Figure 3. Differentially expressed genes overlapping in healthy aging rat iliac arteries and aortas. A. Venn diagram shows differentially expressed genes determined by our analysis of healthy aging rat iliac arteries and healthy aging rat thoracic aortas. The two gene lists were compared to find common differentially expressed transcripts. A 2.0 fold change and p,.05 significance cut-offs were used. No multiple correction was employed. B. Heat map of differentially expressed genes overlapping in healthy aging rat iliac arteries and aortas. Selected genes, including Myl9, from the 114 transcripts overlapping in healthy aging iliac arteries and thoracic aortas, are shown in a heatmap displaying their expression levels in young and old aortas. Color bar shown in Log2. doi:10.1371/journal.pone.0025855.g003 significance cut-offs were used. No multiple correction was employed. Myl9 protein in over-expressed in aged versus young iliac arteries and is concentrated in the endothelial layer To confirm the over-expression of Myl9 in aged versus young arteries, we performed immunohistochemical staining for Myl9 in young (3 months) and older (22 months) healthy rat iliac arteries. Results confirm over-expression of Myl9 in the older group and point to concentration of Myl9 protein expression in the endothelial layer of the healthy iliac arteries (Figure 4). Arterial injury also induced Myl9 expression with age. Interestingly, the localization of Myl9 was dependent on the post-injury time. Myl9 was highly over-expressed in the smooth muscle cell layer of the injured arteries 3 days post insult (Figure 5), whereas Myl9 was concentrated in the endothelial layer 30 days post injury (Figure 6). Vascular Myl9 with Injury and Aging Figure 4. Myl9 immunostaining of old (22 months) and young (3 months) non-injured rat iliac arteries. Shown are representative images of young and old healthy iliac arteries immunostained for Myl9. Results confirm over-expression of Myl9 in the older group and point to concentration of Myl9 expression in the endothelial layer of the iliac arteries. doi:10.1371/journal.pone.0025855.g004 Discussion The present findings show that vascular remodeling is a dynamic process that is greatly influenced by age and post-injury time. While we found that the transcriptional programs clearly vary among age groups and post-injury times, we identified a common denominator for aging healthy arteries and aging injured arteries at various post-injury times. The contractile regulatory gene myosin light chain (Myl9) was consistently upregulated in healthy aging aortas and was the only differentially expressed gene Figure 5. Myl9 immunostaining of old (22 months) and young (3 months) injured rat iliac arteries. Shown are representative images of young and old iliac arteries 3 days post injury, immuno- stained for Myl9. Results confirm over-expression of Myl9 in the older injured group and point to concentration of Myl9 expression in the smooth muscle layer of the iliac arteries during this post injury transition period. doi:10.1371/journal.pone.0025855.g005 PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e25855