Electric Cell-substrate Impedance Sensing Research Articles

BSBM-13 MELANOMA CELLS PRODUCE A SUSPENSION SUB-POPULATION THAT DRASTICALLY DISRUPTS THE BLOOD-BRAIN BARRIER ENDOTHELIUM

Abstract Melanoma metastasizes to the brain with high propensity. Brain metastasis requires cancer cells to breach the blood-brain barrier (BBB) endothelium, which forms the first line of defence against blood-borne pathogens. An increasing number of molecules are discussed in BBB disruption, and their importance varies across melanoma and endothelial cell lines. This diversity in literature proposes that cancer cells of the same cancer type employ different modes of metastasis. This study shows an example of the difference in migration capabilities of adherent melanoma cells and a suspension sub-population. We used Electric Cell-substrate Impedance Sensing (ECIS) to assess changes in human brain endothelial barrier function upon adding human melanoma lines. Several melanoma lines induced brain endothelial barrier disruption within the first few hours of addition. Intriguingly, the suspension population of melanoma cells disrupted the endothelial barrier faster and more drastically than the adherent counterpart. Time-lapse imaging revealed that the suspension population used a transcellular route of invasion compared to the paracellular route used by the adherent cells. Confocal microscopy supported this, showing clear differences in the physical interaction of the suspension subtype with the brain endothelium, compared to the adherent cells. Flow cytometry and NanoString technology allowed characterization of the melanoma lines, showing that the suspension subtype protein composition differs from their adherent counterpart. We hypothesize that melanoma cells may produce specific subtypes that are more invasive, which can accelerate migration through the brain endothelium. We stress the importance of investigating the composition of melanoma cells and their bi-products when researching cancer metastasis. Once successfully within the brain parenchyma, these cancer cells are effectively separated from the peripheral adaptive immune system, designed to detect and eliminate them. Therefore, preventing cancer cell migration into the brain is an attractive consideration, as they are considerably more targetable whilst still in the blood.

#5930 ENDOTHELIAL CELL PROTEOME AND SECRETOME ALTERATION UNDER CHRONIC UREMIC EXPOSURE

Abstract Background and Aims Dialysis therapies including peritoneal dialysis (PD) are described to lead to premature vascular aging, atherosclerosis and associated cardiomyopathies, which are associated with high cardiovascular morbidity and mortality. Chronic inflammation, incomplete clearance of uremic toxins, and dyslipidaemia, are factors affecting the vasculature system. However, how these factors affect the endothelium, the fist cell layer in contact with these molecules, and the molecular mechanisms triggered by uremic toxins remain poorly understood. In vitro and in vivo studies have shown that cytoprotective additives (e.g. dipeptide alanyl-glutamine (AlaGln) or kinase inhibitor lithium chloride (LiCl)) to PD fluids reduce peritoneal damage in mesothelial and endothelial (EC) cells. Their potential systemic effects were not studied so far. Here, we demonstrate, in a newly developed model system, the changes induced by chronic exposure to serum of PD patients in the proteome and secretome of endothelial cells, and the effects of cytoprotective additives. Method For modelling systemic conditions of PD patients, primary EC were cultured for 5 passages in medium containing 10% serum collected from PD patients (n = 26) during regular PET tests, or from healthy donors (n = 12). Cytoprotective additives were added in parallel experiments. Cells were stably labelled (SILAC) to differentiate cell and donor serum proteins and the cellular proteome and secretome profiles were analysed by quantitative mass spectrometry. Prior to analysis of the secretome, equalizer beads, to enrich low abundant and deplete high abundant proteins, were used. ECIS (Electric Cell-substrate Impedance Sensing), was used to measure barrier function, growth rate, and permeability. Results Proteome analysis revealed perturbation of major cellular processes by serum of PD patients including inflammatory related processes such TLR regulation and complement activation, as well membrane related processes such as extracellular matrix interactions and junctions, and plasma lipoprotein remodelling. Addition of LiCl counteracted cell-adhesion related proteins. ECIS analysis showed that in uremic conditions the EC monolayer has a decreased barrier function compared to healthy conditions, and LiCl partially restores the tightness of the membrane. Secretome analysis showed differentially regulated proteins related to oxidative stress, senescence-associated secretory phenotype (SASP), and apoptosis. Interestingly, in the secretome, LiCl addition counter regulated INHBA and tissue factor pathway inhibitor 2, both proteins related to vascular calcification. Conclusion Surprisingly few studies have analysed uremic effects on EC using proteomics approaches, and no reports of chronic settings, modelling the patient situation, are available. Our data demonstrates that EC react to serum factors of PD patients with increased inflammation, permeability and a secretory profile. Interestingly, our model reflects many of the known effects on the vasculature, but unravels the molecular mechanism that may induce those processes. We have also identified potential mechanisms by which the addition of cytoprotective additives may counteract some of the uremic effects systemically. Secretomics data identified several proteins secreted by EC that are regulated in uremia with potential for cellular crosstalk with other cells of the vasculature, showing the potential to identify therapeutic targets to reduce the cardiovascular risk of PD patients and current limitations of the therapy.

