It is widely generally accepted that high-density lipoprotein cholesterol (HDL) is anti-atherogenic (Gordon et al., 1989), via effects on reverse cholesterol transport, oxidation of low-density lipoprotein cholesterol and inflammation (Cockerill et al., 2001) both in vitro and in vivo. There is considerable interest therefore in the direct therapeutic potential of appropriately formulated HDL in the treatment of coronary heart disease with attendant hypercholesterolemia (Black, 2003). The extent and the molecular mechanisms underlying these effects remain unclear. In this study we used transcriptional profiling to test the hypothesis that the anti-inflammatory effects of HDL lead to the activation of atheroprotective gene networks in activated endothelial cells.

Human umbilical vein endothelial cells were pre-treated for 16 h with native HDL (nHDL) or reconstituted HDL (rHDL) preparations (l mg/ml of apo AI) prior to activation with TNF(10 ng/ml) for 2 h. RNA populations were characterised by real time RT-PCR measurements of E-selectin mRNA expression in order to establish the inhibitory effects of both nHDL and rHDL. The effects of both HDL preparations on activated global gene expression were then assessed using Affymetrix HU-133A microarrays. (Fodor et al., 1991). Data was analysed using GeneSpring® analysis software for hierarchical and functional clustering.

The complex expression patterns generated highlighted various potential new atheroprotective mechanisms of action of HDL. In two separate experiments, TNF treatment altered gene expression (>2-fold) in 1326 genes. nHDL and rHDL pre-treatment normalised 35% and 35.4% of these changes respectively. A significant overlap was demonstrated in the transcriptional profile generated by nHDL or rHDL pre-treatment (193 genes). In particular, unsupervised hierarchical clustering analysis demonstrated differential gene expression in pathways related to vascular cell migration and proliferation, as well as control of vascular tone. The activated expression of endothelin-1 was increased (>2-fold) by both preparations of HDL. However, while rHDL normalised TNF-activated expression of phospholipase C-ß1, a downstream effector of the endothelin-1 receptor (>1.5-fold increase), nHDL did not. These differences were confirmed by real-time RT-PCR , n=3 (p<0.05, t-test).

In conclusion, the results of this study of the effects of HDL on activated endothelial cells have identified pathways potentially important to its anti-inflammatory/atherogenic properties. This suggests separate physiological roles for HDL subclass components in the regulation of endothelial cell migration and proliferation and regulation of vascular tone.

Black, D. M. (2003) Am. J. Cardiol. 91, 40E-43E.
Cockerill, G. W. et al., (2001) Circ. 103, 108-112.
Fodor, S. P. et al., (1991) Science 251, 767-773.
Gordon, D. J. et al., (1989) New Engl. J. Med. 321, 1311-1316.

This work was supported by the Wellcome Trust, the British Cardiac Society and the National Heart Research Fund.