Electric Cell-substrate Impedance Sensing Research Articles

P90RSK-MAGI1 Module Controls Endothelial Permeability by Post-translational Modifications of MAGI1 and Hippo Pathway.

Previously, we reported that post-translational modifications (PTMs) of MAGI1, including S741 phosphorylation and K931 de-SUMOylation, both of which are regulated by p90RSK activation, lead to endothelial cell (EC) activation. However, roles for p90RSK and MAGI1-PTMs in regulating EC permeability remain unclear despite MAGI1 being a junctional molecule. Here, we show that thrombin (Thb)-induced EC permeability, detected by the electric cell-substrate impedance sensing (ECIS) based system, was decreased by overexpression of dominant negative p90RSK or a MAGI1-S741A phosphorylation mutant, but was accelerated by overexpression of p90RSK, siRNA-mediated knockdown of magi1, or the MAGI1-K931R SUMOylation mutant. MAGI1 depletion also increased the mRNA and protein expression of the large tumor suppressor kinases 1 and 2 (LATS1/2), which inhibited YAP/TAZ activity and increased EC permeability. Because the endothelial barrier is a critical mediator of tumor hypoxia, we also evaluated the role of p90RSK activation in tumor vessel leakiness by using a relatively low dose of the p90RSK specific inhibitor, FMK-MEA. FMK-MEA significantly inhibited tumor vessel leakiness at a dose that does not affect morphology and growth of tumor vessels in vivo. These results provide novel insights into crucial roles for p90RSK-mediated MAGI1 PTMs and the Hippo pathway in EC permeability, as well as p90RSK activation in tumor vessel leakiness.

Impedance-based drug-resistance characterization of colon cancer cells through real-time cell culture monitoring

Interest in impedance-based cellular assays is rising due to their remarkable advantages, including label-free, low cost, non-invasive, non-destructive, quantitative and real-time monitoring. In order to test their potential in cancer treatment decision and early detection of chemoresistance, we devised a new custom-made impedance measuring system based on electric cell-substrate impedance sensing (ECIS), optimized for long term impedance measurements. This device was employed in a proof of concept cell culture impedance analysis for the characterization of chemo-resistant colon cancer cells. Doxorubicin-resistant HT-29 cells were used for this purpose and monitored for 140 h. Analysis of impedance-based curves reveal different trends from chemo-sensitive and chemo-resistant cells. An impedance-based cytoxicity assay with different concentrations of doxorubicin was also performed using ECIS. The obtained results confirm the feasibility of ECIS in the study of drug resistance and show promises for studies of time-dependent factors related to physiological and behavioral changes in cells during resistance acquisition. The methodology presented herein, allows the continuous monitoring of cells under normal culture conditions as well as upon drug exposure. The ECIS device used, sets the basis for high-throughput early detection of resistance to drugs, administered in the clinical practice to cancer patients, and for the screening of new drugs in vitro, on patient-derived cells.

Abstract 552: Bradykinin Receptor B1/ Matrix Metalloprotease 3 Pathway is a Novel Target in Preventing Cardiac Ischemia-reperfusion Injury

Introduction: Impaired endothelial function leads to the progression of heart failure after Ischemia-reperfusion (IR). Kinin activation of bradykinin receptor 1 (B1R), a G protein-coupled receptor that has been found to induce capillary leakage, may serve as a critical mediator in cardiac microvascular barrier dysfunction. However, the underlying mechanisms are not clear. We found that B1R inhibition abolished IR-induced endothelial matrix metalloprotease (MMP3) expression and improved endothelial barrier formation. Thus, we hypothesized that B1R antagonist protects against cardiac IR injury through an MMP3 pathway. Methods and Results: MMP3-/- mice and their littermate controls (WT) were subjected to either cardiac IR or sham control. The baseline characteristics of these mice showed minimal phenotypes. Cardiac function was determined at 3, 7 and 24 days post-IR by echocardiography. The MMP3-/- mice displayed improved cardiac function compared to the control mice, as determined by fractional shortening (26% ± 1.1 MMP3-/- vs. 21% ± 0.9 WT, p<0.05, n=5) and ejection fraction (48% ± 1.9 MMP3-/- vs. 42% ± 2.8.1 WT, p<0.05, n=5), and treating with B1R antagonist (300 μg/Kg) significantly reduced serum MMP3 levels (p<0.01). Compared to the control mice, MMP3-/- mice had significantly less infarction/area at risk 24 hours post-IR demonstrated through TTC staining. In vitro studies revealed that cellular hypoxia-reoxygenation (HR) injury significantly increased both B1R and MMP3 expression levels in primary isolated cardiac mice microvascular endothelial cells (mCMVEC). MMP3 levels were measured via ELISA. Moreover, B1R agonist treatment (1uM) increased MMP3 levels, while the use of the antagonist attenuated the increase of MMP3 in response to HR. Finally, the use of B1R antagonist improved MMP3 induced endothelial barrier dysfunction, which was measured by the electric cell-substrate impedance sensing (ECIS) system. Taken together, the results demonstrated that B1R antagonist abolished IR induced MMP3 induction and that the deletion of MMP3 is protective of cardiac function upon IR injury. Conclusions: MMP3 is a critical regulator of cardiac microvascular barrier function, and B1R/MMP3 could potentially serve as a novel therapeutic target for heart failure in response to IR injury.

