Reproducible determination of transpulmonary pressures.

Oesophageal pressures, as measured in an oesophageal balloon catheter, are a validated substitute for pleural pressures. Transpulmonary pressures, indispensable to improve our understanding of ventilatory physiology, are therefore typically calculated as the difference between airway and oesophageal pressures. The oesophageal pressure signal, however, features a superimposed oscillation due to cardiac motion, not representative for pleural pressure. Additionally, oesophageal contractions or surgical manipulation can alter the signal. In practice, transpulmonary pressures are therefore manually determined from the pressure-time graphic by visual inspection of the waves and averaging a limited number of samples. We suggest an approach to extract the end-expiratory transpulmonary pressure from the raw monitoring data.•Our approach reproducibly determines end-expiratory transpulmonary pressures at a given level of set positive end-expiratory pressure at the ventilator.•Our approach ignores surgical disturbance and cardiac oscillations in the oesophageal pressure signal.

[18F]ZCDD083: A PFKFB3-Targeted PET Tracer for Atherosclerotic Plaque Imaging.

PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [18F]ZCDD083 was synthesized, radiolabeled in 17 ± 5% radiochemical yield and >99% radiochemical purity, and formulated for preclinical PET/CT imaging in mice. In vivo stability analysis showed no significant metabolite formation. Biodistribution studies showed high blood pool activity and slow hepatobiliary clearance. Significant activity was detected in the lung 2 h postinjection (pi) (11.0 ± 1.5%ID/g), while at 6 h pi no pulmonary background was observed. Ex vivo autoradiography at 6 h pi showed significant high uptake of [18F]ZCDD083 in the arch region and brachiocephalic artery of atherosclerotic mice, and no uptake in control mice, matching plaques distribution seen by lipid staining along with PFKFB3 expression seen by immunofluorescent staining. In vivo PET scans showed higher aortic region uptake of [18F]ZCDD083 in atherosclerotic ApoE-/-Fbn1C1039G+/- than in control mice (0.78 ± 0.05 vs 0.44 ± 0.09%ID/g). [18F]ZCDD083 was detected in aortic arch and brachiocephalic artery of ApoE-/- (with moderate atherosclerosis) and ApoE-/-Fbn1C1039G+/- (with severe, advanced atherosclerosis) mice, suggesting this tracer may be useful for the noninvasive detection of atherosclerotic plaques in vivo.

[Frequency and nature of drug related problems with corticosteroids in Belgian community pharmacies]. / Fréquence et nature des problems liés aux corticosteroïdes dans les officines publiques de Belgique.

Aim Aims are: 1] Identify causes of Drug Related Problems (DRPs), interventions performed by pharmacists and results of corticosteroid- related problems and 2] distinguish between problems related to inhaled and general corticosteroids. Methods During 5 days of their internship, 534 final year students of pharmaceutical sciences in six Belgian universities collected DRPs encountered in community pharmacies, as well as related interventions performed by pharmacists and the result of the intervention. The DRPs' electronic registration was done through an adapted tool for Belgium based on the classification of Pharmaceutical Care Network Europe [PCNE- v 6.2]. Findings The frequency of DRPs is 24,8%. 766 DRPs (4,8%) related to corticosteroids, of which 351 were inhaled corticosteroids. The most common causes of corticosteroid-related problems (53- 59%) were technical causes. The most represented category of clinical causes was the inappropriate choice of drug [33-41%]. Pharmacists' intervention was similar for inhaled and general corticosteroids. Pharmacists intervened orally with patients in 38-40% of total interventions, and in writing in 16% of interventions. Pharmacists did not react in 16% of corticosteroid-related problems. 81-83% of PLMS were resolved partially or completely. Conclusion In conclusion, DRPs detected in community pharmacies related to corticosteroid are infrequent (4,8% of DRPs) but 82% of detected problems have been resolved. Furthermore, the study shows the importance for the Belgian health system to introduce an official DRPs classification and software facilitating their documentation in community pharmacies.

