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Article

COVID-19 Causes Ferroptosis and Oxidative Stress in Human Endothelial Cells

by
Stanislovas S. Jankauskas
1,
Urna Kansakar
1,
Celestino Sardu
2,
Fahimeh Varzideh
1,
Roberta Avvisato
1,3,
Xujun Wang
1,
Alessandro Matarese
4,
Raffaele Marfella
2,
Marcello Ziosi
5,
Jessica Gambardella
1,3 and
Gaetano Santulli
1,3,6,*
1
Department of Medicine, Division of Cardiology, Wilf Family Cardiovascular Research Institute, Einstein Institute for Aging Research, Albert Einstein College of Medicine, New York, NY 10461, USA
2
University of Campania “Luigi Vanvitelli”, 81100 Caserta, Italy
3
“Federico II” University, 80131 Naples, Italy
4
Cardarelli Hospital, 80131 Naples, Italy
5
New York Genome Center, New York, NY 10013, USA
6
Department of Molecular Pharmacology, Einstein Institute for Neuroimmunology and Inflammation (INI), Einstein-Mount Sinai Diabetes Research Center (ES-DRC), Fleischer Institute for Diabetes and Metabolism (FIDAM), Albert Einstein College of Medicine, New York, NY 10461, USA
*
Author to whom correspondence should be addressed.
Antioxidants 2023, 12(2), 326; https://doi.org/10.3390/antiox12020326
Submission received: 1 December 2022 / Revised: 17 January 2023 / Accepted: 30 January 2023 / Published: 31 January 2023
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Abstract

:
Oxidative stress and endothelial dysfunction have been shown to play crucial roles in the pathophysiology of COVID-19 (coronavirus disease 2019). On these grounds, we sought to investigate the impact of COVID-19 on lipid peroxidation and ferroptosis in human endothelial cells. We hypothesized that oxidative stress and lipid peroxidation induced by COVID-19 in endothelial cells could be linked to the disease outcome. Thus, we collected serum from COVID-19 patients on hospital admission, and we incubated these sera with human endothelial cells, comparing the effects on the generation of reactive oxygen species (ROS) and lipid peroxidation between patients who survived and patients who did not survive. We found that the serum from non-survivors significantly increased lipid peroxidation. Moreover, serum from non-survivors markedly regulated the expression levels of the main markers of ferroptosis, including GPX4, SLC7A11, FTH1, and SAT1, a response that was rescued by silencing TNFR1 on endothelial cells. Taken together, our data indicate that serum from patients who did not survive COVID-19 triggers lipid peroxidation in human endothelial cells.

1. Introduction

We and others have evidenced the fundamental role of endothelial cells in the pathobiology of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17]. Equally important, oxidative stress has been shown to be a key player in COVID-19 [18,19,20,21,22,23,24,25]. Chronic inflammation and oxidative stress are associated with endothelial dysfunction, and have been linked to the pathogenesis of atherosclerosis, hypertension, and other cardiovascular and cerebrovascular disorders [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48].
The overproduction of reactive oxygen species (ROS) is known to induce endothelial dysfunction through different mechanisms, including ferroptosis [49,50], a non-apoptotic type of programmed cell death characterized by the accumulation of lipid peroxides [51,52,53,54,55]. However, to the best of our knowledge, the association of endothelial dysfunction with ferroptosis has never been investigated in the context of COVID-19.
We hypothesized that oxidative stress and subsequent lipid peroxidation induced by COVID-19 in endothelial cells could be linked with the disease outcome. To test this hypothesis, we collected serum from COVID-19 patients on hospital admission, and we incubated these sera with human endothelial cells, comparing the effects on ROS generation and lipid peroxidation between patients who survived and patients who did not survive.

2. Results

2.1. COVID-19 Induces Oxidative Stress in Human Endothelial Cells

We evaluated the effects on ROS production in human umbilical endothelial cells (HUVECs) of serum obtained from patients who did not survive COVID-19, comparing these effects with the serum of patients who did survive. The main characteristics of the patients are shown in Supplementary Table S1. We observed a significantly (p < 0.0001) increased production of both cellular (Figure 1) and mitochondrial (Figure 2) ROS in HUVECs incubated for 24 h with 10% serum from non-survivors.

2.2. Serum from COVID-19 Non-Survivors Causes Lipid Peroxidation

Oxidative stress has been linked to lipid peroxidation, a process that has been recently suggested to play a critical role in COVID-19 [56]; on these grounds, we assessed lipid oxidation in endothelial cells, measuring malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and C11-BODIPY [57,58,59]. We found that all these markers were significantly increased in HUVECs incubated for 24 h with 10% serum from non-survivors compared to serum from COVID-19 survivors (Figure 3), whereas there were no significant differences between normal serum (from healthy donors) and serum from COVID-19 survivors (Figure 3).

2.3. Serum from COVID-19 Non-Survivors Reduces the Expression of the Antioxidant Enzyme Glutathione Peroxidase 4 (GPX4) in HUVECs

Since the selenoprotein glutathione peroxidase 4 (GPX4) has been shown to be a major modulator of lipid peroxidation [60,61,62], we reasoned that COVID-19 serum could regulate its expression. Thus, we measured GPX4 in HUVECs, both at the mRNA and at the protein level, and we found that a 24 h incubation with serum obtained from patients who did not survive COVID-19 significantly decreased GPX4 expression (Figure 4).

2.4. Serum from COVID-19 Non-Survivors Triggers Ferroptosis in Human Endothelial Cells

Increased lipid peroxidation and reduced levels of GPX4 represent two hallmarks of ferroptosis [63,64]; thus, to verify whether this process was actually involved in our model, we measured the levels of other markers of ferroptosis and their response to Ferrostatin-1 (Fer-1), a well-established inhibitor of ferroptosis [64,65,66]. We observed that in addition to GPX4, suppressors of ferroptosis, such as Solute Carrier Family 7 Member 11 (SLC7A11, also known as Calcium Channel Blocker Resistance Protein, CCBR1, and Cystine/Glutamate Transporter, a subunit of the Amino Acid Transport System Xc−) [67,68,69,70] and Ferritin heavy chain (FTH1) [71,72,73], were significantly downregulated, whereas the ferroptosis inducer spermidine/spermine N1-acetyltransferase 1 (SAT1) [74,75,76] was upregulated, in HUVECs incubated with serum from COVID-19 non-survivors; strikingly, these regulations were all prevented by Fer-1 (Figure 5).

2.5. COVID-19-Induced Ferroptosis in HUVECs Is Mediated by TNFα

Several studies have consistently shown that increased TNFα serum levels on hospital admission are associated with the risk of mortality in COVID-19 patients [77,78,79]. Similarly, increased serum levels of soluble TNFα Receptor 1 (TNFR1) are associated with mortality [80]. A recent meta-analysis [81] has confirmed that TNFα significantly augments the risk of COVID-19 mortality. Therefore, we measured TNFα in our samples, observing significantly elevated levels in serum from non-survivors compared to serum from survivors (10.7 ± 3.3 * vs. 6.3 ± 4.1 pg/mL, p < 0.001).
Then, to mechanistically prove that TNFα was involved in the COVID-19 induced ferroptotic response, we measured by immunoblot GPX4, SLC7A11, FTH1, and SAT1 after having treated HUVECs with a siRNA for TNFR1 or scramble (the efficiency of TNFR1 silencing is shown in Supplementary Figure S1). Strikingly, we observed that knocking-down TNFR1 (200 nM siRNA) rescued the regulation of all the ferroptosis markers induced by incubation with serum obtained from patients who did not survive COVID-19 (Figure 5A–E).

3. Discussion

The essential contribution of endothelial dysfunction to the pathobiology of COVID-19 was proposed at the beginning of the pandemic in an attempt to explain the systemic manifestations of the disease, and was later verified by investigations on autoptic samples [1,3,4,5,6,7,8,82,83,84,85]. Recent analyses using single-cell atlases have confirmed that endothelial cells in different organs are indeed deeply affected by COVID-19 [86], although the exact mechanisms are still not fully understood.
The association between oxidative stress and programmed cell death, known as oxytosis [87], had been described in neuronal cells years before the term ferroptosis had been coined by Scott Dixon, Brent Stockwell, and collaborators in 2012 [88] to describe a form of non-apoptotic cell death induced by the small molecule erastin by inhibiting SLC7A11. SLC7A11 is a member of a heteromeric, Na+-independent, anionic amino acid transport system that is highly specific for glutamate and cysteine [89,90]; in such a system, designated Xc−, the anionic form of cysteine is transported in exchange for glutamate [91,92,93,94,95]. When inhibited, the transportation into the cytosol of cystine, a precursor of glutathione, in exchange for glutamate, is blocked, leading to an impaired function of the housekeeping enzyme GPX4 [96]. The subsequent accumulation of lipid peroxides eventually compromises the plasma membrane integrity [97,98]. It is notable that previous studies exploring cellular metabolism had described similar phenomena, although less detailed from a molecular point of view, in human cells [99].
In our experiments, we were able to demonstrate that the main actors involved in the ferroptotic process, namely, GPX4, SLC7A11, FTH1, and SAT1, were all regulated by serum from COVID-19 non-survivors and, in the opposite direction, by TNFR1 silencing, strongly suggesting that TNFα is functionally involved in the association between COVID-19 and ferroptosis in endothelial cells. Although TNFα has been shown in several studies to be a reliable predictor of mortality in COVID-19 [77,81,100,101,102,103], we reckon that other mechanisms could be at play, including the main component of the so-called cytokine storm [103,104,105,106,107,108,109,110,111]. Our observation that not only cellular oxidative stress but also mitochondrial ROS are increased after incubation of HUVECs with COVID-19 serum for non-survivors is consistent with previous reports, which indicate that mitochondria are not simple spectators but active participants in oxytosis and ferroptosis [87,112,113,114,115,116].
In the last years, lipid peroxidation has been implied in a series of disorders, including cancer [117,118,119], neurodegeneration [120,121,122], autoimmune diseases [123,124,125,126], diabetes [127,128,129,130], and ischemia-reperfusion injuries [131,132,133]. With COVID-19, we add another small piece to this jigsaw puzzle, which is in agreement with the functional contribution of ferroptosis to a complex process, such as fibrosis of the lung [134,135,136]. Intriguingly, our results are consistent with the autoptic observations of accumulated oxidized phospholipids in both renal and myocardial tissue in a patient who died because of COVID-19 [137], further indicating that lipid peroxidation and ferroptosis could be playing major roles in the pathophysiology of COVID-19.
Our study is not exempt from limitations, including having performed the assays only in vitro, and not having explored in detail the exact signaling pathway linking TNFα and ferroptosis. In this sense, further dedicated experiments, including in vivo assays, are warranted to corroborate our findings. We did not measure another marker of ferroptosis, heme-oxygenase 1 (HO-1), which has been recently reported both as a suppressor and as an inductor of ferroptosis in a series of conflicting data [138,139].