An in vitro model of chronic wounding and its implication for age-related macular degeneration.

Degeneration of the retinal pigment epithelium (RPE) plays a central role in age-related macular degeneration (AMD). Throughout life, RPE cells are challenged by a variety of cytotoxic stressors, some of which are cumulative with age and may ultimately contribute to drusen and lipofuscin accumulation. Stressors such as these continually damage RPE cells resulting in a state of chronic wounding. Current cell-based platforms that model a state of chronic RPE cell wounding are limited, and the RPE cellular response is not entirely understood. Here, we used the electric cell-substrate impedance sensing (ECIS) system to induce a state of acute or chronic wounding on differentiated human fetal RPE cells to analyze changes in the wound repair response. RPE cells surrounding the lesioned area employ both cell migration and proliferation to repair wounds but fail to reestablish their original cell morphology or density after repetitive wounding. Chronically wounded RPE cells develop phenotypic AMD characteristics such as loss of cuboidal morphology, enlarged size, and multinucleation. Transcriptomic analysis suggests a systemic misregulation of RPE cell functions in bystander cells, which are not directly adjacent to the wound. Genes associated with the major RPE cell functions (LRAT, MITF, RDH11) significantly downregulate after wounding, in addition to differential expression of genes associated with the cell cycle (CDK1, CDC6, CDC20), inflammation (IL-18, CCL2), and apoptosis (FAS). Interestingly, repetitive wounding resulted in prolonged misregulation of genes, including FAS, LRAT, and PEDF. The use of ECIS to induce wounding resulted in an over-representation of AMD-associated genes among those dysregulated genes, particularly genes associated with advanced AMD. This simple system provides a new model for further investigation of RPE cell wound response in AMD pathogenesis.

Bone Morphogenetic Protein (BMP)4 But Not BMP2 Disrupts the Barrier Integrity of Retinal Pigment Epithelia and Induces Their Migration: A Potential Role in Neovascular Age-Related Macular Degeneration.

Disruption of retinal pigment epithelial (RPE) barrier integrity and RPE migration are hallmark features in neovascular age-related macular degeneration (nAMD), but the underlying causes and pathophysiology are not completely well-defined. Herein, we aimed to evaluate the effect of bone morphogenetic proteins (BMPs) on the barrier function and migration of RPE. In particular, we investigated the role of BMP2 and BMP4 in these processes as our analysis of RNA-sequencing (seq) data from human donor eyes demonstrated that they are highly differentially expressed BMP members in macular RPE/choroid versus macular retina. We used electrical cell-substrate impedance sensing (ECIS) system to monitor precisely in real time the barrier integrity and migration of ARPE-19 after treatment with various concentrations of BMP2 or BMP4. Immunofluorescence was also used to assess the changes in the expression and the organization of the key tight junction protein, zona occludens (ZO)-1, in ARPE-19 cells under BMP2 or BMP4 treatment. This was followed by measuring the activity of matrix metalloproteinases (MMPs). Finally, RNA-seq and ELISA were used to determine the local and circulating levels of BMP2 and BMP4 in retinas and serum samples from nAMD donors. Our ECIS results showed that BMP4 but not BMP2 decreased the transcellular electrical resistance (TER) of ARPE-19 and increased their migration in comparison with control (vehicle-treated cells). Furthermore, immunofluorescence showed a disorganization of ZO-1 in BMP4-treated ARPE-19 not in BMP2-treated cells or vehicle-treated controls. This effect of BMP4 was associated with significant increases in the activity of MMPs, specifically MMP2. Lastly, these results were corroborated by additional findings that circulating but not local BMP4 levels were significantly higher in nAMD donor samples compared to controls. Collectively, our results demonstrated unreported effects of BMP4 on inducing RPE dysfunction and suggest that BMP4 but not BMP2 may represent a potential therapeutic target in nAMD.

