cooperation between cyb5r3 and nox4 via coenzyme q ......2021/08/12 · 12 coq redox cycling allows...

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Cooperation between CYB5R3 and NOX4 via coenzyme Q 1

mitigates endothelial inflammation 2

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Shuai Yuan1, Scott A. Hahn1, Megan P. Miller1, Subramaniam Sanker1, Michael J 4

Calderon3, Mara Sullivan3, Atinuke M. Dosunmu-Ogunbi1,2, Marco Fazzari2, Yao Li1, 5

Michael Reynolds1, Katherine C Wood1, Claudette M. St. Croix3, Donna Stolz3, Eugenia 6

Cifuentes-Pagano 1,2, Placido Navas4, Sruti Shiva1,2, Francisco J. Schopfer1,2,5, Patrick J. 7

Pagano1,2, Adam C. Straub1,2,6. 8

9 1Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, 10

Pennsylvania; 11 2Department of Pharmacology and Chemical Biology, University of Pittsburgh, 12

Pittsburgh, Pennsylvania; 13 3Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, Pennsylvania; 14

4Centro Andaluz de Biología del Desarrollo and CIBERER, Instituto de Salud Carlos III, 15

Universidad Pablo de Olavide-CSIC-JA, Sevilla, Spain, Spain; 16 5Pittsburgh Liver Research Center (PLRC), University of Pittsburgh, Pittsburgh, 17

Pennsylvania; 18 6Center for Microvascular Research, University of Pittsburgh, Pittsburgh, Pennsylvania. 19

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Running Title: Endothelial CYB5R3 regulates NOX4 activity 22

Key Words: CYB5R3, NOX4, CoQ, ROS, inflammation 23

Word Count: 5667 (main body only) 24

25

Address correspondence to: 26

Adam C. Straub, Ph.D. 27

Associate Professor Pharmacology and Chemical Biology 28

Director of the Center for Microvascular Research 29

University of Pittsburgh School of Medicine, 30

Department of Pharmacology and Chemical Biology 31

The Heart, Lung, Blood and Vascular Medicine Institute 32

E1254 Biomedical Science Tower, 200 Lothrop St. 33

Pittsburgh, PA 15216 34

Tel: 412-648-7097 35

Fax: 412-648-5980 36

[email protected] 37

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted August 12, 2021. ; https://doi.org/10.1101/2021.08.12.456058doi: bioRxiv preprint

https://doi.org/10.1101/2021.08.12.456058

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ABSTRACT 1

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing reactive 2

oxygen species. Since coenzyme Q (CoQ) mimics affect NOX4 activity, we hypothesize 3

that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular 4

endothelial cells, modulates inflammatory activation. 5

Mice lacking endothelial CYB5R3 (R3 KO), under lipopolysaccharides (LPS) challenge, 6

showed exacerbated hypotension, decreased acetylcholine-induced vasodilation, and 7

elevated vascular adhesion molecule 1 (Vcam-1) mRNA in aorta. In vitro, silencing 8

Cyb5r3 enhanced LPS-induced VCAM-1 protein in a NOX4 dependent manner. APEX2-9

based electron microscopy and proximity biotinylation demonstrated CYB5R3's 10

localization on the mitochondrial outer membrane and its interaction with NOX4, which 11

was further confirmed by the proximity ligation assay. Notably, Cyb5r3 silenced HAECs 12

had less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound 13

active CYB5R3, we found CYB5R3 activity and membrane translocation were needed 14

for optimal generation of H2O2 by NOX4. Lastly, CoQ deficient cells showed decreased 15

NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. 16

In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in 17

NOX4-dependent H2O2 generation via CoQ. 18

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NOVELTY AND SIGNIFICANCE 1

What Is Known? 2

NADPH oxidase 4 (NOX4) reportedly produces primarily hydrogen peroxide 3

(H2O2) and, to a lesser extent, superoxide (O2•-) and has been shown to have 4

both beneficial and deleterious effects in the cardiovascular system. 5

NOX4 activity can be affected by NAD(P)H quinone oxidoreductase 1 (NQO1), a 6

CoQ reductase, and synthetic quinone compounds used to mimic CoQ. 7

Cytochrome b5 reductase 3 (CYB5R3) is known to reduce CoQ and is highly 8

expressed in endothelial cells. 9

What New Information Does This Article Contribute? 10

In vivo, the lack of endothelial CYB5R3 causes exacerbated lipopolysaccharides 11

(LPS)-induced inflammatory signaling, endothelial dysfunction, and hypotension. 12

Endothelial CYB5R3 mitigates inflammatory signaling by LPS and tumor necrosis 13

factor (TNF-) in a NOX4 dependent manner. 14

In endothelial cells, CYB5R3 and NOX4 reside in close proximity on the 15

mitochondrial outer membrane. 16

NOX4's ability to generate H2O2 depends on the membrane translocation and 17

activity of CYB5R3 and the presence of endogenous CoQ. 18

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NONSTANDARD Abbreviations and Acronyms 1

CoQ coenzyme Q

CYB5R3 cytochrome b5 reductase 3

R3

GFP green fluorescent protein

H2O2 hydrogen peroxide

HAEC human aortic endothelial cell

HEK human embryonic kidney cell

ICAM-1 Intercellular cell adhesion molecule 1

KO knockout

LPS lipopolysaccharides

NF-κB Nuclear factor kappa B

NO nitric oxide

NOS2 nitric oxide synthase 2, inducible

NOS3 nitric oxide synthase 3, endothelial

NOX4 NADPH oxidase 4

NQO1 NAD(P)H quinone oxidoreductase 1

O2•- superoxide anion

PLA proximity ligation assay

siNOX4 Nox4 siRNA

siNT non-targeting siRNA

siR3 Cyb5r3 siRNA

TLR4 toll-like receptor 4

TNF-α tumor necrosis factor

TOM20 translocase of outer mitochondrial membrane 20

UQ ubiquinone

UQH• semiubiquinone

VCAM-1 vascular cell adhesion molecule 1

WT wild-type 2

Protein names are abbreviated as capital letters (e.g., CYB5R3), while the 3

corresponding gene names are annotated as in italic lower cases (e.g., Cyb5r3). 4


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INTRODUCTION 1

In the vascular wall, the endothelium governs leukocyte recruitment during inflammation 2

to help detain and remediate pathogens1. Pathogenic stimuli such as endotoxin activate 3

endothelium to rapidly deploy adhesion molecules to the surface to entrap leukocytes 4

and facilitate diapedesis2. Vascular cell and intercellular adhesion molecules, VCAM-1 5

and ICAM-1, are major endothelial recruitment factors for leukocytes at sites of 6

infection3-5. In severe infection, endotoxin released by circulating pathogens 7

exacerbates this process, causing systemic hypotension, reduced peripheral blood flow, 8

and multi-organ failure6, 7. One important element of the inflammatory signaling 9

response is reactive oxygen species. Reactive oxygen species are being increasingly 10

appreciated for their essential role in cellular signal transduction and conditioning. On 11

the one hand, phagosomal superoxide (O2•-) in leukocytes kills bacteria, whereas 12

excessive reactive species such as O2•-, hydrogen peroxide (H2O2), and peroxynitrite 13

can contribute to host endothelial overactivation and dysfunction8, 9. 14

Endothelial cells employ a diversity of machinery, under physiological and pathological 15

conditions, to generate reactive species such as O2•- and H2O2. NADPH oxidases (NOX) 16

are a family of enzymes dedicated to reducing ambient oxygen for O2•- and H2O2 17

formation. In the NOX family, NOX4 is unique on several levels: 1) it is constitutively 18

activated, 2) it predominantly produces H2O2 versus O2•-, 3) it is compartmentalized on 19

intracellular membranes and in particular the mitochondrion and 4) it serves divergent 20

roles in the cardiovascular system10-12. Although NOX4 is protective in animal models of 21

chronic inflammation, including atherosclerosis and peripheral arterial disease13, 14, it 22

exacerbates the acute inflammatory response induced by lipopolysaccharides and 23


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tumor necrosis factor (TNF-) in endothelial cells15-17. However, it remains 1

incompletely understood how NOX4 causes these opposing cardiovascular outcomes. 2

One explanation for NOX4's dichotomous functions in cardiovascular outcomes likely 3

stems from its capacity to generate two different primary products, H2O2 and O2•-. 4

Coenzyme Q (CoQ) has been suggested to augment NOX4's production of H2O2, but 5

the evidence is limited to biochemical studies using synthetic quinone compounds and a 6

CoQ reductase, NAD(P)H quinone oxidoreductase 1 (NQO1)18. Ubiquitously 7

synthesized by mammalian cells, CoQ has a long hydrophobic side chain embedded in 8

the lipid bilayer and a benzoquinone head group. The head group is redox-sensitive 9

and can receive or donate up to two electrons to cycle between three distinct states: the 10

fully oxidized ubiquinone, the fully reduced ubiquinol, and the semiubiquinone radical. 11

CoQ redox cycling allows it to shuttle electrons between enzymes maintaining their 12

activity19. Therefore, endogenous CoQ may modulate NOX4 activity with the assistance 13

of a CoQ reductase. 14

Cytochrome b5 reductase 3 (CYB5R3) is a CoQ reductase highly expressed in 15

endothelial cells. CYB5R3-dependent CoQ reduction is essential in the membrane 16

antioxidation pathway20-22. In addition to CoQ reduction, CYB5R3 mediates the 17

reduction of hemeproteins such as cytochrome b5 and hemoglobin, which is required 18

for fatty acid metabolism and the prevention of methemoglobinemia23, 24. We have 19

previously demonstrated that endothelial cells in small arteries and arterioles use 20

CYB5R3 to modulate nitric oxide signaling via α-globin heme redox cycling25. However, 21

the role of endothelial cell CYB5R3 in large arteries, such as the aorta, has not been 22

investigated. This constitutes a critical research gap since endothelial cells of large 23


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arteries abundantly express CYB5R3 but do not express α-globin or use fatty acid as a 1

primary source of energy. Therefore, considering the critical role of NOX4 in 2

inflammation and the potential interaction between NOX4 and CYB5R3, we 3

hypothesized that endothelial CYB5R3 regulates inflammatory activation by modulating 4

NOX4 oxidant formation. 5

METHODS AND MATERIALS 6

Animals 7

All animal studies were approved by and in compliance with the University of Pittsburgh 8