4. Materials and Methods

4.1. Cell Culture and Reagents

All reagents were purchased from Merck (Darmstast, Germany), unless otherwise stated. HUVECs were obtained from ThermoFisher Scientific (Waltham, MA, USA; Catalog number: #C0035C). Cells were cultured in a standard humidified atmosphere (37 °C) containing 5% CO2, in EGM-2 medium (Lonza, Basel, Switzerland; Catalog number: #CC4147), as we described [12,13,47,140]. Experiments on HUVECs were performed at passages 3–7. HUVECs were plated on glass-bottom culture dishes (MatTek Corporation, Mashland, MA, USA; Catalog number: #P35GCOL-0-10-C). When 70–80% confluent, the cells were treated with 10% human serum for 24 h under normal conditions (37 °C and 5% CO2).
The sera from fully de-identified COVID-19 patients were provided by the Montefiore–Einstein COVID-19 biorepository; the protocol was approved by the Institutional Ethical Committee (IRB #202011756). Normal human serum was purchased from ThermoFisher Scientific. The absence of SARS-CoV-2 in the serum was confirmed by RT-qPCR. TNFα was quantified via ELISA (ThermoFisher Scientific; Catalog number #KHC3011) following the manufacturer’s protocol.

4.2. Assays Measuring Reactive Oxygen Species (ROS) and Lipid Oxidation

ROS production was quantified using 2′-7′-dichlorofluorescin diacetate (H2DCF-DA, ThermoFisher Scientific; Catalog number #D399), as described previously [141,142]. Incubation for both fluorescent probes, as well as washing and imaging, were conducted in a Krebs-Ringer solution (NaCl 115 mM, KCl 5 mM, NaHCO3 10 mM, MgCl2 2.5 mM, CaCl2 2.5 mM, HEPES 20 mM) supplemented with 10 mM glucose. After 24 h of treatment with 10% patients’ serum, HUVECs were incubated with 2.5 μg/mL Hoechst 33342, trihydrochloride, trihydrate (ThermoFisher Scientific; Catalog number #H21492) for 30 min, in the dark, at room temperature. Then, HUVECs were washed and incubated with 10 μM H2DCF-DA for another 15 min and then washed 3 more times and incubated without any fluorescent probes for another 15 min, in the dark, at room temperature. Immediately after this step, cells were imaged using a Nikon CSU-W1 Spinning Disk confocal microscope, using a 40x objective (Nikon Corporation, Minato City, Tokyo, Japan). The non-fluorescent dye H2DCF-DA is a chemically reduced form of fluorescein and is cell-permeable; once intracellular esterases cleave off the diacetate (DA) moiety, H2DCF becomes sensitive to oxidation by ROS: in its oxidized form, dichlorofluorescein (DCF) is highly fluorescent and easily detectable. Thus, cells were excited with a laser at wavelengths 405 nm and 488 nm for Hoechst and DCF, respectively. Light emission was detected using 455/50 and 520/40 filters for Hoechst and DCF, respectively. The same settings (including laser intensity, exposure time, and pinhole width) were used for imaging in all experimental groups. In order to prevent DCF photodynamic reactions, fields of view search and focusing were performed using a Hoechst signal. Quantification of DCF fluorescence was performed using ImageJ software.
Mitochondrial ROS production was quantified by MitoSOX™ Red (ThermoFisher Scientific, #M36008) as previously described and validated [43,140,142,143,144]. Briefly, after 24 h of treatment with 10% serum, HUVECs were incubated with 200 nM MitoTracker™ Green FM (MTG, ThermoFisher Scientific; Catalog number #M7514) in EGM-2 medium in the dark for 1 h at 37 °C and 5% CO2. Then, the cells were washed with EGM-2 medium and incubated with 5 µM MitoSOX™ Red in EGM-2 medium in the dark for 10 min at 37 °C and 5% CO2. After incubation, cells were washed with EGM-2 medium and imaged at the Nikon CSU-W1 Spinning Disk confocal microscope using a 100× objective (Nikon Corporation). Cells were excited with a laser at wavelengths 488 nm and 561 nm for MTG and MitoSOX, respectively. Light emission was detected using 520/40 and 605/52 filters for MTG and MitoSOX, respectively. The same settings (including laser intensity, exposure time, and pinhole width) were used for imaging all experimental groups. In order to prevent MitoSOX photodynamic reaction, fields of view search and focusing were performed using a MTG signal. MitoSOX fluorescence intensity was quantified using NIS-Elements software (Nikon Corporation).
MDA and 4-HNE levels were measured by using commercially available kits (both from Abcam, Cambridge, UK), following the manufacturer’s instructions [57]. We quantified lipid peroxidation using C11-BODIPY581/591 (ThermoFisher Scientific) as previously described [59,145,146]. Briefly, HUVECs were incubated with 2 μM C11-BODIPY581/591 for 30 minutes (37 °C in the dark) and then fluorometrically measured (with an excitation wavelength of 581 nm and an emission wavelength of 591 nm) using a microplate reader.

4.3. Immunoblotting and RT-qPCR

Immunoblotting assays were performed as previously described by our group [144,147]; the intensity of the bands was quantified using the open-source image processing package FIJI (Fiji is Just ImageJ). The antibody for GPX4 was purchased from GeneTex (Irvine, CA, USA; Catalog number: #GT1282); the antibody for SCL7A11 was purchased from Cell Signaling (Danvers, MA, USA; Catalog number: #12691); the antibody for FTH1 was purchased from ABclonal Technology (Woburn, MA, USA; Catalog number: #A19544); the antibody for SAT1 was purchased from Cell Signaling (Catalog number: #61586); the antibody for GAPDH was purchased from Novus Biologicals (Centennial, CO, USA; Catalog number: #NB300-221). RT-qPCR was performed using a SYBR Green mix as we previously described and validated [148,149,150] using GAPDH as an internal standard; primer sequences are listed in Table 1.

4.4. Statistical Analysis

Statistical analyses were performed using GraphPad 9 (Dotmatics, Boston, MA, USA). Statistical significance, set at p < 0.05, was tested using the two-way ANOVA followed by Tukey–Kramer multiple comparison test or the non-parametric Mann–Whitney U test, as appropriate.

5. Conclusions

Taken together, our results indicate that the serum from COVID-19 patients who did not survive induces oxidative stress, lipid peroxidation, and ferroptosis in human endothelial cells through a mechanism that depends on TNF-α.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12020326/s1, Figure S1: Successful knock-down of TNFR1 in human endothelial cells. Table S1: Main characteristics of COVID-19 patients from whom the serum was obtained.

Author Contributions

Conceptualization, S.S.J. and G.S.; methodology, S.S.J., U.K., C.S., F.V., R.A., A.M., R.M., M.Z. and J.G.; formal analysis, S.S.J., U.K., C.S., R.A. and R.M.; investigation, S.S.J., U.K., C.S., R.A., X.W., A.M. and J.G.; writing—original draft preparation, S.S.J., U.K., C.S. and J.G.; writing—review and editing, G.S. All authors have read and agreed to the published version of the manuscript.

Funding

The Santulli’s Lab is supported in part by the National Institutes of Health (NIH): National Heart, Lung, and Blood Institute (NHLBI: R01-HL159062, R01-HL164772, R01-HL146691, T32-HL144456), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK: R01-DK123259, R01-DK033823) (to G.S.), by the National Center for Advancing Translational Sciences (NCATS: UL1TR002556-06) (to G.S.), by the Diabetes Action Research and Education Foundation (to G.S.), and by the Monique Weill-Caulier and Irma T. Hirschl Trusts (to G.S.). S.S.J. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA-21POST836407); U.K. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA-23POST1026190); F.V. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA-22POST995561); and J.G. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA-20POST35211151).