A portable standalone wireless electric cell-substrate impedance sensing (ECIS) system for assessing dynamic behavior of mammalian cells

In this study, we have developed a wireless, portable, standalone, and simple electric cell-substrate impedance sensing (ECIS) system to analyze in-depth functional aspects of cellular functions on the surface of a co-planar metal electrode coated on conventional glass substrate using a low-cost circuitry and correlated it with an equivalent electrical circuit (EEC) model. Low-cost circuitry was used for studying the dynamic behavior of the mouse myoblast cells (C2C12) in a culture chamber. Further, the developed ECIS system was connected with 8-bit Arduino UNO microcontroller board for establishing a compact sized measuring unit, which can be placed inside a CO2 incubator to provide proper environmental condition for the biological cells during the entire measuring time. Integrating ZigBee RF module with the 8-bit Arduino UNO microcontroller board provides a wireless communication network. Theoretical calculation of the lumped-elemental electrical parameters associated with cell-electrolyte interface and metal-electrolyte interface was calculated. The calculation was performed by fitting the experimental impedance data to EEC model using least mean square method to determine the dynamic and vital functions of the mammalian cells such as proliferation (in real-time) with a change in intrinsic electrical parameters associated at any particular time point. Impedance measurements and the lumped-elemental electrical parameter were correlated with the respective microscopic images. The developed ECIS system was found to enable measuring of the extent of cellular proliferation over time. The compactness of the developed ECIS system integrated with the ZigBee RF module and the 8-bit Arduino UNO microcontroller board facilitates its utilization even when placed in the CO2 incubator for a prolonged time.

Three-dimensional analyses of cells’ positioning on the quadrupole-electrode microfluid chip considering the coupling effect of nDEP, ACEO, and ETF

Microfluidic chips integrated with negative dielectrophoresis (nDEP) and electrochemical impedance spectroscopy have wide applications in cell sensing. Accurate analysis of the kinematics and dynamics of cells in the nDEP process is crucial to improve the positioning accuracy and electric cell-substrate impedance sensing (ECIS) performance. This paper reports employing the three-dimensional (3D) finite element model to analyze the coupling effects of electrokinetic flows (EF) such as alternating current electroosmosis (ACEO) and the electrothermal flow (ETF) on the nDEP positionings. On the quadrupole ECIS microfluid chip, three typical nDEP results are observed in the frequency range of 100 Hz-25 MHz and the amplitude range of 1-20 Vp-p. Simulations Based on the 3D hybrid model provide abundant kinematic information and show clear dynamic processes. Based on the discussion, the mechanisms of nDEP localizations and phase-tuning manipulations are proposed. It is found that the drag force could affect the particle's movement through the vortex of the flow field induced by ACEO and ETF, while the nDEP forces dominate the particles' locations on the substrate. Thus, the 3D dynamic-coupling analyses could help design the quadrupole-electrode microfluid chip and optimize the manipulation parameters in the experiment.

Use of Discrete Wavelet Transform to Assess Impedance Fluctuations Obtained from Cellular Micromotion.

Electric cell–substrate impedance sensing (ECIS) is an attractive method for monitoring cell behaviors in tissue culture in real time. The time series impedance fluctuations of the cell-covered electrodes measured by ECIS are the phenomena accompanying cellular micromotion as cells continually rearrange their cell–cell and cell–substrate adhesion sites. Accurate assessment of these fluctuations to extract useful information from raw data is important for both scientific and practical purposes. In this study, we apply discrete wavelet transform (DWT) to analyze the concentration-dependent effect of cytochalasin B on human umbilical vein endothelial cells (HUVECs). The sampling rate of the impedance time series is 1 Hz and each data set consists of 2048 points. Our results demonstrate that, in the Daubechies (db) wavelet family, db1 is the optimal mother wavelet function for DWT-based analysis to assess the effect of cytochalasin B on HUVEC micromotion. By calculating the energy, standard deviation, variance, and signal magnitude area of DWT detail coefficients at level 1, we are able to significantly distinguish cytotoxic concentrations of cytochalasin B as low as 0.1 μM, and in a concentration-dependent manner. Furthermore, DWT-based analysis indicates the possibility to decrease the sampling rate of the micromotion measurement from 1 Hz to 1/16 Hz without decreasing the discerning power. The statistical measures of DWT detail coefficients are effective methods for determining both the sampling rate and the number of individual samples for ECIS-based micromotion assays.

