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Cited by 111Nitric Oxide and Endothelin EffectsAtherosclerosis and Cardiovascular DiseasesNeutrophil, Myeloperoxidase and Oxidative Mechanisms

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Endothelial-specific Nox2 overexpression increases vascular superoxide and macrophage recruitment in ApoE−/− mice

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Douglas, Gillian, Bendall, Jennifer K., Crabtree, Mark J., Tatham, Amy L., Carter, Emma E., Hale, Ashley B., Channon, Keith M.·2012·Cardiovascular Research
DOI 10.1093/cvr/cvs026PMC PMC3307381
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Abstract

AIMS: Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species derived from NADPH oxidases. However, it remains unclear whether a primary increase in superoxide production specifically in the endothelium alters the initiation or progression of atherosclerosis. METHODS AND RESULTS: Mice overexpressing Nox2 specifically in the endothelium (Nox2-Tg) were crossed with ApoE(-/-) mice to produce Nox2-Tg ApoE(-/-) mice and ApoE(-/-) littermates. Endothelial overexpression of Nox2 in ApoE(-/-) mice did not alter blood pressure, but significantly increased vascular superoxide production compared with ApoE(-/-) littermates, measured using both lucigenin chemiluminescence and 2-hydroxyethidium production (ApoE(-/-), 19.9 ± 6.3 vs. Nox2-Tg ApoE(-/-), 47.0 ± 7.0 nmol 2-hydroxyethidium/aorta, P< 0.05). Increased endothelial superoxide production increased endothelial levels of vascular cell adhesion protein 1 and enhanced macrophage recruitment in early lesions in the aortic roots of 9-week-old mice, indicating increased atherosclerotic plaque initiation. However, endothelial-specific Nox2 overexpression did not alter native or angiotensin II-driven atherosclerosis in either the aortic root or the descending aorta. CONCLUSION: Endothelial-targeted Nox2 overexpression in ApoE(-/-) mice is sufficient to increase vascular superoxide production and increase macrophage recruitment possible via activation of endothelial cells. However, this initial increase in macrophage recruitment did not alter the progression of atherosclerosis. These results indicate that Nox-mediated reactive oxygen species signalling has important cell-specific and distinct temporal roles in the initiation and progression of atherosclerosis.

