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Hidde Bult

Publications and source records attributed to Hidde Bult.

16 recordsLinked to original sources
PubMed2006

Endothelium-dependent relaxation evoked by ATP and UTP in the aorta of P2Y2-deficient mice.

Based on pharmacological criteria, we previously suggested that in the mouse aorta, endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (adenosine diphosphate (ADP)), P2Y2 (adenosine triphosphate (ATP)) and P2Y6 (uridine diphosphate (UDP)) receptors. For UTP, it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists, we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild-type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice, indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and adenosine 5'[gamma-thio] triphosphate (ATPgammaS)-evoked relaxation, suggesting that in WT mice, ATP and ATPgammaS activate predominantly the P2Y2 subtype. The ATP/ATPgammaS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, as they were sensitive to 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS2179), a P2Y1-selective antagonist. In contrast to ATP, the UTP-evoked relaxation was not different between knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6(like) receptor subtype. In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6(like) receptor.

Adenosine Diphosphate↗
PubMed2006

Decreased number of circulating plasmacytoid dendritic cells in patients with atherosclerotic coronary artery disease.

BACKGROUND: Dendritic cells are potent antigen-presenting and immune modulating cells that have been implicated in the development of atherosclerosis. In human blood, two distinct lineages are distinguished: plasmacytoid dendritic cells and myeloid dendritic cells. Although dendritic cells have been described in atherosclerotic plaques, no information exists concerning circulating blood dendritic cells in atherosclerosis. This study aims to evaluate the number of circulating dendritic cells in patients with coronary artery disease. The relation with the extent of coronary artery disease, the clinical syndrome and with a marker of inflammation will be documented. METHODS: Patients with angiographically proven coronary artery disease (n=18) and age and sex-matched controls (n=18) were included. Myeloid dendritic cells and plasmacytoid dendritic cells were detected with the specific blood dendritic cell antigens, blood dendritic cell antigen-1 and blood dendritic cell antigen-2, respectively. RESULTS: Absolute and relative numbers of circulating plasmacytoid dendritic cells were significantly lower in patients with coronary artery disease (5722+/-601/ml and 0.08+/-0.01%) than in controls (12,640+/-1289/ml and 0.21+/-0.02%). Plasmacytoid dendritic cells were more decreased in patients with troponin-positive unstable coronary syndromes than in patients with low troponin values, and tended to be lower in more extensive coronary artery disease. Absolute myeloid dendritic cells numbers tended to be reduced in patients, whereas relative numbers were significantly decreased: 11,857+/-1895/ml versus 15,226+/-928/ml and 0.17+/-0.03% versus 0.26+/-0.01% in controls. CONCLUSIONS: The present study shows a significant decrease of circulating blood dendritic cell antigen-2 positive plasmacytoid dendritic cells in patients with coronary artery disease. The decrease tended to be more pronounced in unstable coronary syndromes and extensive coronary artery disease, suggesting a possible role of dendritic cells in plaque progression and rupture.

Adult↗
PubMed2005

Effects of cytochalasin D-eluting stents on intimal hyperplasia in a porcine coronary artery model.

