Vasodilator efficacy of nitric oxide depends on mechanisms of intracellular calcium mobilization in mouse aortic smooth muscle cells.

BACKGROUND AND PURPOSE: Reduction of intracellular calcium ([Ca(2+)](i)) in smooth muscle cells (SMCs) is an important mechanism by which nitric oxide (NO) dilates blood vessels. We investigated whether modes of Ca(2+) mobilization during SMC contraction influenced NO efficacy. EXPERIMENTAL APPROACH: Isometric contractions by depolarization (high potassium, K(+)) or alpha-adrenoceptor stimulation (phenylephrine), and relaxations by acetylcholine chloride (ACh), diethylamine NONOate (DEANO) and glyceryl trinitrate (GTN) and SMC [Ca(2+)](i) (Fura-2) were measured in aortic segments from C57Bl6 mice. KEY RESULTS: Phenylephrine-constricted segments were more sensitive to endothelium-derived (ACh) or exogenous (DEANO, GTN) NO than segments contracted by high K(+) solutions. The greater sensitivity of phenylephrine-stimulated segments was independent of the amount of pre-contraction, the source of NO or the resting potential of SMCs. It coincided with a significant decrease of [Ca(2+)](i), which was suppressed by sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) inhibition, but not by soluble guanylyl cylase (sGC) inhibition. Relaxation of K(+)-stimulated segments did not parallel a decline of [Ca(2+)](i). However, stimulation (BAY K8644) of L-type Ca(2+) influx diminished, while inhibition (nifedipine, 1-100 nM) augmented the relaxing capacity of NO. CONCLUSIONS AND IMPLICATIONS: In mouse aorta, NO induced relaxation via two pathways. One mechanism involved a non-cGMP-dependent stimulation of SERCA, causing Ca(2+) re-uptake into the SR and was prominent when intracellular Ca(2+) was mobilized. The other involved sGC-stimulated cGMP formation, causing relaxation without changing [Ca(2+)](i), presumably by desensitizing the contractile apparatus. This pathway seems related to L-type Ca(2+) influx, and L-type Ca(2+) channel blockers increase the vasodilator efficacy of NO.

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.

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.

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.

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.

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.

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.

Biochemical and biological characterization of neutrophil chemotactic protein, a novel rabbit CXC chemokine from alveolar macrophages.

The role of interleukin-8 (IL-8) and related CXC chemokines has been demonstrated in many human diseases. However, more profound studies, e.g., by blocking the effect of these inflammatory mediators, request animal models and hence the identification of all human counterparts for commonly used laboratory animals. In this study, we describe the identification of a novel neutrophil chemotactic protein (NCP) of the rabbit. Intact and NH(2)-terminally truncated NCP forms and IL-8 were isolated from LPS-stimulated rabbit alveolar macrophages and purified to homogeneity by a four-step purification procedure. Determination of the complete primary structure of NCP by mass spectrometry and NH(2)-terminal sequencing of natural protein revealed high structural homology with human epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2), two related ELR(+)CXC chemokines. Intact NCP(1-76) was found to be 10-fold less potent than truncated NCP(7, 8-76) at inducing neutrophil chemotaxis. NCP(7,8-76) was equally potent as intact rabbit IL-8 at chemoattracting human neutrophils and at inducing calcium fluxes in rabbit neutrophils, 1 ng/mL being the minimal effective concentration. However, like IL-8, NCP failed to induce monocyte or eosinophil migration at 300-fold higher concentrations. IL-8 desensitized the calcium increase induced by NCP and vice versa. Finally, intradermal injection of NCP induced a dose-dependent and significant infiltration of neutrophils in mice skin. It can be concluded that NCP is a novel rabbit CXC chemokine that is, like IL-8, implicated in animal models used to study various human disorders in which neutrophils play an important role.

Heterogeneity in relaxation mechanisms in the carotid and the femoral artery of the mouse.

The participation of prostanoids, nitric oxide and non-prostanoid non-nitric oxide factors in endothelium-dependent relaxations was investigated in phenylephrine (PE)-constricted carotid and femoral arteries of C57BL6 mice. The carotid artery was more sensitive to acetylcholine as compared to the femoral artery, and cyclooxygenase inhibition did not influence the relaxation in either vessel. In the carotid artery, high doses of acetylcholine caused transient constrictions, which were abolished by indomethacin or piroxicam. In the carotid but not the femoral artery, N(omega)-nitro-L-arginine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) enhanced PE-induced contractions enormously, suggesting that endogenous nitric oxide production is much higher in the carotid artery. While in the carotid artery all relaxation was abolished by N(omega)-nitro-L-arginine or ODQ, a residual response (34+/-5% and 74+/-4%, respectively) but with a different shape, was maintained in the femoral artery. This N(omega)-nitro-L-arginine-resistant relaxation was abolished by the combination of apamin and charybdotoxin. In both arteries, ODQ abolished relaxation to S-nitroso-N-acetyl-D-penicillamine, while N(omega)-nitro-L-arginine enhanced the sensitivity to this donor of exogenous nitric oxide. In 30 mM KCl, the relaxation to acetylcholine was abolished by N(omega)-nitro-L-arginine or ODQ in either artery. In conclusion, in the carotid artery endothelium-dependent relaxation is mediated predominantly by nitric oxide acting via cyclic GMP-dependent pathways, while in the femoral artery part of the relaxation can be attributed to a non-prostanoid non-nitric oxide factor operating via apamin/charybdotoxin-sensitive potassium channels.

