Yuqing E. Chen

Cardiomyocyte-restricted peroxisome proliferator-activated receptor-|[delta]| deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy

Fatty acid oxidation (FAO) is a primary energy source for meeting the hearts energy requirements. Peroxisome proliferator-activated receptor-δ (PPAR-δ) may have important roles in FAO. But it remains unclear whether PPAR-δ is required for maintaining basal myocardial FAO. We show that cre-loxP-mediated cardiomyocyte-restricted deletion of PPAR-δ in mice downregulates constitutive expression of key FAO genes and decreases basal myocardial FAO. These mice have cardiac dysfunction, progressive myocardial lipid accumulation, cardiac hypertrophy and congestive heart failure with reduced survival. Thus, chronic myocardial PPAR-δ deficiency leads to lipotoxic cardiomyopathy. Together, our data show that PPAR-δ is a crucial determinant of constitutive myocardial FAO and is necessary to maintain energy balance and normal cardiac function. We suggest that PPAR-δ is a potential therapeutic target in treating lipotoxic cardiomyopathy and other heart diseases.

Nitrated Fatty Acids: Endogenous Anti-inflammatory Signaling Mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-γ activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-κB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-κB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-α-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.

IQGAP1, a Novel Vascular Endothelial Growth Factor Receptor Binding Protein, Is Involved in Reactive Oxygen Species—Dependent Endothelial Migration and Proliferation

Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)–mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.

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.

Generation of an Adult Smooth Muscle Cell–Targeted Cre Recombinase Mouse Model

To the Editor: The smooth muscle cell (SMC)–targeted Cre recombination mice are critical tools for in vivo analysis of gene function in the vasculature and for establishing animal models for cardiovascular diseases. Therefore, there is a continuing effort to generate SMC-targeted Cre recombinase mice for in vivo loss-of-gene function studies. Currently, several genetically engineered mice express the Cre-recombinase under the control of SMC-specific promoters such as SM22α (also known as transgelin, a 22-kDa protein that is abundantly and exclusively expressed in SMCs of adult animals) promoters and smooth muscle myosin heavy chain promoters.1–6 However, there are potential limitations in their uses for knockout studies; some of them show relatively low excision efficiency and potential embryonic lethality, which prevent subsequent in vivo analyses in adult SMCs. In our effort to generate an SMC-targeted Cre recombination mouse line that effectively excises loxP-flanked target …

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.