Electric Cell-substrate Impedance Sensing Research Articles

Electrical cell‐substrate impedance sensing (ECIS) in lung biology and disease

Electrical cell‐substrate impedance sensing (ECIS) in lung biology and disease

Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit

Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and β1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (β1/β1B) mimetic peptide, βadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore βadp1's mechanism and develop novel SCN1B mimetic peptides affecting β1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in β1-expressing cells, we observed βadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of βadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and β1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. βadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, βadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (β1/β1B) mimetic peptides are reported with the potential to modulate intercellular VGSC β1-mediated adhesion, potentially through β1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.

Cytoprotective 3K3A-activated protein C and plasma: A comparison of therapeutics for the endotheliopathy of trauma.

Both healthy plasma and cytoprotective aPC (3K3A-aPC) have been shown to mitigate the endotheliopathy of trauma (EoT), but optimal therapeutics remain unknown. Our aim was therefore to determine optimal therapies to mitigate EoT by investigating the effectiveness of 3K3A-aPC with and without plasma-based resuscitation strategies. Electric cell-substrate impedance sensing (ECIS) was used to measure real-time permeability changes in endothelial cells. Cells were treated with a 2 μg/mL solution of aPC 30 minutes prior to stimulation with plasma taken from severely injured trauma patients (ISS > 15 and BD < -6) (TP). Healthy plasma, or plasma frozen within 24 hours (FP24), was added concomitantly with TP. Cells treated with thrombin and untreated cells were included in this study as control groups. A dose-dependent difference was found between the 5% and 10% plasma-treated groups when HUVECs were simultaneously stimulated with TP (μd 7.346 95%CI 4.574 to 10.12). There was no difference when compared to TP alone in the 5% (μd 5.713 95%CI -1.751 to 13.18) or 10% group (μd -1.633 95%CI -9.097 to 5.832). When 3K3A-aPC was added to plasma and TP, the 5% group showed improvement in permeability compared to TP alone (μd 10.11 95%CI 2.642 to 17.57), but there was no difference in the 10% group (μd -1.394 95%CI -8.859 to 6.070). The combination of 3K3A-aPC, plasma, and TP at both the 5% plasma (μd -28.52 95%CI-34.72 to -22.32) and 10% plasma concentrations (μd -40.02 95%CI -46.22 to -33.82) had higher inter-cellular permeability than the 3K3A-aPC pre-incubation group. Our data shows that FP24, in a post-trauma environment, pre-treatment with 3K3A-aPC can potentially mitigate the EoT to a greater degree than FP24 with or without 3K3A-aPC. Although further exploration is needed, this represents a potentially ideal and perhaps superior therapeutic treatment for the dysregulated thromboinflammation of injured patients. Prognostic/Epidemiological, Therapeutic/Care Management, Level III.

Platelet releasates mitigate the endotheliopathy of trauma.

Platelets are well known for their roles in hemostasis, but they also play a key role in thromboinflammatory pathways by regulating endothelial health, stimulating angiogenesis, and mediating host defense through both contact dependent and independent signaling. When activated, platelets degranulate releasing multiple active substances. We hypothesized that the soluble environment formed by trauma platelet releasates attenuates thromboinflammation via mitigation of trauma induced endothelial permeability and metabolomic reprogramming. Blood was collected from injured and healthy patients to generate platelet releasates and plasma in parallel. Permeability of endothelial cells when exposed to trauma platelet releasates (TPR) and plasma (TP) was assessed via resistance measurement by Electric Cell-substrate Impedance Sensing (ECIS). Endothelial cells treated with TPR and TP were subjected to mass spectrometry-based metabolomics. TP increased endothelial permeability, whereas TPR decreased endothelial permeability when compared to untreated cells. When TP and TPR were mixed ex vivo, TPR mitigated TP-induced permeability, with significant increase in AUC compared to TP alone. Metabolomics of TPR and TP demonstrated disrupted redox reactions and anti-inflammatory mechanisms. TPRs provide endothelial barrier protection against TP-induced endothelial permeability. Our findings highlight a potential beneficial action of activated platelets on the endothelium in injured patients through disrupted redox reactions and increased antioxidants. Our findings support that soluble signaling from platelet degranulation may mitigate the endotheliopathy of trauma. The clinical implications of this are that activated platelets may prove a promising therapeutic target in the complex integration of thrombosis, endotheliopathy, and inflammation in trauma. Prognostic/Epidemiological, Level III.