Electrochemical Assessment of Anticancer Compounds on the Human Tongue Squamous Carcinoma Cells.

The most common oral cancer is squamous cell carcinoma (SCC) and its highest occurrence is in the tongue. Almost 30% of patients with one primary head and neck tumor will have a second primary malignancy. In recent studies, two novel plant extracts, andrographolide and cannabidiol (CBD), have been exploited for their anticancer effects. Here, we investigated the cytotoxic effects of these two compounds on SCC-25 cells, a human tongue squamous carcinoma cell line, and compared the outcomes with two chemotherapeutic drugs, cisplatin and fluorouracil. Electric cell substrate impedance sensing (ECIS) system was applied to measure frequency- and time-dependent impedance of SCC-25 cell-covered electrodes and to further assess subtle changes in cell morphology and micromotion in response to different concentrations (0, 10, 30, 100, and 300 µM) of these compounds. AlamarBlue and Annexin V/7-AAD binding assays were used to measure the concentration dependent changes in viability and apoptosis of SCC-25 cells. Our results demonstrate that 24 hours after exposure to 30 µM CBD can significantly decrease the micromotion rate, damage the integrity of cell morphology, reduce cell viability, and induce higher apoptosis in treated SCC-25 cells, while the other three drugs attain similar effects at the concentration of 100 µM or higher. The apoptosis-induced changes in cell morphology and micromotion monitored by ECIS correlate well with biochemical assays. Thus, both frequency- and time-dependent impedance measurements using ECIS can be used to real-time follow cancer cell activities in response to anticancer drugs with different temporal cytotoxicity profiles.

Using Electric Cell-Substrate Impedance Sensing (ECIS) as a novel method to evaluate bacterial urinary tract infections

Abstract Urinary tractinfections (UTIs) are commonly acquired bacterial infections that cause significant morbidity, worsened by the rise of antimicrobial resistance among uropathogenic bacteria. Understanding the dynamics of bacterial UTIs is essential to the proper use of antimicrobial therapies in patient treatment. In this study, Electric Cell-Substrate Impedance Sensing (ECIS) technology was used to monitor bacterial-mediated cytotoxicity in-vitro enabling evaluation of antibiotics. Using the ECIS system, real-time changes of barrier function were recorded for human bladder epithelial cells infected with either antibiotic sensitive or resistant bacteria. This study employed four common uropathogens: Pseudomonas aeruginosa, Escherichia coli, Enterococcus species, and Klebsiella pneumoniae. The effect of different antimicrobials, antibiotic titers, and organism concentrations were evaluated. The addition of an antibiotic helped confirm and differentiate sensitive and resistant bacterial strains. Antibiotic concentrations at sensitive and resistant breakpoints were not as effective at high organism concentrations but were completely effective at lower more clinically relevant bacterial loads. The data showed bacterial strains resistant to co-cultured antibiotic took longer on average to degrade the cell monolayer than sensitive strains. Overall, these results indicate that antibiotic resistance is often associated with a fitness cost. Our findings support the use of ECIS to augment routine susceptibility data allowing the direct phenotypic measurement of bacterial virulence and the effects of antibiotic treatment on these strains.

Laying Siege to the Castle: The Role of Sepsis Related Cytokines and Inflammation in Bringing Down the Barriers

Abstract The endothelium plays a critical role in the progression from sepsis to organ failure. The endothelium lines all blood vessels and organs in the body. As such, it provides a protective barrier against pathologic micro-organisms. The endothelium is also responsible for dynamically regulating the passage of small molecules, fluid, and cells between the bloodstream and tissues. In sepsis, many factors play a role in compromising the endothelium. Dysfunctional endothelium often results in development of capillary leak, tissue hypoperfusion, hypoxia, edema, and shock. This study evaluates the individual contributions of sepsis-related immunomodulatory cytokines as well as a potential synergistic effect of cytokine combinations on vascular endothelial permeability. The Electric Cell-substrate Impedance Sensor was employed to evaluate the barrier function (Rb) and cell membrane capacitance (CMC) of endothelial cell monolayers treated with an array of cytokines including TNF-α IFN-γ, TGF-β, IL1-β, IL-6, IL-8, IL-10, and IL-12. The ECIS results revealed significant differences in the Rb of cell monolayers exposed to sepsis related cytokines compared to cytokine-free control samples. Components of intercellular tight junctions and associated cell adhesion molecules also revealed differences in the endothelial cell monolayer under immunofluorescence microscopy. Thus, cytokines as individual entities or in combinations that may be produced during episodes of sepsis significantly alter the endothelial morphology and permeability.