OBJECTIVE: To investigate whether cytochalasin D-eluting stents (CDES) suppress intimal hyperplasia in porcine coronary arteries and to compare the efficacy of paclitaxel and cytochalasin D as inhibitors of vascular smooth muscle cell (SMC) proliferation and platelet aggregation in vitro. METHODS: Rabbit platelet-rich plasma and SMC cultures derived from rabbit aortas were exposed to 10(-8)-10(-5) M cytochalasin D or paclitaxel. Stents directly coated with 2 microg cytochalasin D (low-dose CDES, n=12) and bare stents (n=12) were randomly deployed in the right and left coronary artery of 12 pigs. Six weeks later, neointima was studied using quantitative coronary angiography (QCA) and morphometry. To examine a ten-fold higher dose, polybutyl methacrylate/polyvinyl acetate-coated stents were loaded with 20 microg cytochalasin D. High-dose CDES (n=10) and polymer-only stents (n=11) were deployed in 11 pigs. RESULTS: After 7 days, cytochalasin D (IC(50) 9.9+/-0.4 10(-8) M) and paclitaxel (IC(50) 1.1+/-0.4 10(-8) M) inhibited SMC proliferation in vitro (n=4). In contrast, cytochalasin D (10(-6)-10(-5) M, n=5), but not paclitaxel, attenuated platelet shape change and aggregation induced by ADP. In vivo QCA showed less late lumen loss in low-dose CDES (0.08+/-0.07 vs. 0.32+/-0.08 mm, P=0.05), but morphometry demonstrated only a tendency toward a decreased intimal area. High-dose CDES inhibited both late lumen loss (0.31+/-0.08 vs. 0.91+/-0.06 mm, P<0.01) and intimal area (1.57+/-0.20 vs. 2.46+/-0.22 mm(2), P<0.01). Immunohistochemistry revealed that CDES suppressed peri-strut macrophage recruitment (CD68, P=0.04) and cell proliferation (Ki67, P=0.03) as compared to polymer-only stents without interfering with endothelial cell recovery or the density of alpha-SMC actin staining. Thromboses or edge effects were not observed in either study. CONCLUSIONS: CDES inhibited in-stent hyperplasia. The reduction (39%) with 20 mug CDES was equivalent to that reported for paclitaxel-eluting stents in pigs. Interference with platelet aggregation, SMC migration, SMC proliferation, and leukocyte recruitment could contribute to the benefit. The data indicate that targeting of actin microfilaments has a potential to suppress in-stent restenosis.

Animals↗
PubMed2005

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta.

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5' triphosphate (ATP), uridine 5' triphosphate (UTP), uridine 5' diphosphate (UDP); >90%) or partial (adenosine 5' diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP approximately UTP approximately ADP>adenosine 5'-[gamma-thio] triphosphate (ATPgammaS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53+/-0.07) was compatible with literature, but the pKb for UTP (5.19+/-0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2'-deoxy-N6-methyladenosine3',5'-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation.

Animals↗
PubMed2004

7-Ketocholesterol induces reversible cytochrome c release in smooth muscle cells in absence of mitochondrial swelling.

OBJECTIVE: 7-Ketocholesterol, a major oxysterol in oxidized low-density lipoproteins in advanced atherosclerotic plaques, induces vascular smooth muscle cell (SMC) death. We investigated whether cytochrome c release participated in SMC death induced by 7-ketocholesterol and whether the processes were reversible. METHODS: SMC cultures derived from the rabbit aorta were exposed to 25 microM 7-ketocholesterol. Cytochrome c and Bax were studied by means of immunofluorescence and immunoblotting, apoptosis by the TUNEL technique and mitochondrial structure by transmission electron microscopy. RESULTS: 7-Ketocholesterol induced rapid upregulation of the proapoptotic protein Bax and its translocation from cytosol into the mitochondria (4 h). This was followed by mitochondrial cytochrome c release (65% at 8 h) into the cytosol, which was almost complete at 16 h. The mitochondria became spherical and ultracondensed, without showing signs of lysis. They clustered around the nucleus and were wrapped by wide cisternae of the rough endoplasmic reticulum. Cytochrome c release was not blocked by the pan-caspase inhibitor zVAD-fmk, in contrast to DNA fragmentation and SMC loss. Interestingly, upon removal of 7-ketocholesterol after 16 h and re-exposure to serum for 24 h, the mitochondrial cytochrome c content, their transmembrane potential and TUNEL labelling normalised and SMC loss decreased. However, none of these cell death markers was rescued when the SMCs had been exposed to the oxysterol for 24 h. CONCLUSION: The results indicate that cytochrome c release during oxysterol-induced SMC apoptosis is not caspase-dependent and occurs as a result of a reversible mitochondrial conformational change rather than swelling and rupture of the outer membrane. The reversibility of these events suggests that the apoptotic cascade could be arrested before a point of no return.

Animals↗
PubMed2004

Effect of non-steroidal anti-inflammatory drugs on amyloid-beta formation and macrophage activation after platelet phagocytosis.

