Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.

Impaired fibrillin-1 function promotes features of plaque instability in apolipoprotein E-deficient mice.

BACKGROUND: Arterial stiffness has been associated with an increased cardiovascular risk. The aim of this study was to investigate the interaction between arterial stiffness and atherosclerosis. METHODS AND RESULTS: Mice with a mutation C1039G+/-) in the fibrillin-1 gene leading to fragmentation of the elastic fibers were crossbred with apolipoprotein E-deficient (ApoE-/-) mice. Subsequently, ApoE-/- and ApoE-/-C1039G+/- mice were fed a Western-type diet for 10 or 20 weeks. Our results show that the interaction between arterial stiffness and atherosclerosis is bidirectional. On the one hand, arterial stiffness in ApoE-/-C1039G+/- mice increased more rapidly in the presence of atherosclerotic plaques. On the other hand, arterial stiffness promoted the development of larger and more unstable plaques in ApoE-/-C1039G+/- mice. The plaque area at the aortic root was increased 1.5- and 2.1-fold in ApoE-/-C1039G+/- mice after 10 and 20 weeks of Western-type diet, respectively. After 10 weeks of Western-type diet, plaques of ApoE-/-C1039G+/- mice showed increased apoptosis of smooth muscle cells, which was associated with a decrease in collagen content, an enlargement of the necrotic core, and an increase in macrophages. After 20 weeks of Western-type diet, the number of buried fibrous caps was increased in atherosclerotic lesions of ApoE-/-C1039G+/- mice, not only at the level of the aortic valves but also in the brachiocephalic artery and in the upper, middle, and lower thoracic aorta. Furthermore, acute plaque rupture was observed. CONCLUSIONS: These results indicate that fragmentation of the elastic fibers leads to increased vascular stiffness, which promotes features of multifocal plaque instability.

Proteasome inhibitor bortezomib promotes a rupture-prone plaque phenotype in ApoE-deficient mice.

The ubiquitin-proteasome system is involved in the development and progression of atherosclerosis. The aim of this study was to investigate whether plaque composition is affected by proteasome function. In vitro, the potent and selective proteasome inhibitor bortezomib induced apoptosis in both cultured smooth muscle cells (SMCs) and activated macrophages. This effect was associated with increased expression of C/EBP homologous protein and cleavage of caspase-12, indicative of endoplasmic reticulum stress. The sensitivity to the proapoptotic effects of proteasome inhibition correlated with the protein synthesis rate. Proteasome inhibition in explanted atherosclerotic plaques of ApoE-deficient mice resulted in a significant decrease in SMCs and macrophages, indicating that both cell types in the atherosclerotic plaque were susceptible to the proapoptotic effects of proteasome inhibition. In vivo proteasome inhibition in ApoE-deficient mice did not affect plaque size or composition of early atherosclerotic plaques, but resulted in a significant decrease in collagen content as well as a significant enlargement of the necrotic core in advanced atherosclerotic plaques. In conclusion, our results indicate that an impaired proteasome function promotes features of a more rupture-prone plaque phenotype.

Multi-slice computed tomography with N1177 identifies ruptured atherosclerotic plaques in rabbits.

Rupture-prone and ruptured plaques are characterized by the presence of large numbers of macrophages. N1177 is a contrast agent consisting of iodinated nanoparticles that are selectively phagocytosed by macrophages. The aim of this study was to investigate the effect of N1177 on the CT attenuation of rupture-prone and ruptured plaques in rabbits. In addition, we examined in vitro whether uptake of N1177 occurred without cytotoxic or pro-inflammatory effects on macrophages. In vitro, the viability of J774 macrophages was not affected by treatment with N1177. Moreover, N1177 had no effect on the phagocytic capacity or cytokine production of macrophages. For the in vivo experiments, 6 New Zealand White rabbits were fed a cholesterol-supplemented diet for 12-15 months, resulting in the development of large atherosclerotic plaques that resembled rupture-prone plaques in humans. In three rabbits, mechanical plaque rupture was induced by retrograde pullback of an embolic protection device. N1177 had no effect on the median density of rupture-prone plaques [35 HU (range 3-85) before injection vs. 32 HU (range 1-93) 2 h after injection of N1177; P > 0.05]. However, after induction of mechanical plaque rupture, the median density of the atherosclerotic plaques increased from 40 HU (range 6-86) before injection to 74 HU (range 14-111) 2 h after injection of N1177 (P < 0.001). Using time-of-flight static secondary ion mass spectrometry, the presence of N1177 nanoparticles was demonstrated in macrophage-rich areas of ruptured plaques, but not of non-ruptured plaques. In conclusion, our results show that N1177 is a contrast agent that can identify ruptured atherosclerotic plaques.

