Electric Cell-substrate Impedance Sensing Research Articles

Activating transcription factor 6 protects against endothelial barrier dysfunction

BackgroundEndothelial hyperpermeability is associated with sepsis and acute respiratory distress syndrome (ARDS). The identification of molecular pathways involved in barrier dysfunction; may reveal promising therapeutic targets to combat ARDS. Unfolded protein response (UPR) is a highly conserved molecular pathway, which ameliorates endoplasmic reticulum stress. The present work focuses on the effects of ATF6, which is a UPR sensor, in lipopolysaccharides (LPS)-induced endothelial hyperpermeability. MethodsThe in vitro effects of AA147 and Ceapin-A7 in LPS-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Small interfering (si) RNA was utilized to “silence” ATF6, and electric cell-substrate impedance sensing (ECIS) measured transendothelial resistance. Fluorescein isothiocyanate (FITC)-dextran assay was utilized to assess paracellular permeability. Protein expression levels were evaluated with Western blotting, and cell viability with MTT assay. ResultsWe demonstrated that AA147 prevents LPS-induced barrier disruption by counteracting Cofilin and myosin light chain 2 (MLC2) activation, as well as VE-Cadherin phosphorylation. Moreover, this ATF6 inducer opposed LPS-triggered decrease in transendothelial resistance (TEER), as well as LPS-induced paracellular hyperpermeability. On the other hand, ATF6 suppression due to Ceapin-A7 or small interfering RNA exerted the opposite effects, and potentiated LPS-induced endothelial barrier disruption. Moderate concentrations of both ATF6 modulators did not affect cell viability. ConclusionsATF6 activation protects against endothelial barrier function, suggesting that this UPR sensor may serve as a therapeutic target for sepsis and ARDS.

In-Depth Characterization of Zika Virus Inhibitors Using Cell-Based Electrical Impedance.

ABSTRACTIn this study, we use electric cell-substrate impedance sensing (ECIS), an established cell-based electrical impedance (CEI) technology, to decipher the kinetic cytopathic effect (CPE) induced by Zika virus (ZIKV) in susceptible human A549 lung epithelial cells and to evaluate several classes of compounds with reported antiviral activity (two entry inhibitors and two replication inhibitors). To validate the assay, we compare the results with those obtained with more traditional in vitro methods based on cell viability and viral yield readouts. We demonstrate that CEI can detect viral infection in a sensitive manner and can be used to determine antiviral potency. Moreover, CEI has multiple benefits compared to conventional assays: the technique is less laborious and better at visualizing the dynamic antiviral activity profile of the compounds, while also it has the ability to determine interesting time points that can be selected as endpoints in assays without continuous readout. We describe several parameters to characterize the compounds’ cytotoxicity and their antiviral activity profile. In addition, the CEI patterns provide valuable additional information about the presumed mechanism of action of these compounds. Finally, as a proof of concept, we used CEI to evaluate the antiviral activity of a small series of compounds, for which we demonstrate that the sulfonated polymer PRO2000 inhibits ZIKV with a response profile representative for a viral entry inhibitor. Overall, we demonstrate for the first time that CEI is a powerful technology to evaluate and characterize compounds against ZIKV replication in a real-time, label-free, and noninvasive manner.IMPORTANCE Zika virus can cause serious disease in humans. Unfortunately, no antiviral drugs are available to treat infection. Here, we use an impedance-based method to continuously monitor virus infection in—and damage to—human cells. We can determine the Zika viral dose with this technique and also evaluate whether antiviral compounds protect the cells from damage caused by virus replication. We also show that this technique can be used to further unravel the characteristics of these compounds, such as their toxicity to the cells, and that it might even give further insight in their mechanism of antiviral action. Finally, we also find a novel Zika virus inhibitor, PRO2000. Overall, in this study, we use the impedance technology to—for the first time—evaluate compounds with anti-Zika virus properties, and therefore it can add valuable information in the further search for antiviral drugs.