A simplified workflow with end-point validation of real-time electrical cell-substrate impedance sensing of retinoic acid stimulated neurogenesis in human SH-SY5Y cells in vitro

ObjectiveRetinoic acid (RA) is known to transition proliferating SH-SY5Y neuroblastoma cells towards functional neurons. However, the activity of RA is restricted due to its photolability where any findings from prolonged time course observations using microscopy may alter outcomes. The aim of the study was to establish a real-time, long-term (9-day) protocol for the screening of differentiation events using Electrical cell-substrate impedance sensing (ECIS).Results and discussionA differentiation baseline for SH-SY5Y cells was established. Cells were seeded and exposed to repeated spikes of RA using the xCELLigence real-time cell analyser single plate (RTCA-SP) for real-time monitoring and identification of differentiation activity over a 9 day period in order to be more representative of differentiation over a prolonged timeline. Specific features associated with differentiation (growth inhibition, neurite outgrowths) were confirmed by end-point analysis.RA-induced growth inhibition and assumed phenotypic changes (i.e. neurite outgrowth) were identified by the xCELLigence analysis and further confirmed by end-point metabolic and phenotypic assays. Change in cellular morphology and neurite outgrowth length was identified by end-point fluorescence detection followed by computational analysis. Based on this it was possible to identify SH-SY5Y phenotypic differentiation with distinct phases observed over 9 days using Electric cell-substrate impedance sensing (ECIS) cell index traces providing a path to application in larger scale neurotrophic factor screening using this scalable technology.

Optimal interdigitated electrode sensor design for biosensors using multi-objective particle-swarm optimization

<p>Interdigitated electrodes (IDEs) are commonly employed in biological cellular characterization techniques such as electrical cell-substrate impedance sensing (ECIS). Because of its simple production technique and low cost, interdigitated electrode sensor design is critical for practical impedance spectroscopy in the medical and pharmaceutical domains. The equivalent circuit of an IDE was modeled in this paper, it consisted of three primary components: double layer capacitance, C<sub>dl</sub>, solution capacitance, C<sub>Sol</sub>, and solution resistance, R<sub>Sol</sub>. One of the challenging optimization challenges is the geometric optimization of the interdigital electrode structure of a sensor. We employ metaheuristic techniques to identify the best answer to problems of this kind. multi-objective optimization of the IDE using multi-objective particle swarm optimization (MOPSO) was achieved to maximize the sensitivity of the electrode and minimize the Cut-off frequency. The optimal geometrical parameters determined during optimization are used to build the electrical equivalent circuit. The amplitude and phase of the impedance versus frequency analysis were calculated using EC-LAB® software, and the corresponding conductivity was determined.</p>

PEDOT:PSS-Based Bioelectrodes for Multifunctional Drug Release and Electric Cell-Substrate Impedance Sensing.

Electric cell-substrate impedance sensing (ECIS) is an innovative approach for the label-free and real-time detection of cell morphology, growth, and apoptosis, thereby playing an essential role as both a viable alternative and valuable complement to conventional biochemical/pharmaceutical analysis in the field of diagnostics. Constant improvements are naturally sought to further improve the effective range and reliability of this technology. In this study, we developed poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) conducting polymer (CP)-based bioelectrodes integrated into homemade ECIS cell-culture chamber slides for the simultaneous drug release and real-time biosensing of cancer cell viability under drug treatment. The CP comprised tailored PEDOT:PSS, poly(ethylene oxide) (PEO), and (3-glycidyloxypropyl)trimethoxysilane (GOPS) capable of encapsulating antitumor chemotherapeutic agents such as doxorubicin (DOX), docetaxel (DTX), and a DOX/DTX combination. This device can reliably monitor impedance signal changes correlated with cell viability on chips generated by cell adhesion onto a predetermined CP-based working electrode while simultaneously exhibiting excellent properties for both drug encapsulation and on-demand release from another CP-based counter electrode under electrical stimulation (ES) operation. Cyclic voltammetry curves and surface profile data of different CP-based coatings (without or with drugs) were used to analyze the changes in charge capacity and thickness, respectively, thereby further revealing the correlation between their drug-releasing performance under ES operation (determined using ultraviolet-visible (UV-vis) spectroscopy). Finally, antitumor drug screening tests (DOX, DTX, and DOX/DTX combination) were performed on MCF-7 and HeLa cells using our developed CP-based ECIS chip system to monitor the impedance signal changes and their related cell viability results.

