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Cited by 155Nitric Oxide and Endothelin EffectsBarrier Structure and Function StudiesAcute Ischemic Stroke Management

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NOX4-dependent neuronal autotoxicity and BBB breakdown explain the superior sensitivity of the brain to ischemic damage

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Casas, Ana I., Geuss, Eva, Kleikers, Pamela W. M., Mencl, Stine, Herrmann, Alexander M., Buendia, Izaskun, Egea, Javier, Meuth, Sven G., Lopez, Manuela G., Kleinschnitz, Christoph, Schmidt, Harald H. H. W.·2017·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1705034114PMC PMC5699031
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Abstract

Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood-brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

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Figures

Fig. 1. Role of NOX4 in brain ischemia (blue), hindlimb ischemia (green), and MI (red). (A) Infarct volume in Nox4 KO mice (blue) was reduced compared with the WT mice (*P < 0.05, n = 8). (B) Quantified number of capillaries in the ischemic gastrocnemius muscle was not different between Nox4 KO (green, n = 16) and WT mice (white, n = 22). (C) Quantified number of capillaries in the ischemic adductor muscle did not show a difference between Nox4 KO (green, n = 15) and WT mice (white, n = 19). (D) Blood flow restoration decreased in the Nox4 KO mice (green, n = 17) at day 3, an effect that was not seen in the long-term with Nox4 KO mice (green, n = 17) and WT mice (black, n = 23) (*P < 0.05), showing the same blood flow at 28 d after ischemia. (E) No significant differences in infarct size 4 wk after MI between Nox4 KO (red, n = 25) and WT mice (white, n = 26). (F) Ejection fraction decreased after MI with no significant change between Nox4 KO (red, n = 23) and WT mice (black, n = 21). (G) Left ventricular function was not different between Nox4 KO (red, n = 22) and WT mice (black, n = 20). Representative staining pictures are shown above each graph.
Fig. 1. Role of NOX4 in brain ischemia (blue), hindlimb ischemia (green), and MI (red). (A) Infarct volume in Nox4 KO mice (blue) was reduced compared with the WT mice (*P < 0.05, n = 8). (B) Quantified number of capillaries in the ischemic gastrocnemius muscle was not different between Nox4 KO (green, n = 16) and WT mice (white, n = 22). (C) Quantified number of capillaries in the ischemic adductor muscle did not show a difference between Nox4 KO (green, n = 15) and WT mice (white, n = 19). (D) Blood flow restoration decreased in the Nox4 KO mice (green, n = 17) at day 3, an effect that was not seen in the long-term with Nox4 KO mice (green, n = 17) and WT mice (black, n = 23) (*P < 0.05), showing the same blood flow at 28 d after ischemia. (E) No significant differences in infarct size 4 wk after MI between Nox4 KO (red, n = 25) and WT mice (white, n = 26). (F) Ejection fraction decreased after MI with no significant change between Nox4 KO (red, n = 23) and WT mice (black, n = 21). (G) Left ventricular function was not different between Nox4 KO (red, n = 22) and WT mice (black, n = 20). Representative staining pictures are shown above each graph.CC BY-NC-ND · PubMed Central
Fig. 2. Validation of cell-specific NOX4 deletion in mice in endothelial, neuronal, and SMCs. (A, Left) Main cellular components of the neurovascular unit are included in the scheme. NOX4 is predominantly expressed in some components of the neurovascular unit such as endothelial cells (green), neurons (green), and pericytes (green) rather than astrocytes (gray), macrophages (gray), and microglia (gray). In fact, NOX4 play an important role in both BBB leakage after stroke (red circle on left) and neuron-derived excitotoxicity (red circle on right). Neuronal (NeuN) and endothelial (CD31) makers are also included in red. (A, Right) Due to the lack of SMCs (green) in the brain, carotid artery tissue was needed for the cell-specific Nox4 KO mice validation. SMC-specific marker (αSMA) is in red. (B, Left, first row) Immunohistochemistry (IHC) of brain tissue from a WT animal shows NOX4 expression (green) in neurons (top and bottom arrowheads) and endothelial cells (middle arrow). (B, Left, second row) IHC shows no expression of endothelial NOX4 in eNox4 KO (see merged panels) while NOX4 is still expressed in neurons (arrows). (B, Left, third row) Neuronal NOX4 is not expressed (green, all arrows) where neurons are detected by the specific tissue marker NeuN (red). (B, Left, fourth row) sNOX4 KO presents NOX4 expression (green) in both neuronal (arrow) and endothelial cells (arrowhead). Since specific expression of NOX4 in SMCs is not possible to detect in brain tissue, carotid tissue is used. (B, Right, first row) Carotid artery sections from WT mice show SMCs (red) and NOX4 (green) expression. (B, Right, second row) Carotid tissue from sNOX4 KO animals show no expression of NOX4 (green) in SMCs in comparison with WT sections. DNA was visualized using Hoechst dye.
Fig. 2. Validation of cell-specific NOX4 deletion in mice in endothelial, neuronal, and SMCs. (A, Left) Main cellular components of the neurovascular unit are included in the scheme. NOX4 is predominantly expressed in some components of the neurovascular unit such as endothelial cells (green), neurons (green), and pericytes (green) rather than astrocytes (gray), macrophages (gray), and microglia (gray). In fact, NOX4 play an important role in both BBB leakage after stroke (red circle on left) and neuron-derived excitotoxicity (red circle on right). Neuronal (NeuN) and endothelial (CD31) makers are also included in red. (A, Right) Due to the lack of SMCs (green) in the brain, carotid artery tissue was needed for the cell-specific Nox4 KO mice validation. SMC-specific marker (αSMA) is in red. (B, Left, first row) Immunohistochemistry (IHC) of brain tissue from a WT animal shows NOX4 expression (green) in neurons (top and bottom arrowheads) and endothelial cells (middle arrow). (B, Left, second row) IHC shows no expression of endothelial NOX4 in eNox4 KO (see merged panels) while NOX4 is still expressed in neurons (arrows). (B, Left, third row) Neuronal NOX4 is not expressed (green, all arrows) where neurons are detected by the specific tissue marker NeuN (red). (B, Left, fourth row) sNOX4 KO presents NOX4 expression (green) in both neuronal (arrow) and endothelial cells (arrowhead). Since specific expression of NOX4 in SMCs is not possible to detect in brain tissue, carotid tissue is used. (B, Right, first row) Carotid artery sections from WT mice show SMCs (red) and NOX4 (green) expression. (B, Right, second row) Carotid tissue from sNOX4 KO animals show no expression of NOX4 (green) in SMCs in comparison with WT sections. DNA was visualized using Hoechst dye.CC BY-NC-ND · PubMed Central
