1. Arterioscler Thromb Vasc Biol
Systemic Regulation of Vascular NAD(P)H Oxidase Activity and Nox Isoform Expression in Human Arteries and Veins
by Tomasz J. Guzik, Jerzy Sadowski, Boguslaw Kapelak, Andrzej Jopek, Pawel Rudzinski, Ravi Pillai, Richard Korbut, and Keith M. Channon
Arterioscler Thromb Vasc Biol
Volume 24(9):
September 1, 2004
Copyright © American Heart Association, Inc. All rights reserved.

2. Figure 1. Sources of vascular superoxide generation in human saphenous veins and mammary arteries.
Figure 1. Sources of vascular superoxide generation in human saphenous veins and mammary arteries. Superoxide production was determined by lucigenin-enhanced chemiluminescence (20 μmol/L lucigenin) in saphenous vein (HSV) and internal mammary artery (IMA) segments (n=21). Vessels were incubated for 30 minutes before and during superoxide determination with oxidase inhibitors: diphenyleneiodonium (50 μmol/L), apocynin (500 μmol/L), oxypurinol(100 μmol/L), rotenone (100 μmol/L), or l-NMMA (100 μmol/L). Oxygen consumption was measured to ensure that mitochondrial respiration was inhibited by rotenone. Superoxide generation was expressed as RLU/mg dry weight (mean±SEM). *P<0.05 versus basal; **P<0.01 versus basal; ¶P<0.01 versus HSV.
Tomasz J. Guzik et al. Arterioscler Thromb Vasc Biol. 2004;24:
Copyright © American Heart Association, Inc. All rights reserved.

3. Figure 2. NAD(P)H oxidase protein subunit levels and activity in human saphenous veins and mammary arteries.
Figure 2. NAD(P)H oxidase protein subunit levels and activity in human saphenous veins and mammary arteries. A, Western blots showing NAD(P)H oxidase subunits, p22phox, p67phox, and p47phox in saphenous vein (HSV) and IMA. α-Actin band shows equal loading of protein. B, Densitometric analysis of bands in HSV and IMA (mean±SEM; paired n=8), expressed in relation to actin. C, Superoxide production measured from vascular homogenates in response to 100 μmol/L NADPH or NADH measured using SOD-inhibitable ferricytochrome c reduction assay. Bars show mean±SEM (n=10 patients). *P<0.01 versus HSV.
Tomasz J. Guzik et al. Arterioscler Thromb Vasc Biol. 2004;24:
Copyright © American Heart Association, Inc. All rights reserved.

4. Figure 3. Relationships between human arteries and veins in vascular superoxide production and endothelial function.
Figure 3. Relationships between human arteries and veins in vascular superoxide production and endothelial function. Basal superoxide (O2−) production (A), maximal NAD(P)H oxidase activity (B), and endothelial function (C) were determined in paired segments of IMA and HSV. Superoxide production and NAD(P)H oxidase activity in intact vascular rings were measured using lucigenin-enhanced chemiluminescence (5 μmol/L) and endothelial function was assessed using maximal acetylcholine-dependent vasorelaxations (ACh max); n=31 patients.
Tomasz J. Guzik et al. Arterioscler Thromb Vasc Biol. 2004;24:
Copyright © American Heart Association, Inc. All rights reserved.

5. Figure 4. Molecular composition of NAD(P)H oxidases in saphenous veins and mammary arteries.
Figure 4. Molecular composition of NAD(P)H oxidases in saphenous veins and mammary arteries. A, RT-PCR of NADPH oxidase subunits of HSV and IMA. Equivalent RNA amounts (20 ng) were used. Inflammatory cell content was assessed by MCSFR mRNA expression. THP-1 RNA (nox2; p22phox; MCSFR) or hMEC RNA (nox1, nox4) were used as positive control. B, Quantitative RT-PCR of NADPH oxidase subunits of HSV and IMA (n=10 pairs). Results were normalized to mRNA levels of the housekeeping gene MLN-51. *P<0.05 versus HSV.
Tomasz J. Guzik et al. Arterioscler Thromb Vasc Biol. 2004;24:
Copyright © American Heart Association, Inc. All rights reserved.

6. Figure 5. Relationships between NAD(P)H oxidase subunit mRNA expression in arteries and veins.
Figure 5. Relationships between NAD(P)H oxidase subunit mRNA expression in arteries and veins. Quantitative RT-PCR of NADPH oxidase subunit mRNA (p22phox, nox2, and nox4) of HSV and IMA (n=10 patients) was performed as described in Methods; 20 ng of total RNA was used and results were normalized to expression of the housekeeping gene MLN-51. Relationships were analyzed using Pearson correlation analysis.
Tomasz J. Guzik et al. Arterioscler Thromb Vasc Biol. 2004;24:
Copyright © American Heart Association, Inc. All rights reserved.