Iloprost up-regulates vascular endothelial growth factor expression in human dental pulp cells in vitro and enhances pulpal blood flow in vivo.

INTRODUCTION: Prostacyclin (PGI2) is a biomolecule capable of enhancing angiogenesis and cellular proliferation. METHODS: We investigated the influence of a PGI2 analogue (iloprost) on dental pulp revascularization in vitro and in vivo by using human dental pulp cells (HDPCs) and a rat tooth injury model, respectively. Iloprost stimulated the human dental pulp cell mRNA expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF) in a significant dose-dependent manner. This mRNA up-regulation was significantly inhibited by pretreatment with a PGI2 receptor antagonist and forskolin (a protein kinase A activator). In contrast, a protein kinase A inhibitor significantly enhanced the iloprost-induced mRNA expression of VEGF, FGF-2, and PDGF. Pretreatment with a fibroblast growth factor receptor inhibitor attenuated the VEGF, FGF-2, and PDGF mRNA expression, indicating opposing regulatory mechanisms. RESULTS: The effect of iloprost on the dental pulp was investigated in vivo by using a rat molar pulp injury model. The iloprost-treated group exhibited a significant increase in pulpal blood flow at 72 hours compared with control. CONCLUSIONS: The present study indicates that iloprost may be a candidate agent to promote neovascularization in dental pulp tissue, suggesting the potential clinical use of iloprost in vital pulp therapy.

Effects of iloprost on calvarial sutures.

Premature fusion of calvarial sutures is the result of a long and complex reaction, and several growth factors including transforming growth factor beta and basic fibroblast growth factor have important role in this event. Several prostaglandins have important functions in local bone modeling and remodeling by autocrine and paracrine mechanisms. Although effects of prostaglandins on long bones were studied both experimentally and clinically, there are limited data about cranial bones and sutures. In this study, we investigated the effect of iloprost-a stable prostacyclin analogue, which is widely used for the treatment of pulmonary arterial hypertension even in early pregnancy, to rat calvarial sutures. In 2 study groups, iloprost was injected intraperitoneally 10 and 15 microg kg d, respectively. In the third group, dexamethasone 2 mg kg d + iloprost 15 microg kg d was injected intraperitoneally to antagonize the effects of iloprost. In every group, 4 rats were killed at the postoperative 15, 30, and 45 days, and specimens including the sagittal and frontal sutures were excised immediately. Routine histological and immunohistological staining were performed on the specimens. Morphological measurements were performed on the skulls, also. In histological evaluation, bone formation in the both frontal and sagittal suture area was increased and accelerated in iloprost groups. Dexamethasone inhibited the effects of iloprost on the third group. Expressions of transforming growth factor beta and basic fibroblast growth factor were also increased in immunohistological staining. In morphological measurements, statistically significant differences were found between control and study groups. Iloprost did not fused the rat calvarial sutures prematurely, but it narrowed the sagittal and frontal sutures especially after the second week of the study. This situation might effect the sutures of the babies of the pregnant patients with pulmonary arterial hypertension treated with iloprost. Cranial sutures, calvarial bones, and cranial shape of the babies of the pregnant patients who were treated with iloprost should be monitored to clarify the topic.

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

Evidence that Ca2+-activated K+ channels play a major role in mediating the vascular effects of iloprost and cicaprost.

The role of K+ channels in mediating vasorelaxation induced by two prostacyclin analogues was investigated in guinea-pig aorta. Iloprost caused substantial relaxation of tissues contracted with phenylephrine or 25 mM K+ but not 60 mM K+. In endothelial-denuded tissues, maximal relaxations to iloprost, cicaprost or isoprenaline were inhibited by approximately 40-50% with tetraethylammonium or iberiotoxin, both blockers of large conductance Ca2+-activated K+ (BKCa) channels. In contrast, the response to forskolin, an activator of adenylate cyclase was marginally inhibited by tetraethylammonium. The K(ATP) channel blocker, glibenclamide significantly augmented the response to iloprost but not cicaprost. These effects were largely inhibited by the EP1 receptor antagonist, 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid 2-[1-oxo-3(4-pyridinyl)propyl]hydrazide, monohydrochloride (SC-51089) and partially by indomethacin, suggesting that iloprost relaxation is counterbalanced by activation of EP1 receptors, in part through a constrictor prostaglandin. We conclude that BKCa channels play an important role in mediating the effects of iloprost and cicaprost and raises the possibility that cyclic AMP-independent pathways might be involved.

Iloprost, a stable analogue of PGI2, potentiates the hyperthermic effect of PGE2 in rats.

