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

References

[1] Sies H., Belousov VV., Chandel N.S., Davies M.J., Jones D.P., Mann G.E., Murphy M.P., Yamamoto M., Winterbourn C., Defining roles of specific reactive oxygen species (ROS) in cell biology and physiology, Nat Rev Mol Cell Biol., 23(7) (2022) 499-515
[2] Panth N., Paudel K.R., Parajuli K., Reactive Oxygen Species: A Key Hallmark of Cardiovascular Disease, Adv Med., 2016 (2016) 9152732
[3] Rana J.S., Khan S.S., Lloyd-Jones D.M., Sidney S., Changes in Mortality in Top 10 Causes of Death from 2011 to 2018, J. Gen. Intern Med., 36(8) (2021) 2517-2518.
[4] Senoner T., Dichtl W., Oxidative Stress in Cardiovascular Diseases: Still a Therapeutic Target?, Nutrients, 11(9) (2019) 2090
[5] Abdul-Muneer P.M., Chandra N., Haorah J., Interactions of oxidative stress and neurovascular inflammation in the pathogenesis of traumatic brain injury, Mol. Neurobiol., 51(3) (2015) 966-79
[6] Sharma P., Jha A. B., Dubey R. S., Pessarakli M., Reactive Oxygen Species, Oxidative Damage, and Antioxidative Defense Mechanism in Plants under Stressful Conditions, Journal of Botany, 217037 (2012) 2012
[7] Sies H., Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress, Redox Biol., 11 (2017) 613-619.
[8] Veal E., Day A., Hydrogen peroxide as a signaling molecule, Antioxid Redox Signal., 15 (1) (2011) 147-51