Institutional Animal Care and Use Committee. Cyb5r3 floxed mice (Cyb5r3fl/fl) were 9

generated as previously described26. Both wild-type C57BL/6J and Cyb5r3fl/fl mice were 10

crossed with the tamoxifen-inducible Cdh5(PAC)-CreERT2 mice kindly provided by Dr. 11

Ralf Adams27. Male mice at the age of 10-12 weeks were treated with tamoxifen (10 12

mg/kg in corn oil) intraperitoneally for five consecutive days and allowed to rest for 13

seven days before experiments. All mice were assigned a number randomly at birth, 14

and the genotype was concealed from the technician during LPS treatment and data 15

acquiring. All mice used in this study were reported. 16

Cell culture 17

Human aortic endothelial cells (HAEC) were purchased from Lonza and maintained in 18

endothelial growth medium (EGM-2, Lonza, CC-3162), with 5% CO2, at 37°C. 19

According to Lonza's suggestion, population doubling was used to track the age of cells 20

in culture instead of the conventional passage number. Cells less than 13 times of 21

population doublings were used for experiments. In an experiment, HAECs were 22


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allowed to grow fully confluent before they were challenged with LPS (1 or 10 ug/ml) for 1

indicated incubation time. 2

HEK293FT cells were obtained from Thermo Fisher Scientific (R70007) and grew in 3

Dulbecco's Modified Eagle Medium (DMEM, 12430062) with 10% fetal bovine serum. 4

COQ6 gene deletion was achieved in HEK293 cells with the CRISPR-Cas9 technology 5

and kindly offered by Dr. Placido Navas28. The COQ6 knockout cell line and 6

corresponding control cell line were maintained in DMEM with 10% fetal bovine serum. 7

Additionally, 10 µM uridine was supplied for both cell lines to compensate for the loss of 8

CoQ. 9

Cloning of expression vectors. 10

The wild-type (WT) human Cyb5r3 (NM_000398.6) was cloned from a pINCY vector 11

(LIFESEQ1901142, Open Biosystems) into the pcDNA3.1 expression vector (Invitrogen, 12

V86020). The nucleotides encoding the first 23 amino acids for membrane anchoring 13

were deleted (ΔAA1-23) by PCR. Two inactive variants, K111A and K111A/G180V, 14

were created by point mutations on the pcDNA3.1-Cyb5r3 (WT) plasmid using 15

QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, 200521). Mutagenesis 16

primers were designed using Agilent's online program. Additionally, human Nox4 17

(AF254621.1) and p22 (NM_000101.4) in pcDNA3.1 were kindly provided by Dr. Patrick 18

Pagano. 19

APEX2 related electron microscopy and affinity pulldown 20

Transient expression of CYB5R3-APEX2 was achieved by using lentivirus as described 21

above. Our bicistronic vector coexpresses CYB5R3-APEX2 and green fluorescent 22

protein (GFP) with an internal ribosome entry site (IRES). To avoid arbitrary effects due 23


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to a high level of expression, we used a low concentration of virus to obtain 10% GFP 1

positive cells. APEX2 based electron microscopy and affinity enrichment were 2

performed according to previous publications29, 30. 3

For electron microscopy, HAECs were fixed with 2% (v/v) glutaraldehyde in 100 mM 4

cacodylate solution. Fixed cells were loaded with 0.5 mg/ml 3,3′-Diaminobenzidine 5

(DAB) and treated with 10 mM H2O2. Once brown precipitates form in CYB5R3-APEX2 6

positive cells, cells were rinsed with 100 mM cacodylate solution, post-fixed in 1% 7

osmium tetroxide with 1% potassium ferricyanide, rinsed in PBS, dehydrated through a 8

graded series of ethanol, and embedded in Poly/Bed® 812 (Luft formulations). Semi-9

thin (300 nm) sections were cut on a Leica Reichart Ultracut, stained with 0.5% 10

Toluidine Blue in 1% sodium borate, and examined under the light microscope. 11

Ultrathin sections (65 nm) were examined on a JEOL 1400 Plus transmission electron 12

microscope with a side mount AMT 2k digital camera (Advanced Microscopy 13

Techniques, Danvers, MA). 14

For affinity pulldown, HAECs were incubated with 500 µM biotin-phenoxyl radical 15

(Cayman, 26997) for 30 minutes. To activate APEX2, H2O2 was added at the final 16

concentration of 100 µM for 60 seconds. Biotin-phenoxyl radical were immediately 17

neutralized by repeated washing with the quench buffer (10 mM sodium ascorbate, 10 18

mM sodium azide, 5 mM Trolox [Cayman, 10011659] in phosphate-buffered saline, pH 19

8). Biotinylated proteins were enriched using Dynabeads™ M-280 Streptavidin 20

(Invitrogen, 11205D) and eluted by heating the beads in 2X Laemmli buffer for 21

immunoblotting. 22

Quantification of H2O2 and NOX4 activity 23


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Cellular production of H2O2 was evaluated by its fluorescent reaction product with 1

coumarin boronate (Cayman, 14051) in the presence of L-NAME (Cayman, 80210) and 2

taurine (Cayman, 27031)31-33. For endothelial cells, cells were plated and transfected in 3

a 96-well plate. To measure H2O2, the medium was changed to the assay buffer (20 mM 4

HEPES pH 7.5, 10 µM EDTA, 100 µM L-NAME, 1 mM taurine, and 0.01% bovine serum 5

albumin in DMEM). For each group, negative controls were included by adding catalase 6

to the final concentration of 500 µg/ml. Reactions were started by adding 20 µM CBA 7

probe. Fluorescence (350/450 nm) was measured kinetically at 37 °C for four hours. At 8

the end of the assay, endothelial cells were stained with crystal violet to determine the 9

number of cells. To calculate the reaction rate, the log phase slope was normalized with 10

the crystal violet staining results. For HEK293(FT) cells, since they did not withstand 11

repeated rinsing during crystal violet staining, a different strategy was used for 12

normalization. After transfection, HEK293(FT) cells were dislodged, counted, and 13

resuspended in the assay buffer so that each well in a 96-well plate contains 120,000 14

cells at the time of the assay. Otherwise, the CBA assay was performed the same as for 15

HAECs. Additionally, cells were pelleted from the remaining cell suspension for protein 16

quantification to normalize the result. Importantly, for each treatment, the fluorescent 17

signal from a corresponding catalase control was included, and the catalase-inhibitable 18

amount was designated as H2O2. 19

Mitochondrial O2•- measurement 20

Cells were transfected in 10-cm dishes for siRNA transfection. To measure 21

mitochondrial O2•-, cells were trypsinized and resuspended in Ca2+ and Mg2+ negative 22

Hank's balanced salt solution (HBSS). In a 96-well plate, 100,000 cells were added to 23


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each well with 5 µM MitoSOX™ Red (Invitrogen, M36008). Immediately after the 1

addition of MitoSOX, the fluorescent signal (510/580 nm) was recorded at 37 °C for two 2

hours. To reduce variation, fluorescent intensities within every five minutes were 3

averaged before the log phase slope was calculated to represent the reaction rate. The 4

results were normalized with the protein concentrations measured in the remaining cell 5

suspension. 6

Statistics 7

Data were reported as mean ± standard error of the mean unless specified otherwise. 8

The n value represents the number of animals or independent experiments repeated on 9

different days. Statistical analysis was performed with Graph Pad Prism 8 using Student 10

t-test, one-way ANOVA, and two-way ANOVA with Sidak or Tukey post-hoc test. P-11

values less than 0.05 were considered as statistically significant. Specific tests used for 12

experiments are shown in figure legends. 13

Extended methods can be found in the supplementary material. 14


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RESULTS 1

Endothelial CYB5R3 protects against LPS-induced inflammatory responses in 2

vivo. 3

Cyb5r3 floxed mice were previously generated by our group and crossed with Cdh5-4

CreERT2 mice to create the tamoxifen-inducible, endothelium-specific Cyb5r3 knockout 5

mice (R3 KO)26 (Figure 1a). After five consecutive days of intraperitoneal tamoxifen 6

injections (10 mg/kg), endothelial CYB5R3 protein was absent in the aorta from R3 KO 7

mice (Figure 1a). To determine whether endothelial CYB5R3 affects LPS induced 8

systemic hypotension, we implanted radiotelemetry units in mice to monitor real-time 9

changes in arterial blood pressure. At baseline, there was no difference in resting blood 10

systolic pressure between R3 KO mice and WT controls during a full 24-hour cycle 11

(Figure 1b). However, significantly lower heart rates were observed in the R3 KO mice 12

(Supplemental Table 1). A single sublethal dose of LPS (5 mg/kg) injected 13

intraperitoneally caused a drop in systolic blood pressure in WT (Figure 1c). Importantly, 14

R3 KO mice suffered a more significant loss of systolic blood pressure (34.4±17 vs. 15

20.2±19.5 mmHg at the 6-hour time point). In a separate cohort of mice, aortae were 16

collected from WT and R3 KO mice 8 hours after LPS (5 mg/kg) injections. Ex vivo wire 17

myography showed inhibited acetylcholine-induced vasodilation in R3 KO aortic rings, 18

suggesting LPS resulted in more severe endothelial dysfunction in the Cyb5r3 deficient 19

endothelium (Figure 1d). It is worth noting that the vasodilation induced by 20

acetylcholine ex vivo depends on endothelial nitric oxide synthase (NOS3) function34. In 21

contrast, LPS-induced vasodilation in vivo is known to be mediated by nitric oxide 22

derived from inducible nitric oxide synthase (NOS2) in leukocytes35, 36. Indeed, 23


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inflammatory genes such as Nos2 and vascular cell adhesion molecule 1 (Vcam1), but 1

not intercellular adhesion molecule 1 (Icam1), were expressed at significantly higher 2

levels in R3 KO mice relative to WT (Figure 1e-g). These animal experiments indicate 3

that CYB5R3 deficiency in endothelial cells exacerbates LPS induced inflammatory 4

activation, endothelial dysfunction, and systemic hypotension. 5

To examine whether endothelial CYB5R3 deficiency is directly responsible for the 6

enhanced LPS signaling, we challenged cultured human aortic endothelial cells (HAEC) 7

with LPS overnight. Using 1 g/ml of LPS, we observed significantly higher VCAM-1 8

and ICAM-1 protein expression in Cyb5r3 siRNA-treated HAECs (Figure 2a-c). It is 9

known that nuclear factor kappa B (NF-κB) is a major transcription factor responsible for 10

VCAM-1 and ICAM-1 expression37. A time course of LPS treatment showed over-11

activation of NF-κB at the one-hour time point, indicated by augmented phosphorylation 12

of the p65 subunit and enhanced VCAM-1 and ICAM-1 expression at the 4-hour time 13

