR) was performed to measure changes in EC expression of 84 genes. Immunofluorescent (IF) cytostaining was performed to validate qPCR findings. RESULTS: Fragment X treatment significantly increased endothelial permeability over time (P < 0.05). There was also a significant reduction in VE-cadherin mRNA expression in fragment X-treated HLMVECs compared to control (P = 0.01), which was confirmed by IF staining. CONCLUSIONS: Fragment X may induce EC hyperpermeability by reducing VE-cadherin expression. This suggests that a targeted approach to disrupting EC-fragment X interactions could mitigate EC barrier disruption, organ edema, and MOF associated with major trauma.

Asunto(s)

RESUMEN

Capillary leak syndrome (CLS) represents a phenotype of increased fluid extravasation, resulting in intravascular hypovolemia, extravascular edema formation and ultimately hypoperfusion. While endothelial permeability is an evolutionary preserved physiological process needed to sustain life, excessive fluid leak-often caused by systemic inflammation-can have detrimental effects on patients' outcomes. This article delves into the current understanding of CLS pathophysiology, diagnosis and potential treatments. Systemic inflammation leading to a compromise of endothelial cell interactions through various signaling cues (e.g., the angiopoietin-Tie2 pathway), and shedding of the glycocalyx collectively contribute to the manifestation of CLS. Capillary permeability subsequently leads to the seepage of protein-rich fluid into the interstitial space. Recent insights into the importance of the sub-glycocalyx space and preserving lymphatic flow are highlighted for an in-depth understanding. While no established diagnostic criteria exist and CLS is frequently diagnosed by clinical characteristics only, we highlight more objective serological and (non)-invasive measurements that hint towards a CLS phenotype. While currently available treatment options are limited, we further review understanding of fluid resuscitation and experimental approaches to target endothelial permeability. Despite the improved understanding of CLS pathophysiology, efforts are needed to develop uniform diagnostic criteria, associate clinical consequences to these criteria, and delineate treatment options.

RESUMEN

Introduction: Anaphylaxis is among the most severe manifestations of allergic disorders, but its molecular basis remains largely unknown and reliable diagnostic markers are not currently available. MicroRNAs (miRNAs) regulate several pathophysiological processes and have been proposed as non-invasive biomarkers. Therefore, this study aims to evaluate their involvement in anaphylactic reaction and their value as biomarkers. Methods: Acute (anaphylaxis) and baseline (control) serum samples from 67 patients with anaphylaxis were studied. Among them, 35 were adults with drug-induced anaphylaxis, 13 adults with food-induced anaphylaxis and 19 children with food-induced anaphylaxis. The circulating serum miRNAs profile was characterized by next-generation sequencing (NGS). For this purpose, acute and baseline samples from 5 adults with drug-induced anaphylaxis were used. RNA was extracted, retrotranscribed, sequenced and the readings obtained were mapped to the human database miRBase_20. In addition, a system biology analysis (SBA) was performed with its target genes and revealed pathways related to anaphylactic mediators signaling. Moreover, functional and molecular endothelial permeability assays were conducted with miR-375-3p-transfected cells in response to cAMP. Results: A total of 334 miRNAs were identified, of which 21 were significant differentially expressed between both phases. Extracellular vesicles (EVs) were characterized by Western blot, electron microscopy and NanoSight. A decrease of miR-375-3p levels was determined by qPCR in both serum and EVs of patients with anaphylaxis (****p<.0001). Precisely, the decrease of miR-375-3p correlated with the increase of two inflammatory cytokines: monocyte chemoattractant protein-1 (MCP-1) and granulocyte macrophage colony-stimulating factor (GM-CSF). On the other hand, functional and molecular data obtained showed that miR-375-3p partially blocked the endothelial barrier maintenance and stabilization by disassembly of cell-cell junctions exhibiting low Rac1-Cdc42 levels. Discussion: These findings demonstrate a differential serum profile of circulating miRNAs in patients with anaphylaxis and exhibit the miR-375-3p modulation in serum and EVs during drug- and food-mediated anaphylactic reactions. Furthermore, the in silico and in vitro studies show a negative role for miR-375-3p/Rac1-Cdc42 in the endothelial barrier stability.