Transglutaminase 2 deficiency decreases plaque fibrosis and increases plaque inflammation in apolipoprotein-E-deficient mice.

AIM: Transglutaminase 2 (TG2) is important for the deposition and stability of the extracellular matrix via effects on cross-linking of matrix proteins and transforming growth factor beta (TGFbeta) activity. The purpose of this study was to investigate the effect of TG2 deficiency on the composi- tion of atherosclerotic plaques. METHODS: Apolipoprotein E (ApoE)(-/-) mice were crossbred with TG2(-/-) mice to obtain ApoE(-/-)TG2(-/-) mice. ApoE(-/-) and ApoE(-/-)TG2(-/-) mice were fed a Western-type diet for 16 or 30 weeks to determine the effect of TG2 deficiency on early and advanced atherosclerosis, respectively. RESULTS: Atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice showed decreased cross-linking of matrix proteins, as well as decreased nuclear staining for phospho-Smad2/-Smad3, indicative of decreased TGFbeta activity. Compared to ApoE(-/-) mice, plaque area was decreased by 45 and 48% in ApoE(-/-)TG2(-/-) mice after 16 and 30 weeks, respectively. Sirius red staining showed a significant decrease in collagen content in early and advanced atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice. Furthermore, there was a significant increase in macrophages in advanced atherosclerotic plaques of ApoE(-/-)TG2(-/-) mice. CONCLUSION: TG2 deficiency resulted in a decreased collagen content and increased inflammation, which are features of a more unstable plaque.

Non-alcoholic steatohepatitis induces non-fibrosis-related portal hypertension associated with splanchnic vasodilation and signs of a hyperdynamic circulation in vitro and in vivo in a rat model.

INTRODUCTION: Steatosis, without fibrosis, may lead to changes in liver blood flow, which are poorly understood, and to date have not been correlated to portal pressure and related haemodynamics. AIMS: To study the temporal relation between progressive steatosis, portal pressure, systemic haemodynamics, vascular responsiveness, mesenteric and portal blood flow in methionine-choline-deficient diet (MCDD)-fed rats. METHODS: Male Wistar rats fed the MCDD were examined at week (w) 0-1-2-3-4-5-6-7-8, respectively, including systemic haemodynamics and portal pressure. At w0-4-8, in vivo blood flow was measured in the portal vein and the superior mesenteric artery. Dose-response curves to phenylephrine (PE) were established in abdominal aortic rings. RESULTS: Histology showed 100% steatosis from w3 on. Fibrosis was absent. Significant inflammation was nearly absent upon w4. Portal pressure slightly increased at w2, reached a maximum at w4 [9.4 +/- 0.3 vs 2.9 +/- 0.6 mmHg at w0 (P=0.003)] and remained stable upon w8. Mean arterial blood pressure (MABP) decreased from w2 on [98.7 +/- 5.7 mmHg on w4 compared with 123.8 +/- 1.8 on w0 (P=0.002)]. Portal flow increased from 1.85 +/- 0.11 to 3.07 +/- 0.44 ml/min/100 g on w0 and w8 respectively (P=0.039). Mesenteric artery flow increased from 3.40 +/- 0.26 to 4.56 +/- 0.30 ml/min/100 g on w0 and w8 respectively (P=0.043). Vascular responsiveness to PE gradually decreased from 138 +/- 3% on w0 to 110 +/- 5% on w4 (P=0.013). CONCLUSION: Steatohepatitis induces significant portal hypertension (PHT) in the absence of fibrosis, associated with an increase in mesenteric arterial and portal venous flow, arterial hyporesponsiveness to vasoconstrictors and a decrease in MABP, indicating the presence of splanchnic vasodilation and hyperdynamic circulation. These alterations resemble those seen in cirrhotic PHT.

Effect of statins on the viability of macrophages and smooth muscle cells.

