Marina Braun

Platelet CD40 ligand (CD40L) – subcellular localization, regulation of expression, and inhibition by clopidogrel

This study compares the subcellular localization and the regulation of expression of the platelet activation markers CD62P and CD63 with CD40 ligand (CD40L) on the surface of washed human platelets. CD40L was expressed upon stimulation with a wide range of platelet activators. However, quantitative flow cytometry demonstrated that, as compared with CD62P and CD63, CD40L expression was low. Upon stimulation with thrombin receptor-activating peptide (TRAP-6), all activation markers were expressed. In contrast, upon stimulation with low concentrations of collagen (1-3 w g/ml), CD40L, but not the granule proteins (CD62P, CD63), were expressed. Using immunofluorescence microscopy, a cytoplasmic staining was observed for CD40L, and cytoplasmic localization of CD40L was verified by Western blotting of subcellular platelet fractions. The staining of CD40L was different from that of filamentous actin and only little association of CD40L with platelet cytoskeleton was found. Surface expression of CD40L was dependent on internal Ca 2+ stores and protein kinase C, while the mitogen-activated protein kinases (ERK, p38) or tyrosine kinases were not involved. ADP (30 w M)-induced CD40L expression was not inhibited by aspirin. In contrast, clopidogrel treatment completely abolished ADP-induced expression of CD40L. Finally, the expression level of CD40L was shown to be upregulated by phorbol myristate acetate (PMA) in the promegakaryocytic cell line MEG-01.

Cellular adhesion molecules on vascular smooth muscle cells

Several studies during the last years have shown that, in addition to endothelial cells, vascular smooth muscle cells also express the cellular adhesion molecules ICAM-1 and VCAM-1 in atherosclerosis, restenosis and transplant vasculopathy. In vitro studies have characterized stimulatory and inhibitory factors that regulate the expression of ICAM-1 and VCAM-1 on cultured smooth muscle cells. There is evidence for a role of adhesion molecules on smooth muscle cells for leukocyte accumulation and activation of mononuclear cells. Some recent data suggest that the expression of adhesion molecules on smooth muscle cells are cell cycle-dependent and influence smooth muscle cell proliferation and differentiation. Therefore, ICAM-1 and VCAM-1 on smooth muscle cells may contribute to the inflammatory reaction in the vascular wall and may actively be involved in the progression and stability of atherosclerotic plaques.

Factor Xa Releases Matrix Metalloproteinase-2 (MMP-2) From Human Vascular Smooth Muscle Cells and Stimulates the Conversion of Pro–MMP-2 to MMP-2: Role of MMP-2 in Factor Xa–Induced DNA Synthesis and Matrix Invasion

Pro–matrix metalloproteinase-2 (pro–MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro–MMP-2 (72 kDa) into MMP-2. Pro–MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro–MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). FX at concentrations up to 300 nmol/L was ineffective. The conversion of pro–MMP-2 to MMP-2 was inhibited by a selective FXa inhibitor (DX-9065a) at 3 to 10 &mgr;mol/L. There was a concentration-dependent induction of an intermediate MMP-2 form (68 kDa) in lysates of FXa-treated cells. This indicates that cellular mechanisms are involved in FXa-induced conversion of pro–MMP-2. As a possible biological consequence of MMP-2 activation by FXa, DNA synthesis and matrix invasion of SMCs were determined. Both were stimulated by FXa and inhibited by the selective FXa inhibitor DX-9065a and the MMP inhibitor GM 6001 but not by hirudin or aprotinin. It is concluded that stimulation of SMCs by FXa increases the levels of MMP-2 in the extracellular space and that two different mechanisms are involved: release of active MMP-2 and cleavage of secreted pro–MMP-2. Both might contribute to the mitogenic potency of FXa and FXa-stimulated matrix invasion of SMCs.

Evidence for functionally active protease‐activated receptor‐4 (PAR‐4) in human vascular smooth muscle cells

This study investigates, whether in addition to the protease‐activated receptor‐1 (PAR‐1), PAR‐4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR‐1 and PAR‐4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. mRNAs for both, PAR‐1 and PAR‐4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT – PCR). Treatment of the SMC with GYPGQV (200 μM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. Stimulation of the SMC with 10 nM thrombin or 200 μM SFLLRN caused a time‐dependent activation of the extracellular signal‐regulated kinases‐1/2 (ERK‐1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 μM of GYPGQV induced a prolonged phosphorylation of ERK‐1/2 with a maximum at 60 min. These data suggest that PAR‐1 and PAR‐4 are activated by thrombin at distinct concentrations and with distinct kinetics. GYPGQV stimulated [3H]‐thymidine incorporation in SMC. At 500 μM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 μM SFLLRN, respectively. These data indicate that a functionally active PAR‐4 is present in SMC and, in addition to PAR‐1, might contribute to thrombin‐induced mitogenesis.

