Lung Microvascular Endothelial Cells Research Articles

Neutrophil‐Derived Microvesicles Enhance Pulmonary Vascular Inflammation via a Toll‐like Receptor 4 Signaling‐Dependent Mechanism

BackgroundCirculating neutrophil‐derived microvesicles (NMVs) are markedly elevated during sepsis and therefore could have a role in the development of indirect acute lung injury (ALI). We recently found that NMV‐enriched CD11b+ MVs, immunoaffinity isolated from lipopolysaccharide (LPS)‐stimulated healthy volunteer blood, have potent pro‐inflammatory activity in a human peripheral blood mononuclear cell (PBMC) and lung microvascular endothelial cell (HLMEC) coculture model of pulmonary vascular inflammation (1). By contrast, immunoaffinity isolated platelet‐MVs (CD61+) produced negligible responses in these assays, suggesting specificity of the NMV‐enriched CD11b+ MV, activity. Here, we investigated the signaling mechanisms responsible for NMV‐mediated activation of monocytes and HLMECs in this model.MethodsHeparinized blood from healthy donors was treated with LPS (100 ng/ml, 3 h) and NMVs (CD66b+) were isolated by positive immunoaffinity selection. NMVs were then incubated in PBMC‐HLMEC cocultures for 4 h. NMV‐induced responses were determined by flow cytometric quantification of cell surface activation markers (ICAM‐1 for HLMECs, ICAM‐1 and tissue factor for monocytes) and pro‐inflammatory cytokine release by ELISA (TNFα, IL‐8, IL‐6, MCP‐1).ResultsNMV‐induced HLMEC activation required the presence of monocytes and was completely attenuated by anti‐TNFα neutralizing antibody (ICAM‐1: isotype control 302±103 vs anti‐TNFα 66±29; p<0.01). Pharmacological inhibition of mitogen‐activated protein kinases p38, MEK1‐MEK2 and phosphoinositide‐3 kinase ablated monocyte and HLMEC activation. Monocyte activation was prevented in the presence of the Toll‐like receptor 4 (TLR4) inhibitor TAK‐242 (e.g., TNFα release: vehicle 1108±257 vs TAK‐242 16±12; p<0.001) and partially inhibited by an anti‐TLR4 neutralizing antibody. However, the LPS inhibitor polymyxin‐B had no effect on these responses, indicating that LPS contamination was unlikely. Furthermore, NMV activity was found to be sensitive to proteinase K and heat‐treatment (55 °C, 15 min) (e.g., TNFα release: untreated 474±267 vs proteinase K 12±15; p<0.0001 vs heat‐treatment 200±84; p<0.01).ConclusionsWe have demonstrated that NMVs produced within LPS‐stimulated blood are potent mediators of lung microvascular endothelial cells via monocyte‐derived TNFα signaling. NMV‐induced monocyte activation involves TLR4 signaling and is protease sensitive, suggesting that MVs serve as vehicles of damage‐associated molecular pattern (DAMP) activity, mediated by surface proteins. Together these findings support a major role for NMVs as novel long‐range mediators for systemic propagation of inflammation, contributing to the pathogenesis of sepsis‐induced indirect acute lung injury.(1) Tsiridou et al. European Respiratory Journal. 2020;56:4468

Abstract 388: Role Of The Antiapoptotic C-iap2 On The Generation Of An Endothelial Memory In Schistosomiasis-associated Pulmonary Arterial Hypertension

Introduction: Schistosomiasis-associated Pulmonary Arterial Hypertension (Sch-PAH) is a life-threatening complication of chronic S. mansoni infection, which can evolve to heart failure and death. During PAH, hyperproliferation of apoptosis-resistant endothelial cells (ECs) has been extensively reported, but therapeutic approaches to reverse this pathological phenotype remain clinically challenging. Hypothesis: Thus, our hypothesis is that antigenic molecules from S. mansoni eggs amplify the secretion of inhibitor of apoptosis protein 2 (c-IAP2), leading to generation of an abnormal EC memory of survival and the development of PAH. Methods/Results: Previously, we showed that depletion of the anti-apoptotic protein Caveolin-1 (Cav-1) via shedding of extracellular vesicles (EVs) leads to the survival of an abnormal EC phenotype through an unclear mechanism. Analysis of survival-associated genes in isolated murine lung ECs from Flk1 +/GFP ;Cav1 -/- mice, revealed high expression of the anti-apoptotic genes BIRC3 and BIRC5 (which encodes cIAP2 and survivin, respectively) when compared to control ECs. Interesting, exposure to 1 μg/mL of the major S. mansoni egg antigen Sm-p40 induced time-dependent phosphorylation of Cav-1 at residue Tyr14 in human lung microvascular EC (HMVEC-L: 228.2 +/- 38.28% of control; p<0.05; n=4), culminating in c-IAP2 expression with no difference on the constitutive expression of c-IAP1. Furthermore, Sm-p40 treatment of HMVEC-L in the presence of pro-inflammatory mediators IL-6, TNF-α, and ATP induced a significant release of EVs containing c-IAP2. Finally, in vivo analysis of lungs from S. mansoni -infected mice confirmed a significant decrease in Cav-1 expression unique to the egg-dependent granuloma area, indicating that local inflammatory response due to egg-derived antigenic molecules is essential for reprogramming and survival of an abnormal EC phenotype during Sch-PAH. Conclusions: Our data suggest that S. mansoni -induced Cav-1 depletion promotes the secretion of the anti-apoptotic c-IAP2 protein, which may be critical for prolonged survival of a pathogenic EC phenotype during the development of Sch-PAH.

