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. doi:10.1371/journal.pone.0025855.g006 in injured iliac arteries at all 3 post-injury time points explored (30 minutes, 3 days, and 7days). Myosins are a diverse superfamily of actin-dependent molecular motors consisting of distinct structural and functional classes and are implicated in contraction, cell shape, migration, adhesion, intracellular transport of organelles, and signal transduction [9]. Conventional myosin II-complexes are hexamers consisting of 2 heavy chains, 2 regulatory MLCs, and 2 essential light chains. The activity of myosin is regulated by reversible phosphorylation of specific amino acids in the regulatory MLC [9]. In the vascular muscle, the interaction of myosin with actin is the primary determinant of force production (contraction). Arterial vascular smooth muscle cells contain abundant amounts of smooth muscle myosin heavy chain (gene MYH11) that are generated by alternative splicing of exons in the head (SM-A,B) and tail (SM1,2) of the motor protein [10].The consequences of the differential distribution of muscle and non- muscle MLC isoforms in vascular tissues are unknown though it has been proposed that MLCs influence myosin ATPase activity and velocity of shortening [11]. We found that Myl9 expression transiently increased with age in the smooth muscle cell layers of injured arteries (3 d post- injury). Our finding is in agreement with previously published work demonstrating that aging rats showed an increased vascular negative remodeling (constriction) after balloon dilatation com- pared with adult animals[12]. This agrees with the aggressive circumferential coronary constriction (‘recoil’) observed after balloon angioplasty in patients with coronary artery diseases and animal models [12,13]. Our observation is also in accordance with previous findings from the literature reporting that the effect of phosphorylation and de-phosphorylation of Myl9 protein to be at the essence of altered vascular contractility [14]. Interestingly, we found that in healthy and late injured (30 days post injury) iliac arteries, Myl9 expression increased with age in the endothelial layers. Our findings are in line with those recently published by Licht et al [15], describing Jun-b dependent expression of Myl9 in primary cultures of endothelial cells (ECs). The effect of contractility proteins on aged-related vascular remodeling and their localization in the EC versus SMC layers is not well understood. Zeng et al had also found Myl9 expressed in ECs, which upon phosphorylation regulated endothelial cytoskeletal remodeling, and increased endothelial cell contraction resulting in increased paracellular gap formation and endothelial hyperpermeability [16]. If Myl9 ectopic expression causes endothelial dysfunction, it may explain the increased signs of arterial constriction in the aging vasculature. Vascular Myl9 with Injury and Aging For instance, age-related endothelial dysfunction in aged rat aortas was found associated with decreased vasorelaxation likely caused by a decline in NO and endothelium-derived hyperpo- larizing factor [17,18]. Myosin light chain phosphorylation may play an important role in endothelial function, including reorganization of the cytoskel- eton, changing cell shape, and controlling endothelial cell isometric tension [19,20]. Interestingly, endothelial permeability appears quite early in the progress of aging. In fact aortas of 30- month-old rats had a 2-fold increase in endothelial permeability to albumin compared with 10-month-old rats [21]. Endothelial cells lining blood vessels form a continuous layer that constrains proteins and blood elements to the vascular lumen. An increase in endothelial cell isometric tension (contraction) may disrupt the continuous endothelial barrier leading to an increase in perme- ability and development of edema, a hallmark of acute and chronic inflammation. It is believed that elevated permeability of the endothelium allows entry of lipoproteins into the vessel wall, which become oxidized and propagate endothelial dysfunction [22]. The relevance of vascular permeability in the development of age-related vascular diseases have been extensively discussed [23,24]. Of note, endothelial leakage favors the passage of plasma macromolecules across the endothelium and their trapping in the intima, which could contribute to the development of restenosis and atherosclerosis. The increased endothelial Myl9 may also explain the morphological changes of endothelial cells associated with aging which could account for the altered endothelial permeability [25]. Conversely, phosphorylation of myosin regula- tory light chain (MLC) not only increases actomyosin ATPase activity, but also destabilizes the endothelial cell2cell junctions leading to increased monolayer cell permeability [26], [16,27].Our results are encouraging and they warrant future research on demonstrating whether ectopic expression of Myl9 in the aged endothelium causes endothelial dysfunction. In conclusion, our findings suggest that the amount and localization of Myl9 is critical in the vascular function and remodeling process with age. The analysis presented here sets the basis for future studies in the field of vascular aging, in particular for those aimed at preventing age-related vascular dysfunction. Based on the knowledge of the role of Myl9 in endothelium degeneration, strategies to prevent this process could be designed and tested. New therapeutic interventions to prevent vascular aging might have enormous medical consequences given the strong age dependency of cardiovascular diseases. Methods Rat Balloon Injury Model Aged Fisher (.22-month-old, F344) rats were purchased from the National Institute of Aging (Bethesda, MD). Young (2-month- old) rats were obtained from Harlan Laboratories (Indianapolis, IN). Animal work was revised and approved by the Institutional Committee for Use and Care of Laboratory Animals at the University of Miami protocol approval ID 05-069 entitled ‘‘The role of vascular senescence in age-related vasculopathies’’. All operative procedures were under isoflurane anesthesia (Baxter, IL, USA). Balloon injury in the right iliac artery was inflicted with a 2F Fogarty catheter (Baxter Corp., Irvine, CA, USA) adapted to a custom angiographic kit (Boston Scientific, Scimed) [28]. The balloon catheter was always inflated to yield a constant pressure between 1.5–1.6 atmospheres. Arterial specimens were collected 30 min, 3, 7 and 30 days after injury, cut in two fragments that were fixed in 4% formalin-PBS or submerged in RNA later until RNA isolation. All animal procedures were previously approved PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e25855