(Figure 2d-g). Moreover, BMS 345541, an inhibitor targeting inhibitor of nuclear factor-14

κB kinase (IKK) prevented VCAM-1 and ICAM-1 upregulation in Cyb5r3 knockdown 15

cells, indicating the overactivated inflammatory signaling was NF-B dependent. 16

Together, these data indicate that CYB5R3 is critical for LPS induced endothelial 17

inflammatory activation both in vivo and in vitro. 18

CYB5R3 mitigates LPS signaling in a NOX4 dependent manner. 19

It is well established that LPS signals through toll-like receptor-4 (TLR-4) to elevate 20

ICAM and VCAM-1 expression38. Additionally, NOX4 has been shown to mediate LPS-21

TLR4 signaling in endothelial cells39. Considering the regulatory effect of NQO1, a CoQ 22

reductase, on NOX4, we tested whether CYB5R3 modulates NOX4 activity to affect 23


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LPS-induced inflammatory activation using cultured HAECs treated with siRNA 1

targeting Cyb5r3 and Nox4 mRNA, separately or in combination. Non-targeting siRNA 2

was added as a control for the total siRNA load. As shown in Figure 3a, 3d, and 3e, 3

both CYB5R3 and NOX4 protein expression were significantly suppressed by the 4

corresponding siRNA. Interestingly, Nox4 siRNA slightly but significantly increased 5

CYB5R3 protein expression (Figure 3d), suggesting a potential compensatory effect 6

between NOX4 and CYB5R3. More importantly, the loss of NOX4 completely prevented 7

the upregulation of LPS-induced VCAM-1 and ICAM-1 expression in Cyb5r3 deficient 8

HAECs (Figure 3a-c). To examine whether the protective effects of Cyb5r3 and Nox4 9

occur at the transcriptional level, we measured mRNA expression at 2 and 4 hours after 10

LPS treatment. Indeed, both LPS-stimulated Vcam-1 and Icam-1 mRNA were increased 11

in Cyb5r3 knockdown HAECs at the 4-hour time point (Figure 4a-b). When HAECs 12

were treated with Nox4 siRNA, augmentation of Vcam-1 mRNA in the Cyb5r3 deficient 13

HAECs was ablated (Figure 4a). By contrast, Nox4 gene silencing did not significantly 14

affect Icam1 mRNA at either time point (Figure 4b). It is possible that LPS-induced 15

Vcam-1 and Icam-1 expression follow different time courses. Knockdown efficiencies of 16

Cyb5r3 and Nox4 were verified at the mRNA level (Figure 4c-d). Surprisingly, the 17

compensatory upregulation of CYB5R3 protein in Nox4 knockdown cells was not 18

significantly reflected at the mRNA level (Figure 4c). However, Nox4 mRNA was 19

significantly increased with Cyb5r3 gene silencing (Figure 4d). 20

CYB5R3 and NOX4 modulate TNF-α induced VCAM-1 expression. 21

LPS-induced inflammatory signaling in vivo can be magnified and propagated via TNF-22

α40, 41. Therefore, we extended our studies to test if the protective effects of CYB5R3 23


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involved TNF-α signaling. In cultured HAECs, in which Cyb5r3 was silenced using 1

siRNA prior to overnight treatment with TNF-α (10 ng/ml), Cyb5R3 deficiency 2

exacerbated both VCAM-1 and ICAM-1 protein expression. When Nox4 was silenced 3

alongside Cyb5r3, only the previously seen VCAM-1 upregulation with Cyb5r3 4

deficiency was prevented, not ICAM-1. (Figure 5a-c). Therefore, CYB5R3 protects, in 5

part, against TNF-α mediated inflammatory signaling. Moreover, we observed the same 6

compensatory effects on CYB5R3 and NOX4 expression (Figure 4d-e, 5d-e). 7

CYB5R3 and NOX4 interact on the mitochondrial outer membrane. 8

Since our data indicated a protective role for CYB5R3 in LPS and TNF-α-activated 9

endothelial inflammation mediated by NOX4, we hypothesized that CYB5R3 may 10

regulate NOX4 activity through ubiquinone reduction on the mitochondrial membrane. 11

Mitochondria are a primary location for both CYB5R3 and NOX411, 42-44. To test this 12

hypothesis, we first performed immunostaining for CYB5R3 and NOX4 in cultured 13

HAECs. Both CYB5R3 and NOX4 colocalized with TOM20, a mitochondrial marker 14

(Figure 6a). Their mitochondrial localization was better demonstrated by the super-15

resolution images stained with the same antibodies (Figure 6b). 16

Moreover, we sought to determine whether CYB5R3 and NOX4 were expressed on the 17

outer or inner mitochondrial membrane by exposing isolated mitochondria to proteinase 18

K. As shown in Figure 6c, CYB5R3 and NOX4 were fully digested at 10 and 3 ug/ml 19

proteinase K, respectively, which similarly occurred with the mitochondrial outer 20

membrane protein TOM20 (Figure 6c). In comparison, cytochrome c oxidase subunit 4 21

(COX IV), an inner membrane protein, was resistant to proteinase K (30 g/ml). These 22

findings indicated that CYB5R3 and NOX4 reside on the mitochondrial outer membrane. 23


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Additional studies were performed to investigate CYB5R3's spatial localization and 1

protein-protein interactions. APEX2, a modified soybean peroxidase used to examine 2

subcellular localization and protein interacting partners, was fused to the C-terminus of 3

CYB5R3 (Figure 6d)29, 30. In the presence of hydrogen peroxide (H2O2), CYB5R3-4

APEX2 converts 3,3′-Diaminobenzidine into brown precipitates that can be visualized in 5

electron microscopy as an electron-dense signal. In HAECs overexpressing CYB5R3-6

APEX2, the electron-dense signal accumulated in the outer periphery of mitochondria, 7

suggesting CYB5R3 resides on the outside of mitochondria (Figure 6e), consistent with 8

the long-standing hypothesis on CYB5R3's orientation on the mitochondrial outer 9

membrane45. Since APEX2 can be activated by high concentrations of H2O2 to 10

biotinylate proximal proteins with biotin-phenoxyl radical, we also used it to pull down 11

biotinylated proteins from CYB5R3-APEX2 expressing HAECs. Our pulldown results 12

included TOM20 and NOX4 (Figure 6f), suggesting both proteins are in close proximity 13

to CYB5R3. Proximity ligation assay was also used and revealed endogenous CYB5R3 14

and NOX4 to be spatially close to each other in endothelial cells (Figure 6g). 15

Membrane-bound CYB5R3 regulates NOX4 activity. 16

Since CYB5R3 and NOX4 reside in close proximity to each other on the mitochondrial 17

outer membrane, we hypothesized that CYB5R3 interacts with NOX4 to regulate its 18

activity. NOX4 is known to produce predominantly H2O2 and a portion of O2•-12. HAECs 19

treated with Cyb5r3 siRNA showed decreased H2O2 production and increased 20

mitochondrial O2•- (Figure 7a-b). Electron leakage from mitochondrial respiration is a 21

major source of O2•-. Therefore, we examined mitochondrial function to distinguish the 22

source of reactive oxygen species in Cyb5r3 knockdown HAECs. The results showed 23


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that the lack of CYB5R3 did not affect protein expression levels of respiration 1

complexes (Supplementary Figure 2), mitochondrial respiration (Supplementary 2

Figure 3a-g), or mitochondrial membrane potential (Supplementary Figure 3h) with or 3

without LPS stimulation. Furthermore, by quantifying the superoxide specific DHE 4

oxidation product, 2OHE+, using HPLC-MS, we confirmed increased superoxide anion 5

levels in Cyb5r3 knockdown HAECs was NOX4 dependent (Figure 7c). To better 6

understand how CYB5R3 affects NOX4 activity, we created expression vectors carrying 7

wild-type CYB5R3, inactive CYB5R3 (K111A or K111A/G180V), and non-membrane 8

bound active CYB5R3 missing the N-terminal membrane anchor (ΔAA1-23). To exclude 9

the effect of endogenous CYB5R3, we generated Cyb5r3 deficient HEK293FT cell lines 10

using lentiviral shRNA (Supplementary Figure 3a). CYB5R3 activity was verified in 11

Cyb5r3 deficient HEK293FT cells forced to express similar amounts of mutant CYB5R3 12

(Figure 7d). Additionally, to test NOX4 activity, Cyb5r3 deficient HEK293FT cells were 13

co-transfected with NOX4 and its cofactor p22 (Supplementary Figure 3b). These 14

experiments revealed significantly increased H2O2 produced from NOX4 in cells with 15

wild-type CYB5R3, while inhibitory effects were observed in cells transfected with 16

inactive CYB5R3. (Figure 7e). The activity of NOX4 did not appear to be affected in 17

cells transfected with the non-membrane bound active CYB5R3 (AA 1-23), which lacks 18

membrane binding. (Figure 7e). These data suggest that both the activity and 19

membrane localization of CYB5R3 are required for NOX4 to produce H2O2. 20

Endogenous CoQ plays a role in NOX4-dependent H2O2 production. 21

To test if the regulation of CYB5R3 on NOX4 activity is mediated by CoQ, we measured 22

H2O2 generated from NOX4 in COQ6 knockout HEK293 cells unable to synthesize 23


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endogenous CoQ28. Consistent with our hypothesis, NOX4's activity to generate H2O2 1

was compromised in CoQ deficient cells (Figure 7f). Since catalase expression was 2

higher in the COQ6 knockout HEK293 cells than the wild-type control cells 3

(Supplementary Figure 4a-c), we tested whether the apparent loss of H2O2 production 4

from NOX4 was due to greater H2O2 consumption by catalase. The use of 1,3-5

aminotriazole (ATZ), a catalase inhibitor, failed to increase H2O2 steady-state levels 6

arising from NOX4 in COQ6 knockout cells, indicating that the compromised phenotype 7

was most likely independent of catalase activity (Supplemental Figure 4d). Our data 8

confirmed that endogenous CoQ is required for optimal NOX4-dependent H2O2 9

production. 10

DISCUSSION 11

Vascular wall inflammation is allied to an imbalance of reduction-oxidation (redox) 12

signaling. However, the fundamental mechanisms that modulate redox homeostasis in 13

the setting of vascular inflammation are not fully understood. In this study, we uncover 14

significant breakthroughs that advance the understanding of redox signaling and 15

endothelial inflammatory activation. First, by generating a novel endothelium-specific 16

