Chronic intermittent hypoxia (CIH) causes atherosclerosis in mice fed a high cholesterol diet (HCD). The mechanisms by which CIH promotes atherosclerosis are incompletely understood. This study defined the mechanistic role of NF-κB pathway in CIH+HCD induced atherosclerosis. Wild type (WT) and mice deficient in the p50 subunit of NF-κB (p50-KO) were fed normal chow diet (ND) or HCD, and exposed to sham or CIH. Atherosclerotic lesions on the en face aortic preparation and cross-sections of aortic root were examined. In WT mice, neither CIH nor HCD exposure alone caused, but CIH+HCD caused evident atherosclerotic lesions on both preparations after 20weeks of exposure. WT mice on ND and exposed to CIH for 35.6weeks did not develop atherosclerotic lesions. P50 gene deletion diminished CIH+HCD induced NF-κB activation and abolished CIH+HCD induced atherosclerosis. P50 gene deletion inhibited vascular wall inflammation, reduced hepatic TNF-α level, attenuated the elevation in serum cholesterol level and diminished macrophage foam cell formation induced by CIH+HCD exposure. These results demonstrate that inhibition of NF-κB activation abrogates the activation of three major atherogenic mechanisms associated with an abolition of CIH+HCD induced atherosclerosis. NF-κB may be a central common pathway through which CIH+HCD exposure activates multiple atherogenic mechanisms, leading to atherosclerosis.
e14526 Background: We developed a novel technology, Mutant-enriched liquidchip (MEL), which integrates the sensitive mutant enriched PCR and quantitative high throughput liquidchip (suspension array), to detect circulating EGFR mutations (Exon 19 deletion and exon 21 L858R mutation) in patients with advanced non-small cell lung cancer (NSCLC). Methods: To enrich mutant EGFR, a unique restriction site is introduced into the mutation alleles so that the wild type sequence can be selectively removed by restriction digestion, and the undigested mutated DNA is amplified by PCR. The product is then hybridized to complementary probes (including 15 types of exon 19 deletion and exon 21 L858R mutation) which had been conjugated to beads coding with different fluorescent dye, followed by measuring through Luminex 200 system. Plasmid DNA mixture with different EGFR genotypes was applied to determine the sensitivity and accuracy of MEL. Afterwards, the MEL was validated in 49 patients whose EGFR genotypes of tissue specimen had been tested with direct sequencing The circulating genomic DNA was obtained from serum sample of other 201 Chinese stage IIIB or IV NSCLC patients without EGFR-TKI administration, and the EGFR mutation status was analyzed by using of MEL. Results: The results shows that MEL is capable of detecting as few as 20 copies of mutant EGFR alleles with a sensitivity limit of at least mutant/wild-type ratio of 0.1%. It also shows that MEL can not only confirm EGFR mutations status in tissue specimens already known by direct sequencing (13/49), but also detect mutations in some of those showing wild type by sequencing (16/49). Overall, 54% of patients had circulating EGFR mutation. 34% of patients had an exon 19 deletion and 29.6% had L858R. 63.1% of mutations were found in females and 67.6% in never-smokers. Conclusions: This novel MEL method allows for highly sensitive and reproducible detection of human somatic mutations in heterogeneous specimens, and could be applicable to test EGFR mutations non-invasively in advanced NSCLC patients for predicting response to targeted therapy. No significant financial relationships to disclose.
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