Figure 1.
L/E-interactions analysed by intravital microscopy.
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).
Figure 2.
eNOS deletion increases VCAM-1 expression.
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).
Figure 3.
NO from eNOS influences macrophage infiltration in the vascular wall.
a) Real time PCR analysis of CD14 showed significantly increased expression of CD14 mRNA in apoE−/−/eNOS−/− (n = 12) carotids, compared to apoE−/− (n = 17, *p<0.00002). b) Immunohistochemistry for MOMA-2 showed elevated vascular macrophage infiltration in carotid arteries of apoE−/−/eNOS−/− (n = 10), compared to apoE−/− (n = 9, *p<0.05).
Figure 4.
Unaltered vascular resistance index in eNOS deficiency.
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.
Figure 5.
eNOS is a significant source of vascular wall NO production and circulating NO.
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).
Figure 6.
eNOS is uncoupled and contributes to vascular production of superoxide in apoE−/− mice.
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.
Figure 7.
Vascular expression of NOS isoforms.
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.