Interactions between cell death induced by statins and 7-ketocholesterol in rabbit aorta smooth muscle cells.

BACKGROUND AND PURPOSE: 7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects. EXPERIMENTAL APPROACH: Cultured rabbit aorta SMCs (passage 2-6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined. KEY RESULTS: NR uptake declined upon 18 h exposure to 25 microM 7-ketocholesterol (-41+/-3%, n=13), 100 microM fluvastatin (-59%) or 30-100 microM simvastatin (-28 to -74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10-30 microM, simvastatin 1-10 microM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10-100 microM, 70-545% increase) and simvastatin (3-100 microM 43-322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 microM was always inactive. CONCLUSIONS AND IMPLICATIONS: 7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings.

Paraoxonase 1 gene transfer lowers vascular oxidative stress and improves vasomotor function in apolipoprotein E-deficient mice with pre-existing atherosclerosis.

BACKGROUND AND PURPOSE: Transgenesis of human paraoxonase 1 (PON1), a HDL-associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E-deficient (apoE(-/-)) mice with advanced atherosclerosis. EXPERIMENTAL APPROACH: ApoE(-/-) mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n=10) or control adenovirus (AdRR5, n=10). Non-transfected apoE(-/-) (n=9) and C57Bl/6J (WT, n=6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta. KEY RESULTS: PON1 gene transfer raised total PON1 serum activity 13-15 fold during the 3-week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque-free thoracic aorta rings from apoE(-/-) mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86+/-2%), ATP (90+/-2%) or UTP (83+/-3%). In contrast, in plaque-bearing segments amplitude (55+/-7%, 68+/-8%, 52+/-8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE(-/-) SMCs released less intracellular calcium than WT upon sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection. CONCLUSIONS AND IMPLICATIONS: These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca(2+) homeostasis in segments with pre-existing atherosclerosis, independently of an effect on plaque size.

Altered Ca2+ handling of smooth muscle cells in aorta of apolipoprotein E-deficient mice before development of atherosclerotic lesions.

To study the effect of hypercholesterolemia on vascular smooth muscle cell (VSMC) function, atherosclerosis-prone but plaque-free endothelium-denuded aortic rings (width 2mm) from C57Bl6 Wild Type (WT) and apolipoprotein E-deficient (apoE(-/-)) mice (age 4 months) were mounted in a myograph and loaded with Fura-2 AM to simultaneously measure free Ca(2+) ([Ca(2+)](i)) and force development. In comparison with WT, apoE(-/-) mice displayed higher basal [Ca(2+)](i). Moreover, the time constant of the second phase of the biphasic high K(+)-induced [Ca(2+)](i) response was significantly increased in apoE(-/-) compared to WT mice. This phase was abolished by treatment with cyclopiazonic acid (CPA), depleting sarcoplasmic reticulum (SR). Further investigation of SR dependent [Ca(2+)](i) handling with CPA and caffeine revealed no alteration of maximal SERCA or ryanodine receptor function. Inositol (1,4,5)-triphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) release was, however, significantly increased in apoE(-/-) mice compared to WT mice as established with phenylephrine and ATP. In Ca(2+)-free conditions the ATP-induced [Ca(2+)](i) was not altered. The ATP-induced store-operated Ca(2+) entry was, however, significantly increased in apoE(-/-) compared to WT mice. The results demonstrate that basal [Ca(2+)](i) levels and IP(3)R-mediated store-operated [Ca(2+)](i) release over the plasma membrane were elevated in hypercholesterolemic but plaque-free apoE(-/-) mice.

Gene expression profiling of LPS-stimulated murine macrophages and role of the NF-kappaB and PI3K/mTOR signaling pathways.