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Figure 1 Characterization of NADPH oxidase expression in the Nox2-Tg ApoE−/− mouse. (A) mRNA expression of human Nox2, mouse Nox2 and mouse Nox4 in whole aortas from Nox2-Tg ApoE−/− and ApoE−/− mice. Data were quantified using the Delta ct method and GAPDH used as the house-keeping gene. (B) Western blot of global Nox2 in lung homogenates from Nox2-Tg ApoE−/− and ApoE−/− mice. Data are expressed as the mean ± SEM of n= 3–6 mice per group. *P< 0.05; ND, not detectable.
Figure 1 Characterization of NADPH oxidase expression in the Nox2-Tg ApoE−/− mouse. (A) mRNA expression of human Nox2, mouse Nox2 and mouse Nox4 in whole aortas from Nox2-Tg ApoE−/− and ApoE−/− mice. Data were quantified using the Delta ct method and GAPDH used as the house-keeping gene. (B) Western blot of global Nox2 in lung homogenates from Nox2-Tg ApoE−/− and ApoE−/− mice. Data are expressed as the mean ± SEM of n= 3–6 mice per group. *P< 0.05; ND, not detectable.CC BY-NC · PubMed Central
Figure 2 Characterization of NADPH oxidase expression in primary vascular cells from the Nox2-Tg ApoE−/− mouse. Human Nox2, mouse Nox2 and cell-specific markers in primary endothelial cells, VSMCs and primary macrophages from Nox2-Tg ApoE−/− and ApoE−/− mice. Tissue-specific markers were PECAM for primary endothelial cells, myosin heavy chain (MHC) III for primary VSMCs, and CD68 for resident macrophages. Data were quantified using the Delta ct method, and GAPDH used as the house-keeping gene. Data are expressed as the mean ± SEM of n= 3–6 mice per group. *P< 0.05. ND, not detectable.
Figure 2 Characterization of NADPH oxidase expression in primary vascular cells from the Nox2-Tg ApoE−/− mouse. Human Nox2, mouse Nox2 and cell-specific markers in primary endothelial cells, VSMCs and primary macrophages from Nox2-Tg ApoE−/− and ApoE−/− mice. Tissue-specific markers were PECAM for primary endothelial cells, myosin heavy chain (MHC) III for primary VSMCs, and CD68 for resident macrophages. Data were quantified using the Delta ct method, and GAPDH used as the house-keeping gene. Data are expressed as the mean ± SEM of n= 3–6 mice per group. *P< 0.05. ND, not detectable.CC BY-NC · PubMed Central
Figure 3 Quantification of aortic superoxide production in Nox2-Tg ApoE−/− and ApoE−/− mice. Quantification of NADPH-dependent thoracic aortic superoxide production by (A) 2-hydroxyethidium fluorescence HPLC showed that superoxide production was significantly increased in aortas from 24-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice (*P< 0.05, n= 9–13). Data are presented as box plots of the tiron-inhibitable fraction of 2-hydroxyethidium production. (B) Lucigenin-enhanced chemiluminescence showed that superoxide production was significantly increased in aortas from 9 to 18-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice (*P< 0.05, n= 13–17). (C) 2-Hydroxyethidium fluorescence in frozen sections of the aortic root from 9-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice demonstrated that endothelial superoxide production was significantly increased in aortic roots from 9-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice (*P< 0.05, n= 8–5). Data are presented as box plots. Area of 2-hydroxyethidium fluorescence in frozen sections was normalized to vessel length.
Figure 3 Quantification of aortic superoxide production in Nox2-Tg ApoE−/− and ApoE−/− mice. Quantification of NADPH-dependent thoracic aortic superoxide production by (A) 2-hydroxyethidium fluorescence HPLC showed that superoxide production was significantly increased in aortas from 24-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice (*P< 0.05, n= 9–13). Data are presented as box plots of the tiron-inhibitable fraction of 2-hydroxyethidium production. (B) Lucigenin-enhanced chemiluminescence showed that superoxide production was significantly increased in aortas from 9 to 18-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice (*P< 0.05, n= 13–17). (C) 2-Hydroxyethidium fluorescence in frozen sections of the aortic root from 9-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice demonstrated that endothelial superoxide production was significantly increased in aortic roots from 9-week-old Nox2-Tg ApoE−/− mice vs. ApoE−/− mice (*P< 0.05, n= 8–5). Data are presented as box plots. Area of 2-hydroxyethidium fluorescence in frozen sections was normalized to vessel length.CC BY-NC · PubMed Central
Figure 4 Quantification of aortic root VCAM-1 and macrophage levels in Nox2-Tg ApoE−/− and ApoE−/− mice at 9 weeks of age. (A) Aortic root VCAM-1 staining (VCAM-1 positive cells stain red) was significantly greater in aortic roots from Nox2-Tg ApoE−/− mice compared with ApoE−/− littermates (P< 0.05, n= 5). (B) Aortic root macrophage content was quantified by immunohistochemical staining in frozen sections for CD68 (macrophages stained red); Nox2-Tg ApoE−/− mice had significantly greater macrophage content compared with ApoE−/− littermates (P< 0.05, n= 5–8). (C) Aortic root macrophage content was quantified by immunohistochemical staining in paraffin sections for Mac-3 (macrophages stained red); Nox2-Tg ApoE−/− mice had significantly greater macrophage recruitment compared with ApoE−/− littermates (P< 0.05, n= 5–8). For quantification of VCAM-1 and CD68 staining, the area of positive staining was normalized to vessel length.
Figure 4 Quantification of aortic root VCAM-1 and macrophage levels in Nox2-Tg ApoE−/− and ApoE−/− mice at 9 weeks of age. (A) Aortic root VCAM-1 staining (VCAM-1 positive cells stain red) was significantly greater in aortic roots from Nox2-Tg ApoE−/− mice compared with ApoE−/− littermates (P< 0.05, n= 5). (B) Aortic root macrophage content was quantified by immunohistochemical staining in frozen sections for CD68 (macrophages stained red); Nox2-Tg ApoE−/− mice had significantly greater macrophage content compared with ApoE−/− littermates (P< 0.05, n= 5–8). (C) Aortic root macrophage content was quantified by immunohistochemical staining in paraffin sections for Mac-3 (macrophages stained red); Nox2-Tg ApoE−/− mice had significantly greater macrophage recruitment compared with ApoE−/− littermates (P< 0.05, n= 5–8). For quantification of VCAM-1 and CD68 staining, the area of positive staining was normalized to vessel length.CC BY-NC · PubMed Central
Figure 5 Quantification of aortic root and aortic atherosclerosis in Nox2-Tg ApoE−/− and ApoE−/− mice at 16 and 24 weeks of age. (A) Aortic root plaque area was quantified using Masson–Goldner staining of aortic root sections. No difference in plaque area was observed between Nox2-Tg ApoE−/− and ApoE−/− mice at 16 or 24 weeks of age (P> 0.05). (B) Aortic root macrophage content was quantified by immunohistochemical staining for Mac-3 (macrophages stained red). No difference in macrophage content was observed between Nox2-Tg ApoE−/− mice and there ApoE−/− littermates (P> 0.05). (C) Aortic atherosclerosis was assessed by en face analysis of Oil red O staining in the aorta of Nox2-Tg ApoE−/− mice and their ApoE−/− littermates. No difference in aortic atherosclerosis was observed between Nox2-Tg ApoE−/− mice and ApoE−/− mice (P> 0.05). A time-dependent increase in aortic root plaque area, macrophage content, and aortic plaque area was observed in both groups (P< 0.05). Each point is representative of one animal.
Figure 5 Quantification of aortic root and aortic atherosclerosis in Nox2-Tg ApoE−/− and ApoE−/− mice at 16 and 24 weeks of age. (A) Aortic root plaque area was quantified using Masson–Goldner staining of aortic root sections. No difference in plaque area was observed between Nox2-Tg ApoE−/− and ApoE−/− mice at 16 or 24 weeks of age (P> 0.05). (B) Aortic root macrophage content was quantified by immunohistochemical staining for Mac-3 (macrophages stained red). No difference in macrophage content was observed between Nox2-Tg ApoE−/− mice and there ApoE−/− littermates (P> 0.05). (C) Aortic atherosclerosis was assessed by en face analysis of Oil red O staining in the aorta of Nox2-Tg ApoE−/− mice and their ApoE−/− littermates. No difference in aortic atherosclerosis was observed between Nox2-Tg ApoE−/− mice and ApoE−/− mice (P> 0.05). A time-dependent increase in aortic root plaque area, macrophage content, and aortic plaque area was observed in both groups (P< 0.05). Each point is representative of one animal.CC BY-NC · PubMed Central
Figure 6 AngII-induced atherosclerosis in the aortic root and descending aorta of Nox2-Tg ApoE−/− and ApoE−/− mice. Atherosclerosis was assessed in the aortic root and descending aorta of Nox2-Tg ApoE−/− and ApoE−/− littermates at 24 weeks of age after 4 weeks of treatment with either 0.4 or 0.8 mg/kg/day AngII or no treatment. (A) Aortic root plaque area was quantified in Masson-Goldner stained sections. No difference in plaque size was observed between Nox2-Tg ApoE−/− and ApoE−/− mice (P> 0.05). (B) Aortic atherosclerosis was assessed by en face analysis of Oil red O staining. No difference in aortic atherosclerosis was observed between Nox2-Tg ApoE−/− and ApoE−/− mice (P> 0.05). A significant dose-dependent increase in aortic root plaque area and aortic atherosclerosis was observed in both groups (P< 0.05). Each point is representative of one animal.
Figure 6 AngII-induced atherosclerosis in the aortic root and descending aorta of Nox2-Tg ApoE−/− and ApoE−/− mice. Atherosclerosis was assessed in the aortic root and descending aorta of Nox2-Tg ApoE−/− and ApoE−/− littermates at 24 weeks of age after 4 weeks of treatment with either 0.4 or 0.8 mg/kg/day AngII or no treatment. (A) Aortic root plaque area was quantified in Masson-Goldner stained sections. No difference in plaque size was observed between Nox2-Tg ApoE−/− and ApoE−/− mice (P> 0.05). (B) Aortic atherosclerosis was assessed by en face analysis of Oil red O staining. No difference in aortic atherosclerosis was observed between Nox2-Tg ApoE−/− and ApoE−/− mice (P> 0.05). A significant dose-dependent increase in aortic root plaque area and aortic atherosclerosis was observed in both groups (P< 0.05). Each point is representative of one animal.CC BY-NC · PubMed Central

