Nikolaus Marx

Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells

Migration of vascular smooth muscle cells (VSMCs) plays an important role in atherogenesis and restenosis after arterial interventions. The expression of matrix metalloproteinases (MMPs), particularly MMP-9, contributes to VSMC migration. This process requires degradation of basal laminae and other components of the arterial extracellular matrix. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor family, regulate gene expression after activation by various ligands. Recent studies have suggested opposing effects of PPAR gamma (PPARgamma) activation on atherogenesis. The present study tested the hypotheses that human VSMCs express PPAR alpha (PPARalpha) and PPARgamma and that PPAR agonists in VSMCs modulate MMP-9 expression and activity, as well as VSMC migration. Human VSMCs expressed PPARalpha and PPARgamma mRNA and protein. Treatment of VSMCs with the PPARgamma ligands troglitazone and the naturally occurring 15-deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) decreased phorbol 12-myristate 13-acetate-induced MMP-9 mRNA and protein levels, as well as MMP-9 gelatinolytic activity in the supernatants in a concentration-dependent manner. Six different PPARalpha activators lacked such effects. Addition of prostaglandin F2alpha, known to limit PPARgamma activity, diminished the MMP-9 inhibition seen with either troglitazone or 15d-PGJ2, further implicating PPARgamma in these effects. Finally, troglitazone and 15d-PGJ2 inhibited the platelet-derived growth factor-BB-induced migration of VSMCs in vitro in a concentration-dependent manner. PPARgamma activation may regulate VSMC migration and expression and activity of MMP-9. Thus, PPARgamma activation in VSMCs, via the antidiabetic agent troglitazone or naturally occurring ligands, may act to counterbalance other potentially proatherosclerotic PPARgamma effects.

PPARα Activators Inhibit Cytokine-Induced Vascular Cell Adhesion Molecule-1 Expression in Human Endothelial Cells

Background—Adhesion molecule expression on the endothelial cell (EC) surface is critical for leukocyte recruitment to atherosclerotic lesions. Better understanding of transcriptional regulation of adhesion molecules in ECs may provide important insight into plaque formation. Peroxisome proliferator–activated receptor-α (PPARα), a member of the nuclear receptor family, regulates gene expression in response to certain fatty acids and fibric acid derivatives. The present study investigated PPARα expression in human ECs and their regulation of vascular cell adhesion molecule-1 (VCAM-1). Methods and Results—Immunohistochemistry revealed that human carotid artery ECs express PPARα. Pretreatment of cultured human ECs with the PPARα activators fenofibrate or WY14643 inhibited TNF-α–induced VCAM-1 in a time- and concentration-dependent manner, an effect not seen with PPARγ activators. Both PPARα activators decreased cytokine-induced VCAM-1 mRNA expression without altering its mRNA half-life. Transient transfection...

Macrophages in Human Atheroma Contain PPARγ: Differentiation-Dependent Peroxisomal Proliferator-Activated Receptor γ (PPARγ) Expression and Reduction of MMP-9 Activity through PPARγ Activation in Mononuclear Phagocytes in Vitro

Mononuclear phagocytes play an important role in atherosclerosis and its sequela plaque rupture in part by their secretion of matrix metalloproteinases (MMPs), including MMP-9. Peroxisomal proliferator-activated receptor γ (PPARγ), a transcription factor in the nuclear receptor superfamily, regulates gene expression in response to various activators, including 15-deoxy-Δ 12,14 -prostaglandin J 2 and the antidiabetic agent troglitazone. The role of PPARγ in human atherosclerosis is unexplored. We report here that monocytes/macrophages in human atherosclerotic lesions ( n = 12) express immunostainable PPARγ. Normal artery specimens ( n = 6) reveal minimal immunoreactive PPARγ. Human monocytes and monocyte-derived macrophages cultured for 6 days in 5% human serum expressed PPARγ mRNA and protein by reverse transcription-polymerase chain reaction and Western blotting, respectively. In addition, PPARγ mRNA expression in U937 cells increased during phorbol 12-myristate 13 acetate-induced differentiation. Stimulation of PPARγ with troglitazone or 15-deoxy-Δ 12,14 -prostaglandin J 2 in human monocyte-derived macrophages inhibited MMP-9 gelatinolytic activity in a concentration-dependent fashion as revealed by zymography. This inhibition correlates with decreased MMP-9 secretion as determined by Western blotting. Thus, PPARγ is present in macrophages in human atherosclerotic lesions and may regulate expression and activity of MMP-9, an enzyme implicated in plaque rupture. PPARγ is likely to be an important regulator of monocyte/macrophage function with relevance for human atherosclerotic disease.