Abstract 390: Influenza A Virus Infection Increases Magi1 Expression In Endothelial Cells And Its Depletion Inhibits Virus Replication Through Increased Expression Of Mx1

When influenza virus infects cells, it changes cellular metabolism in such a way that allows virus particles to replicate efficiently. This metabolic engineering takes place soon after virus infects cells, for which the PSD95/DiscLarge/ZO-1 (PDZ) domain of certain proteins is known to play a role. Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) is a scaffold protein with 6 PDZ domains, and we have shown that it is involved in the regulation of endothelial cell (EC) activation and atherosclerosis in mice. Since recent studies indicate that the vascular endothelium can be infected by influenza A virus (IAV) and plays a role in the influenza-induced pathogenesis and cardiovascular disease (CVD), we investigated the role of MAGI1 in IAV infection using cultured human umbilical vein endothelial cells (HUVECs) as well as human lung microvascular endothelial cells (HULECs). We found increased MAGI1 mRNA expression in IAV-infected cells. Conversely, when MAGI1 depleted ECs were infected with IAV, virus infection and replication was greatly suppressed. Our microarray studies revealed that depletion of MAGI1 in HUVECs increased the protein expression and signaling networks involved in interferon production. Specifically, we found that the MAGI1 null condition induced expression of anti-viral response genes including interferon-induced GTP-binding protein MX1, an antiviral protein, interferon beta1, a cytokine promotor STAT1 (signal transducer and activator of transcription 1), and also increased protein expression levels of STAT1, phosphorylated STAT5 and MX1. Co-transfection of HUVECs with siMX1 and siMAGI1 impaired MAGI1 depletion-induced suppression of IAV infection. Furthermore, we found nuclear localization of interferon regulatory factor 3 (IRF3) in MAGI1 depleted cells, indicating that MAGI1 depletion elicits the interferon production and signaling. Taken together, we conclude that IAV infection and replication occurs in ECs in a MAGI1 expression dependent manner. Thus, MGAI1 depletion in ECs suppresses IAV replication, and this suppression is due to increased MX1 expression, which induces IRF3 activation and interferon production. MAGI1 can be a potential therapeutic target for influenza-induced CVD.

Effects of Viral Infection on Endothelial Activation and Barrier Function

Endothelial cells (ECs) are in the inner layer of blood vessels, and it controls the transportation of nutrition and essential molecules from the extracellular matrix to the intracellular space making a homeostasis environment. Vascular endothelium can be disrupted with viruses, which are among the main reasons for epidemic and pandemic outbreaks, and it leads to health issues such as cardiovascular‐related diseases. Wide range of viruses can potentially target the endothelium including Dengue virus estimated to infect 390 million people per year. Zika virus leads to pregnancy complications such as preterm birth and miscarriage. Tick‐borne encephalic damaged the central nervous system and, Hantavirus has a mortality rate of 38% through hantavirus pulmonary syndrome. Reviewing these several diseases, including SARS‐CoV‐2, has given an in‐depth look at what the endothelial cells are going through when these viruses are infecting the host bodies. Following the virus infection, ECs are going through activation mode, which is accompanied with glycocalyx degradation, plasma leakage, phosphorylation of P120, and cadherin, increased proinflammatory responses and, disruption of barrier integrity.The SARS‐CoV‐2 pandemic has taken a global toll; With a total of 362 million cases, there are 5.63 million deaths occurring. With the usage of immunofluorescence and vascular permeability assay, we monitor how Human Umbilical Vascular Endothelial Cells (HUVEC) and Human Lung Microvascular Endothelial Cells (HLMVEC) are impacted when treated with UV‐inactivated & heat‐inactivated SARS‐CoV‐2, and tumor necrosis factor (TNF)‐α. TNF‐α is an inflammatory cytokine prominent during inflammation and its known to increase the permeability of endothelium. Like the TNF‐α, both cells treated with UV‐inactivated and heat‐inactivated SARS‐CoV2 demonstrated boosted permeability. With the considerable effect of SARS‐CoV2 on the permeability of ECs, it is essential to understand the mechanisms and pathways behind the boosted leak, including the impact of the virus on the expression of adhesion molecules like platelet endothelial cell adhesion molecule (PECAM‐1). One can see in the images of HUVEC and HLMVEC that SARS‐CoV‐2 diminish the localization of PECAM‐1. On a non‐treated negative control, cells are spaced evenly and are close to another since their junctions are not targeted. Once the cells are treated with either heat‐inactivated or UV‐inactivated SARS‐CoV‐2, PECAM‐1 localization is disrupted, which can be seen from the space between cells since the PECAM‐1 has been altered and is no longer holding the cells together. From those exact images, one can detect the enlarged and irregular shapes of nucleuses and change in morphology of these cells since the SARS‐CoV‐2 is not only altering the PECAM‐1 complex but it can also modify various proteins and mRNAs. By combining information of various viruses and knowledge from our studies, we can understand the mechanisms of ECs and take measurements to protect ECs from viruses. In future experiments, we will detect if the PECAM‐1 expression is lowered when treated with SARS‐CoV‐2 and TNF‐α or if PECAM‐1 is internalized in the cell. Moreover, we can do mRNA and immunoblotting analysis to elaborate the changes in protein pathways as a consequence of COVID‐19.