Institutional Review Board Statement

Sera from fully de-identified COVID-19 patients were provided by the Montefiore–Einstein COVID-19 biorepository; the protocol was approved by the Institutional Ethical Committee (IRB #202011756).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data supporting reported results are contained within this article and its supplementary material.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Sardu, C.; Gambardella, J.; Morelli, M.B.; Wang, X.; Marfella, R.; Santulli, G. Hypertension, Thrombosis, Kidney Failure, and Diabetes: Is COVID-19 an Endothelial Disease? A Comprehensive Evaluation of Clinical and Basic Evidence. J. Clin. Med. 2020, 9, 1417. [Google Scholar] [CrossRef] [PubMed]
  2. Matarese, A.; Gambardella, J.; Sardu, C.; Santulli, G. miR-98 Regulates TMPRSS2 Expression in Human Endothelial Cells: Key Implications for COVID-19. Biomedicines 2020, 8, 462. [Google Scholar] [CrossRef] [PubMed]
  3. Bonaventura, A.; Vecchié, A.; Dagna, L.; Martinod, K.; Dixon, D.L.; Van Tassell, B.W.; Dentali, F.; Montecucco, F.; Massberg, S.; Levi, M.; et al. Endothelial dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19. Nat. Rev. Immunol. 2021, 21, 319–329. [Google Scholar] [CrossRef] [PubMed]
  4. Siddiqi, H.K.; Libby, P.; Ridker, P.M. COVID-19—A vascular disease. Trends Cardiovasc. Med. 2020, 31, 1–5. [Google Scholar] [CrossRef]
  5. Perico, L.; Benigni, A.; Casiraghi, F.; Ng, L.F.P.; Renia, L.; Remuzzi, G. Immunity, endothelial injury and complement-induced coagulopathy in COVID-19. Nat. Rev. Nephrol. 2020, 17, 46–64. [Google Scholar] [CrossRef]
  6. Gu, S.X.; Tyagi, T.; Jain, K.; Gu, V.W.; Lee, S.H.; Hwa, J.M.; Kwan, J.M.; Krause, D.S.; Lee, A.I.; Halene, S.; et al. Thrombocytopathy and endotheliopathy: Crucial contributors to COVID-19 thromboinflammation. Nat. Rev. Cardiol. 2020, 18, 194–209. [Google Scholar] [CrossRef]
  7. Ionescu, M.; Stoian, A.P.; Rizzo, M.; Serban, D.; Nuzzo, D.; Mazilu, L.; Suceveanu, A.I.; Dascalu, A.M.; Parepa, I.R. The Role of Endothelium in COVID-19. Int. J. Mol. Sci. 2021, 22, 11920. [Google Scholar] [CrossRef]
  8. Flaumenhaft, R.; Enjyoji, K.; Schmaier, A.A. Vasculopathy in COVID-19. Blood 2022, 140, 222–235. [Google Scholar] [CrossRef]
  9. Faria, D.; Moll-Bernardes, R.J.; Testa, L.; Moniz, C.M.; Rodrigues, E.C.; Rodrigues, A.G.; Araujo, A.; Alves, M.J.; Ono, B.E.; Izaias, J.E.; et al. Sympathetic Neural Overdrive, Aortic Stiffening, Endothelial Dysfunction, and Impaired Exercise Capacity in Severe COVID-19 Survivors: A Mid-Term Study of Cardiovascular Sequelae. Hypertension 2023, 80, 470–481. [Google Scholar] [CrossRef]
  10. Montiel, V.; Lobysheva, I.; Gérard, L.; Vermeersch, M.; Perez-Morga, D.; Castelein, T.; Mesland, J.-B.; Hantson, P.; Collienne, C.; Gruson, D.; et al. Oxidative stress-induced endothelial dysfunction and decreased vascular nitric oxide in COVID-19 patients. Ebiomedicine 2022, 77, 103893. [Google Scholar] [CrossRef]
  11. Trimarco, V.; Izzo, R.; Zanforlin, A.; Tursi, F.; Scarpelli, F.; Santus, P.; Pennisi, A.; Pelaia, G.; Mussi, C.; Mininni, S.; et al. Endothelial dysfunction in long-COVID: New insights from the nationwide multicenter LINCOLN Study. Pharmacol. Res. 2022, 185, 106486. [Google Scholar] [CrossRef] [PubMed]
  12. Kansakar, U.; Gambardella, J.; Varzideh, F.; Avvisato, R.; Jankauskas, S.S.; Mone, P.; Matarese, A.; Santulli, G. miR-142 Targets TIM-1 in Human Endothelial Cells: Potential Implications for Stroke, COVID-19, Zika, Ebola, Dengue, and Other Viral Infections. Int. J. Mol. Sci. 2022, 23, 10242. [Google Scholar] [CrossRef] [PubMed]
  13. Mone, P.; Gambardella, J.; Wang, X.; Jankauskas, S.S.; Matarese, A.; Santulli, G. miR-24 Targets the Transmembrane Glycoprotein Neuropilin-1 in Human Brain Microvascular Endothelial Cells. Non-Coding RNA 2021, 7, 9. [Google Scholar] [CrossRef]
  14. Gambardella, J.; Santulli, G. What is linking COVID-19 and endothelial dysfunction? Updates on nanomedicine and bioengineering from the 2020 AHA Scientific Sessions. Eur. Heart J. Cardiovasc. Pharmacother. 2021, 7, e2–e3. [Google Scholar] [CrossRef]
  15. Gambardella, J.; Kansakar, U.; Sardu, C.; Messina, V.; Jankauskas, S.S.; Marfella, R.; Maggi, P.; Wang, X.; Mone, P.; Paolisso, G.; et al. Exosomal miR-145 and miR-885 Regulate Thrombosis in COVID-19. J. Pharmacol. Exp. Ther. 2023, 384, 109–115. [Google Scholar] [CrossRef]
  16. Jiang, Z.; Wu, L.; van der Leeden, B.; van Rossum, A.C.; Niessen, H.W.; Krijnen, P.A. NOX2 and NOX5 are increased in cardiac microvascular endothelium of deceased COVID-19 patients. Int. J. Cardiol. 2022, 370, 454–462. [Google Scholar] [CrossRef] [PubMed]
  17. Plazak, W.; Drabik, L. SARS-CoV-2 infection and SLE: Endothelial dysfunction, atherosclerosis, and thrombosis. Clin. Rheumatol. 2023, 1–12. [Google Scholar] [CrossRef]
  18. Silvagno, F.; Vernone, A.; Pescarmona, G.P. The Role of Glutathione in Protecting against the Severe Inflammatory Response Triggered by COVID-19. Antioxidants 2020, 9, 624. [Google Scholar] [CrossRef]
  19. Vardakas, P.; Skaperda, Z.; Tekos, F.; Kouretas, D. ROS and COVID. Antioxidants 2022, 11, 339. [Google Scholar] [CrossRef]
  20. Chernyak, B.V.; Popova, E.N.; Prikhodko, A.S.; Grebenchikov, O.A.; Zinovkina, L.A.; Zinovkin, R.A. COVID-19 and Oxidative Stress. Biochem. (Moscow) 2020, 85, 1543–1553. [Google Scholar] [CrossRef]
  21. Carnevale, R.; Cammisotto, V.; Bartimoccia, S.; Nocella, C.; Castellani, V.; Bufano, M.; Loffredo, L.; Sciarretta, S.; Frati, G.; Coluccia, A.; et al. Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection. Circ. Res. 2023, in press. [Google Scholar] [CrossRef]
  22. Jana, S.; Heaven, M.R.; Stauft, C.B.; Wang, T.T.; Williams, M.C.; D’Agnillo, F.; Alayash, A.I. HIF-1α-Dependent Metabolic Reprogramming, Oxidative Stress, and Bioenergetic Dysfunction in SARS-CoV-2-Infected Hamsters. Int. J. Mol. Sci. 2022, 24, 558. [Google Scholar] [CrossRef] [PubMed]
  23. Munguía, L.; Nájera, N.; Martínez, F.D.J.; Díaz-Chiguer, D.; Jiménez-Ponce, F.; Ortiz-Flores, M.; Villarreal, F.; Ceballos, G. Correlation of Biomarkers of Endothelial Injury and Inflammation to Outcome in Hospitalized COVID-19 Patients. J. Clin. Med. 2022, 11, 7436. [Google Scholar] [CrossRef] [PubMed]
  24. Aparisi, Á.; Martín-Fernández, M.; Ybarra-Falcón, C.; Gil, J.F.; Carrasco-Moraleja, M.; Martínez-Paz, P.; Cusácovich, I.; Gonzalo-Benito, H.; Fuertes, R.; Marcos-Mangas, M.; et al. Dyslipidemia and Inflammation as Hallmarks of Oxidative Stress in COVID-19: A Follow-Up Study. Int. J. Mol. Sci. 2022, 23, 15350. [Google Scholar] [CrossRef] [PubMed]
  25. Tavassolifar, M.J.; Aghdaei, H.A.; Sadatpour, O.; Maleknia, S.; Fayazzadeh, S.; Mohebbi, S.R.; Montazer, F.; Rabbani, A.; Zali, M.R.; Izad, M.; et al. New insights into extracellular and intracellular redox status in COVID-19 patients. Redox Biol. 2023, 59, 102563. [Google Scholar] [CrossRef] [PubMed]
  26. Higashi, Y. Roles of Oxidative Stress and Inflammation in Vascular Endothelial Dysfunction-Related Disease. Antioxidants 2022, 11, 1958. [Google Scholar] [CrossRef] [PubMed]
  27. Odegaard, A.O.; Jacobs, D.R., Jr.; Sanchez, O.A.; Goff, D.C., Jr.; Reiner, A.P.; Gross, M.D. Oxidative stress, inflammation, endothelial dysfunction and incidence of type 2 diabetes. Cardiovasc. Diabetol. 2016, 15, 51. [Google Scholar] [CrossRef] [Green Version]