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, activates a broad spectrum of signaling pathways in immune cells. In this article, RAW264.7 cells have been stimulated for 4 h with 1 microg/mL of LPS in the presence or not of specific inhibitors of the NF-kappaB pathway (BAY 11-7082) and the PI3K pathway (LY294002). Gene expression profiles were characterized using the DNA microarray "Dual Chip Mouse Inflammation." This array monitors the expression of 233 genes encoding proteins playing a role in inflammation. Both signaling pathways exert an important role in the response to LPS, but they are not completely overlapping. For example, genes encoding the PAF receptor, PAI-1, PlA2 (group V), IL-13 receptor (alpha2), and GTP cyclohydrolase 1, were upregulated after LPS treatment, but this upregulation was counteracted by LY294002. The same was observed for BAY 11-7082: genes encoding the kit ligand, TLR2, or TNFRSF5 were mainly under the control of NF-kappaB. NF-kappaB plays an important role in the macrophage response to LPS, but we have also shown that the PI3K pathway partially contributes to it. Further experiments with the specific inhibitor of mTOR (rapamycin) will provide more information on the specific contribution of the PI3K/mTOR pathway in the inflammatory response in LPS-stimulated macrophages.

Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E-deficient mice. METHODS AND RESULTS: Intravenous administration of an adenovirus (5 x 10(8) plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P<0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P<0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P<0.001), macrophages by 69% (P=0.006), and smooth muscle cells by 84% (P=0.002) in the arterial wall. This resulted in a 77% reduction (P<0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P=0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P=0.01 and P=0.04, respectively). CONCLUSIONS: Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

Restenosis: a challenge for pharmacology.

The quest for an anti-restenotic drug continues to be a major challenge in the field of cardiovascular pharmacology because most therapies with proven efficacy in experimental neointima models have failed to limit restenosis. Some drug classes, including glycoprotein IIb/IIIa antagonists, nitric oxide donors and the antioxidant probucol, have recently demonstrated potential benefits in clinical trials. Progress in the development of local delivery systems for administration of drugs, antisense oligonucleotides or genes, in combination with an improved understanding of the pathogenesis of restenosis holds promise for ultimate pharmacotherapy of this condition.

Impaired endothelium-dependent cholinergic coronary vasodilation in patients with angina and normal coronary arteriograms.

The coronary vasomotor responses to selective infusion of graded concentrations (10(-6) to 10(-4) M) of acetylcholine into the left anterior descending artery were assessed by quantitative coronary arteriography in 24 patients with normal coronary arteriograms (12 patients with atypical symptoms and 12 patients with typical anginal pain) and 36 patients with coronary artery disease with different degrees of atherosclerosis of the left anterior descending artery. In the patients with normal coronary arteries and atypical chest pain, acetylcholine induced predominantly a vasodilator response, which was maximal during a 10(-5) M acetylcholine infusion. In contrast, in patients with coronary artery disease, acetylcholine caused dose-dependent vasoconstriction, which was observed even if the left anterior descending artery itself was smooth. Marked vasoconstriction was also induced in the patients with typical anginal pain and angiographically normal coronary arteries. In nine of these patients, this constrictor response was associated with anginal pain and electrocardiographic evidence of myocardial ischemia. Intracoronary administration of isosorbide dinitrate (1 mg) relieved the anginal pain and dilated all vessels. These data suggest that 1) patients with normal coronary arteriograms and angina pectoris manifest impairment of endothelium-dependent vasodilation similar to that observed in patients with overt coronary atherosclerosis; and 2) abnormal coronary vasoconstrictor responses resulting from this impairment may contribute to the pathogenesis of myocardial ischemia and angina in these patients.

Local application of advanced glycation end products and intimal hyperplasia in the rabbit collared carotid artery.