Extracellular vesicles from ALI models induce endothelial dysfunction

One distinguishing characteristic of acute respiratory distress syndrome (ARDS) or its lesser form, acute lung injury (ALI), is dysfunction of the lung endothelium. This dysfunction leads to increased permeability and eventual pulmonary edema. Despite the globally climbing rates of ARDS, the mechanisms of endothelial dysfunction are poorly understood. Recently, extracellular vesicles (EVs) have been shown to induce various microvascular disease states, as they contain various endothelial‐targeting cargo in the form of mRNA, proteins, lipids, and miRNAs. Therefore, we hypothesized that EVs play a role in the endothelial dysfunction observed in ARDS/ALI. EVs were isolated from the plasma of male Sprague Dawley rats which were given saline or P. aeruginosa (PA103) via intratracheal (IT) injection for 48h. ALI was confirmed by determining the wet to dry weights of the rodent lungs. We then tested the effect of the EVs isolated from saline‐ and ALI treated animals (sEVs and ALI‐EVs, respectively) on endothelial barrier function and modifications in gene expression. We found that treatment with ALI‐EVs, but not sEVs, increased rat lung microvascular endothelial cell (RLMVEC) permeability in the presence of lipopolysaccharide (LPS). Interestingly, ALI‐EVs did not induce endothelial dysfunction on their own, rather exacerbated the damage induced by LPS. We further noted ALI‐EVs, but not sEVs, increased gene expression of toll‐like receptor 4 (TLR4), which is the main pattern recognition receptor (PRR) for LPS and its downstream signaling molecule, MyD88 in the presence of LPS in RLMVEC. Further, we found that ALI‐EVs, together with LPS, increased gene expression of intracellular adhesion molecule 1 (ICAM‐1), vascular adhesion molecule 1 (VCAM‐1), and interleukin 6 (IL‐6), which are all involved in inflammatory signaling. Thus, it is likely that through the modulation of inflammatory signaling mediators, ALI‐EVs contain select cargo that worsen the endothelial barrier in settings where LPS is present, such as bacterial‐ARDS.

Cell‐Free Hemoglobin Increases Leukocyte Adhesion and Mitochondrial Oxidative Damage in the Pulmonary Microvascular Endothelium

Sepsis is a critical problem in intensive care units globally, and accounts for 20% of all annual deaths. During septic shock, the highly inflammatory state leads to the release of cell‐free hemoglobin (CFH) from lysed red blood cells. Once released into the vascular circulation, CFH can oxidize from the normal ferrous 2+ state to methemoglobin (3+, ferric). We have shown previously that CFH is a key driver of acute respiratory distress syndrome due to the breakdown of the alveolar capillary barrier. However, the underlying mechanism of how CFH disrupts the pulmonary endothelium remains unknown. Originally, it was believed hemoglobin acted only in an oxidative damage fashion. We hypothesized that oxidized CFH disrupts the pulmonary endothelium due to mitochondrial oxidative damage and leads to increased leukocyte adhesion. We utilized primary human lung microvascular endothelial cells (HLMVECs) to probe the mechanism underlying CFH‐induced barrier dysfunction. Endothelial barrier function was analyzed using electric cell‐substrate impedance sensing (ECIS) to quantify the transendothelial resistance changes in real‐time following treatment with CFH2+ and CFH3+ (1.0 and 0.5 mg/mL). Permeability was assessed using the SynVivo idealized network coculture system using HLMVECs and small airway epithelial cells and was quantified using fluorescent 60 kDa dextrans mixed with CFH2+,3+ (1.0 mg/mL) in media flowed through the channels while images were taken every 15 minutes for 4 h. Cytokines were measured via ELISA post CFH3+ treatment from conditioned culture media at 1, 3, 6, and 24 h. Adhesion of leukocytes was quantified using immunofluorescence by counting the number of fluorescent polymorphonuclear cells (PMNs) adhered to an endothelial monolayer following pretreatment with CFH3+ of the endothelial cells, the PMNs, or both. Mitochondrial superoxide production was quantified by MitoSOX staining followed by flow cytometry analysis of CFH‐treated HLMVECs. We saw a dose‐dependent decrease in transendothelial resistance following oxidized CFH3+ treatment but not with CFH2+ treatment (‐1270 vs 262 max TER drop, p<0.0001). Real‐time permeability assays using the SynVivo system showed an increase in barrier permeability from CFH3+ treatment compared to CFH2+ (47 vs 6 relative permeability). Surface expression of E‐selectin (p=0.037) and ICAM‐1 (p=0.0497) was increased following oxidized CFH3+ treatment versus vehicle. In addition, pretreatment of HLMVECs with CFH3+ increased adhesion of polymorphonuclear cells (PMNs) to the endothelium (1.69 x 108 vs 7.81 x 109 GFP intensity, p=0.0014), while pretreatment of the PMNs did not increase adhesion. Treatment of HLMVECs with CFH3+ for 24 h increased secretion of IL‐1b, IL‐6, and IL‐8 (p<0.05). HLMVECs exposed to CFH3+ for 6 h have significantly increased mitochondrial superoxide compared to vehicle treated cells (608 vs 283 adjusted MFI, p=0.0014). These data demonstrate that oxidized CFH decreases pulmonary endothelial function through both immune and oxidative damage pathways. We also show that CFH induces endothelial dysfunction in an oxidation‐state dependent manner. In summary, these studies provide new evidence to the potential mechanism in which CFH harms the endothelium during sepsis beyond the conventionally attributed oxidative cycling of the heme moiety.

