eNOS Protects from Atherosclerosis Despite Relevant Superoxide Production by the Enzyme in apoE−/− Mice

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme. Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE(-/-)/eNOS(-/-)), while P/E-interactions did not differ, compared to apoE(-/-). eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE(-/-) vessels. Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE(-/-)/eNOS(-/-) vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE(-/-) atherosclerosis but does not negate the enzyme's strong protective effects

The inhibition of leukocyte-endothelial cell-interactions by endothelial nitric oxide

Ziel dieser Arbeit war es, die Rolle der endothelialen Stickstoffmonoxid-Synthase in Hinblick auf die Leukozyten-Endothelzell-Interaktionen bei der spontanen Arterioskleroseentwicklung zu untersuchen. Apolipoprotein E knockout-Mäuse und Apolipoprotein E knockout/endotheliale Stickstoffmonoxid-Synthase knockout-Mäuse dienten dabei als genetisches Modell. Durch IVM-, Real-time PCR-, Western Blot und immunhistochemische Versuche konnte gezeigt werden, dass apoE/eNOS dko-Tiere im Vergleich zu apoE ko-Kontrollen signifikant erhöhte L/E-Interaktionen, eine verstärkte endotheliale Adhäsionsmolekülexpression und eine gesteigerte Makrophagen-Infiltration in die Gefäßwand aufweisen. Duplexsonographisch vergleichbare Widerstands-Indices bei beiden Genotypen belegen eine ähnliche Hämodynamik und schließen veränderte Flussbedingungen als Ursache der erhöhten L/E-Interaktionen aus. Zusammenfassend kann man sagen, dass die verminderte NO-Produktion in apoE/eNOS dko-Mäusen die gesteigerten Leukozyten-Endothelzell-Interaktionen in diesem Modell bedingt und andere Quellen vaskulären Stickstoffmonoxids, genauer gesagt nNOS und iNOS, dies nicht zu kompensieren vermögen. Überraschenderweise nahm die eNOS-Deletion keinen Einfluss auf die Thrombozyten-Endothelzell-Interaktionen, was gegen eine bedeutende Rolle von Blutplättchen bei der beschleunigten Arterioskleroseentwicklung von apoE/eNOS dko-Mäusen spricht.It was the aim of the study to analyse the role of the endothelial nitric oxide synthase with regard to the leukocyte-endothelial cell-interactions in spontaneous arteriosclerosis development. Apolipoprotein E knockout mice and apolipoprotein E/endothelial nitric oxide synthase double knockout mice served as genetic models. Intravital microscopy, real-time PCR, western blot and immunohistochemical experiments revealed significantly increased leukocyte-endothelial cell (L/E)-interactions, an elevated expression of endothelial surface adhesion molecules and a heightened macrophage infiltration in carotid arteries in apoE/eNOS dko mice compared to apoE ko control animals. Comparable resistance indexes in duplex ultrasonography of both genotypes show similar hemodynamics and exclude altered flow conditions as cause of increased L/E-interactions. In summary you can say that reduced NO-production in apoE/eNOS dko mice causes the increased L/E-interactions and that other sources of vascular nitric oxide, more precisely nNOS and iNOS, are unable to compensate for that. Surprisingly, eNOS deletion didn’t influence platelet-endothelial cell-interactions, which argues against an important role of platelets in accelerated arteriosclerosis development of apoE/eNOS dko mice

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

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

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 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

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

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

Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase

Genetic and pharmacological inhibition of inducible nitric oxide synthase (iNOS) decreases atherosclerosis development. Potential proatherogenic effects of iNOS include iNOS mediated oxidative stress and iNOS expression in different cellular compartments. Lesional iNOS can potentially produce nitric oxide radicals (NO), superoxide radicals (O2−), or both; these radicals may then react to form peroxynitrite. Alternatively, O2− radicals from oxidases co-expressed with iNOS could react with NO to produce peroxynitrite. Therefore, the expression profiles of the genes that modulate the redox system in different iNOS-expressing cell compartments may determine the role of iNOS in atherosclerosis. We used apoE (apoE−/−) and apoE/iNOS double knockout (apoE−/−/ iNOS−/−) mice to assess vascular NO, O2−, and peroxynitrite formation by electron spin resonance spectroscopy, high performance liquid chromatography, and 3-nitrotyrosine staining. The relevance of the iNOS expressing cell compartment was tested by bone marrow transplantation. We show that iNOS significantly contributes to vascular NO production and itself produces O2−, leading to peroxynitrite formation in atherosclerotic lesions. Our bone marrow transplantation experiments show that bone marrow derived cells exclusively mediate the proatherosclerotic effects of iNOS in males, while both parenchymal and bone marrow derived iNOS equally contribute to atherosclerosis in females. Moreover, iNOS expression affects vascular remodeling. These findings establish for the first time that the proatherosclerotic effects of iNOS vary with sex in addition to the compartment of its expression