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.

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.

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.

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.

Antiatherosclerotic activity of drugs in relation to nitric oxide function.

Many studies have shown that loss of endothelium-derived nitric oxide is a major factor of ischemic episodes in patients with coronary artery disease and there is increasing evidence to suggest that nitric oxide might exert antiatherosclerotic actions. Based on these concepts, the results of animal studies on the effects of lipid lowering drugs, antioxidants, angiotensin converting enzyme inhibitors, Ca2+ channel blockers, estrogens and agents which modulate nitric oxide bioavailability are presented and compared to the results of patient studies and clinical trials. In spite of encouraging results obtained with antioxidants in animals, clinical trials could only show a clear positive effect of vitamin E treatment on the outcome of cardiovascular disease. Angiotensin converting enzyme inhibitors can ameliorate endothelial dysfunction in coronary heart disease, but their impact on disease progression remains unclear. There is evidence that estrogen replacement therapy in post-menopausal women may increase the bioavailability of nitric oxide. Finally, improved endothelial function and plaque stability clearly contribute to the clinical benefits of lipid lowering interventions, statins in particular. Taken together, these studies lend support to the concept that improving endothelial function and nitric oxide release might serve as valuable elements in the prevention or therapy of cardiovascular disease.

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.

Oxidized low-density lipoprotein enhances intimal thickening and alters vascular reactivity.

Oxidized low density lipoprotein (oxLDL) is present in atherosclerotic lesions and has been implicated in the etiopathogenesis of atherosclerosis mainly based on in vitro studies. In view of the lack of data on the activity of oxLDL in vivo, we decided to study its effects in the rabbit by local application at the level of the vascular wall. Intimal thickening was evoked by the placement of a silicone collar around the carotid arteries during 2 weeks. The collar was connected to an osmotic minipump containing human oxLDL (7 micrograms h-1), LDL (7 micrograms h-1) or phosphate-buffered saline. Collar placement resulted in a thickening of the intima thereby increasing the thickness from 5 +/- 1 to 26 +/- 5 microns with the appearance of alpha-actine positive smooth muscle cells. Perivascular infusion of LDL or oxLDL significantly enhanced the intima, containing large amounts of T-lymphocytes, collagen and smooth muscle cells. The placement of the collar and the infusion of oxLDL during 14 days resulted in an increased sensitivity to serotonin and a decreased sensitivity to acetylcholine. The maximal relaxation to acetylcholine was reduced by 50% whereas the endothelium-independent relaxation to nitroglycerin were not affected. These results show for the first time that the local application of oxLDL in vivo promotes intimal thickening and impairs the endothelium-dependent relaxations thereby supporting the suggestion that oxLDL plays an important role in the morphological and functional changes present in atherosclerotic blood vessels.

Continuous perivascular L-arginine delivery increases total vessel area and reduces neointimal thickening after experimental balloon dilatation.

The aim of this study was to evaluate whether vascular remodeling and neointimal thickening occur after balloon dilatation of the nonatherosclerotic rabbit carotid artery, and whether both processes are influenced by continuous perivascular delivery of L-arginine or the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In the first experiment, histological and morphometric evaluation of arteries was performed at different time points after balloon dilatation: 10 minutes (n=7), and 1 (n=7), 2 (n=9), 3 (n=20), or 10 (n=5) weeks. Neointimal thickening progressively contributed to luminal narrowing for at least 10 weeks after angioplasty. During the first 2 weeks after dilatation, a significant decrease of the total vessel area was measured. Ten weeks after dilatation, both the neointimal and total vessel area were increased without further changing of the luminal area. In the second experiment, endothelial injured rabbits were randomly assigned to receive 2 weeks of continuous local perivascular physiological salt solution (n=6), L-arginine (n=8), or L-NAME (n=7), starting immediately after balloon dilatation (ie, local drug delivery during the first phase of the biphasic vascular remodeling process). Perivascular L-arginine delivery significantly reduced the neointimal area, despite an increased number of neointimal Ki-67-positive smooth muscle cells. Both the luminal area and total vessel area were significantly increased. Serum L-arginine levels remained unchanged. L-NAME administration had no effect on the neointimal area, nor on the luminal and total vessel area. Neointimal formation and biphasic vascular remodeling occur after experimental balloon dilatation of the nonatherosclerotic rabbit carotid artery, and can be influenced by continuous local perivascular delivery of L-arginine.

Regression of cholesterol induced venous plaques after cholesterol withdrawal in rabbits.

Rabbits receiving a dietary cholesterol supplement of 0.5% develop atheromatous plaques in the systemic arteries, the pulmonary artery, the pulmonary veins and the venous cavities of the plexus pampiniformis in the funiculus spermaticus. Six months after the withdrawal of the cholesterol supplement the arterial lesions are still present, and show a fibrous transformation. This study is the first report of the total regression of the lesions in the particular venous localizations of the lungs and the plexus pampiniformis. The level of intraluminal pressure is discussed as the possible mechanism responsible for the diverging vascular reactivity.