Fibrillin-1 impairment enhances blood-brain barrier permeability and xanthoma formation in brains of apolipoprotein E-deficient mice.

We recently reported that apolipoprotein E (ApoE)-deficient mice with a mutation in the fibrillin-1 gene (ApoE(-/-)Fbn1(C1039G+/-)) develop accelerated atherosclerosis with enhanced inflammation, atherosclerotic plaque rupture, myocardial infarction and sudden death. In the brain, fibrillin-1 functions as an attachment protein in the basement membrane, providing structural support to the blood-brain barrier (BBB). Here, we investigated whether fibrillin-1 impairment affects the permeability of the BBB proper and the blood-cerebrospinal fluid barrier (BCSFB), and whether this leads to the accelerated accumulation of lipids (xanthomas) in the brain. ApoE(-/-) (n=61) and ApoE(-/-)Fbn1(C1039G+/-) (n=73) mice were fed a Western-type diet (WD). After 14 weeks WD, a significantly higher permeability of the BBB was observed in ApoE(-/-)Fbn1(C1039G+/-) mice compared to age-matched ApoE(-/-) mice. This was accompanied by leukocyte infiltration, enhanced expression of pro-inflammatory cytokines, matrix metalloproteinases and transforming growth factor-ß, and by decreased expression of tight junction proteins claudin-5 and occludin. After 20 weeks WD, 83% of ApoE(-/-)Fbn1(C1039G+/-) mice showed xanthomas in the brain, compared to 23% of their ApoE(-/-) littermates. Xanthomas were mainly located in fibrillin-1-rich regions, such as the choroid plexus and the neocortex. Our findings demonstrate that dysfunctional fibrillin-1 impairs BBB/BCSFB integrity, facilitating peripheral leukocyte infiltration, which further degrades the BBB/BCSFB. As a consequence, lipoproteins can enter the brain, resulting in accelerated formation of xanthomas.

[Drug related problems in Belgian community pharmacies]. / Les problèmes liés aux médicaments dans les officines belges.

INTRODUCTION: The identification, the management and if possible the prevention of drug related problems (DRP), are the main responsibilities of pharmacists. AIM: The aims of the study were 1/to investigate the frequency and nature of drug related problems detected by community pharmacists, 2/to inventories the frequency and nature of the interventions by community pharmacists on prescribed medicines, and 3/to evaluate whether there is a difference between DRP detection at the moment of dispensing versus in a quiet setting (a posteriori detection). METHOD: All trainees of the participating universities of Belgian were asked to contribute to a observational study. Participating pharmacists quantified DRP's and their interventions on prescribed medicines for 5 days. Registrations were made by using a web tool based on an adapted version of the classification list of PCNE. The registration took place in two phases, at the time of delivery as well as in an a posteriori verification of the prescriptions with the pharmaceutical record file of the patients. RESULTS: The study was conducted from November 2012 to April 2013 in 534 community-pharmacies with internship. During this period 9.869 prescriptions (15%) with at least one DRP were detected on a total of 64.962 prescriptions treated by tutor pharmacists. Since there could be more than one problem on a prescription, 15.952 DRP's were registered. 2.597 of the DRP's were detected by a posteriori verification. 75% of all problems had a technical cause and 37% were clinical in nature. Under the technical causes an incomplete prescription was the most common. The most frequently registered clinical causes were a drug interaction, an inopportune time of intake, a too high or too low dose and an unsuitable drug. Participating pharmacists solved almost 3 of the 4 detected DRP's. In more than half of the DRP's, the patient was verbally and/or written informed. In 44% of the a posteriori discovered problems, the pharmacist intervened. CONCLUSION: Pharmacist detected one or more DRP's with 15% of the prescriptions. Analysis of a prescription prior to dispensing the medicines therefore appears necessary. The active intervention of the pharmacist in 83% of the problems indicates that he contributes to the optimization of drug therapy with a potential increase in the quality of life of the patient and a reduction in the cost of healthcare. The a posteriori discovered DRP's demonstrate the need for pharmacist lead meditation reviews possibly together with the physician and/or patient.