OBJECTIVE: Accumulation of advanced glycation end products (AGEs) in the vessel wall has been implicated in atherogenesis. The aim of our study was to examine the effects of local application of glycated bovine serum albumin (AGE-BSA) on collar-induced intimal hyperplasia in a diabetes-free setting. METHODS: Intimal thickening was induced by placing a collar around the carotid artery of rabbits. Via a catheter attached to an osmotic minipump, control BSA or AGE-BSA was administered locally to the vessel wall in a dose of 1.5 or 15 microg h(-1) during 14 days. Vessels receiving phosphate buffered saline (PBS, 5 microl h(-1)) were used as controls. RESULTS: Infusion of AGE-BSA 15 microg h(-1) significantly enhanced intimal thickening as compared to control BSA or PBS. Positive remodelling, measured as an increase in the area comprised by the external elastic lamina and preservation of lumen size, was only significant after treatment with the higher dose of AGE-BSA. In all other groups, intimal thickening was accompanied by a decrease of the lumen without outward displacement. Infusion of control BSA or AGE-BSA changed the cell composition of the neointima, with a significant enhancement in the number of T-lymphocytes and macrophages and a reduction in the percentage of intimal area occupied by smooth muscle cells. These effects were however similar for control BSA as well as AGE-BSA. CONCLUSIONS: It is concluded that infusion of control BSA or AGE-BSA may aggravate collar-induced intimal thickening by evoking an inflammatory response. This supports the concept that inflammation contributes to atherogenesis. Further, the significant enhancement in intimal hyperplasia by AGE-BSA suggests that glycated proteins provide an additional stimulus for the development of atherosclerotic lesions.

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.

Exposure to oxidized low-density lipoprotein in vivo enhances intimal thickening and selectively impairs endothelium-dependent dilation in the rabbit.

OBJECTIVES: Based on in vitro studies, oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis and the associated deficiency in endothelium-dependent relaxation. The aim of this study was to investigate the effects of in vivo exposure to oxLDL on intimal thickening and relaxing behaviour. METHODS: Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of the rabbit for 3 or 14 days. OxLDL (Cu(2+)-oxidized, 7 micron/h) or the vehicle phosphate-buffered saline (PBS) was infused in the collars via subdermally implanted osmotic minipumps. RESULTS: The collared vessels receiving PBS developed discrete intimal thickening after 14 days (intima/media (I/M) ratio 11 +/- 2%). OxLDL infusion resulted in intimal thickening after 3 days and significantly enhanced the intimal thickness by 14 days (I/M ratio 98 +/- 16%). Collaring alone for 3 or 14 days and 3 days exposure to oxLDL did not impair the endothelium-dependent relaxations to acetylcholine or calcium ionophore, nor to the NO donors glyceryl trinitrate (GTN) and S-nitroso-N-acetylpenicillamine (SNAP). However, the sensitivity to acetylcholine was decreased after exposure to oxLDL for 14 days (-logEC50 oxLDL 6.95 +/- 0.11 vs. 7.52 +/- 0.11 collar alone) and the maximal relaxation to the endothelium-dependent agonist was reduced by 50%, this in the presence of a virtually intact endothelium. Complete relaxation was still obtained with the nitric oxide donors. CONCLUSION: Our results show for the first time that local vascular exposure to oxLDL in vivo promotes intimal thickening and inhibits endothelium-dependent dilation, thereby supporting an active role for oxLDL in the morphological and functional changes observed in atherosclerotic blood vessels.

Distribution of cell replication and apoptosis in atherosclerotic plaques of cholesterol-fed rabbits.

In human atherosclerosis the development of a cell-poor lipid-rich core is an important feature of atheromatous plaque formation. The core is characterized by extracellular lipid deposition, cholesterol crystals and cell death and is situated in the deep layer of the plaque. The aim of the present study was to localize apoptotic cell death and cell replication in atherosclerotic plaques of cholesterol-fed rabbits in order to examine the hypothesis that core formation is a consequence of an imbalance between cell replication and apoptosis. New Zealand White male rabbits were fed a diet supplemented with 0.3% cholesterol for 16 (n = 5) and 27 weeks (n = 9). Cell replication and cell types were demonstrated by immunohistochemistry and apoptotic cell death was demonstrated by DNA in situ end-labeling (ISEL) and transmission electron microscopy. Quantification was done using a colour image analysis system. The plaques showed a clear distinction between a luminal layer composed of numerous lipid-rich foam cells of macrophage origin and a deep layer which was fibrous, containing extracellular lipid deposits and few smooth muscle cells. Cell replication (expressed as percentage of total number of nuclei) in the superficial layer was higher then in the deep layer at both 16 (5.1 +/- 1.8% vs. 1.2 +/- 0.8%) and 27 weeks (11.3 +/- 2.1% vs. 4.4 +/- 1.0%). This was also the case for the total number of nuclei per 50000 microns2 cross-sectional intimal area (numerical density): 235 +/- 13 vs. 147 +/- 7 at 16 weeks and 130 +/- 10 vs. 89 +/- 11 at 27 weeks. Apoptotic cell death (expressed as percentage of total number of nuclei) was low and there was no difference between the superficial and the deep layers of the plaques (0.8% +/- 0.2% vs. 0.4% +/- 0.2% at 16 weeks and 0.6 +/- 0.2% vs. 1.7% +/- 0.6% at 27 weeks). Our results indicate that the control of cell number in superficial vs. deep regions of the plaque is mainly a consequence of differences in cell replication. This may be due to a gradient of endothelial and plasma-derived growth factors. Cells can disappear by apoptosis, albeit at a relatively low level, throughout the lesion. This process may contribute to the pronounced cell loss in more advanced human atherosclerotic plaques, setting the base for plaque rupture.