Because macrophages play an important role in the destabilization of atherosclerotic plaques, and smooth muscle cells (SMCs) contribute to plaque stability, selective clearance of macrophages in atherosclerotic plaques is a promising strategy for plaque stabilization. In the present study, we investigated the effects of fluvastatin, simvastatin, lovastatin, and pravastatin on the viability of macrophages and SMCs. All statins, except pravastatin, induced cell death of J774A.1 macrophages after 24 hours, albeit with different sensitivity. The viability of rabbit aortic SMCs was hardly affected. Fluvastatin-induced macrophage cell death was characterized as apoptosis and could be reversed by isoprenoid intermediates of the mevalonate pathway. Peritoneal macrophages from male or female mice were much more resistant to statin-induced cell death. The high sensitivity of J774A.1 macrophages to statin-induced cell death was related to the strong 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in these cells. Macrophage and SMC content of rabbit atherosclerotic plaques was not affected after in vitro treatment with fluvastatin or lovastatin for 3 days. In conclusion, fluvastatin, simvastatin, and lovastatin, but not pravastatin, can selectively induce apoptosis in J774A.1 macrophages, but not in SMCs, primary macrophages or rabbit atherosclerotic plaques. This effect was related to the degree of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the different cell types.

The protein synthesis inhibitor anisomycin induces macrophage apoptosis in rabbit atherosclerotic plaques through p38 mitogen-activated protein kinase.

Because macrophages play a major role in atherosclerotic plaque destabilization, selective removal of macrophages represents a promising approach to stabilize plaques. We showed recently that the protein synthesis inhibitor cycloheximide, in contrast to puromycin, selectively depleted macrophages in rabbit atherosclerotic plaques without affecting smooth muscle cells (SMCs). The mechanism of action of these two translation inhibitors is dissimilar and could account for the differential effects on SMC viability. It is not known whether selective depletion of macrophages is confined to cycloheximide or whether it can also be achieved with translation inhibitors that have a similar mechanism of action. Therefore, in the present study, we investigated the effect of anisomycin, a translation inhibitor with a mechanism of action similar to cycloheximide, on macrophage and SMC viability. In vitro, anisomycin induced apoptosis of macrophages in a concentration-dependent manner, whereas SMCs were only affected at higher concentrations. In vivo, anisomycin selectively decreased the macrophage content of rabbit atherosclerotic plaques through apoptosis. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole] prevented anisomycin-induced macrophage death, without affecting SMC viability. SB202190 decreased anisomycin-induced p38 MAPK phosphorylation, did not alter c-Jun NH(2)-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. The latter effect was abolished by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynyltio)butadiene ethanolate], although the prevention of anisomycin-induced macrophage death by SB202190 remained unchanged. The JNK phosphorylation inhibitor SP600125 did not affect anisomycin-induced macrophage or SMC death. In conclusion, anisomycin selectively decreased the macrophage content in rabbit atherosclerotic plaques, indicating that this effect is not confined to cycloheximide. p38 MAPK, but not ERK1/2 or JNK, plays a major role in anisomycin-induced macrophage death.

Mitochondrial uncoupling protein 2 mediates temperature heterogeneity in atherosclerotic plaques.

AIMS: Rupture-prone atherosclerotic plaques show an elevated temperature, but a molecular explanation for this phenomenon is unknown. Here, we investigated whether mitochondrial uncoupling protein 2 (UCP2) could be involved because this protein is a macrophage homologue of thermogenin in brown fat tissue. METHODS AND RESULTS: Immunohistochemistry, western blotting, and real-time quantitative polymerase chain reaction were used to detect UCP2 expression in human and rabbit atherosclerotic plaques. Temperature was measured in plaques with thermography catheters and in cultured cells with precision thermometers. UCP2 was abundantly expressed in subendothelial macrophages of atherosclerotic plaques but not in deeper layers of the plaque. Ex vivo temperature measurements in atherosclerotic rabbit thoracic aorta demonstrated a correlation between local plaque temperature, total macrophage mass, and UCP2 expression. In vitro, chemical uncoupling of macrophages with sodium cyanide resulted in heat production (DeltaT = 0.13 +/- 0.04 degrees C vs. controls). Also, overexpression of UCP2 in cultured cells led to a similar increase in temperature. CONCLUSION: Our findings provide evidence that temperature heterogeneity in atherosclerotic plaques is at least in part attributed to UCP2 expression in macrophages. The heat generated might be used to detect unstable, macrophage-rich, atherosclerotic plaques via thermography.