Transient changes in endothelial and epithelial cell cytoskeleton and morphology upon olfactory receptor signaling

Olfactory receptors (ORs) are expressed in various non-nasal tissues; however, their physiological roles are largely unknown. ORs are G-protein coupled receptors (GPCRs), and there is a well-established link between different classes of G-proteins and cytoskeletal structure changes affecting cellular morphology that has been unexplored after odorant binding. Thus, the present study was conducted to determine if odorant binding in non-olfactory cells causes cytoskeletal changes that lead to cell morphological changes. To this end, human umbilical vein endothelial cells (HUVECs), which express OR10J5, and human keratinocyte (HaCaT) cells, which express OR2AT4, were used to conduct this study. Using these two different cell lines, it was shown that odorant addition, lyral and Sandalore, respectively, caused a dose-dependent increase in cAMP. Furthermore, HUVECs exposed to lyral exhibited a disassembly of the tubulin-containing microtubules and an increase in cortical actin microfilaments, whereas HaCaT cell exposure to Sandalore exhibited both a disassembly of the tubulin-containing microtubules and cortical actin microfilaments. Even though the endothelial and epithelial cell lines had different cytoskeletal responses to odorant exposure, both exhibited a change in cellular morphology as detected by a decrease in electrical resistance using Electric Cell-substrate Impedance Sensing (ECIS; Applied Biophysics, Inc., Troy, NY). Interestingly, these odorant-induced cytoskeletal and morphological changes occurred rapidly (under 1 hour) but returned to normal after 20 hours, highlighting the complexity of olfactory signaling, which may involve desensitization of the signaling pathway and/or internalization of the odorant receptor. Future experiments will further elucidate the olfactory receptor signaling mechanism in endothelial and epithelial cells so that their physiological role can be fully understood. Funded by Army Research Office STTR #W911NF-19-P-0014 and W911NF-20-C-0014, the SUNY Cortland Undergraduate Research Council, and Research and Sponsored Programs. This is the full abstract presented at the American Physiology Summit 2023 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-mediated oxidation of low-density lipoprotein (oxLDL) contributes to lung microvascular endothelial dysfunction during sepsis

Introduction: Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis-induced acute lung injury. Plasma cell-free hemoglobin (CFH) is elevated in approximately 80% of patients with sepsis and is independently associated with development of acute respiratory distress syndrome (ARDS) and mortality. Oxidized CFH (ferric, 3+) can oxidize low-density lipoprotein (oxLDL), which signals through its major endothelial receptor, the lectin-like oxidized LDL receptor 1 (LOX-1), to cause endothelial dysfunction through several pro-inflammatory pathways including activation of mitogen-activated protein kinases (MAPKs). However, little is known regarding whether LOX-1 receptor signaling leads to microvascular endothelial hyperpermeability or acute lung injury, especially in the context of sepsis. We hypothesized that oxidation of LDL by CFH contributes to lung microvascular endothelial barrier dysfunction and worse outcomes during sepsis through LOX-1 and downstream p38 MAPK. Methods: To test whether generation of oxLDL by CFH is associated with endothelial injury in clinical sepsis, circulating levels of CFH, oxLDL, and vascular injury marker sVE-cadherin were measured in 24 sepsis patients via ELISA and tested for association with development of ARDS. LDL was oxidized by combining LDL with CFH in a test tube overnight at 37°C and oxLDL was quantified by TBARS assay. In primary human lung microvascular endothelial cells (HLMVEC) transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell-substrate Impedance Sensing (ECIS). LOX-1 receptor was blocked using BI-0115 (Boehringer Ingelheim, 20 μM) and p38 MAPK was inhibited using NiPp (100 μM). Results: In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686, p=0.016) and sVE-cadherin (r=0.603, p=0.012), and tended to be higher in those who developed ARDS (38 U/L [IQR 27, 45] vs. 27 U/L [IQR 19, 35], p=0.1). Oxidation of LDL by CFH exacerbated HLMVEC barrier dysfunction compared to control, LDL, or CFH. Barrier dysfunction induced by CFH or oxLDL was attenuated by blocking the LOX-1 receptor or p38 MAPK. Conclusions: Increased plasma CFH and oxLDL are associated with vascular injury during clinical sepsis; one mechanism by which CFH may cause vascular hyperpermeability and sepsis-mediated lung injury is through oxidation of LDL which can drive signaling through the endothelial LOX-1 receptor and activation of p38 MAPK. NIH R35HL150783, R21GM144915, R01HL158906, R01HL164937, T32HL094296; Parker B. Francis Fellowship This is the full abstract presented at the American Physiology Summit 2023 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.