Inflammatory Conditions Disrupt Constitutive Endothelial Cell Barrier Stabilization by Alleviating Autonomous Secretion of Sphingosine 1-Phosphate.

The breakdown of the endothelial cell (EC) barrier contributes significantly to sepsis mortality. Sphingosine 1-phosphate (S1P) is one of the most effective EC barrier-stabilizing signaling molecules. Stabilization is mainly transduced via the S1P receptor type 1 (S1PR1). Here, we demonstrate that S1P was autonomously produced by ECs. S1P secretion was significantly higher in primary human umbilical vein endothelial cells (HUVEC) compared to the endothelial cell line EA.hy926. Constitutive barrier stability of HUVEC, but not EA.hy926, was significantly compromised by the S1PR1 antagonist W146 and by the anti-S1P antibody Sphingomab. HUVEC and EA.hy926 differed in the expression of the S1P-transporter Spns2, which allowed HUVEC, but not EA.hy926, to secrete S1P into the extracellular space. Spns2 deficient mice showed increased serum albumin leakage in bronchoalveolar lavage fluid (BALF). Lung ECs isolated from Spns2 deficient mice revealed increased leakage of fluorescein isothiocyanate (FITC) labeled dextran and decreased resistance in electric cell-substrate impedance sensing (ECIS) measurements. Spns2 was down-regulated in HUVEC after stimulation with pro-inflammatory cytokines and lipopolysaccharides (LPS), which contributed to destabilization of the EC barrier. Our work suggests a new mechanism for barrier integrity maintenance. Secretion of S1P by EC via Spns2 contributed to constitutive EC barrier maintenance, which was disrupted under inflammatory conditions via the down-regulation of the S1P-transporter Spns2.

Simvastatin in Endothelial Cell Barrier Regulation: A Novel Role for SAMD4A

IntroductionAcute lung injury (ALI) is a critical illness which results in significant morbidity and mortality. Endothelial barrier disruption is a key pathologic feature of ALI and serves as the main cause of life‐threatening microvascular leakage and alveolar flooding. Efforts to develop novel therapeutics have been futile with only a handful of agents available, none of which prevent or reverse endothelial barrier disruption. We believe that the answer may lie in the most unlikely of places: a cholesterol‐lowering drug. Simvastatin confers vascular protection through modulation of the endothelial barrier, attenuation of vascular leak, and promotion of endothelial repair following lung inflammation. Integrin β4 (ITGβ4, gene code: ITGB4), a transmembrane receptor protein, plays a key role in endothelial barrier regulation during ALI, and its upregulation is necessary for the enhanced barrier function seen following administration of simvastatin.RationaleThe mechanism by which simvastatin up‐regulates ITGβ4 expression has not been elucidated. We sought to define the mechanistic pharmacology that controls the regulation of ITGβ4 mRNA and protein expression by simvastatin.MethodsIn this study, we identified potential RNA binding proteins regulated by simvastatin and inflammatory mediators that target ITGB4 using genomic analysis and molecular biology methods (western blotting and silencing), and further validated with endothelial cell biology approaches. Endothelial barrier function modulation is also investigated by electric cell‐substrate impedance sensing (ECIS).ResultsWe have identified that Sterile Alpha Motif Domain‐Containing Protein 4A (SAMD4A), an ITGB4 RNA binding protein, is up‐regulated by LPS or TNFa, and downregulated by simvastatin. Additionally, intracellular SAMD4A levels are negatively correlated with ITGB4 mRNA levels. Silencing of SAMD4A in endothelial cells (ECs) results in increased mRNA expression of ITGB4 and enhances ITGB4‐mediated barrier function in ECs.ConclusionOur results suggest that simvastatin upregulates ITGB4 via the suppression of SAMD4A, an ITGB4 RNA binding protein, leading to enhanced endothelial cell barrier function. These findings have re‐defined the pharmacology of simvastatin on ITGB4 regulation. Furthermore, SAMD4A may serve as a novel therapeutic target for ALI via the modulation of EC barrier.Support or Funding InformationThis study is supported by NIH grants HL134610 and T32HL007249.

