Comparative population genomics in animals uncovers the determinants of genetic diversity.

Genetic diversity is the amount of variation observed between DNA sequences from distinct individuals of a given species. This pivotal concept of population genetics has implications for species health, domestication, management and conservation. Levels of genetic diversity seem to vary greatly in natural populations and species, but the determinants of this variation, and particularly the relative influences of species biology and ecology versus population history, are still largely mysterious. Here we show that the diversity of a species is predictable, and is determined in the first place by its ecological strategy. We investigated the genome-wide diversity of 76 non-model animal species by sequencing the transcriptome of two to ten individuals in each species. The distribution of genetic diversity between species revealed no detectable influence of geographic range or invasive status but was accurately predicted by key species traits related to parental investment: long-lived or low-fecundity species with brooding ability were genetically less diverse than short-lived or highly fecund ones. Our analysis demonstrates the influence of long-term life-history strategies on species response to short-term environmental perturbations, a result with immediate implications for conservation policies.

Positive selection on sperm ion channels in a brooding brittle star: consequence of life-history traits evolution.

Closely related species are key models to investigate mechanisms leading to reproductive isolation and early stages of diversification, also at the genomic level. The brittle star cryptic species complex Ophioderma longicauda encompasses the sympatric broadcast-spawning species C3 and the internal brooding species C5. Here, we used de novo transcriptome sequencing and assembly in two closely related species displaying contrasting reproductive modes to compare their genetic diversity and to investigate the role of natural selection in reproductive isolation. We reconstructed 20 146 and 22 123 genes for C3 and C5, respectively, and characterized a set of 12 229 orthologs. Genetic diversity was 1.5-2 times higher in C3 compared to C5, confirming that species with low parental investment display higher levels of genetic diversity. Forty-eight genes were the targets of positive diversifying selection during the evolution of the two species. Notably, two genes (NHE and TetraKCNG) are sperm-specific ion channels involved in sperm motility. Ancestral sequence reconstructions show that natural selection targeted the two genes in the brooding species. This may result from an adaptation to the novel environmental conditions surrounding sperm in the brooding species, either directly affecting sperm or via an increase in male/female conflict. This phenomenon could have promoted prezygotic reproductive isolation between C3 and C5. Finally, the sperm receptors to egg chemoattractants differed between C3 and C5 in the ligand-binding region. We propose that mechanisms of species-specific gamete recognition in brittle stars occur during sperm chemotaxis (sperm attraction towards the eggs), contrary to other marine invertebrates where prezygotic barriers to interspecific hybridization typically occur before sperm-egg fusion.

Influence of the larval phase on connectivity: strong differences in the genetic structure of brooders and broadcasters in the Ophioderma longicauda species complex.

Closely related species with divergent life history traits are excellent models to infer the role of such traits in genetic diversity and connectivity. Ophioderma longicauda is a brittle star species complex composed of different genetic clusters, including brooders and broadcasters. These species diverged very recently and some of them are sympatric and ecologically syntopic, making them particularly suitable to study the consequences of their trait differences. At the scale of the geographic distribution of the broadcasters (Mediterranean Sea and northeastern Atlantic), we sequenced the mitochondrial marker COI and genotyped an intron (i51) for 788 individuals. In addition, we sequenced 10 nuclear loci newly developed from transcriptome sequences, for six sympatric populations of brooders and broadcasters from Greece. At the large scale, we found a high genetic structure within the brooders (COI: 0.07 < F(ST) < 0.65) and no polymorphism at the nuclear locus i51. In contrast, the broadcasters displayed lower genetic structure (0 < F(ST) < 0.14) and were polymorphic at locus i51. At the regional scale, the multilocus analysis confirmed the contrasting genetic structure between species, with no structure in the broadcasters (global F(ST) < 0.001) and strong structure in the brooders (global F(ST) = 0.49), and revealed a higher genetic diversity in broadcasters. Our study showed that the lecithotrophic larval stage allows on average a 50-fold increase in migration rates, a 280-fold increase in effective size and a threefold to fourfold increase in genetic diversity. Our work, investigating complementary genetic markers on sympatric and syntopic taxa, highlights the strong impact of the larval phase on connectivity and genetic diversity.