Senomorphic properties of miR-181b in aged human aortic smooth muscle cells

Introduction: Age associated large artery stiffness (LAS) is an independent predictor of adverse cardiovascular health outcomes in humans and has also been shown to be associated with the development of heart failure, kidney disease, and even dementia. Furthermore, the development of cellular senescence in the aorta, the permanent arrest of the cell cycle and development of pro-inflammatory, pro-fibrotic senescence associated secretory phenotype (SASP), has also been shown to contribute to age associated LAS. MicroRNAs (miR) are small, non-coding RNAs that down-regulate protein expression in a semi-specific manner. miR-181b has been shown to be downregulated with aging, targets many of the secreted factors associated with the aortic SASP and has previously been shown to have a beneficial effect on vascular health. With smooth muscle cells being the predominant cell type in the large arteries, we wanted to address the hypothesis that miR-181b has the potential to act as a senomorphic in human aortic smooth muscle cells (HASMCs) and promote beneficial changes in individual cells that influence LAS. Methods: To test our hypothesis, we obtained young and old HASMCs and transfected them with miR-181b or a scrambled miR control utilizing a lipofectamine transfection protocol. Total RNA was isolated from the HASMCs and quantitative PCR was performed to assess changes of the SASP profile. Cell proliferation was measured using electric cell-substrate impedance sensing (ECIS) technology. Lastly, individual HASMC stiffness measurements were made using twisting magnetic microscopy. Results: Transfection of HASMCs with miR-181b resulted in reduced expression of markers of cell cycle arrest (p16 and p21) and the SASP profile (IL-6, TNF-alpha, MCP1, TGF-b1, IL-1alpha, and MMP2) when compared to scrambled control (p<0.05). HASMC proliferation was decreased in old HASMCs compared to young (p<0.05) and was increased with miR-181b transfection when compared to scramble control (p<0.05). Lastly, individual HASMC stiffness was lower in young cells compared to old (0.4382 ± 0.29 vs. 0.9959 ± 0.72 Pa/nm, n=120, p<0.001). Cell stiffness was decreased in old HASMCs when treated with miR-181b when compared with control (0.7856 ± 0.144 vs. 1.18 ± 0.288 Pa/nm, n=76, p<0.001), but there was no difference in young HASMCs. Conclusions: miR-181b appears to act as a senomorphic agent when transfected in old HASMCs. This is evidenced by noticeable changes in the expression of markers of cellular senescence which corresponded with positive changes in cellular characteristics that are associated with LAS, namely the improvement in cellular proliferation and reduction of individual cell stiffness in the old HASMC groups. Together, these results suggest the modulation of miR-181b may be a potential therapeutic target to improve age associated LAS through an effect on cellular senescence. K08 AG070281 (ET), Western Institute for Veterans Research (ET), U-CARES (ET), R01 AG048366 (LAL), R01 AG050238 (AJD), R01 AG060395 (AJD) This is the full abstract presented at the American Physiology Summit 2023 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.

Donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells

Abstract The Cholinergic Anti-inflammatory Pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Donepezil, a centrally acting reversible acetylcholinesterase inhibitor, has been used in the palliative treatment of Alzheimer’s disease (AD). We hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Endothelial permeability was evaluated by using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. Our data show that 25mM glucose concentration increased endothelial permeability and abrogated the wound healing process. Donepezil at the dose of 5 μM and 10 μM significantly prevented alteration of endothelial barrier function and enhances wound healing process in the presence of high glucose concentration.

SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process.