Reduction of Reactive Oxygen Species by a Growth Hormone‐Releasing Hormone Antagonist in Endothelial Cells

INTRODUCTIONGrowth hormone‐releasing hormone (GHRH) is a hypothalamic hormone regulating the secretion of Growth Hormone (GH) from the anterior pituitary gland. The expression of GHRH and its receptors (full length pituitary‐type and splice variants) in tissues such as breast, prostate and lung, suggested that the activities of that hypothalamic hormone is not limited to the GH ‐ insulin‐like growth factor‐I axis. Reactive Oxygen Species (ROS) are involved in inflammation, disrupt inter‐endothelial junctions, promote cytoskeletal remodeling and induce pro‐inflammatory cytokines (e.g. interleukin [IL]‐1β, IL‐6). Those events have been associated to endothelial hyper‐permeability, the hallmark of Acute Respiratory Distress Syndrome (ARDS). The purpose of the present study is to investigate the effects of GHRH and GHRH antagonists (GHRHAnt) in ROS generation of three different endothelial cell lines.METHODSBovine pulmonary artery endothelial cells (BPAEC), human lung microvascular endothelial cells (HMVEC‐L), human cerebral microvascular endothelial cells (hCMEC/D3) and mouse embryonic fibroblasts (NIH/3T3) were maintained at 37°C in a humidified atmosphere of 5% CO2‐ 95% air per suppliers’ instructions. Cells seeded in 96‐well plates (1×104 cells/well, n=4 wells per group) were treated with GHRH (1µM), or GHRHAnt (1µM), or GHRHAnt (1µM) prior to H2O2 (0.1mM) exposure. To measure ROS levels, the cells were incubated with 25µM of DCFDA for 45 min, and fluorescence intensity was measured by a fluorescence microplate reader using excitation and emission wavelength of 485 nm and 535 nm respectively. Protein expression was measured utilizing Western Blot analysis. The densitometry of immunoblots was accomplished by using Image J software (NIH), and Student's t test was used to determine the statistically significant differences among the groups. A value of p < 0.05 was considered significant.RESULTSThe expression of GHRH receptors in BPAEC, hCMEC/D3 and HMVEC‐L was confirmed by Western Blot. NIH/3T3 cells were used as negative controls, since they do not express receptors responding to GHRH. All cells were exposed to vehicle (0.1% DMSO), GHRH (1µM), or GHRHAnt (1µM) for 8 hours. Our results demonstrate that GHRH significantly increases ROS generation in all three endothelial cell lines, and GHRHAnt reduces ROS production. We also measured the effects of GHRHAnt against H2O2‐induced oxidative stress. Cells were treated with vehicle (0.1% DMSO), or GHRHAnt (1µM) for 8 hours, prior to vehicle (PBS) or H2O2 (0.1mM) exposure (8 hours). H2O2 potentiated ROS generation, and that effect was counteracted by GHRHAnt. Those peptides failed to suppress the H2O2‐induced ROS production in NIH/3T3 cells, since they do not express GHRH receptors.CONCLUSIONSOur results reveal that GHRHAnt suppress ROS production in the endothelium, suggesting that those compounds may be of therapeutic value in diseases related to endothelial barrier dysfunction and oxidative stress (e.g. Acute Respiratory Distress Syndrome and sepsis).

SMAD3 is a Mediator of TGF‐β ‐induced Barrier Dysfunction and Hyperpermeability in Microvascular Endothelial Cells

The endothelial transcription factor ETS (Erythroblast‐transformation specific)‐related gene (ERG) regulates canonical TGFβ‐SMAD signaling, by repressing SMAD3 activity. SMAD proteins function as important mediators of intracellular signal transduction of TGF‐β. Following their phosphorylation by TGF‐β receptor‐I, Receptor‐regulated SMADs (including SMAD2 and SMAD3 proteins) form a heteromeric complex with co‐SMAD and then translocate into the nucleus where they bind and regulate the expression of multiple target genes. Recent studies from our lab have demonstrated that ERG is a critical regulator of permeability in human lung microvascular endothelial cells (HLMECs) where ERG gene activation provided protection to the barrier. We have hypothesized that the protective effects of ERG on barrier integrity may be mediated via inhibiting TGF‐β/SMAD3 signaling pathway. To study the role of SMAD3 in microvascular endothelial barrier functions and permeability, HLMECs were grown on Transwell inserts and other cell culture platforms. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. TGF‐β (1‐50 ng/ml) induced a dose‐dependent increase in monolayer hyperpermeability. TGF‐β‐induced hyperpermeability was attenuated by a SMAD3 pharmacological inhibitor (SIS3) or CRISPR/cas9‐mediated knockdown of SMAD3. Immunoblot analysis demonstrated highly efficient SMAD3 knockdown by SMAD3 CRISPR/Cas9. TGF‐β treatment resulted in significant increase in matrixmetalloproteinase‐9 (MMP‐9) activity and the effect was decreased by SMAD3 inhibition. MMP‐9 is known to proteolytically breakdown the adherens junction proteins in microvascular endothelial cells, resulting in hyperpermeability. These results demonstrate that SMAD3 is a mediator of TGF‐β signaling‐induced loss of endothelial barrier integrity and permeability in microvascular endothelial cells. It also suggests that one of the downstream effects of TGFβ‐SMAD signaling is the activation of MMP‐9 which mediates loss of adherens junctional integrity.

Stimulation of Neutrophils in Whole Blood Enhances the Pro‐inflammatory Activity of Neutrophil‐Derived Microvesicles