PDGF-induced Akt phosphorylation does not activate NF-κB in human vascular smooth muscle cells and fibroblasts

A recent report suggested that platelet‐derived growth factor (PDGF) activates nuclear factor‐κB (NF‐κB) by phosphorylation of the protein kinase Akt [Romashkova and Makarov, Nature 401 (1999) 86–90]. The present study investigates the role of Akt in the activation of NF‐κB by tumor necrosis factor‐α (TNFα, 10 ng/ml) and PDGF‐BB (20 ng/ml) in human vascular smooth muscle cells (SMC), skin and foreskin fibroblasts. TNFα stimulated serine phosphorylation and degradation of the inhibitory protein IκBα and strongly induced nuclear NF‐κB translocation and binding activity. PDGF did not induce serine phosphorylation or degradation of IκBα and did not enhance binding activity of NF‐κB. In contrast, stimulation with PDGF resulted in a marked phosphorylation of Akt, but no Akt phosphorylation occurred after stimulation with TNFα. These data suggest that Akt phosphorylation is not involved in NF‐κB activation in human SMC and fibroblasts.

Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells.

This study investigates, whether in addition to the thrombin receptor (PAR‐1), the proteinase‐activated receptor‐2 (PAR‐2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR‐2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA‐synthesis. A similar effect was observed with 10 nM thrombin. Trypsin‐induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. The specific PAR‐2‐activating peptide SLIGRL or the PAR1‐activating peptide SFFLRN did not elicit mitogenesis. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). Trypsin (2 nM) as well as SLIGRL (100 μM) activated the nuclear factor κB (NFκB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR‐2. Maximum activation of NFκB by thrombin (10 nM) was detected after 4–5 h. These data suggest that PAR‐2 is present in coronary SMC and mediates a mitogenic response. Activation of NFκB via either PAR‐1 or PAR‐2 does not predict mitogenesis.

Absorption, Disposition, Metabolic Fate, and Elimination of the Dopamine Agonist Rotigotine in Man: Administration by Intravenous Infusion or Transdermal Delivery

The dopamine agonist rotigotine was developed for the treatment of Parkinsons disease and restless legs syndrome. Disposition, metabolism, elimination, and absolute bioavailability of rotigotine were determined in six healthy male subjects by using two different forms of administration in a randomized sequence with a crossover design. Treatment A (continuous infusion) consisted of a single radiolabeled 12-h intravenous infusion of 1.2 mg of rotigotine (0.6 mg of [14C] and 0.6 mg of unlabeled rotigotine, 3.7 MBq) solution. Treatment B (transdermal application) consisted of a single 10-cm2 patch containing 4.5 mg of unlabeled rotigotine with a patch-on period of 24 h. During the 12 h-infusion, total radioactivity concentration rapidly increased within 2 h; there was a slight additional increase toward the end of infusion. Plasma concentrations of total radioactivity declined by 75% within 12 h after completion of infusion. More than 94% of the radioactivity was excreted 216 h after the start of infusion, 71% by the kidneys and 23% by feces. Renal elimination of the parent compound was <1%. Systemically absorbed rotigotine was rapidly metabolized. The major rotigotine biotransformation pathway was conjugation of the parent compound, mainly by sulfation; a second pathway was the formation of phase 1 metabolites (N-desalkylation) with subsequent conjugation. Plasma concentration-time profiles of unchanged rotigotine during and after infusion and during and after patch administration were comparable. Absolute bioavailability of transdermally applied rotigotine was 37%.

Prostaglandin D2 relaxes bovine coronary arteries by endothelium-dependent nitric oxide-mediated cGMP formation.

This study investigates the vasomotor activities of prostaglandin (PG) D2 in bovine coronary arteries in relation to endothelial function. Isolated segments of bovine coronary arteries with intact endothelium were concentration-dependently relaxed by PGD2 (0.01-1 microM), a reaction that was blocked by a selective PGD receptor antagonist (BW A868C). There was a tight correlation between PGD2- and acetylcholine-induced relaxations (r = 0.894, n = 96, p < 0.001). Removal of endothelium abolished the PGD2-induced relaxation and unmasked a contractile activity of the compound. Inhibition of endogenous PGI2 formation by indomethacin did not modify these responses, whereas inhibition of endogenous nitric oxide generation by NG-nitro-L-arginine and NG-monomethyl L-arginine (10 or 100 microM) or scavenging of released nitric oxide by oxyhemoglobin (3 microM) considerably (> 50%) antagonized the PGD2-induced relaxation. The vessel relaxation by PGD2 was associated with a threefold to fourfold increase in vascular cGMP. A considerable reduction in vascular cGMP was measured after removal of the endothelium (by 53%) and inhibition of endogenous nitric oxide generation by NG-nitro-L-arginine (by 70%). This also resulted in a complete inhibition of PGD2-induced cGMP accumulation. Similar results were obtained with the stable PGD2 mimetic ZK 110.841, suggesting that these biological activities of PGD2 were due to the compound itself and not caused by any PGD2 metabolite. A slight but significant increase in cAMP was observed in arteries with intact endothelium as well as after removal of endothelium. Because the relaxing effect of PGD2 was strictly endothelium dependent, the observed relaxation cannot be explained by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Tumor Necrosis Factor-α and Interleukin-1β Induced Adhesion Molecule Expression in Human Vascular Smooth Muscle Cells by cAMP