Abstract Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as diabetes and atherosclerosis. Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions. Vascular injury is associated with activation of the coagulation cascade and release of thrombin, which increases endothelial permeability by activating endothelial cell surface thrombin receptor. This increase in endothelial permeability is typically followed by a recovery period of ≈2 hours, during which barrier integrity is restored. It has been surmised that thrombin signaling stimulates intrinsic repair mechanisms that restore barrier function. To validate our finding we determined endothelial barrier function using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Our data show that 50mM thrombin increase endothelial permeability followed by complete recovery and reannealing of adherens junction in control endothelial cells. However, SPHK1 depleted cells showed significantly decrease in barrier disruption, and barrier failed recover completely compared to control monolayer. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease. CTRE Seed Grant to Professor Nadeem Fazal, MD, PhD

Abstract 712: IL-19 Is A Lymphokine That Induces Prox1-dependent Lymphangiogenesis And Permeability

Introduction: Atherosclerosis is a vascular inflammatory disease associated with lymphangiogenesis, but whether lymphangiogenesis contributes to disease progression or is simply a compensatory attempt at disease resolution is currently unresolved. Recent studies are revealing a role for lymphatic vessels in reverse cholesterol transport (RCT), which could attenuate atherosclerosis. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our hypothesis that one mechanism whereby IL-19 is atheroprotective is by promoting functional lymphangiogenesis and lymphatic flux. Methods & Results: IL-19 can significantly induce human dermal lymphatic endothelial cell (hdLEC) proliferation, migration, and tube formation, to the same degree as VEGFC, a potent inducer of lymphangiogenesis. RNA sequencing analysis indicates that IL-19 induces an angiogenic transcriptional program through Prox1, a master regulator of lymphangiogenesis. Knockdown of Prox1 led to a significant decrease in IL-19-associated proliferation and migration. IL-19 also increased expression of Angpt2, an endothelial cell protein that induces permeability through regulation of tight junctions, through a Prox1-dependent mechanism. Electric Cell-substrate Impedance Sensing assays showed that IL-19 treatment increased hdLEC permeability and mitigated oxLDL-associated decreased permeability, suggestive of maintaining lymphatic barrier function during atherosclerosis. IL-19 treatment also increased dil-HDL transport across an hdLEC barrier. Preliminary in vivo data suggests injection of rmIL-19 in Il19 -/- mice increases lymphatic permeability and flux through increased lymphatic uptake of Evans blue dye. Conclusion: These data suggest that IL-19 increases lymphangiogenesis, permeability, and may increase RCT to plausibly decrease atherosclerosis. These results can influence our understanding of the association between the lymphatic system and atherosclerosis.

Bone Morphogenetic Protein-4 Impairs Retinal Endothelial Cell Barrier, a Potential Role in Diabetic Retinopathy.

Bone Morphogenetic Protein 4 (BMP4) is a secreted growth factor of the Transforming Growth Factor beta (TGFβ) superfamily. The goal of this study was to test whether BMP4 contributes to the pathogenesis of diabetic retinopathy (DR). Immunofluorescence of BMP4 and the vascular marker isolectin-B4 was conducted on retinal sections of diabetic and non-diabetic human and experimental mice. We used Akita mice as a model for type-1 diabetes. Proteins were extracted from the retina of postmortem human eyes and 6-month diabetic Akita mice and age-matched control. BMP4 levels were measured by Western blot (WB). Human retinal endothelial cells (HRECs) were used as an in vitro model. HRECs were treated with BMP4 (50 ng/mL) for 48 h. The levels of phospho-smad 1/5/9 and phospho-p38 were measured by WB. BMP4-treated and control HRECs were also immunostained with anti-Zo-1. We also used electric cell-substrate impedance sensing (ECIS) to calculate the transcellular electrical resistance (TER) under BMP4 treatment in the presence and absence of noggin (200 ng/mL), LDN193189 (200 nM), LDN212854 (200 nM) or inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2; SU5416, 10 μM), p38 (SB202190, 10 μM), ERK (U0126, 10 μM) and ER stress (Phenylbutyric acid or PBA, 30 μmol/L). The impact of BMP4 on matrix metalloproteinases (MMP2 and MMP9) was also evaluated using specific ELISA kits. Immunofluorescence of human and mouse eyes showed increased BMP4 immunoreactivity, mainly localized in the retinal vessels of diabetic humans and mice compared to the control. Western blots of retinal proteins showed a significant increase in BMP4 expression in diabetic humans and mice compared to the control groups (p < 0.05). HRECs treated with BMP4 showed a marked increase in phospho-smad 1/5/9 (p = 0.039) and phospho-p38 (p = 0.013). Immunofluorescence of Zo-1 showed that BMP4-treated cells exhibited significant barrier disruption. ECIS also showed a marked decrease in TER of HRECs by BMP4 treatment compared to vehicle-treated HRECs (p < 0.001). Noggin, LDN193189, LDN212854, and inhibitors of p38 and VEGFR2 significantly mitigated the effects of BMP4 on the TER of HRECs. Our finding provides important insights regarding the role of BMP4 as a potential player in retinal endothelial cell dysfunction in diabetic retinopathy and could be a novel target to preserve the blood-retinal barrier during diabetes.