Fig. 3. Endothelial and neuronal, but not SMC-specific NOX4 KO reduces brain infarct and neurological scores. (A) eKO (dark blue) and nKO (light blue) show smaller infarct sizes compared with WT mice (white) (***P < 0.001, n = 23), sKO mice (cyan) did not show decreased infarcts (n = 12). Complete sets of brain slices from a representative animal (TTC staining) are shown above the graph. (B) Neurological outcome was improved in eKO and nKO in comparison with WT mice (**P < 0.01, n = 23). However, no protection on neurological function was observed in sKO (n = 12). (C) Motor function was also enhanced in eKO and nKO in comparison with WT mice (***P < 0.001, n = 23), but not in sKO mice (*P < 0.05). The n/sWT mice were treated with tamoxifen based on their respective n/sKO mice. The eWT mice were not treated since eKO have a constitutive deletion.
Fig. 3. Endothelial and neuronal, but not SMC-specific NOX4 KO reduces brain infarct and neurological scores. (A) eKO (dark blue) and nKO (light blue) show smaller infarct sizes compared with WT mice (white) (***P < 0.001, n = 23), sKO mice (cyan) did not show decreased infarcts (n = 12). Complete sets of brain slices from a representative animal (TTC staining) are shown above the graph. (B) Neurological outcome was improved in eKO and nKO in comparison with WT mice (**P < 0.01, n = 23). However, no protection on neurological function was observed in sKO (n = 12). (C) Motor function was also enhanced in eKO and nKO in comparison with WT mice (***P < 0.001, n = 23), but not in sKO mice (*P < 0.05). The n/sWT mice were treated with tamoxifen based on their respective n/sKO mice. The eWT mice were not treated since eKO have a constitutive deletion.CC BY-NC-ND · PubMed Central
Fig. 4. Contribution of endothelial and neuronal NOX4 to BBB breakdown, neuronal apoptosis, and ROS formation. (A) BBB integrity as assessed by Evans blue extravasation was preserved in the eKO (dark blue) but not in the nKO (light blue) at day 1 after 1 h of tMCAO (*P < 0.05, n = 6). Complete set of brain slices from a representative animal are shown above the graph. (B) nKO presented fewer apoptotic cells in comparison with nWT (*P < 0.05, n = 5) while no effect was shown in eKO mice (n = 5). Representative staining panels are shown above the bar graph. (C) Both eKO and nKO mice showed decreased oxidative stress compared with their respective WT littermates (***P < 0.001, n = 5). Representative staining pictures are shown above the graph.
Fig. 4. Contribution of endothelial and neuronal NOX4 to BBB breakdown, neuronal apoptosis, and ROS formation. (A) BBB integrity as assessed by Evans blue extravasation was preserved in the eKO (dark blue) but not in the nKO (light blue) at day 1 after 1 h of tMCAO (*P < 0.05, n = 6). Complete set of brain slices from a representative animal are shown above the graph. (B) nKO presented fewer apoptotic cells in comparison with nWT (*P < 0.05, n = 5) while no effect was shown in eKO mice (n = 5). Representative staining panels are shown above the bar graph. (C) Both eKO and nKO mice showed decreased oxidative stress compared with their respective WT littermates (***P < 0.001, n = 5). Representative staining pictures are shown above the graph.CC BY-NC-ND · PubMed Central
Fig. 5. Validation of NOX4 in a second animal species by using tMCAO in NOX4 KO rats and NOX4 inhibitors in hippocampal brain slices subjected to OGD. (A) Similarly to the mouse model, occlusion of the middle cerebral artery was performed in WT and Nox4 KO rats. Rat hippocampal brain slices were subjected to oxygen and glucose deprivation. After treatment, cell viability and ROS formation (CA3) were measured. (B) Infarct volume in Nox4 KO rats was decreased compared with the WT littermate [*P < 0.05, n = 11 (WT) and n = 9 (KO)]. Complete sets of brain slices from a representative animal (TTC staining) are shown above the graph. (C) Cell death (#P < 0.05) was significantly reduced in hippocampal brain slices treated with 0.1 μM GKT136901 and 10 μM VAS2870 as NOX inhibitors (*P < 0.05, **P < 0.01, n = 6). (D) ROS formation (###P < 0.001) was also decreased after NOX inhibition (*P < 0.05, n = 5).
Fig. 5. Validation of NOX4 in a second animal species by using tMCAO in NOX4 KO rats and NOX4 inhibitors in hippocampal brain slices subjected to OGD. (A) Similarly to the mouse model, occlusion of the middle cerebral artery was performed in WT and Nox4 KO rats. Rat hippocampal brain slices were subjected to oxygen and glucose deprivation. After treatment, cell viability and ROS formation (CA3) were measured. (B) Infarct volume in Nox4 KO rats was decreased compared with the WT littermate [*P < 0.05, n = 11 (WT) and n = 9 (KO)]. Complete sets of brain slices from a representative animal (TTC staining) are shown above the graph. (C) Cell death (#P < 0.05) was significantly reduced in hippocampal brain slices treated with 0.1 μM GKT136901 and 10 μM VAS2870 as NOX inhibitors (*P < 0.05, **P < 0.01, n = 6). (D) ROS formation (###P < 0.001) was also decreased after NOX inhibition (*P < 0.05, n = 5).CC BY-NC-ND · PubMed Central

