Simvastatin modulates the heat shock response and cytotoxicity mediated by oxidized LDL in cultured human endothelial smooth muscle cells.

Oxidized low density lipoproteins (OxLDL) are toxic to cells of the arterial wall and trigger the expression of the inducible form of hsp 70 in cultured endothelial cells (EAhy-926) and smooth muscle cells (HUVSMC). The latter response is believed to protect cells from toxicity since heat shock protein 70 (hsp70) is synthesized by cells under stress condition to protect proteins from irreversible denaturation. Simvastatin (10(-8) M to 10(-5) M), a competitive inhibitor of hydroxy methyl glutaryl coenzyme A reductase (HMG-CoA reductase), a key enzyme in cholesterol biosynthesis, enhanced the toxicity of OxLDL (300 micrograms/mL) to endothelial cells and smooth muscle cells in a dose-dependent manner, as detected by 3H-adenine release and the MTT test. In EAhy, 3H-adenine release with OxLDL was 0.419 +/- 0.048 (ratio of radioactivity released in the medium to total radioactivity) versus 0.337 +/- 0.008 of control; in the presence of simvastatin and OxLDL this value increased from 0.49 +/- 0.01 at 10(-8) M to 0.918 +/- 0.001 at 10(-5) M with simvastatin alone (10(-5) M) this value was 0.463 +/- 0.025. Furthermore simvastatin reduced in a dose-dependent manner the expression of hsp 70 triggered by OxLDL, as detected by immunoblotting. To address whether this finding was due to the effect of simvastatin on the cholesterol pathway, mevalonate (100 microM) was used to bypass the HMG-CoA reductase block. This compound completely prevented the enhancement of OxLDL toxicity by simvastatin and restored the expression of hsp70. To verify whether cholesterol synthesis was required for the induction of hsp70 by OxLDL, squalestatin I (25 nM to 100 nM), an inhibitor of squalene synthase, another key enzyme of the cholesterol pathway, was used: OxLDL toxicity and hsp70 expression were not affected by this compound. These results indicate that simvastatin increases OxLDL cytotoxicity in vitro with a concomitant decrease of hsp70 expression triggered by OxLDL and that the key step in the cholesterol synthesis responsible for these effects must be between mevalonate and squalene formation.