Characterization by Electric Impedance Sensing of Normal and Cancer Cell Attachment and the Effect of Ginseng

This research reestablishes an application of an Electric Cell Substrate Impedance Sensing (ECIS) system to examine the effects of Ginseng on the attachment of epithelial cells; HaCAT (healthy human skin cells) and A431 (human carcinoma cells). The study is functionally a continuation of the authors’ previously published paper, where an identical set of experiments were carried out for the putative homeopathic Reishi. The purpose for this study was two-fold: (1) to investigate variations in results between a roundly discredited homeopathic (Reishi) and a studied, well-accepted homeopathic (Gensing), and (2) to justify the experimental technique from the results - as the electric impedance sensing technique has heretofore been considered itself experimental; specifically, impedance measurements reflecting cell-to-cell binding and their associated binding electric field strength and the high correlation to shape (normal vs. cancer) and cluster size (as measured by increased field strength in greater clusters). Here the impedance analysis shows that Ginseng at applied doses does not alter the impedance of HaCAT colonies but that Ginseng does significantly increase the strength of cell-to-cell attachments in A431 colonies and may confer previously suggested anti-inflammatory properties. This result is not seen with the previous set of Reishi experiments, and this correct distinction provides more ammunition for the electrical impedance sensing technique as a valid differentiation in cell attachment.

Electric Cell-Substrate Impedance Sensing (ECIS) as a Platform for Evaluating Barrier-Function Susceptibility and Damage from Pulmonary Atelectrauma.

Biophysical insults that either reduce barrier function (COVID-19, smoke inhalation, aspiration, and inflammation) or increase mechanical stress (surfactant dysfunction) make the lung more susceptible to atelectrauma. We investigate the susceptibility and time-dependent disruption of barrier function associated with pulmonary atelectrauma of epithelial cells that occurs in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). This in vitro study was performed using Electric Cell-substrate Impedance Sensing (ECIS) as a noninvasive evaluating technique for repetitive stress stimulus/response on monolayers of the human lung epithelial cell line NCI-H441. Atelectrauma was mimicked through recruitment/derecruitment (RD) of a semi-infinite air bubble to the fluid-occluded micro-channel. We show that a confluent monolayer with a high level of barrier function is nearly impervious to atelectrauma for hundreds of RD events. Nevertheless, barrier function is eventually diminished, and after a critical number of RD insults, the monolayer disintegrates exponentially. Confluent layers with lower initial barrier function are less resilient. These results indicate that the first line of defense from atelectrauma resides with intercellular binding. After disruption, the epithelial layer community protection is diminished and atelectrauma ensues. ECIS may provide a platform for identifying damaging stimuli, ventilation scenarios, or pharmaceuticals that can reduce susceptibility or enhance barrier-function recovery.

Real-Time Monitoring the Cytotoxic Effect of Andrographolide on Human Oral Epidermoid Carcinoma Cells.

Andrographolide is an active diterpenoid compound extracted from Andrographis paniculata. It exhibits antiinflammatory and anticancer effects. Previous studies show that it is non-toxic to experimental animals. The leading causes of cancer are chronic inflammation and high blood glucose. This study determines the cytotoxic effect of andrographolide on cellular morphology, viability, and migration for human oral epidermoid carcinoma cell Meng-1 (OEC-M1). We use electric cell-substrate impedance sensing (ECIS) to measure the subsequent overall impedance changes of the cell monolayer in response to different concentrations of andrographolide for 24 h (10–100 µM). The results for exposure of OEC-M1 cells to andrographolide (10–100 µM) for 24 h show a concentration-dependent decrease in the overall measured resistance at 4 kHz. AlamarBlue cell viability assay and annexin V also show the apoptotic effect of andrographolide on OEC-M1 cells. A reduction in wound-healing recovery rate is observed for cells treated with 30 μM andrographolide. This study demonstrates that ECIS can be used for the in vitro screening of anticancer drugs. ECIS detects the cytotoxic effect of drugs earlier than traditional biochemical assays, and it is more sensitive and shows more detail.

A Cell Co-Culture Taste Sensor Using Different Proportions of Caco-2 and SH-SY5Y Cells for Bitterness Detection

