Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells
Introduction
Statins are potent inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase via blocking the substrate accessibility to the enzyme and thereby effectively subverting cholesterol metabolism. Several large trials have revealed an impressive clinical benefit of statins which has led to a widespread use of these drugs in the primary and secondary prevention of coronary artery disease (for review see [1]). In the last 5 years compelling evidence has accumulated suggesting a beneficial effect of statins beyond their inhibition of cholesterol synthesis (for review see [2], [3], [4]). Indeed, for example, simvastatin and lovastatin have been reported to increase the half-life of the mRNA for eNOS. Atorvastatin, pravastatin and cerivastatin were able to scavenge oxygen derived free radicals and simvastatin and atorvastatin have been shown to decrease the precursor for endothelin-1 [2], [3], [4]. Thus, statins may exert directly vasculoprotective, perhaps cholesterol-independent effects.
Vascular endothelial growth factor (VEGF) has been proven to be an important growth factor critical for blood vessel formation [5]. Recently, it has been suggested that statins may also modulate VEGF synthesis and consequently angiogenesis. Indeed, lovastatin at micromolar concentrations inhibited the production of VEGF in transformed fibroblasts [6] and cytokine-induced VEGF synthesis in rat vascular smooth muscle cells (VSMC) [7], while mevastatin suppressed the VEGF synthesis in rat primary aortic endothelial cells [8]. In contrast, statins at nanomolar concentrations have been recently demonstrated to enhance angiogenesis via activation of Akt kinase in human umbilical vein endothelial cells [9] or endothelial progenitor cells [10], [11]. However, it is not known so far whether the decreasing effect is dependent on the dose of statins or associated to its efficacy on different cell types. Therefore, the aim of this study was to investigate whether the effect of statins on endothelial cell angiogenic activity and on VEGF expression is dependent on the concentration of the drug and/or on the cell type.
Section snippets
Reagents
Lovastatin was purchased from Calbiochem (Vienna, Austria), atorvastatin and simvastatin were gifts from Pfizer and MSD, respectively (Vienna, Austria). SNAP (S-nitroso-N-acetyl-d,l-penicillamine) was obtained from Alexis Biochemicals (Laufelfingen, Switzerland). Total RNA isolation kit, AMV reverse transcriptase and PCR core kit were purchased from Promega (Madison, USA). All other chemicals were obtained from Sigma (St. Louis, USA). Concentration of VEGF in the cell culture media or in cell
Baseline synthesis of VEGF is cell-dependent (Fig. 1)
Untreated HVSMC, cultured in 24-well plates (∼105 cells), generate during 24 h 100–200 pg of VEGF per ml of culture supernatant (Fig. 1). Under the same conditions, HMEC-1 produce only about 20 pg/ml, while no detectable VEGF was found in HUVEC conditioned media (Fig. 1). However, up to a few pg of VEGF could be detected in the cell lysates of unstimulated HUVEC (Fig. 1). RT–PCR demonstrated that VEGF mRNA expression is also cell-dependent (Fig. 1A) and related to the amount of VEGF protein
Discussion
The crucial finding of this study is the demonstration of differential effects of statins on VEGF synthesis, which appear to be cell type dependent. At micromolar concentrations, statins inhibited the generation of VEGF in HVSMC and HMEC-1 which constitutively produce this growth factor. In contrast, HUVEC under basal conditions do not release VEGF and produce only very small amounts of intracellular stored VEGF. Interestingly, statins at micromolar concentration can enhance the formation of
Acknowledgements
We are grateful to Prof. Aleksander Koj for useful comments. J. Dulak was a recipient of a fellowship from the Austrian Society of Cardiology (1999–2001). The study was in part supported by the Grant 3 PO4 049 22 awarded by the Polish State Committee for Scientific Research and by the Polish–Austrian Collaborative Grant (17/2002).
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These authors contributed equally to this study.