BackgroundAcute increases in circulating levels of neutrophil‐derived microvesicles (N‐MVs) during sepsis combined with evidence of enhanced uptake of MVs within the pulmonary microvasculature during endotoxemia in mice, suggests a role for N‐MVs in development of indirect acute lung injury (ALI). However, evidence for the pro‐inflammatory activity of N‐MVs is unclear, with various in vitro studies demonstrating anti‐inflammatory effects. Most in vitro investigations of N‐MV activity involve stimulation of purified neutrophils with single agonists such as formyl‐methionyl‐leucyl‐phenylalanine (fMLP). This reductionist approach excludes the complex in vivo interplay between different stimuli and other vascular cells contributing to neutrophil responses during sepsis. Here we investigated the proinflammatory activities of N‐MVs under more clinically relevant in vitro conditions, treating healthy volunteer whole blood with lipopolysaccharide (LPS) as a sepsis‐relevant inflammatory stimulus and fMLP as an acute N‐MV inducing stimulus. The activities of N‐MVs derived from whole blood as well as isolated neutrophils were evaluated in a monocyte‐neutrophil‐endothelial cell ‘tri‐culture’ model of pulmonary vascular inflammation.MethodsN‐MVs were generated from whole blood or neutrophils isolated by two‐step density gradient centrifugation, by stimulation with fMLP (1 µM, 30 min) alone or combined with LPS pre‐treatment (100 ng/ml, 1 h). N‐MVs were isolated from blood by anti‐CD66b immunomagnetic‐bead selection. For the tri‐culture assay, isolated neutrophils and immunomagnetic‐bead sorted monocytes were cultured with human lung microvascular endothelial cells. N‐MVs were incubated in the tri‐cultures for 3 h, and MV‐induced responses were determined by ELISA and flow cytometric quantification of cell activation markers on monocytes and neutrophils.ResultsN‐MVs derived from LPS‐fMLP‐stimulated (but not fMLP‐stimulated) whole blood induced significant responses in the tri‐culture assay, i.e. substantive tumour necrosis factor (TNF) release (375.2±251.4 vs untreated 2.2±1.4 pg/ml, p<0.01), increased tissue factor expression on monocytes (MFI: 231.1±90.0 vs untreated 9.2±2.5, p<0.001), and increased expression of CD45 (MFI: 478.3±131.2 vs untreated 281.4±37.5, p<0.05), CD11b (MFI: 208.0±33.0 vs untreated 112.7±20.1, p<0.05) and CD63 (MFI: 77.3±36.5 vs untreated 16.23±5.3, p<0.05) on neutrophils. In contrast, N‐MVs derived from isolated neutrophils showed no activity, with either the LPS‐fMLP or the fMLP stimulation protocols.ConclusionsWe demonstrated that LPS inflammatory ‘priming’ of neutrophils within a whole blood environment was necessary for generation of leukocyte‐activating N‐MVs in our in vitro model of pulmonary vascular inflammation. These findings support a role for circulating N‐MVs in the pathogenesis of indirect ALI, and also highlight the crucial importance of using physiologically relevant in vitro models to evaluate the function of circulating intravascular cell‐derived MVs.

Cell‐free Hemoglobin‐Oxidized LDL Axis Contributes to Microvascular Endothelial Barrier Dysfunction and Poor Outcomes During Sepsis

IntroductionSepsis, a dysregulated host response to infection with high morbidity and mortality, is characterized by a systemic inflammatory response and widespread vascular hyperpermeability leading to edema, organ dysfunction, and death with no specific treatments. Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis‐induced organ dysfunction driven by circulating inflammatory mediators, oxidants, and proteolytic enzymes. Cell‐free hemoglobin (CFH) is one such factor, released from damaged red blood cells during sepsis due to their increased deformability and fragility. Though CFH is not detectable in healthy subjects, plasma CFH is elevated in patients with sepsis and independently associated with mortality. CFH contributes to endothelial dysfunction, but the signaling mechanisms are incompletely understood. CFH can oxidize circulating low‐density lipoproteins (oxLDL), which could potentiate endothelial barrier disruption. We hypothesized that elevated levels of CFH during sepsis are associated with increased oxLDL and contribute to microvascular endothelial barrier dysfunction through the oxLDL receptor, lectin‐like oxidized LDL receptor 1 (LOX‐1).MethodsCirculating levels of CFH and oxLDL were measured in sepsis patients. In human lung microvascular endothelial cells (HLMVECs), transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell‐substrate Impedance Sensing (ECIS).Results (Figure)In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686; p=0.016, n=12) and were higher in patients who died in hospital (33.24 U/L [IQR 28.08, 48.28] vs. 27.19 U/L [IQR 16.24, 38.24]; p=0.045, n=25). HLMVEC barrier dysfunction induced by CFH (p<0.0001, n=6‐7) or oxLDL (p=0.002, n=4‐6) was attenuated by blocking the LOX‐1 receptor with small molecule inhibitor BI‐0115 (Boehringer Ingelheim, 20 µM; p=0.018 vs CFH, n=5; p=0.0002 vs oxLDL, n=6).ConclusionIncreased plasma CFH and oxLDL are associated with HLMVEC barrier dysfunction and poor clinical outcomes during sepsis; one mechanism by which CFH may cause vascular hyperpermeability during sepsis is through oxidation of LDL which can drive signaling through the LOX‐1 receptor.

A Protein Tyrosine Phosphatase 4A3 (PTP4A3) inhibitor prevents and repairs pulmonary endothelial barrier dysfunction and ameliorates acute lung injury provoked by the SARS‐CoV‐2 Spike protein subunit 1 in k18‐hACE2 transgenic mice

PTP4A3 regulates various inflammatory and cytoskeletal signaling pathways (e.g., STAT3, RhoA, ERK, AKT) that are involved in endothelial barrier dysfunction and acute lung injury (ALI). We have investigated the actions of JMS‐053, a small PTP4A3 inhibitor, in both in vitro and in vivo models of SARS‐CoV‐2 Spike protein subunit 1 (S1SP)‐induced endothelial inflammation and acute lung injury. S1SP (10nM) caused a time‐dependent endothelial barrier dysfunction in human lung microvascular endothelial cell monolayers that was ameliorated by JMS‐053 (12.5µM) administered either as pre‐treatment (4 hours prior to S1SP) or post‐treatment (5 hours after S1SP). In K18‐hACE2 transgenic mice, which express the human ACE2 receptor for SARS‐CoV‐2 S1SP, we instilled S1SP intratracheally, post‐treated with JMS‐053 (10mg/kg i.p. at 1, 24 and 48 hours after S1SP) or vehicle, and investigated molecular, functional and histological outcomes 72 hours later. JMS‐053 mice displayed lower alveolar cellularity, proteinosis and “cytokine storm” (i.e., IL‐6, IL‐1β, IL‐17), and lower levels of phosphorylated (activated) STAT3 and NF‐kB, compared to mice instilled with S1SP and treated with vehicle. Moreover, JMS‐053 greatly reduced quantifiable histological evidence of injury, compared to vehicle‐treated animals. This data suggests that inhibition of PTP4A3 could represent a critical target for novel therapeutic approaches towards endothelial dysfunction, inflammation and acute lung injury.

