15-deoxy-delta(12-14)-prostaglandin-j2 and Arteriosclerosis

Thiazolidinediones, a new class of antidiabetic drugs that increase insulin sensitivity, have been shown to be ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Recent studies demonstrating that PPARgamma occurs in macrophages have focused attention on its role in macrophage functions. In this study, we investigated the effect of thiazolidinediones on monocyte proliferation and migration in vitro and the mechanisms involved. In addition, we examined the therapeutic potentials of thiazolidinediones for injured atherosclerotic lesions. Troglitazone and pioglitazone, the two thiazolidinediones, as well as 15-deoxy-delta12,14-prostaglandin J2 inhibited in a dose-dependent manner the serum-induced proliferation of THP-1 (human monocytic leukemia cells) and of U937 (human monoblastic leukemia cells), which permanently express PPARgamma. These ligands for PPARgamma also significantly inhibited migration of THP-1 induced by monocyte chemoattractant protein-1 (MCP-1). Troglitazone and 15-deoxy-delta12,14-prostaglandin J2 significantly suppressed the mRNA expression of the MCP family-specific receptor CCR2 (chemokine CCR2 receptor) in THP-1 at the transcriptional level. Furthermore, troglitazone significantly inhibited MCP-1 binding to THP-1. Oral administration of troglitazone to Watanabe heritable hyperlipidemic (WHHL) rabbits after balloon injury suppressed acute recruitment of monocytes/macrophages and accelerated re-endothelialization. These results suggest that thiazolidinediones have therapeutic potential for the treatment of diabetic vascular complications.

Topics: Alitretinoin; Angioplasty, Balloon; Animals; Aorta; Arteriosclerosis; Binding, Competitive; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CCL2; Chromans; Dose-Response Relationship, Drug; Endothelium, Vascular; Gene Expression Regulation; Humans; Macrophages; Male; Monocytes; Pioglitazone; PPAR gamma; Prostaglandin D2; Rabbits; Receptors, CCR2; Receptors, Chemokine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Time Factors; Tretinoin; Troglitazone; Vascular Endothelial Growth Factor A; Wound Healing

A functional myeloperoxidase (MPO) promoter polymorphism, -463GA, has been associated with incidence or severity of inflammatory diseases, including atherosclerosis and Alzheimer's disease, and some cancers. The polymorphism is within an Alu element encoding four hexamer repeats recognized by nuclear receptors (AluRRE). Here we show that peroxisome proliferator-activated receptor gamma (PPARgamma) agonists strongly regulate MPO gene expression through the AluRRE. Opposite effects were observed in granulocyte/macrophage colony-stimulating factor (GMCSF)- versus macrophage colony-stimulating factor (MCSF)-derived macrophages (Mphi): Expression was markedly up-regulated (mean 26-fold) in MCSF-Mphi and down-regulated (34-fold) in GMCSF-Mphi. This was observed with rosiglitazone and three other PPARgamma ligands of the thiazolidinedione class, as well as the natural prostaglandin metabolite 15-deoxy-Delta(12,14) prostaglandin J(2). The selective PPARgamma antagonist, GW9662, blocked both the positive and negative effects on MPO expression. Gel retardation assays showed PPARgamma bound hexamers 3/4, and estrogen receptor-alpha bound hexamers 1/2, with -463A in hexamer 1 enhancing binding. Estrogen blocked PPARgamma effects on MPO expression, especially for the A allele. Charcoal filtration of fetal calf serum eliminated the block of PPARgamma, whereas replenishing the medium with 17beta-estradiol reinstated the block. These findings suggest a model in which estrogen receptor binds the AluRRE, preventing PPARgamma binding to the adjacent site. The positive and negative regulation by PPARgamma ligands, and the block by estrogen, was also observed in transgenic mice expressing the G and A alleles. The mouse MPO gene, which lacks the primate-specific AluRRE, was unresponsive to PPARgamma ligands, suggesting the human MPO transgenes will enhance the utility of mouse models for diseases involving MPO, such as atherosclerosis and Alzheimer's.

Topics: Alu Elements; Animals; Arteriosclerosis; Binding Sites; Bone Marrow Cells; Cells, Cultured; Estradiol; Estrogen Receptor alpha; Estrogens; Gene Expression Regulation, Enzymologic; Genotype; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Peroxidase; Polymorphism, Genetic; Promoter Regions, Genetic; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, LDL; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Transcription Factors

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues, readily undergoes dehydration to yield the cyclopentenone-type PGs of the J(2) series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), which have been suggested to exert anti-inflammatory effects in vivo. Meanwhile, the mechanism of these effects is not well understood and the natural site and the extent of its production in vivo remain unclear. In the present study, we raised a monoclonal antibody specific to 15d-PGJ(2) and determined its production in inflammation-related events. The monoclonal antibody (mAb11G2) was raised against the 15d-PGJ(2)-keyhole limpet hemocyanin conjugate and was found to recognize free 15d-PGJ(2) specifically. The presence of 15d-PGJ(2) in vivo was immunohistochemically verified in the cytoplasm of most of the foamy macrophages in human atherosclerotic plaques. In addition, the immunostaining of lipopolysaccharide-stimulated RAW264.7 macrophages with mAb11G2 demonstrated an enhanced intracellular accumulation of 15d-PGJ(2), suggesting that the PGD(2) metabolic pathway, generating the anti-inflammatory PGs, is indeed utilized in the cells during inflammation. The activation of macrophages also resulted in the extracellular production of PGD(2), which was associated with a significant increase in the extracellular 15d-PGJ(2) levels, and the extracellular 15d-PGJ(2) production was reproduced by incubating PGD(2) in a cell-free medium and in phosphate-buffered saline. Moreover, using a chiral high performance liquid chromatography method for separation of PGD(2) metabolites, we established a novel metabolic pathway, in which PGD(2) is converted to 15d-PGJ(2) via an albumin-independent mechanism.