Recently, we showed that platelet phagocytosis occurs in human atherosclerotic plaques and leads to foam cell formation. Platelet phagocytosis, resulting in macrophage activation and iNOS induction, was associated with the formation of amyloid-beta peptide (Abeta) via proteolytic cleavage of platelet-derived amyloid precursor protein (APP), possibly by secretases. To test the involvement of gamma-secretase in this process, we used indomethacin, ibuprofen, and sulindac sulfide, non-steroidal anti-inflammatory drugs (NSAIDs) known to alter the gamma-secretase cleaving site of APP, on their ability to inhibit macrophage activation evoked by platelet phagocytosis. J774 macrophages were incubated with human platelets or lipopolysaccharide (LPS) with or without NSAIDs. Nitrite was quantified as a measure for inducible nitric oxide synthase (iNOS) activity. Indomethacin, ibuprofen, sulindac sulfide, and meloxicam concentration-dependently reduced nitrite production by macrophages incubated with platelets, but did not alter LPS-induced iNOS activity or platelet uptake. However, acetylsalicylic acid and naproxen, two NSAIDs without effect on the gamma-secretase cleaving site of APP, did not affect nitrite production in either platelet- or LPS-stimulated macrophages. Surface-enhanced laser desorption/ionization time-of-flight mass-spectrometry demonstrated time-dependent formation of Abeta-containing peptides after platelet phagocytosis, which could be inhibited by indomethacin. In conclusion, these results point to the involvement of gamma-secretase in macrophage activation following platelet phagocytosis.

Amyloid beta-Peptides↗
PubMed2003

Plaque-associated endothelial dysfunction in apolipoprotein E-deficient mice on a regular diet. Effect of human apolipoprotein AI.

OBJECTIVE: Apolipoprotein E-deficient mice (apoE(-/-)) on a regular diet become hypercholesterolemic and develop atherosclerosis, but endothelium-dependent relaxation remains undisturbed for up to 6 months. We investigated whether vasomotor dysfunction develops in aged apoE(-/-), whether the defect was systemic (hypercholesterolemia-dependent) or focal (plaque-related), and the effect of human apolipoprotein AI transgenesis (apoAI/E(-/-)). METHODS: Arteries of apoE(-/-) (n=5), apoAI/E(-/-) (n=6) and C57Bl/6J (WT, n=4) mice (18 months) were systematically dissected for isometric tension recording and subsequent morphometry. RESULTS: Acetylcholine (ACh)-induced relaxation was impaired (P<0.01) in atherosclerotic segments of apoE(-/-) (26+/-14%) as compared to WT mice (93+/-2%). Similar reduced (P<0.01) responses to adenosine 5'-triphosphate (apoE(-/-) 38+/-14, WT 94+/-3%) and the calcium ionophore A23187 (apoE(-/-) 19+/-6%, WT 97+/-2%) pointed to a post-receptor defect. Indeed, responses to exogenous nitric oxide were impaired in atherosclerotic segments as well (apoE(-/-) 71+/-7%, WT 92+/-1%, P<0.05). Furthermore, relaxations inversely correlated with plaque size (ACh r(s)=-0.74, P<0.01). In adjacent plaque-free segments however, responses to ACh (apoE(-/-) 92+/-3%, WT 97+/-1%) and all other agents were preserved, despite the prolonged hypercholesterolemia. ApoAI improved vasomotor responses in atherosclerotic segments. However, negative correlations between maximal relaxation and plaque area remained in apoAI/E(-/-) mice (ACh r(s)=-0.67, P<0.01). Indeed, covariate analysis of variance did not point to direct protection of vasomotor function by apoAI when the smaller lesions were taken into account. CONCLUSIONS: Endothelial dysfunction in apoE(-/-) mice is not affected by hypercholesterolemia alone, but is strictly associated with plaque formation. Human apoAI transgenesis-known to raise HDL-attenuated atherogenesis, thereby indirectly improving relaxation responses in apoE(-/-) mice.

Animals↗
PubMed2003

Phagocytosis and macrophage activation associated with hemorrhagic microvessels in human atherosclerosis.

