When validated by two highly reliable mouse transcript databases, this MMES based empirical Angoroside-C method is shown to be remarkably more accurate than readcounting method, and also better than the logistic regression method used in Pan et al. In general, there were much more reads aligned to ESJ than to ERJ. Reads aligned to ESJ were almost uniformly Glycoside-O-4 distributed regardless of MMES score. In contrast, reads mapped to negative control ERJ presented a skewed distribution, i.e. the number of mapped reads drops dramatically with the increase of MMES score, which indicated that there were only a few reads having their centers close to exon junctions. Furthermore, for exact match, several orders of magnitude more reads map to ESJ than to ERJ regardless of the MMES scores, which implied that exact match reads were the most reliable to detect spicing junction, no matter which part of the junction the read was aligned to. However, if there were mismatches especially 2-mismatches, nearly the same number of reads will map to ESJ as well as ERJ. This strongly indicated that the reliability of 2-mismatch read alignment largely depended on the corresponding MMES score, i.e. those with large MMES score were more likely to be real ones while those with small MMES score were non-specific mapping artifacts. The latter cannot be applied to identify splicing junctions without further calibration. We also performed the same analysis on another RNA-seq dataset from human embryonic kidney and B cell line, and obtained very similar results. We further demonstrated that this non-uniform distribution of MMES is not due to the uniqueness of read mapping, as similar distributions were observed in both uniquely and non-uniquely matched junction reads, for both human and mouse datasets. The vascular wall is a complex construct composed of different cell types including the inner layer of endothelial cells, surrounded by smooth muscle cells within the media and finally the adventitia composed of fibroblasts. For function of the vascular wall, interdependency between endothelial cells and mural cells is required.