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

Insight into oxidative stress mediated by nitric oxide synthase (NOS) isoforms in atherosclerosis

The principle product of each NOS is nitric oxide. However, under conditions of substrate and cofactor deficiency the enzymes directly catalyze superoxide formation. Considering this alternative chemistry of each NOS, the effects of each single enzyme on key events of atherosclerosis are difficult to predict. Here, we evaluate nitric oxide and superoxide production by all three NOS isoforms in atherosclerosis. ESR measurements of circulating and vascular wall nitric oxide production showed significantly reduced nitric oxide levels in apoE/eNOS double knockout (dko) and apoE/iNOS dko animals but not in apoE/nNOS dko animals suggesting that eNOS and iNOS majorly contribute to vascular nitric oxide production in atherosclerosis. Pharmacological inhibition and genetic deletion of eNOS and iNOS reduced vascular superoxide production suggesting that eNOS and iNOS are uncoupled in atherosclerotic vessels. Though genetic deletion of nNOS did not alter superoxide production, acute inhibition of nNOS showed that nNOS contributes significantly to superoxide production. In conclusion, uncoupling of eNOS occurs in apoE ko atherosclerosis but eNOS mediated superoxide production does not outweigh the protective effects of eNOS mediated nitric oxide production. We show that although nNOS is not a major contributor of the vascular nitric oxide formation, it prevents atherosclerosis development. Acute inhibition of nNOS showed a significant reduction of superoxide formation suggesting that nNOS is uncoupled. The exact mechanism of action of nNOS in atheroprotection is yet to be elucidated. Genetic deletion of iNOS reduced NADPH oxidase activity. Thus, iNOS has both direct and indirect proatherosclerotic effects, as it directly generates both nitric oxide and superoxide simultaneously resulting in peroxynitrite formation and indirectly modulates NADPH oxidase activity. We hypothesize that eNOS is coupled in the disease free regions of the vessel and contributes to nitric oxide generation whereas in the diseased region of the vessel it is uncoupled to produce superoxide (Figure 16). nNOS expressed in the smooth muscle cells of the plaque contributes to the local superoxide generation. iNOS expressed in smooth muscle cells and leukocytes of the plaque generates superoxide and nitric oxide simultaneously to produce the strong oxidant peroxynitrite.Stickstoffmonoxid (NO) ist das prinzipielle Produkt aller Stickstoffmonoxid-Synthasen (NOS). Im Falle eines Mangels an Substrat (L-arginin) und Kofaktoren (Tetrahydrobiopterin, BH4) katalysieren die NOS-Enzyme direkt Superoxid (O2-). Diese Veränderung in der Radikalproduktion wird auch als Entkopplung der NOS bezeichnet. Die alternative Produktion von NO oder O2- durch die NOS bedingen, dass eine Voraussage über die Schlüsselfunktion der einzelnen Enzyme in der Entstehung der Atherosklerose schwierig ist. In unserer Studie evaluieren wir die Produktion von NO sowie O2- in atherosklerotischen Läsionen von apoE ko Mäusen und apoE/NOS doppel knockout (dko) Mäusen denen jeweils eine NOS-Isoform fehlt. Elektronen Spin Resonanz (ESR) Messungen konnten eine signifikante Reduktion sowohl des zirkulierenden, als auch der Gefäßwand eigenen Produktion von NO in apoE/eNOS dko und apoE/iNOS dko Mäusen zeigen, nicht jedoch in apoE/nNOS dko Mäusen. Dies lässt darauf schließen, dass eNOS und iNOS den hauptsächlichen Anteil der vaskulären NO-Produktion in atherosklerotischen Läsionen bewerkstelligen. Die pharmakologische Inhibierung wie auch die genetische Deletion von eNOS und iNOS führten ebenfalls zu einer reduzierten vaskulären O2- produktion, was die partielle Entkopplung beider Enzyme in atherosklerotisch veränderten Gefäßen nahe legt. Obwohl die chronische genetische Deletion von nNOS in apoE/nNOS dko die O2- Produktion nicht verändert, zeigte sich bei der akuten pharmakologischen Inhibierung von nNOS (durch L-NAANG) eine maßgebliche Beteiligung von nNOS an der O2- produktion in apoE ko Mäusen. Schlussfolgernd lässt sich sagen, dass in atherosklerotischen Gefäßen von apoE ko Tieren eine Entkopplung von eNOS statt findet, diese jedoch zu keinem Ausgleich der protektiven Effekte der eNOS vermittelten NO-Produktion führt. Unsere Ergebnisse in apoE/nNOS dko Mäusen zeigen eine atheroprotektive Rolle der nNOS, die sich nicht allein durch eine lokale, vaskuläre NO-Produktion durch das Enzym erklären lässt. Wir postulieren weitere systemisch atheroprotektive Eigenschaften der nNOS. Die signifikante Reduktion der Superoxidproduktion durch eine akute Inhibierung der nNOS weist auf eine Entkopplung der nNOS hin. Der exakte Wirkungsmechansimus von nNOS in der Atheroskleroseprävention ist weiterhin noch zu eruieren. Die genetische Deletion von iNOS führt zu einer reduzierten Aktivität der NADPH-Oxidase. Demnach sind für iNOS direkte sowie indirekte atherosklerosefördernde Effekte anzunehmen, da sie auf direktem Wege gleichzeitig NO und O2- produziert, was in einer Peroxynitritbildung resultiert. Wir stellen die Hypothese auf, dass eNOS in den läsionsfreien Gefäßregionen gekoppelt ist und dort seine atheroprotektiven Effekte durch die NO-Produktion vermittelt, während die eNOS in atherosklerotischen Läsionen entkoppelt vorliegt und hier O2- produziert (Fig. 16). iNOS, welches vor allem in den Plaques, in glatten Muskelzellen und Leukozyten zu finden ist, produziert gleichzeitig hohe Konzentrationen von O2- und NO, die als gemeinsames Endprodukt das stark oxidierende Peroxynitrit ergeben und die von uns dokumentierte proatherosklerotische Wirkung der iNOS vermittelt

Comprehensive approach for identification of functional FCGR2C alleles resulting in protein expression as a determinant for predicting predisposition to autoimmunity

Abstract The balance of activating and inhibitory signals from the low affinity Fc gamma receptors modulates immune responses triggered by IgG antibody‐immune complexes. In homeostasis, this leads to antigen clearance, while in autoimmune diseases to unwanted immune response. Besides the activating receptors FcɣRIIa, FcɣRIIIa, and the inhibitory FcɣRIIb receptor, a third activating receptor, FcɣRIIc, was shown to be expressed on several immune cell types, however, only in the presence of a functional FCGR2C‐ORF allele. FcɣRIIc expression is associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus or systemic sclerosis. Thus, the determination of the functional FCGR2C gene resulting in protein expression on immune cells becomes highly relevant, particularly in the context of unwanted immune responses through inadvertent FcɣRIIc activation by molecules targeting stimulation of the inhibitory receptor FcɣRIIb, currently pursued by several pharmaceutical companies. The high degree of homology within the FCGR2/3 gene cluster complicates development of an accurate method for identification of FcɣRIIc expression. Here we describe a comprehensive approach to characterize genetic status of the FCGR2C gene locus consisting of cDNA sequencing, SNaPshot genotyping and low‐coverage next‐generation sequencing. This might enable Mendelian randomization hypothesis testing across autoimmune diseases to personalize therapies and enhance treatment outcomes

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

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

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

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