Metal-organic frameworks as an active substrate for cell-interaction studies and cell-on-a-chip platforms

Metal-organic frameworks (MOFs) are an emerging class of nanomaterials with immense biomedical potential for their unique interactions with biological and organic materials. In this work, we select two candidate two-dimensional (2D) MOF systems based on Fe3+ and Ni2+ metal centers and 2-aminoterephthalate acid ligand (Fe-MOF and Ni-MOF) and evaluate their performance as an active interface for study of cell-interactions. 2D Fe-MOF and Ni-MOF were synthesized onto hydroxyl-modified gold and glass substrates using a layer-by-layer liquid-phase-epitaxy (LbL-LPE) growth at room temperature and used as active substrates (Fe-MOF/glass, Fe-MOF/Au, Ni-MOF/glass and Ni-MOF/Au, respectively) for MTT cell-proliferation and reactive oxygen species tests using the PC-12 cell-line in order to investigate the biocompatibility. Immunostaining and morphological analyses of PC-12 cells on MOF interfaces suggested a stronger cell-substrate interaction in comparison to glass and were further characterized using the Electrical Cell-substrate Impedance Sensing (ECIS) technique, here for the first time, employed to study cell attachment, spreading and proliferation on 2D Fe-MOF. The 2D Fe-MOF showed superior long-term stability in cell culture medium by recording impedance over 24 h, crucial to monitor cell-dynamics at a solid-liquid interface. A significant increase of interfacial impedance was observed in ECIS, due to PC-12 cells adhering onto 2D Fe-MOF, which was also confirmed by the focused ion beam etching followed by scanning electron microscopy. Our novel findings, therefore, suggest 2D MOFs as highly suitable platform for the study of cell-related interactions using electrical techniques and potentially pave the way for future use of MOFs for bioelectronics and biosensor applications.

Development of a label‐free, impedance‐based biosensor to identify harmful effects of pesticides on insect cells

AbstractInsects are a major part of the planet's ecosystem and their vital role as pollinators for agriculture is undisputed. Alongside factors as climate change or loss of habitats, rising use of pesticides emerges as a key threat to insect populations. For fighting this man‐made problem, development of an easy, fast, sensitive, and non‐invasive biosensor for determining pesticide toxicity may help to ban harmful substances and formulations. Here, a biosensor based on Sf21 (Spodoptera frugiperda) insect cells as sensors and electric cell‐substrate impedance sensing (ECIS) as physical transducer is described. Sf21 cell suspensions and well‐defined pesticide solutions were mixed immediately before seeding on planar gold‐film electrodes. The capacitance at 20 kHz was recorded as a function of time as a measurand for cell adhesion providing dose–response profiles of pesticide impact. For future in‐field applications, decoupling of the cell culture routines from the actual cytotoxicity assay is mandatory. Thus, suspensions of Sf21 cells were cryopreserved at –80°C in the wells of multielectrode arrays and thawed anytime for conducting the assays. Five pesticides were tested for their concentration‐dependent cytotoxicity expressed as EC50 values by ECIS and validated using the well‐established WST‐1 cell viability assay. Results were found to be in good agreement. Our studies revealed cytotoxic effects of some pesticides sold for home usage far below the recommended concentration and were found to be more toxic than formulations sold for agricultural industry only.

Modulation of the Blood-Brain Barrier by Sigma-1R Activation.

