Genetic Response of Angiogenesis Markers to Embryonic Mouse Masseter Muscle Development

Objectives: Angiogenesis markers are an important factor in the angiogenesis of masseter muscle (MM). However, it has many other functions in addition to the regulation of muscle. We try to clearly the relationship between angiogenesis and morphological markers in terminating the differentiation of the MM. Principal component analysis (PCA) was performed for mouse MM in different early postnatal stages and the expression of the following 6 mRNA CD31, LYVE-1, VEGF, embryonic MyHC, Collagen I, and MMP-2.
Methods: We analyzed the expression and localization of VEGF by in situ hybridization in the developing mouse MM during embryonic days 12.5 (E12.5), E14.5, E17.5, and E18.5. We also detected the mRNA abundance of MM various markers (Collagen I, VEGF, CD31, LYVE-1, MMP-2, embryonic MyHC) from embryonic stages by real-time RT-PCR.
Results: The antisense probe for VEGF was detected in mesenchymal cells surrounding blood vessels in the masseter–like muscular area at E12.5, and clearly localization was seen at E18.5 in the embryonic mouse MM by in situ hybridization. The VEGF abundance levels almost constant compared with other angiogenesis markers and morphogenesis markers from E14.5 to E18.5. The two principal components significantly explained 73.38%(component 1, 38.0%; component 2, 35.3%) of the information in the data set in the MM. Cluster analyses identified the following distinct clusters for mRNA abundance in the masseter: cluster 1, E12.5, E14.5; cluster 2, E17.5 and E18.5; cluster 3, day 0 and days 5. The positive correlation between VEGF and MMP2 (Pearson's r=0.754; p<0.01), CD31 (Pearson's r= 0.476; p<0.05), and negative correlation between VEGF and embryonic MyHC (Pearson's r=-0.501; p<0.05) were analyzed. The positive correlation between CD31 and LYVE-1 (Pearson's r=0.736; p<0.01), and negative correlation between embryonic MyHC and MMP-2 (Pearson's r=-0.487; p<0.05) were analyzed.
Conclusions: These data suggested that angiogenesis markers may effect to influence MyHCs expresssion during mouse MM development and morphogenesis. VEGF also affects the morphological markers in specific stages and areas, terminating the differentiation of the MM.