The sigma‐1 agonist PRE‐084 protects against energy depletion induced barrier disruption and also enhances glycolysis in HUVECs

The sigma‐1 receptor (σ1) is a well‐known endoplasmic reticulum (ER) chaperone that resides at the mitochondria‐associated ER membrane, having an important role in calcium signaling between ER and mitochondria. Expression of σ1 is present in all human tissues with specific predominance in arteries, liver, fibroblasts and brain. Our laboratory has shown that σ1 activation in lymphatic vessels elicits nitric oxide‐dependent lymphatic relaxation, a finding that suggested the potential important role of σ1 in endothelium. Other recent work has highlighted that σ1 may contribute to healthy endothelial barrier function, although little is known about the mechanisms involved. We hypothesized that σ1 helps protect the endothelial barrier disruption by stabilizing cellular bioenergetics. Cultured human umbilical vein endothelial cell (HUVEC) monolayers were used to model the endothelial barrier. Electric cell‐substrate impedance sensing (ECIS) was used to study the contribution of σ1 to endothelial barrier function. Western blotting was used to quantify σ1 protein expression. Endothelial bioenergetics were determined with extracellular flux analysis using glycolytic rate and ATP rate assays. The endothelial‐specific contribution of σ1 was assessed using with the σ1‐selective agonist PRE‐084 and by specific siRNA knockdown of σ1. The mitochondrial un‐coupler Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) was used to model mitochondrial dysfunction in HUVEC. Our results show that activation of σ1 with PRE‐084 50 μM, 100 μM, 150 μM, 200 μM significantly enhances barrier function of HUVEC monolayers in a concentration‐dependent manner. Knockdown of σ1 with siRNA reduces baseline barrier function and diminishes the ability of PRE‐084 to enhance TER. The decrease in TER elicited by CCCP (10 μM) is significantly amplified in cells with siRNA‐mediated knockdown of σ1 expression, and is reversed by PRE‐084 (100 μM) in cells with normal σ1 expression. HUVEC treated with PRE‐084 display higher levels of glycolysis and glycolytic ATP production than control cells. This response to PRE‐084 was not evident in the presence of σ1 knockdown. Collectively, our results suggest that σ1 plays an important role in maintenance of baseline endothelial barrier function, and that activation of σ1 can enhance barrier function. Moreover, σ1 activation also can rescue endothelial barrier dysfunction caused by disruption of mitochondrial ATP production, likely due to its ability enhance glycolysis. Thus, σ1 may represent a future therapeutic target to rescue endothelial barrier dysfunction.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.

Endothelial focal adhesion kinase contributes to intestinal microvascular leakage during ischemia/reperfusion injury

Ischemia/reperfusion (I/R) injury triggers a series of inflammatory responses in the microcirculation characterized by vascular leakage and leukocyte activation leading to organ dysfunction. In this study, we examine whether focal adhesion kinase (FAK) contributes to endothelial barrier dysfunction in response to I/R in splanchnic tissues. Using an in vivo model of intestinal I/R injury, we showed that the increase of microvascular leakage induced by I/R injury was greatly attenuated in endothelial specific, tamoxifen inducible, FAK knockout animals as well as wild type animals given pharmacological inhibition of FAK with PF‐573228. Using murine intestinal endothelial cells, we performed electric cell‐substrate impedance sensing (ECIS) assays with or without FAK inhibitor in the presence of H2O2. Results showed that FAK inhibition significantly attenuated endothelial barrier dysfunction induced by H2O2. Western blot analysis showed that H2O2 promoted FAK phosphorylation in a time course similar to that of endothelial barrier dysfunction. Confocal imaging analysis demonstrated that H2O2 increases the disruption of junctional proteins. The effect was blocked by FAK inhibitor. Taken together, the results suggested that FAK plays an important role in mediating intestinal vascular leakage during I/R injury.Support or Funding InformationNIH HL120954, VA BX000799

Effect of Entresto (valsartan+sacubitril) in the cardiac function and contractile response of detrusor and corpus cavernosum smooth muscles of heart failure rats.

