Mingui Fu

Egr-1 target genes in human endothelial cells identified by microarray analysis.

Early growth response factor 1 (Egr-1) is a key transcriptional factor to mediate gene expression after vascular injury. To better understand the role of Egr-1 in vasculature, we globally profiled Egr-1 target genes in human endothelial cells using adenoviral gene transfer and Affymetrix oligonucleotide-based microarray technology. More than 300 genes regulated by >/=3-fold with Egr-1 overexpression were identified and, partially, confirmed by Northern and Western blotting, including genes for transcriptional regulators, signaling proteins, cell cycle regulatory proteins, growth factors, and cytokines. Among them, thymus-expressed chemokine (TECK) and IP-30 were dramatically induced by Egr-1, but TNFalpha-related apoptosis inducing ligand (TRAIL) was significantly repressed by Egr-1, suggesting that Egr-1 is a key mediator of inflammation and apoptosis in vascular cells. These data provide novel Egr-1 target genes and contribute to the understanding of the role of Egr-1 in vasculature.

PDGF induces osteoprotegerin expression in vascular smooth muscle cells by multiple signal pathways.

Osteoprotegerin (OPG) is a key regulator of osteoclastogenesis. Recent reports suggest that OPG may function as a protector of arterial calcification and survival of endothelial cells. However, the role and expression of OPG in vascular wall is unclear. Here we report that OPG was highly expressed in vascular smooth muscle cells (VSMC) but not in endothelial cells. Platelet‐derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, tumor necrosis factor α and interleukin‐1β upregulated OPG expression in VSMC. Moreover, inhibition of phosphatidylinositol 3‐kinase/Akt or P38‐signal pathway abrogated PDGF‐induced OPG expression. Our results suggest that OPG may be an important determinant of vascular homeostasis.

Peroxisome Proliferator-activated Receptor δ Is Up-regulated during Vascular Lesion Formation and Promotes Post-confluent Cell Proliferation in Vascular Smooth Muscle Cells

Although peroxisome proliferator-activated receptor (PPAR) δ is widely expressed in many tissues, the role of PPARδ is poorly understood. In this study, we report that PPARδ was up-regulated in vascular smooth muscle cells (VSMC) during vascular lesion formation. By using Northern blot analysis, we demonstrated that PPARδ was increased by 3–4-fold in VSMC treated with platelet-derived growth factor (PDGF) (20 ng/ml). In addition, PDGF-induced PPARδ mRNA expression neither needs de novo protein synthesis nor affects the stability of PPARδ mRNA in VSMC. Preincubation of VSMC with phosphatidylinositol 3-kinase inhibitor (LY294002, 50 μmol/liter) or infection of VSMC with an adenovirus carrying the gene for a dominant negative form of Akt abrogated PDGF-induced PPARδ mRNA expression, suggesting that phosphatidylinositol 3-kinase/Akt signaling pathway is involved in the regulation of PDGF-induced PPARδ mRNA expression in VSMC. To explore the role of PPARδ in VSMC, we generated rat vascular smooth muscle cells (A7r5) stably overexpressing PPARδ and the control green fluorescent protein. Overexpression of PPARδ in VSMC increased post-confluent cell proliferation by increasing the cyclin A and CDK2 as well as decreasing p57 kip2 . Taken together, the results suggest that PPARδ plays an important role in the pathology of diseases associated with VSMC proliferation, such as primary atherosclerosis and restenosis.

Early stimulation and late inhibition of peroxisome proliferator-activated receptor gamma (PPAR gamma) gene expression by transforming growth factor beta in human aortic smooth muscle cells: role of early growth-response factor-1 (Egr-1), activator protein 1 (AP1) and Smads.

Transforming growth factor beta (TGF beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) play major roles in the development of vascular diseases. It has been documented that PPAR gamma activation inhibits the TGF beta signal pathway in vascular smooth muscle cells (VSMC). Here we examined whether TGF beta can regulate PPAR gamma expression. Northern blot analyses revealed that both TGF beta 1 and 2 exert a biphasic effect (early stimulation and late repression) on PPAR gamma gene expression in VSMC. TGF beta rapidly and transiently induced early growth-response factor-1 (Egr-1) expression through the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK1)/ERK-mediated pathway. Inhibition of MEK1/ERK by PD98059 not only abrogated the induction of Egr-1 but also abolished the rapid and transient induction of PPAR gamma by TGF beta. Furthermore, overexpression of NAB2, a repressor of Egr-1 activation, also blocked the induction of PPAR gamma by TGF beta in VSMC, suggesting that Egr-1 mediates the rapid and transient induction of PPAR gamma by TGF beta. With regard to the TGF beta repression of PPAR gamma expression, activator protein 1 (AP1) and Smad3/4 dramatically inhibited the PPAR gamma promoter activity in transient-transfection studies. In contrast, adenovirus-mediated overexpression of a dominant-negative form of c-Jun partially rescued the TGF beta-induced PPAR gamma repression in VSMC. Taken together, our data demonstrate that Egr-1, AP1 and Smad are part components of the TGF beta signal transduction pathway that regulates PPAR gamma expression.