Dipyridamole potentiates platelet inhibition by nitric oxide.

In a placebo-controlled double blind cross-over experiment the adenosine uptake inhibitor dipyridamole (400 mg/day) did not affect ex vivo platelet aggregation induced by collagen or adenosine-diphosphate (ADP) in an electronic whole blood aggregometer (WBA). Dipyridamole was also inactive in vitro, unless red blood cell injury was deliberately enhanced, thereby increasing the level of free adenine nucleotides. Since dipyridamole also inhibits cyclic guanosine monophosphate (GMP) phosphodiesterase (PDE), we used platelet rich plasma (PRP) to study its interaction with authentic and endothelium-derived nitric oxide (NO). The latter inhibits platelets by increasing cyclic GMP. Dipyridamole (1 to 30 microM), either alone or in combination with a subthreshold concentration of prostacyclin (PGI2), was inactive. However, when combined with a subthreshold concentration of NO, dipyridamole caused a concentration-dependent platelet suppression, which became more pronounced when PGI2 was present as well. It is concluded that dipyridamole could reduce the threshold for platelet suppression by NO through inhibition of cyclic GMP PDE.

Platelet adhesion to subendothelial structures under flow conditions: no effect of the lipoxygenase product 13-HODE.

It has been shown that endothelial cells can convert linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) and it has been suggested that 13-HODE has non-thrombogenic properties. However, no direct evidence has been presented that indicates that 13-HODE indeed modulates platelet-vessel wall interaction. In this study we have bound a purified 13-HODE to a thrombogenic surface and its effect on platelet adhesion was studied and compared to the effects of an analogous hydroxy fatty acid, 15-hydroxyeicosatetraenoic acid (15-HETE). The effect of 13-HODE on platelet adhesion was studied both under static and flow conditions. In this report we show that binding of up to 40 times the physiological concentration to a thrombogenic surface has no inhibitory effect on platelet adhesion under static or flow conditions. We conclude that 13-HODE is not an important regulatory substance in platelet-subendothelium interaction, although this does not exclude it has a putative anti-adhesive role on intact endothelium.

Effects of local cytochalasin D delivery on smooth muscle cell migration and on collar-induced intimal hyperplasia in the rabbit carotid artery.

1. Smooth muscle cell (SMC) migration has been implicated in neointima formation after angioplasty. Therefore, we investigated whether cytochalasin D, a fungal metabolite that inhibits actin filament formation, suppressed SMC migration and collar-induced intimal hyperplasia in the rabbit carotid artery. 2. To establish effective concentrations, contractions of carotid artery rings to phenylephrine were determined after incubation with cytochalasin D (10(-8) - 10(-6) M) for 30 min or 3 days. In vitro cell migration was studied using carotid artery explants and a modified Boyden chamber with SMCs isolated from the rabbit aorta. The in vivo effect was tested after infusion of 10(-8) - 10(-4) M cytochalasin D into collars placed around the left carotid artery; collars placed around the right artery served as controls. 3. Contractions to phenylephrine decreased after 30 min or 3 days exposure to 10(-7) and 10(-6) M cytochalasin D; the effect was partly reversible. These concentrations also inhibited cellular outgrowth and SMC migration in the in vitro assays. 4. Immunohistochemistry showed that local delivery of 10(-5) or 10(-4) M cytochalasin D for 2 weeks suppressed collar-induced alpha-SMC actin expression in the intima by 68% and 84% respectively. However, the cross-sectional area of the intima was not reduced due to an influx of T-lymphocytes and macrophages. 5. It is concluded that cytochalasin D suppressed SMC contractility and migration in vitro. Although perivascular infusion of cytochalasin D inhibited collar-induced SMC migration from media to intima in vivo as well, the intimal hyperplasia was not reduced due to concomitant development of an inflammatory response.