[Opinions and attitudes of Flemish pharmacists and general practitioners towards INN prescribing. A survery in Antwerp and East Flanders]. / Avis et attitudes des pharmaciens et des médecins généralistes vis-à-vis de la prescription en DCI. Enquête réalisée à Anvers et en Flandre Orientate.

INTRODUCTION: Since 2002 in Belgium, physicians are allowed to prescribe by International Non-proprietary Name (INN). In 2005, the conditions for this decree were set. Examples from other countries have shown that INN prescribing can significantly contribute to controlling pharmaceutical expenditures. The share of INN prescriptions remains low in Belgium (7% in 2011). OBJECTIVE: To formulate an answer to the question: what are the opinions and attitudes of pharmacists and general practitioners [GP's] with regards to INN prescribing? METHOD: In the winter of 2011-2012, a questionnaire with closed-ended questions was send to pharmacists and GP's in the provinces of Antwerp and East-Flanders, through training days and personal visits. Pharmacists and GP's scored a list of statements with a 5-point Likert scale. The themes of the statements related to: delivering INN prescriptions, legislation, impact on expenditures, choices regarding patient concerns and interprofessional relations. RESULTS: In total, 353 questionnaires were completed and returned of which 228 165%1 were by pharmacists and 125 (35%1 by GP's. Although both declared to be sufficiently up to date with regulations to prescribe (84%) or to deliver (95%] a INN prescription, only 13% of the pharmacists said all prescription they receive contain the correct information. Less GP's [36%) than pharmacists (82%] feel aided by their software program when prescribing or delivering an INN prescription. GP's rely mostly on NIHDI (National Institute for Health and Disability Insurance) as the main source for information on INN prescribing, pharmacists rely on the [Local) pharmacists association. The pharmacists and GP's in the study who relied on NIHDI as main information source, were less aware of legislation concerning INN [N2, p<0,05] than those who rely on the local professional association [N2, p<0,0001]. All pharmacists in the study said to consider the patients medication history when delivering an INN prescription for chronic treatment. However, 57% of the GP's preferred not to prescribe by INN for the reason that they are not sure whether the pharmacist will always consider the patients medication history in case of an INN prescription. Although the GP's showed certain motivation to prescribe by INN, it was no greater than for generic prescribing. And INN prescribing has no added value compared to generic prescribing, according to the GP's. For the pharmacists, INN prescribing does contain an opportunity. With the increase in numbers of dosages and sorts of packaging of generic products, it becomes more and more difficult for pharmacists to manage their stock. In case of an INN prescription, the pharmacist can choose between the different packages in his stock. This offers opportunities especially for acute conditions. CONCLUSION: INN prescribing is a good example of where the collaboration between pharmacists and GP's still contains a lot of opportunities, as well for the two professions, as the government and the patient in terms of controlling the pharmaceutical expenditures. Also the education for pharmacist or GP can further contribute to the sensitization of INN prescribing. In practice, there remain a number of issues and differences in opinions between pharmacists and general practitioners regarding INN prescribing. GP's feel few motivation to prescribe by INN and the government has put no imperative demands towards prescribers. Further evaluation of the practicaL feasibility of the current conditions for prescribing and delivering INN prescriptions is needed.

Potential impact of policy regulation and generic competition on sales of cholesterol lowering medication, antidepressants and acid blocking agents in Belgium.

