Inhibitory non-adrenergic non-cholinergic (NANC) nerves are thought to be important in the autonomic innervation of the gastrointestinal tract and other organ systems. The nature of their neurotransmitter is still debated. Speculation that nitric oxide (NO), formed from L-arginine in neuronal structures and other cells, could act as a neurotransmitter, is not yet supported by demonstration of its release upon nerve stimulation. Using a superfusion bioassay, we report the release of a vasorelaxant factor upon stimulation of the NANC nerves in the canine ileocolonic junction. Several pieces of evidence, including the selectivity of the bioassay tissues, chemical instability, inactivation by superoxide anion and haemoglobin, inhibition by NG-nitro-L-arginine (L-NNA) and potentiation by L-arginine all indicated that NO accounted for the biological activity of this transferable NANC factor.

Background-The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions. Methods and Results-Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (nϭ18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (PϽ0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8Ϯ3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles. Conclusions-We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques. (Circulation. 1998;97:2307-2315.)

P2Y receptors are G-protein-coupled receptors activated by extracellular nucleotides. The P2Y 6 receptor is selectively activated by UDP, and its transcript has been detected in numerous organs, including the spleen, thymus, intestine, blood leukocytes, and aorta. To investigate the biological functions of this receptor, we generated P2Y 6 -null mice by gene targeting. The P2Y 6 knockout (KO) mice are viable and are not distinguishable from the wild-type (WT) mice in terms of growth or fertility. In thioglycollate-elicited macrophages, the production of inositol phosphate in response to UDP stimulation was lost, indicating that P2Y 6 is the unique UDP-responsive receptor expressed by mouse macrophages. Furthermore, the amount of interleukin-6 and macrophage-inflammatory protein-2, but not tumor necrosis factor-␣, released in response to lipopolysaccharide stimulation was significantly enhanced in the presence of UDP, and this effect was lost in the P2Y 6 KO macrophages. The endothelium-dependent relaxation of the aorta by UDP was abolished in KO P2Y 6 mice. The contractile effect of UDP on the aorta, observed when endothelial nitric-oxide synthase is blocked, was also abolished in P2Y 6 -null mice. In conclusion, we generated P2Y 6 -deficient mice and have shown that these mice have a defective response to UDP in macrophages, endothelial cells, and vascular smooth muscle cells. These observations might be relevant to several physiopathological conditions such as atherosclerosis or hypertension.

Our study underscores the importance of elastin fragmentation in the vessel wall as an accelerator of atherosclerosis with enhanced inflammation and increased neovascularization, thereby promoting the development of unstable plaques that eventually may rupture. The present mouse model offers the opportunity to further investigate the role of key factors involved in plaque destabilization and potential targets for therapeutic interventions.

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.

Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8+/-0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.

A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90±0J6% of the medial smooth muscle cells (SMCs) were replicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for or-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76±0.22% at day 7). In the third phase the number of subendothelial cells increased (104±15 SMC nuclei per section at day 7, of which 8.89±2.26% were PCNA-positive) and was associated with deposition of collagen type IV and flbronectin. At 14 days a complete, circular neointima was present and contained 2.13 ±0.28% replicating SMCs. The media showed 0.44±0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage. While the factors responsible for intimal development in humans are largely unknown, numerous methods of injury have been applied to produce intimal lesions in animals. These methods can be divided into two broad categories: those that use intraluminal (e.g., balloon denudation) and those that use perivascular manipulation. Examples of the latter are external electrical stimulation, 6 external compression, 7 stripping the adventitia from the arteries, 8 and positioning of a cuff around an artery. 9 -" Perivascular cuff placement is used to avoid direct injury to the vessel wall, particularly