Abstract This study investigates the hypothesis that the elevation of intracellular cAMP may affect cytokine-induced expression of adhesion molecules on human vascular smooth muscle cells. In cultured human smooth muscle cells from coronary arteries and saphenous veins, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) induced the expression of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), whereas interferon-γ (INF-γ) selectively stimulated the expression of ICAM-1. Adenylyl cyclase was stimulated either by the stable prostacyclin mimetic cicaprost or by forskolin. Adhesion molecules were detected by a cell surface enzyme immunoassay and the respective mRNA by reverse transcriptase polymerase chain reaction (rt-PCR). Cicaprost as well as forskolin significantly inhibited TNF-α- and IL-1β-induced cell surface expression of ICAM-1 and VCAM-1. Semiquantitative rt-PCR measurements showed a marked decrease of TNF-α- and IL-1β-induced mRNA levels of both adhesion molecules after preincubation with cicaprost. The stability of TNF-α-induced ICAM-1 and VCAM-1 expression at mRNA and protein level was not altered by cicaprost. The IFN-γ-induced increase of cell surface expression of ICAM-1 and the respective mRNA levels, however, were not significantly altered by elevation of intracellular cAMP. Basal and stimulated cAMP levels, measured by radioimmunoassay, did not differ in TNF-α- and IFNγ-treated cells. The present results demonstrate that the expression of adhesion molecules on human smooth muscle cells induced by cytokines is differentially modulated by activation of adenylyl cyclase.

Influence of domperidone on pharmacokinetics, safety and tolerability of the dopamine agonist rotigotine

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Rotigotine transdermal patch is a new non-ergolinic dopamine agonist developed for the treatment of Parkinsons disease and restless legs syndrome. Peripheral dopaminergic side-effects of dopamine agonists such as nausea and vomiting can be prevented by the antiemetic agent domperidone. WHAT THIS STUDY ADDS The study results show no evidence for an interaction of domperidone on bioavailability and steady-state pharmacokinetics of transdermal rotigotine. Co-administration of domperidone and rotigotine does not require dose adjustments for rotigotine transdermal patch. AIMS To evaluate the influence of the antiemetic agent domperidone on steady-state pharmacokinetics, safety and tolerability of multiple-dose treatment of the transdermally applied non-ergolinic dopamine agonist rotigotine. METHODS Sixteen healthy male subjects (mean age 30.3 years) participated in a randomized, two-way crossover clinical trial. Treatment A consisted of transdermal rotigotine patch (2 mg (24 h)(-1), 10 cm(2), total drug content 4.5 mg) applied daily for 4 days, and concomitant oral domperidone (10 mg t.i.d.) for 5 days. For treatment B, subjects received only transdermal rotigotine treatment (daily for 4 days). Pharmacokinetic variables describing systemic exposure and renal elimination of rotigotine and metabolites, and safety and tolerability of the treatment were assessed. RESULTS The primary steady-state pharmacokinetic parameters (C(max,ss) and AUC((0-24),ss)) were similar with or without co-administration of domperidone. Geometric mean ratios were close to 1 and respective 90% confidence intervals were within the acceptance range of bioequivalence (0.8, 1.25): C(max,ss) 0.96 (0.86, 1.08) and AUC((0-24),ss) 0.97 (0.87, 1.08). t(max,ss), t(1/2), secondary parameters calculated on days 4/5 after repeated patch application (C(min,ss), C(ave,ss), AUC((0-tz))) and renal elimination for unconjugated rotigotine and its metabolites were also similar with and without comedication of domperidone. A reduction in the dopaminergic side-effect nausea was seen with domperidone comedication. CONCLUSIONS No changes of pharmacokinetic parameters describing systemic exposure and renal elimination of rotigotine were observed when domperidone was administered concomitantly with rotigotine. The lack of pharmacokinetic interactions indicates that a dose adjustment of rotigotine transdermal patch is not necessary with concomitant use of domperidone.