CYB5R3 knockout mouse, we discover that knockout animals subjected to endotoxin 17

exhibit exacerbated hypotension, endothelial cell dysfunction, and increased vascular 18

wall inflammation, identifying a previously unrecognized role of CYB5R3 in the 19

endothelium. Second, we discover for the first time that CYB5R3 regulates 20

inflammation through an NADPH oxidase 4 (NOX4) -dependent mechanism to facilitate 21

the formation of H2O2. Lastly, we show that mitochondrial membrane-bound CYB5R3 22

regulates NOX4's ability to produce hydrogen peroxide via endogenous CoQ. 23

Collectively, these new findings highlight the functional significance of endothelial 24


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CYB5R3 in vascular wall inflammation and a previously unrecognized partnership 1

between CYB5R3 and NOX4 via CoQ to produce H2O2 in order to control inflammatory 2

signaling (Figure 8). 3

NOX4 is a member of the family of NADPH oxidases that reduces O2 to reactive oxygen 4

species. Among its family members, NOX4 is unique as it is constitutively active and 5

generates predominantly H2O2. Different mechanisms have been proposed to explain 6

NOX4's ability to produce H2O2 rather than O2•-10, 12. One theory is that NOX4 facilitates 7

the dismutation of newly formed O2•- to H2O2 (Eq 1)10. On the plasma membrane, NOX4 8

is a six-transmembrane protein with both N and C-termini in the cytosol46. Among 9

NOX4's three extracellular loops, the third loop is 28 amino acids longer than the 10

corresponding loops in NOX1 and NOX2 that are dedicated to O2•- production. Takac 11

and colleagues demonstrated that NOX4, lacking the third extracellular loop or with 12

substitution of two essential cysteines in that loop (Cys 226 and Cys270), switched to 13

preferentially producing O2•- versus H2O2

10. The authors hypothesized that Cys226 and 14

Cys270 may form a disulfide bond to retain a newly formed O2•-, increasing the rate of 15

spontaneous dismutation into H2O2. Moreover, the authors suggested that His222 in this 16

loop helps donate protons to the retained O2•-. 17

Contradictory to the hypothesized intrinsic dismutase activity of NOX4, breaking the 18

Cys226-Cys270 by β-mercaptoethanol enhanced NOX4's ability to produce H2O218. 19

Interestingly, NOX4 possesses a putative quinone binding site (AA203-209) that can be 20

used to interact with CoQ. Exposing NOX4 to certain quinone compounds or NQO1, a 21

major CoQ reductase, induced H2O2 production from NOX4. Furthermore, Nisimoto Y. 22

et al. analyzed the kinetics of the H2O2 formation by NOX4, which did not support the 23


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intrinsic dismutase activity. Instead, their data suggested a one-electron reduction of 1

O2•- to H2O2 (Eq 2)12. 2

2O2•- + 2H+ → O2 + H2O2 (1) 3

O2•- + 2H+ + e- → H2O2 (2) 4

In our results, endothelial cells with Cyb5r3 gene silencing had decreased H2O2 and 5

increased O2•- production (Figure 7a-b). Since mitochondrial respiration was not 6

affected by knocking down Cyb5r3, the elevated O2•- was not likely due to electron 7

leakage from the respiratory chain (Supplementary Figure 1-2). Indeed, 8

simultaneously silencing Nox4 prevented O2•- from Cyb5r3 knockdown endothelial cells 9

(Figure 7c). Furthermore, NOX4-derived H2O2 can be promoted by coexpressing the 10

wild-type CYB5R3 but is suppressed when inactive mutants are expressed (Figure 7e). 11

Additionally, NOX4 activity was not affected by the enzymatically active CYB5R3 12

missing its membrane binding capacity (Figure 7e). These findings pointed to a crucial 13

role for both the activity and subcellular location of CYB5R3 in the optimal function of 14

NOX4. Therefore, we hypothesized that NOX4 generates H2O2 with O2•- reductase 15

activity that was fulfilled at least partially in combination with CYB5R3. 16

In support of this hypothesis, we found that CoQ deficiency in COQ6 knockout HEK293 17

cells resulted in diminished H2O2 production from NOX4 (Figure 7f). To our knowledge, 18

this is the first time that endogenous CoQ has been shown to regulate NOX4 activity, 19

which is in agreement with previous findings for synthetic quinone compounds and 20

NQO1 overexpression18. Importantly, although NQO1 is known to regulate 21

mitochondrial function, its localization on the mitochondrial membrane remains 22

controversial47-51. While NQO1 can be detected in mitochondria isolated from multiple 23


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mouse tissues, it was not found in mitochondria in a human cell line50. This 1

disagreement in NQO1's subcellular localization may be rooted in the difference in 2

species, tissues, and antibody sensitivity. Nonetheless, we clearly demonstrate that 3

CYB5R3 is enriched on the mitochondrial membrane using immunofluorescent imaging 4

and electron microscopy in primary endothelial cells (Figure 6a-band 6e), which is 5

consistent with previous reports42, 43. Specifically, our data indicate both NOX4 and 6

CYB5R3 colocalize on the mitochondrial outer membrane (Figure 6c and 6e). We 7

further show with proximity ligation assay that NOX4 and CYB5R3 are close neighbors, 8

permitting the two enzymes to interact (Figure 6g). Therefore, it is possible that 9

CYB5R3, like NQO1, reduces ubiquinone to regulate NOX4 activity. 10

To further understand the interaction of CYB5R3 and NOX4, the orientation of both 11

enzymes on the mitochondrial outer membrane needs to be defined. Similar to NOX4, 12

CYB5R3 can also be found on the plasma membrane. While the former is 13

transmembrane, the latter a cytosolic protein anchored to the membrane by a short N-14

terminal sequence52. Although we found that endothelial CYB5R3 and NOX4 15

predominantly reside on the outer mitochondrial membrane, their orientations on the 16

plasma membrane may help with understanding their protein assembly on the 17

mitochondrial membrane. By creating a chimeric CYB5R3 with APEX2 fused to the 18

reductase's C-terminus, we found that CYB5R3, NOX4, and TOM20 are all in close 19

proximity (Figure 6f). APEX2-catalyzed biotin-phenoxyl radicals rapidly react with 20

electron-rich amino acids on proteins in close proximity, 20-nm, because the radicals 21

are short-lived and thus have a short radius of diffusion53. Since TOM20 is a 22

transmembrane protein on the mitochondrial outer membrane, our APEX2 data indicate 23


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that CYB5R3 and NOX4 are on the outer membrane. Moreover, the biotin-phenoxyl 1

radicals are not membrane permeable, limiting biotinylation of proteins to one side of 2

the membrane, as shown by our electron microscopy image in which APEX2-fused 3

CYB5R3 accumulation occurs outside mitochondria and not in cristae. Therefore, the 4

evidence points to CYB5R3 being on the cytosolic side of the outer membrane. If we 5

can assume that the orientation of CYB5R3 and NOX4 on the mitochondrial outer 6

membrane is the same as on the plasma membrane, NOX4 should have both its termini 7

in the cytosol and its "extracellular loops" in the intermembrane space (Figure 8). 8

The orientation of CYB5R3 and NOX4 is crucial for their collaboration with respect to 9

O2•- reduction. As a one-electron reductase, CYB5R3 reduces ubiquinone to 10

semiubiquinone at the expense of NADH. On the mitochondrial outer membrane, 11

CYB5R3 is close to NOX4, allowing physical interaction between them or by association 12

with a third player, ultimately enabling NOX4 to access semiubiquinone produced by 13

CYB5R3. Meanwhile, NOX4 uses NADPH to produce O2•- that is transiently trapped at 14

the third "extracellular loop". With the electron donated by semiubiquinone and the 15

proton donated by His222, O2•- is reduced to H2O2. Furthermore, the high proton 16

concentration in the intermembrane space, maintained by the electron transport chain, 17

can also favor such a reaction. 18

O2•- dismutase (SOD) and reductase (SOR) represent two distinct systems for 19

regulating O2•- in cells. Although SORs have been found in lower organisms, their 20

existence in mammalian cells is still under debate54. By contrast, three human isoforms 21

of SODs (SOD1-3) have been identified in mammalian cells, having abundant 22

distribution in the cytosol, mitochondria, and extracellular space. Since SOD is 23


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extremely effective at converting O2•- to H2O2 (~109 M-1s-1)55, 56, an immediate question 1

is why human endothelial cells would require an additional SOR to detoxify O2•-. A 2

closer examination of the SOD isoform subcellular location predicates the necessity for 3

such an alternative reductase system. On the one hand, SOD2 has a mitochondrial 4

targeting sequence that leads it to the mitochondrial matrix57. On the other hand, SOD1 5

is synthesized, matured, and accumulated in the cytosol. Although there is some 6

evidence that in yeast, SOD1 is imported to mitochondria, there is no evidence of SOD1 7

in mammalian cells translocating to mitochondria unless associated with 8

neurodegenerative disorders58. Furthermore, we have confirmed the absence of SOD1 9

in isolated mitochondria (Supplementary Figure 4a). With this current understanding of 10

the SOD isoforms' subcellular locations, the mitochondrial intermembrane space seems 11

to be devoid of any SOD. Since NOX4 resides on the mitochondrial outer membrane 12

with its critical loop in the intermembrane space, O2•- can be trapped there and reduced 13

to H2O2. It is possible that the semiubiquinone radical produced by CYB5R3, which 14

forms on the cytosolic side of the membrane, flips across the outer mitochondrial 15

membrane to get oxidized by intermembrane O2•- to form ubiquinone and H2O2. This 16

"flip-flop" action of CoQ is fast enough to support a biological function such as the 17

electron transport chain59. 18

The interaction between CYB5R3 and NOX4 has significant implications for endothelial 19

inflammatory activation. In vitro, LPS induced expression of VCAM-1 and ICAM-1 was 20

enhanced by the loss of CYB5R3 in endothelial cells on both protein and mRNA levels 21

(Figure 3a-c and 4a-c). The overactivation of VCAM-1 and ICAM-1 by LPS in CYB5R3 22

deficient endothelial cells is through the NF-B pathway and was completely blunted by 23


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simultaneous knockdown of NOX4 (Figure 2h-i, 3e, and 4d). A similarly enhanced 1

induction effect by TNF-α was seen for VCAM-1 (Figure 5a-b) but not ICAM-1 (Figure 2

5c). A plausible explanation is that temporal regulation of CYB5R3 on TNF-α induced 3