In vitro model of pulmonary candidiasis for testing novel therapeutics

Invasive candidiasis is one of the most common causes of hospital‐acquired blood infections, with the most common cause being Candida albicans. These bloodstream infections often lead to long hospital stays and have a high mortality rate. Once C. albicansenters the bloodstream it can disseminate and invade many organs of the body, including the lungs, where it can inhibit function and cause further complications. Furthermore, C. albicansis commonly isolated from the lungs of cystic fibrosis patients and often causes coinfection in patients with severe COVID‐19. Current antifungal drugs exhibit high toxicity, poor water solubility, and are ineffective against some strains of C. albicans due to antifungal resistance, creating an urgent need for new antifungal therapeutics. Here, we have developed a customizable in vitroplatform to study the interaction between rat lung microvascular endothelial cells (ECs), C. albicansSC5314, and novel liposomes encapsulating the antifungal drug anidulafungin. Liposome formulations composed of hydrogenated soy phosphatidylcholine, phosphatidylglycerol, cholesterol, and α‐tocopherol were fabricated containing three different % wt/wt concentrations of anidulafungin (0.31, 1.55, and 3.10, or 1x, 5x and 10x, respectively). The minimum inhibitory concentrations (MICs) of the different liposome formulations for C. albicans were determined using a microdilution assay. After 24 hours of exposure, the MICs were found to be 12.50, 6.25, and 1.56 μg/mL for 1x, 5x, and 10x liposome formulations, respectively. Cell Counting Kit 8 was used to determine EC viability after 24‐hour treatment with the different liposome formulations. The 1x and 5x anidulafungin liposome formulations showed no significant decrease in viability up to the maximum liposome concentration tested (50 μg/mL), with the 10x formulation causing a slight but significant decrease in viability. The 1x and 5x anidulafungin liposomes were then assessed at 6.25 and 12.5 μg/mL in a co‐culture setting. C. albicans were seeded onto established EC monolayers and were allowed to form hyphae, which is the invasive form. After the two hours, the co‐cultures were treated with the antifungal liposomes. Treatment with the liposomes reduced hyphae formation and prevented C. albicansovergrowth within four hours of exposure, with the 5x anidulafungin liposome at 12.5 μg/mL showing the strongest change. These data suggest that these novel antifungal liposomes have the potential to treat pulmonary candidiasis without the toxic side effects associated with current antifungal drugs.

Rabeprazole Promotes Vascular Repair and Resolution of Sepsis-Induced Inflammatory Lung Injury through HIF-1α.

There are currently no effective treatments for sepsis and acute respiratory distress syndrome (ARDS). The repositioning of existing drugs is one possible effective strategy for the treatment of sepsis and ARDS. We previously showed that vascular repair and the resolution of sepsis-induced inflammatory lung injury is dependent upon endothelial HIF-1α/FoxM1 signaling. The aim of this study was to identify a candidate inducer of HIF-1α/FoxM1 signaling for the treatment of sepsis and ARDS. Employing high throughput screening of a library of 1200 FDA-approved drugs by using hypoxia response element (HRE)-driven luciferase reporter assays, we identified Rabeprazole (also known as Aciphex) as a top HIF-α activator. In cultured human lung microvascular endothelial cells, Rabeprazole induced HIF1A mRNA expression in a dose-dependent manner. A dose-response study of Rabeprazole in a mouse model of endotoxemia-induced inflammatory lung injury identified a dose that was well tolerated and enhanced vascular repair and the resolution of inflammatory lung injury. Rabeprazole treatment resulted in reductions in lung vascular leakage, edema, and neutrophil sequestration and proinflammatory cytokine expression during the repair phrase. We next used Hif1a/Tie2Cre knockout mice and Foxm1/Tie2Cre knockout mice to show that Rabeprazole promoted vascular repair through HIF-1α/FoxM1 signaling. In conclusion, Rabeprazole is a potent inducer of HIF-1α that promotes vascular repair and the resolution of sepsis-induced inflammatory lung injury via endothelial HIF-1α/FoxM1 signaling. This drug therefore represents a promising candidate for repurposing to effectively treat severe sepsis and ARDS.

CircEXOC5 promotes ferroptosis by enhancing ACSL4 mRNA stability via binding to PTBP1 in sepsis-induced acute lung injury

BackgroundSepsis causes severe acute lung injury (ALI). Circular RNA is involved in the regulation of sepsis-related ALI progression. The regulation mechanism of circEXOC5 in sepsis-induced ALI is still unclear. Whether circEXOC5 is involved in the regulation of ferroptosis remains to be explored. MethodsWe constructed a mouse model of sepsis through cecal ligation and puncture (CLP). LPS induced mouse lung microvascular endothelial cells (MPVECs) to construct a sepsis cell model. The expression of circEXOC5 in the sepsis model was detected by qPCR. The extent of lung injury in mice was analyzed by HE staining. The contents of GSH/GSSG, iron, MDA and 4HNE in mice lung tissues and cells were detected by the kit. And further the ROS content was detected in the cells. Finally, the binding relationship between circEXOC5 and PTBP1 was detected by RIP and RNA pulldown. ResultsOur results showed that the circEXOC5 expression was significantly increased in the in vivo and in vitro models of sepsis. And after inhibiting circEXOC5, it improved the lung injury of septic mice. It was confirmed in cell models that ROS levels and ferroptosis in cells were reduced after knocking down circEXOC5. In addition, the expressions of ACSL4 and Gpx4 proteins were regulated by the level of circEXOC5. Finally, we also found that circEXOC5 had a direct binding relationship with PTBP1. ConclusionOur study found that the expression of cell ferroptosis and circEXOC5 increased in ALI induced by sepsis, and circEXOC5 aggravated ferroptosis in septic cells by regulating the PTBP1/ACSL4 axis.

