Electric Cell-substrate Impedance Sensing Research Articles

Investigating the Potential for Na+‐H+ Exchanger Isoform 1 (NHE1) Inhibitors as Adjuvant Therapies in the Treatment of Ovarian Cancer

Each year in the United States over 200,000 women are diagnosed with ovarian cancer leading to more than 130,000 deaths. A major challenge in ovarian cancer treatment is the development of drug resistance to the therapeutic agents. Increases in the expression and activity Na+‐H+ Exchanger Isoform 1 (NHE1) was recently demonstrated to regulate cell proliferation in ovarian cancer patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. We hypothesize that combining common ovarian cancer therapeutic agents along with compounds that directly or indirectly inhibit NHE1 will enhance the ability of the therapeutic agents to slow ovarian cancer cell proliferation and migration. Three ovarian adenocarcinoma cell lines; CAOV‐3, SKOV‐3, and OVCAR‐3 are used in this research. Using a proliferation assay, we assessed the impact of different ovarian cancer therapeutics (Rucaparib, Olaparib, Paclitaxel and Cisplatin) in these three cell lines in the presence and absence of inhibitors that decrease NHE1 activity directly or indirectly. Our previous work demonstrated that the NHE1 inhibitor cariporide and a number of protein kinase inhibitors decreased cell proliferation rates in all three cell types. Treatment with Cisplatin reduced cell proliferation most significantly in the CAOV‐3 line. Paclitaxel and Cisplatin each had significant and comparable impacts on the SKOV‐3 and OVCAR‐3 cell lines. Cell migration was evaluated using electric cell‐substrate impedance sensing (ECIS). In the SKOV‐3 and CAOV‐3 cells, inhibition with cariporide and the Rsk inhibitor BID1870 each dramatically reduced the rate of cell migration. The research presented here supports the role of NHE1 in cell proliferation, and for the first time implicates NHE1 in the regulation of cell migration in ovarian cancer cells. Data will be presented that evaluated the potential for the inhibition of NHE1 as an adjuvant therapy in ovarian cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tissue Factor Enhances the Alveolar Epithelial Barrier Integrity during Acute Lung Injury

IntroductionWe have previously reported that lung epithelial tissue factor (TF) knockout (TFΔEpi) mice have increased alveolar capillary barrier permeability. Ultrastructurally, these mouse lungs have profound morphological defects in epithelial attachment to the basement membrane. Adhesion/migration are regulated in part by integrins which bind the extracellular matrix proteins to modulate cytoskeletal dynamics. In cancer cells, TF binds β1 integrin and increases cell migration, a finding that suggests a role for non‐coagulative mechanisms of TF in cell adhesion and migration. We hypothesize that TF‐β1 integrin binding promotes alveolar epithelial barrier integrity through cell adhesion and migration during acute lung injury (ALI).MethodTF overexpressing (TF+Epi) and knockout (TFΔEpi) mice, along with TF knockout A549 cells (A549ΔTF) were used to test whether lung epithelial TF is critical for the integrity of the alveolar epithelial barrier. TF+Epi mice were treated with intratracheal PBS or LPS and lung permeability was measured by bronchoalveolar lavage (BAL) fluid protein level. Alveolar type II epithelial cells (ATII) were isolated from TFΔEpi and TF+Epi mice to measure β1 integrin protein levels. A549ΔTF cells were treated with LPS (100ug/mL) or PBS for 24 hours before western blot of β1 integrin expression. Migration was assessed in wild‐type and A549ΔTF cells using Electric Cell‐substrate Impedance Sensing (ECIS) on cell monolayers. Adhesion was assayed by plating wild‐type and A549ΔTF cells on matrigel and quantifying the absorbance of crystal violet stained adherent cells.ResultsTF+Epi mice had decreased lung permeability (BAL protein 303 μg/mL +/− 62 versus 416 +/− 137 in control, p=0.025) following LPS challenge. A549ΔTF cells had significantly decreased cell adhesion (Figure) (absorbance 0.308 +/− 0.119 versus 0.603 +/− 0.246 in wild type, *p<0.001) along with decreased migration (p=0.017) compared to control A549 cells with wild type TF expression levels. A549ΔTF cells had decreased β1 integrin protein levels (relative expression 0.017 +/− 0.01 versus 0.61 +/− 0.02 control, p=0.036) following LPS challenge. ATII cells isolated from TFΔEpi mice also have decreased β1 integrin protein levels.ConclusionsTF contributes to alveolar barrier integrity in part through a mechanism dependent on β1 integrin. These data provide support that lung epithelial TF may play a non‐coagulative role in protection during ALI. This novel role for TF in maintaining alveolar epithelial barrier integrity offers a potential therapeutic target during ALI.Support or Funding InformationVanderbilt University Initiative for Maximizing Student Diversity funding through the NIH, Nashville Veterans Affairs Medical Center, Funding: HL090785, HL103836This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating the Inhibition of Palmitoylation and NHE1 on Cell Proliferation and Migration

