Ca2+ dependence and kinetics of cell membrane repair after electropermeabilization

Bhattacharya, Sayak; Šilkūnas, Mantas; Gudvangen, Emily; Mangalanathan, Uma; Pakhomova, Olga N.; Pakhomov, Andrei G.

doi:10.1016/j.bbamem.2021.183823

Cited by 14 publications

(6 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, according to immunofluorescence images and mean fluorescence intensity of VE-cadherin cell-cell junctions maintained their structural integrity. This is consistent with observed Ca 2+ -dependent membrane repair after electropermeabilization, where perfusion with 2 mM external Ca 2+ advanced membrane resealing, which could have a positive effect on cell-cell junction preservation [ 66 ]. Although immunofluorescence of VE-cadherin 1 h after treatment shows that both cell lines are similarly affected, the higher mean fluorescence intensity of VE-cadherin in HMEC-1 cells at 4 and 24 h time points may indicate more pronounced Ca 2+ -mediated membrane resealing compared to Ea.hy926 cells (Fig.…”

Section: Discussionsupporting

confidence: 89%

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles

Lisec,

Bozic,

Santek

et al. 2024

Cell Commun Signal

View full text Add to dashboard Cite

Background Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. Methods CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. Results EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926’s actin filaments, microtubules, and cell–cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. Conclusions Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

show abstract

Section: Discussionsupporting

confidence: 89%

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles

Lisec,

Bozic,

Santek

et al. 2024

Cell Commun Signal

View full text Add to dashboard Cite

show abstract

“…This result suggests that the degree of membrane damage is not the sole predictor of cell survival, and that damaged SMC are less capable of recovering from PEF-induced damage than similarly damaged pre-malignant epithelial cells. The exact mechanisms responsible for the poorer survival of SMC are not known, but may include less efficient membrane repair [50] and the reduced capacity to restore the cytosolic Ca 2+ level and osmotic balance [35,49,51].…”

Section: Discussionmentioning

confidence: 99%

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study

Gudvangen

Mangalanathan

Semenov

et al. 2023

IJMS

Self Cite

View full text Add to dashboard Cite

Cancer ablation therapies aim to be efficient while minimizing damage to healthy tissues. Nanosecond pulsed electric field (nsPEF) is a promising ablation modality because of its selectivity against certain cell types and reduced neuromuscular effects. We compared cell killing efficiency by PEF (100 pulses, 200 ns–10 µs duration, 10 Hz) in a panel of human esophageal cells (normal and pre-malignant epithelial and smooth muscle). Normal epithelial cells were less sensitive than the pre-malignant ones to unipolar PEF (15–20% higher LD50, p < 0.05). Smooth muscle cells (SMC) oriented randomly in the electric field were more sensitive, with 30–40% lower LD50 (p < 0.01). Trains of ten, 300-ns pulses at 10 kV/cm caused twofold weaker electroporative uptake of YO-PRO-1 dye in normal epithelial cells than in either pre-malignant cells or in SMC oriented perpendicularly to the field. Aligning SMC with the field reduced the dye uptake fourfold, along with a twofold reduction in Ca2+ transients. A 300-ns pulse induced a twofold smaller transmembrane potential in cells aligned with the field, making them less vulnerable to electroporation. We infer that damage to SMC from nsPEF ablation of esophageal malignancies can be minimized by applying the electric field parallel to the predominant SMC orientation.

show abstract

“…Smaller pores stabilize and can increase in size to allow larger molecules, such as pDNA, to enter the cell [ 127 ]. Under the right electrical conditions, this permeabilization is temporary and reversible, and after the electrical pulses are complete, the membrane seals within minutes [ 128 , 129 , 130 ]. By creating pores to facilitate movement of DNA, EP significantly increases DNA uptake by cells, therefore increasing the total expression yield when compared to injections without EP [ 16 , 123 , 131 ].…”

Section: Electroporation As a Methods For Pdna Deliverymentioning

confidence: 99%

Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases

Sussman,

Liberatore,

Drozdz

2024

Pharmaceutics

View full text Add to dashboard Cite

Gene therapy and its role in the medical field have evolved drastically in recent decades. Studies aim to define DNA-based medicine as well as encourage innovation and the further development of novel approaches. Gene therapy has been established as an alternative approach to treat a variety of diseases. Its range of mechanistic applicability is wide; gene therapy has the capacity to address the symptoms of disease, the body’s ability to fight disease, and in some cases has the ability to cure disease, making it a more attractive intervention than some traditional approaches to treatment (i.e., medicine and surgery). Such versatility also suggests gene therapy has the potential to address a greater number of indications than conventional treatments. Many DNA-based therapies have shown promise in clinical trials, and several have been approved for use in humans. Whereas current treatment regimens for chronic disease often require frequent dosing, DNA-based therapies can produce robust and durable expression of therapeutic genes with fewer treatments. This benefit encourages the application of DNA-based gene therapy to manage chronic diseases, an area where improving efficiency of current treatments is urgent. Here, we provide an overview of two DNA-based gene therapies as well as their delivery methods: adeno associated virus (AAV)-based gene therapy and plasmid DNA (pDNA)-based gene therapy. We will focus on how these therapies have already been utilized to improve treatment of chronic disease, as well as how current literature supports the expansion of these therapies to treat additional chronic indications in the future.

show abstract

Ca2+ dependence and kinetics of cell membrane repair after electropermeabilization

Cited by 14 publications

References 55 publications

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles

Characterization of two distinct immortalized endothelial cell lines, EA.hy926 and HMEC-1, for in vitro studies: exploring the impact of calcium electroporation, Ca2+ signaling and transcriptomic profiles

Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study

Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases

Contact Info

Product

Resources

About