Modeling a nonlinear biophysical trend followed by long-memory equilibrium with unknown change point

Measurements of many biological processes are characterized by an initial trend period followed by an equilibrium period. Scientists may wish to quantify features of the two periods as well as the timing of the change point. Specifically, we are motivated by problems in the study of electrical cell-substrate impedance sensing (ECIS) data. ECIS is a popular new technology which measures cell behavior noninvasively. Previous studies using ECIS data have found that different cell types can be classified by their equilibrium behavior. However, it can be challenging to identify when equilibrium has been reached and to quantify the relevant features of cells’ equilibrium behavior. In this paper we assume that measurements during the trend period are independent deviations from a smooth nonlinear function of time, and that measurements during the equilibrium period are characterized by a simple long memory model. We propose a method to simultaneously estimate the parameters of the trend and equilibrium processes and locate the change point between the two. We find that this method performs well in simulations and in practice. When applied to ECIS data, it produces estimates of change points and measures of cell equilibrium behavior which offer improved classification of infected and uninfected cells.

Abstract P6-12-01: ALCAM (Activated Leukocyte Cell Adhesion Molecule) is associated with HGF/MET signalling pathway in different subtypes of breast cancer

Abstract Introduction. ALCAM (Activated Leukocyte Cell Adhesion Molecule), also known as CD166, is a cell adhesion molecule which belongs to the immunoglobulin superfamily and is widely expressed in various human tissues. It has been demonstrated that ALCAM plays an important role in the progression of malignant diseases and tumour metastasis in multiple cancer types including breast cancer. However, the molecular mechanism of ALCAM and cancer progression is currently unclear. The present study performed protein array analyses using ALCAM genetically manipulated cell models to select potential protein partners of ALCAM. The study focused on MET (Hepatocyte Growth Factor (HGF) Receptor), a prominent ALCAM interacting protein kinase and a protooncogene contributing to cancer progression and spread. Method. Human breast cancer cell lines MCF-7 and MDA-MB-231 were selected to create ALCAM knockdown cell models. A range of other breast cancer cell line with differing hormone receptor status were also used. Protein samples of transfected cells were used to perform Kinexus protein kinase microarray analysis. The protein interaction between ALCAM and other prospective protein kinases including MET was verified by the method of immunoprecipitation. Additionally, the ALCAM knockdown model was also assessed for the impact of ALCAM and HGF/MET on the biological functions by Electric Cell-substrate Impedance Sensing (ECIS). Results. MCF-7 and MDA MB-231 cells both were strongly expressed ALCAM. Cells models with ALCAM knocking down were successfully created by way of anti-ALCAM shRNA. We have shown on the protein kinase microarray analysis that the hepatocyte growth factor (HGF) receptor, MET was one of the kinases significantly affected following ALCAM knocking down. It was also found that the alteration of protein interaction between the MET protein kinase and ALCAM protein showed an opposite pattern between ER positive and ER negative ALCAM knocking down cells. This protein interaction was observed by immunoprecipitation in MDA-MB-361 and MDA-MB-231 cell lines, but not in MCF-7 cells. The biological analyses using the ECIS technologies showed that MET kinase inhibitors and exogenous recombinant HGF affected the ALCAM-mediated cell adhesion in different breast cancer subtypes. Conclusion. ALCAM is associated with HGF/MET signalling and the interaction between ALCAM and MET is different amongst subtypes of breast cancer which have different hormone receptor status. Citation Format: Yiming Yang, Andrew J. Sanders, Wen G. Jiang. ALCAM (Activated Leukocyte Cell Adhesion Molecule) is associated with HGF/MET signalling pathway in different subtypes of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-01.