Asunto(s)

RESUMEN

Tumor cell extravasation across endothelial barrier has been recognized as a pivotal event in orchestrating metastasis formation. This event is initiated by the interactions of extravasating tumor cells with endothelial cells (ECs). Therefore, targeting the crosstalk between tumor cells and ECs might be a promising therapeutic strategy to prevent metastasis. In this study, we demonstrated that Rh1, one of the main ingredients of ginseng, hindered the invasion of breast cancer (BC) cells as well as diminished the permeability of ECs both in vitro and in vivo, which was responsible for the attenuated tumor cell extravasation across endothelium. Noteworthily, we showed that ECs were capable of inducing the epithelial-mesenchymal transition (EMT) and invadopodia of BC cells that are essential for tumor cell migration and invasion through limiting the nuclear translocation of hematopoietically expressed homeobox (HHEX). The decreased nuclear HHEX paved the way for initiating the CCL20/CCR6 signaling axis, which in turn contributed to damaged endothelial junctions, uncovering a new crosstalk mode between tumor cells and ECs. Intriguingly, Rh1 inhibited the kinase activity of casein kinase II subunit alpha (CK2α) and further promoted the nuclear translocation of HHEX in the BC cells, which resulted in the disrupted crosstalk between chemokine (C-C motif) ligand 20 (CCL20) in the BC cells and chemokine (C-C motif) receptor 6 (CCR6) in the ECs. The prohibited CCL20-CCR6 axis by Rh1 enhanced vascular integrity and diminished tumor cell motility. Taken together, our data suggest that Rh1 serves as an effective natural CK2α inhibitor that can be further optimized to be a therapeutic agent for reducing tumor cell extravasation.

Asunto(s)

RESUMEN

Background: Increased endothelial permeability of pulmonary vessels is a primary pathological characteristic of septic acute lung injury (ALI). Previously, elevated lysophosphatidic acid (LPA) levels and LPA2 (an LPA receptor) expression have been found in the peripheral blood and lungs of septic mice, respectively. However, the specific role of LPA2 in septic ALI remains unclear. Methods: A lipopolysaccharide (LPS)-induced model of sepsis was established in wild-type (WT) and global LPA2 knockout (Lpar2-/-) mice. We examined mortality, lung injury, assessed endothelial permeability through Evans blue dye (EBD) assay in vivo, and transendothelial electrical resistance (TEER) of mouse lung microvascular endothelial cells (MLMECs) in vitro. Enzyme-linked immunosorbent assay (ELISA), histopathological, immunofluorescence, immunohistochemistry, and Western blot were employed to investigate the role of LPA2 in septic ALI. Results: Lpar2 deficiency increased vascular endothelial permeability, impaired lung injury, and increased mortality. Histological examination revealed aggravated inflammation, edema, hemorrhage and alveolar septal thickening in the lungs of septic Lpar2-/- mice. In vitro, loss of Lpar2 resulted in increased permeability of MLMECs. Pharmacological activation of LPA2 by the agonist DBIBB led to significantly reduced inflammation, edema and hemorrhage, as well as increased expression of the vascular endothelial tight junction (TJ) protein zonula occludens-1 (ZO-1) and claudin-5, as well as the adheren junction (AJ) protein VE-cadherin. Moreover, DBIBB treatment was found to alleviate mortality by protecting against vascular endothelial permeability. Mechanistically, we demonstrated that vascular endothelial permeability was alleviated through LPA-LPA2 signaling via the PLC-PKC-FAK pathway. Conclusion: These data provide a novel mechanism of endothelial barrier protection via PLC-PKC-FAK pathway and suggest that LPA2 may contribute to the therapeutic effects of septic ALI.