Sigma non-opioid intracellular receptor 1 (Sigma-1R) is an intracellular chaperone protein residing on the endoplasmic reticulum at the mitochondrial-associated membrane (MAM) region. Sigma-1R is abundant in the brain and is involved in several physiological processes as well as in various disease states. The role of Sigma-1R at the blood-brain barrier (BBB) is incompletely characterized. In this study, the effect of Sigma-1R activation was investigated in vitro on rat brain microvascular endothelial cells (RBMVEC), an important component of the blood-brain barrier (BBB), and in vivo on BBB permeability in rats. The Sigma-1R agonist PRE-084 produced a dose-dependent increase in mitochondrial calcium, and mitochondrial and cytosolic reactive oxygen species (ROS) in RBMVEC. PRE-084 decreased the electrical resistance of the RBMVEC monolayer, measured with the electric cell-substrate impedance sensing (ECIS) method, indicating barrier disruption. These effects were reduced by pretreatment with Sigma-1R antagonists, BD 1047 and NE 100. In vivo assessment of BBB permeability in rats indicates that PRE-084 produced a dose-dependent increase in brain extravasation of Evans Blue and sodium fluorescein brain; the effect was reduced by the Sigma-1R antagonists. Immunocytochemistry studies indicate that PRE-084 produced a disruption of tight and adherens junctions and actin cytoskeleton. The brain microcirculation was directly visualized in vivo in the prefrontal cortex of awake rats with a miniature integrated fluorescence microscope (aka, miniscope; Doric Lenses Inc.). Miniscope studies indicate that PRE-084 increased sodium fluorescein extravasation in vivo. Taken together, these results indicate that Sigma-1R activation promoted oxidative stress and increased BBB permeability.

Liraglutide protects against microvascular dysfunction and sepsis-mediated inflammation and organ injury

Introduction: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection characterized by increased systemic inflammation and microvascular injury. Recently, commonly used diabetes and obesity medications, glucagon-like peptide-1 (GLP-1) receptor agonists, have demonstrated unexpected anti-inflammatory effects. We tested GLP-1 receptor agonist liraglutide in a clinically relevant two-hit murine model of polymicrobial abdominal sepsis and hyperoxia and microvascular endothelial injury in vitro, hypothesizing that GLP-1 receptor agonists exert protective effects during sepsis, limiting microvascular permeability, inflammation, organ injury and death. Methods: Sepsis was induced in male and female mice by intraperitoneal (IP) injection of cecal contents (CS;1.8 mg/g body weight) collected from euthanized donor mice. Mice were also exposed to hyperoxia (FiO2 90-96%) for 24 hours. Six and 18 hours after CS injection, mice were administered saline or liraglutide (0.1 mg/kg). At 12 hours, mice were given fluids and antibiotics. At 24 hours, mice were euthanized for plasma and bronchoalveolar lavage (BAL; right lung) collection. Plasma cytokines, organ injury markers, and BAL immune cells and protein were measured. The left lung was tied off and excised to measure wet-to-dry lung weight ratios. Additionally, for in vitro studies, primary human lung microvascular endothelial cells (HLMVECs) were treated with saline or liraglutide for 24 hours prior to saline (control) or LPS (100 ng/mL). HLMVEC barrier dysfunction was quantified using express permeability testing (XPerT) assay or Electric Cell-Substrate Impedance Sensing (ECIS) to measure transendothelial electrical resistance (TER). Results: In murine sepsis, illness severity scores and lung injury were improved in mice pretreated with liraglutide (N=12). Plasma blood urea nitrogen (BUN; P=0.0581), alanine transaminase (ALT; P=0.0311) and aspartate aminotransferase (AST; P=0.0263), and cytokines IL-1β (P=0.0078), IL-6 (P=0.0038), and TNF-α (P=0.0183) in plasma were reduced in mice treated with liraglutide. In HLMVECs, liraglutide (1.5 nM) significantly reduced LPS-induced barrier dysfunction measured by XPerT assay (P=0.0030) and ECIS (P=0.0075). Conclusion: Liraglutide reduces illness severity, inflammation, and organ injury in a two-hit model of sepsis-induced ALI. Liraglutide has direct effects in the microvascular endothelium, limiting LPS-mediated barrier dysfunction. These findings support a protective role for GLP-1 receptor agonism in sepsis, mediated through the microvasculature. HL150783, AG075341, HL158906. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of miR-181b Treatment on Senescent Human Aortic Smooth Muscle Cells

