Prostacyclin Mimetic Iloprost Research Articles

An Innovative Therapy for the Treatment of Severe Frostbite Using the Prostacyclin Mimetic Iloprost

An Innovative Therapy for the Treatment of Severe Frostbite Using the Prostacyclin Mimetic Iloprost

Thrombospondin-1 promotes hemostasis through modulation of cAMP signaling in blood platelets

AbstractThrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1–deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1−/− platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1–deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1−/− platelets. By contrast, incubation of TSP-1−/− platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1−/− platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1−/− mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.

PGE2 reverses Gs-mediated inhibition of platelet aggregation by interaction with EP3 receptors, but adds to non-Gs-mediated inhibition of platelet aggregation by interaction with EP4 receptors

Prostaglandin E2 (PGE2) has intriguing effects on platelet function in the presence of agents that raise cyclic adenosine 3′5′-monophosphate (cAMP). PGE2 reverses inhibition of platelet aggregation by agents that stimulate cAMP production via a Gs-linked receptor, but adds to the inhibition of platelet function brought about by agents that raise cAMP through other mechanisms. Here, we used the EP receptor antagonists DG-041 (which acts at the EP3 receptor) and ONO-AE3-208 (which acts at the EP4 receptor) to investigate the role of these receptors in mediating these effects of PGE2. Platelet aggregation was measured in platelet-rich plasma obtained from healthy volunteers in response to adenosine diphosphate (ADP) using single platelet counting. The effects of a range of concentrations of PGE2 were determined in the presence of (1) the prostacyclin mimetic iloprost, which operates through Gs-linked IP receptors, (2) the cAMP PDE inhibitor DN9693 and (3) the direct-acting adenylate cyclase stimulator forskolin. Vasodilator-stimulated phosphoprotein (VASP) phosphorylation was also determined as a measure of cAMP. PGE2 reversed the inhibition of aggregation brought about by iloprost; this was prevented in the presence of the EP3 antagonist DG-041, indicating that this effect of PGE2 is mediated via the EP3 receptor. In contrast, PGE2 added to the inhibition of aggregation brought about by DN9693 or forskolin; this was reversed by the EP4 antagonist ONO-AE3-208, indicating that this effect of PGE2 is mediated via the EP4 receptor. Effects on aggregation were accompanied by corresponding changes in VASP phosphorylation. The dominant role of EP3 receptors circumstances where cAMP is increased through a Gs-linked mechanism may be relevant to the situation in vivo where platelets are maintained in an inactive state through constant exposure to prostacyclin, and thus the main effect of PGE2 may be prothrombotic. If so, the results described here further support the potential use of an EP3 receptor antagonist in the control of atherothrombosis.

Regulation of Human Vascular Protease-Activated Receptor-3 through mRNA Stabilization and the Transcription Factor Nuclear Factor of Activated T Cells (NFAT)

Thrombin promotes vascular smooth muscle cell (SMC) proliferation and inflammation via protease-activated receptor (PAR)-1. A further thrombin receptor, PAR-3, acts as a PAR-1 cofactor in some cell-types. Unlike PAR-1, PAR-3 is dynamically regulated at the mRNA level in thrombin-stimulated SMC. This study investigated the mechanisms controlling PAR-3 expression. In human vascular SMC, PAR-3 siRNA attenuated thrombin-stimulated interleukin-6 expression and extracellular signal-regulated kinases 1/2 phosphorylation, indicating PAR-3 contributes to net thrombin responses in these cells. Thrombin slowed the decay of PAR-3 but not PAR-1 mRNA in the presence of actinomycin D and induced cytosolic shuttling and PAR-3 mRNA binding of the mRNA-stabilizing protein human antigen R (HuR). HuR siRNA prevented thrombin-induced PAR-3 expression. By contrast, forskolin inhibited HuR shuttling and destabilized PAR-3 mRNA, thus reducing PAR-3 mRNA and protein expression. Other cAMP-elevating agents, including the prostacyclin-mimetic iloprost, also down-regulated PAR-3, accompanied by decreased HuR/PAR-3 mRNA binding. Iloprost-induced suppression of PAR-3 was reversed with a myristoylated inhibitor of protein kinase A and mimicked by phorbol ester, an inducer of cyclooxygenase-2. In separate studies, iloprost attenuated PAR-3 promoter activity and prevented binding of nuclear factor of activated T cells (NFAT2) to the human PAR-3 promoter in a chromatin immunoprecipitation assay. Accordingly, PAR-3 expression was suppressed by the NFAT inhibitor cyclosporine A or NFAT2 siRNA. Thus human PAR-3, unlike PAR-1, is regulated post-transcriptionally via the mRNA-stabilizing factor HuR, whereas transcriptional control involves NFAT2. Through modulation of PAR-3 expression, prostacyclin and NFAT inhibitors may limit proliferative and inflammatory responses to thrombin after vessel injury.