Impact of a large dam on reproduction of a non-migratory teleost species, Acestrorhynchus lacustris (Characiformes: Acestrorhynchidae).

The release of water from the reservoir hypolimnion, lower concentration of oxygen and the anthropogenic regulation of the river flow, could affect the reproduction of fish, especially migratory species. However, little is known about the effects of these changes in water on non-migratory species. In this sense, the reproduction of Acestrohynchus lacustris was evaluated in two sections of São Francisco River, Minas Gerais, Brazil. Section 1, located immediately downstream from Três Marias Dam (18°09'31.65"S and 45°13'36.00"W) and section 2, located at the confluence of the São Francisco and the Abaeté Rivers (18°02'47.78"S and 45°10'57.95"W). For this, we obtained the physico-chemical parameters of water of each study section. Additionally, biometric data and biological indices of all specimens were measured. Fecundity and follicles diameters were measured in females. Temperature, dissolved oxygen and flow showed lower values in section 1. Fish captured in this section, had lower values of GSI in both sexes, and females presented decreased values of fecundity and follicles diameter. This species showed reproductive activity in the two sections analyzed, however, in section 1 where the temperature and dissolved oxygen presented significant lower values, the reproductive capacity of A. lacustris, was negatively affected.

Epac inhibits migration and proliferation of human prostate carcinoma cells.

BACKGROUND: It was recently found that cAMP mediates protein kinase A-independent effects through Epac proteins. The aim of this study was to investigate the role of Epac in migration and proliferation of prostate carcinoma cells. METHODS: The effect of Epac activation was determined by [(3)H]thymidine incorporation and scratch assays in PC-3 and DU 145 cells. Furthermore, cytoskeletal integrity was analysed by phalloidin staining. The participation of intracellular Epac effectors such as mitogen-activated protein (MAP) kinases, Rap1- and Rho-GTPases was determined by immunoblotting and pull-down assay. RESULTS: The specific Epac activator 8-pCPT-2'-O-Me-cAMP (8-pCPT) interfered with cytoskeletal integrity, reduced DNA synthesis, and migration. Although 8-pCPT activated Rap1, it inhibited MAP kinase signalling and RhoA activation. These findings were translated into functional effects such as inhibition of mitogenesis, cytoskeletal integrity, and migration. CONCLUSION: In human prostate carcinoma cells, Epac inhibits proliferative and migratory responses likely because of inhibition of MAP kinase and RhoA signalling pathways. Therefore, Epac might represent an attractive therapeutic target in the treatment of prostate cancer.

Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis.

BACKGROUND: Anti-inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug-drug interaction at the level of platelet cyclooxygenase-1 (COX-1). OBJECTIVE: We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. METHODS: Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. RESULTS: 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. CONCLUSION: This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

Effects of fibroblast extracellular matrix calcification on platelet adhesion in vitro.

Calcified atherosclerotic lesions are more prone to rupture during angioplasty than non-calcified lesions and are associated with an increased risk of thrombotic complications following angioplasty. This study investigates the possible role of extracellular matrix (ECM) calcification for platelet adhesion. Human cultured fibroblasts (CRL-1635) were subjected to beta-glycerophosphate (10 mM) for 10 to 16 days. Calcification was visualized by von Kossa staining and quantified by the O-cresolphthalein complexone method. Adhesion of calcein-labelled platelets was measured by fluorescence microscopy at static conditions and in a parallel-flow chamber at a shear rate of 1000 s(-1). beta-glycerophosphate treatment resulted in a marked calcification of the ECM. In parallel, a small, albeit significant increase in platelet adhesion under static conditions was observed. In contrast, at flow conditions, the area covered by thrombi was significantly lower when calcified ECM was used. The number of thrombi was not significantly different which is compatible with a smaller thrombus size. Taken together, it appears unlikely that calcification of atherosclerotic lesions contributes to thrombotic complications by an increased platelet adhesion.

