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Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation
Published on: April 3, 2017
Mitochondrial dysfunction and metabolic reprogramming induce macrophage pro-inflammatory phenotype switch and
Aleksandr E Vendrov1, Andrey Lozhkin1, Takayuki Hayami1
1Frankel Cardiovascular Center, Division of Cardiovascular Medicine, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, United States.
Introduction:
Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages.
Methods:
We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet.
Results:
Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1β secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis.
Discussion:
Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.
Insights
Aging increases NADPH oxidase 4 (NOX4) expression, promoting mitochondrial dysfunction and inflammation in macrophages, which drives atherosclerosis. Inhibiting NOX4 could reduce vascular inflammation and preserve plaque integrity.
Area of Science:
- Cardiovascular Biology
- Immunology
- Aging Research
Background:
- Aging exacerbates atherosclerotic vascular disease risk, with macrophages playing a key role in vascular aging, inflammation, and atherosclerosis.
- NADPH oxidase 4 (NOX4) expression rises with age, linked to mitochondrial dysfunction, inflammation, and atherosclerosis.
- Increased NOX4 is hypothesized to promote aging-associated atherosclerosis via NOX4-dependent mitochondrial oxidative stress, metabolic dysfunction, and macrophage inflammatory phenotype switching.
Purpose of the Study:
- To investigate the role of NOX4 in aging-associated atherosclerosis.
- To determine if NOX4-driven mitochondrial oxidative stress in macrophages contributes to metabolic dysfunction and inflammatory phenotype switching.
- To assess the therapeutic potential of NOX4 inhibition in mitigating aging-related atherosclerosis.
Main Methods:
- Comparative analysis of atherosclerotic lesion morphology and macrophage phenotype in young and aged Apoe-/- and Nox4-/-/Apoe-/- mice fed a Western diet.
- Immunofluorescence, flow cytometry, and mitochondrial respiration assessments were used to analyze macrophage populations and function.
- Pharmacological inhibition of NOX4 using GKT137831 in aged Apoe-/- mice.
Main Results:
- Aged Nox4-/- mice exhibited significantly reduced atherosclerotic lesion areas, lower oxidative stress, and a shift towards pro-resolving macrophages compared to aged Apoe-/- mice.
- Macrophages from aged Apoe-/- mice displayed impaired mitochondrial function and a glycolytic metabolic switch, whereas Nox4-/- macrophages maintained aerobic metabolism.
- NOX4 inhibition in aged Apoe-/- mice attenuated atherosclerosis by reducing macrophage mitochondrial ROS, improving mitochondrial function, and altering macrophage phenotype.
Conclusions:
- Increased NOX4 in aging drives macrophage mitochondrial dysfunction, metabolic reprogramming towards glycolysis, and a pro-inflammatory phenotype, thereby accelerating atherosclerosis.
- Targeting NOX4 or mitochondrial dysfunction presents a promising therapeutic strategy to alleviate vascular inflammation and preserve plaque integrity in aging individuals.
- The study highlights NOX4 as a critical mediator of macrophage dysfunction in vascular aging and atherosclerosis.
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