  28. Sardu, C.; Santulli, G.; Savarese, G.; Trotta, M.C.; Sacra, C.; Santamaria, M.; Volpicelli, M.; Ruocco, A.; Mauro, C.; Signoriello, G.; et al. Endothelial Dysfunction Drives CRTd Outcome at 1-Year Follow-Up: A Novel Role as Biomarker for miR-130a-5p. Int. J. Mol. Sci. 2023, 24, 1510. [Google Scholar] [CrossRef]
  29. Scioli, M.G.; Storti, G.; D’Amico, F.; Guzmán, R.R.; Centofanti, F.; Doldo, E.; Miranda, E.M.C.; Orlandi, A. Oxidative Stress and New Pathogenetic Mechanisms in Endothelial Dysfunction: Potential Diagnostic Biomarkers and Therapeutic Targets. J. Clin. Med. 2020, 9, 1995. [Google Scholar] [CrossRef]
  30. Ciccarelli, M.; Santulli, G.; Campanile, A.; Galasso, G.; Cervèro, P.; Altobelli, G.G.; Cimini, V.; Pastore, L.; Piscione, F.; Trimarco, B.; et al. Endothelial α1 -adrenoceptors regulate neo-angiogenesis. Br. J. Pharmacol. 2008, 153, 936–946. [Google Scholar] [CrossRef]
  31. Avvisato, R.; Jankauskas, S.S.; Varzideh, F.; Kansakar, U.; Mone, P.; Santulli, G. Sortilin and Hypertension. Curr. Opin. Nephrol. Hypertens. 2023, 32, 134–140. [Google Scholar] [CrossRef] [PubMed]
  32. Gui, F.; You, Z.; Fu, S.; Wu, H.; Zhang, Y. Endothelial Dysfunction in Diabetic Retinopathy. Front. Endocrinol. 2020, 11, 591. [Google Scholar] [CrossRef] [PubMed]
  33. Varzideh, F.; Jankauskas, S.S.; Kansakar, U.; Mone, P.; Gambardella, J.; Santulli, G. Sortilin drives hypertension by modulating sphingolipid/ceramide homeostasis and by triggering oxidative stress. J. Clin. Investig. 2022, 132, e156624. [Google Scholar] [CrossRef]
  34. Wang, M.; Li, Y.; Li, S.; Lv, J. Endothelial Dysfunction and Diabetic Cardiomyopathy. Front. Endocrinol. 2022, 13, 851941. [Google Scholar] [CrossRef]
  35. Santulli, G. Endothelial cells: The heart attack of the clones. Sci. Transl. Med. 2018, 10, 427. [Google Scholar] [CrossRef] [PubMed]
  36. Saenz-Medina, J.; Muñoz, M.; Rodriguez, C.; Contreras, C.; Sánchez, A.; Coronado, M.J.; Ramil, E.; Santos, M.; Carballido, J.; Prieto, D. Hyperoxaluria Induces Endothelial Dysfunction in Preglomerular Arteries: Involvement of Oxidative Stress. Cells 2022, 11, 2306. [Google Scholar] [CrossRef] [PubMed]
  37. Wilson, S.; Mone, P.; Kansakar, U.; Jankauskas, S.S.; Donkor, K.; Adebayo, A.; Varzideh, F.; Eacobacci, M.; Gambardella, J.; Lombardi, A.; et al. Diabetes and restenosis. Cardiovasc. Diabetol. 2022, 21, 1–14. [Google Scholar] [CrossRef] [PubMed]
  38. Ronconi, K.D.S.; Stefanon, I.; Junior, R.F.R. Tributyltin and Vascular Dysfunction: The Role of Oxidative Stress. Front. Endocrinol. 2018, 9, 354. [Google Scholar] [CrossRef]
  39. Iaccarino, G.; Ciccarelli, M.; Sorriento, D.; Cipolletta, E.; Cerullo, V.; Iovino, G.L.; Paudice, A.; Elia, A.; Santulli, G.; Campanile, A.; et al. AKT Participates in Endothelial Dysfunction in Hypertension. Circulation 2004, 109, 2587–2593. [Google Scholar] [CrossRef] [Green Version]
  40. Liu, P.; Li, Y.; Li, M.; Zhou, H.; Zhang, H.; Zhang, Y.; Xu, J.; Xu, Y.; Zhang, J.; Xia, B.; et al. Endothelial Shp2 deficiency controls alternative activation of macrophage preventing radiation-induced lung injury through notch signaling. Iscience 2022, 25, 103867. [Google Scholar] [CrossRef]
  41. Gambardella, J.; Sorriento, D.; Bova, M.; Rusciano, M.; Loffredo, S.; Wang, X.; Petraroli, A.; Carucci, L.; Mormile, I.; Oliveti, M.; et al. Role of Endothelial G Protein-Coupled Receptor Kinase 2 in Angioedema. Hypertension 2020, 76, 1625–1636. [Google Scholar] [CrossRef] [PubMed]
  42. McElwain, C.; Tuboly, E.; McCarthy, F.P.; McCarthy, C.M. Mechanisms of Endothelial Dysfunction in Pre-eclampsia and Gestational Diabetes Mellitus: Windows into Future Cardiometabolic Health? Front. Endocrinol. 2020, 11, 655. [Google Scholar] [CrossRef] [PubMed]
  43. Mone, P.; Varzideh, F.; Jankauskas, S.S.; Pansini, A.; Lombardi, A.; Frullone, S.; Santulli, G. SGLT2 Inhibition via Empagliflozin Improves Endothelial Function and Reduces Mitochondrial Oxidative Stress: Insights from Frail Hypertensive and Diabetic Patients. Hypertension 2022, 79, 1633–1643. [Google Scholar] [CrossRef] [PubMed]
  44. Murdaca, G.; Colombo, B.M.; Cagnati, P.; Gulli, R.; Spanò, F.; Puppo, F. Endothelial dysfunction in rheumatic autoimmune diseases. Atherosclerosis 2012, 224, 309–317. [Google Scholar] [CrossRef]
  45. Mone, P.; Lombardi, A.; Kansakar, U.; Varzideh, F.; Jankauskas, S.S.; Pansini, A.; Marzocco, S.; De Gennaro, S.; Famiglietti, M.; Macina, G.; et al. Empagliflozin Improves the MicroRNA Signature of Endothelial Dysfunction in Patients with Heart Failure with Preserved Ejection Fraction and Diabetes. J. Pharmacol. Exp. Ther. 2023, 384, 116–122. [Google Scholar] [CrossRef]
  46. Duică, F.; Dănilă, C.A.; Boboc, A.E.; Antoniadis, P.; Condrat, C.E.; Onciul, S.; Suciu, N.; Creţoiu, S.M.; Varlas, V.N.; Creţoiu, D. Impact of Increased Oxidative Stress on Cardiovascular Diseases in Women with Polycystic Ovary Syndrome. Front. Endocrinol. 2021, 12, 614679. [Google Scholar] [CrossRef]
  47. Ciccarelli, M.; Cipolletta, E.; Santulli, G.; Campanile, A.; Pumiglia, K.; Cervero, P.; Pastore, L.; Astone, D.; Trimarco, B.; Iaccarino, G. Endothelial β2 adrenergic signaling to AKT: Role of Gi and SRC. Cell. Signal. 2007, 19, 1949–1955. [Google Scholar] [CrossRef]
  48. Jankauskas, S.S.; Mone, P.; Santulli, G. From resveratrol to ISIDE11: How to activate SIRT1 and improve endothelial function? New therapeutic insights for methylenetetrahydrofolate reductase deficiency. Cell. Mol. Life Sci. 2022, 79, 451. [Google Scholar] [CrossRef]
  49. Yuan, W.; Xia, H.; Xu, Y.; Xu, C.; Chen, N.; Shao, C.; Dai, Z.; Chen, R.; Tao, A. The role of ferroptosis in endothelial cell dysfunction. Cell Cycle 2022, 21, 1897–1914. [Google Scholar] [CrossRef]
  50. Zhang, H.; Zhou, S.; Sun, M.; Hua, M.; Liu, Z.; Mu, G.; Wang, Z.; Xiang, Q.; Cui, Y. Ferroptosis of Endothelial Cells in Vascular Diseases. Nutrients 2022, 14, 4506. [Google Scholar] [CrossRef]
  51. Agmon, E.; Solon, J.; Bassereau, P.; Stockwell, B.R. Modeling the effects of lipid peroxidation during ferroptosis on membrane properties. Sci. Rep. 2018, 8, 5155. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Farooqi, A.A.; Kapanova, G.; Kalmakhanov, S.; Kussainov, A.Z.; Datkhayeva, Z. Regulation of Ferroptosis by Non-Coding RNAs: Mechanistic Insights. J. Pharmacol. Exp. Ther. 2023, 384, 20–27. [Google Scholar] [CrossRef] [PubMed]
  53. Chou, H.-C.; Chen, C.-M. Hyperoxia Induces Ferroptosis and Impairs Lung Development in Neonatal Mice. Antioxidants 2022, 11, 641. [Google Scholar] [CrossRef] [PubMed]
  54. Musheshe, N.; Oun, A.; Sabogal-Guáqueta, A.M.; Trombetta-Lima, M.; Mitchel, S.C.; Adzemovic, A.; Speek, O.; Morra, F.; van der Veen, C.H.J.T.; Lezoualc’H, F.; et al. Pharmacological Inhibition of Epac1 Averts Ferroptosis Cell Death by Preserving Mitochondrial Integrity. Antioxidants 2022, 11, 314. [Google Scholar] [CrossRef] [PubMed]
  55. Dang, D.; Zhang, C.; Meng, Z.; Lv, X.; Li, Z.; Wei, J.; Wu, H. Integrative analysis links ferroptosis to necrotizing enterocolitis and reveals the role of ACSL4 in immune disorders. iScience 2022, 25, 105406. [Google Scholar] [CrossRef]
  56. Martín-Fernández, M.; Aller, R.; Heredia-Rodríguez, M.; Gómez-Sánchez, E.; Martínez-Paz, P.; Gonzalo-Benito, H.; Prada, L.S.-D.; Gorgojo, Ó.; Carnicero-Frutos, I.; Tamayo, E.; et al. Lipid peroxidation as a hallmark of severity in COVID-19 patients. Redox Biol. 2021, 48, 102181. [Google Scholar] [CrossRef]