[Methods to evaluate aspirin and clopidogrel resistance]. / Methoden zur Messung der Azetylsalizylsäure- bzw. Clopidogrelresistenz.

Based on the concept that the so-called resistance to anti-platelet drugs is meant to describe a phenomenon where the drug does not hit its direct pharmacodynamic target, assays, used to evaluated the effects of anti-platelet drugs, should as closely as possible measure the direct pharmacodynamic effect of a particular drug. Thus, for the detection of aspirin effects, thromboxane concentrations or arachidonic acid-induced responses (light aggregometry, whole-blood aggregometry) should be measured. For the detection of clopidogrel actions, VASP phosphorylation (flow cytometry) or ADP-induced responses (light aggregometry, whole blood aggregometry) should be analysed.

Impact of a large dam on reproduction of a non-migratory teleost species, Acestrorhynchus lacustris (Characiformes: Acestrorhynchidae)

Abstract The release of water from the reservoir hypolimnion, lower concentration of oxygen and the anthropogenic regulation of the river flow, could affect the reproduction of fish, especially migratory species. However, little is known about the effects of these changes in water on non-migratory species. In this sense, the reproduction of Acestrohynchus lacustris was evaluated in two sections of São Francisco River, Minas Gerais, Brazil. Section 1, located immediately downstream from Três Marias Dam (18°0931.65S and 45°1336.00W) and section 2, located at the confluence of the São Francisco and the Abaeté Rivers (18°0247.78S and 45°1057.95W). For this, we obtained the physico-chemical parameters of water of each study section. Additionally, biometric data and biological indices of all specimens were measured. Fecundity and follicles diameters were measured in females. Temperature, dissolved oxygen and flow showed lower values in section 1. Fish captured in this section, had lower values of GSI in both sexes, and females presented decreased values of fecundity and follicles diameter. This species showed reproductive activity in the two sections analyzed, however, in section 1 where the temperature and dissolved oxygen presented significant lower values, the reproductive capacity of A. lacustris, was negatively affected.

Resumo A liberação de água do hipolímnio do reservatório, baixa concentração de oxigênio e a regulação antropogênica do fluxo do rio podem afetar a reprodução de peixes, principalmente espécies migradoras. No entanto, pouco se sabe sobre os efeitos dessas mudanças nas condições da água em espécies não migradoras. A reprodução de Acestrohynchus lacustris foi avaliada em duas seções do rio São Francisco. Seção 1, localizada imediatamente a jusante da barragem de Três Marias e seção 2, localizada na confluência dos rios São Francisco e Abaeté. Para isso, foram obtidos os parâmetros físico-químicos da água de cada seção do estudo. Além disso, dados biométricos e índices biológicos de todos os peixes capturados foram obtidos. Adicionalmente, nas fêmeas foram medidos os diâmetros dos folículos vitelogênicos e a fecundidade. Temperatura, oxigênio dissolvido e fluxo apresentaram valores mais baixos na seção 1. Os peixes capturados nesta seção apresentaram menores valores de IGS em ambos os sexos, e as fêmeas apresentaram menores valores de fecundidade e diâmetro dos folículos. Essa espécie apresentou atividade reprodutiva nas duas seções analisadas, porém, na seção 1, onde os parâmetros da água apresentam piores condições para o processo reprodutivo de peixes, a capacidade reprodutiva de A. lacustris foi afetada negativamente.

