Dennis Bruemmer

Peroxisome proliferator-activated receptor gamma: implications for cardiovascular disease.

Abstract—Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. PPAR&ggr; is expressed by macrophages, endothelial cells, and vascular smooth muscle cells. It regulates gene expression of key proteins involved in lipid metabolism, vascular inflammation, and proliferation contributing to atherogenesis and postangioplasty restenosis. The discovery of synthetic ligands for PPAR&ggr; has led to significant enhancement of our understanding of the mechanism of their ligand-dependent activation and subsequent biological effects, particularly with respect to the role of PPAR&ggr; in vascular pathophysiology. The thiazolidinedione PPAR&ggr; agonists not only improve insulin resistance in patients with type II diabetes but also exert a broad spectrum of antiatherogenic effects in vitro and in animal models of atherosclerosis. In this review, we summarize the important role of PPAR&ggr; as a molecular target for thiazolidinediones and its implications for the control of vascular inflammation and proliferation for the cardiovascular system.

Peroxisome Proliferator-Activated Receptor γ: Implications for Cardiovascular Disease

Abstract—Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. PPAR&ggr; is expressed by macrophages, endothelial cells, and vascular smooth muscle cells. It regulates gene expression of key proteins involved in lipid metabolism, vascular inflammation, and proliferation contributing to atherogenesis and postangioplasty restenosis. The discovery of synthetic ligands for PPAR&ggr; has led to significant enhancement of our understanding of the mechanism of their ligand-dependent activation and subsequent biological effects, particularly with respect to the role of PPAR&ggr; in vascular pathophysiology. The thiazolidinedione PPAR&ggr; agonists not only improve insulin resistance in patients with type II diabetes but also exert a broad spectrum of antiatherogenic effects in vitro and in animal models of atherosclerosis. In this review, we summarize the important role of PPAR&ggr; as a molecular target for thiazolidinediones and its implications for the control of vascular inflammation and proliferation for the cardiovascular system.

PGE2 is generated by specific COX-2 activity and increases VEGF production in COX-2-expressing human pancreatic cancer cells

Abstract In some cancers cyclooxygenase (COX) inhibition appears to be anti-mitogenic and anti-angiogenic, but the actions of COX-derived prostaglandins in pancreatic cancer (PaCa) are unknown. In this study COX-2 was detected in three of six PaCa cell lines while COX-1 was identified in all cell lines. COX-2 expression correlated with basal and arachidonic acid (AA) stimulated PGE2 production. PGE2 production was inhibited by the COX-2 inhibitor nimesulide. In COX-2 expressing cells, exogenous AA and PGE2 increased VEGF synthesis via the EP2 receptor. Whereas PGE2 stimulated intracellular cAMP formation in COX-2 positive and negative cells, 8-bromo cAMP stimulated VEGF production only in COX-2 expressing cells. Stimulating COX-2 expressing PaCa cell lines with AA enhanced migration of endothelial cells, an effect which was inhibited by a COX-2 inhibitor and EP2 receptor antagonist. These data identify a subset of human PaCa cell lines that express functional COX-2 enzyme. PGE2 generated by specific COX-2 activity increases VEGF secretion in human PaCa cells through an autocrine mechanism.

Signaling pathways involved in induction of GADD45 gene expression and apoptosis by troglitazone in human MCF-7 breast carcinoma cells

We previously reported that the PPARγ agonist troglitazone (TRO) inhibits proliferation and induces apoptosis in human MCF-7 breast carcinoma cells. To understand the mechanisms of antiproliferative and pro-apoptotic effects of TRO, we screened a limited DNA array containing 23 genes involved in regulating either the cell cycle and/or apoptosis. Four of the 23 genes screened exhibited regulation by TRO, with growth arrest and DNA damage-inducible gene 45 (GADD45) being the most strongly upregulated. TRO induced GADD45 mRNA expression in a time- and dose-dependent manner. Depletion of GADD45 by siRNA abrogated TRO-induced apoptosis in MCF-7 cells demonstrating the physiological relevance of GADD45 upregulation. Signaling pathways mediating TRO-induced GADD45 were also investigated. Several mitogen-activated protein kinase (MAPK) pathways were involved in the induction of GADD45 by TRO. Inhibition of the c-jun N-terminal kinase MAPK pathway by SP600125 partially abolished TRO-induced GADD45 mRNA, and protein expression and apoptosis. In contrast, inhibition of the p38 MAPK pathway by SB203580, or through overexpression of a dominant-negative mutant of p38 MAPK, augmented GADD45 mRNA induction and GADD45 promoter activation as well as cell apoptosis by TRO. Blockade of the extracellular signal-regulated kinase MAPK pathway by PD98059 also enhanced TROs effects on GADD45 and apoptosis. Two other PPARγ agonists pioglitazone and rosiglitazone did not induce GADD45 expression. Our finding of GADD45 induction by TRO may provide a new insight concerning the mechanisms for TROs antiproliferative and pro-apoptotic effects in breast cancer cells.