Bitter taste receptors (T2Rs) are involved in bitter taste perception, which is one of the five basic taste modalities in mammals. In this study, a cell co-culture taste sensor using different proportions of Caco-2 cells and SH-SY5Y cells was proposed. Caco-2 cells, which endogenously expressed the human T2R38 receptor, and SH-SY5Y cells, which endogenously expressed the human T2R16 receptor, were co-cultured. Using Caco-2 cells and SH-SY5Y cells at a constant total concentration of 40 K/mL, we designed seven mixtures with [Caco-2]/([Caco-2] + [SH-SY5Y]) ratios of 0, 20, 40, 50, 60, 80, and 100%. These mixtures were then seeded on the 16 E-plates of the electric cell-substrate impedance sensor (ECIS) for bitterness detection. Theoretically, after T2R38 ligands activation, continuous evolution profiles (CEP), with [Caco-2]/([Caco-2] + [SH-SY5Y]) ratios as the x-axis and ΔCI (Max) as the y-axis, would exhibit positive correlation property. After T2R16 ligands activation, the CEP would show negative correlation property. However, when stimulated with compounds that could activate both T2R16 and T2R38, it would show different response patterns.

MO618: Influence of Butyrate and Acetate on Behavior and Metabolism of Human Glomerular Microvascular Endothelial Cells

Abstract BACKGROUND AND AIMS The gut microbiota has emerged as an important modulator of cardiovascular and renal diseases, including diabetic nephropathy (DN), one of the most common complications caused by diabetes mellitus. Importantly, diabetic patients display a reduction in bacteria producing short chain fatty acids (SCFAs) when compared with healthy individuals. SCFAs are generally associated with beneficial effects on the vascular system, as they help to reduce both oxidative stress and pro-inflammatory stimuli [1]. Although SCFAs help to protect against DN, their mechanisms of action at cell level remain largely unknown. For this reason, we aimed to study the effect of two SCFAs, butyrate and acetate, on human glomerular microvascular endothelial cells (hgMVECs) that represent one of the main components of the glomerular filtration barrier. In case of DN, glomerular endothelium shows multiple signs of dysfunction, such as uncontrolled proliferation [2], mitochondrial fragmentation [3] and monolayer disruption [4]. METHOD hgMVECs have been exposed to different concentrations of butyrate and acetate for 24 h and/or 48 h. After treatment with SCFAs, cells have been collected for qPCR, stained for mitochondrial mass or fixated for immunocytochemistry. For the latter, we have used antibodies for the proliferation marker ki-67 and cell-to-cell junction proteins. To measure the resistance of the endothelial monolayer, electric cell-substrate impedance sensing (ECIS) was used. This system lets a current go over and between cells and the tighter the monolayer, the higher the resistance. For this type of experiment, hgMVECs have been cultured on a specific plate that is located in the ECIS machine throughout the whole experiments (before and after treatment with SCFAs). RESULTS Butyrate decreases the number of ki-67 positive nuclei in a concentration-dependent manner, which implies lower cellular proliferation. On the other hand, at the highest concentration tested (500 uM), butyrate increases mitochondrial mass and elongation, as opposite to acetate, which does not show any effect on mitochondrial mass. These results are also supported by qPCR analysis, where it is possible to observe a decrease of genes involved in mitochondrial fragmentation upon butyrate exposure. MOreover, butyrate increases the resistance of the endothelial monolayer, as we can see in the results of the ECIS experiments. After an initial decrease, butyrate induces a steady increase of the resistance of the monolayer, which does not happen with acetate. We believe that the increase in resistance is due to a higher expression of genes and proteins involved in the formation of tight junctions. For instance, in cells treated with butyrate, gene expression of ve-cadherin, ZO-1, claudin 5 and occludin is upregulated and especially for ve-cadherin this is also observed by immunocytochemistry. CONCLUSION Our results show that in hgMVECs, on the one hand butyrate especially can increase monolayer resistance and influence mitochondrial morphology and possibly its metabolism, while on the other hand it decreases cellular proliferation. Therefore, at cellular level, butyrate can help against several hallmarks of endothelial dysfunction Of DN.

ECIS Based Electric Fence Method for Measurement of Human Keratinocyte Migration on Different Substrates

Electric Cell-substrate Impedance Sensing (ECIS) is an impedance-based, real-time, and label-free measuring system for monitoring cellular activities in tissue culture. Previously, ECIS wound healing assay has been used to wound cells with high electric current and monitor the subsequent cell migration. In this study, we applied ECIS electric fence (EF) method, an alternative to electrical wounding, to assess the effects of different surface coatings on human keratinocyte (HaCaT) migration. The EF prevents inoculated cells from attaching or migrating to the fenced electrode surface while maintaining the integrity of the surface coating. After the EF is turned off, cells migrate into the cell-free area, and the increase in measured impedance is monitored. We cultured HaCaT cells on gold electrodes without coating or coated with poly-L-lysin (PLL), poly-D-lysine (PDL), or type-I collagen. We quantified migration rates according to the different slopes in the impedance time series. It was observed that either poly-L-lysine (PLL) or poly-D-lysine (PDL) limits cell adhesion and migration rates. Furthermore, the surface charge of the coated substrate in the culture condition positively correlates with the cell adhesion and migration process. Our results indicate that the EF method is useful for determining cell migration rates on specific surface coatings.

