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

Azaindoles as Zinc-Binding Small-Molecule Inhibitors of the JAMM Protease CSN5

Cullin-RING ligases (CRLs) represent the largest family of E3 ubiquitin ligases. CSN5 is the zinc metalloprotease subunit of the COP9 signalosome, an important regulator of CRLs. Elevated expression of CSN5 has been found in several types of cancers. Altmann and coworkers describe the discovery of azaindoles as a new class of CSN5 inhibitors, which interact with the active-site zinc ion through an unprecedented binding mode. Nanomolar inhibitors led to degradation of the substrate recognition subunit Skp2 and reduced the viability of HCT116 cells. The study provides a proof-of-concept for the potential of CSN5 inhibitors as anticancer agents

Azaindoles as zinc-binding small molecule inhibitors of the JAMM protease CSN5

The COP9 signalosome (CSN) is an eight-subunit protein complex which is an important regulator of Cullin-Ring E3 ubiquitin ligases (CRLs). CSN5 is the Zinc metalloprotease subunit of CSN and is responsible for the cleavage of the ubiquitin-like protein NEDD8 from CRLs. Blocking deconjugation of NEDD8 traps the CRLs in a hyperactive state leading to their inactivation by inducing auto-ubiquitination and subsequent degradation. Consequently CRL substrates (e.g. tumor suppressors p27 and p21) are stabilized resulting in inhibition of cell proliferation. Thus pharmacological inhibition of CSN5 has the potential to offer a new therapeutic strategy for an efficacious treatment of CSN5 dependent cancers. A high-throughput screen (HTS) with the entire CSN complex led to the identification of an azaindole hit as a micromolar CSN5 inhibitor. Optimization of this hit resulted in a series of potent CSN5 inhibitors which stabilized neddylated Cullin-1 and led to the degradation of Skp2 in HCT116 cells. Furthermore these inhibitors demonstrated the expected functional effect on inhibiting viability of cancer cells. In addition a X-ray cocrystal structure elucidated the binding mode and revealed the N7 of the azaindole as a monodate ligand of the active site Zn2+ ion. The 7-azaindole motif represents a novel Zinc-binding scaffold for metalloproteases

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 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

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

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 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

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