Centrally mediated effects of iloprost, a stable analogue of PGI2, on rectal temperature have been investigated in conscious rats. ICV administration of iloprost (100-1,000 ng, ICV) produced a dose-dependent, monophasic hyperthermic response that was not inhibited by indomethacin. When injected into the preoptic anterior hypothalamic (POAH) region, iloprost (2-50 ng/POAH) induced a biphasic increase in rectal temperature. While the first phase was inhibited by AH 6809, an E1-type prostaglandin (EP1) receptor antagonist, the second phase was abolished by indomethacin pretreatment. Iloprost was found not to alter rectal temperature when injected into the ventromedial hypothalamic area. Administration of iloprost into the POAH in a dose that had no effect on rectal temperature significantly potentiated the hyperthermic effect of PGE2 (50 ng, ICV). These findings suggest that the pyrogenic effect of iloprost is partly mediated by EP1 receptors located on the POAH. Regarding the similarities of iloprost and PGI2, it is further proposed that endogenous PGI2 might act to modulate hyperthermic effect of PGE2 released during arachidonic acid- or endogenous pyrogen-induced fever.

Stimulatory (EP1 and EP3) and inhibitory (EP2) prostaglandin E2 receptors in isolated ileal smooth muscle cells.

Isolated smooth muscle cells from the circular layer of pig and guinea-pig ileum were used to study the effect of prostaglandin E2 (PGE2) and three PGE2 receptor (EP) agonists; iloprost (EP1), butaprost (EP2) and enprostil (EP3). In pig cells, PGE2 and enprostil induced cell contraction (22.1 and 21.5% shortening of cell length, obtained at 10 nM for PGE2 and 1 nM for enprostil, respectively). Iloprost and butaprost had no contractile effect. However, the cholecystokinin octapeptide (CCK-8; 10 nM)-induced contraction was inhibited when cells were preincubated with iloprost or butaprost. In guinea-pig cells, PGE2, butaprost and iloprost induced cell contraction, whereas enprostil had no effect (23.1% for 10 nM PGE2, 22.8% for 1 nM butaprost and 22.6% for 10 nM iloprost). Preincubation with SC19220 (EP1 antagonist) inhibited the PGE2-, butaprost- and iloprost-induced contractions. When the contractile effect of PGE2, butaprost and iloprost was inhibited by addition of SC19220, these agents inhibited the cell contraction induced by CCK-8 (1 nM). Smooth muscle cells from guinea-pig and pig ileum express two PGE2 receptor subtypes that induce opposite effect. EP1 and EP3 receptors mediate cell contraction in guinea-pig and pig, respectively, whereas EP2 receptors mediate cell relaxation in both species.

Prostacyclin-induced vasodilation in rabbit heart is mediated by ATP-sensitive potassium channels.

We tested the hypothesis that prostacyclin and its stable analogue iloprost act as agonists of ATP-sensitive potassium channels (KATP) to induce vasodilation of the coronary circulation. The selective blocker of KATP, glibenclamide, was used as a probe for vasodilation mediated by KATP in saline-perfused rabbit hearts (constant flow, Langendorff preparation). Glibenclamide (10-300 nM) significantly increased coronary perfusion pressure and inhibited vasodilation induced by iloprost (1-30 nM), prostacyclin (10 nM), adenosine (0.3 microM), and cromakalim (0.1 microM), a known agonist of KATP. This potassium channel antagonist also inhibited vasodilation of rabbit hearts in response to 10 nM bradykinin in the presence of an inhibitor of nitric oxide synthase (30 microM NG-nitro-L-arginine). Because bradykinin-induced vasodilation is mediated by prostacyclin released from endothelial cells when nitric oxide synthesis is inhibited, these data indicate that glibenclamide is also effective against endogenous prostacyclin. The inhibitory effects of glibenclamide were selective: vasodilation induced by sodium nitroprusside (1-10 microM) or acetylcholine (1 microM) were not inhibited by this potassium channel antagonist. In addition, basal and bradykinin-stimulated release of 6-ketoprostaglandin F1 alpha was not affected by this antagonist of KATP. Glibenclamide also did not inhibit the activation of adenylate cyclase, as indicated by its lack of effect on adenosine 3',5'-cyclic monophosphate accumulation induced by iloprost (10 nM-1 microM) in bovine coronary arterial segments, a tissue in which iloprost-induced vascular smooth muscle relaxation is inhibited by glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase.

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64:1057-1068, 1991) that recognizes alpha-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal ("tethered-ligand") that activates the receptor. We report here that this receptor is involved in alpha-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by alpha- and zeta-thrombin addition and, to a much lesser extent, by gamma-thrombin. The alpha- and zeta-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the "tethered-ligand" thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the "tethered-ligand" receptor. Moreover, this data is consistent with the "tethered-ligand" receptor mediating the high affinity actions of alpha-thrombin.