  57. Tang, X.-H.; Gambardella, J.; Jankauskas, S.S.; Wang, X.; Santulli, G.; Gudas, L.J.; Levi, R. A Retinoic Acid Receptor β2 Agonist Improves Cardiac Function in a Heart Failure Model. J. Pharmacol. Exp. Ther. 2021, 379, 182–190. [Google Scholar] [CrossRef]
  58. Mihalas, B.P.; De Iuliis, G.N.; Redgrove, K.A.; McLaughlin, E.A.; Nixon, B. The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte. Sci. Rep. 2017, 7, 6247. [Google Scholar] [CrossRef] [Green Version]
  59. Wiernicki, B.; Dubois, H.; Tyurina, Y.Y.; Hassannia, B.; Bayir, H.; Kagan, V.E.; Vandenabeele, P.; Wullaert, A.; Berghe, T.V. Excessive phospholipid peroxidation distinguishes ferroptosis from other cell death modes including pyroptosis. Cell Death Dis. 2020, 11, 922. [Google Scholar] [CrossRef]
  60. Doll, S.; Freitas, F.P.; Shah, R.; Aldrovandi, M.; da Silva, M.C.; Ingold, I.; Grocin, A.G.; da Silva, T.N.X.; Panzilius, E.; Scheel, C.H.; et al. FSP1 is a glutathione-independent ferroptosis suppressor. Nature 2019, 575, 693–698. [Google Scholar] [CrossRef]
  61. Ufer, C.; Wang, C.C.; Fähling, M.; Schiebel, H.; Thiele, B.J.; Billett, E.E.; Kuhn, H.; Borchert, A. Translational regulation of glutathione peroxidase 4 expression through guanine-rich sequence-binding factor 1 is essential for embryonic brain development. Genes Dev. 2008, 22, 1838–1850. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Pabisz, P.; Bazak, J.; Girotti, A.W.; Korytowski, W. Anti-steroidogenic effects of cholesterol hydroperoxide trafficking in MA-10 Leydig cells: Role of mitochondrial lipid peroxidation and inhibition thereof by selenoperoxidase GPx4. Biochem. Biophys. Res. Commun. 2022, 591, 82–87. [Google Scholar] [CrossRef] [PubMed]
  63. Chen, X.; Comish, P.B.; Tang, D.; Kang, R. Characteristics and Biomarkers of Ferroptosis. Front. Cell Dev. Biol. 2021, 9, 637162. [Google Scholar] [CrossRef] [PubMed]
  64. Turchi, R.; Faraonio, R.; Lettieri-Barbato, D.; Aquilano, K. An Overview of the Ferroptosis Hallmarks in Friedreich’s Ataxia. Biomolecules 2020, 10, 1489. [Google Scholar] [CrossRef] [PubMed]
  65. Miotto, G.; Rossetto, M.; Di Paolo, M.L.; Orian, L.; Venerando, R.; Roveri, A.; Vučković, A.-M.; Travain, V.B.; Zaccarin, M.; Zennaro, L.; et al. Insight into the mechanism of ferroptosis inhibition by ferrostatin-1. Redox Biol. 2019, 28, 101328. [Google Scholar] [CrossRef] [PubMed]
  66. Zilka, O.; Shah, R.; Li, B.; Angeli, J.P.F.; Griesser, M.; Conrad, M.; Pratt, D.A. On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death. ACS Central Sci. 2017, 3, 232–243. [Google Scholar] [CrossRef]
  67. Zhang, W.; Sun, Y.; Bai, L.; Zhi, L.; Yang, Y.; Zhao, Q.; Chen, C.; Qi, Y.; Gao, W.; He, W.; et al. RBMS1 regulates lung cancer ferroptosis through translational control of SLC7A11. J. Clin. Investig. 2021, 131, e152067. [Google Scholar] [CrossRef]
  68. Wang, Y.; Zhao, Y.; Ye, T.; Yang, L.; Shen, Y.; Li, H. Ferroptosis Signaling and Regulators in Atherosclerosis. Front. Cell Dev. Biol. 2021, 9, 809457. [Google Scholar] [CrossRef]
  69. Yu, Y.; Yan, Y.; Niu, F.; Wang, Y.; Chen, X.; Su, G.; Liu, Y.; Zhao, X.; Qian, L.; Liu, P.; et al. Ferroptosis: A cell death connecting oxidative stress, inflammation and cardiovascular diseases. Cell Death Discov. 2021, 7, 193. [Google Scholar] [CrossRef]
  70. Ong, Y.T.; Andrade, J.; Armbruster, M.; Shi, C.; Castro, M.; Costa, A.S.H.; Sugino, T.; Eelen, G.; Zimmermann, B.; Wilhelm, K.; et al. A YAP/TAZ-TEAD signalling module links endothelial nutrient acquisition to angiogenic growth. Nat. Metab. 2022, 4, 672–682. [Google Scholar] [CrossRef]
  71. Xie, S.-S.; Deng, Y.; Guo, S.-L.; Li, J.-Q.; Zhou, Y.-C.; Liao, J.; Wu, D.-D.; Lan, W.-F. Endothelial cell ferroptosis mediates monocrotaline-induced pulmonary hypertension in rats by modulating NLRP3 inflammasome activation. Sci. Rep. 2022, 12, 3056. [Google Scholar] [CrossRef] [PubMed]
  72. Zhang, Q.; Bai, X.; Lin, T.; Wang, X.; Zhang, B.; Dai, L.; Shi, J.; Zhang, Y.; Zhao, X. HMOX1 Promotes Ferroptosis in Mammary Epithelial Cells via FTH1 and Is Involved in the Development of Clinical Mastitis in Dairy Cows. Antioxidants 2022, 11, 2221. [Google Scholar] [CrossRef] [PubMed]
  73. Fang, X.; Cai, Z.; Wang, H.; Han, D.; Cheng, Q.; Zhang, P.; Gao, F.; Yu, Y.; Song, Z.; Wu, Q.; et al. Loss of Cardiac Ferritin H Facilitates Cardiomyopathy via Slc7a11-Mediated Ferroptosis. Circ. Res. 2020, 127, 486–501. [Google Scholar] [CrossRef] [PubMed]
  74. Dang, Y.; He, Q.; Yang, S.; Sun, H.; Liu, Y.; Li, W.; Tang, Y.; Zheng, Y.; Wu, T. FTH1- and SAT1-Induced Astrocytic Ferroptosis Is Involved in Alzheimer’s Disease: Evidence from Single-Cell Transcriptomic Analysis. Pharmaceuticals 2022, 15, 1177. [Google Scholar] [CrossRef] [PubMed]
  75. Ou, Y.; Wang, S.-J.; Li, D.; Chu, B.; Gu, W. Activation of SAT1 engages polyamine metabolism with p53-mediated ferroptotic responses. Proc. Natl. Acad. Sci. USA 2016, 113, E6806–E6812. [Google Scholar] [CrossRef] [Green Version]
  76. Mou, Y.; Zhang, L.; Liu, Z.; Song, X. Abundant expression of ferroptosis-related SAT1 is related to unfavorable outcome and immune cell infiltration in low-grade glioma. BMC Cancer 2022, 22, 215. [Google Scholar] [CrossRef]
  77. Del Valle, D.M.; Kim-Schulze, S.; Huang, H.-H.; Beckmann, N.D.; Nirenberg, S.; Wang, B.; Lavin, Y.; Swartz, T.H.; Madduri, D.; Stock, A.; et al. An inflammatory cytokine signature predicts COVID-19 severity and survival. Nat. Med. 2020, 26, 1636–1643. [Google Scholar] [CrossRef]
  78. Chen, G.; Wu, D.; Guo, W.; Cao, Y.; Huang, D.; Wang, H.; Wang, T.; Zhang, X.; Chen, H.; Yu, H.; et al. Clinical and immunological features of severe and moderate coronavirus disease 2019. J. Clin. Investig. 2020, 130, 2620–2629. [Google Scholar] [CrossRef] [Green Version]
  79. Huang, C.; Wang, Y.; Li, X.; Ren, L.; Zhao, J.; Hu, Y.; Zhang, L.; Fan, G.; Xu, J.; Gu, X.; et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020, 395, 497–506. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  80. Mortaz, E.; Tabarsi, P.; Jamaati, H.; Roofchayee, N.D.; Dezfuli, N.K.; Hashemian, S.M.; Moniri, A.; Marjani, M.; Malekmohammad, M.; Mansouri, D.; et al. Increased Serum Levels of Soluble TNF-α Receptor Is Associated With ICU Mortality in COVID-19 Patients. Front. Immunol. 2021, 12, 592727. [Google Scholar] [CrossRef]
  81. Halim, C.; Mirza, A.F.; Sari, M.I. The Association between TNF-α, IL-6, and Vitamin D Levels and COVID-19 Severity and Mortality: A Systematic Review and Meta-Analysis. Pathogens 2022, 11, 195. [Google Scholar] [CrossRef]
  82. Kaur, S.; Tripathi, D.M.; Yadav, A. The Enigma of Endothelium in COVID-19. Front. Physiol. 2020, 11, 989. [Google Scholar] [CrossRef] [PubMed]
  83. Gupta, A.; Madhavan, M.V.; Sehgal, K.; Nair, N.; Mahajan, S.; Sehrawat, T.S.; Bikdeli, B.; Ahluwalia, N.; Ausiello, J.C.; Wan, E.Y.; et al. Extrapulmonary manifestations of COVID-19. Nat. Med. 2020, 26, 1017–1032. [Google Scholar] [CrossRef] [PubMed]
  84. Evans, P.C.; Rainger, G.E.; Mason, J.C.; Guzik, T.J.; Osto, E.; Stamataki, Z.; Neil, D.; Hoefer, I.E.; Fragiadaki, M.; Waltenberger, J.; et al. Endothelial dysfunction in COVID-19: A position paper of the ESC Working Group for Atherosclerosis and Vascular Biology, and the ESC Council of Basic Cardiovascular Science. Cardiovasc. Res. 2020, 116, 2177–2184. [Google Scholar] [CrossRef]