Large Artery Stiffness (LAS) is independently associated with increased cardiovascular morbidity and mortality and its development is multifactorial and has been associated with the onset of cellular senescence (CS). CS is defined as a stable cell cycle arrest, characterized by an altered cell phenotype with impaired function and detrimental secretory profile. Vascular smooth muscle cells (VSMCs) are present in two main phenotypes: the contractile phenotype seen in young populations, and the synthetic phenotype related to aging and senescent VSMCs. Micro-RNAs (miR) are non-coding RNAs that can alter protein expression. miR-181b has been demonstrated to undergo downregulation with aging with a close to 80% decrease, this correlates with an upregulation of markers of senescence. To test the hypothesis that miR-181b could modulate the senescent phenotype in VSMCs and promote a more contractile phenotype, a cellular model comprising young and old human aortic smooth muscle cells (HASMCs) was employed. These cells were transfected with either a miR-181b mimic or a scrambled miR control (SCR), resulting in an 11-fold increase in the young and a 3.3-fold increase in the old. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology, where it was found to be lower in older HASMCs (1.05±0.001) compared to the young (1.1±0.001, p&lt;0.05). When miR-181b was introduced, there was a significant increase in proliferation for both age groups by 0.03-fold compared to SCR (p&lt;0.05). Old HASMCs demonstrated a decreased average ionomycin-induced contractile response (3.65%±0.46) when compared to the young (7.17%±0.60, p&lt;0.05). Lipofectamine-mediated transfection of HASMCs with miR-181b resulted in an increased contractility response in the old HASMCs (5.46%±0.91) compared to scrambled control (1.41%±0.39, p&lt;0.05), while there was no significant difference for the young cells. The percentage of Senescence-Associated-β-galactosidase (Saβ-gal) positive cells, a marker of senescence, was higher in old (39.74%±3.04) vs young (4.35%±0.72, p&lt;0.05). The percentage of Saβ-gal positive cells tended to decrease in old VSMCs after miR-181b transfection (SCR 79.65%±4.38 vs Old181b 70.39±4.03, p=0.2.02), with no effect on young cells. Immunofluorescence in situ hybridization (IF-FISH) staining for the double-stranded DNA break marker (53BP1) was lower in the young (4.65%) compared to old HASMCs (32.69%). Likewise, the percentage of cells with one or more 53BP1 foci colocalization in telomeres, referred to as Telomere dysfunction-induced foci (TIF), was higher in old (11.54%) compared to young (2.33%) VSMCs (p=0.002). However, miR-181b transfection did not impact these markers regardless of age (p=0.94). Transfection of HASMCs with miR-181b did not impact senescent cell abundance, but improved contractility and cellular proliferation. Combined with our prior data demonstrating a senomorphic effect of miR-181b transfection to improve cellular stiffness and favorably modulate of senescence-associated secretory factors, our results demonstrate an enhancement in the phenotypic characteristics of the old HASMCs group, suggesting that miR-181b participates in the modulation of the senescent VSMCs phenotype. This translates to a potential improvement in HASMC health that could reduce the development of LAS. National Institutes of Health Awards: K08 AG070281 (ET), T32 HL139451 (DM), R01 AG048366 (LAL), R01 AG060395 (AJD), and Western Institute for Veterans Research (ET). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell-Free Hemoglobin Induces Vascular and Mitochondrial Dysfunction in the Pulmonary Endothelium in an Oxidation-State Dependent Manner