Impact of a large dam on reproduction of a non-migratory teleost species, Acestrorhynchus lacustris (Characiformes: Acestrorhynchidae) / Impacto de uma grande barragem na reprodução de uma espécie de teleósteo não migradora, Acestrorhynchus lacustris (Characiformes: Acestrorhynchidae)

The release of water from the reservoir hypolimnion, lower concentration of oxygen and the anthropogenic regulation of the river flow, could affect the reproduction of fish, especially migratory species. However, little is known about the effects of these changes in water on non-migratory species. In this sense, the reproduction of Acestrohynchus lacustris was evaluated in two sections of São Francisco River, Minas Gerais, Brazil. Section 1, located immediately downstream from Três Marias Dam (18°09'31.65"S and 45°13'36.00"W) and section 2, located at the confluence of the São Francisco and the Abaeté Rivers (18°02'47.78"S and 45°1057.95"W). For this, we obtained the physico-chemical parameters of water of each study section. Additionally, biometric data and biological indices ofall specimens were measured. Fecundity and follicles diameters were measured in females. Temperature, dissolvedoxygen and flow showed lower values in section 1. Fish captured in this section, had lower values of GSI in bothsexes, and females presented decreased values of fecundity and follicles diameter. This species showed reproductiveactivity in the two sections analyzed, however, in section 1 where the temperature and dissolved oxygen presentedsignificant lower values, the reproductive capacity of A. lacustris, was negatively affected.

A liberação de água do hipolímnio do reservatório, baixa concentração de oxigênio e a regulação antropogênica do fluxo do rio podem afetar a reprodução de peixes, principalmente espécies migradoras. No entanto, pouco se sabe sobre os efeitos dessas mudanças nas condições da água em espécies não migradoras. A reprodução de Acestrohynchus lacustris foi avaliada em duas seções do rio São Francisco. Seção 1, localizada imediatamente a jusante da barragem de Três Marias e seção 2, localizada na confluência dos rios São Francisco e Abaeté. Para isso, foram obtidos os parâmetros físico-químicos da água de cada seção do estudo. Além disso, dados biométricos e índices biológicos de todos os peixes capturados foram obtidos. Adicionalmente, nas fêmeas foram medidos os diâmetros dos folículos vitelogênicos e a fecundidade. Temperatura, oxigênio dissolvido e fluxo apresentaram valores mais baixos na seção 1. Os peixes capturados nesta seção apresentaram menores valores de IGS em ambos os sexos, e as fêmeas apresentaram menores valores de fecundidade e diâmetro dos folículos. Essa espécie apresentou atividade reprodutiva nas duas seções analisadas, porém, na seção 1, onde os parâmetros da água apresentam piores condições para o processo reprodutivo de peixes, a capacidade reprodutiva de A. lacustris foi afetada negativamente.

Impact of a large dam on reproduction of a non-migratory teleost species, Acestrorhynchus lacustris (Characiformes: Acestrorhynchidae) / Impacto de uma grande barragem na reprodução de uma espécie de teleósteo não migradora, Acestrorhynchus lacustris (Characiformes: Acestrorhynchidae)

The release of water from the reservoir hypolimnion, lower concentration of oxygen and the anthropogenic regulation of the river flow, could affect the reproduction of fish, especially migratory species. However, little is known about the effects of these changes in water on non-migratory species. In this sense, the reproduction of Acestrohynchus lacustris was evaluated in two sections of São Francisco River, Minas Gerais, Brazil. Section 1, located immediately downstream from Três Marias Dam (18°09'31.65"S and 45°13'36.00"W) and section 2, located at the confluence of the São Francisco and the Abaeté Rivers (18°02'47.78"S and 45°10'57.95"W). For this, we obtained the physico-chemical parameters of water of each study section. Additionally, biometric data and biological indices of all specimens were measured. Fecundity and follicles diameters were measured in females. Temperature, dissolved oxygen and flow showed lower values in section 1. Fish captured in this section, had lower values of GSI in both sexes, and females presented decreased values of fecundity and follicles diameter. This species showed reproductive activity in the two sections analyzed, however, in section 1 where the temperature and dissolved oxygen presented significant lower values, the reproductive capacity of A. lacustris, was negatively affected.