  85. Ackermann, M.; Verleden, S.E.; Kuehnel, M.; Haverich, A.; Welte, T.; Laenger, F.; Vanstapel, A.; Werlein, C.; Stark, H.; Tzankov, A.; et al. Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19. N. Engl. J. Med. 2020, 383, 120–128. [Google Scholar] [CrossRef] [PubMed]
  86. Delorey, T.M.; Ziegler, C.G.K.; Heimberg, G.; Normand, R.; Yang, Y.; Segerstolpe, Å.; Abbondanza, D.; Fleming, S.J.; Subramanian, A.; Montoro, D.T.; et al. COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets. Nature 2021, 595, 107–113. [Google Scholar] [CrossRef]
  87. Tan, S.; Schubert, D.; Maher, P. Oxytosis: A Novel Form of Programmed Cell Death. Curr. Top. Med. Chem. 2001, 1, 497–506. [Google Scholar] [CrossRef]
  88. Dixon, S.J.; Lemberg, K.M.; Lamprecht, M.R.; Skouta, R.; Zaitsev, E.M.; Gleason, C.E.; Patel, D.N.; Bauer, A.J.; Cantley, A.M.; Yang, W.S.; et al. Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death. Cell 2012, 149, 1060–1072. [Google Scholar] [CrossRef] [Green Version]
  89. Kan, X.; Yin, Y.; Song, C.; Tan, L.; Qiu, X.; Liao, Y.; Liu, W.; Meng, S.; Sun, Y.; Ding, C. Newcastle-disease-virus-induced ferroptosis through nutrient deprivation and ferritinophagy in tumor cells. Iscience 2021, 24, 102837. [Google Scholar] [CrossRef]
  90. Maschalidi, S.; Mehrotra, P.; Keçeli, B.N.; De Cleene, H.K.L.; Lecomte, K.; Van der Cruyssen, R.; Janssen, P.; Pinney, J.; van Loo, G.; Elewaut, D.; et al. Targeting SLC7A11 improves efferocytosis by dendritic cells and wound healing in diabetes. Nature 2022, 606, 776–784. [Google Scholar] [CrossRef]
  91. Wang, W.; Green, M.; Choi, J.E.; Gijón, M.; Kennedy, P.D.; Johnson, J.K.; Liao, P.; Lang, X.; Kryczek, I.; Sell, A.; et al. CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy. Nature 2019, 569, 270–274. [Google Scholar] [CrossRef]
  92. Giraudi, P.J.; Bellarosa, C.; Coda-Zabetta, C.D.; Peruzzo, P.; Tiribelli, C. Functional Induction of the Cystine-Glutamate Exchanger System Xc− Activity in SH-SY5Y Cells by Unconjugated Bilirubin. PLoS ONE 2011, 6, e29078. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Lee, J.; Roh, J.-L. SLC7A11 as a Gateway of Metabolic Perturbation and Ferroptosis Vulnerability in Cancer. Antioxidants 2022, 11, 2444. [Google Scholar] [CrossRef] [PubMed]
  94. Huang, C.-Y.; Chen, L.-J.; Chen, G.; Chao, T.-I.; Wang, C.-Y. SHP-1/STAT3-Signaling-Axis-Regulated Coupling between BECN1 and SLC7A11 Contributes to Sorafenib-Induced Ferroptosis in Hepatocellular Carcinoma. Int. J. Mol. Sci. 2022, 23, 11092. [Google Scholar] [CrossRef] [PubMed]
  95. Ananth, S.; Miyauchi, S.; Thangaraju, M.; Jadeja, R.N.; Bartoli, M.; Ganapathy, V.; Martin, P.M. Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina. Antioxidants 2020, 10, 9. [Google Scholar] [CrossRef] [PubMed]
  96. Pasini, A.M.F.; Stranieri, C.; Girelli, D.; Busti, F.; Cominacini, L. Is Ferroptosis a Key Component of the Process Leading to Multiorgan Damage in COVID-19? Antioxidants 2021, 10, 1677. [Google Scholar] [CrossRef]
  97. Bersuker, K.; Hendricks, J.M.; Li, Z.; Magtanong, L.; Ford, B.; Tang, P.H.; Roberts, M.A.; Tong, B.; Maimone, T.J.; Zoncu, R.; et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis. Nature 2019, 575, 688–692. [Google Scholar] [CrossRef]
  98. Jiang, X.; Stockwell, B.R.; Conrad, M. Ferroptosis: Mechanisms, biology and role in disease. Nat. Rev. Mol. Cell Biol. 2021, 22, 266–282. [Google Scholar] [CrossRef]
  99. Eagle, H. Nutrition Needs of Mammalian Cells in Tissue Culture. Science 1955, 122, 501–504. [Google Scholar] [CrossRef]
  100. Guo, Y.; Hu, K.; Li, Y.; Lu, C.; Ling, K.; Cai, C.; Wang, W.; Ye, D. Targeting TNF-α for COVID-19: Recent Advanced and Controversies. Front. Public Health 2022, 10, 833967. [Google Scholar] [CrossRef]
  101. Rarani, F.Z.; Rashidi, B.; Abadi, M.H.J.N.; Hamblin, M.R.; Hashemian, S.M.R.; Mirzaei, H. Cytokines and microRNAs in SARS-CoV-2: What do we know? Mol. Ther.—Nucleic Acids 2022, 29, 219–242. [Google Scholar] [CrossRef]
  102. Ramasamy, S.; Subbian, S. Critical Determinants of Cytokine Storm and Type I Interferon Response in COVID-19 Pathogenesis. Clin. Microbiol. Rev. 2021, 34, e00299-20. [Google Scholar] [CrossRef] [PubMed]
  103. Karki, R.; Sharma, B.R.; Tuladhar, S.; Williams, E.P.; Zalduondo, L.; Samir, P.; Zheng, M.; Sundaram, B.; Banoth, B.; Malireddi, R.K.S.; et al. Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes. Cell 2021, 184, 149–168.e17. [Google Scholar] [CrossRef] [PubMed]
  104. Ruhl, L.; Pink, I.; Kühne, J.F.; Beushausen, K.; Keil, J.; Christoph, S.; Sauer, A.; Boblitz, L.; Schmidt, J.; David, S.; et al. Endothelial dysfunction contributes to severe COVID-19 in combination with dysregulated lymphocyte responses and cytokine networks. Signal Transduct. Target. Ther. 2021, 6, 1–15. [Google Scholar] [CrossRef] [PubMed]
  105. Fara, A.; Mitrev, Z.; Rosalia, R.A.; Assas, B.M. Cytokine storm and COVID-19: A chronicle of pro-inflammatory cytokines. Open Biol. 2020, 10, 200160. [Google Scholar] [CrossRef] [PubMed]
  106. Costela-Ruiz, V.J.; Illescas-Montes, R.; Puerta-Puerta, J.M.; Ruiz, C.; Melguizo-Rodríguez, L. SARS-CoV-2 infection: The role of cytokines in COVID-19 disease. Cytokine Growth Factor Rev. 2020, 54, 62–75. [Google Scholar] [CrossRef]
  107. Attiq, A.; Yao, L.J.; Afzal, S.; Khan, M.A. The triumvirate of NF-κB, inflammation and cytokine storm in COVID-19. Int. Immunopharmacol. 2021, 101, 108255. [Google Scholar] [CrossRef]
  108. Morris, G.; Bortolasci, C.C.; Puri, B.K.; Marx, W.; O’Neil, A.; Athan, E.; Walder, K.; Berk, M.; Olive, L.; Carvalho, A.F.; et al. The cytokine storms of COVID-19, H1N1 influenza, CRS and MAS compared. Can one sized treatment fit all? Cytokine 2021, 144, 155593. [Google Scholar] [CrossRef]
  109. Rabaan, A.A.; Al-Ahmed, S.H.; Muhammad, J.; Khan, A.; Sule, A.A.; Tirupathi, R.; Mutair, A.A.; Alhumaid, S.; Al-Omari, A.; Dhawan, M.; et al. Role of Inflammatory Cytokines in COVID-19 Patients: A Review on Molecular Mechanisms, Immune Functions, Immunopathology and Immunomodulatory Drugs to Counter Cytokine Storm. Vaccines 2021, 9, 436. [Google Scholar] [CrossRef]
  110. Cure, E.; Cure, M.C. Prolonged NHE Activation may be Both Cause and Outcome of Cytokine Release Syndrome in COVID-19. Curr. Pharm. Des. 2022, 28, 1815–1822. [Google Scholar] [CrossRef]
  111. Peter, A.E.; Sandeep, B.V.; Rao, B.G.; Kalpana, V.L. Calming the Storm: Natural Immunosuppressants as Adjuvants to Target the Cytokine Storm in COVID-19. Front. Pharmacol. 2021, 11, 583777. [Google Scholar] [CrossRef] [PubMed]
  112. Tan, S.; Sagara, Y.; Liu, Y.; Maher, P.; Schubert, D. The Regulation of Reactive Oxygen Species Production during Programmed Cell Death. J. Cell Biol. 1998, 141, 1423–1432. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  113. Gao, M.; Yi, J.; Zhu, J.; Minikes, A.; Monian, P.; Thompson, C.B.; Jiang, X. Role of Mitochondria in Ferroptosis. Mol. Cell 2018, 73, 354–363.e3. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  114. Wang, H.; Liu, C.; Zhao, Y.; Gao, G. Mitochondria regulation in ferroptosis. Eur. J. Cell Biol. 2019, 99, 151058. [Google Scholar] [CrossRef]
  115. Battaglia, A.M.; Chirillo, R.; Aversa, I.; Sacco, A.; Costanzo, F.; Biamonte, F. Ferroptosis and Cancer: Mitochondria Meet the “Iron Maiden” Cell Death. Cells 2020, 9, 1505. [Google Scholar] [CrossRef]
  116. Gan, B. Mitochondrial regulation of ferroptosis. J. Cell Biol. 2021, 220, e202105043. [Google Scholar] [CrossRef]