Epigallocatechin Gallate (EGCG) Mitigates The Effect of Camptothecin‐Induced Oxidative Stress on Bone Cancer‐like cells (UMR 106‐01 BSP) by Inhibiting Apoptosis

Many medical treatments have been proposed and utilized to mitigate the effects of oxidants on mammalian cells. Some studies have demonstrated the antioxidative effects of EGCG (an active ingredient in green tea) against cellular oxidants such as camptothecin (CAMPT). However, not many studies have shown how EGCG mitigates the effects of CAMPT on UMR cells. We hypothesized that EGCG mitigates the effects of CAMPT on cell proliferation and biophysical parameters in UMR cells by inhibiting CAMPT‐induced apoptosis. Briefly, UMR cells were routinely passaged, counted, plated at 4.8 x 105 cells/mL in six‐well culture plates, then treated with varying concentrations of CAMPT. The cultures were incubated for 72 hours at 37°C + 5% CO₂ infusion. Cells were subsequently counted to establish the proliferation and viability of the induced cells as compared to controls. In another experiment, designated UMR cells were co‐treated with CAMPT and different concentrations of EGCG then processed, as mentioned above, to evaluate whether or not the EGCG treatment was able to mitigate the oxidative effect of CAMPT through proliferation / viability studies. Apoptosis markers were assessed for each experiment by using a Caspase‐3/CPP32 Colorimetric Assay Kit. Lastly, designated cells were treated with CAMPT alone, or CAMPT +EGCG then assessed using electric cell‐substrate impedance sensing (ECIS) technology to explore the effects of the chemicals used on key biophysical parameters of UMR cells (i.e. cell membrane resistance, rate of cell spreading and cell attachment). Briefly, CAMPT‐treated and CAMPT+EGCG‐treated UMR cells were plated at 2.5 x 105 cells/mL in eight‐well ECIS array plates. The plates were placed into the ECIS array holder and incubated at 37°C with 5% CO₂. The biophysical parameters (cell membrane resistance/impedance, cell spreading, cell attachment) were monitored and measured in real‐time for 48 hours, using the ECIS instrument. F test ANOVA was used to compare variances between all trials and all values in the results were expressed as means ±SD. The results of the proliferation study showed a decrease in CAMPT‐treated UMR cell proliferation in a concentration‐dependent manner (Figure 1). Cells co‐treated with CAMPT + EGCG revealed that EGCG was able to reverse the oxidative effects of CAMPT on the UMR cells in a concentration‐dependent manner *Figure 2). The Caspase‐3/CPP32 Colorimetric Assay showed an increase in apoptotic marker activity from the CAMPT‐induced samples in contrast with the uninduced controls. The ECIS technology documented decreases in biophysical parameters in the UMR cells when CAMPT alone was used. Co‐treatment of the cells with CAMPT+EGCG showed a reverse effect on the biophysical parameters. F‐Test ANOVA yielded statistically significant values with p values <0.05. The results of this research suggest that EGCG is effective in protecting UMR cells from oxidative stress by inhibiting apoptosis. Moreover, results from the ECIS instrument validates this technology as a corroborator of the biological effects of CAMPT and EGCG on UMR cells. This study suggests that EGCG has a protective role in mitigating oxidative stress in mammalian cells.