A liberação de água do hipolímnio do reservatório, baixa concentração de oxigênio e a regulação antropogênica do fluxo do rio podem afetar a reprodução de peixes, principalmente espécies migradoras. No entanto, pouco se sabe sobre os efeitos dessas mudanças nas condições da água em espécies não migradoras. A reprodução de Acestrohynchus lacustris foi avaliada em duas seções do rio São Francisco. Seção 1, localizada imediatamente a jusante da barragem de Três Marias e seção 2, localizada na confluência dos rios São Francisco e Abaeté. Para isso, foram obtidos os parâmetros físico-químicos da água de cada seção do estudo. Além disso, dados biométricos e índices biológicos de todos os peixes capturados foram obtidos. Adicionalmente, nas fêmeas foram medidos os diâmetros dos folículos vitelogênicos e a fecundidade. Temperatura, oxigênio dissolvido e fluxo apresentaram valores mais baixos na seção 1. Os peixes capturados nesta seção apresentaram menores valores de IGS em ambos os sexos, e as fêmeas apresentaram menores valores de fecundidade e diâmetro dos folículos. Essa espécie apresentou atividade reprodutiva nas duas seções analisadas, porém, na seção 1, onde os parâmetros da água apresentam piores condições para o processo reprodutivo de peixes, a capacidade reprodutiva de A. lacustris foi afetada negativamente.

Differential effects of vasodilatory prostaglandins on focal adhesions, cytoskeletal architecture, and migration in human aortic smooth muscle cells.

OBJECTIVE: Cyclooxygenases 1 and 2 are expressed in atherosclerotic arteries, and local generation of prostacyclin and prostaglandin E2 (PGE2) occurs. However, the role of cyclooxygenases and individual prostaglandins during plaque progression is currently uncertain. The present study characterizes the effect of vasodilatory prostaglandins on morphology, focal adhesion (FA) function, and migration in human aortic smooth muscle cells (SMCs). METHODS AND RESULTS: The stable prostacyclin analog iloprost transiently induced: (1) disassembly of FA and stress fibers, (2) partial retraction and rounding of SMCs, (3) hypophosphorylation of FA kinase (FAK) and paxillin, and (4) inhibition of platelet-derived growth factor-BB-induced migration. Inhibition of FAK phosphorylation and morphological changes were mimicked by forskolin, inhibited by H89, and prevented by the protein tyrosine phosphatase inhibitor vanadate and by calpeptin. PGE2 was by far less efficient with respect to all parameters investigated. This difference correlated with the respective cAMP induction in response to iloprost and PGE2. CONCLUSIONS: Inhibition of FAK phosphorylation and FA function is a new target of vasodilatory prostaglandins, which might be causally involved in the antimigratory effects of prostaglandins. Importantly, prostacyclin analogs and PGE2 differ dramatically with respect to dephosphorylation of FAK and inhibition of migration, which might be of relevance for their respective functions in atherosclerosis.

Cyclooxygenase-2 expression in human esophageal carcinoma.

On the basis of epidemiological observations that nonsteroidal antiinflammatory drugs reduce the risk of esophageal carcinoma, we studied the expression of cyclooxygenase-2 (COX-2) in esophageal squamous cell carcinomas (SCCs; n = 172) and in esophageal adenocarcinomas (ADCs; n = 27). Using immunohistochemistry, we observed COX-2 expression in 91% of the SCCs and in 78% of the ADCs. Western blot analysis showed enhanced expression of the COX-2 protein in some tumors as compared with normal esophageal squamous epithelium, whereas similar amounts of the COX-1 protein were found in normal and cancerous tissues. COX expression was also studied in two esophageal cancer cell lines (OSC-1 and OSC-2) to evaluate the functional relevance of COX-2-derived prostaglandins (PGs). OSC-2 cells expressed COX-2 but not COX-1, whereas OSC-1 cells expressed high levels of COX-1 but showed only a very weak COX-2 expression. Accordingly, PGE2 synthesis was 600 times higher in the OSC-2 cells as compared with the OSC-1 cells. Treatment of OSC-2 cells with the selective COX-2 inhibitors flosulide and NS-398 concentration dependently suppressed PGE2 synthesis and proliferation and also induced apoptosis. In contrast, no effect of the COX-2 inhibitors was seen in OSC-1 cells. Our data demonstrate that COX-2 is expressed in the majority of esophageal SCCs and ADCs and that COX-2-derived PGs play an important role in the regulation of proliferation and apoptosis of esophageal tumor cells. It is concluded that inhibition of COX-2 may be useful in the therapy of esophageal cancer.