Activation of peroxisome proliferator-activated receptor γ inhibits osteoprotegerin gene expression in human aortic smooth muscle cells

Abstract Increasing evidence indicates an important role of PPARγ activation in modulating the development and progression of atherosclerosis, however, the mechanisms involved in these effects are not well understood since the PPARγ-regulated genes in vascular smooth muscle cells (VSMC) are poorly defined. Here we reported that PPARγ ligands, GW7845, ciglitazone and troglitazone had the effect of inhibiting osteoprotegerin (OPG) expression in human aortic smooth muscle cells (HASMC). The effect of GW7845 and ciglitazone on OPG expression was completely abolished by GW9662, a PPARγ antagonist. Overexpression of PPARγ in HASMC by the infection of a PPARγ adenovirus dramatically decreased OPG expression. In addition, PPARγ activation inhibited OPG promoter activity. Taken together, our data suggest that OPG expression is a novel PPARγ target gene in VSMC and downregulation of OPG expression by PPARγ activation provides a new insight into the understanding of the role of PPARγ in atheroscelrosis and hypertension.

Peroxisome proliferator-activated receptors and the cardiovascular system

Insulin resistance syndrome (also called syndrome X) includes obesity, diabetes, hypertension, and dyslipidemia and is a complex phenotype of metabolic abnormalities. The disorder poses a major public health problem by predisposing individuals to coronary heart disease and stroke, the leading causes of mortality in Western countries. Given that hypertension, diabetes, dyslipidemia, and obesity exhibit a substantial heritable component, it is postulated that certain genes may predispose some individuals to this cluster of cardiovascular risk factors. Emerging data suggest that peroxisome proliferator-activated receptors (PPARs), including alpha, gamma, and delta, are important determinants that may provide a functional link between obesity, hypertension, and diabetes. It has been well documented that hypolipidemic fibrates and antidiabetic thiazolidinediones are synthetic ligands for PPAR alpha and PPAR gamma, respectively. In addition, PPAR natural ligands, such as leukotriene B4 for PPAR alpha, 15-deoxy-delta 12,14-prostaglandin J2 for PPAR gamma, and prostacyclin for PPAR delta, are known to be eicosanoids and fatty acids. Studies have documented that PPARs are present in all critical vascular cells: endothelial cells, vascular smooth muscle cells, and monocyte-macrophages. These observations suggest that PPARs not only control lipid metabolism but also regulate vascular diseases such as atherosclerosis and hypertension. In this review, we present structure and tissue distribution of PPAR nuclear receptors, discuss the mechanisms of action and regulation, and summarize the rapid progress made in this area of study and its impact on the cardiovascular system.

15-Deoxy-prostaglandin J2 inhibits PDGF-A and -B chain expression in human vascular endothelial cells independent of PPARγ

15-Deoxy-prostaglandin J(2)(15d-PGJ(2)) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARgamma) and plays an important role in the regulation of endothelial cell growth and apoptosis. However, the detailed mechanisms are poorly understood. We hypothesized that 15d-PGJ(2) might affect PDGF expression in endothelial cells through activating PPARgamma. Here we documented that 15d-PGJ(2) dose-dependently inhibited phorbol-12-myristate-13-acetate (PMA)-stimulated expression of the PDGF-A and PDGF-B chain in human umbilical vein endothelial cells (HUVEC) by Northern blot and Western blot analyses. In contrast, the synthetic and high-affinity PPARgamma agonists, including ciglitazone and GW7845, did not affect PMA-induced PDGF expression. In addition, we found that the PPARgamma antagonist GW9662 did not block the effects of 15d-PGJ(2) on PDGF expression. Furthermore, Northern blot analysis showed that 15d-PGJ(2) inhibited the expression of Sp1, which is a well-known positive regulator of PDGF transcription. Taken together, our results demonstrate that the inhibition of PDGF expression by 15d-PGJ(2) in HUVEC is independent of PPARgamma, but may be through the downregulation of Sp1.

Thiazolidinediones, a class of anti-diabetic drugs, inhibit Id2 expression through a PPARγ-independent pathway in human aortic smooth muscle cells

Abstract. Inhibitor of DNA binding (Id2) is a member of the helix-loop-helix family of transcription regulators that is known to play important roles in the proliferation and differentiation of many cell types. Overexpression of Id2 has been reported to result in significant enhancement of vascular smooth muscle cell growth via increased S phase entry. We hypothesized that downregulation of Id2 gene expression by thiazolidinediones (TZDs), a class of anti-diabetic drugs and peroxisome proliferator-activated receptor γ (PPARγ) activators, might contribute to the anti-atherosclerotic and anti-hypertensive effects of the PPARγ. Here we document that TZDs, including troglitazone and ciglitazone, repress Id2 gene expression in a doses- and time-dependent manner. However, GW7845, a high-affinity and non-TZD PPARγ activator, had no inhibitory effect on Id2 gene expression. In addition, PPAR& ggr; antagonist GW9662 did not rescue TZD-induced Id2 repression. Taken together, our data suggest that TZDs repress Id2 expression through a PPARγ-independent pathway.