Decreased neointimal formation in Nox2-deficient mice reveals a direct role for NADPH oxidase in the response to arterial injury

Abstract

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced, in part, from NADPH oxidase in response to host invasion and tissue injury. Defects in NADPH oxidase impair host defense; however, the role of ROS and RNS in the response to tissue injury is not known. We addressed this issue by subjecting leukocyte oxidase (Nox2)-deficient (Nox2-/-) mice to arterial injury. Femoral artery injury was associated with increased Nox2 expression, ROS/RNS production, and oxidative protein and lipid modification in wild-type mice. In Nox2-/- mice, RNS-mediated protein oxidation, as monitored by protein nitrotyrosine content, was significantly diminished. This was accompanied by reduced neointimal proliferation, as monitored by intimal thickness and intimal/medial ratio, in Nox2-/- compared to wild-type mice. In addition, Nox2 deficiency led to reduced cellular proliferation and leukocyte accumulation. These data indicate that Nox2-mediated oxidant production has a requisite role in the response to tissue injury.

Fig. 1.

Nox2 expression after carotid injury. Immunohistochemical staining of femoral artery 7 d after sham operation (a) or wire injury (b) showing enhanced Nox2 expression (reddish-brown color with hematoxylin counterstain delineating nuclei) within the media from wild-type mice (original magnification, ×150). Diminished staining is observed with nonimmune IgG (c) or in Nox2^-/- vessel (d) 7 d after injury. Expression of Nox2 is also evident in the media and intima 21 d after injury in wild-type mice (e). Double staining without hematoxylin counterstaining 7 d after injury in wild-type mice for Nox2 (reddish-brown) and CD45 (blue, f) or smooth muscle cell α-actin (blue, g) shows both inflammatory cell and smooth muscle expression of Nox2, respectively. Arrow designates the internal elastic lamina; arrowheads designate double-positive cells.

Fig. 2.

Superoxide production after wire injury. Superoxide production was detected in situ by staining femoral arteries with the superoxide-sensitive dye DHE (red fluorescence). (a) Sham-operated 7 d; (b) wild-type, wire injury 7 d; (c) Nox2^-/-, wire injury 7 d; (d) sham-operated 21 d, (e) wild-type, wire injury 21 d; and (f) Nox2^-/-, wire injury 21 d (original magnification, ×38). Each image is representative of results from three different animals.

Fig. 3.

Femoral artery content of nitrotyrosine, dityrosine, and lipid peroxidation products. Protein-bound nitrotyrosine, dityrosine, and lipid peroxidation products, 8- and 15-HETE, were measured in femoral arteries before (n = 9 per group, open bars) and 3 d after (n = 19 per group, shaded bars) injury in wild-type and Nox2^-/- mice by using electrospray ionization tandem mass spectrometry as described in Methods. Nitrotyrosine and dityrosine content in samples is expressed as the molar ratio between the oxidized tyrosine species and the precursor amino acid tyrosine; lipid peroxidation product content is expressed as the molar ratio between oxidized fatty acid and the precursor fatty acid arachidonate (mean ± SD).

Fig. 4.

Photomicrographs of mouse femoral arteries after injury. (a-d) VerHoeff elastin stain 28 d after injury: (a) wild-type; (b) Nox2^-/- (original magnification, ×38); (c) wild-type; (d) Nox2^-/- (×150). Neointima separates the internal elastic lamina (arrows) from the lumen. (e and f) Proliferating (BrdUrd-positive) cells 7 d after injury: (e) wild-type; (f) Nox2^-/- (×150).