BACKGROUND: Pharmaceutical expenditures are increasing as a proportion of health expenditures in most rich countries. Antidepressants, acid blocking agents and cholesterol lowering medication are major contributors to medicine sales around the globe. METHODS: We aimed to document the possible impact of policy regulations and generic market penetration on the evolution of sales volume and average cost per unit (Defined Daily Doses and packages) of antidepressants, acid blocking agents and cholesterol lowering medication. We extracted data from the IMS health database regarding the public price and sales volume of the antidepressants (selective serotonin reuptake inhibitors (SSRI's), monoamine oxidase inhibitors (MAOl's) and tricyclic and remaining antidepressants (TCA's)), acid blocking agents (proton pump inhibitors (PPl's) and H2 receptor antagonists) and cholesterol lowering medication (statins and fibrates) in Belgium between 1995 and 2009. We describe these sales data in relation to various national policy measures which were systematically searched in official records. RESULTS: Our analysis suggests that particular policy regulations have had immediate impact on sales figures and expenditures on pharmaceuticals in Belgium: changes in reimbursement conditions, a public tender and entry of generic competitors in a reference pricing system. However, possible sustainable effects seem to be counteracted by other mechanisms such as marketing strategies, prescribing behaviour, brand loyalty and the entry of pseudogenerics. It is likely that demand-side measures have a more sustainable impact on expenditure. CONCLUSION: Compared with other European countries, generic penetration in Belgium remains low. Alternative policy regulations aimed at enlarging the generic market and influencing pharmaceutical expenditures deserve consideration. This should include policies aiming to influence physicians' prescribing and a shared responsibility of pharmacists, physicians and patients towards expenditures.

[Critical evaluation of interaction screening software available in pharmacies]. / Evaluation critique des logiciels d'interactions médicamenteuses a l'officine.

Due to the increasing complexity of medication regimens it is not always easy for the pharmacist to quickly and effectively screen the drug use of a particular patient for interactions. By means of a survey and a comparison, the advantages and disadvantages of the most common software packages available in Flanders were analysed. Major stumbling blocks of the currently available software are the high number of false positive signals, the absence of a history regarding the management of interactions, the lack of timely updates of the database and the absence of clear guidelines for the management of an interaction. Based on this research, we make the following recommendations: (1) signal fatigue should be reduced by interaction screening based on the duration of therapy in addition to the ability to suppress signals, (2) a log, coupled with the prescription-register, should be implemented, (3) software companies should help pharmacists more in configuring software preferences and provide them with better information bout the available options, (4) the underlying databases must be updated more quickly. (5) OTC medications, especially in the context of polypharmacy, should be registered in the patient record by the pharmacist, (6) note that food supplements are not included in the interaction screening software, unlike registered medication. (7) the knowledge of pharmacists regarding interactions should be maintained and improved.

Nitric oxide selectively depletes macrophages in atherosclerotic plaques via induction of endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism. EXPERIMENTAL APPROACH: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis. KEY RESULTS: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin. CONCLUSIONS AND IMPLICATIONS: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.

Processing of amyloid precursor protein as a biochemical link between atherosclerosis and Alzheimer's disease.

Macrophage activation in atherosclerotic plaques plays a role in plaque destabilization, rupture and subsequent atherothrombosis. Platelet phagocytosis that occurs within human atherosclerotic plaques can activate macrophages and it has been suggested that the platelet constituent amyloid precursor protein (APP) is involved. Recent studies show that amyloid beta (Abeta), a peptide extensively studied in Alzheimer's disease and that is cleaved from APP by beta- and gamma-secretase, and/or Abeta-like peptides are also present in human atherosclerotic plaques, in particular in activated, inducible nitric oxide synthase (iNOS) expressing perivascular macrophages that had phagocytized platelets. In vitro studies confirm that platelet phagocytosis leads to macrophage activation and suggest that platelet-derived APP is proteolytically processed to Abeta-like peptides, resulting in iNOS induction. In addition, non-steroidal anti-inflammatory drugs (NSAIDs) and HMG-CoA reductase inhibitors (statins), two classes of drugs reported to affect APP processing and Abeta formation in Alzheimer's disease, have been evaluated for their capacity to inhibit macrophage activation evoked by platelet phagocytosis. Remarkably, the same NSAIDs reported to alter gamma-secretase activity in Alzheimer's disease also reduce macrophage activation after platelet phagocytosis and inhibit formation of Abeta-containing peptides. From the statins investigated (fluvastatin, atorvastatin, simvastatin, pravastatin, lovastatin and rosuvastatin) only fluvastatin and atorvastatin selectively inhibit macrophage activation after platelet phagocytosis, possibly through inhibition of Rho activity. Taken together, these new findings point to the involvement of platelet-derived APP in macrophage activation in atherosclerosis and suggest a biochemical link between atherosclerosis and Alzheimer's disease. Accordingly, drugs interfering with APP processing might have an impact on both diseases.