Regulation of protease-activated receptor-1 by vasodilatory prostaglandins via NFAT

We recently reported that prostacyclin suppresses protease-activated receptor-1 (PAR-1) in human vascular smooth muscle cells (VSMC) via cyclic AMP and protein kinase A. This study examines the downstream mechanisms, particularly the role of nuclear factor of activated T-cells (NFAT). Human saphenous vein VSMC were exposed to phorbol 12-myristate 13-acetate (PMA) to induce endogenous cyclooxygenase-2-dependent prostaglandin generation. This was found to attenuate PAR-1 expression; similar suppression was seen with the EP2-prostaglandin receptor agonist butaprost. Stimulation of the 'exchange protein directly activated by cyclic AMP' (EPAC) was without effect. The NFAT inhibitor cyclosporin A (CsA) or NFAT2 siRNA both reduced PAR-1 mRNA and protein expression and prevented the stimulatory effects of thrombin or PAR-1 activating peptide (TFLLRN) on ERK1/2 phosphorylation and interleukin-6 expression. CsA or mutation of the NFAT binding motif in the PAR-1 promoter also blunted PAR-1 promoter activity (luciferase reporter assay). These inhibitory actions of CsA were comparable to those of the prostacyclin-mimetic iloprost, and both CsA and iloprost similarly attenuated nuclear NFAT2 localization and binding to the PAR-1 promoter (chromatin immunoprecipitation assay). This study provides the first evidence that NFAT2 contributes to the transcriptional control of PAR-1 in human VSMC and that PKA-dependent NFAT2 inhibition represents a mechanism by which vasodilatory prostaglandins regulate the vascular actions of thrombin.

Impaired glycocalyx barrier properties and increased capillary tube haematocrit

Impaired glycocalyx barrier properties and increased capillary tube haematocrit

Regulation of Thrombomodulin Expression in Human Vascular Smooth Muscle Cells by COX-2–Derived Prostaglandins

There is concern that cyclooxygenase (COX)-2 inhibitors may promote atherothrombosis by inhibiting vascular formation of prostacyclin (PGI2) and an increased thrombotic risk of COX-2 inhibitors has been reported. It is widely accepted that the prothrombotic effects of COX-2 inhibitors can be explained by the removal of platelet-inhibitory PGI2. Using microarray chip technology, we have previously demonstrated that thrombomodulin (TM) mRNA is upregulated in cultured human coronary artery smooth muscle cells by the stable prostacyclin mimetic iloprost. This study is the first to demonstrate a stimulation of the expression of functionally active thrombomodulin in human smooth muscle cells by prostaglandins, endogenously formed via the COX-2 pathway. Because TM is an important inhibitor of blood coagulation, these findings provide a novel platelet-independent mechanism to explain the prothrombotic effects of COX-2 inhibitors. The full text of this article is available online at http://circres.ahajournals.org.

Iloprost down-regulates the expression of the growth regulatory gene Cyr61 in human vascular smooth muscle cells

Prostacyclin and its mimetics have repeatedly been shown to act antiatherogenic and to inhibit neointima formation in several animal models of vascular injury. Treatment of human vascular smooth muscle cells with the prostacyclin mimetic iloprost (100 nm) drastically reduces expression of Cyr61, encoding the growth-regulatory cystein-rich angiogenic protein, without affecting the degradation rate of Cyr61 mRNA. Thrombin-induced Cyr61 expression was inhibited completely in the presence of iloprost. It is concluded that vasoprotective actions of prostacyclin in vivo may in part be due to inhibition of expression of the growth regulatory gene Cyr61 at sites of vascular lesions.

