eNOS is a significant source of vascular wall NO production and circulating NO.

<p>a) ESR spectrum of NO-Fe-(DETC)₂ in aortas of apoE^−/− and apoE^−/−/eNOS^−/−. Bold lines indicate apoE^−/−, stripped lines apoE^−/−/eNOS^−/− and patterned lines buffer/spin trap alone. Arrows show the typical 3 peaks NO-Fe-(DETC)₂ signal. b) Vascular NO production in C57BL/6J (n = 12), eNOS^−/− (n = 8), apoE^−/− (n = 14) and apoE^−/−/eNOS^−/− (n = 15), *p≤0.01, **p<0.001, ***p<0.0001). c) Vascular NO production with NOS inhibition using L-NAME in apoE^−/− (n = 11) and apoE^−/−/eNOS^−/− mice (n = 16), *p<0.01, ***p<0.0001. d) Nitrosyl hemoglobin concentration of blood samples from apoE^−/−/eNOS^−/− (n = 11) vs. apoE^−/− controls (n = 13, *p = 0.01).</p

Vascular expression of NOS isoforms.

<p>Significantly increased expression of iNOS protein in the aorta of apoE^−/−/eNOS^−/− (n = 10) compared to apoE^−/− mice (n = 10). The protein levels of nNOS did not differ between apoE^−/− (n = 10) apoE^−/−/eNOS^−/− (n = 11). * p<0.05, NS denotes non-significance.</p

eNOS deletion increases VCAM-1 expression.

<p>a) Real time PCR analysis showed four fold increased expression of VCAM-1 mRNA in apoE^−/−/eNOS^−/− (n = 9) carotids, compared to apoE^−/− (n = 20, *p<0.01). b) Immunohistochemistry confirmed increased endothelial VCAM-1 expression in carotid arteries of apoE^−/−/eNOS^−/−, compared to apoE^−/−. Arrows indicate positive DAB staining (internal carotid artery, location of IVM). c) Double immunofluorescence staining of VCAM-1 protein in atherosclerotic lesions. Sections of the aortic arch of apoE^−/− and apoE^−/−/eNOS^−/− animals were incubated with anti-VCAM-1 antibody (red) and anti-CD31 antibody (endothelial cells, green). Arrows indicate localization of VCAM-1 in endothelial cells in the overlay (yellow). Increased endothelial expression of VCAM-1 was observed in apoE^−/−/eNOS^−/− compared to apoE^−/−. d) Increased medial smooth muscle cell expression of VCAM-1 was observed in advanced plaques in the aortic arch in apoE^−/−/eNOS^−/− compared to apoE^−/−, as shown in yellow (arrows) by the double immunofluorescence staining of VCAM-1 (red) and smooth muscle cells (green).</p

eNOS is uncoupled and contributes to vascular production of superoxide in apoE^−/− mice.

<p>a) HPLC measurements showed lower levels of superoxide production in apoE^−/−/eNOS^−/− (n = 13) vs. apoE^−/− (n = 23). Superoxide levels were higher in apoE^−/− (n = 23) compared to C57BL/6J (n = 14). Interestingly, superoxide levels were significantly lower in apoE^−/−/eNOS^−/− (n = 13) compared to eNOS^−/− (n = 12). b) L-NAME inhibited superoxide production in apoE^−/− (n = 15) but not in C57BL/6J (n = 17) and apoE^−/−/eNOS^−/− (n = 12) aortas. c) Specific inhibition of eNOS using L-NIO resulted in significant reduction of superoxide production in apoE^−/− (n = 19). d) Total ROS production using ESR showed a significant increase in ROS levels in apoE^−/− (n = 23) compared to C57BL/6J (n = 12) and apoE^−/−/eNOS^−/− (n = 15). e) Consistently, SOD inhibitable superoxide production measured by ESR also showed significant increase in superoxide levels in apoE^−/− (n = 23) compared to C57BL/6J (n = 12) and apoE^−/−/eNOS^−/− (n = 15). ^§p<0.05, *p<0.01, **p<0.001, ***p<0.0001, NS denotes non-significance. f) Uncoupling of eNOS in apoE^−/− compared to C57BL/6J aorta shown by western blot of eNOS protein dimer/monomer.</p

Unaltered vascular resistance index in eNOS deficiency.

<p>a) Representative picture of duplex ultrasonography in carotid arteries. b) Equal resistance index of carotid arteries from apoE^−/−, n = 17, vs. apoE^−/−/eNOS^−/−, n = 10, p = 0.88, by duplex ultrasonography. NS denotes non-significance.</p

L/E-interactions analysed by intravital microscopy.

<p>The number of rolling, transiently adherent and firmly adherent leukocytes was significantly increased in the common carotid artery of apoE^−/−/eNOS^−/− (n = 16), vs. apoE^−/− controls (n = 23), a) *p<0.01; b) ***p<0.0001; c) **p<0.001).</p