  117. Lei, G.; Zhuang, L.; Gan, B. Targeting ferroptosis as a vulnerability in cancer. Nat. Rev. Cancer 2022, 22, 381–396. [Google Scholar] [CrossRef]
  118. Chen, X.; Kang, R.; Kroemer, G.; Tang, D. Broadening horizons: The role of ferroptosis in cancer. Nat. Rev. Clin. Oncol. 2021, 18, 280–296. [Google Scholar] [CrossRef]
  119. Fang, K.; Du, S.; Shen, D.; Xiong, Z.; Jiang, K.; Liang, D.; Wang, J.; Xu, H.; Hu, L.; Zhai, X.; et al. SUFU suppresses ferroptosis sensitivity in breast cancer cells via Hippo/YAP pathway. Iscience 2022, 25, 104618. [Google Scholar] [CrossRef]
  120. Jakaria, M.; Belaidi, A.A.; Bush, A.I.; Ayton, S. Ferroptosis as a mechanism of neurodegeneration in Alzheimer’s disease. J. Neurochem. 2021, 159, 804–825. [Google Scholar] [CrossRef]
  121. Mahoney-Sánchez, L.; Bouchaoui, H.; Ayton, S.; Devos, D.; Duce, J.A.; Devedjian, J.-C. Ferroptosis and its potential role in the physiopathology of Parkinson’s Disease. Prog. Neurobiol. 2020, 196, 101890. [Google Scholar] [CrossRef] [PubMed]
  122. Ryan, S.K.; Zelic, M.; Han, Y.; Teeple, E.; Chen, L.; Sadeghi, M.; Shankara, S.; Guo, L.; Li, C.; Pontarelli, F.; et al. Microglia ferroptosis is regulated by SEC24B and contributes to neurodegeneration. Nat. Neurosci. 2022, 26, 12–26. [Google Scholar] [CrossRef] [PubMed]
  123. Lai, B.; Wu, C.-H.; Wu, C.-Y.; Luo, S.-F.; Lai, J.-H. Ferroptosis and Autoimmune Diseases. Front. Immunol. 2022, 13, 916664. [Google Scholar] [CrossRef] [PubMed]
  124. Zhang, D.; Li, Y.; Du, C.; Sang, L.; Liu, L.; Li, Y.; Wang, F.; Fan, W.; Tang, P.; Zhang, S.; et al. Evidence of pyroptosis and ferroptosis extensively involved in autoimmune diseases at the single-cell transcriptome level. J. Transl. Med. 2022, 20, 363. [Google Scholar] [CrossRef]
  125. Hao, L.; Mi, J.; Song, L.; Guo, Y.; Li, Y.; Yin, Y.; Zhang, C. SLC40A1 Mediates Ferroptosis and Cognitive Dysfunction in Type 1 Diabetes. Neuroscience 2021, 463, 216–226. [Google Scholar] [CrossRef]
  126. Chen, Q.; Wang, J.; Xiang, M.; Wang, Y.; Zhang, Z.; Liang, J.; Xu, J. The Potential Role of Ferroptosis in Systemic Lupus Erythematosus. Front. Immunol. 2022, 13, 855622. [Google Scholar] [CrossRef]
  127. Yang, X.-D.; Yang, Y.-Y. Ferroptosis as a Novel Therapeutic Target for Diabetes and Its Complications. Front. Endocrinol. 2022, 13, 853822. [Google Scholar] [CrossRef]
  128. Ye, H.; Wang, R.; Wei, J.; Wang, Y.; Zhang, X.; Wang, L. Bioinformatics Analysis Identifies Potential Ferroptosis Key Gene in Type 2 Diabetic Islet Dysfunction. Front. Endocrinol. 2022, 13, 904312. [Google Scholar] [CrossRef]
  129. Yin, M.; Zhou, L.; Ji, Y.; Lu, R.; Ji, W.; Jiang, G.; Ma, J.; Song, X. In silico identification and verification of ferroptosis-related genes in type 2 diabetic islets. Front. Endocrinol. 2022, 13, 946492. [Google Scholar] [CrossRef]
  130. Du, S.; Shi, H.; Xiong, L.; Wang, P.; Shi, Y. Canagliflozin mitigates ferroptosis and improves myocardial oxidative stress in mice with diabetic cardiomyopathy. Front. Endocrinol. 2022, 13, 1011669. [Google Scholar] [CrossRef]
  131. Zhao, W.-K.; Zhou, Y.; Xu, T.-T.; Wu, Q. Ferroptosis: Opportunities and Challenges in Myocardial Ischemia-Reperfusion Injury. Oxidative Med. Cell. Longev. 2021, 2021, 9929687. [Google Scholar] [CrossRef] [PubMed]
  132. Lee, H.; Zandkarimi, F.; Zhang, Y.; Meena, J.K.; Kim, J.; Zhuang, L.; Tyagi, S.; Ma, L.; Westbrook, T.F.; Steinberg, G.R.; et al. Energy-stress-mediated AMPK activation inhibits ferroptosis. Nature 2020, 22, 225–234. [Google Scholar] [CrossRef] [PubMed]
  133. Pan, Y.; Wang, X.; Liu, X.; Shen, L.; Chen, Q.; Shu, Q. Targeting Ferroptosis as a Promising Therapeutic Strategy for Ischemia-Reperfusion Injury. Antioxidants 2022, 11, 2196. [Google Scholar] [CrossRef] [PubMed]
  134. Yu, S.; Jia, J.; Zheng, J.; Zhou, Y.; Jia, D.; Wang, J. Recent Progress of Ferroptosis in Lung Diseases. Front. Cell Dev. Biol. 2021, 9, 789517. [Google Scholar] [CrossRef]
  135. He, J.; Li, X.; Yu, M. Bioinformatics Analysis Identifies Potential Ferroptosis Key Genes in the Pathogenesis of Pulmonary Fibrosis. Front. Genet. 2022, 12, 919187. [Google Scholar] [CrossRef]
  136. Li, Y.; Yang, Y.; Yang, Y. Multifaceted Roles of Ferroptosis in Lung Diseases. Front. Mol. Biosci. 2022, 9, 919187. [Google Scholar] [CrossRef]
  137. Jacobs, W.; Lammens, M.; Kerckhofs, A.; Voets, E.; Van San, E.; Van Coillie, S.; Peleman, C.; Mergeay, M.; Sirimsi, S.; Matheeussen, V.; et al. Fatal lymphocytic cardiac damage in coronavirus disease 2019 (COVID-19): Autopsy reveals a ferroptosis signature. ESC Hear. Fail. 2020, 7, 3772–3781. [Google Scholar] [CrossRef]
  138. Kwon, M.-Y.; Park, E.; Lee, S.-J.; Chung, S.W. Heme oxygenase-1 accelerates erastin-induced ferroptotic cell death. Oncotarget 2015, 6, 24393–24403. [Google Scholar] [CrossRef] [Green Version]
  139. Sun, X.; Ou, Z.; Chen, R.; Niu, X.; Chen, D.; Kang, R.; Tang, D. Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology 2015, 63, 173–184. [Google Scholar] [CrossRef] [Green Version]
  140. Mone, P.; Gambardella, J.; Pansini, A.; de Donato, A.; Martinelli, G.; Boccalone, E.; Matarese, A.; Frullone, S.; Santulli, G. Cognitive Impairment in Frail Hypertensive Elderly Patients: Role of Hyperglycemia. Cells 2021, 10, 2115. [Google Scholar] [CrossRef]
  141. Plotnikov, E.Y.; Chupyrkina, A.A.; Jankauskas, S.S.; Pevzner, I.B.; Silachev, D.N.; Skulachev, V.P.; Zorov, D.B. Mechanisms of nephroprotective effect of mitochondria-targeted antioxidants under rhabdomyolysis and ischemia/reperfusion. Biochim. et Biophys. Acta (BBA)—Mol. Basis Dis. 2011, 1812, 77–86. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  142. Xie, W.; Santulli, G.; Reiken, S.R.; Yuan, Q.; Osborne, B.W.; Chen, B.-X.; Marks, A.R. Mitochondrial oxidative stress promotes atrial fibrillation. Sci. Rep. 2015, 5, 11427. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  143. Mone, P.; Pansini, A.; Jankauskas, S.S.; Varzideh, F.; Kansakar, U.; Lombardi, A.; Trimarco, V.; Frullone, S.; Santulli, G. L-Arginine Improves Cognitive Impairment in Hypertensive Frail Older Adults. Front. Cardiovasc. Med. 2022, 9, 868521. [Google Scholar] [CrossRef]
  144. Santulli, G.; Xie, W.; Reiken, S.R.; Marks, A.R. Mitochondrial calcium overload is a key determinant in heart failure. Proc. Natl. Acad. Sci. USA 2015, 112, 11389–11394. [Google Scholar] [CrossRef] [Green Version]
  145. Tadokoro, T.; Ikeda, M.; Ide, T.; Deguchi, H.; Ikeda, S.; Okabe, K.; Ishikita, A.; Matsushima, S.; Koumura, T.; Yamada, K.-I.; et al. Mitochondria-dependent ferroptosis plays a pivotal role in doxorubicin cardiotoxicity. J. Clin. Investig. 2020, 5, e132747. [Google Scholar] [CrossRef] [PubMed]
  146. Kushnir, A.; Santulli, G.; Reiken, S.R.; Coromilas, E.; Godfrey, S.J.; Brunjes, D.L.; Colombo, P.C.; Yuzefpolskaya, M.; Sokol, S.I.; Kitsis, R.N.; et al. Ryanodine Receptor Calcium Leak in Circulating B-Lymphocytes as a Biomarker in Heart Failure. Circulation 2018, 138, 1144–1154. [Google Scholar] [CrossRef] [PubMed]
  147. Amgalan, D.; Garner, T.P.; Pekson, R.; Jia, X.F.; Yanamandala, M.; Paulino, V.; Liang, F.G.; Corbalan, J.J.; Lee, J.; Chen, Y.; et al. A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy. Nat. Cancer 2020, 1, 315–328. [Google Scholar] [CrossRef]