Egr-1 is a major vascular pathogenic transcription factor in atherosclerosis and restenosis.

The zinc finger transcription factor Egr-1 (early growth response factor-1), also known as nerve growth factorinduced-A (NGFI-A), zif268, Krox-24 and TIS8 was first identified by Sukhatme and colleagues [1]. It is the prototype of a family of zinc-finger transcription factors, that includes Egr-2, Egr-3 and Egr-4. Egr-1 is rapidly and transiently expressed in many different cell types in response to a variety of extracellular stimuli, including growth factors, cytokines and injurious stimuli [2,3]. Among the vascular cells known to express Egr-1 are endothelial cells (ECs), vascular smooth muscle cells (VSMC), monocytes and macrophages. Multiple stimuli associated with the development of vascular diseases like shear stress [4,5], mechanical stress [6], hypoxia [7,8], angiotensin II (AngII), and acute tissue injury [9] are capable of inducing Egr-1 expression. This review will survey the emerging evidence that Egr-1, like nuclear factor kappaB (NF-κB), orchestrates the transcriptional response to vascular injury.

A non-thiazolidinedione partial peroxisome proliferator-activated receptor γ ligand inhibits vascular smooth muscle cell growth

Several peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione class inhibit vascular smooth muscle cell proliferation. It is not known whether the antiproliferative activity of PPARgamma agonists is limited to the thiazolidinedione class and/or is directly mediated through PPARgamma-dependent transactivation of target genes. We report here that a novel non-thiazolidinedione partial PPARgamma agonist (nTZDpa) attenuates rat aortic vascular smooth muscle cell proliferation. In a transfection assay for PPARgamma transcriptional activation, the non-thiazolidinedione partial PPARgamma agonist elicited approximately 25% of the maximal efficacy of the full PPARgamma agonist rosiglitazone. In the presence of the non-thiazolidinedione partial PPARgamma agonist, the transcriptional activity of the full agonist, rosiglitazone, was blunted, indicating that the non-thiazolidinedione partial PPARgamma agonist inhibits rosiglitazone-induced PPARgamma activity. The non-thiazolidinedione partial PPARgamma agonist (0.1-10 microM) inhibited vascular smooth muscle cell growth which was accompanied by an inhibition of retinoblastoma protein phosphorylation. Mitogen-induced downregulation of the cyclin-dependent kinase (CDK) inhibitor p27(kip1), and induction of the G1 cyclins cyclin D1, cyclin A, and cyclin E were also attenuated by the non-thiazolidinedione partial PPARgamma agonist. Maximal antiproliferative activity of the non-thiazolidinedione partial PPARgamma agonist required functional PPARgamma as adenovirus-mediated overexpression of a dominant-negative PPARgamma mutant partially reversed its inhibition of vascular smooth muscle cell growth. In contrast, overexpression of dominant-negative PPARgamma did not reverse the inhibitory effect of the non-thiazolidinedione partial PPARgamma agonist on cyclin D1. As the full PPARgamma agonist rosiglitazone exhibited no effect on cyclin D1, inhibition of that G1 cyclin by the non-thiazolidinedione partial PPARgamma agonist likely occurred through a PPARgamma-independent mechanism. These data demonstrate that a non-thiazolidinedione partial PPARgamma agonist may constitute a novel therapeutic for proliferative vascular diseases and could provide additional evidence for the important role of PPARgamma in regulating vascular smooth muscle cell proliferation.

Rapamycin inhibits E2F-dependent expression of minichromosome maintenance proteins in vascular smooth muscle cells.

Rapamycin inhibits vascular smooth muscle cell (VSMC) proliferation and rapamycin-eluting stents represent a novel therapeutic strategy for preventing postangioplasty restenosis. The precise molecular mechanism, for rapamycin-mediated inhibition of VSMC cell cycle progression and DNA replication remain to be elucidated. Minichromosome maintenance proteins (MCM) are essential regulators of DNA replication and the objective of this study was to examine the effect of rapamycin on their expression in rat aortic VSMC. Rapamycin substantially inhibited mitogen-induced MCM6 and MCM7 mRNA and protein expression in a dose-dependent fashion. Transient transfection experiments revealed that rapamycin inhibited MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. MCM6 and MCM7 transcriptional activation is regulated by E2F and activity of a luciferase reporter plasmid driven by four E2F elements was also significantly inhibited by rapamycin. The inhibitory effect of rapamycin on MCM6 and MCM7 was reversed by overexpression of E2F, indicating that their downregulation by rapamycin involves an E2F-dependent mechanism. These observations suggest that rapamycin inhibits MCM6 and MCM7 expression by blocking their E2F-dependent transactivation which may contribute importantly to the inhibition of VSMC DNA synthesis by rapamycin.

