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

Abstract

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.

Keywords: Calcium electroporation; Calcium kinetics; Endothelial cells; Transcriptomic profiling.