The Na+‐ H+ Exchanger Isoform I (NHE1) is a key regulator of cell proliferation and migration in a range of cell types from both healthy and diseased tissues. Activation of NHE1 is an initial step in the development of the transformed phenotypes of cancer cells and therefore a key driver for tumor development and cancer progression. NHE1 also contributes to two of the hallmarks of cancer, the activation of invasion and metastasis and sustained proliferative growth. We have identified for the first time, that NHE1 undergoes reversible palmitoylation, the addition of a palmitic acid residue to the cytoplasmic regulatory domain of NHE1. We hypothesize that this process regulates NHE1 activation and will play a role in the control of cell proliferation and migration. This suggests that targeting the potential palmitoylation sites on NHE1 may provide a therapeutic target for cancer treatment. Our work characterizes the role of the palmitoylation of NHE1 in cell growth and migration. Using the palmitoylation inhibitor, 2‐bromopalmitate (2BP), we evaluated cell growth and migration in the presence and absence of palmitoylation. In proliferation assays, the rate of cell growth increased approximately 5‐fold when the serum present in the growth media was increased from 0.5% to 10% and approximately 2‐fold when 50 μM lysophosphatidic acid was added to the growth media. Under both conditions, the addition of 15 μM 2BP or 10 μM cariporide dramatically reduced or eliminated the increased proliferation. Cell migration was evaluated using Electric Cell‐substrate Impedance Sensing (ECIS). Once again, the rate of cell migration was reduced by 50% or more in the presence of 15 μM 2BP or 10 μM cariporide. These data demonstrated that inhibiting the palmitoylation of NHE1 decreases both cell proliferation and the rate of cell migration.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of the Impact of the Traditional Medicine Formulation Goreisan and Its Components on Mesenteric Lymphatic Vessel Contraction and Lymphatic Endothelial Barrier Function

Kampo medicines are traditional herbal formulas used to treat and prevent various diseases. The Kampo medicine Goreisan is a known diuretic and is currently being tested in the clinic to reduce symptoms of surgically induced lymphedema in OB/GYN patients in Japan. The present study was aimed to clarify how Goreisan and its five different components affect lymphatic pump function. Mesenteric collecting lymphatics were isolated from anesthetized male Sprague Dawley rats, and mounted on resistance‐matched glass micropipettes in a 37 °C physiological salt solution bath for studies. The luminal pressure was set at 2 cm H2O and vessels that established intrinsic phasic contractions were studied. Contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD) were measured. From these, amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF) were calculated. Goreisan was applied at concentrations of 1–30 μg/ml. Its components, Cinnamomi Cortex, Atractilodys Rhizoma, Alismatis Rhizoma, Polyporus, and Poria were also studied at concentrations of 1–30 μg/ml. To measure barrier function, human dermal lymphatic endothelial cells (LEC) were treated with 1–100 μg/ml of each compound and transendothelial electrical resistance (TER) was determined by electric cell‐substrate impedance sensing (ECIS). The results show that treatment with Goreisan causes no significant changes in all parameters compared to baseline. However, 30ug/ml Alismatis Rhizoma caused a significant reduction in CF and FPF. The other components did not affect pumping. Goreisan also does not significant affect TER. Polyporus significantly increased TER compared to baseline, while the other components elicited no change. Collectively, the results suggest that Goreisan does not directly affect lymphatic pumping or barrier function, although at its component Alismatis Rhizoma can influence pump activity and Polyporus can enhance barrier function. By extension, in the lymphedema patients treated with Goreisan, its pharmacological action is more likely to be on renal mechanism to reduce excess body fluids. Additional work is needed to determine whether Goreisan may have direct effect to rescue impaired lymphatic vessel functions under inflammatory conditions.Support or Funding InformationThis work was supported by Rotary International Global Grants Scholarship Grant Number GG1746618.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and characterization of kaempferol-based ruthenium (II) complex: A facile approach for superior anticancer application