Modification of endotoxin-induced haemodynamic and haematological changes in the rabbit by methylprednisolone, F(ab')2 fragments and rosmarinic acid.

The effects of methylprednisolone, F(ab')2 fragments of human gamma globulins and rosmarinic acid, an inhibitor of complement activation, were tested on endotoxin-induced haemodynamic and haematological changes in the rabbit. Their effects were compared with complement depletion by cobra venom factor (CVF) pretreatment. The results provide further evidence for the role of complement activation and the concomitant triggering of the arachidonic acid cascade in the early phase of shock. The formation of vasoactive prostanoids (prostacyclin and thromboxane A2), the arterial hypotension and the thrombocytopenia were largely dependent on the presence of the intact complement system. F(ab')2 fragments (150 mg kg-1, i.v.) diminished the second fall in blood pressure to some extent but failed to alter any of the other endotoxin-induced changes. Methylprednisolone (40 mg kg-1, i.v.) given 10 min before endotoxin significantly reduced the activation of complement, the second rise of prostacyclin and the secondary hypotension, but was without effect on the early thromboxane peak of the haematological features of endotoxin shock. Rosmarinic acid (20 mg kg-1, i.v.) may be of potential interest for treatment of septic shock, since the drug suppressed the endotoxin-induced activation of complement, the formation of prostacyclin, both hypotensive phases, the thrombocytopenia and the concomitant release of thromboxane A2. The role of leukocytes and their arachidonic acid metabolites in plasma exudation deserves further investigation, because leukopenia and pulmonary oedema were not complement-dependent and were not affected by any of the treatments. Our results indicate that drugs, interfering with complement activation and/or prostaglandin biosynthesis, may be beneficial in endotoxin shock, provided that they are administered at an early stage.

Influence of chronic treatment with a nitric oxide donor on fatty streak development and reactivity of the rabbit aorta.

1. The influence of chronic treatment with molsidomine, pro-drug of the nitric oxide (NO) donor, 3-morpholino-sydnonimine (SIN-1), on fatty streak development and release of NO and prostacyclin (PGI2) was studied in the aorta of normal and cholesterol-fed rabbits. 2. Groups of 10 rabbits received standard diet (150 g day-1), or diets with 0.3% cholesterol, with 0.02% molsidomine or with the combination of cholesterol and molsidomine for 16 weeks. Lesion area and thickness, maximum change in isometric force (Emax) and sensitivity (-log EC50 or pD2) to constricting and relaxing agonists were assessed in segments of arch, thoracic and abdominal aorta. Bioassay was used to assess NO release. 3. Cholesterol-induced fatty streaks tapered off towards the abdominal aorta. Area, thickness, weight and cholesterylester content of the lesions were augmented by the NO donor, whereas the hypercholesterolaemia remained unchanged. The exacerbation was attributed to co-release of superoxide anion from the sydnonimine. 4. As fatty streaks progressed, amplitude and pD2 of acetylcholine (ACh)-induced relaxations decreased, whereas cyclic GMP and cyclic AMP second messenger systems were not influenced, since Emax and sensitivity to SIN-1 and forskolin remained unchanged. However, extensive lesions apparently trapped some NO, as the pD2 of authentic NO decreased. 5. The fatty streaks curtailed the biosynthesis of PGI2 and the overflow of NO from the perfused thoracic aorta. The latter defect was not restored by L-arginine and appears to be consistent with a functional change of the endothelial muscarinic receptors. 6. The NO donor desensitized the aorta to cyclic GMP-mediated relaxations (ACh, SIN-1 and NO), without affecting cyclic AMP-mediated relaxation to forskolin or constrictor responses to phenylephrine and 5-hydroxytryptamine. 7. The drug also suppressed the ACh-induced overflow of NO, without changing PGI2 release. This selective reduction of endothelial NO release and the desensitization of cyclic GMP-mediated relaxations occurred independently of fatty streak formation. 8. The results indicate that chronic exposure to exogenous NO downregulates endothelial NO release and cyclic GMP-mediated relaxations, and provide evidence for the existence of negative feed-back regulations of the L-arginine NO pathway under in vivo conditions.

