The NADPH organizers NoxO1 and p47phox are both mediators of diabetes-induced vascular dysfunction in mice

Abstract

Aim: NADPH oxidases are important sources of reactive oxygen species (ROS). Several Nox homologues are present together in the vascular system but whether they exhibit crosstalk at the activity level is unknown. To address this, vessel function of knockout mice for the cytosolic Nox organizer proteins p47phox, NoxO1 and a p47phox-NoxO1-double knockout were studied under normal condition and during streptozotocin-induced diabetes.

Results: In the mouse aorta, mRNA expression for NoxO1 was predominant in smooth muscle and endothelial cells, whereas p47phox was markedly expressed in adventitial cells comprising leukocytes and tissue resident macrophages. Knockout of either NoxO1 or p47phox resulted in lower basal blood pressure. Deletion of any of the two subunits also prevented diabetes-induced vascular dysfunction. mRNA expression analysis by MACE (Massive Analysis of cDNA ends) identified substantial gene expression differences between the mouse lines and in response to diabetes. Deletion of p47phox induced inflammatory activation with increased markers of myeloid cells and cytokine and chemokine induction. In contrast, deletion of NoxO1 resulted in an attenuated interferon gamma signature and reduced expression of genes related to antigen presentation. This aspect was also reflected by a reduced number of circulating lymphocytes in NoxO1-/- mice.

Innovation and conclusion: ROS production stimulated by NoxO1 and p47phox limit endothelium-dependent relaxation and maintain blood pressure in mice. However, NoxO1 and p47phox cannot substitute each other despite their similar effect on vascular function. Deletion of NoxO1 induced an anti-inflammatory phenotype, whereas p47phox deletion rather elicited a hyper-inflammatory response.

Keywords: NADPH oxidase; Nox1; NoxO1; Reactive oxygen species; Superoxide; p47phox.

Graphical abstract

Fig. 1

Canonical (Nox1/NoxA1/NoxO1) and hybrid (Nox1/NoxA1/p47phox) activation of Nox1 in overexpression system using HEK cells. A: HEK cells were transfected with Nox1 and its activating subunits as indicated. ROS were measured by chemiluminescence with L012 (200 µmol/L). Stimuli-dependent activation of Nox1 was triggered by PMA (phorbol myristate acetate, 100nmol/L). B: quantification of the signal upon normalization to GFP control. n = 5, *p < 0.05 without PMA vs. with PMA.

Fig. 2

In situ hybridization (RNAscope®) showing the expression of NoxO1 and p47phox in aortic tissue. A: (upper panel) detection with fast red and (lower panel) detection with DAB substrate (brown kit). NoxO1 and p47phox are indicated by arrows. (+) CTR: positive control: peptidylprolyl isomerase B. (-) CTR: negative control: B. subtilis dihydrodipicolinate reductase. B: duplex staining for NoxO1 or p47phox with Adgre1 (F4/80, a macrophage marker). The arrows indicate each of the activators labeled in blue. Zoom in of the adventitia layer shows that p47phox but not NoxO1 co-localizes with Adgre1. C: quantification of the expression of NoxO1 and p47phox in aortic tissue as dots by area (mm²) of either media or adventitia. * p < 0.05 media vs. adventitia for each individual activator. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

NoxO1 and p47phox contribute to cardiovascular homeostasis. A: systolic (*p < 0.05 NO-/-, p47-/-, O1/47-/- vs. WT), (B) diastolic blood pressure (*p < 0.05 p47-/-, O1/47-/- vs. WT), and (C) heart rate measured by tail cuff. D: organ bath experiment of isolated mesenteric artery pre-constricted with phenylephrine and relaxed with acetylcholine. * p < 0.05 wt vs. p47-/-, # p < 0.05 wt vs. O1/47-/-. n ≥ 8.

Fig. 4

NoxO1 and p47phox do not contribute to disease onset but to endothelial dysfunction induced by diabetes. A: blood glucose concentration during STZ-induced diabetes (70 mg/kg/day). B: body weight loss over diabetes. C-E: organ bath experiments of mesenteric vessel pre-constricted with phenylephrine and relaxed with acetylcholine. n ≥ 8, black circles: WT CTR and black squares: WT +STZ. * p < 0.05 wt vs. WT +STZ, # p < 0.05 wt +STZ vs. p47-/- +STZ, + p < 0.05 wt +STZ vs. O1/47-/- +STZ.

Fig. 5

Expression and localization of NoxO1 and p47phox upon diabetes. A: RNAscope (DAB detection, brown kit) for NoxO1 and p47phox (shown by the arrows) in aortic tissue without hematoxylin counter staining. The aortic media comprises the elastic fibers and is delimited by full lines whereas the adventitia by dotted lines. B: quantification of the expression of NoxO1 and p47phox in diabetic aortic tissue represented as relative to CTR * p < 0.05 CTR vs. STZ.

Fig. 6

Gene expression profiling by MACE. Significantly differentially expressed genes of three contrasts (NoxO1-STZ/NoxO1-CTR, WT-STZ/WT-CTR, NoxO1-p47-CTR/NoxO1-p47-STZ) were sorted for descending mean DESeq normalized expression. The top 20 genes of each contrast were merged and transformed to z-scores per gene. Asterisks indicated those genes related to immune response. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

Fig. 7

Interferon gamma and myeloid cells are changed in NO1-/- and p47-/-. A: Cytometric Bead array from plasma showing changes in interferon gamma concentrations n ≥ 8, *p < 0.05 as indicated. B: blood cell counting (*p < 0.05 NO1-/- vs. WT, #p<0.05 p47-/- vs. WT). C: blood cell profiling.