Functional and biochemical evaluation of platelet aspirin resistance after coronary artery bypass surgery.

BACKGROUND: Aspirin inhibits platelet activation and reduces atherothrombotic complications in patients at risk of myocardial infarction and stroke. However, a sufficient inhibition of platelet function by aspirin is not always achieved. The causes of this aspirin resistance are unknown. METHODS AND RESULTS: Patients undergoing coronary artery bypass grafting (CABG) have a high incidence of aspirin resistance. To evaluate functional and biochemical responses to aspirin, platelet-rich plasma was obtained before and at days 1, 5, and 10 after CABG. Thromboxane formation, aggregation, and alpha-granule secretion were effectively inhibited by 30 or 100 micromol/L aspirin in vitro before CABG, but this inhibition was prevented or attenuated after CABG. Whereas the inhibition of thromboxane formation and aggregation by aspirin in vitro partly recovered at day 10 after CABG, oral aspirin (100 mg/d) remained ineffective. The inducible isoform of cyclooxygenase in platelets, COX-2, has been suggested to confer aspirin resistance. In fact, immunoreactive COX-2 was increased 16-fold in platelets at day 5 after CABG, but the COX-2 selective inhibitor celecoxib did not alter aspirin-resistant thromboxane formation. By contrast, the combined inhibitor of thromboxane synthase and thromboxane receptor antagonist terbogrel equally prevented thromboxane formation of platelets obtained before (control) and after CABG. CONCLUSIONS: Platelet aspirin resistance involves an impairment of both in vivo and in vitro inhibition of platelet functions and is probably due to a disturbed inhibition of platelet COX-1 by aspirin.

Cholesterol enhances platelet-derived growth factor-BB-induced [Ca2+]i and DNA synthesis in rat aortic smooth muscle cells.

In the present study, we describe possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular diseases. Treatment of rat aortic smooth muscle cells for 20 hours with cholesterol-rich liposomes (500 micrograms/mL cholesterol, 100 micrograms/mL low-density lipoprotein) resulted in a 76 +/- 12% increase in total cholesterol content. The effects of cholesterol enrichment were examined by determination of changes in cell membrane fluidity. Fluidity of the cholesterol-enriched cell membranes was decreased at all temperatures between 15 degrees C and 40 degrees C. Changes in membrane fluidity in whole cell membranes represented changes in fluidity of microsomal membranes isolated by Percoll gradient ultracentrifugation. The basal [Ca2+]i and the maximal platelet-derived growth factor (PDGF)-BB-induced [Ca2+]i was elevated by 30% and 90% in cholesterol-enriched cells, respectively. In contrast, the resting pH, and the PDGF-BB-induced stimulation of the Na+/H+ exchange were not affected in cholesterol-enriched cells. The effect of PDGF-BB on [3H]thymidine incorporation in cholesterol-enriched cells was elevated by 40% in comparison with untreated cells. Our findings show that cellular cholesterol may be involved in the development of vascular diseases via modulation of the PDGF-induced increase in [Ca2+]i and DNA synthesis in vascular smooth muscle cells.

Antiatherosclerotic effects of oral cicaprost in experimental hypercholesterolemia in rabbits.