Involvement of 5-HT1B receptors in collar-induced hypersensitivity to 5-hydroxytryptamine of the rabbit carotid artery.

In humans intimal thickening is aprerequisite of atherosclerosis. Application of a silicone collar around the rabbit carotid artery induces an intimal thickening but in addition it increases the sensitivity to the vasoconstrictor action of serotonin (5-hydroxytryptamine, 5-HT). The 5-HT receptors involved in collar-induced hypersensitivity to 5-HT were investigated using several agonists and antagonists. One week after placement of collars around both carotid arteries of anaesthetized rabbits, rings (2 mm width) from inside (=collar) and outside (=sham) the collars were mounted in organ baths (10 ml) for isometric force measurements at 6 g loading tension. Collared rings were more sensitive to the contractile effect of 5-HT (7.6 fold) and 5-carboxamidotryptamine (31 fold, 5-CT, 5-HT1 agonist) in cumulative concentration response curves. Sumatriptan (5-HT1B/1D agonist) caused concentration-dependent constrictions in collared rings only. Collar placement did not significantly alter pA2 values (Schild regression) or apparent pKb values (non-linear regression) of spiperone and methysergide (mixed 5-HT2A/5-HT1 antagonists) or ketanserin and ritanserin (5-HT2A antagonists), indicating unchanged binding characteristics of the 5-HT2A receptor. However, the reduced slope of the Schild regression pointed to a heterogeneous receptor population in collared rings. In contrast, the apparent pKb value of methiothepin (5-HT1B antagonist) was significantly reduced by collar placement, and its antagonism shifted from non-surmountable in sham rings to surmountable in collared segments. Taken together, this study demonstrates that the serotonergic receptor involved in the hypersensitivity to 5-HT of rabbit collared carotid artery is a 5-HT1B receptor subtype.

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.

Formation of prostanoids during intravascular complement activation in the rabbit.

Plasma concentrations of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and thromboxane B2 (TXB2) were measured by radioimmunoassay in arterial blood before and after injections of the complement activator, cobra venom factor (CVF). During the control period, the concentration of 6-oxo-PGF1 alpha, which gives the sum of prostacyclin plus 6-oxo-PGF1 alpha, and TXB2 were, respectively, less than 20 pg ml-1 and 70 +/- 15 pg ml-1. Intravenous injections of CVF induced dose-dependent, reversible elevations in the plasma levels of both prostanoids. The time courses for the increases of 6-oxo-PGF1 alpha and TXB2 paralleled the arterial hypotension and thrombocytopenia, suggesting the existence of a causal relationship between these parameters. The results further support our hypothesis that complement-dependent formation of arachidonic acid metabolites contributes to some of the haemodynamic and haematological changes occurring during endotoxin shock.

Effect of dipyridamole on the formation of 6-oxo-prostaglandin F1 alpha by the rat isolated aorta and ram seminal vesicle microsomes.

1 Dipyridamole (1 and 10 muM) enhanced the production of 6-oxo-prostaglandin F1 alpha by rat aortic tissue. 2 Dipyridamole (5 to 40 muM) did not influence the PGI2-synthetase activity in ram seminal vesicle microsomes whereas, in concentrations ranging from 100 to 200 muM, it reduced the metabolism of exogenously added arachidonic acid. The latter effect may be due to an inhibition of the cyclooxygenase.