Odorant Binding Causes Cytoskeletal Rearrangement, Leading to Detectable Changes in Endothelial and Epithelial Barrier Function and Micromotion

Non-olfactory cells have excellent biosensor potential because they express functional olfactory receptors (ORs) and are non-neuronal cells that are easy to culture. ORs are G-protein coupled receptors (GPCRs), and there is a well-established link between different classes of G-proteins and cytoskeletal structure changes affecting cellular morphology that has been unexplored for odorant sensing. Thus, the present study was conducted to determine if odorant binding in non-olfactory cells causes cytoskeletal changes that will lead to cell changes detectable by electric cell-substrate impedance sensing (ECIS). To this end, we used the human umbilical vein endothelial cells (HUVECs), which express OR10J5, and the human keratinocyte (HaCaT) cells, which express OR2AT4. Using these two different cell barriers, we showed that odorant addition, lyral and Sandalore, respectively, caused an increase in cAMP, changes in the organization of the cytoskeleton, and a decrease in the integrity of the junctions between the cells, causing a decrease in cellular electrical resistance. In addition, the random cellular movement of the monolayers (micromotion) was significantly decreased after odorant exposure. Collectively, these data demonstrate a new physiological role of olfactory receptor signaling in endothelial and epithelial cell barriers and represent a new label-free method to detect odorant binding.

The Influence of Electrode Design on Detecting the Effects of Ferric Ammonium Citrate (FAC) on Pre-Osteoblast through Electrical Cell-Substrate Impedance Sensing (ECIS)

Detection sensitivity is a crucial factor in the application of ECIS sensors. For these biosensors, the electrode configuration has a direct impact on sensitivity, yet few studies on monopolar electrodes have been reported. In this study, ECIS sensor arrays, which have a series of working electrode configuration with a wide diameter range and different electrode number, were fabricated to monitor living osteoblast-like MC3T3-E1 cells. The experimental results revealed that when the electrode diameter was larger than 25 μm, electrodes with smaller diameter and number yielded higher impedance values and generated more impedance shift to cell status change. The membrane capacitance obtained by equivalent circuit fitting was at the same level. When the electrode diameter was even smaller, the results in detection of cell monolayer were opposite, and there was no distinct relationship between impedance and membrane capacitance shift to cell status change and electrode geometry. The proposed sensor chip, allowing for a sustained and stable detection of cellular impedance, provides the basis for the selection of the electrode configuration of monopolar electrodes. The test results of electrodes with a diameter of 25 μm and lower indicated the possibility of single cell impedance measurement, which can provide unique insight into the heterogeneous electrical behavior of cells, and, in this case, the electrode size should be close to the cell size.

Combining Electrostimulation with Impedance Sensing to Promote and Track Osteogenesis within a Titanium Implant

(1) Background: Electrical stimulation is a promising alternative to promote bone fracture healing but with the limitation of tracking the osteogenesis progress in vivo. To overcome this issue, we present an opportunity to combine the electrical stimulation of a commercial titanium implant, which promotes osteogenesis within the fracture, with a real-time readout of the osteogenic progress by impedance sensing. This makes it possible to adjust the electrical stimulation modalities to the individual patient's fracture healing process. (2) Methods: In detail, osteogenic differentiation of several cell types was monitored under continuous or pulsatile electrical stimulation at 0.7 V AC/20 Hz for at least seven days on a titanium implant by electric cell-substrate impedance sensing (ECIS). For control, chemical induction of osteogenic differentiation was induced. (3) Results: The most significant challenge was to discriminate impedance changes caused by proliferation events from those initiated by osteogenic differentiation. This discrimination was achieved by remodeling the impedance parameter Alpha (α), which increases over time for pulsatile electrically stimulated stem cells. Boosted α-values were accompanied by an increased formation of actin stress fibers and a reduced expression of the focal adhesion kinase in the cell periphery; morphological alterations known to occur during osteogenesis. (4) Conclusions: This work provided the basis for developing an effective fracture therapy device, which can induce osteogenesis on the one hand, and would allow us to monitor the induction process on the other hand.

IL-33 via PKCμ/PRKD1 Mediated α-Catenin Phosphorylation Regulates Endothelial Cell-Barrier Integrity and Ischemia-Induced Vascular Leakage.

