Characterization of the NPC1L1 gene and proteome from an exceptional responder to ezetimibe.

BACKGROUND: Strategies to reduce LDL-cholesterol involve reductions in cholesterol synthesis or absorption. We identified a familial hypercholesterolemia patient with an exceptional response to the cholesterol absorption inhibitor, ezetimibe. Niemann-Pick C 1-like 1 (NPC1L1) is the molecular target of ezetimibe. METHODS AND RESULTS: Sequencing identified nucleotide changes predicted to change amino acids 52 (L52P), 300 (I300T) and 489 (S489G) in exceptional NPC1L1. In silico analyses identified increased stability and cholesterol binding affinity in L52P-NPC1L1 versus WT-NPC1L1. HEK293 cells overexpressing WT-NPC1L1 or NPC1L1 harboring amino acid changes singly or in combination (Comb-NPC1L1) had reduced cholesterol uptake in Comb-NPC1L1 when ezetimibe was present. Cholesterol uptake was reduced by ezetimibe in L52P-NPC1L1, I300T-NPC1L1, but increased in S489G-NPC1L1 overexpressing cells. Immunolocalization studies found preferential plasma membrane localization of mutant NPC1L1 independent of ezetimibe. Flotillin 1 and 2 expression was reduced and binding to Comb-NPC1L1 was reduced independent of ezetimibe exposure. Proteomic analyses identified increased association with proteins that modulate intermediate filament proteins in Comb-NPC1L1 versus WT-NPC1L1 treated with ezetimibe. CONCLUSION: This is the first detailed analysis of the role of NPC1L1 mutations in an exceptional responder to ezetimibe. The results point to a complex set of events in which the combined mutations were shown to affect cholesterol uptake in the presence of ezetimibe. Proteomic analysis suggests that the exceptional response may also lie in the nature of interactions with cytosolic proteins.

Atorvastatin modulates matrix metalloproteinase expression, activity, and signaling in abdominal aortic aneurysms.

Statins may reduce abdominal aortic aneurysm (AAA) progression. We sought to measure how atorvastatin (AT) treatment might modulate matrix metalloproteinase (MMP) expression and/or activity in human AAA. Tissue from human AAAs at surgical repair was obtained from patients who were either not on statins (NST, n = 19) or treated with AT (n = 19). Immunoblots measured expression and zymography measured activity. Expression of most proteins was greater in the central compared with distal AAA region. Matrix metalloproteinase 1, MMP2, MMP3, MMP9, Tissue Inhibitor of Metalloproteinase (TIMP2), TIMP3, TIMP4, or total Sma Mothers Against Decapentaplegia (SMAD2) expression did not differ with treatment. There was a trend toward reduced MMP8 and TIMP1 expression and MMP2 zymographic activity in the AT-treatment group. In contrast, AT-treated samples had significantly reduced MMP13 (P = .02), latent-transforming growth factor (TGF)-beta (P = .02), and phospho-SMAD2 (P = .029) expression than NST-treated samples. We conclude that the AT-mediated decrease in MMP expression and activity reduces TGF-beta signaling in the central region of human AAAs.

Atorvastatin mediates increases in intralesional BAX and BAK expression in human end-stage abdominal aortic aneurysms.

Chronic apoptosis activation may participate in abdominal aortic aneurysm (AAA) expansion. Statin treatment slows AAA progression independent of cholesterol lowering. We hypothesized that Atorvastatin treatment alters apoptosis protein expression and activation in AAAs. Protein was isolated from the central and distal portions of end-stage human AAA tissue obtained during surgical repair from non-statin (NST) and Atorvastatin-treated (AT) patients. Expression was compared using immunoblots. Bcl-2 expression was unchanged but Bak (4-fold, p < 0.013) and Bax (3-fold, p < 0.035) expression was increased in AT (n = 12) versus NST (n = 15) patients. No cytochrome c release or caspase 3 activation was detected and Clusterin, GRP78, and BNIP1 expression was similar in NST and AT samples. Bcl-2 and Bax cDNA sequences from AAA tissue (n = 10) and the general population were identical. Thus, the increase in Bax and Bak in AT-treated AAAs did not activate the mitochondria or endoplasmic reticulum mediated apoptosis pathways. Bcl-2, Bax, and Bak have non-apoptosis related functions that include maintenance of endoplasmic reticulum (ER), homeostasis, and adaptation to stress. We speculate that Atorvastatin-mediated increases in Bax and Bak may positively affect their non-apoptosis related cell functions to account for the beneficial effect of statins to slow AAA expansion.