In this study, we synthesized a novel metal flavonoid complex and investigated its effects on the non-small cell lung cancer cell lines, A549 and toxicity on the human dermal fibroblast cell lines, HDFa. 1H, 13C NMR, single crystal X-ray diffraction and elemental micro analysis (C,H,N,S/O) were used to characterize the synthesized kaempferol-based Ru (II) complex. Cell toxicity was studied using MTT assay and electric cell substrate impedance sensing (ECIS). It was evident from the MTT results that no significant cytotoxicity in HDFa cells occurs with the synthesized complex, but in case of A549 cells, significant cytotoxicity was observed even at low concentrations (10-20 μm). In addition, the effect of the newly synthesized complex on the A549 cell line was studied by investigating the cellular damage via atomic force microscopy and DNA fragmentation assay. The obtained results revealed that the synthesized complex was able to inhibit the cancer cells and have shown moderate anticancer activity against A549 cancer cell lines. In addition, it was evident that the complex was more active than kaempferol and well tolerated by normal cell lines.

A cell viability assessment method based on area-normalized impedance spectrum (ANIS)

Impedance measurement of cells using electric cell-substrate impedance sensing (ECIS) is widely accepted as an effective method to assess cell status. However, the sensitive frequency drifts over time with the changes of culture condition according to the built circuit model and experimental results. The area-normalized impedance spectrum (ANIS) method, which uses normalized area of impedance spectrum in a certain interval to assess cell viability, was proposed in this paper to solve the problem. The certain interval is calculated due to the threshold Zth, which is determined by 2% decline of the maximum impedance. Stabilities of two methods were analyzed by normalizing the area and impedance, showing that the normalized impedance fluctuated like a wave, while the normalized area was smoother. In addition, Cell Count Kit-8 (CCK-8) assay was carried out proving that the correlation index of ANIS method increases by 2.4% compared with impedance sensing method, and the maximum error of ANIS method decreases by 4%. Comparison analysis of two methods with random measurement noise was also discussed in this paper, and the results showed that the ANIS method was less affected by measurement noise than impedance sensing method. It demonstrated that the ANIS method is a more stable and accurate method to assess cell viability.

Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients

BackgroundMulti-organ failure occurs during critical illness and is mediated in part by destructive neutrophil-to-endothelial interactions. The β2 integrin receptor, CR3 (complement receptor 3; Mac-1; CD11b/CD18), which binds endothelial intercellular adhesion molecule-1 (ICAM-1), plays a key role in promoting the adhesion of activated neutrophils to inflamed endothelia which, when prolonged and excessive, can cause vascular damage. Leukadherin-1 (LA-1) is a small molecule allosteric activator of CR3 and has been shown to promote adhesion of blood neutrophils to inflamed endothelium and restrict tissue infiltration. Therefore, LA-1 offers a novel mechanism of anti-inflammatory action by activation, rather than inhibition, of the neutrophil CR3 integrin. However, whether promotion of neutrophil-to-endothelial interaction by this novel therapeutic is of benefit or detriment to endothelial barrier function is not known.MethodsCritically ill septic and trauma patients were prospectively enrolled from the surgical and the trauma ICU. Blood was collected from these patients and healthy volunteers. Neutrophils were isolated by dextran sedimentation and adhered to TNF-α (tumor necrosis factor-α)-activated human umbilical vein endothelial (HUVEC) monolayers in the presence or absence of fMLP (formylmethionine-leucine-phenylalanine) and/or LA-1. Electric cell-substrate impedance sensing (ECIS) and exposure of underlying collagen were used to quantify endothelial barrier function and permeability.ResultsNeutrophils from critically ill trauma and septic patients caused similar degrees of endothelial barrier disruption which exceeded that caused by cells obtained from healthy controls both kinetically and quantitatively. LA-1 protected barrier function in the absence and presence of fMLP which served as a secondary stimulant to cause maximal loss of barrier function. LA-1 protection was also observed by quantifying collagen exposure underlying endothelial cells challenged with fMLP-stimulated neutrophils. LA-1 treatment resulted in decreased migration dynamics of neutrophils crawling on an endothelial monolayer with reduced speed (μm/s = 0.25 ± 0.01 vs. 0.06 ± 0.01, p < 0.05), path length (μm = 199.5 ± 14.3 vs. 42.1 ± 13.0, p < 0.05), and displacement (μm = 65.2 ± 4.7 vs. 10.4 ± 1.3; p < 0.05).ConclusionNeutrophils from patients with trauma or sepsis cause endothelial barrier disruption to a similar extent relative to each other. The CR3 agonist LA-1 protects endothelial barrier function from damage caused by neutrophils obtained from both populations of critically ill patients even when exposed to secondary stimulation.