Background: Sepsis results in significant organ damage due in part to a breakdown in endothelial vascular function. Elevated levels of cell-free hemoglobin (CFH) drive endothelial dysfunction in the lungs. CFH generates reactive oxygen species (ROS) via the iron present in the heme moiety, which can be oxidized from ferrous (CFH2+) to ferric (CFH3+). Recent studies suggest mitochondrial dysfunction may be a key pathway leading to microvascular dysfunction. We hypothesize that CFH induces mitochondrial dysfunction and microvascular endothelial barrier disruption in the lung in an oxidative-state dependent manner. Methods: CFH was converted from CFH2+ to CFH3+ by UV irradiation (validated by spectrophotometry). Primary human lung microvascular endothelial cells (HLMVECs) were treated with 1.0 mg/mL CFH2+, CFH3+, or vehicle control. Barrier dysfunction was measured via electric cell-substrate impedance sensing (ECIS) and express permeability test (XPerT) at 24h. We quantified mitochondrial superoxide (MitoSOX Red; 5 uM) by flow cytometry (1, 6, 24h) and total cellular ROS production (CellROX Deep Red; 5 uM) by fluorescence microscopy (6 and 24 h). Mitochondrial morphology (electron density, roundness, area, circularity, and aspect ratio) was assessed by transmission electron micrographs at 6h and quantified by a blinded reviewer (ImageJ). Activation of the mitochondrial permeability transition pore (mPTP) was assessed by flow cytometry using calcein-AM with cobalt chloride. DNA was purified from patient plasma and the copy number of mitochondrial genes ND4L and CYB was quantified by digital droplet PCR. Results: In HLMVECs, CFH generated excess ROS (CellROX and MitoSOX) at 6h compared to vehicle (522 vs 265 MFI, p &lt; 0.01). Morphology analysis demonstrated increased electron density and aspect ratio and decreased circularity and roundness. mPTP activity was increased by CFH3+ (1295 vs 2197 MFI, p &lt; 0.01) compared to vehicle or CFH2+. Barrier dysfunction caused by CFH3+ measured by ECIS peaked at 6h (-1270 vs 262 max TER drop, p &lt; 0.0001), while XPerT confirmed dysfunction at 24h (9.41e+06 vs 6.53e+05 total fluorescent area, p &lt; 0.01). Quantification of mtDNA copy number in patient plasma revealed that circulating mt-ND4L copies correlate with CFH (r = 0.44, p &lt; 0.01). Conclusions: The results demonstrate significant perturbations to mitochondrial function and network dynamics caused by oxidized CFH (3+). The novel findings point towards the ferric (3+) form of hemoglobin as the primary driver of endothelial dysfunction, and the underlying mechanism is directly related to disruption of the mitochondrial network. The association of mitochondrial and vascular biomarkers demonstrates an urgent need to better understand how mitochondrial dysfunction can be prevented to limit vascular damage. This work was funded by the National Institutes of Health NHLBI 5R35HL150783 and 1F31HL167471. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Autotaxin-Lysophosphatidic Acid Axis: Key Regulator of Blood-Brain Barrier Integrity and Stroke Outcomes during Cerebral Ischemia-Reperfusion

Background: Endothelial hyperpermeability subsequent to ischemic stroke poses severe ramifications for patient outcomes, with the preservation of blood-brain barrier (BBB) integrity being paramount. The pathological role of the autotaxin-lysophosphatidic acid (ATX-LPA) axis in various disorders is established. While LPA's detrimental effects in stroke pathology have been documented, the mechanistic role of its enzymatic precursor, autotaxin (ATX), especially in the context of BBB disruption and mitochondrial dysfunction, necessitates deeper exploration.Objective: Our research aimed to dissect the intricate interplay of the ATX-LPA axis in modulating BBB integrity and mitochondrial bioenergetics during cerebral ischemia-reperfusion injury.Methods: Employing well-characterized murine models, ischemic strokes were meticulously induced, succeeded by reperfusion. A gamut of advanced assays—including the electrical cell-substrate impedance sensor, Seahorse XF24 analyzer, and AR-2 probe fluorescence—were systematically deployed. Moreover, to attain a nuanced understanding, we genetically engineered a mouse model exhibiting endothelial-specific ATX deletion, facilitating a direct interrogation of ATX's endothelial role.Results: Post-ischemic reperfusion culminated in a statistically significant upregulation of ATX mRNA (1.7-fold, p&lt;0.05) and a quadrupled surge in its enzymatic activity (p&lt;0.01). Disturbingly, the I/R cohort manifested augmented LPA concentrations, concomitant with a pronounced mitochondrial bioenergetic collapse, evidenced by metrics such as the oxygen consumption rate and ATP synthesis (both p&lt;0.01). BBB integrity was critically compromised, a derangement that ATX inhibitors ameliorated (p&lt;0.05). Intriguingly, mice with endothelial-specific ATX ablation exhibited remarkably attenuated permeability (p&lt;0.05) and infarct volumetric reductions (p&lt;0.01), while preserving cerebral perfusion (p&lt;0.05).Conclusion: Our findings underscore the ATX-LPA axis as a linchpin in the pathophysiological cascade following ischemic stroke, specifically impinging on BBB and mitochondrial health. Deciphering and subsequently targeting this nexus could herald a paradigm shift in therapeutic strategies for ischemic stroke. This research was supported by National Institutes of Health grants HL141998, HL141998-01S1, AA025744, AA026708, and AA025744-02S1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of Impedance Based Sensor to Discover the Invasive Nature of A549 Lung Cancer Cell