ICAM-1 is different, which was not captured after overnight treatment. More importantly, 4

although both Vcam-1 and Icam-1 are NF-κB target genes, it is not a surprise that they 5

can be differentially modulated by other transcription factors and upstream signaling 6

pathways60, 61. The exact mechanism defining how CYB5R3 and NOX4 affect LPS and 7

TNF-α induced inflammatory signaling remains a question, and future studies are 8

needed to address specific targeting in the pathway. 9

As an LPS-induced cytokine, TNF-α contributes to the inflammatory cascade initiated by 10

LPS in vivo40, 41. To investigate this in vivo role of LPS, we created inducible endothelial 11

cell-specific Cyb5r3 knockout mice to investigate the role of endothelial CYB5R3 in the 12

LPS induced inflammatory cascade (Figure 1a). Circulating LPS, as seen in sepsis, 13

activates vascular endothelial cells to express adhesion molecules such as VCAM-1 14

and ICAM-1, which recruit leukocytes to the vessel. Leukocytes, such as monocytes, 15

express NOS2 that produces nitric oxide and causes extensive vasodilation resulting in 16

systemic hypotension (Figure 8). We found that LPS-induced hypotension was 17

exacerbated in mice lacking CYB5R3 in the endothelium (Figure 1c). Moreover, without 18

endothelial CYB5R3, aortae isolated from LPS challenged mice showed compromised 19

acetylcholine-induced vasodilation and augmented Nos2 and Vcam-1 mRNA 20

expression, indicating potentiated inflammatory activation and deteriorated endothelial 21

function (Figure 1d-f). Interestingly, endothelial Cyb5r3 deficiency did not affect LPS 22

induced Icam-1 transcription, which was similar to the effect of CYB5R3 on TNF-α 23


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signaling. Based on our previous work, α-globin is not expressed in large artery 1

endothelium but rather small arteries and arterioles at the myoendothelial junction and 2

modulates endothelial NOS3-derived NO signaling25. Therefore, it is unlikely that α-3

globin contributes to endothelial cell dysfunction in the aorta. However, we cannot rule 4

out the possibility that α-globin may also contribute to the hypotensive effects that may 5

arise from the dilation of smaller resistance arteries. Future studies are needed to 6

elucidate a likely nuanced and relative impact of endothelial CYB5R3 on the LPS 7

signaling cascade in various segments of the vascular tree. We recognize that the 8

increase in O2•- by Cyb5r3 knockdown is inconsistent with its effect in smooth muscle 9

cells62. However, the seemingly incongruous results can be caused by different 10

expression levels of NOX isoforms and cellular reliance on mitochondrial respiration for 11

bioenergetics. Therefore, the role of CYB5R3 in redox signaling should be examined 12

tissue by tissue. 13

The regulation of inflammatory signaling by CYB5R3 can significantly impact the 14

pathological progression of inflammatory disease in patients. CYB5R3 T117S, a mutant 15

form encoded by a polymorphism (CYB5R3 c.350C>G), has a high allele frequency 16

(23%) in the African American population23. An important question is whether patients 17

carrying CYB5R3 T117S develop more severe symptoms in infectious diseases, such 18

as sepsis, or chronic inflammatory diseases, such as atherosclerosis. It is also worth 19

noting that CYB5R3 is the best-defined reductase among its family members (CYB5R1-20

4 and CYB5RL). The endothelial cell expression levels of these proteins vary across 21

tissues according to the Human Protein Atlas single-cell RNA seq database (RNA single 22

cell type tissue cluster data available from https://www.proteinatlas.org). It is not clear 23


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whether other reductases may also regulate the NOX family of enzymes in the 1

cardiovascular system. 2

In conclusion, by carefully examining the subcellular locations of CYB5R3 and NOX4, 3

we determined their subcellular orientation on the mitochondrial outer membrane in 4

primary endothelial cells. Their spatial proximity favors an interaction permitting 5

CYB5R3-derived semiubiquinone radical to facilitate the reduction of O2•- generated by 6

NOX4. This novel mechanism contributes to NOX4's ability to produce H2O2 rather than 7

O2•-. Collectively, our current work provides important and novel insight regarding the 8

modulation of NOX4 activity that may have significant translational relevance for the 9

management of inflammatory diseases in patients who are carriers of the CYB5R3 loss-10

of-function variants. 11

ACKNOWLEDGMENT 12

We thank Simon Watkins at the Center for Biologic Imaging, University of Pittsburgh, for 13

the use of light and electron microscopes. 14

SOURCES OF FUNDING 15

This work was supported by National Institutes of Health (NIH) R01 awards [R01 HL 16

133864 (A.C.S), R01 HL 128304, R01 HL 149825, R01 HL 153532 (A.C.S), R01 GM 17

125944 and R01 DK 112854 (F.J.S.)]. American Heart Association (AHA) Established 18

Investigator Award 19EIA34770095 (A.C.S.)], American Heart Association Post-doctoral 19

Fellowship 19POST34410028 (S.Y.), American Society of Hematology (ASH) Minority 20

Hematology Graduate Award (A.M.D-O.) Junta de Andalucía grant BIO-177 (P.N.), the 21

FEDER Funding Program from the European Union and Spanish Ministry of Science, 22

Innovation and Universities grant RED2018-102576-T (P.N.), NIH 1S10OD021540-01 23


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to Center for Biologic Imaging, University of Pittsburgh, NIH 1S10RR019003-01 (Simon 1

Watkins [S.W.]), NIH 1S10RR025488-01 (S.W.), NIH 1S10RR016236-01 (S.W) 2

AUTHOR CONTRIBUTIONS 3

S.Y. and A.S. conceived and designed the study. S.H., S.Y., and A.M.D-O. performed 4

and interpreted animal experiments. S.Y., M.P.M., S.S., and Y.L. performed and 5

interpreted cell culture, molecular biology, and biochemistry experiments. M.J.C., M.S., 6

C.M.S, and D.S. designed, performed, and interpreted super-resolution confocal 7

microscopy and electron microscopy. M.F. and F.J.S. designed, performed, and 8

interpreted O2•- measurements. M.R. and S.S. designed, performed, and interpreted 9

experiments on mitochondrial function. P.N. designed, performed, and interpreted CoQ-10

related experiments. E.C-P. and P.J.P. designed and interpreted NOX4-related 11

experiments. S.Y. K.C.W., F.J.S., P.J.P., and A.C.S. wrote the manuscript, contributed 12

to the writing, or critically reviewed the manuscript. C.M.S., D.S., P.N., S.S., F.J.S., 13

P.J.P., and A.C.S. provided materials for the study. All authors reviewed and approved 14

the manuscript.s 15

CONFLICT OF INTEREST 16

The authors declare that they have no conflict of interest. 17


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1 Figure 1. LPS induced vascular dysfunction is exacerbated in endothelium-2

specific Cyb5r3 knockout mice. (a) Cdh5-CreERT2/Cyb5r3wt/wt (WT) and Cdh5-3

CreERT2/Cyb5r3fl/fl (R3 KO) mice were treated with tamoxifen. Endothelial CYB5R3 4


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expression was examined by immunofluorescence staining in the aorta. (b) Mice 1

received five daily tamoxifen (10 mg/kg) injections and subsequent radiotelemetry unit 2

implantation as indicated in the timeline. Systolic pressure was monitored using 3

radiotelemetry for 24 hours immediately before LPS treatment. N= 8 (WT) and 14 (R3 4

KO). (c) LPS (5 mg/kg) intraperitoneal injections stimulated hypotension in both WT and 5

R3 KO mice. By comparing pre- and post-LPS pressures at similar times of the day, the 6

change in systolic pressure was significantly greater in R3 KO mice relative to WT. N= 8 7

(WT) and 14 (R3 KO); * indicates p <0.05 between WT and R3 KO with 2-way ANOVA. 8

(d) In a different cohort, tamoxifen (10 mg/kg) treated mice were injected 9

intraperitoneally with LPS (5 mg/kg) as indicated in the timeline. Aortae were isolated 10

from a different cohort of mice 8 hours after LPS (5 mg/kg) intraperitoneal injections. In 11

ex vivo wire myography, R3 KO aortae showed less vasorelaxation with cumulative 12

doses of acetylcholine when compared to WT. N=5; * indicates p<0.05 with 2-way 13

ANOVA. (e-g). The mRNA expression levels of Nos2, Vcam-1, and Icam-1 were 14

examined in the same aorta samples. N=5; * indicates p<0.05 between WT and R3 KO 15

with unpaired parametric t-test. 16

17


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1

Figure 2. Cyb5r3 gene silencing potentiates LPS induced NF-kB activation and 2

VCAM-1 and ICAM-1 expression in endothelial culture. HAECs were transfected 3


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with non-targeting (siNT) and Cyb5r3-targeting (siR3) siRNA for 48 hours before 1

challenge with LPS. (a-c) HAECs were treated with 0.1 or 1 µg/ml LPS for 16 hours. At 2

the 1 µg/ml concentration, both LPS-induced VCAM-1 and ICAM-1 protein expressions 3

were significantly higher in the siR3 group. N=5; * indicates p<0.05 between siNT and 4

siR3 with 2-way ANOVA and Sidak post-hoc test. (d-g) Cells were treated with 1 µg/ml 5

LPS for up to four hours. VCAM-1 was increased to a significantly higher level in siR3 6

treated cells relative to siNT and was accompanied by more p65 phosphorylation on 7

S546. N=4; * indicates p<0.05 between siR3 and siNT with 2-way ANOVA and Sidak 8

post-hoc test. (h-i) Cells were pretreated with BMS 345541 (10 µM) for 20 minutes and 9

challenged with LPS (1 µg/ml) overnight. N=3; * indicates p<0.05 between linked groups 10

with 2-way ANOVA and Sidak post-hoc test. 11

12


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1

Figure 3. The anti-inflammatory effects of CYB5R3 are NOX4 dependent. (a) 2

HAECs were co-transfected with siRNA targeting Cyb5r3 (siR3) and Nox4 (siNox4); 3

non-targeting siRNA (siNT) was used to keep the siRNA load the same between groups. 4

At 48 hours following transfection, cells were treated with 1 µg/ml LPS for 16 hours. (b-e) 5

Protein expression levels of VCAM-1, ICAM-1, CYB5R3, and NOX4 were quantified at 6

the 16-hour time point. N=7; * indicates p<0.05 between linked groups with 2-way 7

ANOVA and Tukey's multiple comparisons. 8

9


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1

Figure 4. NOX4 regulates Vcam1 and Icam1 expression at the transcript level. 2

HAECs were co-transfected with siRNA targeting Cyb5r3 (siR3) and Nox4 (siNox4); 3

non-targeting siRNA (siNT) was used to keep the siRNA load the same between groups. 4