RESUMEN

The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage of vascular components into the surrounding tissues. Sphingosine-1-phosphate (S1P) is an active lysophospholipid that enhances the endothelial cell (EC) barrier via activation of its receptor S1PR1. Here, we delineate the effect of non-lethal doses of RSL3, an inhibitor of glutathione peroxidase 4 (GPX4), on EC barrier function. Low doses of RSL3 (50-100 nM) attenuated S1P-induced human lung microvascular barrier enhancement and the phosphorylation of AKT. To investigate the molecular mechanisms by which RSL3 attenuates S1P's effect, we examined the S1PR1 levels. RSL3 treatment reduced S1PR1 levels in 1 h, whereas the effect was attenuated by the proteasome and lysosome inhibitors as well as a lipid raft inhibitor. Immunofluorescence staining showed that RSL3 induced S1PR1 internalization from the plasma membrane into the cytoplasm. Furthermore, we found that RSL3 (100 and 200 nM) increased EC barrier permeability and cytoskeletal rearrangement without altering cell viability. Taken together, our data delineates that non-lethal doses of RSL3 impair EC barrier function via two mechanisms. RSL3 attenuates S1P1-induced EC barrier enhancement and disrupts EC barrier integrity through the generation of 4-hydroxynonena (4HNE). All these effects are independent of ferroptosis.

RESUMEN

BACKGROUND: Myocardial microvascular injury is the key event in early diabetic heart disease. The injury of myocardial microvascular endothelial cells (CMECs) is the main cause and trigger of myocardial microvascular disease. Mitochondrial calcium homeostasis plays an important role in maintaining the normal function, survival and death of endothelial cells. Considering that mitochondrial calcium uptake 1 (MICU1) is a key molecule in mitochondrial calcium regulation, this study aimed to investigate the role of MICU1 in CMECs and explore its underlying mechanisms. METHODS: To examine the role of endothelial MICU1 in diabetic cardiomyopathy (DCM), we used endothelial-specific MICU1ecKO mice to establish a diabetic mouse model and evaluate the cardiac function. In addition, MICU1 overexpression was conducted by injecting adeno-associated virus 9 carrying MICU1 (AAV9-MICU1). Transcriptome sequencing technology was used to explore underlying molecular mechanisms. RESULTS: Here, we found that MICU1 expression is decreased in CMECs of diabetic mice. Moreover, we demonstrated that endothelial cell MICU1 knockout exacerbated the levels of cardiac hypertrophy and interstitial myocardial fibrosis and led to a further reduction in left ventricular function in diabetic mice. Notably, we found that AAV9-MICU1 specifically upregulated the expression of MICU1 in CMECs of diabetic mice, which inhibited nitrification stress, inflammatory reaction, and apoptosis of the CMECs, ameliorated myocardial hypertrophy and fibrosis, and promoted cardiac function. Further mechanistic analysis suggested that MICU1 deficiency result in excessive mitochondrial calcium uptake and homeostasis imbalance which caused nitrification stress-induced endothelial damage and inflammation that disrupted myocardial microvascular endothelial barrier function and ultimately promoted DCM progression. CONCLUSIONS: Our findings demonstrate that MICU1 expression was downregulated in the CMECs of diabetic mice. Overexpression of endothelial MICU1 reduced nitrification stress induced apoptosis and inflammation by inhibiting mitochondrial calcium uptake, which improved myocardial microvascular function and inhibited DCM progression. Our findings suggest that endothelial MICU1 is a molecular intervention target for the potential treatment of DCM.

