prostaglandin-d2 and 9-(tetrahydro-2-furyl)-adenine

Cycloxygenase inhibitors have been shown to prevent angiogenesis in some circumstances, suggesting that growth of capillary pericytes or endothelial cells may be regulated by prostaglandins (PGs). The present study tests the effects of PGs on the growth of human retinal capillary pericytes.. Cell growth was assayed by formazan formation and 5-bromo-2'-deoxyuridine (BrdU) incorporation. The expression of mRNAs corresponding to c-fos, PG receptors, and VEGF was examined by RT-PCR. Signal transduction was evaluated by immunoblot analysis using phosphospecific antibodies against mitogen-activated protein kinases (MAPKs) and cAMP response element-binding protein (CREB). Synthesis of cAMP was inhibited with the adenyl cyclase inhibitor SQ22536. A reporter gene (luciferase) assay was conducted using the expression vector pSVOADelta5' containing the 379-bp c-fos promoter with and without a mutation in cAMP response element (CRE). RESULTS. PGD2 treatment induced c-fos mRNA, stimulated pericyte growth, and increased expression of VEGF mRNA. PGE2 and -F(2alpha) had similar effects on c-fos induction and pericyte growth, whereas PGI2 was ineffective. RT-PCR confirmed that mRNAs corresponding to the receptors for PGD2, -E2, -F(2alpha), and -I(2) were expressed in human retinal pericytes. Stimulation by PGD2 led to phosphorylation of CREB, but had negligible effect on phosphorylation of p44/42 MAPK. The adenylyl cyclase inhibitor inhibited CREB activation and c-fos induction by PGD2. In a reporter gene assay, c-fos induction occurred only with wild-type c-fos promoter. Mutation in CRE eliminated the response to PGD2.. PGD2 promotes the growth of retinal capillary pericytes by signaling through cAMP and CREB. The findings underscore the importance of PGs in the growth of human retinal capillary pericytes and raise the possibility that PGs may play a role in proliferative retinopathies.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Cell Division; Cyclic AMP Response Element-Binding Protein; DNA; Electrophoresis, Polyacrylamide Gel; Endothelial Growth Factors; Enzyme Inhibitors; Humans; Lymphokines; Male; Middle Aged; Pericytes; Phosphorylation; Prostaglandin D2; Proto-Oncogene Proteins c-fos; Receptors, Prostaglandin; Retinal Vessels; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of a large group of nuclear receptors controlling the proliferation of peroxisomes that is involved in the downregulation of macrophage functions. Here, we report that PPAR-gamma was constitutively expressed in rat primary microglial cultures and that such expression was downregulated during microglial activation by endotoxin (LPS). The presence of the PPAR-gamma natural ligand 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) counteracted the repression of PPAR-gamma expression caused by LPS. In microglial cultures stimulated by LPS, interferon-gamma (IFN-gamma) or by their combination, 15d-PGJ2 reduced the production of nitric oxide (NO) and the expression of inducible NO synthase (iNOS). The inhibitory effect was dose-dependent and did not involve an elevation of cyclic AMP, a second messenger known to inhibit NOS expression in microglia. In addition, 15d-PGJ2 down-regulated other microglial functions, such as tumour necrosis factor-alpha (TNF-alpha) synthesis and major histocompatibility complex class II (MHC class II) expression. The effects of 15d-PGJ2 occurred, at least in part, through the repression of two important transcription factors, the signal transducer and activator of transcription 1 and the nuclear factor kappaB, known to mediate IFN-gamma and LPS cell signalling. Our observations suggest that 15d-PGJ2, the synthesis of which is likely to occur within the brain, could play an important role in preventing brain damage associated with excessive microglial activation.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Blotting, Western; Cells, Cultured; Dinoprostone; DNA-Binding Proteins; Enzyme Inhibitors; Interferon-gamma; Lipopolysaccharides; Microglia; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin D2; Rats; Receptors, Cytoplasmic and Nuclear; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transcription Factors; Tumor Necrosis Factor-alpha

This study shows that dazoxiben, a selective inhibitor of thromboxane A2-synthetase in human platelets, inhibited arachidonic acid-induced platelet aggregation in platelet-rich plasma samples from four out of 16 healthy volunteers. In these four "responder" samples, the anti-aggregating effect of dazoxiben was prevented by the compound SQ 22536, a 9-substituted adenine analogue, endowed with an inhibitory activity on adenylate-cyclase. The compound SQ 22536 also counteracted the antiaggregating effect of prostaglandin D2, a known activator of platelet adenylate-cyclase. When platelet thromboxane A2-synthetase was blocked by dazoxiben, a marked increase of prostaglandin D2 was concomitantly observed both in "responder" and "non responder" samples. The compound SQ 22536 blunted the increase in platelet cAMP caused by either dazoxiben and sodium arachidonate or prostaglandin D2. It is suggested that the antiaggregating effect of dazoxiben is mediated by newly synthesized prostaglandin D2. The latter acts by stimulating adenylate-cyclase and increasing cAMP levels. The compound SQ 22536 prevents both phenomena. In "non responder" samples some factors--still to be defined--might counteract similarly to the compound SQ 22536 the antiaggregating activity of PGD2.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Blood Platelets; Cyclic AMP; Humans; Imidazoles; Oxidoreductases; Platelet Aggregation; Prostaglandin D2; Prostaglandins D; Thromboxane A2; Thromboxane-A Synthase

Dazoxiben, a thromboxane synthase inhibitor, inhibits arachidonic acid induced aggregation in platelet-rich plasma from some donors only ("responders"). We have studied the effect of dazoxiben in vitro on platelet aggregation and prostaglandin (PG) metabolism and the influence of the incubation period and of exogenously added serum albumin (SA). SA, which increases the production of anti-aggregatory PGD2 from cyclic endoperoxides, induced "non-responder" human platelets to respond. With rabbit platelets, however, that are insensitive to PGD2, exogenous SA failed to potentiate dazoxiben-induced inhibition. The ratio between PGD2 and TXB2 + PGE2 formed was crucial in determining the response of human platelets to dazoxiben: whenever this ratio was high, platelet aggregation was inhibited. SQ 22536, an adenylate cyclase inhibitor, and NO164, a PGD2 antagonist, reversed the inhibition by dazoxiben in human platelet-rich plasma, stressing the importance of a PGD2 mediated rise of cyclic AMP for the effectiveness of a thromboxane synthase inhibitor.

Topics: Adenine; Adult; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Female; Humans; Imidazoles; Male; Middle Aged; Oxidoreductases; Platelet Aggregation; Prostaglandin D2; Prostaglandins D; Serum Albumin; Thromboxane-A Synthase