Regulation of cyclooxygenase-2 expression by iloprost in human vascular smooth muscle cells: Role of transcription factors CREB and ICER

Prostaglandin-endoperoxide synthase-2 (PGH-synthase) or cyclooxygenase-2 (COX-2) is inducible by a variety of stimuli, e.g. inflammatory mediators, growth factors and hormones and is believed to be responsible for the majority of inflammatory prostanoid production. Moreover, it has been demonstrated that COX-2 contributes substantially to prostacyclin-synthesis in patients with atherosclerosis. In this study, we demonstrate an up-regulation of COX-2 mRNA, protein and product formation by the prostacyclin-mimetic iloprost in human vascular smooth muscle cells (hSMC). COX-2 mRNA expression was induced transiently between 1 and 6 hr and returned to basal levels after 16 hr of iloprost stimulation. COX-2 protein was induced concomitantly between 3 and 6 hr of iloprost stimulation. This was accompanied by an increase in PGI 2 formation. Forskolin, a direct activator of adenylyl cyclase, and dibutyryl cAMP, a cell-permeable cAMP analogue-induced COX-2 mRNA, suggesting a cAMP-dependent COX-2 expression in hSMC. Iloprost-induced COX-2 protein expression and PGI 2 formation was synergistically elevated by co-stimulation with the phorbolester PMA (phorbol-12-myristate-13-acetate). It is concluded, that the observed up-regulation of COX-2 with subsequent release of newly synthesized PGI 2 and the synergistic effect of iloprost and phorbolester on PGI 2 formation provide a positive feedback of prostaglandins on their own synthesizing enzyme. This might be important for control of hSMC proliferation, migration and differentiation as well as inhibition of platelet aggregation.

Activation of IP and EP 3 receptors alters cAMP-dependent cell migration

Migration of vascular smooth cells from the media to the intima essentially contributes to neointima formation after percutaneous transluminal angioplasty and stent implantation. The stable prostacyclin mimetic iloprost has been shown to inhibit neointima formation in experimental restenosis, but it is currently unknown whether this may be caused by an antimigratory effect. Hence, the present study analyses (i) the influence of G s-coupled prostacyclin (IP) receptors on cell migration and (ii) verifies whether EP 3 receptors with opposite (i.e., G i) coupling may conversely stimulate cell migration. In a modified Boyden chamber model, it was shown that iloprost dose-dependently inhibits the migration of primary human arterial smooth muscle cells, which constitutively express the IP receptor. On the other hand, human arterial smooth muscle cell migration was stimulated by the EP 3 receptor agonist M&B 28.767. To independently study the effects of these receptors, IP or EP 3 receptors were stably overexpressed in chinese hamster ovary cells (CHO-IP and CHO-EP 3). Chemotaxis of CHO cells transfected with G s-coupled IP receptors was concentration-dependently inhibited by iloprost (2–100 nM), while there was no effect of iloprost on mock-transfected CHO. By contrast, CHO-cells that overexpressed EP 3 receptors showed a significant, concentration dependent (1–100 nM) increase of cell migration in presence of the selective EP 3 agonist M&B 28.767. It is concluded that the prostacyclin mimetic iloprost inhibits vascular cell migration, which probably depends on a G s-mediated increase of intracellular cAMP. EP 3 receptors conversely stimulate CHO migration.

Endothelial dysfunction of coronary resistance vessels in apoE-/- mice involves NO but not prostacyclin-dependent mechanisms.

We have analyzed the extent of endothelial dysfunction in cardiac resistance vessels of hyperlipidaemic apoE-/- mice and explored whether NO and/or prostacyclin dependent pathways are involved. Coronary resistance was measured in isolated perfused hearts from WT and apoE-/- mice. To discriminate between NO and PGI(2)-dependent flow responses, we made use of the finding that acetylcholine (ACh) predominantly activates the prostaglandin pathway whereas bradykinin (Bk) mainly acts via NO in murine cardiac resistance vessels. Basal coronary flow as well as the ACh induced vasodilation (0.1-1 microM) were not different between WT and apoE-/- hearts (flow increase+100%). Similarly, vasodilation in response to the prostacyclin mimetic iloprost reached the same levels. In contrast, the Bk-stimulated [3.3 microM Bk] coronary flow was reduced from 31.6+/-4.2 in WT to 19.2+/-2.7 ml min(-1) g(-1) in apoE-/- hearts. NOS inhibition by ethylisothiourea (ETU, 10 microM) reduced basal as well as Bk-stimulated coronary flow in WT and apoE-/- hearts to the same extent. RT-PCR and Western analysis demonstrated that neither eNOS expression nor protein levels were reduced. Similarly, the flow response to the NO donor SNAP (0.3-33 microM) was not altered suggesting that soluble guanylyl cyclase was not affected. Intracoronary application of superoxide dismutase augmented the Bk-induced vasodilation of apoE-/- hearts almost back to WT levels (26.6+/-3.3 ml min(-1) g(-1)). In line with this finding the NADPH induced O(2)(-) formation was enhanced in cardiac extracts from apoE-/- hearts. apoE-/- hearts develop a hemodynamically relevant endothelial dysfunction at the level of coronary resistance vessels most likely via inactivation of bioavailable NO by superoxide anions. The function of the prostacyclin system is not altered.