324 The role of CCN3 in lung endothelial identity and function

OBJECTIVES/GOALS: Idiopathic Pulmonary Fibrosis (IPF) is a fatal disease of lung scarring. Aberrant vascular remodeling is a contributor to IPF progression. We have identified CCN3 as an endothelial gene that is upregulated in resolving but not in persistent lung fibrosis in mice. Here we tested the role of CCN3 in lung microvascular endothelial function. METHODS/STUDY POPULATION: RNAi loss of function experiments were used to evaluate the role of CCN3 in lung endothelial biology. Human lung microvascular endothelial cells (HLMECs) were transfected with human CCN3 siRNA and analyzed via qPCR to assess expression of endothelial transcripts, via wound healing assay for assessment of migratory function, and via 2D tube formation assay for assessment of angiogenic function. To ascertain the effect of HLMECs on Normal Human Lung Fibroblasts (NHLFs), conditioned media (CM) from endothelial cells with control and CCN3 siRNA was applied to TGFβ primed fibroblasts and qPCR was used to measure expression levels of pro-fibrotic transcripts. Recombinant human CCN3 protein was subsequently used to confirm the gain of function role of CCN3 in lung endothelial biology using a subset of these assays. RESULTS/ANTICIPATED RESULTS: CCN3 is a secreted matricellular protein thought to be involved in angiogenesis, cell adhesion, cell migration, and inflammatory responses in endothelial cells. In other organs, CCN3 suppresses expression of fellow matricellular protein, CCN2 (CTGF); importantly, CCN2 is a known pro-fibrotic mediator of aberrant tissue remodeling in the fibrotic lung. Upon CCN3 knockdown in HLMECs, we observed reduced transcripts for inflammatory and pro-fibrotic genes, along with impaired endothelial function in wound healing and angiogenesis assays. CM from CCN3 knockdown endothelial cells enhanced the pro-fibrotic effects of TGFβ in NHLFs. Addition of recombinant CCN3 to HLMECs generated, conditioned media that reduced fibroblast pro-fibrotic activation. DISCUSSION/SIGNIFICANCE: We have shown that matricellular protein- CCN3 plays a fundamental role in endothelial identity and function and could be a promising therapeutic target in IPF. A future goal is to restore levels of genes such as CCN3 in the aged vasculature in the setting of lung fibrosis to test their capacity to promote vascular repair and fibrosis resolution.

A Model of Human Small Airway on a Chip for Studies of Subacute Effects of Inhalation Toxicants.

Testing for acute inhalation hazards is conducted in animals; however, a number of robust in vitro human cell-based alternatives have been developed and tested. These models range in complexity from cultures of cell lines or primary cells in air-liquid interface on Transwells, to more complex and physiologically relevant flow- and mechanical stimulation-enabled tissue chips. Although the former models are relatively straightforward to establish and can be tested in medium/high throughput, the latter require specialized equipment and lack in throughput. In this study, we developed a device that can be easily manufactured while allowing for the production of a differentiated lung tissue. This multilayered microfluidic device enables coculture of primary human small airway epithelial cells and lung microvascular endothelial cells under physiological conditions for up to 18 days and recreates the parenchymal-vascular interface in the distal lung. To explore the potential of this airway on a chip for applications in inhalation toxicology, we also devised a system that allows for direct gas/aerosol exposures of the engineered airway epithelium to noxious stimuli known to cause adverse respiratory effects, including dry flowing air, lipopolysaccharide, particulate matter, and iodomethane. This study generated quantitative, high-content data that were indicative of aberrant changes in biochemical (lactate dehydrogenase), barrier (dextran permeability), functional (ciliary beating), and molecular (imaging for various markers) phenotypes of the small airway epithelium due to inhalational exposures. This study is significant because it established an in vitro model of human small airway on a chip that can be used in medium/high-throughput studies of subacute effects of inhalation toxicants.

SARS-CoV-2 Infects Human ACE2-Negative Endothelial Cells through an αvβ3 Integrin-Mediated Endocytosis Even in the Presence of Vaccine-Elicited Neutralizing Antibodies.

Integrins represent a gateway of entry for many viruses and the Arg-Gly-Asp (RGD) motif is the smallest sequence necessary for proteins to bind integrins. All Severe Acute Respiratory Syndrome Virus type 2 (SARS-CoV-2) lineages own an RGD motif (aa 403–405) in their receptor binding domain (RBD). We recently showed that SARS-CoV-2 gains access into primary human lung microvascular endothelial cells (HL-mECs) lacking Angiotensin-converting enzyme 2 (ACE2) expression through this conserved RGD motif. Following its entry, SARS-CoV-2 remodels cell phenotype and promotes angiogenesis in the absence of productive viral replication. Here, we highlight the αvβ3 integrin as the main molecule responsible for SARS-CoV-2 infection of HL-mECs via a clathrin-dependent endocytosis. Indeed, pretreatment of virus with αvβ3 integrin or pretreatment of cells with a monoclonal antibody against αvβ3 integrin was found to inhibit SARS-CoV-2 entry into HL-mECs. Surprisingly, the anti-Spike antibodies evoked by vaccination were neither able to impair Spike/integrin interaction nor to prevent SARS-CoV-2 entry into HL-mECs. Our data highlight the RGD motif in the Spike protein as a functional constraint aimed to maintain the interaction of the viral envelope with integrins. At the same time, our evidences call for the need of intervention strategies aimed to neutralize the SARS-CoV-2 integrin-mediated infection of ACE2-negative cells in the vaccine era.