[9] Pizzino G., Irrera N., Cucinotta M., Pallio G., Mannino F., Arcoraci V., Squadrito F., Altavilla D., Bitto A., Oxidative Stress: Harms and Benefits for Human Health. Oxid Med Cell Longev., 2017 8416763 (2017)
[10] Chen Q., Wang Q., Zhu J., Xiao Q., Zhang L., Reactive oxygen species: key regulators in vascular health and diseases, Br. J. Pharmacol., 175(8) (2018) 1279-1292.
[11] Carvalho C., Moreira P.I., Oxidative Stress: A Major Player in Cerebrovascular Alterations Associated to Neurodegenerative Events, Front Physiol., 9 (2018) 806.
[12] Münzel T., Camici G.G., Maack C., Bonetti N.R., Fuster V., Kovacic J.C., Impact of Oxidative Stress on the Heart and Vasculature: Part 2 of a 3-Part Series, J Am Coll Cardiol, 70(2) (2017) 212-229.
[13] Sies H., On the history of oxidative stress: Concept and some aspects of current development, Current Opinion in Toxicology, 7 (2018)
[14] Eroglu E., Gottschalk B., Charoensin S., Blass S., Bischof H., Rost R., Madreiter-Sokolowski C.T., Pelzmann B., Bernhart E., Sattler W., Hallström S., Malinski T., Waldeck-Weiermair M., Graier W.F., Malli R., Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics, Nat Commun., 4 (2016) 7:10623.
[15] Pak V.V., Ezeriņa D., Lyublinskaya O.G., Pedre B., Tyurin-Kuzmin P.A., Mishina N.M., Thauvin M., Young D., Wahni K., Martínez Gache S.A., Demidovich A.D., Ermakova Y.G., Maslova Y.D., Shokhina A.G., Eroglu E., Bilan D.S., Bogeski I., Michel T., Vriz S., Messens J., Belousov V.V., Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function, Cell Metab.,31(3) (2020) 642-653.e6
[16] Depaoli M.R., Bischof H., Eroglu E., Burgstaller S., Ramadani-Muja J., Rauter T., Schinagl M., Waldeck-Weiermair M., Hay J.C., Graier W.F., Malli R., Live cell imaging of signaling and metabolic activities, Pharmacol Ther., 202 (2019) 98-119
[17] Eroglu E., Charoensin S., Bischof H., Ramadani J., Gottschalk B., Depaoli M.R., Waldeck-Weiermair M., Graier W.F., Malli R., Genetic biosensors for imaging nitric oxide in single cells, Free Radic Biol Med., 128 (2018) 50-58.
[18] Collins J.A., Kapustina M., Bolduc J.A., Pike J.F.W., Diekman B.O., Mix K., Chubinskaya S., Eroglu E., Michel T., Poole L.B., Furdui C.M., Loeser R.F., Sirtuin 6 (SIRT6) regulates redox homeostasis and signaling events in human articular chondrocytes, Free Radic Biol. Med., 166 (2021) 90-103.
[19] Eroglu E., Saravi S.S.S., Sorrentino A., Steinhorn B., Michel T., Discordance between eNOS phosphorylation and activation revealed by multispectral imaging and chemogenetic methods, Proc. Natl. Acad. Sci. USA., 116(40) (2019) 20210-20217
[20] Saravi S.S.S., Eroglu E., Waldeck-Weiermair M., Sorrentino A., Steinhorn B., Belousov V., Michel T., Differential endothelial signaling responses elicited by chemogenetic H2O2 synthesis, Redox Biol., 36 (2020) 101605.
[21] Sorrentino A., Steinhorn B., Troncone L., Saravi S.S.S., Badole S., Eroglu E., Kijewski M.F., Divakaran S., Di Carli M., Michel T., Reversal of heart failure in a chemogenetic model of persistent cardiac redox stress, Am. J. Physiol. Heart Circ. Physiol., 317(3) (2019) H617-H626.
[22] Ghaffari Zaki A., Erdoğan Y.C., Akgul Caglar T., Eroglu E., Chemogenetic approaches to dissect the role of H2O2 in redox-dependent pathways using genetically encoded biosensors, Biochem. Soc. Trans., 50(1) (2022) 335-345.
[23] Steinhorn B., Eroglu E., Michel T., Chemogenetic Approaches to Probe Redox Pathways: Implications for Cardiovascular Pharmacology and Toxicology, Annu. Rev. Pharmacol. Toxicol., 62 (2022) 551-571.
[24] Erdogan Y.C., Altun H.Y., Secilmis M., Ata B.N., Sevimli G., Cokluk Z., Zaki A.G., Sezen S., Akgul Caglar T., Sevgen İ., Steinhorn B., Ai H., Öztürk G., Belousov VV., Michel T., Eroglu E., Complexities of the chemogenetic toolkit: Differential mDAAO activation by d-amino substrates and subcellular targeting, Free Radic. Biol. Med., 177 (2021) 132-142.
[25] Steinhorn B., Sorrentino A., Badole S., Bogdanova Y., Belousov V., Michel T., Author Correction: Chemogenetic generation of hydrogen peroxide in the heart induces severe cardiac dysfunction, Nat. Commun., 12(1) (2021) 357.
[26] Smolyarova D.D., Podgorny O.V., Bilan D.S., Belousov V.V., A guide to genetically encoded tools for the study of H2O2, FEBS J., 289(18) (2021) 5382-5395.
[27] Secilmis M., Altun H.Y., Pilic J., Erdogan Y.C., Cokluk Z., Ata B.N., Sevimli G., Zaki A.G., Yigit E.N., Öztürk G., Malli R., Eroglu E., A Co-Culture-Based Multiparametric Imaging Technique to Dissect Local H2O2 Signals with Targeted HyPer7, Biosensors (Basel)., 11(9) (2021) 338.
[28] Kovala A.T., Harvey K.A., McGlynn P., Boguslawski G., Garcia J.G., English D., High-efficiency transient transfection of endothelial cells for functional analysis. FASEB J., 14(15) (2000) 2486-94.
[29] Eroglu E., Rost R., Bischof H., Blass S., Schreilechner A., Gottschalk B., Depaoli M.R., Klec C., Charoensin S., Madreiter-Sokolowski C.T., Ramadani J., Waldeck-Weiermair M., Graier W.F., Malli R., Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells, J. Vis. Exp., (121) (2017) 55486.
[30] Yockell-Lelièvre J., Riendeau V., Gagnon S.N., Garenc C., Audette M., Efficient transfection of endothelial cells by a double-pulse electroporation method, DNA Cell Biol., 28(11) (2009) 561-6.
[31] Charoensin S., Eroglu E., Opelt M., Bischof H., Madreiter-Sokolowski C.T., Kirsch A., Depaoli M.R., Frank S., Schrammel A., Mayer B., Waldeck-Weiermair M., Graier W.F., Malli R., Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS), Free Radic. Biol. Med., 102 (2017) 248-259.
[32] Opelt M., Eroglu E., Waldeck-Weiermair M., Russwurm M., Koesling D., Malli R., Graier W.F., Fassett J.T., Schrammel A., Mayer B., Formation of Nitric Oxide by Aldehyde Dehydrogenase-2 Is Necessary and Sufficient for Vascular Bioactivation of Nitroglycerin, J. Biol. Chem., 291(46) (2016) 24076-24084.
[33] Pollegioni L., Caldinelli L., Molla G., Sacchi S., Pilone M.S., Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources, Biotechnol. Prog., 20(2) (2004) 467-73
[34] Fraga S., Pinho M.J., Soares-da-Silva P., Expression of LAT1 and LAT2 amino acid transporters in human and rat intestinal epithelial cells, Amino Acids, 29(3) 2005) 229-33.
[35] Sorrentino A., Michel T., Redox à la carte: Novel chemogenetic models of heart failure, Br. J. Pharmacol, 177(14) (2020) 3162-3167.
[36] Faulkner A., Trans-endothelial trafficking of metabolic substrates and its importance in cardio-metabolic disease, Biochem. Soc. Trans., 49(1) (2021) 507-517.
[37] Kurutas E.B., The importance of antioxidants which play the role in cellular response against oxidative/nitrosative stress: current state, Nutr. J., 15(1) (2016) 71.
[38] Sevimli G., Smith M.J., Caglar T.A., Bilir Ş., Secilmis M., Altun H.Y., Yigit E.N., Yang F., Keeley T.P., Malli R., Öztürk G., Mann G.E., Eroglu E., Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels, Redox Biol., 53 (2022) 102319.
[39] Yang Y., Liu N., He Y., Liu Y., Ge L., Zou L., Song S., Xiong W., Liu X., Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP, Nat. Commun., 9(1) (2018) 1504.
[40] Arai S., Kriszt R., Harada K., Looi L.S., Matsuda S., Wongso D., Suo S., Ishiura S., Tseng Y.H., Raghunath M., Ito T., Tsuboi T., Kitaguchi T., RGB-Color Intensiometric Indicators to Visualize Spatiotemporal Dynamics of ATP in Single Cells, Angew Chem. Int. Ed. Engl., 57(34) (2018) 10873-10878

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 1970 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

Cited By

Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions

Free Radical Biology and Medicine

https://doi.org/10.1016/j.freeradbiomed.2024.05.021

Development of an Oxygen-Insensitive Nrf2 Reporter Reveals Redox Regulation under Physiological Normoxia

ACS Sensors

https://doi.org/10.1021/acssensors.4c03167