Dynamic regulation of vascular myosin light chain (MYL9) with injury and aging

Abstract

Background: Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels.

Methodology/principal findings: We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBI's GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers.

Conclusions/significance: The consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty.

Figure 1. Differentially expressed genes overlapping in multiple time points in old versus young injured rat iliac arteries.

A. Venn diagram shows the number of differentially expressed genes determined by our analysis of 3 different time points in old versus young injured rat iliac arteries. 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 Myl9 gene expression levels in multiple time points in old versus young injured rat iliac arteries. Myosin Light chain 9, Myl9, was significantly downregulated at 30 mins (−2.2 fold), highly upregulated at 3 days (4.6 fold), and upregulated at 7 days (2.9 fold). Color bar shown in Log₂.

Figure 2. Differentially expressed genes overlapping in different age groups of 30 min-injured rat iliac arteries.

A. Venn diagram shows lists of differentially expressed genes determined by our analysis of 3 different pairs of age groups of 30 min-injured rat iliac arteries. 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 different age groups of 30 min-injured rat iliac arteries.12 genes overlapped in old versus young, old versus old, and young versus young, 30 min-injured rat iliac arteries. Color bar shown in Log₂.

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.

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.

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, immunostained 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.

Figure 6. Myl9 immunostatining of old (22 months) and young (3 months) injured rat iliac arteries.

Shown are representative images of young and old iliac arteries 30 days post injury, immunostained for Myl9. Results confirm over-expression of Myl9 in the older injured group and point to concentration of Myl9 expression in the endothelial layer of the iliac arteries during this post injury period.