Topics: Animals; Antibodies, Monoclonal; Arteriosclerosis; Cell Line; Cell-Free System; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Cytoplasm; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Immunoblotting; Immunohistochemistry; Immunologic Factors; Inflammation; Isoenzymes; Macrophages; Membrane Proteins; Mice; Models, Biological; Models, Chemical; Phosphates; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Serum Albumin; Time Factors

Endothelial dysfunction or activation, elicited by oxidized low-density lipoprotein (OxLDL), has been implicated in the initiation and progression of atherosclerosis. We elucidated whether tumor necrosis factor-alpha (TNF-alpha)-induced endothelial OxLDL receptor, lectin-like OxLDL receptor-1 (LOX-1), mRNA expression is modified by peroxisome proliferator-activated receptor (PPAR) activators in cultured bovine aortic endothelial cells (BAEC). We confirmed that both PPARalpha and PPARgamma were expressed in BAEC by reverse transcription-polymerase chain reaction analysis. Natural PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and the thiazolidinediones, pioglitazone and troglitazone, decreased TNF-alpha-induced LOX-1 mRNA expression in BAEC. LOX-1 expression induced by phorbol 12-myristrate 13-acetate was also inhibited by 15d-PGJ(2). In contrast, PPARalpha ligands, Wy14643 and fenofibric acid, did not alter TNF-alpha-induced LOX-1 expression. TNF-alpha-induced immunohistochemical staining of LOX-1 was suppressed by 15d-PGJ(2) but not Wy14643. Taken together, PPARgamma activators inhibit TNF-alpha-induced LOX-1 expression in cultured BAEC, which may beneficially influence inflammatory responses in atherosclerosis.

Topics: Animals; Arteriosclerosis; Cattle; Cells, Cultured; Chromans; Drug Antagonism; Endothelium; Immunohistochemistry; Pioglitazone; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Receptors, LDL; Receptors, Oxidized LDL; RNA, Messenger; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation; Troglitazone; Tumor Necrosis Factor-alpha

Plasminogen activator inhibitor type-1 (PAI-1) is a major physiological inhibitor of fibrinolysis, with its plasma levels correlating with the risk for myocardial infarction and venous thrombosis. The regulation of PAI-1 transcription by endothelial cells (ECs), a major source of PAI-1, remains incompletely understood. Adipocytes also produce PAI-1, suggesting possible common regulatory pathways between adipocytes and ECs. Peroxisomal proliferator-activated receptor-gamma (PPAR)gamma is a ligand-activated transcription factor that regulates gene expression in response to various mediators such as 15-deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) and oxidized linoleic acid (9- and 13-HODE). The present study tested the hypotheses that human ECs express PPARgamma and that this transcriptional activator regulates PAI-1 expression in this cell type. We found that human ECs contain both PPARgamma mRNA and protein. Immunohistochemistry of human carotid arteries also revealed the presence of PPARgamma in ECs. Bovine ECs transfected with a PPAR response element (PPRE)-luciferase construct responded to stimulation by the PPARgamma agonist 15d-PGJ2 in a concentration-dependent manner, suggesting a functional PPARgamma in ECs. Treatment of human ECs with 15d-PGJ2, 9(S)-HODE, or 13(S)-HODE augmented PAI-1 mRNA and protein expression, whereas multiple PPARalpha activators did not change PAI-1 levels. Introduction of increasing amounts of a PPARgamma expression construct in human fibroblasts enhanced PAI-1 secretion from these cells in proportion to the amount of transfected DNA. Thus, ECs express functionally active PPARgamma that regulates PAI-1 expression in ECs. Our results establish a role for PPARgamma in the regulation of EC gene expression, with important implications for the clinical links between obesity and atherosclerosis.

Topics: Adipose Tissue; Animals; Arteriosclerosis; Blood Coagulation; Carotid Arteries; Cattle; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endothelium, Vascular; Enzyme Activation; Fibroblasts; Gene Expression; Genes, Reporter; Humans; Luciferases; Plasminogen Activator Inhibitor 1; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Saphenous Vein; Signal Transduction; Transcription Factors; Transfection

The formation of foam cells from macrophages in the arterial wall is characterized by dramatic changes in lipid metabolism, including increased expression of scavenger receptors and the uptake of oxidized low-density lipoprotein (oxLDL). We demonstrate here that the nuclear receptor PPARgamma is induced in human monocytes following exposure to oxLDL and is expressed at high levels in the foam cells of atherosclerotic lesions. Ligand activation of the PPARgamma:RXRalpha heterodimer in myelomonocytic cell lines induces changes characteristic of monocytic differentiation and promotes uptake of oxLDL through transcriptional induction of the scavenger receptor CD36. These results reveal a novel signaling pathway controlling differentiation and lipid metabolism in monocytic cells, and suggest that endogenous PPARgamma ligands may be important regulators of gene expression during atherogenesis.

Topics: Alitretinoin; Animals; Arteriosclerosis; CD36 Antigens; Cell Differentiation; Dimerization; Foam Cells; HL-60 Cells; Humans; Ligands; Lipoproteins, LDL; Macrophages; Membrane Proteins; Mice; Mice, Transgenic; Monocytes; Promoter Regions, Genetic; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Retinoic Acid; Receptors, Scavenger; Retinoid X Receptors; Rosiglitazone; Scavenger Receptors, Class B; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation; Tretinoin