The cardioprotective actions of iloprost in myocardial ischemia of the rabbit can be separated from its vasodilatory effects mediated by KATP(+)-channel opening.

The modification of cardioprotective actions of iloprost by K(+)-channel blockade was studied in ischemic rabbit hearts. Glibenclamide, a blocker of ATP-dependent K(+)-channels, prevented coronary vasodilation mediated by the prostacyclin mimetic iloprost. In contrast, the cardioprotective effects of iloprost which were determined from prevention of ischemia induced rise in left ventricular enddiastolic pressure and loss of cytosolic troponin T in hearts made globally ischemic for two hours were not affected by glibenclamide. It is concluded that the cardioprotective action of iloprost can be separated from ist coronary vasodilator effect mediated by opening KATP(+)-channels.

Inhibition of human platelets and polymorphonuclear neutrophils by the potent and metabolically stable prostaglandin D 2 analog ZK 118.182

The actions of the novel metabolically stable and selective prostaglandin D 2 receptor agonist ZK 118.182 (( 5Z,13E)- (9R,11R,15S)-9-chloro-15-cyclohexyl-16,17,18,19,20-pentanor-3-oxa-5,13-prostadienoic acid) were studied in human platelets and polymorphonuclear neutrophils in vitro and compared to the naturally occuring agonist prostaglandin D 2. ZK 118.182 inhibited collagen and ADP induced platelet aggregation more potently than prostaglandin D 2 (IC 50: 15 nM versus 60 nM) but was less effective than the stable prostacyclin mimetic iloprost (IC 50: 3 nM). The same rank order of potencies was observed for the inhibition of collagen-induced platelet ATP secretion. A dose-dependent activation of adenylate cyclase could be demonstrated by ZK 118.182 which was comparable to that of prostaglandin D 2 with respect to the concentration needed for half maximal stimulation (ED 50) maximal cAMP level achievable. ZK 118.182 also dose dependendly reduced the formyl methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF) induced activation of polymorphonuclear neutrophils. Both, the oxygen burst resulting in the generation of superoxide anions and the degranulation of polymorphonuclear neutrophils accompanied by release of the lysosomal enzyme β-glucuronidase, were significantly and dose dependently inhibited. ZK 118.182 was more potent than prostaglandin D 2 in inhibiting polymorphonuclear neutrophil activation in all tests performed. In summary, ZK 118.182 is a prostaglandin D 2 mimetic exerting potent inhibitory effects on human platelets and polymorphonuclear neutrophils.

Oral naftidrofuryl prevents platelet hyperreactivity ex vivo and inhibits functional desensitization to prostacyclin in hypercholesterolemic rabbits.

Among other mediators, platelet-derived serotonin (5-HT) may contribute to thromboembolic complications of atherosclerosis. We determined whether long-term oral treatment with the 5-HT2 antagonist naftidrofuryl (NAF, 50 mg/kg daily for 12 weeks) alters platelet function in cholesterol-fed (1%) rabbits. Hypercholesterolemia resulted in marked platelet hyperreactivity to collagen and ADP. This included increased aggregation, ATP secretion, and thromboxane formation; e.g., collagen-induced (1.2 micrograms/ml) platelet aggregation was stimulated to 210 +/- 10 mm/30 s in cholesterol-fed rabbits as compared with 108 +/- 9 mm/30 s in rabbits fed a standard diet (p < 0.05). Inhibition of ADP-stimulated platelet activation by the prostacyclin mimetic iloprost was significantly reduced. NAF did not reduce plasma cholesterol in hypercholesterolemia, but prevented enhanced platelet aggregation, thromboxane formation, and ATP secretion. NAF treatment significantly reduced collagen-induced (1.2 micrograms/ml) aggregation to 81 +/- 20 mm/30 s in these animals (p < 0.05). NAF also inhibited functional desensitization of platelets to iloprost, but did not alter the impaired binding of [3H]iloprost to platelet membranes in hypercholesterolemic animals. NAF also did not change any of these parameters in normocholesterolemic rabbits. These data suggest beneficial effects of NAF on platelet hyperreactivity in experimental hypercholesterolemia which may also be relevant for its clinical use.