OBJECTIVE: Previously, we demonstrated that activated inducible NO synthase (iNOS)-expressing foam cells in human carotid plaques often produce autofluorescent (per)oxidized lipids (ceroid). Here, we investigate whether intraplaque microvessels can provide foam cells with lipids and trigger macrophage activation. METHODS AND RESULTS: Microvessels (von Willebrand factor [vWf] immunoreactivity), activated macrophages (iNOS immunoreactivity), and ceroid were systematically mapped in longitudinal sections of 15 human carotid endarterectomy specimens. An unbiased hierarchical cluster analysis classified vascular regions into 2 categories. One type with normal vWf expression and without inflammatory cells was seen, and another type with cuboidal endothelial cells, perivascular vWf deposits, and iNOS and ceroid-containing foam cells was seen in 4 (27%) of 15 plaques. The perivascular foam cells frequently contained platelets (glycoprotein Ibalpha) and erythrocytes (hemoglobin, iron), pointing to microhemorrhage/thrombosis and subsequent phagocytosis. Similar lipid-containing cells, expressing both ceroid and iNOS, were generated in atherosclerosis-free settings by incubating murine J774 macrophages with platelets or oxidized erythrocytes and also in vivo in organizing thrombi in normocholesterolemic rabbits. CONCLUSIONS: Focal intraplaque microhemorrhages initiate platelet and erythrocyte phagocytosis, leading to iron deposition, macrophage activation, ceroid production, and foam cell formation. Neovascularization, besides supplying plaques with leukocytes and lipoproteins, can thus promote focal plaque expansion when microvessels become thrombotic or rupture prone.

Aged↗
PubMed2003

Nitric oxide donor molsidomine favors features of atherosclerotic plaque stability during cholesterol lowering in rabbits.

Rupture-prone atherosclerotic plaques are characterized by a thin fibrous cap containing numerous macrophage-derived foam cells and few smooth muscle cells (SMC). Decreasing the ratio between macrophages and SMC might favor plaque stabilization. Macrophages expressing inducible nitric oxide (NO) synthase become hypersensitive to killing by exogenous NO donors. Therefore, we investigated in cholesterol-fed rabbits (20 weeks 0.3% cholesterol) the effect of 4 weeks cholesterol withdrawal alone and in combination with the NO donor molsidomine on plaque size, cell composition, superoxide production and extracellular superoxide dismutase (ecSOD) mRNA expression in the atherosclerotic plaques in the aorta. Cholesterol withdrawal alone did not alter atherosclerotic plaque size, the increased superoxide production or the decreased ecSOD mRNA, but led to the formation of a thin subendothelial macrophage-free layer and reduced both vascular cell adhesion molecule-1 expression and cell replication in the luminal part of the plaques. Treatment with molsidomine (1 mg/kg/day) during cholesterol withdrawal did not affect plaque size but increased the thickness of the subendothelial macrophage-free layer consisting of SMC, and normalized both superoxide production and ecSOD mRNA expression. The latter findings demonstrate that molsidomine, when combined with cholesterol lowering, decreases signs of oxidative stress and increases features of stable atherosclerotic plaques.

Acetonitriles↗
PubMed2003

Addition of cytochalasin D to a biocompatible oil stent coating inhibits intimal hyperplasia in a porcine coronary model.

BACKGROUND: Polymer-based, drug-eluting stents, are currently under extensive investigation in the conquest against in-stent restenosis. Concern remains, however, about potential long-term lack of biocompatibility of the polymers used in these studies. Therefore, this study aimed to evaluate in porcine coronary arteries (1) the in vivo biocompatibility of a new natural, eicosapentaenoic acid oil stent-coating and (2) the efficacy of this coating in preventing in-stent restenosis when cytochalasin D--an inhibitor of actin filament formation, that interferes with cell proliferation and migration--was added. METHODS AND RESULTS: To assess in vivo biocompatibility of the oil coating, 15 bare and 15 oil-coated stents were randomly deployed in coronary arteries of 15 pigs. No difference in tissue response, regarding inflammation or proliferation, was seen between both groups at five days or at four weeks follow-up. To evaluate the efficacy of the coating in preventing in-stent restenosis by adding a potential anti-restenotic drug, stents were dip-coated in 20 mg cytochalasin D/ml oil solution, resulting in 93 +/- 18 microg cytochalasin D/stent load (n = 3). In vitro drug release studies showed sustained release up to four weeks. Next, 11 oil-coated and 11 cytochalasin D-loaded stents were randomly implanted in coronary arteries of 11 pigs. At four weeks, a 39% decrease in neointimal hyperplasia (p < 0.05, ANCOVA, with injury as covariate) was found in cytochalasin D-loaded stents compared to oil-coated stents. CONCLUSIONS: This new natural oil stent-coating shows excellent biocompatibility to vascular tissue. Local cytochalasin D delivery from this stent-platform significantly inhibits neointimal hyperplasia in a porcine coronary model.

