Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.

RATIONALE: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown. OBJECTIVE: Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene. METHODS AND RESULTS: Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions. CONCLUSIONS: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.

Droplet digital PCR improves urinary exosomal miRNA detection compared to real-time PCR.

OBJECT: Quantification of urinary miRNAs can be challenging especially for low abundance miRNAs. We aimed to optimize the quantification of urinary exosomal miRNAs and compare the performance efficiency between droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR). METHODS: We optimized a number of parameters for ddPCR such as annealing temperatures, annealing time and PCR cycle number. We also compared the performance of ddPCR and qPCR. RESULTS: By comparing the fluorescence amplification separation, the optimal annealing temperature was 59 °C, optimal annealing time was 60s and optimal cycle number was 45 for measuring urinary exosomal miRNAs. ddPCR had much higher technical sensitivity compared to qPCR. The minimal detectable concentration of miR-29a was <50 copies/µL by ddPCR compared to 6473 copies/µL for qPCR. Also, ddPCR generated more consistent results for serially diluted samples compared to qPCR. ddPCR generated smaller within-run variations than qPCR though this did not reach statistical significance. It also resulted in better reproducibility with smaller between-run variations. CONCLUSIONS: Optimization of urinary exosomal miRNA ddPCR assay is dependent on assessing key variables including experimental annealing temperature and time as well as the number of PCR cycles. ddPCR has a higher sensitivity, reproducibility, and accuracy in comparison to qPCR.