Objective-Previously, we demonstrated that activated inducible NO synthase (iNOS)-expressing foam cells in human carotid plaques often produce autofluorescent (per)oxidized lipids (ceroid Key Words: atherosclerosis Ⅲ microvessels Ⅲ inducible NO synthase Ⅲ hemorrhage Ⅲ erythrocytes I ntermittent growth is a characteristic of human atherosclerosis. This could be the consequence of recurrent rupture of the fibrous cap followed by thrombus organization into the plaque. 1,2 Other studies have suggested a causative role of hemorrhages of intraplaque microvessels in carotid plaque rupture. [3][4][5] Paterson et al 6 proposed the vascularization theory of plaque evolution by demonstrating hemosiderin deposition in early atheromatous plaques, and they related this to repeated intraplaque capillary rupture, but it remains unclear how hemorrhages contribute to lipid accumulation. Ceroid is one of these lipid components and consists of insoluble mixtures of oxidized lipids and proteins, which mark sites of previous oxidative events. 7 Macrophages play a central role in the production of this fluorescent pigment, and extracellular ceroid deposits ultimately accumulate in the necrotic core of the plaque. 7,8 Furthermore, the upregulation of inducible NO synthase (iNOS), a major ancillary pathway of host defense by activated macrophages, is a characteristic feature of foam cell-rich plaque regions. 9,10 Recently, we showed that iNOS, which is predominantly expressed in macrophages, 10,11 often colocalizes with ceroid 11 or platelet-derived amyloid ␤ 12 in advanced human plaques. Because the reasons See coverfor these associations are unclear, we investigated the role of microvessels in plaque progression. To this end, the distribution of microvessels, ceroid and iNOS (as a marker of macrophage activation), was systematically mapped in human carotid artery plaques. The expression of von Willebrand factor (vWf) in the endothelial cells of intraplaque microvessels is highly variable, ranging from undetectable to thick perivascular deposits. 5 The latter are due to increased vWf biosynthesis during atherogenesis. 13,14 Therefore, vWf was used as a marker of endothelial cell activation. For an unbiased identification of topographical associations, the results were subjected to a cluster analysis. Finally, the formation of iNOS-expressing ceroid-containing foam cells was demonstrated in normocholesterolemic settings on erythrophagocytosis by macrophages in experimental thrombi in rabbit carotid arteries and in murine J774 macrophages in culture. MethodsThe ethics committees of Middelheim Hospital and Antwerp University approved the studies.

The present study describes

Ceroid, nitrotyrosine and NOS II colocalized in late stages of atherosclerosis and were found around the necrotic core in the plaque. This could suggest that NOS II expression in macrophages is involved in oxidation and peroxidation of lipids, leading to ceroid formation.

Abstract-Positioning a silicone collar around the rabbit carotid artery induces a smooth muscle cell-rich intimal thickening. We investigated the localization of inducible nitric oxide synthase (iNOS) during thickening of the intima, the effect of iNOS inhibition on intimal thickness, and the effect of oxidized LDL (ox-LDL) on iNOS expression in the vessel wall. Collars were positioned for 18 hours or for 3, 7, or 14 days. Arterial cross sections were immunostained for iNOS, including naïve, sham-operated, and carotid arteries in which ox-LDL had been infused locally for 14 days. Furthermore, collars were connected to osmotic minipumps for local delivery (5 L ⅐ h Ϫ1 , 14 days, nϭ12) of saline or the iNOS inhibitor L-N 6 -(1-iminoethyl)-lysine-HCl (L-NIL, 10 mmol/L). In the adventitia and the periadventitial granulation tissue of collared arteries, iNOS-positive macrophages and T lymphocytes were present from 18 hours onward. The media and intima were negative for iNOS. Reverse transcription-polymerase chain reaction revealed iNOS mRNA in collared but not in sham-operated arteries. Local inhibition of iNOS doubled the intimal thickness and decreased nitrotyrosine staining. Ox-LDL-treated arteries, which had a thicker intima, showed a pronounced influx of macrophages and T lymphocytes in all layers of the vessel wall, accompanied by iNOS expression in a subpopulation of these cells. Our study indicates that iNOS was not induced in intimal thickenings predominantly consisting of smooth muscle cells. However, iNOS was expressed in (peri) Key Words: intima Ⅲ oxidized LDL Ⅲ nitric oxide Ⅲ adventitia Ⅲ local delivery E vidence in animals and humans indicates that atherosclerosis and intimal thickening, evoked by mechanical injury of the media, lead to the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells (SMCs) or macrophages within the arterial wall. Verbeuren et al 1 provided functional evidence that nonendothelial NOS is induced in the aortas of hyperlipidemic rabbits. Further studies showed that in rabbits, iNOS was present in cholesterolinduced plaques in T lymphocytes, 2 macrophages, 2,3 and SMCs. 3 Also in human plaques, iNOS was found in association with macrophages, SMCs, endothelial cells (ECs), and mesenchymal-appearing intimal cells [3][4][5][6] but not in normal vessels. Furthermore, arterial SMCs in the neointima formed after a deendothelializing balloon injury to the rat carotid artery expressed iNOS. 7,8 Also, balloon angioplasty led to the induction of NOS in non-ECs of the rabbit carotid artery. 9 Positioning a silicone collar around the carotid artery of rabbits induces thickening of the intima. 10,11 The collar model has the advantage that substances can be infused locally in the space between the collar and the arterial wall. Collar-induced intimal thickening is possibly the consequence of a combination of both vascular injury and hindrance of transmural flow by the collar, leading to retention of cytokines/growth factors within the segment enclosed by the collar. 11 Altho...