Nitric oxide donor molsidomine favors features of atherosclerotic plaque stability and reduces myocardial infarction in mice.

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE-/-Fbn1C1039G+/-) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE-/-Fbn1C1039G+/- mouse model. Female ApoE-/-Fbn1C1039G+/- mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 ±â€¯1.2 vs. 8.1 ±â€¯0.7 µm) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 ±â€¯2% vs. 27 ±â€¯2%) combined with a decreased end diastolic (3.1 ±â€¯0.2 vs. 3.9 ±â€¯0.2 mm) and end systolic diameter (1.9 ±â€¯0.1 vs. 2.9 ±â€¯0.2 mm). Furthermore, perivascular fibrosis (23 ±â€¯2 vs. 30 ±â€¯2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 ±â€¯0.04 vs. 2.91 ±â€¯0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability.

Differential effect of the protein synthesis inhibitors puromycin and cycloheximide on vascular smooth muscle cell viability.

Recent evidence indicates that the protein synthesis inhibitor cycloheximide triggers selective macrophage death in rabbit atheroma-like lesions without affecting smooth muscle cells (SMCs) or the endothelium, thereby favoring a stable plaque phenotype. In this study, we report that puromycin, a protein synthesis inhibitor with a different mode of action but with similar ability to inhibit de novo protein synthesis, did not reveal plaque-stabilizing effects. The macrophage and the SMC content readily decreased in puromycin-treated atheroma-like lesions in rabbit carotid arteries. Moreover, puromycin induced apoptosis in macrophages and SMCs in vitro. Puromycin-treated SMCs showed signs of endoplasmic reticulum (ER) stress, as demonstrated by CCAAT/enhancer-binding protein homologous protein (CHOP) protein expression, splicing of X-box-binding protein 1 mRNA, and phosphorylation of eukaryotic translation initiation factor 2alpha. The ER stress inducer thapsigargin up-regulated CHOP protein expression in SMCs without affecting their viability, indicating that ER stress not necessarily results in cell death. Puromycin, but not thapsigargin, activated the ER stress-related caspase-12. Treatment of SMCs with a combination of cycloheximide and puromycin inhibited ER stress and partially improved SMC viability. In addition, puromycin, but not cycloheximide or thapsigargin, induced intracellular accumulation of polyubiquitinated proteins in SMCs, whereas the proteasome function was not affected. Taken together, puromycin, in contrast to cycloheximide, induces SMC apoptosis, thereby favoring an unstable plaque phenotype. SMC death upon puromycin treatment could only be partially prevented by cycloheximide, which completely blocked ER stress. However, other or additional mechanisms, such as increased polyubiquitination of proteins, might be involved in puromycin-induced SMC death.

Worms and the treatment of inflammatory bowel disease: are molecules the answer?

The lack of exposure to helminth infections, as a result of improved living standards and medical conditions, may have contributed to the increased incidence of IBD in the developed world. Epidemiological, experimental, and clinical data sustain the idea that helminths could provide protection against IBD. Studies investigating the underlying mechanisms by which helminths might induce such protection have revealed the importance of regulatory pathways, for example, regulatory T-cells. Further investigation on how helminths influence both innate and adaptive immune reactions will shed more light on the complex pathways used by helminths to regulate the hosts immune system. Although therapy with living helminths appears to be effective in several immunological diseases, the disadvantages of a treatment based on living parasites are explicit. Therefore, the identification and characterization of helminth-derived immunomodulatory molecules that contribute to the protective effect could lead to new therapeutic approaches in IBD and other immune diseases.

Phagocytosis in atherosclerosis: Molecular mechanisms and implications for plaque progression and stability.

Macrophages play a key role in atherosclerotic plaque destabilization and rupture. In this light, selective removal of macrophages may be beneficial for plaque stability. However, macrophages are phagocytic cells and thus have an important additional role in scavenging of modified lipoproteins, unwanted or dead cells and cellular debris via phagocytosis. The concept of phagocytosis as well as the underlying mechanisms is well defined but the effect of phagocytosis in terms of plaque stability remains poorly understood. Recent findings point towards a complex role of macrophage phagocytosis in atherogenesis. Macrophages are necessary for removal of apoptotic cells from plaques, but exert strong proatherogenic properties upon phagocytosis of lipoproteins, erythrocytes and platelets. Apart from heterophagy, autophagocytosis better known as autophagy may occur in advanced atherosclerotic plaques. Several lines of evidence indicate that autophagy is initiated in plaque smooth muscle cells as a result of cellular distress. Since autophagy is well recognized as a survival mechanism, autophagic smooth muscle cells in the fibrous cap may reflect an important feature underlying plaque stability. All together, phagocytosis is a crucial process involved in atherogenesis that may significantly affect the stability of the atherosclerotic plaque.