Reactive oxygen species induce RNA damage in human atherosclerosis.

BACKGROUND: Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. MATERIALS AND METHODS: The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. RESULTS: Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. CONCLUSION: Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking.

Oxidative DNA damage and repair in experimental atherosclerosis are reversed by dietary lipid lowering.

Increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis. The oxidative environment is mainly created by the production of reactive oxygen species, which are assumed to mediate vascular tissue injury. Oxidative DNA damage resulting from free radical attack remains, however, a poorly examined field in atherosclerosis. Male New Zealand White rabbits were fed a cholesterol-rich diet (0.3%) for 24 weeks. The induced atherosclerotic plaques showed elevated levels of the DNA damage marker 7,8-dihydro-8-oxoguanine (8-oxoG) as demonstrated by immunohistochemistry. 8-oxoG immunoreactivity was found predominantly in the superficial layer of the plaque containing numerous macrophage-derived foam cells but not in the media or in arteries of age-matched control animals. Alkaline single-cell gel electrophoresis revealed that the number of DNA strand breaks was significantly higher in the plaque as compared with control samples of normolipemic animals. These changes were associated with the upregulation of DNA repair enzymes (poly[ADP-ribose] polymerase-1, p53, phospho-p53 [phosphorylated at Ser392], and XRCC1 [x-ray repair cross-complementing 1]). DNA strand breaks normalized after 4 weeks of dietary lipid lowering. However, a significant reduction of 8-oxoG immunoreactivity was only observed after a prolonged period of lipid lowering (12 to 24 weeks). Repair pathways started to decline progressively when cholesterol-fed animals were placed on a normal diet. In conclusion, oxidative DNA damage and increased levels of DNA repair, both associated with diet-induced hypercholesterolemia, are strongly reduced during dietary lipid lowering. These findings may provide a better insight into the benefits of lipid-lowering therapy on plaque stabilization.

Periadventitial inducible nitric oxide synthase expression and intimal thickening.

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 microL. h(-1), 14 days, n=12) 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)adventitial tissue and counteracted the progression of intimal thickening. Ox-LDL treatment was accompanied by an abundant influx of iNOS-positive macrophages and T lymphocytes in the vessel, but this could not prevent the progression of intimal thickening.

Collar-induced elevation of mRNA and functional activity of 5-HT(1B) receptor in the rabbit carotid artery.

Hypersensitivity to serotonin (5-HT) develops in rabbit collared carotid arteries. Previous data demonstrated the involvement of 5-HT(1)-like receptors which are not active in normal carotid arteries. This study investigated the interaction in the rabbit carotid artery between 5-HT and a moderate tone as this can uncover functional 5-HT(1)-like receptors. Furthermore, the expression of messenger RNA (mRNA) and protein of 5-HT(1B), 5-HT(1D) and 5-HT(2A) receptors was addressed. Silicone collars were placed around the carotid arteries of male New Zealand White rabbits for 1 week. Rings from inside (=collar) and outside (=sham) the collar were either mounted in isolated organ baths for isometric force measurements or frozen in liquid nitrogen to isolate total RNA or proteins which were subsequently analysed by respectively reverse transcriptase-polymerase chain reaction and Western blot analysis. In sham and collared rings concentration-response curves (CRC's) to 5-HT were monophasic. Only in collared segments the presence of a 5-HT(2A) antagonist (spiperone or ketanserin, 0.1 microM) revealed a biphasic CRC which was even more pronounced when a moderate tone was induced by KCl pointing to functional 5-HT(1)-like receptors. The rabbit carotid artery constitutively expressed 5-HT(1B) and 5-HT(2A) mRNA, not 5-HT(1D) mRNA. Manipulation of the carotid artery increased the 5-HT(1B) mRNA level. Collar placement raised it even further. The 5-HT(2A) mRNA level remained unchanged. All the anti-5-HT receptor antibodies tested resulted in variable, non specific patterns with multiple bands. In conclusion, collar placement elevates mRNA expression and activity of the 5-HT(1B) receptor in the rabbit carotid artery.