1 The role of Ca2" in nitrergic neurotransmission was studied in the canine ileocolonic junction. 2 The specific N-type voltage-sensitive Ca2" channel blocker w-conotoxin GVIA (CTX, 10-100 nM) significantly reduced the electrically-evoked (2-16 Hz, 1-2 ms pulse width) non-adrenergic noncholinergic (NANC) relaxations, preferentially affecting those to low frequency stimulation, in circular muscle strips of the ileocolonic junction. In contrast, the nerve-mediated NANC-relaxations in response to acetylcholine (30 JAM), y-aminobutyric acid (100IM) and adenosine 5'-triphosphate (100 I1M), as well as the relaxations to nitric oxide (NO) (3-10 IOM) and nitroglycerin (1 JAM), remained unaffected. 3 A NO-related substance (NO-R), released from the ileocolonic junction in response to NANC nerve stimulation (4 and 16 Hz, 2 ms pulse width), was assayed with a superfusion bioassay cascade. CTX (50 nM) reduced the release of NO-R induced by electrical impulses (4 Hz: from 18 ± 4% to 6 ± 4%; 16 Hz: from 33 ± 2% to 14 + 4%, n = 5), but not that in response to the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP, 0.3 mM). In Ca2"-free medium, the release of NO-R evoked by electrical impulses or DMPP was inhibited. The L-type Ca2" channel blockers verapamil (1)(2)(3) and nifedipine (1 JM) had no effect.4 From these results we conclude that the release of NO-R in response to NANC nerve stimulation is Ca2"-dependent. The electrically-evoked release of NO-R results from Ca2" entry through CTX-sensitive N-type voltage-sensitive Ca2" channels, whereas that induced by nicotinic receptor activation involves CTX-insensitive Ca2" channels, different from the L-or N-type.

The aim of this study was to evaluate whether vascular remodeling and neointimal thickening occur after balloon dilatation of the nonatherosclerotic rabbit carotid artery, and whether both processes are influenced by continuous perivascular delivery of L-arginine or the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In the first experiment, histological and morphometric evaluation of arteries was performed at different time points after balloon dilatation: 10 minutes (n=7), and 1 (n=7), 2 (n=9), 3 (n=20), or 10 (n=5) weeks. Neointimal thickening progressively contributed to luminal narrowing for at least 10 weeks after angioplasty. During the first 2 weeks after dilatation, a significant decrease of the total vessel area was measured. Ten weeks after dilatation, both the neointimal and total vessel area were increased without further changing of the luminal area. In the second experiment, endothelial injured rabbits were randomly assigned to receive 2 weeks of continuous local perivascular physiological salt solution (n=6), L-arginine (n=8), or L-NAME (n=7), starting immediately after balloon dilatation (ie, local drug delivery during the first phase of the biphasic vascular remodeling process). Perivascular L-arginine delivery significantly reduced the neointimal area, despite an increased number of neointimal Ki-67-positive smooth muscle cells. Both the luminal area and total vessel area were significantly increased. Serum L-arginine levels remained unchanged. L-NAME administration had no effect on the neointimal area, nor on the luminal and total vessel area. Neointimal formation and biphasic vascular remodeling occur after experimental balloon dilatation of the nonatherosclerotic rabbit carotid artery, and can be influenced by continuous local perivascular delivery of L-arginine.

Rate‐adaptive single chamber pacemakers with accelerometer, closed loop stimulation (CLS), and remote monitoring functionality (Eluna 8 SR‐T, Biotronik, SE & Co, Germany) were implanted in 3 miniature donkeys with third‐degree atrioventricular block and syncope. After recovery, different pacemaker programming modes were tested at rest, during stress without physical exercise and during physical exercise. Pacing rates were compared to actual atrial rates and showed that CLS functionality allowed physiological heart rate adaptation. A transmitter installed in the stable provided wireless connection of the pacemaker to the internet. Home monitoring was activated which performed daily wireless transmission of pacemaker functional measurements to an online server allowing diagnosis of pathological arrhythmias and pacemaker malfunction from a distance. Closed loop stimulation and remote monitoring functionality resulted in nearly physiological rate adaptation and allowed remote “from‐the‐stable” patient follow‐up.

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