Asunto(s)

RESUMEN

AIM: Exercise training exerts protective effects against sepsis-associated multiple organ dysfunction. This study aimed to investigate whether aerobic exercise protected against sepsis-associated acute kidney injury (AKI) via modulating R-spondin 3 (RSPO3) expression. METHODS: To investigate the effects of aerobic exercise on lipopolysaccharide (LPS)-induced AKI, LPS (20 mg/kg) was intraperitoneally injected after six weeks of treadmill training. To investigate the role of RSPO3 in LPS-induced AKI, wild-type (WT) or inducible endothelial cell-specific RSPO3 knockout (RSPO3EC-/- ) mice were intraperitoneally injected with 12 mg/kg LPS. RSPO3 was intraperitoneally injected 30 min before LPS treatment. RESULTS: Aerobic exercise-trained mice were more resistant to LPS-induced body weight loss and hypothermia and had a significant higher survival rate than sedentary mice exposed to LPS. Exercise training restored the LPS-induced decreases in serum and renal RSPO3 levels. Exercise or RSPO3 attenuated, whereas inducible endothelial cell-specific RSPO3 knockout exacerbated LPS-induced renal glycocalyx loss, endothelial hyperpermeability, inflammation, and AKI. Bioinformatics analysis results revealed significant increases in the expression of matrix metalloproteinases (MMPs) in kidney tissues of mice exposed to sepsis or endotoxaemia, which was validated in renal tissue from LPS-exposed mice and LPS-treated human microvascular endothelial cells (HMVECs). Both RSPO3 and MMPs inhibitor restored LPS-induced downregulation of tight junction protein, adherens junction protein, and glycocalyx components, thus ameliorating LPS-induced endothelial leakage. Exercise or RSPO3 reversed LPS-induced upregulation of MMPs in renal tissues. CONCLUSION: Increased renal expression of RSPO3 contributes to aerobic exercise-induced protection against LPS-induced renal endothelial hyperpermeability and AKI by suppressing MMPs-mediated disruption of glycocalyx and tight and adherens junctions.

Asunto(s)

RESUMEN

BACKGROUND: Patients undergoing cardiac surgery are prone to numerous complications. Increased vascular permeability may be associated with morbidity and mortality due to hemodynamic instability, fluid overload, and edema formation. We hypothesized that markers of endothelial injury and inflammation are associated with capillary leak, ultimately increasing the risk of postoperative complications. METHODS: In this prospective, observational, multidisciplinary cohort study at our tertiary academic medical center, we recruited 405 cardiac surgery patients. Patients were assessed daily using body impedance electrical analysis, ultrasound, sublingual intravital microscopy, and analysis of serum biomarkers. Multivariable models, as well as machine learning, were used to study the association of angiopoietin-2 with extracellular water as well as common complications after cardiac surgery. RESULTS: The majority of patients underwent coronary artery bypass grafting, valvular, or aortic surgeries. Across the groups, extracellular water increased postoperatively (20 ± 6 preoperatively to 29 ± 7L on postoperative day 2; P < 0.001). Concomitantly, the levels of the biomarker angiopoietin-2 rose, showing a strong correlation based on the time points of measurements (r = 0.959, P = 0.041). Inflammatory (IL-6, IL-8, CRP) and endothelial biomarkers (VE-Cadherin, syndecan-1, ICAM-1) suggestive of capillary leak were increased. After controlling for common risk factors of edema formation, we found that an increase of 1 ng/mL in angiopoietin-2 was associated with a 0.24L increase in extracellular water (P < 0.001). Angiopoietin-2 showed increased odds for the development of acute kidney injury (OR 1.095 [95% CI 1.032, 1.169]; P = 0.004) and was furthermore associated with delayed extubation, longer time in the ICU, and a higher chance of prolonged dependence on vasoactive medication. Machine learning predicted postoperative complications when capillary leak was added to standard risk factors. CONCLUSIONS: Capillary leak and subsequent edema formation are relevant problems after cardiac surgery. Levels of angiopoietin-2 in combination with extracellular water show promising potential to predict postoperative complications after cardiac surgery. TRIAL REGISTRATION NUMBER: German Clinical Trials Registry (DRKS No. 00017057), Date of registration 05/04/2019, www.drks.de.