The efficacy of the oral prostacyclin mimetic cicaprost in preventing atheromatous plaque formation was studied in an in vivo model of experimental hypercholesterolemia. New Zealand white rabbits were fed either standard chow or a cholesterol-enriched (1%) diet for 12 weeks. Cicaprost was added to the drinking water in a non-hypotensive dose (5 micrograms/kg/day) and withdrawn 3 days prior to studying platelet, leukocyte and endothelial function. In cholesterol-fed rabbits, oral cicaprost reduced the aortic intimal surface covered by atheromatous lesions from 84 to 63% (P < 0.05). There was no major difference in serum lipid composition between cicaprost- and vehicle-treated animals. In hyper-cholesterolemic rabbits there was a significant impairment of endothelium-dependent relaxations. Cicaprost treatment considerably improved this endothelial function but had no effect in rabbits receiving standard diet. In addition, platelet and leukocyte hyperreactivity, as seen in hypercholesterolemic rabbits, were largely reduced by cicaprost treatment. These data are the first to demonstrate marked antiatherosclerotic effects of long-term oral prostacyclin treatment. The mechanism may be related to improved endothelial function and subsequent prevention of secondary platelet and neutrophil hyperreactivity.

Specific inhibition of ADP-induced platelet aggregation by clopidogrel in vitro.

1. The thienopyridine clopidogrel is a specific inhibitor of ADP-induced platelet aggregation ex vivo. No direct effects of clopidogrel (< or = 100 microM) on platelet aggregation in vitro have been described so far. 2. Possible in vitro antiaggregatory effects (turbidimetry) of clopidogrel were studied in human platelet-rich plasma and in washed platelets. 3. Incubation of platelet-rich plasma with clopidogrel (< or = 100 microM) for up to 8 h did not result in any inhibition of ADP (6 microM)-induced platelet aggregation. 4. Incubation of washed platelets with clopidogrel resulted in a time- (maximum effects after 30 min) and concentration-dependent (IC50 1.9+/-0.3 microM) inhibition of ADP (6 microM)-induced platelet aggregation. Clopidogrel (30 microM) did not inhibit collagen (2.5 microg ml(-1))-, U46619 (1 microM)- or thrombin (0.1 u ml(-1))-induced platelet aggregation. The inhibition of ADP-induced aggregation by clopidogrel (30 microM) was insurmountable indicating a non-equilibrium antagonism of ADP actions. The R enantiomer SR 25989 C (30 microM) was significantly less active than clopidogrel (30 microM) in inhibiting platelet aggregation (32+/-5% vs 70+/-1% inhibition, P < 0.05, n = 5). 5. In washed platelets, clopidogrel (< or = 30 microM) did not significantly reverse the inhibition of prostaglandin E1 (1 microM)-induced platelet cyclic AMP formation by ADP (6 microM). 6. The antiaggregatory effects of clopidogrel were unchanged when the compound was removed from the platelet suspension. However, platelet inhibition by clopidogrel was completely abolished when albumin (350 mg ml(-1)) was present in the test buffer. 7. It is concluded that clopidogrel specifically inhibits ADP-induced aggregation of washed platelets in vitro without hepatic bioactivation. Inhibition of ADP-induced platelet aggregation by clopidogrel in vitro occurs in the absence of measurable effects on the reversal of PGE1-stimulated cyclic AMP by ADP.

Inhibition of glycosphingolipid synthesis by threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and the modulation of IL-1beta-stimulated expression of inducible nitric oxide synthase in rat aortic smooth muscle cells.