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Metadata

Authors
Douglas, G., Bendall, J. K., Crabtree, M. J., Tatham, A. L., Carter, E. E., Hale, A. B., & Channon, K. M.
Affiliations
  • John Radcliffe Hospital (GB)
  • University of Oxford (GB)
Corresponding author
Douglas, Gillian
ORCID
Venue
Cardiovascular Research
Publisher
Oxford University Press (OUP)
Published
2012-01-27
Volume
94
Issue
1
Pages
20-29
ISSN
  • 0008-6363 (ISSN-L)
  • 1755-3245
DOI
10.1093/cvr/cvs026
PMC
PMC3307381
OpenAlex
W1988097865
Cited by
111
References
28
Citations by year
2026: 5 · 2025: 5 · 2024: 8 · 2023: 7 · 2022: 12 · 2021: 5 · 2020: 1 · 2019: 6 · 2018: 8 · 2017: 13 · 2016: 10 · 2015: 6 · 2014: 10 · 2013: 10 · 2012: 5
FWCI
6.65
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • Cardiovascular Research (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • Wellcome Trust (090532)
  • British Heart Foundation (PG/05/141/20098, RG/07/003/23133, RG/12/5/29576)
Topics
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
  • Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
Keywords
NOX2, NADPH oxidase, atherosclerosis initiation, macrophage recruitment, ApoE-deficient mice, VCAM-1, reactive oxygen species, endothelial dysfunction, angiotensin II, aortic root lesions
MeSH terms
NADPH Oxidase 2, Angiotensin II, Animals, Aortic Diseases, Apolipoproteins E, Blood Pressure, Chemotaxis, Disease Models, Animal, Humans, Macrophages, Membrane Glycoproteins, Mice, Transgenic, Superoxides, Time Factors, Signal Transduction, Up-Regulation, Mice, Knockout, Disease Progression, Vascular Cell Adhesion Molecule-1, NADPH Oxidases, Endothelial Cells, Atherosclerosis, Mice

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