IntroductionHeart failure (HF) has been closely related to the development of lower urinary tract symptoms (LUTS) and erectile dysfunction (ED). New pharmacological therapies aims to restore the neurohumoral balance, among these new proposals, stands out LCZ696 (Entresto), which consists of the association of the Valsartan (ANG‐II receptor antagonist, AT1R) with a Sacubitril (neprilysin inhibitor). However, there is no evidence evaluating the neurohumoral balance and the impact of Entresto on ED and LUTS associated with HF.ObjectiveWe aimed to Investigated the effect of in vitro treatment with Entresto (valsartan+sacubitril) on the contractile response of detrusor and corpus cavernosum (CC) muscles of control rats, as well as the effect of long‐term treatment with Entresto in the cardiovascular, urinary and erectile dysfunctions of HF rats.MethodsFor in vitro protocols, response concentration curves were constructed to phenylephrine (PHE; CC) and carbachol (CCh; detrusor), as well as, response frequency curves to electrical field stimulation (EFS; CC and detrusor) in the absence or presence of valsartan (10μM), sacubitril (10μM) or both drugs. For the chronic protocols, HF was surgically induced through the FAC model, and after 8 weeks, these animals were submitted to cardiac function evaluation (echocardiogram) and subdivided into 3 groups: Sham, HF and HF/Entresto (60mg/day for more 4 weeks). Response concentration curve were constructed to PHE (CC) and CCh (detrusor), and response frequency curve to EFS (CC and detrusor) for all groups. Culture human umbilical vein endothelial cell (HUVEC) monolayers were used to model the endothelial barrier. Electric cell‐substrate impedance sensing (ECIs) was used to study the contribution of valsartan, sacubitril or both drugs (10μM, respect.) to endothelial barrier function.ResultsIn vitro, valsartan, sacubitril or both drugs did not change the contractile responses in the CC or detrusor, independent of stimulated used in control animals. The HF animals presented both reduced of ejection and shortening fraction (p<0.05), as well present cardiac hypertrophy (p<0.05) when compared to the Sham group. Long‐term treatment with Entresto restored the ejection and shortening fraction in the HF/Entresto group (p<0.05). In the CC of HF group the contractile response mediated by PE and EFS (p<0.05) were increased compared to Sham group, being this hypercontractility restored by the Entresto treatment (p<0.05). CCh and EFS induced detrusor smooth muscle contractions, which were greater in HF rats than in Sham rats. The treatment with Entresto restore the detrusor hypercontractility of HF rats (p<0.05). Additionally, only the valsartan enhance the barrier function (P<0.05) of HUVEC monolayer.ConclusionOur data provide evidence that Entresto chronic treatment, restore the detrusor‐ and CC‐contractile response due the improved of endothelial barrier and cardiovascular function in rats with HF, suggesting new multi‐therapeutic properties.Support or Funding InformationFAPESP 2011/21095‐4, CNPq 423987/2016‐0 and NIH R01GM20774

Assessment of Cytotoxicity of Magnesium Oxide and Magnesium Hydroxide Nanoparticles using the Electric Cell-Substrate Impedance Sensing

Magnesium (Mg)-based alloys have the potential for bone repair due to their properties of biodegradation, biocompatibility, and structural stability, which can eliminate the requirement for a second surgery for the removal of the implant. Nevertheless, uncontrolled degradation rate and possible cytotoxicity of the corrosion products at the implant sites are known current challenges for clinical applications. In this study, we assessed in vitro cytotoxicity of different concentrations (0 to 50 mM) of possible corrosion products in the form of magnesium oxide (MgO) and magnesium hydroxide (Mg(OH)2) nanoparticles (NPs) in human fetal osteoblast (hFOB) 1.19 cells. We measured cell proliferation, adhesion, migration, and cytotoxicity using a real-time, label-free, non-invasive electric cell-substrate impedance sensing (ECIS) system. Our results suggest that 1 mM concentrations of MgO/Mg(OH)2 NPs are tolerable in hFOB 1.19 cells. Based on our findings, we propose the development of innovative biodegradable Mg-based alloys for further in vivo animal testing and clinical trials in orthopedics.

Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblasts

Cartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein’s structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein’s biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro.