In situ detection of starvation-induced autophagy.

Autophagy is a regulated bulk degradation process involved in many different human pathologies. Transmission electron microscopy (TEM) is currently the only reliable method for monitoring autophagy in situ. Because TEM is labor intensive, we questioned whether useful marker proteins can be found for unambiguous detection of autophagy in tissue via routinely used colorimetric, immunohistochemical, or fluorescent techniques. Starved HepG2 hepatocytes and nutrient deprived liver tissue were used as a model for the initiation of autophagy. Our findings indicate that starvation-induced autophagy in HepG2 cells was associated neither with differential mRNA gene expression nor with changes in the expression level of known autophagy-related proteins. On the contrary, both transcription and translation were inhibited, suggesting that the identification of autophagy-specific biomarkers for tissue is highly compromised. Light chain 3 (LC3) protein, which is an attractive marker of autophagosomes, revealed a relatively low expression level in tissue and cultured cells, but could be detected via immunohistochemistry in liver from GFP-LC3 transgenic mice. The number of LC3 immunopositive dot-like structures was significantly upregulated in liver tissue from nutrient-deprived GFP-LC3 mice as compared with nonstarved control tissue. Our results suggest that LC3 immunostaining can be used as an alternative detection method for autophagy in situ, but only when this protein is overexpressed.

Platelet phagocytosis and processing of beta-amyloid precursor protein as a mechanism of macrophage activation in atherosclerosis.

In human occluded saphenous vein grafts, we previously demonstrated cytotoxic foam cells, presumably derived from macrophages engulfing platelets. In the present study, we investigated whether platelet phagocytosis occurs in human atherosclerotic plaques, whether this activates macrophages, and whether the platelet constituent, amyloid precursor protein (APP), was involved. Immunohistochemistry documented the presence of APP, beta-amyloid peptide (Abeta, cleaved from APP), and platelets (CD9), along with inducible NO synthase (iNOS) and cyclooxygenase-2, two markers of macrophage activation, around microvessels in advanced human carotid artery plaques (n=18). Abeta colocalized with iNOS-expressing macrophages that were often surrounded by platelets. In vitro, murine J774 and human THP-1 macrophages were incubated with or without washed human platelets. Coincubation of macrophages and platelets led to platelet phagocytosis (electron and confocal microscopy) and formation of lipid-, APP-, and Abeta-containing foam cells. These expressed iNOS mRNA (reverse transcription-polymerase chain reaction) and protein and produced nitrite and tumor necrosis factor-alpha (ELISA). Macrophage pretreatment with 4-(2-aminoethyl)benzenesulfonyl fluoride, a protease inhibitor, reduced APP processing and inhibited NO biosynthesis induced by platelet phagocytosis but not by lipopolysaccharides. Human atherosclerotic plaques and J774 and THP-1 macrophages contained mRNA of the APP-cleaving enzyme beta-secretase. This is the first demonstration of Abeta, a peptide extensively studied in Alzheimer's disease, in human atherosclerotic plaques. It was present in activated iNOS-expressing perivascular macrophages that had phagocytized platelets. In vitro studies indicate that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to Abeta, resulting in iNOS induction. This represents a novel mechanism for macrophage activation in atherosclerosis.

Phagocytosis of apoptotic cells by macrophages is impaired in atherosclerosis.

