Original ArticleBiomedicalIn Vitro Modulation of Peroxisome Proliferator-activated Receptor-γ and Its Genes by C-Reactive Protein. Role of Atorvastatin
Introduction
Inflammation plays a central and pivotal role in atherosclerosis from its inception onwards (1). Nuclear receptors like peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs) have been implicated in the modulation of the expression of genes involved in inflammation and lipid metabolism in macrophages. Activation of macrophages in atheromas lead to release of vasoactive molecules such as nitric oxide (NO), endothelins (ETs), several eicosanoids, reactive oxygen species (ROS) (2) and secretion of proteolytic enzymes (e.g., matrix metalloproteinases (MMPs) that degrade extracellular matrix. The loss of matrix components subsequently leads to destabilization of the plaques and poses an increased risk for plaque rupture and thrombosis 3, 4.
Considerable evidence has emerged to indicate that, in addition to inducing genes involved in reverse cholesterol transport, these nuclear receptors reciprocally repress a set of inflammatory genes after bacterial LPS, TNF-α or IL-1β stimulation 3, 5. Inflammation also regulates the production of the acute phase proteins such as C-reactive protein (CRP), fibrinogen and serum amyloid A (1). CRP is not only a biomarker for atherosclerotic events (6) but a potent vasoactive mediator to promote atherogenesis by activating various inflammatory genes in macrophages including matrix metalloproteinases (MMPs), inflammatory chemokines and cytokines, adhesion molecules, tissue factor, etc. 7, 8, 9, 10, 11.
3-Hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors (statins) are potent inhibitors of cholesterol biosynthesis. Recent in vitro and in vivo studies have shown statins have several pleiotropic effects that include decreasing the production of reactive oxygen species (ROS) (12), enhancing the levels of endothelial cell nitric oxide synthases (eNOS) (13), and inhibiting vascular smooth muscle cell (VSMCs) proliferation, as well as exhibiting anti-thrombotic and anti-inflammatory effects (14). Statins have been shown to stabilize atherosclerotic plaques and prevent coronary artery disease (CAD) events 15, 16. It has also been demonstrated that statins increase PPARγ and LXRα mRNA expression and protein levels in human umbilical vein endothelial cells, hepatocytes and monocytes 17, 18.
In this study we investigated how CRP (as a possible mediator in atherosclerosis/inflammation) modulates the expression of PPARγ and LXRα in human THP-1 monocytic cell line. Keeping in mind the fact that gene coding for MMP-9 is specifically downregulated by these nuclear receptors at the transcriptional level 19, 20, we also determined the effects of CRP on the expression of MMP-9. Further, atorvastatin was explored as a potential therapeutic modality on the expression of these genes in the presence of exogenous CRP as an inflammatory stimulus.
Section snippets
Chemicals
All chemicals and reagents used in the experiments were of cell culture and molecular biology grade and were purchased from the following companies (Sigma, St Louis, MO, USA); Fermentas, Inc, St. Leon Rot Germany); Hi-Media, Mumbai, India), etc. The commercial recombinant E. coli-derived CRP preparation (236608, Calbiochem, La Jolla, CA) was used and sodium azide was removed as described previously (11). Bacterial endotoxin levels were determined using E-toxate kit (ET0100, Sigma). All reagents
Cell Viability and Cell Proliferation
CRP (100 μg/mL) had no significant effect on the viability and proliferation of cells up to 48 h, whereas it was cytotoxic at 72 h. However, CRP at a dose of 150 μg/mL showed a significant decrease in cell viability and proliferation of cells at 48 h. A minimal dose of CRP, i.e., 25 μg/mL, was able to adversely affect cells at 72 h both in terms of viability and morphology. No morphological abnormalities were found in cell cultures up to 48 h with 100 μg/mL CRP (data not shown). Based on these
Discussion
Participation in both metabolic and inflammatory control is a common feature of a number of different nuclear receptor signaling pathways. PPARγ belongs to the nuclear receptor cum transcription factor family and has the unique distinction in that these genes not only control the expression of LXRα, CD36 and NFkB but also of the other effector genes that play a key role in the cooperativity that exists between oxidative and inflammatory processes like MMP-9 23, 24. It is in this context that
Acknowledgments
The authors wish to thank the Indian Council of Medical Research (ICMR), New Delhi for financial assistance. Nitin Mahajan was awarded a Senior Research Fellowship by ICMR, New Delhi.
Competing interests: The authors declare that they have no competing interests.
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