48 hours after the siRNA transfection, cells were treated with 1 µg/ml LPS and collected 5

2 or 4 hours after the LPS treatment to examine mRNA expression. N=5; * indicates 6

p<0.05 between linked groups with 2-way ANOVA and Tukey's multiple comparisons. 7

8


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1

Figure 5. Endothelial CYB5R3 and NOX4 regulate TNF-α induced inflammatory 2

signaling in a similar pattern. (a) HAECs were co-transfected with non-targeting 3

(siNT), Cyb5r3-targeting (siR3), and Nox4-targeting (siNox4) siRNA as indicated. TNF-α 4

was added to the medium 48 hours after transfection, and cells were collected after 16 5

hours for immunoblotting. (b-e) Protein expression levels of VCAM-1, ICAM-1, CYB5R3, 6

and NOX4 were quantified at the 16-hour time point. N=7; * indicates p<0.05 between 7

linked groups with 2-way ANOVA and Tukey's multiple comparisons. 8

9


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1


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Figure 6. CYB5R3 resides in close proximity to NOX4 in mitochondria. (a) 1

Confluent HAECs were stained with CYB5R3 or NOX4 (green), TOM20 (red), and DAPI 2

(blue). Both CYB5R3 and NOX4 colocalize with TOM20 in confocal images. The scale 3

bar represents 10 µm. (b) The mitochondrial localization of CYB5R3 and NOX4 is 4

shown in super-resolution images (STED). The scale bar represents 2 µm. (c) 5

Mitochondria were isolated from HEK293FT cells and digested in various 6

concentrations of proteinase K. Proteins on the outer membrane were more susceptible 7

to digestion compared to inner membrane proteins. (d) Strategy using CYB5R3-APEX2 8

to study protein subcellular localization and protein-protein interactions. (e) The electron 9

microscopic image shows the electron-dense area around mitochondria in CYB5R3-10

APEX2 expressing HAECs only in the presence of H2O2. (f) Biotinylated proteins were 11

captured from CYB5R3-APEX2 expressing HAECs. In the presence of H2O2, both 12

TOM20 and NOX4 were detected in the pulldown samples irrespective of LPS treatment 13

(1 µg/ml for one hour). (g) The proximity ligation assay (PLA, red) confirmed direct 14

interactions between CYB5R3 and NOX4 (CYB5R3 X NOX4). The PLA positive signals 15

were quantified as dots per 10 nuclei (DAPI, blue). The quantification represents 16

average count ± standard deviation from 5 images in each group. * indicates p<0.05 17

with Student's t-test. 18

19


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1

Figure 7. Optimal NOX4 production of H2O2 requires CYB5R3 membrane 2

localization and activity. (a-c) HAECs were transfected with non-targeting (siNT), 3

Cyb5r3-targeting (siR3), and Nox4-targeting (siNox4) siRNA for 48 hours. (a-b) H2O2 4

and mitochondrial O2•- were measured using coumarin boronic acid and MitoSOX, 5

respectively. N=3-4; * indicates p<0.05 between siNT and siR3 with ratio paired t-test. 6


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(c) 2OHE+ was quantified with HPLC-MS. N=5; * indicates p<0.05 between linked 1

groups with 2-way ANOVA and Tukey's multiple comparisons. (d) Cyb5r3 knockdown 2

HEK293FT cells were forced to express wild-type CYB5R3 (R3 WT), K111A CYB5R3 3

(R3 K111A), K111A/G180V CYB5R3 (R3 K111A/G180V), and non-membrane bound 4

active CYB5R3 (R3 ΔAA1-23) to similar levels. CYB5R3 activities were measured with 5

the pseudosubstrate 2,6-Dichlorophenolindophenol (DCPIP). N=3; * indicates p<0.05 6

between indicated groups with one-way ANOVA and Dunnett's multiple comparisons. (e) 7

NOX4 activities were evaluated by the amount of H2O2 produced from NOX4 and p22 8

expressing HEK293FT cells. NOX4 derived H2O2 varied between HEK293FT cells with 9

different forms of coexpressed CYB5R3. N=4; * indicates p<0.05 between indicated 10

groups with one-way ANOVA and Dunnett's multiple comparisons. (f) Wildtype (WT) 11

and COQ6 knockout (COQ6KO) HEK293 cells were forced to express NOX4 and p22, 12

and H2O2 production was measured. N=3; * indicates p<0.05 with ratio paired t-test. 13

14


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1

Figure 8. Schematic for the regulation of CYB5R3 on endothelial inflammatory 2

activation and NOX4 activity. In the right panel, CYB5R3 reduces the fully oxidized 3

coenzyme Q, ubiquinone (UQ), to semiubiquinone (UQH•) at the expense of NADH. 4

Meanwhile, NOX4 reduces a molecule of oxygen (O2) to superoxide (O2•-) in the 5

mitochondrial intermembrane space. The resultant O2•- and UQH• react to generate 6

H2O2 and UQ. CYB5R3 normally facilitates NOX4 predominant production of H2O2 in 7

mitochondria (left panel). During endothelial inflammatory activation, e.g., with LPS 8

challenge, H2O2 derived from NOX4 upregulates VCAM-1 expression on the endothelial 9

surface to recruit monocytes. NOS2 in recruited monocytes produces nitric oxide (NO), 10

causing extensive vasodilation and systemic hypotension. When CYB5R3 activity is 11

impaired in endothelial cells (right panel), NOX4 produces less H2O2 and more O2•-, 12


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which results in elevated VCAM-1 expression, enhanced monocyte recruitment, more 1

NOS2 derived NO, and more severe hypotension. 2

3


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SUPPLEMENTARY METHODS AND MATERIALS 1

Telemetric measurements of blood pressure 2

Mice were anesthetized with inhaled isoflurane before telemetry units (HDX-10, Data 3

Sciences International) were surgically implanted in the left common carotid artery. After 4

recovering for 14 days, systolic blood pressure data were recorded with the 5

manufacturer's software (Ponemah) for 24 hours to determine the baseline LPS 6

(Escherichia coli O111:B4, Sigma, L2630) was injected intraperitoneally at a sublethal 7

dose (5 mg/kg in saline). To control for the circadian variation of blood pressure, all LPS 8

injections were performed at the same time (2-3 PM), and the recorded systolic 9

pressure was compared to the baseline reading at the same time to determine the 10

change of pressure. 11

Ex vivo wire myography 12

After tamoxifen treatments, LPS was administered intraperitoneally at 5 mg/kg. Eight 13

hours after the LPS injection, mice were euthanized, and aorta was collected for 14

myography according to our previous publications26. Briefly, thoracic aortae were rapidly 15

excised placed in room temperature physiological salt solution (PSS), cleaned of fat, cut 16

into 2 mm rings, and placed on a two-pin myograph (DMT 620M) filled with PSS 17

containing (mM): NaCl 119, KCl 4.7, MgSO4 1.17, KH2PO4 1.18, D-glucose 5.5, 18

NaHCO3 25, EDTA 0.027, CaCl2 2.5, pH 7.4 when bubbled with 95% O2 5% CO2 at 19

37°C. Following a 30-minute rest, aortas were incrementally stretched to 500 mg initial 20

tension. Vessels were then constricted with 60mM KCl for 5 minutes to test the viability 21

and then washed three times with PSS and allowed to rest for 30 minutes. A final wash 22

was performed, and vessels rested for additional 10 minutes. 23


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Following the final 10 min rest period, vessels were constricted with a dose-response of 1

phenylephrine (50uM-1uM). After reaching a plateau, a continuous dose-response 2

curve of acetylcholine (10 nM-100uM) was used to assess endothelium-dependent 3

relaxation. Ca2+ free PSS containing 100μM sodium nitroprusside was added to 4

determine maximal dilation. 5

Quantification of gene expression 6

Aorta samples were pulverized in liquid nitrogen with a Kimble Teflone® pestle and 7

motor homogenizer. The pulverized sample was dissolved in TrizolTM reagent (Thermo 8

Fisher Scientific, 15596026). Cultured endothelial cells were directly lysed in the 9

TrizolTM reagent. Total RNA was extracted from TrizolTM with the Direct-zol™ RNA 10

Miniprep Plus kit (Zymo, R2072) according to the manufacturer's instructions. Reverse 11

transcription was performed using SuperScript™ IV VILO™ Master Mix (Thermo Fisher 12

Scientific, 11756050). Quantitative PCR for the cDNA library (RT-PCR) was set up with 13

Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific, 4367659) and 14

measured by QuantStudioTM 5 real-time PCR machine (Thermo Fisher Scientific). The 15

delta-delta Ct method was used to analyze mRNA expression levels. Primer sequences 16

used in this study are as the following. Mouse Actb (housekeeping gene): forward 5'-17

CTTTGCAGCTCCTTCGTTG-3'; reverse 5'-CGATGGAGGGGAATACAGC-3'. Mouse 18

Vcam-1: forward 5'-GCAAAGGACACTGGAAAAGAG-3', reverse 5'-19

TCAAAGGGATACACATTAGGGAC-3'. Mouse Icam-1: forward 5'-20

GCAGAGGACCTTAACAGTCTAC-3', reverse 5'-TGGGCTTCACACTTCACAG-3'. 21

Mouse Nos2: forward 5’-ACATCGACCCGTCCACAGTAT-3', reverse 5’-22

CAGAGGGGTAGGCTTGTCTC-3'. Human Rps13 (housekeeping gene): forward 5'-23


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TCTCCTTTCGTTGCCTGATC-3', reverse 5'-AATCTGCTCCTTCACGTCG-3'. Human 1

Vcam-1: forward 5'-TTCTCATCACGACAGCAACTT-3', reverse 5'-2

GGAGTCACCAATCTGAGCAAG-3'. Human Icam-1: forward 5'-3

TGACCGTGAATGTGCTCTC-3', reverse 5'-CTGTATTTCTTGATCTTCCGCTG-3'. 4

Human Cyb5r3: forward 5’-ATGGGGGCCCAGCTCAG-3', reverse 5’-5

AGCGCTGGAACAGCTTCAT-3'. Human Nox4: forward 5'-6

TCACAGAAGGTTCCAAGCAG-3', reverse 5'-ACTGAGAAGTTGAGGGCATTC-3'. 7

Immunoblotting 8

HAECs were lysed directly in 2X Laemmli buffer. HEK293(FT) cells were lysed in 9

radioimmunoprecipitation (RIPA) buffer with proteinase (P8340-5ML) and phosphatase 10

inhibitors (P5726-5ML). Protein samples in RIPA buffer were quantified using Pierce™ 11