Numerous studies have been conducted to investigate the effectiveness of impedance-based sensors in detecting the invasive behavior of cancer cells, specifically through the use of Electric Cell-substrate Impedance Sensing (ECIS) methodology. However, the current equivalent circuit models used to represent the invasive nature of cancer cells have limitations and inaccuracies, and there has been a lack of utilization of mathematical and data analysis software to better understand the growth and invasive behavior of these cells. To address these gaps, this research aims to measure the impedance of A549 lung cancer cells, develop a simplified equivalent circuit model, and analyze the results using mathematical and data analysis software. The invasive behavior of A549 cells will first be studied through impedance measurements, and then a circuit model will be designed and simulated using software tools to reveal the true invasive nature of these cells. Finally, rigorous mathematical analysis and the use of suitable data analysis software (such as Matlab Powergui and Simulink, EIS Spectrum Analyzer, and Plotly) will be applied to gain a comprehensive understanding of the morphological behavior of the cells. The experimental results of this research are expected to align with previous findings, and a quadratic equation will be derived to predict the body's resistance of the A549 lung cancer cell using mathematical and data analysis approaches.

Modulation of the expression of connexins 37, 40, and 43 in endothelial cells in a culture.

Connexin (Cx) 37, 40, and 43 are implicated in vascular function, specifically in the electrical coupling of endothelial cells and vascular smooth-muscle cells. In the present study, we investigated whether factors implicated in vascular dysfunction can modulate the gene expression of Cx37, Cx40, and Cx43 and whether this is associated with changes in endothelial layer barrier function in human microvascular endothelial cells (HMEC-1). First, HMEC-1 were subjected to stimuli for 4 and 8h. We tested their responses to DETA-NONOate, H2O2, high glucose, and angiotensin II, none of which relevantly affected the transcription of the connexin genes. Next, we tested inflammatory factors IL-6, interferon gamma (IFNγ), and TNFα. IFNγ (10ng/mL) consistently induced Cx40 expression at 4 and 8 h to 10-20-fold when corrected for the control. TNFα and IL-6 resulted in small but significant depressions of Cx37 expression at 4h. Two JAK inhibitors, epigallocatechin-3-gallate (EGCG) (100-250μM) and AG490 (100-250μM), dose-dependently inhibited the induction of Cx40 expression by IFNγ. Subsequently, HMEC-1 were subjected to 10ng/mL IFNγ for 60h, and intercellular and transcellular impedance was monitored by electric cell-substrate impedance sensing (ECIS). In response to IFNγ, junctional-barrier impedance increased more than cellular-barrier impedance; this was prevented by AG490 (5μM). In conclusion, IFNγ can strongly induce Cx40 expression and modify the barrier properties of the endothelial cell membrane through the JAK/STAT pathway. Moreover, the Cx37, Cx40, and Cx43 expression in endothelial cells is stable and, apart from IFNγ, not affected by a number of factors implicated in endothelial dysfunction and vascular diseases.

Porous Membrane Electrical Cell-Substrate Impedance Spectroscopy for Versatile Assessment of Biological Barriers In Vitro.

