Effects of a common human gene variant of extracellular superoxide dismutase on endothelial function after endotoxin in mice

Abstract

A common gene variant in the heparin-binding domain (HBD) of extracellular superoxide dismutase (ECSOD) may predispose human carriers to ischaemic heart disease. We have demonstrated that the HBD of ECSOD is important for ECSOD to restore vascular dysfunction produced by endotoxin. The purpose of this study was to determine whether the gene variant in the HBD of ECSOD (ECSOD(R213G)) protects against endothelial dysfunction in a model of inflammation. We constructed a recombinant adenovirus that expresses ECSOD(R213G). Adenoviral vectors expressing ECSOD, ECSOD(R213G) or beta-galactosidase (LacZ, a control) were injected i.v. in mice. After 3 days, at which time the plasma SOD activity is maximal, vehicle or endotoxin (lipopolysaccharide or LPS, 40 mg kg(-1)) was injected i.p. Vasomotor function of aorta in vitro was examined 1 day later. Maximal relaxation to sodium nitroprusside was similar in aorta from normal and LPS-treated mice. Maximal relaxation to acetylcholine (10(-5)) was impaired after LPS and LacZ (63 +/- 3%, mean +/- s.e.m.) compared to normal vessels (83 +/- 3%) (P < 0.05). Gene transfer of ECSOD improved (P < 0.05) relaxation in response to acetylcholine (76 +/- 5%) after LPS, whereas gene transfer of ECSOD(R213G) had no effect (65 +/- 4%). Superoxide was increased in aorta (measured using lucigenin and hydroethidine) after LPS, and levels of superoxide were significantly reduced following ECSOD but not ECSOD(R213G). Thus, ECSOD reduces superoxide and improves relaxation to acetylcholine in the aorta after LPS, while the ECSOD variant R213G had minimal effect. These findings suggest that, in contrast to ECSOD, the common human gene variant of ECSOD fails to protect against endothelial dysfunction produced by an inflammatory stimulus.

Figure 1. Vasomotor responses to acetylcholine

Relaxation of aorta to acetylcholine in control mice treated with vehicle (Con-LacZ, n= 15), mice given LPS after transfection with LacZ (LPS-LacZ, n= 15), mice given LPS after transfection with ECSOD (LPS-ECSOD, n= 8) and mice given LPS after transfection with ECSOD_R213G (LPS-R213G, n= 6). Values are means ±s.e.m.*P < 0.05 versus control. +P < 0.05 versus LPS – ECSOD.

Figure 2. Relaxation to nitroprusside

Relaxation of aorta to nitroprusside of control mice treated with vehicle (Con-LacZ, n= 15), mice given LPS after transfection with LacZ (LPS-LacZ, n= 15), mice given LPS after transfection with ECSOD (LPS-ECSOD, n= 8) and mice given LPS after transfection with ECSOD_R213G (LPS-R213G, n= 6). Values are means ±s.e.m.

Figure 3. Contraction to prostaglandin F2α (PGF_2α)

Contraction of aorta to F2α PGF_2α in control mice treated with vehicle (Con-LacZ, n= 15), mice given LPS after transfection with LacZ (LPS-LacZ, n= 15), mice given LPS after transfection with ECSOD (LPS-ECSOD, n= 8), and mice given LPS after transfection with ECSOD_R213G (LPS-R213G, n= 6). Values are mean ±s.e.m.*P < 0.05 versus control. +P < 0.05 versus LPS-LacZ.

Figure 4. Detection of superoxide in mouse aorta in situ

Confocal fluorescence photomicrographs of aorta incubated with DHE from control mouse (A) and LPS treated mouse (B). In aortas from control mice, fluorescence is minimal. In contrast, in LPS-treated mice, fluorescence is increased. In mice given LPS after transfection with ECSOD (C), fluorescence is minimal. In mice given LPS after transfection with ECSOD_R213G (D), superoxide is similar to the untreated LPS mice. Similar finding were observed in 3 mice from each group.

Figure 5. Superoxide in aorta

Superoxide levels (lucigenin) in aorta of control mice treated with vehicle (Con-LacZ, n= 15), mice given LPS after transfection with LacZ (LPS-LacZ, n= 15), mice given LPS after transfection with ECSOD (LPS-ECSOD, n= 8) and mice given LPS after transfection with ECSOD_R213G (LPS-R213G, n= 6). Values are means ±s.e.m.*P < 0.05 versus control. +P < 0.05 versus LPS-LacZ.