Lipid phosphate phosphatase‐3 dysfunction exaggerates neointimal hyperplasia following diabetic vascular injury

IntroductionDiabetes mellitus is a major risk factor for cardiovascular diseases and promotes neointimal hyperplasia characterized by occlusive disorders such as atherosclerosis and stenosis, caused by excessive proliferation and migration of vascular smooth muscle cells (VSMCs) following endovascular interventions such as balloon angioplasty and stent placement. Lipid phosphate phosphatase‐3 (LPP3) is an enzyme that regulates the availability of lysophosphatidic acid (LPA) and has been reported to play a significant role in vascular genesis, cellular permeability, tumor progression, vascular remodeling, and myocardial infarction. However, the functional role and mechanism of LPP3 in diabetic neointimal hyperplasia is unclear. LPP3 dysfunction may lead to LPA accumulation at the vascular injury site, leading to vascular inflammation, proliferation, and migration, causing restenosis. Recently, the Receptor for Advanced Glycation End product (RAGE) has been presented as a potential receptor for LPA leading to downstream signaling in vascular smooth muscle cells (VSMCs).MethodsIn this study, we used a streptozocin‐induced diabetes mice model. We made SM‐22 specific LPP3 knock‐out mice and performed endothelial denudation surgery in the carotid artery to induce neointima hyperplasia to tease out the role of LPP3 in restenosis. We isolated aortic VSMCs from LPP3 floxed and LPP3 knock‐out mice and cultured them under normal or high glucose media. The immunoblotting was performed and probed with antibodies for total extracellular signal‐regulated kinase (ERK) and phosphorylated‐ERK. The cell proliferation and migration assay was performed using the Electric Cell substrate Impedance Sensing (ECIS) with or without LPP3 in VSMC under normal or high glucose conditions. Data are mean ± SEM; P‐values were determined by Student t‐test or one‐way ANOVA with Tukey's post‐hoc tests.ResultWe observed a significant increase in proliferation (P<0.01) and exaggerated migration (p<0.01) of VSMC that lack LPP3 under the hyperglycemic condition in real‐time using ECIS. These effects are at least partially a consequence of decreased lysophosphatidic acid hydrolysis due to lack of LPP3 activity. Furthermore, VSMC that lack LPP3 showed increased phospho‐ERK expression (P<0.01) under hyperglycemic conditions. The diabetic LPP3 knock‐out mice exhibited exaggerated neo‐intimal areas after the carotid injury compared to LPP3 floxed mice.ConclusionThese findings implicate that LPP3 plays a vital role as a negative regulator of smooth muscle phenotypic modulation. We speculate that changes in LPP3 expression control the development of neo‐intima by limiting smooth muscle cell proliferation, migration, and vascular inflammation in diabetic restenosis.

In Vitro Screening Identifies TRPV4 and PAR1 as Targets for Endothelial Barrier Stabilization in COVID‐19

Study objectiveEndothelial dysfunction and increased microvascular permeability are hallmarks of severe COVID‐19. At present, the underlying mechanisms of endothelial barrier failure in COVID‑19 remain elusive. Here, we show that increased thrombin activity in plasma from severe COVID‐19 patients activates endothelial protease‐activated receptor (PAR1), which mediates barrier failure by triggering TRPV4‐mediated Ca2+ influx in lung microvascular endothelial cells.MethodsCitrate plasma was sampled as part of the Pa‐COVID‐19 cohort study (ethics approval EA2/066/20) from patients with severe COVID‐19 (high flow O2 or mechanically ventilated; WHO severity score: 5‐7) COVID‑19. Plasma samples were diluted to 10% (v/v) in cell culture medium without FCS and tested for their ability to disrupt barrier integrity of primary human pulmonary microvascular endothelial cells (HPMEC) monolayers by electrical cell‐substrate impedance sensing (ECIS), immunofluorescence for endothelial VE‐cadherin and F‐actin, western blot analyses of PAR‐1 cleavage, and real‐time Ca2+ imaging. Plasma from healthy donors served as control.ResultsCOVID‐19 plasma had elevated thrombin activity while levels of antithrombin III, a key anti‐coagulant with thromboprotective function were decreased. COVID‐19 plasma caused endothelial barrier dysfunction as measured by ECIS and gap formation in HPMEC monolayers. Endothelial barrier disruption and endothelial Ca2+ influx in response to COVID‐19 plasma could be blocked by selective antagonists targeting thrombin (Argatroban), its receptor PAR1 (SCH79797), or TRPV4 (HC‐067047).ConclusionHere, we identify a novel signaling axis involving thrombin, its receptor PAR1, and TRPV4 as mechanism for increased microvascular permeability in COVID‑19. Targeting this signaling axis in endothelial barrier failure may provide a promising adjunctive therapy in COVID‐19.