Intimal deposition of functional von Willebrand factor in atherogenesis.

During the formation of intimal thickening in normocholesterolemic rabbits, von Willebrand factor (vWF) is increased in the endothelial cells (ECs) and deposited in the intima. We investigated whether this also occurs during cholesterol-induced plaque formation, whether the synthesis of vWF increases, and whether this influences platelet adhesion. Rabbits were fed a cholesterol-rich (0.3%) diet for 26 weeks. Thereafter, half of the animals received a normal diet for another 26 weeks (cholesterol withdrawal). To induce intimal thickening in normocholesterolemic rabbits, collars were positioned around the carotid artery. Arterial segments were studied using immunohistochemistry, reverse transcription-polymerase chain reaction, electron microscopy, and platelet adhesion tests. Cholesterol treatment induced plaque formation in the aorta. The ECs had a cuboidal aspect, showed a dense immunoreactivity for vWF, a pronounced rough endoplasmic reticulum, and numerous Weibel-Palade bodies. There were subendothelial vWF deposits in the plaques and vWF mRNA was significantly increased as compared with controls. Similar changes were seen after collar-induced intimal thickening. After cholesterol withdrawal, both vWF mRNA and the ultrastructural morphology of the ECs normalized, and the vWF deposits disappeared from the plaque. Perfusion studies with anticoagulated rabbit blood over cross-sections of atherosclerotic aortas revealed increased vWF-mediated platelet adhesion in the subendothelial plaque region. Whereas rabbit platelets perfused through the lumen adhered to the same extent to de-endothelialized aortas of normocholesterolemic and atherosclerotic rabbits, vWF mediated platelet adhesion to endothelium was observed in atherosclerotic but not in normal aortas. Our results show an increased synthesis and (sub)endothelial presence of vWF after vascular injury, with functional consequences for platelet deposition on the vessel wall.

Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques.

OBJECTIVE: Advanced human atherosclerotic plaques are characterized by the abundant presence of the autofluorescent non-soluble lipid pigment ceroid, consisting of oxidized lipoproteins. The aim of the present study was to examine the topographical and cellular distribution of inducible nitric oxide synthase (iNOS or NOS II) within different stages of atherosclerosis and its colocalization with ceroid deposits and nitrotyrosine. METHODS AND RESULTS: Different stages of atherosclerosis were studied by immunohistochemistry on whole-mount longitudinal sections of carotid endarterectomy specimens. In the adaptive intimal thickening the predominant cell type were smooth muscle cells. The fatty streaks contained both smooth muscle cells and macrophages with an extremely low NOS II immunoreactivity. The advanced atherosclerotic plaques however, showed a very dense infiltration by macrophages, of which a subpopulation expressed NOS II as a vesicular immunoreactivity in their cytoplasm. These were mainly present around the necrotic core, in association with ceroid accumulation and nitrotyrosine. Fluorescence quenching microscopy showed the presence of NOS II on autofluorescent ceroid vesicles in the macrophages. Large extracellular ceroid granules were not NOS II immunoreactive. NOS II mRNA was detected by RT-PCR and the protein by Western blot in the plaque tissue but not in mammary arteries used as controls. CONCLUSION: 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.

Cell composition, replication, and apoptosis in atherosclerotic plaques after 6 months of cholesterol withdrawal.

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.