Cell culture models of endothelial and epithelial barriers typically use porous membrane inserts (e.g., Transwell inserts) as a permeable substrate on which barrier cells are grown, often in coculture with other cell types on the opposite side of the membrane. Current methods to characterize barrier function in porous membrane inserts can disrupt the barrier or provide bulk measurements that cannot isolate barrier cell resistance alone. Electrical cell-substrate impedance sensing (ECIS) addresses these limitations, but its implementation on porous membrane inserts has been limited by costly manufacturing, low sensitivity, and lack of validation for barrier assessment. Here, we present porous membrane ECIS (PM-ECIS), a cost-effective method to adapt ECIS technology to porous substrate-based in vitro models. We demonstrate high fidelity patterning of electrodes on porous membranes that can be incorporated into well plates of a variety of sizes with excellent cell biocompatibility with mono- and coculture set ups. PM-ECIS provided sensitive, real-time measurement of isolated changes in endothelial cell barrier impedance with cell growth and barrier disruption. Barrier function characterized by PM-ECIS resistance correlated well with permeability coefficients obtained from simultaneous molecular tracer permeability assays performed on the same cultures, validating the device. Integration of ECIS into conventional porous cell culture inserts provides a versatile, sensitive, and automated alternative to current methods to measure barrier function in vitro, including molecular tracer assays and transepithelial/endothelial electrical resistance.

Transmembrane Protein CMTM6 Alleviates Ocular Inflammatory Response and Improves Corneal Epithelial Barrier Function in Experimental Dry Eye.

To investigate the impact of transmembrane protein CMTM6 on the pathogenesis of dry eye disease (DED) and elucidate its potential mechanisms. CMTM6 expression was confirmed by database analysis, real-time polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry. Tear secretion was measured using the phenol red thread test. Immune cell infiltration was assessed through flow cytometry. Barrier function was evaluated by fluorescein sodium staining, immunofluorescence staining of zonula occludens 1 (ZO-1), and electric cell-substrate impedance sensing (ECIS) assessment. For silencing CMTM6 expression, siRNA and shRNA were employed, along with lentiviral vector-mediated overexpression of CMTM6. Proinflammatory cytokine levels were analyzed by RT-PCR and cytometric bead array (CBA) analysis. CMTM6 showed high expression in healthy human and mouse corneal and conjunctival epithelium but was notably reduced in DED. Notably, this downregulation was correlated with disease severity. Cmtm6-/- dry eye (DE) mice displayed reduced tear secretion, severe corneal epithelial defects, decreased conjunctival goblet cell density, and upregulated inflammatory response. Additionally, Cmtm6-/- DE mice and CMTM6 knockdown human corneal epithelial cell-transformed (HCE-T) cells showed more severe barrier disruption and reduced expression of ZO-1. Knockdown of CMTM6 in HCE-T cells increased inflammatory responses induced by hyperosmotic stress, which was significantly mitigated by CMTM6 overexpression. Moreover, the level of phospho-p65 in hyperosmolarity-stimulated HCE-T cells increased after silencing CMTM6. Nuclear factor kappa B (NF-κB) p65 inhibition (JSH-23) reversed the excessive inflammatory responses caused by hyperosmolarity in CMTM6 knockdown HCE-T cells. The reduction in CMTM6 expression on the ocular surface contributes to the pathogenesis of DED. The CMTM6-NF-κB p65 signaling pathway may serve as a promising therapeutic target for DED.

Monitoring Intestinal Organoid-Derived Monolayer Barrier Functions with Electric Cell-Substrate Impedance Sensing (ECIS).

The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids. Key features • We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers. • The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.

Correction Notice: Monitoring Intestinal Organoid–Derived Monolayer Barrier Functions with Electric Cell–Substrate Impedance Sensing (ECIS)

Correction Notice: Monitoring Intestinal Organoid–Derived Monolayer Barrier Functions with Electric Cell–Substrate Impedance Sensing (ECIS)

Lidocaine inhibits migrationof tenocytesby downregulating focal adhesion kinaseand paxillin phosphorylation.

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague-Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell-substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F-actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho-focal adhesion kinase (FAK), FAK, phospho-paxillin, paxillin, and F-actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F-actin distribution. Lidocaine reduced F-actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho-FAK, F-actin, and phospho-paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F-actin, phospho-FAK, and phospho-paxillin expression when cells are treated with lidocaine.

Upper glycolytic components contribute differently in controlling retinal vascular endothelial cellular behavior: Implications for endothelial-related retinal diseases.