Effects of Platelet-Activating Factor on Brain Microvascular Endothelial Cells

Platelet-activating factor (PAF) is a potent phospholipid mediator that exerts various pathophysiological effects by interacting with a G protein-coupled receptor. PAF has been reported to increase the permeability of the blood–brain barrier (BBB) via incompletely characterized mechanisms. We investigated the effect of PAF on rat brain microvascular endothelial cells (RBMVEC), a critical component of the BBB. PAF produced a dose-dependent increase in cytosolic Ca2+ concentration; the effect was prevented by the PAF receptor antagonist, WEB2086. The effect of PAF on cytosolic Ca2+ was abolished in Ca2+-free saline or in the presence of L-type voltage-gated Ca2+ channel inhibitor, nifedipine, indicating that Ca2+ influx is critical for PAF-induced increase in cytosolic Ca2+. PAF produced RBMVEC depolarization; the effect was inhibited by WEB2086. In cells loaded with [(4-amino-5-methylamino-2′,7′-difluoro-fluorescein)diacetate] (DAF-FM), a nitric oxide (NO)-sensitive fluorescent dye, PAF increased the NO level; the effect was prevented by WEB2086, nifedipine or by l-NAME, an inhibitor of NO synthase. Immunocytochemistry studies indicate that PAF reduced the immunostaining of ZO-1, a tight junction-associated protein, increased F-actin fibers, and produced intercellular gaps. PAF produced a decrease in RBMVEC monolayer electrical resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS), indicative of a disruption of endothelial barrier function. In vivo studies indicate that PAF increased the BBB permeability, assessed with sodium fluorescein and Evans Blue methods, via PAF receptor-dependent mechanisms, consequent to Ca2+ influx and increased NO levels. Our studies reveal that PAF alters the BBB permeability by multiple mechanisms, which may be relevant for central nervous system (CNS) inflammatory disorders.

DAQ based Impedance Measurement System for Low Cost and Portable Electrical Cell-Substrate Impedance Sensing

Electric cell-substrate impedance sensing (ECIS), a technique for label-free and real-time detection of cells, is emerging as an alternative or assistive method to traditional biochemical assays for diagnostic and pharmaceutical applications. In this work, we developed a DAQ based impedance measurement system for low cost and portable ECIS applications. The alternating input signal for ECIS was generated from the digital-to-analog converter (DAC) channel of the DAQ board and applied through the current limited resistor to the cells on the electrode. The responding signal was recorded by the analog-to-digital converter (ADC) channel of the DAQ board, and its amplitude and phase were analyzed by software lock-in amplifier. The feasibility of the developed ECIS system was evaluated by impedance measurement and DAPI analysis of C2C12 cell growth on the indium tin oxide microelectrode, and by impedimetric and MTT cell viability assay of HEK293 cells exposed to H2O2.

Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma

ABSTRACTThe non-receptor cytoplasmic tyrosine kinase, Focal Adhesion Kinase (FAK) is known to play a key role in a variety of normal and cancer cellular functions such as survival, proliferation, migration and invasion. It is highly active and overexpressed in various cancers including Pancreatic Ductal Adenocarcinoma (PDAC) and Malignant Pleural Mesothelioma (MPM). Here, initially, we demonstrate that FAK is overexpressed in both PDAC and MPM cell lines. Then we analyze effects of two small molecule inhibitors PF-573228, and PF-431396, which are dual specificity inhibitors of FAK and proline rich tyrosine kinase 2 (PYK2), as well as VS-6063, another small molecule inhibitor that specifically inhibits FAK but not PYK2 for cell growth, motility and invasion of PDAC and MPM cell lines. Treatment with PF-573228, PF-431396 and VS-6063 cells resulted in a dose-dependent inhibition of growth and anchorage-independent colony formation in both cancer cell lines. Furthermore, these compounds suppressed the phosphorylation of FAK at its active site, Y397, and functionally induced significant apoptosis and cell cycle arrest in both cell lines. Using the ECIS (Electric cell-substrate impedance sensing) system, we found that treatment of both PF compounds suppressed adherence and migration of PDAC cells on fibronectin. Interestingly, 3D-tumor organoids derived from autochthonous KC (Kras;PdxCre) mice treated with PF-573228 revealed a significant decrease in tumor organoid size and increase in organoid cell death. Taken together, our results show that FAK is an important target for mesothelioma and pancreatic cancer therapy that merit further translational studies.

Cytoprotective effects of atmospheric-pressure plasmas against hypoxia-induced neuronal injuries

Atmospheric pressure plasma jet (APPJ) has recently been the focus of cytoprotective research due to the physiological roles of ROS and RNS. In the current study, we investigated the effect of APPJ treatment on the hypoxia (1% oxygen) induced cell injuries. SH-SY5Y cells were treated by APPJ for different duration and incubated in normoxic condition (20% oxygen) for 5 h followed by 24 h hypoxia treatment. Cell viability was evaluated by lactate dehydrogenase (LDH) release and further monitored using the electric cell-substrate impedance sensing (ECIS) system after APPJ treatment. Results showed that APPJ could reduce cell injuries after 24 h hypoxia, which was consistent with the ECIS results. Furthermore, extracellular NO and H2O2 production was significantly increased with the APPJ treatment. It was also interesting to find that APPJ treatment reduced SH-SY5Y cells proliferation in the hypoxic microenvironment during the first 20 h of hypoxia. Although more work was still need to clarify whether the cell viability maintenance was related to the cell proliferation during hypoxia, our results provide the first evidence of real-time cell viability changes after APPJ treatment under both normoxic and hypoxic conditions, which could provide evidence for the neuroprotective applications of APPJ.

Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier

ABSTRACTA principal function of endothelial cells is the formation of a barrier between the blood and tissues. This barrier arises from the physical connections at cell-cell junctions, which includes cytoskeletal tight junction and adherens junction proteins. Methods that alter barrier function must therefore affect these cell-cell connections. The blood brain barrier (BBB) represents perhaps the most selective endothelial barrier, which arises from endothelial cell interactions with astrocytes and pericytes. Even in non-central nervous system (CNS) endothelial cells, barrier properties can be enhanced, mimicking the BBB, through induction of intercellular junctions, by either direct co-culture with astrocytes, supplementation with astrocyte conditioned medium (ACM) and/or pharmacologic enhancement of cAMP. To understand how cell-cell junctions change during endothelial barrier enhancement, we examined the effects of ACM and/or cAMP donors added to standard media on human umbilical vein endothelial cells (HUVEC). HUVEC cultured with cAMP-elevating agents had the most enhanced barrier function as measured by Electric Cell-substrate Impedance Sensing (ECIS®), a real-time, label-free, impedance based method of studying cell barrier properties. However, subtle differences in actin and cell-cell junction proteins were seen across all four culture conditions. cAMP-elevating agents also triggered the redistribution of ZO-1 and VE-cadherin to cell-cell junctions, and intensified the actin microfilament network at the cell cortex. Using a VE-cadherin FRET-force sensor, we observed a decrease in VE-cadherin force in HUVEC cultured with ACM with cAMP donors. Our data indicate cAMP elevation induces both junctional strengthening and reduced VE-cadherin forces. Additionally, treatment with an inhibitor of formin, which reduced actin stress fibers, enhanced barrier function. These data suggest that barrier function is modulated both through the trafficking of proteins to cell-cell junctions, and through the modulation and a relaxation of mechanical force through adherens junctions as intercellular junctional complexes become established.