BCA Protein Assay Kit (Thermo Fisher Scientific, 23225), diluted, and mixed with an 12

equal amount of 2X Laemmli buffer. All samples were denatured at 95 °C for 10 minutes. 13

Western blotting was performed as we previously described63. Proteins were resolved in 14

NuPAGE Bis-Tris gradient gels (Thermo Fisher Scientific, NP0336BOX) and transferred 15

to 0.2 µm nitrocellulose membranes (BIO-RAD 1620112). Proteins of interest were 16

probed with specific antibodies overnight at 4 °C, followed by infrared fluorescent 17

secondary antibodies for one hour at the room temperature. Blots were imaged with LI-18

COR Odyssey® CLx, and fluorescent intensity was quantified with LI-COR Image 19

Studio. Antibodies and dilutions used in this study are as the following. VCAM-1 (Abcam, 20

ab134047) 1:1,000; ICAM-1 (Santa Cruz, sc-8439) 1:500; α-tubulin (Sigma, T6074) 21

1:10,000; CYB5R3 (Proteintech, 10894-1-AP) 1:2,500; NF-κB p65 (Cell Signaling, 8242) 22

1:1,000; Phospho-NF-κB p65 (Ser536) (Cell Signaling, 3033) 1:1,000; NOX4 23


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(Proteintech, 14347-1-AP; or Abcam, ab133303) 1:1,000; TOM20 (Santa Cruz, SC-1

11415) 1:2,000; COX IV (Abcam, ab14744) 1:1,000; catalase (Cell Signaling, 12980) 2

1:1,000; oxidative phosphorylation complex antibody cocktail (Abcam, ab110411) 3

1:1,000; (SOD1 (Cell Signaling, 2770) 1:1,000; SOD2 (Cell Signaling, ab13533) 1:1,000; 4

IRDye® 800CW Donkey anti-Rabbit IgG Secondary Antibody (LI-COR) 1:15,000; 5

IRDye® 680RD Donkey anti-Mouse IgG Secondary Antibody (LI-COR) 1:15,000. 6

Immunofluorescence staining 7

For tissues, samples were fixed in 4% paraformaldehyde (Santa Cruz, sc-281692) 8

overnight at 4 °C before they were paraffin-embedded and sectioned at a 7-μM 9

thickness. After standard deparaffinization, antigens were retrieved using a heat-10

mediated citric acid-based method (Vector Laboratories, H-3300). After one-hour 11

blocking with 10% horse serum at the room temperature, sections were incubated with 12

primary antibodies overnight at 4 °C and secondary antibodies for one hour at the room 13

temperature. For cell culture experiments, HAECs were plated on coverslips and 14

allowed to grow confluent. Cells were fixed in 4% paraformaldehyde, permeabilized with 15

0.1% Triton X-100 (Sigma, X100-500ML), and blocked with 10% horse serum. 16

Subsequently, coverslips were stained with primary and secondary antibodies. 17

Antibodies and concentrations used for immunofluorescent staining are as the following. 18

CYB5R3 (Proteintech, 10894-1-AP) 2 µg/ml; FITC conjugated α-actin (Sigma, F3777) 19

1:500; NOX4 (Proteintech, 14347-1-AP) 2 µg/ml; TOM20 (Santa Cruz, SC-17764) 2 20

µg/ml. DAPI (Thermo Fisher Scientific, D3571, 1:100) was used for counterstaining 21

along with the Alexa Fluor secondary antibodies. Fluorescent images were captured 22

using a Nikon A1 confocal microscope or a Leica STED super-resolution confocal 23


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microscope at the Center for Biological Imaging at the University of Pittsburgh. For the 1

super-resolution imaging, images were sequentially captured using a 1.4NA 100x oil 2

immersion STED objective with 0.019µm pixels and 0.150 µm z steps. NOX4 and 3

CYB5R3 were excited with a pulsed 555nm laser and depleted with the 775nm STED 4

laser. Emission was captured between 563-651nm and temporally gated between 1-5

6nsec. TOM20 was excited with a 663nm laser and depleted with the 775nm STED 6

laser. Emission was captured between 670-750nm and temporally gated between 1-6 7

nsec. 8

In vitro transfection of siRNA and expression vectors 9

The transfection of siRNA in HAECs and expression vectors in HEK293 cells were 10

achieved using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) according to 11

the manufacturer's instructions. Non-targeting siRNA (siNT) and human Cyb5r3 12

targeting siRNA (siR3) were purchased from Horizon Discovery (D-001810-01-20 and 13

L-009554-00-0005). Human Nox4 targeting siRNA was purchased from Invitrogen 14

(Stealth siRNA, HSS121312). The day before transfection, HAECs were seeded at 15

15,000-20,000 cells/cm2 in 12-well plates. For Cyb5r3 silencing alone, 10 µM of siNT or 16

siR3 was given to HAECs in lipid complex overnight, before the medium was 17

replenished to allow cells to recover. For experiments involving Cyb5r3 and Nox4 18

double knockdown, four groups of cells received 20 µM siNT, 10 µM siNT + 10 µM siR3, 19

10 µM siNT + 10 µM siNox4, or 10 µM siR3 + 10 µM siNox4, respectively. In this way, 20

siRNA loads were kept the same in all treatment groups. 21

HEK293(FT) cells were seeded at 80,000 cell/cm2 in 12-well plates. Plasmids for NOX4, 22

p22, and CYB5R3 WT were used at 0.3 µg per well. For unknown, CYB5R3 mutants 23


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showed different expression efficiency or protein stability. The amount of plasmid used 1

per well was empirically adjusted as the following: K111A 0.15 µg, K111A/G180V 0.2 µg, 2

ΔAA1-23 0.09 µg. When experiments were performed in different cell culture vessels, 3

the plasmid amount is scaled according to the growth area. 4

Two days after the transfection of siRNA or expression vectors, HAECs were treated 5

with LPS, and HEK293(FT) cells were collected for downstream analysis. 6

Lentiviral production and transduction. 7

The shRNA vector against the human Cyb5r3 3’-untranslated region (UTR) was 8

purchased from MISSION® shRNA Library (Sigma, TRCN0000236407). For the 9

Lentiviral expression vector for CYB5R3-APEX, the APEX ORF was amplified from 10

pcDNA3-Connexin43-APEX (Addgene, 49385), fused to CYB5R3 in pcDNA3.1, and 11

cloned into pLV-IRES-GFP. To generate lentivirus, HEK293FT cells were seeded in 15-12

cm dishes at 80,000 cells/cm2. On the following day, for each plate, 8.4 µg pLP1, 4.8 µg 13

pLP2, 6 µg pLP/VSVG (Thermo Fisher Scientific, K497500), and 15 µg shRNA or 14

expression vector were mixed with 171 µg polyethylenimine. After 20 minutes of 15

incubation, the plasmid mixture was spread on HEK293FT cells and left for 8 hours 16

before the medium was changed. Medium containing lentivirus was collected 2-4 days 17

after transfection, and the virus was precipitated by adding 1/3 the volume of a viral 18

concentrator (40% [w/v] polyethylene glycol 8000 and 1.2 M NaCl in phosphate-buffered 19

saline) and reconstituted in phosphate-buffered saline. 20

To establish Cyb5r3 knockdown cell lines, HEK293FT cells were treated with a series of 21

dilutions of lentiviral Cyb5r3 shRNA. The monoclonal selection was performed with 2 22

µg/ml puromycin from cells treated with the least effective dose of the virus. CYB5R3 23


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expression was further examined in selected cell lines by immunoblotting. To transiently 1

express CYB5R3-APEX2 in HAECs, cells were treated with diluted expression lentivirus 2

so that 10% of cells were GFP positive. 3

Mitochondria isolation 4

Mitochondria were isolated from cultured cells as previously described64. Briefly, a 10-5

cm dish of HEK293FT cells was dislodged and resuspended in ice-cold hypotonic buffer 6

(10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.5) for 5-10 minutes. Swollen cells 7

were mechanically broken using a Dounce homogenizer with the tight-fitting pestle. 8

Osmolarity was recovered by adding a 2.5X homogenization buffer (525 mM mannitol, 9

175 mM sucrose, 12.5 mM Tris-HCl, 2.5 mM EDTA, pH 7.5). Nuclei and intact cells 10

were removed by centrifugation at 1,300 g for 5 minutes at 4 °C. Mitochondria were 11

pelleted from the supernatant by centrifugation at 17,000 g for 15 minutes at 4 °C. For 12

immunoblotting, mitochondria were solubilized in RIPA buffer for protein quantification. 13

Subsequently, 20 µg whole cell lysate and 4 µg mitochondrial lysate were loaded on 14

gels. 15

For proteinase K digestion, two 15-cm dishes of HEK293FT cells were used to get a 16

larger amount of mitochondria. Instead of lysing the pellet, it was gently reconstituted in 17

1X homogenizer buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl, 1 mM EDTA, 18

pH 7.5). Proteinase K was added to aliquots of mitochondria homogenate. After 10 19

minutes of incubation at room temperature, proteinase inhibitors and Laemmli buffer 20

were added to each aliquot for downstream immunoblotting. 21

Measurement of CYB5R3 activity 22


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CYB5R3 activity was measured by the reduction of 2,6-Dichlorophenolindophenol 1

(DCPIP). HEK293FT cells were lysed with proteinase and phosphatase inhibitors in 2

RIPA buffer for protein quantification. All samples were diluted to 200 µg/ml in Tris-HCl 3

buffer (100 mM, pH6.8). In a 96-well plate, 50 µl diluted samples, 100 µl DCPIP (450 4

µM in 100 mM Tris-HCl, pH 6.8), and 50 µl NADH (1 mM in 100 mM Tris-HCl, pH 6.8) 5

were added for each reaction. Immediately after adding NADH, dynamic absorbance 6

(600 nm) reading was started at 37 °C until reactions plateau. The log phase slope was 7

used to determine the reaction rate. 8

Proximal ligation assay 9

Proximal ligation assay was performed using Duolink In Situ PLA Probe (Sigma, Anti-10