Animals↗
PubMed2002

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.

Adult↗
PubMedDate not supplied

Stereology: a simplified and more time-efficient method than planimetry for the quantitative analysis of vascular structures in different models of intimal thickening.

OBJECTIVE: Semiautomatic computer-assisted planimetry is currently the standard morphometric technique in models of intimal thickening. We evaluated stereological point counting as an alternative method for the measurement of cross-sectional vascular areas (lumen, intima, and media) by comparing precision, efficiency, and variance components. METHODS: Sections from murine atherosclerotic aorta (n = 21), stented rabbit iliac arteries (n = 30), and porcine coronary arteries (n = 30) were analyzed at two institutes using both techniques. To determine reproducibility, porcine arteries were measured twice. RESULTS: Area measurements showed almost identical means and standard deviations for planimetry and stereology [e.g., intima (mm2): 0.10+/-0.11 vs. 0.12+/-0.12 (mouse), 0.60+/-0.16 vs. 0.60+/-0.15 (rabbit), and 1.92+/-1.52 vs. 1.97+/-1.61 (pig)]. Deming regression and Bland-Altman plots demonstrated a good agreement between both techniques that was not influenced by artery size. Both methods exhibited excellent repeatability, although planimetry (-0.18+/-0.27) was more precise than stereology (-0.17+/-0.47; variance, P < .01, Levene test). In addition, intraoperator variance (error inherent to the technique) was greater for stereology (1.6-15.8% vs. 4.8-33.5%), whereas interoperator variance (error between institutes) was very small for both methods (0.1-0.9% vs. 0.1-1.7%). Indeed, biologic variability was, by far, the most important variance component in all measurements (84-98% vs. 65-95%). Finally, stereology required 20% (mouse; P < .05) to 40% (pig; P < .001) less time to complete analysis. CONCLUSION: The quantification of vascular structures by planimetry and stereology yielded comparable results in all models of intimal thickening, but stereology proved to be less time-consuming. Therefore, study design may dictate the most appropriate choice of technique.

Animals↗
PubMedDate not supplied

A three-dimensional quantitative analysis of restenosis parameters after balloon angioplasty: comparison between semi-automatic computer-assisted planimetry and stereology.

Semi-automatic computer-assisted planimetry is often used for the quantification of restenosis parameters after balloon angioplasty although it is a time-consuming method. Moreover, slicing the artery to enable analysis of two-dimensional (2-D) images leads to a loss of information since the vessel structure is three-dimensional (3-D). Cavalieri's principle uses systematic random sampling allowing 3-D quantification. This study compares the accuracy and efficiency of planimetry versus point-counting measurements on restenosis parameters after balloon angioplasty and investigates the use of Cavalieri's principle for 3-D volume quantification. Bland and Altman plots showed good agreement between planimetry and point counting for the 2-D and 3-D quantification of lumen, internal elastic lamina (IEL) and external elastic lamina (EEL), with a slightly smaller agreement for intima and media. Mean values and induced coefficients of variation were similar for both methods for all parameters. Point counting induced a 6% error in its 3-D quantification, which is negligible in view of the biological variation (>90%) among animals. However, point counting was 3 times faster compared to planimetry, improving its efficiency. This study shows that combining Cavalieri's principle with point counting is a precise and efficient method for the 3-D quantification of restenosis parameters after balloon angioplasty.

Angioplasty, Balloon↗