T-lymphocyte Infiltration in Visceral Adipose Tissue. A Primary Event in Adipose Tissue Inflammation and the Development of Obesity-Mediated Insulin Resistance

Background—Adipose tissue inflammation may play a critical role in the pathogenesis of insulin resistance (IR). The present study examined the role of lymphocytes in adipose tissue inflammation and IR. Methods and Results—In a mouse model of obesity-mediated IR, high-fat diet (HFD) induced IR already after 5 weeks, which was associated with a marked T-lymphocyte infiltration in visceral adipose tissue. In contrast, recruitment of macrophages was delayed with an increase of MAC3-positive staining and F4/80 mRNA expression after 10 weeks of HFD, suggesting a dissociation of macrophage invasion into adipose tissue and IR initiation. In patients with type 2 diabetes, lymphocyte content in adipose tissue biopsies significantly correlated with waist circumference, a marker of IR. Immunohistochemical staining of human adipose tissue revealed the presence of mainly CD4-positive lymphocytes as well as macrophage infiltration. Most macrophages were HLA-DR–positive, reflecting activation through IFN&ggr;, a cytokine released from CD4-positive lymphocytes. Conclusions—Proinflammatory T-lymphocytes are present in visceral adipose tissue and may contribute to local inflammatory cell activation before the appearance of macrophages, suggesting that these cells could play an important role in the initiation and perpetuation of adipose tissue inflammation as well as the development of IR.

Peroxisome Proliferator-Activated Receptors and Atherogenesis Regulators of Gene Expression in Vascular Cells

A large body of data gathered over the past couple of years has identified the peroxisome proliferator-activated receptors (PPAR) alpha, gamma, and beta/delta as transcription factors exerting modulatory actions in vascular cells. PPARs, which belong to the nuclear receptor family of ligand-activated transcription factors, were originally described as gene regulators of various metabolic pathways. Although the PPARalpha, gamma, and beta/delta subtypes are approximately 60% to 80% homologous in their ligand- and DNA-binding domains, significant differences in ligand and target gene specificities are observed. PPARalpha is activated by polyunsaturated fatty acids and oxidized derivatives and by lipid-modifying drugs of the fibrate family, including fenofibrate or gemfibrozil. PPARalpha controls expression of genes implicated in lipid metabolism. PPARgamma, in contrast, is a key regulator of glucose homeostasis and adipogenesis. Ligands of PPARgamma include naturally occurring FA derivatives, such as hydroxyoctadecadienoic acids (HODEs), prostaglandin derivatives such as 15-deoxyDelta12,14-prostaglandin J2, and glitazones, insulin-sensitizing drugs presently used to treat patients with type 2 diabetes. Ligands for PPARbeta/delta are polyunsaturated fatty acids, prostaglandins, and synthetic compounds, some of which are presently in clinical development. PPARbeta/delta stimulates fatty acid oxidation predominantly acting in muscle. All PPARs are expressed in vascular cells, where they exhibit antiinflammatory and antiatherogenic properties. In addition, studies in various animal models as well as clinical data suggest that PPARalpha and PPARgamma activators can modulate atherogenesis in vivo. At present, no data are available relating to possible effects of PPARbeta/delta agonists on atherogenesis. Given the widespread use of PPARalpha and PPARgamma agonists in patients at high risk for cardiovascular disease, the understanding of their function in the vasculature is not only of basic interest but also has important clinical implications. This review will focus on the role of PPARs in the vasculature and summarize the present understanding of their effects on atherogenesis and its cardiovascular complications.

