Fibroblast Nox2 (NADPH Oxidase-2) Regulates ANG II (Angiotensin II)–Induced Vascular Remodeling and Hypertension via Paracrine Signaling to Vascular Smooth Muscle Cells

Figures

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  Figure 1.. Basal phenotype of Fibro-Nox2KO mice. A, Polymerase chain reaction showing Cre-mediated recombination in Fibro-Nox2KO (fibroblast-specific Nox2 knockout) aorta. B, Nox2 protein levels in aorta from Fibro-Nox2KO and control (Nox2Flox) mice. A representative immunoblot is shown along with mean data. n=7 per group. *P<0.05, unpaired Student t test. C, Immunofluorescence for Nox2 (Red) in aortic sections from Fibro-Nox2KO and control mice. Blue, nuclei stained with 4′-6-diamidino-2-phenylindole (DAPI); green, autofluorescence (AF) of the vessel wall. Scale bars, 50 µm. D, Systolic blood pressure (BP) and (E) heart rate measured by ambulatory telemetry in Fibro-Nox2KO and control mice (n=3 per group). F–I, Cardiac structure and function assessed by echocardiography. n=5–7 per group. IVST indicates interventricular septal thickness; L, vessel lumen; LVEDV, left ventricular end-diastolic volume; LVESV, left ventricular end-systolic volume; and Nox2, NADPH oxidase-2.

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  Figure 2.. ANG II (angiotensin II)–induced hypertension is inhibited in Fibro-Nox2KO mice. Telemetric ambulatory blood pressure (BP) in fibroblast-specific Nox2 knockout (Fibro-Nox2KO) and matched control (Nox2Flox) mice treated with chronic ANG II infusion or saline. A, Systolic BP (BP); (B) diastolic BP; (C) mean arterial BP; (D) heart rate. 2-way repeated-measures ANOVA, n=3–7 per group. Nox2 indicates NADPH oxidase-2. *P<0.05 for ANG II–treated FibroNox2KO vs ANG II–treated Nox2Flox.

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  Figure 3.. ANG II (angiotensin II)–induced vascular remodeling is inhibited in Fibro-Nox2KO mice. Control (Nox2Flox) and fibroblast-specific Nox2 knockout (Fibro-Nox2KO) mice were chronically treated with ANG II or vehicle, then perfusion-fixed. A, Representative aortic sections. Scale bars, 100 µm. B, Mean aortic medial area and (C) media/lumen ratio; n=4–6 aortae per group. D, Representative sections of carotid artery. Scale bars, 100 µm. E, Mean medial area of carotid artery sections; n=3–6 arteries per group. F, Representative myocardial sections showing small septal arteries. Scale bars, 50 µm. G, Mean media/lumen ratio of septal arteries; n=4–6 hearts per group and 5 sections per heart. 1-way ANOVA followed by Tukey post-test. Nox2 indicates NADPH oxidase-2. *P<0.05.

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  Figure 4.. Fibroblast Nox2 (NADPH oxidase-2) is required for ANG II (angiotensin II)–induced vascular smooth muscle cell (VSMC) growth. A, Increased VSMC ploidy in aortic media of ANG II–treated control (Nox2Flox) vs fibroblast-specififc Nox2 knockout (Fibro-Nox2KO) mice. Representative aortic sections examined by immunofluorescence are shown to the left. Red, α-smooth muscle actin; blue, 4′-6-diamidino-2-phenylindole (DAPI; nucleus); green, autofluorescence (AF). Scale bars, 50 µm. Mean data for number of DAPI-positive nuclei/100 μm of media shown to the right; n=3–5 aortae per group. B, Effect of ANG II–conditioned cardiac fibroblast medium on VSMC growth, assessed by Ki67 staining or BrdU (bromodeoxyuridine) incorporation, respectively; n=4 per group. C, Knockdown of Nox2 in cardiac fibroblasts by adenoviruses expressing shRNA (short hairpin RNA) targeted against Nox2 (Ad.shNox2) compared with adenoviruses expressing shRNA targeted against GFP (green fluorescent protein) control (Ad.shGFP). Representative immunoblot at the top shows Nox2 knockdown; mean data for Nox2 protein levels are shown below; n=8 per group. D, Conditioned medium from fibroblasts treated with Ad.shNox2 has reduced…

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  Figure 5.. Nox2 (NADPH oxidase-2)-dependent upregulation of GDF6 (growth differentiation factor 6). A, GDF6 levels in conditioned medium of primary cardiac fibroblasts infected with adenoviruses expressing shRNA (short hairpin RNA) targeted against Nox2 (Ad.shNox2) or adenoviruses expressing shRNA targeted against a GFP (green fluorescent protein) control (Ad.shGFP; control) and treated with ANG II (angiotensin II) or vehicle. Representative immunoblot shown at the top and mean data at the bottom; n=3 per group. Ponceau staining of the membrane is shown in Figure XI in the Data Supplement. B, GDF6 protein levels in aorta from Fibro-Nox2KO or matched control (Nox2Flox) mice chronically treated with ANG II or saline vehicle. Representative immunoblot shown at the top and mean data at the bottom; n=4 per group. β-actin was used as a loading control. Densitometry values for GDF6-specific bands were divided by the corresponding densitometry of Ponceau bands in A and β-actin bands in B. Arbitrary units were calculated by normalizing data vs the control group (ie, fold-change). C, GDF6 immunostaining in aorta from Fibro-Nox2KO and control mice treated with ANG II or saline. Representative images…

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  Figure 6.. GDF6 (growth differentiation factor 6) induces vascular smooth muscle cell (VSMC) growth. A, Concentration-dependent effect of recombinant GDF6 (0–4 μg/mL) on BrdU (bromodeoxyuridine) incorporation in VSMC; n=4–6 per group. B, Representative Western blot for GDF6 expression after silencing using siRNA (silencing RNA) against GDF6 or scrambled control (control) in primary cardiac fibroblasts. C and D, VSMC growth assessed by Ki67 staining after incubation with conditioned media of aortic (C) or cardiac (D) fibroblasts treated with ANG II (angiotensin II). Some fibroblasts were treated with siGDF6 (silencing RNA against GDF6) to reduce GDF6 levels. 1-way ANOVA followed by Tukey post-test (A and C) or Kruskal-Wallis followed by Dunn post-test (D). *P<0.05.