Heparan sulfate mimetic fucoidan restores the endothelial glycocalyx and protects against dysfunction induced by serum of COVID-19 patients in the intensive care unit

Accumulating evidence proves that endothelial dysfunction is involved in coronavirus disease 2019 (COVID-19) progression. We previously demonstrated that the endothelial surface glycocalyx has a critical role in maintenance of vascular integrity. Here, we hypothesised that serum factors of severe COVID-19 patients affect the glycocalyx and result in endothelial dysfunction.We included blood samples of 32 COVID-19 hospitalised patients at the Leiden University Medical Center, of which 26 were hospitalised in an intensive care unit (ICU) and six on a non-ICU hospital floor; 18 of the samples were obtained from convalescent patients 6 weeks after hospital discharge, and 12 from age-matched healthy donors (control) during the first period of the outbreak. First, we determined endothelial (angiopoietin 2 (ANG2)) and glycocalyx degradation (soluble thrombomodulin (sTM) and syndecan-1 (sSDC1)) markers in plasma.In the plasma of COVID-19 patients, circulating ANG2 and sTM were elevated in patients in the ICU. Primary lung microvascular endothelial cell (HPMEC) and human glomerular microvascular endothelial cell (GEnC) cultured in the presence of these sera led to endothelial cell glycocalyx degradation, barrier disruption, inflammation and increased coagulation on the endothelial surface, significantly different compared to healthy control and non-ICU patient sera. These changes could all be restored in the presence of fucoidan.In conclusion, our data highlight the link between endothelial glycocalyx degradation, barrier failure and induction of a procoagulant surface in COVID-19 patients in ICU which could be targeted earlier in disease by the presence of heparan sulfate mimetics.

The Heat Shock Protein 90 Inhibitor, AT13387, Protects the Alveolo-Capillary Barrier and Prevents HCl-Induced Chronic Lung Injury and Pulmonary Fibrosis.

Hydrochloric acid (HCl) exposure causes asthma-like conditions, reactive airways dysfunction syndrome, and pulmonary fibrosis. Heat Shock Protein 90 (HSP90) is a molecular chaperone that regulates multiple cellular processes. HSP90 inhibitors are undergoing clinical trials for cancer and are also being studied in various pre-clinical settings for their anti-inflammatory and anti-fibrotic effects. Here we investigated the ability of the heat shock protein 90 (HSP90) inhibitor AT13387 to prevent chronic lung injury induced by exposure to HCl in vivo and its protective role in the endothelial barrier in vitro. We instilled C57Bl/6J mice with 0.1N HCl (2 µL/g body weight, intratracheally) and after 24 h began treatment with vehicle or AT13387 (10 or 15 mg/kg, SC), administered 3×/week; we analyzed histological, functional, and molecular markers 30 days after HCl. In addition, we monitored transendothelial electrical resistance (TER) and protein expression in a monolayer of human lung microvascular endothelial cells (HLMVEC) exposed to HCl (0.02 N) and treated with vehicle or AT13387 (2 µM). HCl provoked persistent alveolar inflammation; activation of profibrotic pathways (MAPK/ERK, HSP90); increased deposition of collagen, fibronectin and elastin; histological evidence of fibrosis; and a decline in lung function reflected in a downward shift in pressure–volume curves, increased respiratory system resistance (Rrs), elastance (Ers), tissue damping (G), and hyperresponsiveness to methacholine. Treatment with 15 mg/kg AT13387reduced alveolar inflammation, fibrosis, and NLRP3 staining; blocked activation of ERK and HSP90; and attenuated the deposition of collagen and the development of chronic lung injury and airway hyperreactivity. In vitro, AT13387 prevented HCl-induced loss of barrier function and AKT, ERK, and ROCK1 activation, and restored HSP70 and cofilin expression. The HSP90 inhibitor, AT13387, represents a promising drug candidate for chronic lung injury that can be administered subcutaneously in the field, and at low, non-toxic doses.

Direct Thrombin Inhibitor Dabigatran Compromises Pulmonary Endothelial Integrity in a Murine Model of Breast Cancer Metastasis to the Lungs; the Role of Platelets and Inflammation-Associated Haemostasis.

Activation of the coagulation cascade favours metastatic spread, but antithrombotic therapy might also have detrimental effects on cancer progression. In this study, we characterized the effects of dabigatran, a direct reversible thrombin inhibitor, on the pulmonary endothelial barrier and metastatic spread in a murine model of breast cancer metastasis. Dabigatran etexilate (100 mg kg−1) was administered to mice twice daily by oral gavage. Pulmonary metastasis, pulmonary endothelium permeability in vivo, and platelet reactivity were evaluated after intravenous injection of 4T1 breast cancer cells into BALB/c mice. The effect of dabigatran on platelet-dependent protection of pulmonary endothelial barrier in the presence of an inflammatory stimulus was also verified in vitro using human lung microvascular endothelial cell (HLMVEC) cultures. Dabigatran-treated mice harbored more metastases in their lungs and displayed increased pulmonary endothelium permeability after cancer cell injection. It was not associated with altered lung fibrin deposition, changes in INFγ, or complement activation. In the in vitro model of the pulmonary endothelial barrier, dabigatran inhibited platelet-mediated protection of pulmonary endothelium. In a murine model of breast cancer metastasis, dabigatran treatment promoted pulmonary metastasis by the inhibition of platelet-dependent protection of pulmonary endothelial barrier integrity.