PPARγ Activation in Human Endothelial Cells Increases Plasminogen Activator Inhibitor Type-1 Expression PPARγ as a Potential Mediator in Vascular Disease

Abstract—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-γ (PPAR)γ is a ligand-activated transcription factor that regulates gene expression in response to various mediators such as 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and oxidized linoleic acid (9- and 13-HODE). The present study tested the hypotheses that human ECs express PPARγ and that this transcriptional activator regulates PAI-1 expression in this cell type. We found that human ECs contain both PPARγ mRNA and protein. Immunohistochemistry of human carotid arteries also revealed the presence of PPARγ in ECs. Bovine ECs transfec...

PPAR Activators as Antiinflammatory Mediators in Human T Lymphocytes Implications for Atherosclerosis and Transplantation-Associated Arteriosclerosis

Abstract— Activation of T lymphocytes and their ensuing elaboration of proinflammatory cytokines, such as interferon (IFN)-&ggr;, represent a critical step in atherogenesis and arteriosclerosis. IFN&ggr; pathways also appear integral to the development of transplantation-associated arteriosclerosis (Tx-AA), limiting long-term cardiac allograft survival. Although disruption of these IFN&ggr; signaling pathways limits atherosclerosis and Tx-AA in animals, little is known about inhibitory regulation of proinflammatory cytokine production in humans. The present study investigated whether activators of peroxisome proliferator-activated receptor (PPAR)&agr; and PPAR&ggr;, with their known antiinflammatory effects, might regulate the expression of proinflammatory cytokines in human CD4-positive T cells. Isolated human CD4-positive T cells express PPAR&agr; and PPAR&ggr; mRNA and protein. Activation of CD4-positive T cells by anti-CD3 monoclonal antibodies significantly increased IFN&ggr; protein secretion from 0 to 504±168 pg/mL, as determined by ELISA. Pretreatment of cells with well-established PPAR&agr; (WY14643 or fenofibrate) or PPAR&ggr; (BRL49653/rosiglitazone or pioglitazone) activators reduced anti-CD3-induced IFN&ggr; secretion in a concentration-dependent manner. PPAR activators also inhibited TNF&agr; and interleukin-2 protein expression. In addition, PPAR activators markedly reduced cytokine mRNA expression in these cells. Such antiinflammatory actions were also evident in cell-cell interactions with medium conditioned by PPAR activator-treated T cells attenuating human monocyte CD64 expression and human endothelial cell major histocompatibility complex class II induction. Thus, activation of PPAR&agr; and PPAR&ggr; in human CD4-positive T cells limits the expression of proinflammatory cytokines, such as IFN&ggr;, yielding potential therapeutic benefits in pathological processes, such as atherosclerosis and Tx-AA.

Peroxisome Proliferator-Activated Receptor-γ Activators Inhibit IFN-γ-Induced Expression of the T Cell-Active CXC Chemokines IP-10, Mig, and I-TAC in Human Endothelial Cells

Peroxisome proliferator-activated receptor-γ (PPARγ), a member of the nuclear hormone receptor superfamily originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate inflammatory responses. Given the importance of endothelial cell (EC)-derived chemokines in regulating leukocyte function and trafficking, we studied the effects of PPARγ ligands on the expression of chemokines induced in ECs by the Th1 cytokine IFN-γ. Treatment of ECs with PPARγ activators significantly inhibited IFN-γ-induced mRNA and protein expression of the CXC chemokines IFN-inducible protein of 10 kDa (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α-chemoattractant (I-TAC), whereas expression of the CC chemokine monocyte chemoattractant protein-1 was not altered. PPARγ activators decreased IFN-inducible protein of 10 kDa promoter activity and inhibited protein binding to the two NF-κB sites but not to the IFN-stimulated response element ISRE site. Furthermore, PPARγ ligands inhibited the release of chemotactic activity for CXC chemokine receptor 3 (CXCR3)-transfected lymphocytes from IFN-γ-stimulated ECs. These data suggest that anti-diabetic PPARγ activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation.