Angiogenesis, neovascularization, and vascular remodeling are highly dynamic processes, where endothelial cell-cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity, and barrier function. The cadherin-catenin adhesion complex is a key contributor to inner blood-retinal barrier (iBRB) integrity and dynamic cell movements. However, the pre-eminent role of cadherins and their associated catenins in iBRB structure and function is not fully understood. Using a murine model of oxygen-induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we try to understand the significance of IL-33 on retinal endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability. Using electric cell-substrate impedance sensing (ECIS) analysis and FITC-dextran permeability assay, we observed that IL-33 at a 20 ng/mL concentration induced endothelial-barrier disruption in HRMVECs. The adherens junction (AJs) proteins play a prominent role in the selective diffusion of molecules from the blood to the retina and in maintaining retinal homeostasis. Therefore, we looked for the involvement of adherens junction proteins in IL-33-mediated endothelial dysfunction. We observed that IL-33 induces α-catenin phosphorylation at serine/threonine (Ser/Thr) residues in HRMVECs. Furthermore, mass-spectroscopy (MS) analysis revealed that IL-33 induces the phosphorylation of α-catenin at Thr654 residue in HRMVECs. We also observed that PKCμ/PRKD1-p38 MAPK signaling regulates IL-33-induced α-catenin phosphorylation and retinal endothelial cell-barrier integrity. Our OIR studies revealed that genetic deletion of IL-33 resulted in reduced vascular leakage in the hypoxic retina. We also observed that the genetic deletion of IL-33 reduced OIR-induced PKCμ/PRKD1-p38 MAPK-α-catenin signaling in the hypoxic retina. Therefore, we conclude that IL-33-induced PKCμ/PRKD1-p38 MAPK-α-catenin signaling plays a significant role in endothelial permeability and iBRB integrity.

The Autotaxin-LPA Axis Emerges as a Novel Regulator of Smooth Muscle Cell Phenotypic Modulation during Intimal Hyperplasia.

Neointimal hyperplasia is characterized by a loss of the contractile phenotype of vascular smooth muscle cells (VSMCs). Our group has recently shown that VSMC proliferation and migration are mediated by lysophosphatidic acid (LPA) during restenosis, but the role of autotaxin (ATX; lysophospholipase D), which produces LPA, remains unclear. Endothelial denudation of the mouse carotid artery was performed to induce neointimal hyperplasia, and the extent of damage caused by the ATX-LPA axis was assessed in VSMCs. We observed the upregulation of ATX activity (p < 0.0002) in the injured carotid artery using an AR2 probe fluorescence assay. Further, the tissue carotid LPA levels were elevated 2.7-fold in carotid vessels, augmenting neointimal hyperplasia. We used an electrical cell-substrate impedance sensor (ECIS) to measure VSMC proliferation and migration. Treatment with an ATX inhibitor (PF8380) or LPA receptor inhibitor (Ki16425) attenuated VSMC proliferation (extracellular signal-regulated kinases) activity and migration in response to recombinant ATX. Indeed, PF8380 treatment rescued the aggravated post-wire injury neointima formation of carotid arteries. The upregulation of ATX following vessel injury leads to LPA production in VSMCs, favoring restenosis. Our observations suggest that inhibition of the ATX-LPA axis could be therapeutically targeted in restenosis to minimize VSMC phenotypic modulation and inflammation after vascular injury.

Impedance Measurement for the Monitoring of In Vitro Cells Cultured in the Presence of Electromagnetic Waves

This paper explores the possibility of using the impedance measurement method used to monitor morphological changes in culture cells for use in cultures in the presence of an electromagnetic field generated by a mobile phone. For this purpose, we used Electric Cell–Substrate Impedance Sensing (ECIS), which is a real-time, label-free, impedance-based method to study cell behaviors in tissue culture. As part of the work, a device enabling the connection in a climatic chamber was prepared without the need to interfere with environmental conditions, and a test culture of mouse fibroblasts was performed. The device based on the Arduino UNO programmable platform worked like a mobile phone. During cell proliferation, it was connected to the device three times and a change in electrical parameters in the measuring system was observed. During the phone call, there was a clear change in the values of the measured parameters. However, analysis of the obtained results indicated that there was little or no effect of the presence of the electromagnetic field on the cell culture, while the observed changes in the values of impedance, resistance, and capacitance are most likely due to the separation of positive and negative medium ions in the electromagnetic field. The application of the presented method seems possible; however, in order to eliminate the separation of ions, a different type of antenna should be designed to emit a homogeneous field to the entire well.