1. The composition of glycosphingolipids is altered in atherosclerotic tissue. In order to study the possible modulation of interleukin-1beta (IL-1beta)-induced expression of inducible nitric oxide synthase (iNOS) by endogenously synthesized glycosphingolipids, we investigated rat aortic vascular smooth muscle cells (VSMC) grown in the presence of the inhibitor of glycosphingolipid synthesis, threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). 2. Depletion of glycosphingolipids by PDMP (20-30 microM) was demonstrated by thin-layer chromatography of D-[1-(14)C]-galactose- or L-[-U14C]-serine-labelled glycosphingolipids. Nitrite generation was measured by the diaminonaphthalene assay, nitric oxide was determined by the oxyhaemoglobin technique and iNOS protein was detected by immunocytochemistry. 3. In VSMC grown in the presence of PDMP, the glycosphingolipid content was reduced by 30-50%. In PDMP-treated VSMC, IL-1beta (3 micro ml[-1])-stimulated release of nitrite (135 +/- 4 nmol mg(-1) protein 48 h[-1]) was significantly increased as compared to IL-1beta-stimulated control cells (40 +/- 3 nmol mg(-1) protein 48 h(-1); n = 6, P < 0.001). Similarly, IL-1beta (3 micro ml(-1), 36 h)-stimulated release of nitric oxide was higher in PDMP-treated VSMC (6.1 +/- 0.5 nmol mg(-1) protein h[-1]) as compared to untreated cells (2.0 +/- 0.6 nmol mg(-1) protein h(-1); n = 3, P < 0.01). These findings were confirmed by the demonstration of increased expression of iNOS protein (14.9 +/- 1.2% vs 6.4 +/- 0.2%; n = 4, P < 0.001), as shown by immunocytochemistry. 4. Evidence is presented that endogenous glycosphingolipids are important modulators of cytokine-induced iNOS expression. In view of an altered glycosphingolipid profile in atherosclerotic arteries, these mechanisms might be of relevance for the pathogenesis of atherosclerosis and restenosis subsequent to vessel injury.

Effects of terbogrel on platelet function and prostaglandin endoperoxide transfer.

The present study describes the platelet-inhibitory effects of terbogrel (5-hexenoic acid, 6-[3-[[(cyanoamino)[(1,1-dimethylethyl)amino]methylene]amino]pheny l]-6-(3-pyridinyl)-, (epsilon)-), a novel combined thromboxane A2 synthase inhibitor and thromboxane A2 receptor antagonist. Terbogrel concentration-dependently inhibited collagen (0.6 microg/ml)- and U46619 (11alpha,9alpha-epoxymethano-15(S)-hydroxy-prosta-5Z,+ ++13E-dienoic acid) (1 microM)-induced aggregation and thromboxane synthesis of washed human platelets. In this system, terbogrel exhibited an equipotent (IC50 of about 10 nM) activity as thromboxane A2 synthase inhibitor and thromboxane A2 receptor antagonist. In addition, the compound favoured prostacyclin synthesis in cultured vascular smooth muscle cells by increasing the transfer of platelet-derived prostaglandin endoperoxides. Terbogrel appears to be a compound with an equipotent molar potency as thromboxane A2 synthase inhibitor and receptor antagonist.

Direct inhibition of platelet function by organic nitrates via nitric oxide formation.

This study investigates the mechanisms of platelet inhibition by the nitrate esters isosorbide dinitrate, isoidide dinitrate, isomannide dinitrate, isosorbide 2-mononitrate and isosorbide 5-mononitrate as compared to the spontaneous nitric oxide (NO)-donor linsidomine, the active metabolite of molsidomine. Nitrates and linsidomine dose-dependently inhibited aggregation, ATP secretion and thromboxane formation of washed human platelets at a rank order of potency, identical with that for stimulation of cyclic GMP in cultured rat lung fibroblasts. While linsidomine (0.1 mM) caused a 3-fold platelet cGMP elevation, there was a weak (< or = 30%) but significant cGMP stimulation by organic nitroesters, which was tightly correlated with inhibition of platelet aggregation (r = 0.926, P = 0.008). Zaprinast (2 microM) potentiated, while methylene blue (1 microM) and oxyhemoglobin (10 microM) reversed the antiaggregatory effects. Linsidomine (0.5 microM-0.1 mM) dose-dependently released NO in a cell-free system. No spontaneous NO release was detected with organic nitroesters (0.1 mM). These data suggest that, to some extent, bioactivation of organic nitroesters occurs in platelets, resulting in platelet inhibition via the NO/cGMP system.