IL‐33/ST2L Signaling Regulate Blood‐Retinal Barrier Integrity via Affecting Cadherin‐Catenin Adheren Junction Complex

PurposeAngiogenesis, neovascularization and vascular remodeling are highly dynamic processes, where endothelial cell‐cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity and barrier function. The cadherin–catenin adhesion complex is a key contributor to blood‐retinal barrier (BRB) integrity and dynamic cell movements. However, the pre‐eminent role of cadherins and their associated catenins in adheren junction structure and function is not fully understood. Also, the role of IL33/ST2L signaling in regulating BRB integrity is not known.MethodsUsing a murine model of oxygen‐induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we are trying to understand the significance of IL‐33/ST2L‐signaling on endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability.ResultsOur in vivo genetic deletion studies revealed that deletion of IL‐33 resulted in reduced vascular tufts and vascular leakage. Therefore, in order to understand the molecular mechanism by which IL‐33 regulates the BRB integrity, we performed some in vitro studies using HRMVECs. The electric cell‐substrate impedance sensing (ECIS) analysis showed that IL‐33 (20 ng/ml) induce endothelial barrier disruption in HRMVECs. Likewise, FITC‐dextran permeability assay also showed that IL‐33 stimulate the endothelial dysfunction in HRMVECs. As we know, tight junctions (TJs) and adheren junctions (AJs) form the adhesion structure and maintain the BRB integrity, therefore we look for the junctional proteins involved in the IL‐33/ST2L mediated endothelial dysfunction. Furthermore, we found that IL‐33 mediates the phosphorylation of VE‐cadherin and a‐catenin in HRMVECs. Furthermore, mass‐spectroscopy (MS) analysis revealed the phosphorylation of specific Ser439 residue of a‐catenin upon IL‐33 inducement in HRMVECs. Consequently, mutation of a‐catenin at Ser439 residue restores the BRB integrity disrupted by IL‐33. In addition, we have observed that IL‐33 induce the generation of reactive oxygen species (ROS), thereby destabilize adheren junctions in HRMVECs.ConclusionsWe conclude that IL‐33/ST2L signaling plays a significant role in angiogenesis and neovascularization by regulating endothelial permeability and BBB integrity.

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.

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.

Electrical cell impedance spectral mesoscopic model applied to experimental data of variable size microelectrodes.

We apply the electric cell-substrate impedance sensing (ECIS) technique to monolayers of Madin-Darby canine kidney type II cells cultured on microelectrodes of different sizes. We analyze the effect of the microelectrode radius on the parameters provided by existing ECIS models. The cellular properties inferred from the models should be invariant to the change in the microelectrode radius used for the measurements, since these properties are inherent to the type of cells studied. The current standard model, the Giaever-Keese (GK) model, derived from electrical balances of a single cell extended to infinity by suitable boundary conditions, assumes an infinite microelectrode. The model is fitted to experimental data acquired with a large-radius microelectrode, which can be considered infinite for practical purposes. We compute the impedance of the other cell-covered microelectrodes from the parameters obtained with the GK model, resulting in values strongly discrepant with the experimental data for small microelectrodes. We repeat the process with the mean field (MF) model, an alternative model that depends on the microelectrode radius but not on the cell radius. In this paper we introduce the mesoscopic model, an analytical model that simultaneously includes the properties of an individual cell and the sizes of the microelectrode and the insulator (region between the microelectrode and the ground). The impedances calculated with the mesoscopic model are in excellent agreement with experimental data. Finally, the mesoscopic model reduces to the MF model when the insulator goes to infinity and to the GK model when it goes to zero.

Differential Effects of Cytopathic Hypoxia on Human Retinal Endothelial Cellular Behavior: Implication for Ischemic Retinopathies.