Modeling and development of screen-printed impedance biosensor for cytotoxicity studies of lung carcinoma cells.

Electrical cell-substrate impedance sensing (ECIS) is a powerful technique to monitor real-time cell behavior. In this study, an ECIS biosensor formed using two interdigitated electrode structures (IDEs) was used to monitor cell behavior and its response to toxicants. Three different sensors with varied electrode spacing were first modeled using COMSOL Multiphysics and then fabricated and tested. The silver/silver chloride IDEs were fabricated using a screen-printing technique and incorporated with polydimethylsiloxane (PDMS) cell culture wells. To study the effectiveness of the biosensor, A549 lung carcinoma cells were seeded in the culture wells together with collagen as an extracellular matrix (ECM) to promote cell attachment on electrodes. A549 cells were cultured in the chambers and impedance measurements were taken at 12-h intervals for 120h. Cell index (CI) for both designs were calculated from the impedance measurement and plotted in comparison with the growth profile of the cells in T-flasks. To verify that the ECIS biosensor can also be used to study cell response to toxicants, the A549 cells were also treated with anti-cancer drug, paclitaxel, and its responses were monitored over 5days. Both simulation and experimental results show better sensitivity for smaller spacing between electrodes. Graphical abstract The fabricated impedance biosensor used screen-printed silver/silver chloride IDEs. Simulation and experimental results show better sensitivity for smaller between electrodes.

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin

BackgroundIncreased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity. MethodsHuman umbilical vein endothelial cells (HUVEC) were grown to confluence and treated with CFH with or without ascorbate. Monolayer permeability was measured by Electric Cell-substrate Impedance Sensing (ECIS) or transfer of 14C-inulin. Viability was measured by trypan blue exclusion. Intracellular ascorbate was measured by HPLC. ResultsCFH increased permeability in a dose- and time-dependent manner with 1 mg/ml of CFH increasing inulin transfer by 50% without affecting cell viability. CFH (1 mg/ml) also caused a dramatic reduction in intracellular ascorbate in the same time frame (1.4 mM without CFH, 0.23 mM 18 h after 1 mg/ml CFH, p < 0.05). Pre-treatment of HUVECs with ascorbate attenuated CFH induced permeability. ConclusionsCFH increases endothelial permeability in part through depletion of intracellular ascorbate. Supplementation of ascorbate can attenuate increases in permeability mediated by CFH suggesting a possible therapeutic approach in sepsis.

CRISPR-mediated deletion of the PECAM-1 cytoplasmic domain increases receptor lateral mobility and strengthens endothelial cell junctional integrity

AimsPECAM-1 is an abundant endothelial cell surface receptor that becomes highly enriched at endothelial cell-cell junctions, where it functions to mediate leukocyte transendothelial migration, sense changes in shear and flow, and maintain the vascular permeability barrier. Homophilic interactions mediated by the PECAM-1 extracellular domain are known to be required for PECAM-1 to perform these functions; however, much less is understood about the role of its cytoplasmic domain in these processes. Main methodsCRISPR/Cas9 gene editing technology was employed to generate human endothelial cell lines that either lack PECAM-1 entirely, or express mutated PECAM-1 missing the majority of its cytoplasmic domain (∆CD-PECAM-1). The endothelial barrier function was evaluated by Electric Cell-substrate Impedance Sensing, and molecular mobility was assessed by fluorescence recovery after photobleaching. Key findingsWe found that ∆CD-PECAM-1 concentrates normally at endothelial cell junctions, but has the unexpected property of conferring increased baseline barrier resistance, as well as a more rapid rate of recovery of vascular integrity following thrombin-induced disruption of the endothelial barrier. Fluorescence recovery after photobleaching analysis revealed that ∆CD-PECAM-1 exhibits increased mobility within the plane of the plasma membrane, thus allowing it to redistribute more rapidly back to endothelial cell-cell borders to reform the vascular permeability barrier. SignificanceThe PECAM-1 cytoplasmic domain plays a novel role in regulating the rate and extent of vascular permeability following thrombotic or inflammatory challenge.