  148. Santulli, G.; Wronska, A.; Uryu, K.; Diacovo, T.G.; Gao, M.; Marx, S.O.; Kitajewski, J.; Chilton, J.M.; Akat, K.M.; Tuschl, T.; et al. A selective microRNA-based strategy inhibits restenosis while preserving endothelial function. J. Clin. Investig. 2014, 124, 4102–4114. [Google Scholar] [CrossRef]
  149. Cipolletta, E.; Rusciano, M.R.; Maione, A.S.; Santulli, G.; Sorriento, D.; Del Giudice, C.; Ciccarelli, M.; Franco, A.; Crola, C.; Campiglia, P.; et al. Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy. PLoS ONE 2015, 10, e0130477. [Google Scholar] [CrossRef] [Green Version]
  150. Gambardella, J.; Coppola, A.; Izzo, R.; Fiorentino, G.; Trimarco, B.; Santulli, G. Role of endothelial miR-24 in COVID-19 cerebrovascular events. Crit. Care 2021, 25, 306. [Google Scholar] [CrossRef]
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Figure 1. Serum from patients who did not survive COVID-19 promotes oxidative stress in human endothelial cells. (A) Representative microphotographs of DCF and Hoechst staining of HUVECs treated for 24 h with 10% serum obtained on hospital admission from patients who survived or succumbed to COVID-19; scale bar: 50 μm. (B) Quantification of DCF fluorescence intensity. Data were obtained from at least three independent experiments and are presented as a box-and-whiskers plot showing the median and the 5th–95th percentiles; A.U.: arbitrary units.
Figure 1. Serum from patients who did not survive COVID-19 promotes oxidative stress in human endothelial cells. (A) Representative microphotographs of DCF and Hoechst staining of HUVECs treated for 24 h with 10% serum obtained on hospital admission from patients who survived or succumbed to COVID-19; scale bar: 50 μm. (B) Quantification of DCF fluorescence intensity. Data were obtained from at least three independent experiments and are presented as a box-and-whiskers plot showing the median and the 5th–95th percentiles; A.U.: arbitrary units.
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Figure 2. Serum from patients who did not survive COVID-19 increases mitochondrial ROS production. (A) Representative microphotographs of MitoSOX Red and MitoTracker Green FM (MTG) staining of HUVECs treated for 24 h with 10% serum from patients who survived or did not survive COVID-19. (B) Quantification of MitoSOX Red fluorescence intensity. Data are from at least three independent experiments and are presented as a box-and-whiskers plot showing the median and the 5th–95th percentiles; A.U.: arbitrary units.
Figure 2. Serum from patients who did not survive COVID-19 increases mitochondrial ROS production. (A) Representative microphotographs of MitoSOX Red and MitoTracker Green FM (MTG) staining of HUVECs treated for 24 h with 10% serum from patients who survived or did not survive COVID-19. (B) Quantification of MitoSOX Red fluorescence intensity. Data are from at least three independent experiments and are presented as a box-and-whiskers plot showing the median and the 5th–95th percentiles; A.U.: arbitrary units.
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Figure 3. Lipid peroxidation in HUVECs is increased by serum from patients who did not survive COVID-19. Three different assays, namely the quantification of MDA equivalents (A), 4HNE (B), and C11-BODIPY (C), confirmed that the incubation of HUVECs for 24 h with serum from COVID-19 patients who did not survive significantly increased lipid peroxidation. All experiments were performed in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Normal Serum, #: p < 0.01 vs. Survivors.
Figure 3. Lipid peroxidation in HUVECs is increased by serum from patients who did not survive COVID-19. Three different assays, namely the quantification of MDA equivalents (A), 4HNE (B), and C11-BODIPY (C), confirmed that the incubation of HUVECs for 24 h with serum from COVID-19 patients who did not survive significantly increased lipid peroxidation. All experiments were performed in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Normal Serum, #: p < 0.01 vs. Survivors.
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Figure 4. GPX4 expression in human endothelial cells is reduced by serum from patients who did not survive COVID-19. GPX4 levels were measured by RT-qPCR (A) and by immunoblot (B) in HUVECs, normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH); panel B shows representative blots from triplicate experiments (top) and their quantification (bottom). All experiments were performed at least in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Normal Serum, #: p < 0.01 vs. COVID-19 Survivors. Sequences of oligonucleotide primers are reported in Table 1.
Figure 4. GPX4 expression in human endothelial cells is reduced by serum from patients who did not survive COVID-19. GPX4 levels were measured by RT-qPCR (A) and by immunoblot (B) in HUVECs, normalizing to glyceraldehyde 3-phosphate dehydrogenase (GAPDH); panel B shows representative blots from triplicate experiments (top) and their quantification (bottom). All experiments were performed at least in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Normal Serum, #: p < 0.01 vs. COVID-19 Survivors. Sequences of oligonucleotide primers are reported in Table 1.
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Figure 5. COVID-19 serum from non-survivors regulates ferroptosis via TNFR1 in HUVECs. Representative immunoblots are shown in panel (A), quantified in panel (B) (GPX4), (C) (SCL7A11), (D) (FTH1), and (E) (SAT1). All experiments were performed at least in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Survivors.
Figure 5. COVID-19 serum from non-survivors regulates ferroptosis via TNFR1 in HUVECs. Representative immunoblots are shown in panel (A), quantified in panel (B) (GPX4), (C) (SCL7A11), (D) (FTH1), and (E) (SAT1). All experiments were performed at least in triplicate; the box-and-whiskers graphs show the median and the 5th–95th percentiles; *: p < 0.01 vs. Survivors.
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Table
Table 1. Oligonucleotide sequences of the primers used for RT-qPCR.
Table 1. Oligonucleotide sequences of the primers used for RT-qPCR.
PrimerSequence (5′-3′)Amplicon (bp)
GPX4ForwardGAG ATC AAA GAG TTC GCC GC102
ReverseCTT CAT CCA CTT CCA CAG CG
TNFR1ForwardTTG TAT GGC CCC AAC TGT CT99
ReverseCTG GCT CAA GTC CTT CCT CA
GAPDHForwardGGC TCC CTT GGG TAT ATG GT94
ReverseTTG ATT TTG GAG GGA TCT CG
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Jankauskas, S.S.; Kansakar, U.; Sardu, C.; Varzideh, F.; Avvisato, R.; Wang, X.; Matarese, A.; Marfella, R.; Ziosi, M.; Gambardella, J.; et al. COVID-19 Causes Ferroptosis and Oxidative Stress in Human Endothelial Cells. Antioxidants 2023, 12, 326. https://doi.org/10.3390/antiox12020326

AMA Style

Jankauskas SS, Kansakar U, Sardu C, Varzideh F, Avvisato R, Wang X, Matarese A, Marfella R, Ziosi M, Gambardella J, et al. COVID-19 Causes Ferroptosis and Oxidative Stress in Human Endothelial Cells. Antioxidants. 2023; 12(2):326. https://doi.org/10.3390/antiox12020326

Chicago/Turabian Style

Jankauskas, Stanislovas S., Urna Kansakar, Celestino Sardu, Fahimeh Varzideh, Roberta Avvisato, Xujun Wang, Alessandro Matarese, Raffaele Marfella, Marcello Ziosi, Jessica Gambardella, and et al. 2023. "COVID-19 Causes Ferroptosis and Oxidative Stress in Human Endothelial Cells" Antioxidants 12, no. 2: 326. https://doi.org/10.3390/antiox12020326

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