Loss of barrier integrity of retinal endothelial cells (RECs) is an early feature of ischemic retinopathies (IRs), but the triggering mechanisms remain incompletely understood. Previous studies have reported mitochondrial dysfunction in several forms of IRs, which creates a cytopathic hypoxic environment where cells cannot use oxygen for energy production. Nonetheless, the contribution of cytopathic hypoxia to the REC barrier failure has not been fully explored. In this study, we dissect in-depth the role of cytopathic hypoxia in impairing the barrier function of REC. We employed the electric cell-substrate impedance sensing (ECIS) technology to monitor in real-time the impedance (Z) and hence the barrier functionality of human RECs (HRECs) under cytopathic hypoxia-inducing agent, Cobalt(II) chloride (CoCl2). Furthermore, data were deconvoluted to test the effect of cytopathic hypoxia on the three key components of barrier integrity; Rb (paracellular resistance between HRECs), α (basolateral adhesion between HRECs and the extracellular matrix), and Cm (HREC membrane capacitance). Our results showed that CoCl2 decreased the Z of HRECs dose-dependently. Specifically, the Rb parameter of the HREC barrier was the parameter that declined first and most significantly by the cytopathic hypoxia-inducing agent and in a dose-dependent manner. When Rb began to fall to its minimum, other parameters of the HREC barrier, including α and Cm, were unaffected. Interestingly, the compromised effect of cytopathic hypoxia on Rb was associated with mitochondrial dysfunction but not with cytotoxicity. In conclusion, our results demonstrate distinguishable dielectric properties of HRECs under cytopathic hypoxia in which the paracellular junction between adjacent HRECs is the most vulnerable target. Such selective behavior could be utilized to screen agents or genes that maintain and strengthen the assembly of HRECs tight junction complex.

The ATX-LPA Axis Regulates Vascular Permeability during Cerebral Ischemic-Reperfusion.

Endothelial permeability is a major complication that must be addressed during stroke treatment. Study of the mechanisms underlying blood–brain barrier (BBB) disruption and management of the hypoxic stress-induced permeability of the endothelium following reperfusion are both urgently needed for stroke management. Lysophosphatidic acid (LPA), a bioactive lipid essential for basic cellular functions, causes unfavorable outcomes during stroke progression. LPA-producing enzyme autotaxin (ATX) is regulated in ischemic stroke. We used an electrical cell-substrate impedance sensor (ECIS) to measure endothelial permeability. Mitochondrial bioenergetics were obtained using a Seahorse analyzer. AR-2 probe fluorescence assay was used to measure ATX activity. LPA increased endothelial permeability and reduced junctional protein expression in mouse brain microvascular endothelial cells (MBMEC). LPA receptor inhibitors Ki16425 and AM095 attenuated the LPA-induced changes in the endothelial permeability and junctional proteins. LPA significantly diminished mitochondrial function in MBMEC. ATX was upregulated (p < 0.05) in brain microvascular endothelial cells under hypoxic reperfusion. ATX activity and permeability were attenuated with the use of an ATX inhibitor in a mouse stroke model. The upregulation of ATX with hypoxic reperfusion leads to LPA production in brain endothelial cells favoring permeability. Inhibition of the ATX–LPA–LPAR axis could be therapeutically targeted in stroke to achieve better outcomes.

Blood-Brain Barrier Disruption Mediated by FFA1 Receptor-Evidence Using Miniscope.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood–brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell–Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro.

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Biological Activity of an Epilobium angustifolium L. (Fireweed) Infusion after In Vitro Digestion.

The biological activity of an in vitro digested infusion of Epilobium angustifolium (fireweed) was examined in a model system of intestinal epithelial and colon cancer tissues. The content of selected phenolic compounds in the digested aqueous extract of fireweed was determined using HPLC-ESI-QTOF-MS/MS. Biological activity was examined using the human colon adenocarcinoma cell lines HT-29 and CaCo-2 and the human colon epithelial cell line CCD 841 CoTr. Cytotoxicity was assessed by an MTT assay, a Neutral Red uptake assay, May-Grünwald-Giemsa staining, and a label-free Electric Cell-Substrate Impedance Sensing cytotoxicity assay. The effect of the infusion on the growth of selected intestinal bacteria was also examined. The extract inhibited the growth of intestinal cancer cells HT-29. This effect can be attributed to the activity of quercetin and kaempferol, which were the most abundant phenolic compounds found in the extract after in vitro digestion. The cytotoxicity of the fireweed infusion was dose-dependent. The highest decrease in proliferation (by almost 80%) compared to the control was observed in HT-29 line treated with the extract at a concentration of 250 μg/mL. The fireweed infusion did not affect the growth of beneficial intestinal bacteria, but it did significantly inhibit E. coli. The cytotoxic effect of the fireweed extract indicates that it does not lose its biological activity after in vitro digestion. It can be concluded that the fireweed infusion has the potential to be used as a supporting agent in colon cancer therapy.