Rabbit PLUS [DUO92002-30rxn], Anti-Mouse MINUS [DUO92004-30rxn]) according to 11

manufacturer's instructions with modifications. HAECs were grown on coverslips to 12

confluence. Following methanol fixation, cells were blocked in Duolink blocking buffer 13

for one hour at 37 °C. Primary antibodies, mouse anti-CYB5R3 (Santa Cruz, sc-398043) 14

and rabbit anti-NOX4 (Proteintech, 14347-1-AP), were diluted to 2 µg/ml and added to 15

coverslips for overnight incubation at 4 °C. The next day, coverslips were rinsed and 16

incubated with anti-rabbit plus and anti-mouse minus secondary antibodies for one hour 17

at 37 °C. Ligation and amplification were setup using provided reagents and lasted 30 18

and 100 minutes, respectively, at 37 °C. Afterward, DAPI was used as the 19

counterstaining. Confocal images were taken with a Nikon A1 microscope at the Center 20

for Biologic Imaging at the University of Pittsburgh and quantified using ImageJ. 21

Detailed method on transmission electron microscopy 22

23


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The specimens were fixed in cold 2.5% glutaraldehyde (25% glutaraldehyde stock EM 1

grade, Taab Chemical, Berks, England) in 0.01 M PBS (sodium chloride, potassium 2

chloride, sodium phosphate dibasic, potassium phosphate monobasic, Fisher, 3

Pittsburgh, PA), pH 7.3. The specimens were rinsed in PBS, post-fixed in 1% osmium 4

tetroxide (Osmium Tetroxide crystals, Electron Microscopy Sciences, Hatfield, PA) with 5

1% potassium ferricyanide (Potassium Ferricyanide, Fisher, Pittsburgh, PA), dehydrated 6

through a graded series of ethanol (30% - 90% - Reagent Alcohol, Fisher, Pittsburgh, 7

PA, and 100% - Ethanol 200 Proof, Pharmco, Brookefield, CT), and embedded in 8

Poly/Bed® 812 (Luft formulations) (Dodecenyl Succinic Anhydride, Nadic Methyl 9

Anhydride, Poly/Bed 812 Resin and Dimethylaminomethyl, Polysciences, Warrington, 10

PA). Semi-thin (300 nm) sections were cut on a Leica Reichart Ultracut (Leica 11

Microsystems, Buffalo Grove, IL), stained with 0.5% Toluidine Blue in 1% sodium borate 12

(Toluidine Blue O and Sodium Borate, Fisher, Pittsburgh, PA) and examined under the 13

light microscope. Ultrathin sections (65 nm) were examined on JEOL 1400 14

transmission electron microscope (grant #1S10RR016236-01 NIH for Simon Watkins) 15

with a side mount AMT 2k digital camera (Advanced Microscopy Techniques, Danvers, 16

MA). 17

HPLC-MS/MS quantification of 2-hydroxyethidium (2OHE+) 18

The HPLC-MS/MS based measurement was performed according to the previous 19

publication65. To prepare the sample, HAECs were transfected in 6-well plates with 20

siRNA for 48 hours. Cells were incubated with 10 M dihydroethidium (DHE) (Cayman) 21

for 1 hour, washed with ice-old PBS, and scraped in PBS. Three wells of cells were 22

pooled together and pelleted by centrifugation at 1000 g for 5 minutes. The pellet was 23


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lysed in PBS containing 0.1% Triton X-100 and 1 M 3,8-diamino-6-1

phenylphenanthridine (DAPP) (Sigma). While an aliquot of lysate was used to 2

determine protein concentration, 0.2 M perchloric acid (HClO4) was added to the 3

remaining lysate for protein precipitation. After centrifugation (20,000 g, 30 minutes at 4

4 °C), an equal volume of 1 M phosphate buffer (pH 2.6) was added to the supernatant. 5

For HPLC-MS/MS analysis, 2-hydroxyethidium (2-OH-E+) was synthetized as previously 6

reported66, and analyzed by HPLC-ESI-MS/MS using an analytical Phenyl-hexyl Luna 7

column (2 × 150 mm, 3 μm, Phenomenex) at a 0.25 ml/min flow rate, with a gradient 8

solvent system consisting of water containing 0.1% formic acid (solvent A) and 9

acetonitrile containing 0.1% formic acid (solvent B). Samples were chromatographically 10

resolved using the following gradient program: 20–50% solvent B (0–7 min); 50-100% in 11

0.25 min and 1 min at 100% solvent B followed by 4.5 minutes of re-equilibration to 12

initial conditions. Detection of 2-OH-E+ was performed using an AB Sciex5000 triple 13

quadrupole mass spectrometer (Applied Biosystems, San Jose, CA) equipped with an 14

ESI probe in positive mode with the following parameters: declustering potential (DP) 60 15

V, entrance potential (EP) 5 V, collision cell exit potential (CXP) 10 V, and a source 16

temperature of 500 ºC. Multiple reaction monitoring (MRM) transitions for 2-OH-E+ and 17

its internal standard DAPP were used with the following optimized collision energies 18

(CE): MRM 330.1/300, 286/208 with CE 45 and 40 eV respectively. Results were 19

reported as ratio of analyte peakarea/ I.S. peak area normalized to the protein 20

concentration. 21

Seahorse Assay 22


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Cellular oxygen consumption rate was measured by with Seahorse Extracellular Flux 1

(XF96) Analyzer (Seahorse Bioscience, Inc., North Billerica, MA) as previously 2

described. HAECs were transfected with siRNA for 48 hours and then seeded in an 3

Agilent Seahorse XF96 cell culture microplate (Agilent, 101085-004) at 200,000 4

cells/cm2. After attachment, cells were treated with LPS (1 µg/ml) overnight. On the day 5

of experiment, medium was changed to DMEM containing 25 mM glucose, 2 mM 6

glutamine and 1 mM pyruvate. In the Seahorse Analyzer, cells were consecutively 7

treated with oligomycin A (2 µM), trifluoromethoxy carbonylcyanide phenylhydrazone 8

(FCCP) (0.5 µM), antimycin A (Ant A) (2 µM), and rotenone (2 µM). After each treatment, 9

two OCR measurements were recorded. The data was reported as fold changes 10

compared to the baseline (DMEM only) level of control group. 11

Mitochondrial membrane potential 12

HAECs were transfected with siRNA for 48 hours. After overnight LPS (1 µg/ml) 13

treatment, cells were trypsinzed, pelleted, and resuspended in 10 µg/ml JC-1 probe 14

(Thermo, T3168). After 20-minute incubation at 37 ºC, cells were subjected to 2-color 15

flow cytometry (514/529 nm and 585/590 nm). The emission ratio of 590/529 was 16

recorded as the measurement of mitochondrial membrane potential. 17


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1

Supplementary Figure 1. HAECs were transfected with non-targeting (siNT) or 2

Cyb5r3-targeting (siR3) siRNA for 48 hours and then treated with LPS (1 µg/ml) 3

overnight. The experiment was repeated 3 times. Representative images were shown in 4

(a), and quantifications of each complex in (b-f). (b-f) share the same figure legend. 5

6


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1

Supplementary Figure 2. HAECs were transfected with non-targeting (siNT) or 2

Cyb5r3-targeting (siR3) siRNA for 48 hours reseeded in XF96 Seahorse plates, and 3

treated with LPS (1 µg/ml) overnight. The experiment was repeated on different days. (a) 4

In each experiment, the oxygen consumption rate (OCR) was normalized to the 5

baseline (Phase 1) level of the control group (siNT vehicle). The curves represent 6


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average values from 4 experiments. Each phase corresponds to the OCR under 1

indicated inhibitor treatments for the ease of description. (b) Non-mitochondrial OCR = 2

Phase 4. (c) Baseline OCR = Phase 1 – Phase 4. (d) ATP-linked OCR = Phase 1 – 3

Phase 2. (e) Proton leakage = Phase 2 – Phase 4. (f) Maximum OCR = Phase 3 – 4

Phase 4. (g) Spare capacity = Phase 3 – Phase 1. (h) Cells were transfected and 5

treated with LPS in the same way but dislodged for JC-1 probe treatment. The data 6

shows the red (585/590 nm) and green (514/529 nm) ratio averaged from 3 7

independent experiments. (b-h) share the same figure legend. 8

9


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1

Supplementary Figure 3. (a) Cyb5r3 deficient HEK293FT cells were generated using 2

lentiviral shRNA targeting the 3' untranslated region. After monoclonal selection, three 3

lines of control (non-targeting shRNA treated, shNT) and knockdown (shR3) cells were 4

used to examine CYB5R3 protein expression and activity. N=3 cell lines; * indicates 5

p<0.05 with unpaired t-test. Error bars represent standard deviations. (b) HEK293FT 6


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cells were transfected with NOX4, p22, or the combination. Only cells expressing both 1

NOX4 and p22 showed elevated H2O2 production. N=3; * indicates p<0.05 between 2

indicated groups with one-way ANOVA and Dunnett's multiple comparisons. 3

4


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1

Supplementary Figure 4. (a) NOX4 and p22 were expressed in wild-type (WT) and 2

Coq6 knockout (KO) HEK293 cells. The protein expression of catalase, SOD1, and 3

SOD2 were examined in the whole cell lysate (WCL) and isolated mitochondria (Mito). 4

(b) The quantification of proteins in WCL normalized to α-tubulin. N=4; * indicates 5

p<0.05 between indicated groups with one-way ANOVA and Tukey’s multiple 6

comparisons. (c) The quantification of proteins in mitochondria normalized to TOM20. 7

N=4. (d) Wildtype (WT) and Coq6 knockout (COQ6KO) HEK293 cells were treated with 8

20 mM 3-amino-1,2,4-triazole (ATZ) immediately prior to H2O2 measurement. The 9


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catalase inhibitor did not increase NOX4-derived H2O2 in COQ6KO cells. N=3 replicates; 1

* indicates p<0.05 with unpaired t-test; error bars represent standard deviations. 2

3


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Supplementary Table 1. Baseline parameters measured by radio telemetry. 1

2

WT R3 KO P-Value

Heart Rate 575.5±25.3 536.5±29.7 0.00542*

Systolic Pressure 121.4±9.1 121.1±7.4 0.93857

Diastolic Pressure 91.4±7.0 91.1±5.6 0.9014

Pulse Pressure 30.0±6.1 30.0±6.6 0.98286

Mean Arterial Pressure 101.4±7.2 101.1±5.4 0.90709

3

Multiple parameters were monitored and recorded by radiotelemetry within the same 4

time-frame, as shown in Figure 1B, and presented as the 24-hour average ± standard 5

deviation. * indicates p<0.05 between tamoxifen-treated wild-type (WT, n=8) and 6

inducible endothelial Cyb5r3 mice (R3 KO, n=14). 7

8


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