Pirillo A, Jacoviello C, Longoni C, Radaelli A, Catapano A L
Institute of Pharmacological Sciences, University of Milano, Italy.
Biochem Biophys Res Commun. 1997 Feb 13;231(2):437-41. doi: 10.1006/bbrc.1997.6117.
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.
氧化型低密度脂蛋白（OxLDL）对动脉壁细胞具有毒性，并能在培养的内皮细胞（EAhy - 926）和平滑肌细胞（HUVSMC）中触发诱导型热休克蛋白70（hsp 70）的表达。后一种反应被认为可保护细胞免受毒性，因为热休克蛋白70（hsp70）是细胞在应激条件下合成的，用于保护蛋白质免于不可逆的变性。辛伐他汀（10⁻⁸ M至10⁻⁵ M）是羟甲基戊二酰辅酶A还原酶（HMG - CoA还原酶）的竞争性抑制剂，HMG - CoA还原酶是胆固醇生物合成中的关键酶，它以剂量依赖的方式增强了OxLDL（300微克/毫升）对内皮细胞和平滑肌细胞的毒性，这通过³H - 腺嘌呤释放和MTT试验检测到。在EAhy细胞中，OxLDL引起的³H - 腺嘌呤释放为0.419±0.048（培养基中释放的放射性与总放射性的比值），而对照组为0.337±0.008；在辛伐他汀和OxLDL存在的情况下，该值从10⁻⁸ M时的0.49±0.01增加到10⁻⁵ M时的0.918±0.001，单独使用辛伐他汀（10⁻⁵ M）时该值为0.463±0.025。此外，通过免疫印迹检测发现，辛伐他汀以剂量依赖的方式降低了OxLDL触发的hsp 70的表达。为了探究这一发现是否是由于辛伐他汀对胆固醇途径的影响，使用甲羟戊酸（100微摩尔）绕过HMG - CoA还原酶的阻断。该化合物完全阻止了辛伐他汀增强OxLDL的毒性，并恢复了hsp70的表达。为了验证OxLDL诱导hsp70是否需要胆固醇合成，使用了鲨烯合酶抑制剂角鲨他汀I（25纳摩尔至100纳摩尔），鲨烯合酶是胆固醇途径中的另一种关键酶：该化合物未影响OxLDL的毒性和hsp70的表达。这些结果表明，辛伐他汀在体外增加了OxLDL的细胞毒性，同时降低了OxLDL触发的hsp70的表达，并且胆固醇合成中导致这些效应的关键步骤一定在甲羟戊酸和鲨烯形成之间。
