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Our previous studies have demonstrated that the Nox2 isoform of NADPH oxidase is essential for the development of cardiac fibrosis in response to either angiotensin II or aldosterone. These in-vivo studies could not, however, identify the critical cell type responsible for this protective effect. As monocytes/macrophages are known to play an important role in the initiation of fibrosis, the current studies investigated whether Nox2 influences the ability of macrophages to migrate in response to the chemoattractant, colony stimulating factor-1 (CSF-1).
CSF-1-dependent bone-marrow derived macrophages (BMM) were isolated from femurs of wild-type (wt) and Nox2 knockout (Nox2−/−) mice. BMM were seeded onto glass coverslips, starved of CSF-1, re-stimulated with CSF-1, fixed and stained for F-actin. Cell images were analysed for shape (elongation) and spread area using Image J software. Mean cell speed and chemotactic potential in a CSF-1 gradient were analysed using the Dunn Chemotaxis chamber.
A number of endpoints were assessed both under growing (basal) conditions and following CSF-1 stimulation. Under growing conditions Nox2−/− BMM had a significantly increased spread area (proportion of the cell in contact with the glass coverslip) compared with wt cells (0.261±0.03 vs 0.237±0.03; p<0.05). However, while CSF-1 stimulation increased the spread area in wt cells above growing conditions (0.261±0.01) Nox2−/− BMM returned only to their initial spread area (0.256±0.01). Nox2−/− BMM were, however, significantly more elongated than wt cells following CSF-1 stimulation (0.670±0.01 vs 0.624±0.01). Importantly, Nox2−/− BMM had a significantly reduced migration speed (0.46±0.03 vs 0.6116±0.02 μm/minute, p<0.0004), a significant reduction in their persistence of direction (0.15±0.02 vs 0.38±0.02 p≪0.0005) and failed to migrate positively towards CSF-1. Nox2−/− cells also had significantly lower increases in Akt and ERK1/2 phosphorylation following CSF-1 stimulation compared with wtT cells.
Nox2−/− BMM display marked abnormalities in morphological and migratory behaviour that may contribute significantly to the ability of the monocyte to differentiate and migrate in vivo in response to pathological stimuli. This phenotype could underlie the protection against fibrosis observed in vivo in Nox2−/− mice.