Abstract P2-20-04: Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease

Abstract Introduction: Brain metastatic disease (BMD) is attributed to significant mortality and morbidity in breast cancer patients. Strict policing of molecular traffic across the blood-brain barrier (BBB) precludes brain parenchymal penetration of most small molecule drugs, chemotherapy included. Consequently, the treatment of BMD remains difficult, particularly if the BBB remains intact during disease progression. Artemisinin has been efficacious in treating multi-drug resistant cerebral malaria likely indicative of an ability to traverse the BBB. Thus, Artemisinin may be able to modulate the restrictive nature of the BBB and provide a potential adjunctive therapy to established drug therapy in the treatment of BMD. Emerging evidence suggests that Artemisinin itself may harbour potent anti-cancer properties. Aim: To investigate in vitro the effect on barrier function in particular paracellular permeability of brain vascular endothelial cells compared to non-brain vascular endothelial cells when treated with Artemisinin. Materials and Methods: Inter-individual BBB differences were modelled using the human brain vascular endothelial cell lines hCMEC-D3, TY09 and TY10. In addition to the brain cell lines, a HUVEC cell line was treated with incremental concentrations of artemisinin. Adhesion, migration and electrical resistance across cellular monolayers were quantified using Electric Cell-Substrate Impedance Sensing (ECIS). Trans- Endo/Epithelial Resistance (TEER) and Paracellular Permeability (PCP) were used to assess barrier function. Immunofluorescence was used to qualitatively assess tight junction (TJ) integrity of all cell lines. RNAseq was used to determine changes in message after treatment with Artemisinin in all 4 cell lines. Results: A significant reduction in electrical resistance across all cell lines was observed when treated with 1mM Artemisinin and may be suggestive of a cytotoxic concentration. In comparison when treated with 0.01mM Artemisinin the electrical resistance decreased which could suggest restrictive barrier properties were enhanced, in addition evidencing Artemisinin may exhibit biphasic activity. The reduction in electrical resistance across the brain endothelial cell lines was significant (p<0.05) but deemed not significant across HUVEC, perhaps mirroring Artemisinin’s pharmacological action at differing vascular interfaces in vivo. Higher Artemisinin concentrations significantly reduced TEER across the brain cell lines and increased paracellular permeability, the same trends were observed in HUVEC. IF demonstrated that Artemisinin was able to effect changes in TJ protein location which supports the hypothesis that Artemisinin reduces barrier function across the brain endothelial lines, suggested by an increased permeability in the presence of Artemisinin. Electrical resistance across all cell lines significantly reduced when treated with 1mM Artemisinin. When compared to negative control, there was a significant decrease in electrical resistance across all cell lines treated with 1mM Artemisinin. RNAseq comparisons revealed that in TY10 cells, 1167 genes were upregulated and 94 downregulated in comparison to HUVEC where 185 and 0 genes were up or downregulated respectively and that in all 3 brain endothelial cell lines that the TJ protein CLDN5 exhibited a significant reduction in expression at gene level (p=0.03). Conclusion: The results demonstrate that Artemisinin may have the capacity to transiently increase or decrease the permeability of the BBB. Therefore, Artemisinin may have a role in facilitating drug delivery to the cerebral parenchyma providing potential therapeutic benefit for the treatment of BMD or other related neurological conditions difficult to treat due to the BBB. Citation Format: Emily Appadurai, Fiona Ruge, Andrew Sanders, Wen G Jiang, Tracey A. Martin. Controlling tight junctions in the blood brain barrier (BBB) with artemisinin as a potential adjuvant in the treatment of metastatic disease [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-20-04.

Using Electric Cell-substrate Impedance Sensing to Characterize Effects of Curcumin on NRK Cells

Electric Cell-substrate Impedance Sensing (ECIS) is a real-time, label-free technique that can monitor a variety of cell behaviors on the same platform, including cell-attachment, spreading, proliferation, migration, and cell-cell junction formation. We demonstrate the use of ECIS to study the dose-dependent effects of curcumin on normal rat kidney cells (NRK) and compare results to traditional cell viability and wound healing assays. Curcumin is a lipophilic polyphenolic compound found in the tuberous root of the Curcuma longa plant (also known as turmeric) and used for centuries in Ayurvedic and traditional Chinese medicine for its suspected anti-inflammatory, anti-oxidant, anti-microbial, anti-carcinogenic, anti-mutagenic, and anti-atherosclerotic properties. Current research is aimed at understanding the effects of curcumin at the cellular level and at identifying its specific molecular targets. Cumulative results generated from different experimental strategies applied to different cell lines suggest curcumin can affect a broad range of cell behaviors and signaling pathways in a cell-type specific manner. Our understanding of the cellular effects of curcumin, as well as other naturopathic compounds, could benefit from the application of a common set of molecular or physiological assays across different cell types. The ability of ECIS to generate a multiparameter data set for any given cell line is well suited to serve as such an assay.