Exosomes and Metabolic Function in Mice Exposed to Alternating Dark-Light Cycles Mimicking Night Shift Work Schedules.

Sleep is an important modulator of metabolic function. Disruptions of sleep in circadian rhythm are common in modern societies and are associated with increased risk of developing cardiometabolic disorders. Exosomes are ubiquitous extracellular vesicles that may play a mechanistic role in metabolic derangements. We hypothesized that alternating dark-light cycles mimicking shift work in mice would alter fecal microbiota and colonic epithelium permeability and alter plasma exosome cargo and metabolic function. C57BL/6 mice were randomly assigned to (i) control day light (CL), or (ii) inverted dark-light every 2 weeks for 8 weeks (IN). Body weight, fat mass and HOMA-IR were measured, along with Tregs, metabolic, and resident macrophages in visceral white adipose tissue (vWAT). Fecal water samples were incubated with confluent colonic epithelium cell cultures in electric cell-substrate impedance sensing (ECIS) arrays, and plasma exosomes were added to differentiated adipocytes and insulin-induced pAKT/AKT expression changes were assessed by western blots. Mice exposed to IN showed elevated HOMA-IR, and their fecal samples showed altered microbiota which promote increased permeability of the colonic epithelial cell barrier. Plasma exosomes decreased pAKT/AKT responses to exogenous insulin compared to CL, and altered expression of circadian clock genes. Inflammatory macrophages (Ly-6chigh) were increased in IN-exposed vWAT, while Tregs were decreased. Thus, gut microbiota and the cargo of plasma exosomes are altered by periodic shifts in environmental lighting, and effectively alter metabolic function, possibly via induction of systemic inflammation and altered clock expression in target tissues. Further exploration of exosomal miRNA signatures in shift workers and their putative metabolic organ cell targets appears warranted.

Fibroblast activation protein-α promotes the growth and migration of lung cancer cells via the PI3K and sonic hedgehog pathways.

A characteristic of the epithelial-to-mesenchymal transition in cancer cells is the upregulation of mesenchymal markers. Fibroblast activation protein α (FAPα) is predominantly expressed by stromal fibroblasts. Previous studies have demonstrated that FAPα is also expressed by certain epithelium-derived cancer cells and is involved in the regulation of certain signaling pathways. One of our previous studies showed that FAPα promoted the proliferation of breast cancer cells via the phosphatidylinositol-3-kinase (PI3K) signaling pathway. In the present study, the A549 adenocarcinoma (AC) and SK-MES-1 squamous cell carcinoma (SCC) lung cancer cell lines were transfected with FAPα. The FAPα-expressing SK-MES-1 cells exhibited an increased growth rate, whereas the FAPα-expressing A549 cells exhibited a similar growth rate, compared with respective empty vector-transfected control cells. Electric cell-substrate impedance sensing (ECIS)-based attachment and wound-healing assays showed that the overexpression of FAPα markedly increased the adhesive and migratory properties of the SK-MES-1 cells but not those of the A549 cells. Additionally, inhibitors of focal adhesion kinase, agonist-induced phospholipase C, neural Wiskott-Aldrich syndrome protein, extracellular signal-regulated kinase, Rho-associated protein kinase, PI3K, and sonic hedgehog (SHH) were used to evaluate the interaction between FAPα and signaling pathways. Only the inhibitors of SHH and PI3K inhibited the increased motility of the FAPα-expressing SK-MES-1 cells. Western blot analysis confirmed the activation of PI3K/AKT and SHH/GLI family zinc finger 1 signaling in the FAPα-expressing SK-MES-1 cells. These results revealed that FAPα promoted the growth, adhesion and migration of lung SCC cells. In addition, FAPα regulated lung cancer cell function, potentially via the PI3K and SHH pathways. Further investigations are required to examine the role of FAPα in lung AC cells.