BAK1 gene variation and abdominal aortic aneurysms.

We sought to examine the role of genetics in the multifactorial disease, abdominal aortic aneurysm (AAA), by studying sequence variation in the BAK1 gene (BAK1) that codes for an apoptotic-promoting protein, as chronic apoptosis activation has been linked to AAA development and progression. BAK1 abdominal aorta cDNA from AAA patients and nondiseased individuals were compared with each other, as well as to the BAK1 genomic sequence obtained from matching blood samples. We found specific BAK1 single nucleotide polymorphism (SNP) containing alleles in both aneurysmic (31 cases) and healthy aortic tissue (5 cases) without seeing them in the matching blood samples. These same BAK1 SNPs have been reported, although rarely (average frequency <0.06%), in reference BAK1 DNA sequences. Based on this and other similar observations, we propose a novel hypothesis postulating that multiple variants of genes may preexist in "minority" forms within specific nondiseased tissues and be selected for, when intra- and/or extracellular conditions change. Therefore, the fact that different BAK1 variants can exist in both diseased and nondiseased AA tissues compared to matching blood samples, together with the rare occurrence of these same SNPs in reference sequences, suggests that selection may be a significant factor in AAA ontogeny.

A comparison of pravastatin and gemfibrozil in the treatment of dyslipoproteinemia in patients with non-insulin-dependent diabetes mellitus.

The effects of pravastatin (pravachol) compared with gemfibrozil on cholesterol-rich and trigylceride-rich lipoproteins were evaluated in this multi-centered trial. Following an 8-12 week prerandomization phase, 136 patients with NIDDM and hypercholesterolemia were randomized to receive either pravastatin 40 mg or gemfibrozil 1200 mg daily for 16 weeks. The reduction of total cholesterol (TC), betaquant LDL and LDL cholesterol (LDL-C) was significantly greater in patients treated with pravastatin than with gemfibrozil. However, gemofibrozil treatment resulted in a significantly greater reduction of triglyceride (TG) levels than did treatment with pravastatin. Pravastatin reduced the concentration of apoB (-19.3%, P<0.001) and cholesterol-rich Lp-B (Lp-B+Lp-B; E) particles (-19%, P<0.001) to a significantly greater extent (P<-0.001) than gemfibrozil (-4.1 and -1%, respectively). Both gemfibrozil and pravastatin reduced the concentrations of trigylceride-rich Lp-Bc (-12.2 and -13.3%, respectively) and Lp-A-II;B;C;D;E (-19 and -12.7%, respectively) particles and their characteristic apoC-III constituent (-10.0 and -7.0%, respectively). In contrast, gemfibozil has a greater lowering effect compared with pravastatin on TG levels (-29.6 vs. -6.3%, respectively). Both pravastatin and gemfibrozil significantly increased the levels of apoA-I and, with both drugs, the elevated concentrations of apoA-I were due to significantly increased levels of Lp-A-I;A-II particles. By decreasing both cholesterol-rich Lp-B and triglyceride-rich Lp-Bc particles and increasing HDL-C and Lp-A-I;A-II particles in addition to proven efficacy in decreasing coronary events in NIDDM patients, pravastatin appears to be an appropriate choice for monotherapy in a broad range of diabetic patients with Type IIA and Type IIB hyperlipoproteinemias. These results also showed that direct measurement of lipoprotein family of particles provides important information not only about the composition but also the type and number of apoA- and apoB-containing lipoprotein particles.