Retinal degenerative diseases such as diabetic retinopathy and diabetic macular edema are characterized by impaired retinal endothelial cells (RECs) functionality. While the role of glycolysis in glucose homeostasis is well-established, its contributions to REC barrier assembly and cell spreading remain poorly understood. This study aimed to investigate the importance of upper glycolytic components in regulating the behavior of human RECs (HRECs). Electric cell-substrate impedance sensing (ECIS) technology was employed to analyze the real-time impact of various upper glycolytic components on maintaining barrier functionality and cell spreading of HRECs by measuring cell resistance and capacitance, respectively. Specific inhibitors were used: WZB117 to inhibit Glut1/3, lonidamine to inhibit hexokinases, PFK158 to inhibit the PFKFB3-PFK axis, and TDZD-8 to inhibit aldolases. Additionally, the viability of HRECs was evaluated using the lactate dehydrogenase (LDH) cytotoxicity assay. The most significant reduction in electrical resistance and increase in capacitance of HRECs resulted from the dose-dependent inhibition of PFKFB3/PFK using PFK158, followed by aldolase inhibition using TDZD-8. LDH level analysis at 24- and 48-hours post-treatment with PFK158 (1 μM) or TDZD-8 (1 and 10 μM) showed no significant difference compared to the control, indicating that the disruption of HRECs functionality was not attributed to cell death. Conversely, inhibiting Glut1/3 with WZB117 had minimal impact on HREC behavior, except at higher concentrations (10 μM) and prolonged exposure. Lastly, inhibiting hexokinase with lonidamine did not noticeably alter HREC cell behavior. This study illustrates the unique impacts of components within upper glycolysis on HREC functionality, emphasizing the crucial role of the PFKFB3/PFK axis in regulating HREC behavior. Understanding the specific contributions of each glycolytic component in preserving normal REC functionality will facilitate the development of targeted interventions for treating endothelial cell dysfunction in retinal disorders while minimizing effects on healthy cells.

Abstract 13070: Interleukin-19 Regulates Lymphatic Permeability and Induces Prox1-Dependent Lymphangiogenesis

Introduction: Increased lymphangiogenesis is associated with atherosclerosis, but whether it is disease-causative or a compensatory attempt to attenuate disease is currently unresolved. Recent studies suggest lymphatic permeability plays a role in reverse cholesterol transport (RCT) as a possible mechanism for attenuating atherosclerosis. We previously reported that that Interleukin-19 (IL-19), an immunomodulatory cytokine, is unique by inducing angiogenesis while reducing atherosclerosis. The purpose of this study was to test the hypothesis that IL-19 could be atheroprotective by inducing functional lymphangiogenesis and promoting lymphatic flux. Methods &amp; Results: RNA sequencing of IL-19-treated human Lymphatic Endothelial Cells (hLEC) identified induction of chemokines, pro-lymphangiogenic, and permeability-associated genes. Prox1, the master regulator of lymphatic formation, was increased by six-fold, validated by qPCR and western blot. Cellular assays further demonstrated IL-19-associated lymphangiogenesis through hLEC proliferation, migration, and tube formation, each mitigated by Prox1 siRNA knockdown. Angpt2, a regulator of permeability through VE-cadherin, is also upregulated by IL-19 and mitigated by Prox1 siRNA knockdown. Electric Cell-substrate Impedance Sensing (ECIS) showed that IL-19 mitigates oxLDL-associated decreases in permeability, suggestive of preventing lymphatic barrier malfunction during atherosclerosis. Preliminary in vivo lymphatic permeability assays suggest injection of rmIL-19 improves lymphatic function in conditional lymphatic-specific Prox1 heterozygous mice. Conversely, similar experiments show that Il19 / LDLR double knockout mice, which are known to have exacerbated atherosclerosis, also have significantly decreased lymphatic permeability compared to LDLR -/- mice after 16-weeks of high fat diet. Conclusion: These data suggest that IL-19 is a previously unrecognized regulator of lymphatic function as it increases lymphangiogenesis and permeability, with the potential to improve RCT. Together, these effects may regulate atherosclerosis, and influence our understanding of the association between the lymphatic system and atherosclerosis.