TGF-β1 induces peroxisome proliferator-activated receptor γ1 and γ2 expression in human THP-1 monocytes

Abstract The nuclear hormone receptor peroxisome proliferator-activated γ (PPARγ) is expressed as two isoforms (PPARγ1 and γ2), and is an important modulator of monocyte gene regulation and function. TGF-β1 is an essential and potent immune modulator and we therefore examined its effect on PPARγ expression in human THP-1 monocytes. TGF-β1 strongly induced PPARγ2 mRNA and protein expression with a lesser effect on PPARγ1. Transcription from a PPARγ2 promoter/luciferase reporter vector was activated approximately 3-fold by TGF-β1. Mutation of two C/EBP elements within the PPARγ2 promoter reduced TGF-β1-induced transcription by approximately 65%. TGF-β1 also induced the expression of three C/EBPisoforms (α, β, and δ). Induction of PPARγ1 and γ2 in monocytes by TGF-β1 may contribute to the anti-inflammatory effects of this growth factor.

Expression of minichromosome maintenance proteins in vascular smooth muscle cells is ERK/MAPK dependent

Proliferation of vascular smooth muscle cells (VSMC) represents a key event for the pathogenesis of postangioplasty restenosis. Minichromosome maintenance proteins (MCM) form essential components of the prereplicative complex at DNA replication origins and are regulated by E2F. The present studies were designed to investigate the signal transduction pathways controlling the expression of MCM6 and MCM7 in VSMC in response to mitogenic stimuli. MCM6 and MCM7 expression was substantially increased after stimulation with platelet-derived growth factor-BB and insulin. Pretreatment with PD98059, a specific inhibitor of the extracellular signal-regulated kinases (ERK)-mitogen-activated protein kinase (MAPK), competely inhibited the mitogen-induced MCM6 and MCM7 mRNA and protein expression, demonstrating a critical role for this pathway in transmitting transmembrane signals required for the initiation of DNA replication. The p38MAPK inhibitor SB203580, the phosphatidylinositol 3 kinase (PI3-kinase) pathway inhibitor wortmannin, and the protein kinase C pathway (PKC) inhibitor Gö 6976 did not significantly affect mitogen-induced MCM6 and MCM7 expression. Transient transfection experiments revealed that PD98059 inhibited mitogen-induced MCM6 and MCM7 transcriptional activation. In addition, blockade of ERK/MAPK signaling with PD98059 strongly inhibited phosphorylation of the retinoblastoma protein (Rb) and activity of a luciferase reporter plasmid driven by multiple E2F elements. Inhibition of mitogen-induced MCM6 and MCM7 expression by PD98059 was reversed by ectopic overexpression of E2F, indicating that ERK/MAPK signaling is required for events that occur upstream of E2F release from phosphorylated Rb. In combination, these data demonstrate that the ERK/MAPK signal transduction pathway plays a central role in regulating E2F-dependent MCM expression and DNA replication in VSMC.

p38 MAP kinase negatively regulates angiotensin II-mediated effects on cell cycle molecules in human coronary smooth muscle cells

Many of the signaling events in VSMC stimulated by angiotensin II (AngII) are mediated by members of the mitogen-activated protein kinase (MAPK) family, including p38 MAPK. The role of p38 MAPK in AngII-mediated cell cycle regulation is poorly understood. Therefore, we examined the involvement of p38 MAPK signaling in AngII-stimulated DNA synthesis, phosphorylation of the retinoblastoma protein (Rb), and expression of the G1-phase cyclin D1 in human coronary artery smooth muscle cells (CASMC). AngII (1 microM) stimulated p38 MAPK and ERK1/2 activation. Pretreatment with the p38 MAPK inhibitors SB203580 (10 microM) (SB) or SKF-86002 (10 microM) (SKF) potently inhibited AngII-induced p38 MAPK activation, but enhanced AngII-mediated ERK1/2 activation. AngII-induced-phosphorylation of Rb (Ser 795 and Ser 807/811), -cyclin D1 expression, and -DNA synthesis was also markedly enhanced by pharmacological inhibition of the p38 MAPK pathway. The present study demonstrates that p38 MAPK negatively regulates AngII-induced ERK1/2 activity, Rb phosphorylation, cyclin D1 expression, and DNA-synthesis in human CASMC. These findings support an important role for p38 MAPK in modulating AngII-mediated VSMC hyperplasia.