Cerebrovascular effects of prostanoids: In Vitro studies in feline middle cerebral and basilar artery

The efect of prostaglandin (PG) E 2, F 2α, the thromboxane-A 2 mimetic U46619 (9,11-dideoxy-9α, 11α-methaneopoxy-prostaglandin F 2α) and the prostacyclin mimetic iloprost was investigated in cat middle cerebral and basilar arteries in vitro precontracted with 5-hydroxytryptamine (5-HT) (50nM) in the absence and presence of the cyclooxygenase inhibitor indomethacin or the thromboxane receptor blocker AH23848b [1α(z), 2β, 5α]-(+)-7-[5-[1,1′-(biphenyl)-4-yl] methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid). PGF 2α and U46619 both produced further concentration-related contractions of basilar and middle cerebral artery, U46619 being approximately 1,000 times more potent than PGF 2α. Iloprost produced concentration-related relaxations of precontracted basilar and middle cerebral artery, the mean maximum relaxations produced at a concentration of 1.3μM being 57.3% and 80.6%, respectively of the contraction produced by 50nM 5-HT. PGE 2, 100nM relaxed the basilar and middle cerebral artery, 46.7% and 38.5% respectively. However, at 1μM, PGE 2 caused contraction. Indomethacin, 2.8μM had no effect on contractile or relaxant responses to any of the prastanoids. Oxyhaemoglobin inhibited the relaxation of both arterial preparations but had no effect on responses to PGE 2 or iloprost. The thromboxane-receptor blocker AH23848B antagonised the contractile responses to U46619, PGF 2α and PGE 2 and had no effect against relaxant responses to PGE 2 or iloprost. It is concluded that both contraction- and relaxation-inducing prostanoid receptors are present in the in vitro preparation of feline basilar and middle cerebral artery. Under sustained tension conditions, endothelial factors do not appear to be involved in the responses to dilating prostanoids.

Cerebrovascular effects of prostanoids

The effect of prostaglandin (PG) E2, F2 alpha, the thromboxane A2 mimetic U46619 (9,11-dideoxy-9 alpha,11 alpha-methanoepoxy-prostaglandin F2 alpha) and the prostacyclin mimetic iloprost was investigated in feline and rat pial arteries in-situ using perivascular microapplication and measurement of vascular diameter. U46619 induced a concentration-dependent vasoconstriction with a maximal response of 24% at 10(-6) mol.l-1 and an EC50 of 4.9 x 10(-8) mol.L-1. This effect was inhibited by the thromboxane receptor blocking drugs AH23848 B [[1 alpha(z),2 beta,5 alpha]-(+/-)-7-[5-[(1,1'-(biphenyl)-4- yl]methoxy]-2-(4-morpholinyl)-3-oxocyclo-pentyl]-4-heptenoic acid)] and EP092 [(+/-)-5-endo-(6'-carboxyhex-2'z-enyl)-6-exo[1'-[N- (phenylthiocarbamoyl)-hydrazano]-ethyl]-bicyclo[2,2,1]-hepta ne], indicating the involvement of thromboxane-prostanoid-receptors. PGF2 alpha produced vasodilatation in cats with an increase in vessel diameter of 30% at 10(-3) mol.l-1, but constricted rat pial arteries concentration-dependently with a maximal response of 23% at 10(-4) mol.l-1. PGE2 and iloprost induced concentration-dependent (10(-9)-10(-5) mol.l-1)) dilatations with apparent maximal responses of 39% and 34% at 10(-5) mol.l-1, respectively. The corresponding EC50 values were 2.45 x 10(-7) mol.l-1 (PGE2) and 3.5 x 10(-7) mol.l-1 (iloprost). These data demonstrate the presence of prostanoid receptors mediating constriction and dilatation under in-situ conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of Oxygen-Centered Free Radical Formation by the Stable Prostacyclin-Mimetic Iloprost (ZK 36 374) in Acute Myocardial Ischemia

Inhibition of Oxygen-Centered Free Radical Formation by the Stable Prostacyclin-Mimetic Iloprost (ZK 36 374) in Acute Myocardial Ischemia