The Residual Risk Reduction Initiative: a call to action to reduce residual vascular risk in dyslipidaemic patients

Despite current standards of care aimed at achieving targets for low-density lipoprotein (LDL) cholesterol, blood pressure and glycaemia, dyslipidaemic patients remain at high residual risk of vascular events. Atherogenic dyslipidaemia, specifically elevated triglycerides and low levels of high-density lipoprotein (HDL) cholesterol, often with elevated apolipoprotein B and non-HDL cholesterol, is common in patients with established cardiovascular disease, type 2 diabetes, obesity or metabolic syndrome and is associated with macrovascular and microvascular residual risk. The Residual Risk Reduction Initiative (R3I) was established to address this important issue. This position paper aims to highlight evidence that atherogenic dyslipidaemia contributes to residual macrovascular risk and microvascular complications despite current standards of care for dyslipidaemia and diabetes, and to recommend therapeutic intervention for reducing this, supported by evidence and expert consensus. Lifestyle modification is an important first step. Additionally, pharmacotherapy is often required. Adding niacin, a fibrate or omega-3 fatty acids to statin therapy improves achievement of all lipid risk factors. Outcomes studies are evaluating whether these strategies translate to greater clinical benefit than statin therapy alone. In conclusion, the R3i highlights the need to address with lifestyle and/or pharmacotherapy the high level of residual vascular risk among dyslipidaemic patients who are treated in accordance with current standards of care.

Pioglitazone Reduces Neointima Volume After Coronary Stent Implantation A Randomized, Placebo-Controlled, Double-Blind Trial in Nondiabetic Patients

Background— Restenosis requiring reintervention limits the long-term success after coronary stent implantation. Thiazolidinediones, like pioglitazone or rosiglitazone, are oral antidiabetic drugs with additional antirestenotic properties. In a randomized, placebo-controlled, double-blind trial, we examined the effect of 6-month pioglitazone therapy on neointima volume after coronary stenting in nondiabetic coronary artery disease patients. Methods and Results— Fifty nondiabetic patients after coronary stent implantation were randomly assigned to pioglitazone (30 mg daily; pio) or placebo (control) treatment in addition to standard therapy, and neointima volume was assessed by intravascular ultrasound at the 6-month follow-up. Both groups were comparable with regard to baseline characteristics, angiographic lesion morphology, target vessel, and length of the stented segment. In addition, there were no statistical differences in minimal lumen diameter before and after intervention, as well as reference diameter after stent implantation. In this study population of nondiabetic patients, pio treatment did not significantly change fasting blood glucose, fasting insulin, or glycosylated hemoglobin levels, as well as lipid parameters. In contrast, pio treatment significantly reduced neointima volume within the stented segment, with 2.3±1.1 mm3/mm in the pio group versus 3.1±1.6 mm3/mm in controls (P=0.04). Total plaque volume (adventitia-lumen area) was significantly lower at follow-up in the pio group (11.2±3.2 mm3/mm) compared with controls (13.2±4.2 mm3/mm; P=0.04). Moreover, the binary restenosis rate was 3.4% in the pio group versus 32.3% in controls (P<0.01). Conclusions— Thus, 6-month treatment with pio significantly reduced neointima volume after coronary stent implantation in nondiabetic patients. These data bolster the hypothesis that antidiabetic thiazolidinediones, in addition to their metabolic effects, exhibit direct antirestenotic effects in the vasculature.