Biosensor Technology Reveals the Disruption of the Endothelial Barrier Function and the Subsequent Death of Blood Brain Barrier Endothelial Cells to Sodium Azide and Its Gaseous Products.

Herein we demonstrate the sensitive nature of human blood-brain barrier (BBB) endothelial cells to sodium azide and its gaseous product. Sodium azide is known to be acutely cytotoxic at low millimolar concentrations, hence its use as a biological preservative (e.g., in antibodies). Loss of barrier integrity was noticed in experiments using Electric Cell-substrate Impedance Sensing (ECIS) biosensor technology, to measure endothelial barrier integrity continuously in real-time. Initially the effect of sodium azide was observed as an artefact where it was present in antibodies being employed in neutralisation experiments. This was confirmed where antibody clones that were azide-free did not mediate loss of barrier function. A delayed loss of barrier function in neighbouring wells implied the influence of a liberated gaseous product. ECIS technology demonstrated that the BBB endothelial cells had a lower level of direct sensitivity to sodium azide of ~3 µM. Evidence of gaseous toxicity was consistently observed at 30 µM and above, with disrupted barrier function and cell death in neighbouring wells. We highlight the ability of this cellular biosensor technology to reveal both the direct and gaseous toxicity mediated by sodium azide. The sensitivity and temporal dimension of ECIS technology was instrumental in these observations. These findings have substantial implications for the wide use of sodium azide in biological reagents, raising issues of their application in live-cell assays and with regard to the protection of the user. This research also has wider relevance highlighting the sensitivity of brain endothelial cells to a known mitochondrial disruptor. It is logical to hypothesise that BBB endothelial dysfunction due to mitochondrial dys-regulation could have an important but underappreciated role in a range of neurological diseases.

A Screening Approach for Identifying Gliadin Neutralizing Antibodies on Epithelial Intestinal Caco-2 Cells

Celiac disease (CD) is a chronic inflammatory condition caused by the ingestion of gliadin-containing food in genetically susceptible individuals. Undigested peptides of gliadin exert various effects, including increased intestinal permeability and inflammation in the small intestine. Although many therapeutic approaches are in development, a gluten-free diet is the only effective treatment for CD. Affecting at least 1% of the population in industrialized countries, it is important to generate therapeutic options against CD. Here, we describe the establishment of a high-throughput screening (HTS) platform based on AlphaLISA and electrical cell–substrate impedance sensing (ECIS) technology for the identification of anti-inflammatory and barrier-protective compounds in human enterocytes after pepsin-trypsin-digested gliadin (PT-gliadin) treatment. Our results show that the combination of these HTS technologies enables fast, reliable, simple, and label-free screening of IgY antibodies against PT-gliadin. Using this platform, we have identified a new chicken anti-PT-gliadin IgY antibody as a potential anti-CD agent.

Love mode surface acoustic wave and impedance sensors for water toxicity sensing

Clean water supply systems and infrastructure require periodic testing of water samples to maintain water quality. To enable this, toxicity tests need to be rapid, accurate, portable, and low cost to provide affordable water security especially in developing countries. Qualitative and quantitative measurement of contaminants in water is classically done by standard analytic chemical methods, which are time‐consuming, labor‐intensive, expensive, and cannot be performed easily outside the laboratory. An alternative to these methods is cell‐based sensors, which can detect the presence of a wide range of toxicants by monitoring the cell states.Although electric cell‐substrate impedance sensing (ECIS) has been demonstrated to be an effective method for water toxicity sensing, the use of ECIS only is not sensitive enough to detect many toxicants at low concentrations. Merging a sensor platform with multiparametric measurement capabilities could increase the security of detection. Love mode surface acoustic wave (SAW) device is considered as one of the powerful candidates for cell‐based toxicity biosensors. In this study, a hybrid Love mode SAW/ECIS sensor was developed. Theoretical analysis and experiments were performed to demonstrate the capability of two sensing techniques (i.e., SAW and ECIS) for toxicity sensing. © 2017 American Institute of Chemical Engineers Environ Prog, 37: 172–179, 2018