OBJECTIVE: Apoptotic cell death has been demonstrated in advanced human atherosclerotic plaques. Apoptotic cells (ACs) should be rapidly removed by macrophages, otherwise secondary necrosis occurs, which in turn elicits inflammatory responses and plaque progression. Therefore, we investigated the efficiency of phagocytosis of ACs by macrophages in atherosclerosis. METHODS AND RESULTS: Human endarterectomy specimens and human tonsils were costained for CD68 (macrophages) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) (apoptosis). Free and phagocytized ACs were counted in both tissues. The ratio of free versus phagocytized AC was 19-times higher in human atherosclerotic plaques as compared with human tonsils, indicating a severe defect in clearance of AC. Impaired phagocytosis of AC was also detected in plaques from cholesterol-fed rabbits and did not further change with plaque progression. In vitro experiments with J774 or peritoneal mouse macrophages showed that several factors caused impaired phagocytosis of AC including cytoplasmic overload of macrophages with indigestible material (beads), free radical attack, and competitive inhibition among oxidized red blood cells, oxidized low-density lipoprotein and ACs for the same receptor(s) on the macrophage. CONCLUSIONS: Our data demonstrate that phagocytosis of ACs is impaired in atherosclerotic plaques, which is at least partly attributed to oxidative stress and cytoplasmic saturation with indigestible material.

The role of tachykinins in circular muscle contractility of the murine ileum: a functional investigation.

We investigated the participation of different tachykinin receptors in contractility of circular muscle strips of the mouse ileum using selective NK receptor agonists and antagonists. The NK1 receptor agonist septide (1-100 nM) induced dose-dependent contractions which were reduced by atropine and augmented by L-NNA. L-NNA increased and TTX consecutively reduced contractions to the NK2 receptor agonist beta-A-NKA (1-100 nM). Senktide, agonist of NK3 receptors, failed to induce contractions. NANC contractions to EFS were decreased after NK1 receptor blockade with RP67580. This inhibitory effect was more pronounced after additional blockade of NK2 and NK3 receptors. NK3 receptor antagonism alone reduced contractions at higher frequencies of stimulation. When the duration of the EFS stimulus was increased, the participation of all NK receptor subtypes became more evident. Our results suggest that excitatory NANC transmission in the circular muscle layer of the mouse ileum is mediated by tachykinins acting principally on NK1 receptors on cholinergic nerves and smooth muscle cells. Also NK2 receptors, located on smooth muscle cells and nitrergic neurons, and NK3 receptors on enteric neurons are involved.

Elevated levels of oxidative DNA damage and DNA repair enzymes in human atherosclerotic plaques.

BACKGROUND: The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated whether reactive oxygen species-induced oxidative damage to DNA occurs in human atherosclerotic plaques and whether this is accompanied by the upregulation of DNA repair mechanisms. METHODS AND RESULTS: We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques contained 160+/-29 8-oxo-dG residues/10(5) dG versus 3+/-1 8-oxo-dG residues/10(5) dG in mammary arteries. Single-cell gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (>90%). This suggests that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohistochemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear antigen. CONCLUSIONS: Our findings provide evidence that oxidative DNA damage and repair increase significantly in human atherosclerotic plaques.

Long-term treatment with the NO-donor molsidomine reduces circulating ICAM-1 levels in patients with stable angina.

Recent clinical evidence has indicated that the severity of atherosclerosis is correlated with the level of soluble ICAM-1 (sICAM-1). Nitric oxide (NO) donors are used to treat patients with stable angina pectoris, and the aim of this study was to investigate the short- and long-term effect of molsidomine on the level of this circulating biochemical marker of endothelial function. We included 172 patients and examined the effect of the NO donor treatment on angina related parameters and on sICAM-1 levels after a 4-week- and a 1-year treatment period. After 4 weeks, angina attacks and sublingual (s.l.) isosorbide dinitrate tablet (ISDN) consumption frequency was significantly (p<0.0001) reduced without altering sICAM-1 levels when compared to the baseline values. The anti-anginal effect of molsidomine 16 mg once a day (o.a.d.) was sustained (s.l. ISDN consumption) or improved (angina attacks frequency; p<0.002) during the following year and a significant decrease in sICAM-1 levels (p<0.0001) was observed. When the sICAM-1 changes during the 1-year treatment period were distributed in four categories (quartiles of the distribution), it was demonstrated that the decrease in s.l. ISDN consumption between the start and the end, was most pronounced in the group with the largest sICAM-1 decrease (fourth quartile of distribution; p=0.038). In conclusion, the reduction in the pro-inflammatory marker sICAM-1 after 1-year daily treatment with molsidomine may indicate that this NO donor besides its anti-anginal function, promotes a less activated state of the endothelium in patients with stable angina.

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum.

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.