Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

Emrah Eroğlu

doi:10.17776/csj.1114125

EN TR

Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

Abstract

Hydrogen peroxide (H2O2) is a critical signaling molecule in vascular cells, which controls signaling events, yet it can cause pathological oxidative stress in excess. The lack of suitable tools undermined experimental approaches to study the role of oxidative eu- and distress in cellular ultra-locales. This study exploits a yeast-derived D-amino acid oxidase (mDAAO) as a chemogenetic tool to induce, visualize and test the cytotoxicity of H2O2 in hardly transfectable endothelial cells. Due to the poor transfectability of endothelial cells, lentiviral vectors have been used to generate cell lines stably expressing mDAAOs. mDAAOs are substrate-based chemogenetic enzymes that convert D-amino acids to their corresponding alpha-keto acids and generate H2O2 as a byproduct, which can be visualized with a novel ultrasensitive, and ratiometric H2O2 biosensor termed HyPer7. This study tested the suitability of two different D-amino acids, including D-alanine and D-methionine, to induce oxidative stress in endothelial cells. Live-cell imaging experiments unveiled that 10 mM D-methionine generated significantly higher and faster H2O2 signals than D-alanine. However, both D-amino acids induced comparable levels of cell death documented by a colorimetric cell metabolic activity assay (MTT). This study provides a guide for manipulating and monitoring the cytotoxic effect of H2O2 in endothelial cells.

Keywords

Zor Transfekte Edilebilir Endotel Hücrelerinde Kemogenetik Olarak İndüklenmiş Hidrojen Peroksitin Eşzamanlı Manipülasyonu ve Görüntülenmesi

Öz

Hidrojen peroksit (H2O2) vasküler hücrelerinde sinyal yolaklarını kontrol eden önemli bir sinyal molekülüdür, ancak yüksek konsantrasyonu hücrede patolojik oksidatif strese neden olabilir. Uygun araçların yetersizliği nedeniyle, faydalı ve zararlı oksidatif stresin hücre altı lokallerde araştırılmasında deneysel zorluklarla karşılaşılmaktadır. Bu çalışmada, zor transfekte edilebilir endotel hücrelerinde H2O2'yi indüklemek, görselleştirmek ve sitotoksisitesini test etmek için bir kemogenetik araç olan maya türevli D-amino asit oksidaz (mDAAO) enzimi kullanıldı. Endotel hücrelerinin transfeksiyon yüzdesinin düşük olması nedeniyle, lentiviral vektörler kullanılarak mDAAO enzimini ifade eden sabit hücre hatları oluşturuldu. Substrat bazlı kemogenetik araç olan mDAAO enzimi, D-amino asitleri çevirerek yan ürün olarak H2O2’yi üretmektedir; ve H2O2 HyPer7 olarak adlandırılan ultra-duyarlı ve orantı-bazlı H2O2 biyosensörü ile görüntülenmektedir. Bu çalışmada, D-alanin ve D-metionin olmak üzere iki farklı D-amino asidin endotel hücrelerinde oksidatif stresi indükleme kapasiteleri test edildi. Canlı hücre görüntüleme deneyleri, 10 mM D-metionin’in D-alanin'den önemli ölçüde daha yüksek ve daha hızlı H2O2 sinyali ürettiğini ortaya çıkardı. Ancak, yapılankolorimetrik hücre metabolik aktivite testi (MTT) ile, her iki amino asidin benzer sitotoksik etkiye sahip olduğu gösterildi. Bu çalışma, endotel hücrelerinde H2O2’nin kontrolü ve sitotoksik etkisinin takibinde başvurulabilecek bir kılavuz niteliğindedir.

Anahtar Kelimeler

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Details

Primary Language

English

Subjects

Structural Biology , Toxicology

Journal Section

Research Article

Authors

Emrah Eroğlu ^*
0000-0002-9373-0808
Türkiye

Publication Date

December 27, 2022

Submission Date

May 9, 2022

Acceptance Date

September 28, 2022

Published in Issue

Year 2022 Volume: 43 Number: 4

DOI

https://doi.org/10.17776/csj.1114125

IZ

https://izlik.org/JA83MH67JH

Cite

RIS / Bibtex

APA

Eroğlu, E. (2022). Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells. Cumhuriyet Science Journal, 43(4), 645-651. https://doi.org/10.17776/csj.1114125

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