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Authors
Casas, A. I., Geuss, E., Kleikers, P. W. M., Mencl, S., Herrmann, A. M., Buendia, I., Egea, J., Meuth, S. G., Lopez, M. G., Kleinschnitz, C., & Schmidt, H. H. H. W.
Affiliations
  • Maastricht University (NL)
  • Universitätsklinikum Würzburg (DE)
  • Essen University Hospital (DE)
  • University of Münster (DE)
  • Instituto Teofilo Hernando, Department of Pharmacology, School of Medicine, Universidad Autónoma de Madrid, 28029 Madrid, Spain;
  • Hospital Universitario de La Princesa (ES)
Corresponding author
  • Kleinschnitz, Christoph
  • Schmidt, Harald H. H. W.
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2017-10-31
Volume
114
Issue
46
Pages
12315-12320
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1705034114
PMC
PMC5699031
OpenAlex
W2766223740
Cited by
152
References
41
Citations by year
2026: 8 · 2025: 13 · 2024: 16 · 2023: 15 · 2022: 19 · 2021: 28 · 2020: 27 · 2019: 17 · 2018: 9
FWCI
10.73
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Research Publications (Maastricht University) (submittedVersion, cc-by-nc-nd)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, other-oa)
  • Research Publications (Maastricht University) (submittedVersion, other-oa)
Funders
  • European Research Council. Advanced Investigator Grant (294683 RedMed)
  • Deutsche Forschungsgemeinschaft (SFB688)
  • Fondo Investigaciones Sanitarias (CP14/00008, PI6/00735)
  • Ministerio Economia y Competencia Español (SAF2015-63935R)
Topics
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Health Sciences › Medicine › Clinical Neurology › Acute Ischemic Stroke Management
Keywords
NOX4, NADPH oxidase, blood-brain barrier disruption, cerebral ischemia, ischemic stroke, hypoxia, reactive oxygen species, endothelial cells, vascular smooth muscle cells, neuroprotection
MeSH terms
NADPH Oxidase 4, Animals, Benzoxazoles, Blood-Brain Barrier, Brain, Brain Ischemia, Enzyme Inhibitors, Female, Femoral Artery, Gene Expression Regulation, Hindlimb, Humans, Male, Neurons, Organ Specificity, Pyrazoles, Pyridones, Triazoles, Signal Transduction, Myocardial Ischemia, Mice, Knockout, Neuroprotective Agents, Endothelial Cells, Mice, Rats

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