Dan Yang and Donald J. McCrann contributed equally to this paper.
Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence
Version of Record online: 15 JAN 2007
Volume 6, Issue 2, pages 257–260, April 2007
How to Cite
Yang, D., McCrann, D. J., Nguyen, H., Hilaire, C. St., DePinho, R. A., Jones, M. R. and Ravid, K. (2007), Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence. Aging Cell, 6: 257–260. doi: 10.1111/j.1474-9726.2007.00274.x
- Issue online: 5 FEB 2007
- Version of Record online: 15 JAN 2007
- Accepted for publication 6 December 2006
- cellular senescence;
- senescence-associated β-galactosidase;
- vascular smooth muscle
We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16INK4a and senescence-associated β-galactosidase activity are detected primarily in the old tissues. VSMC from p16INK4a knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.
Polyploidy represents greater than diploid DNA content (Ravid et al., 2002). In previous studies, we showed that in old Brown Norway rats the majority of aortic vascular smooth muscle cells (VSMC) are polyploid (Jones & Ravid, 2004). Studies with cultured cells suggested that polyploidy may be associated with cellular senescence (Sigal et al., 1999; Wagner et al., 2001; Uryvaeva et al., 2004). This, however, has never been demonstrated in vivo. As to the significance of senescence for cellular physiology in early life, cell cycle arrest and senescence represent an anticancer mechanism (Campisi, 2005); however, in later life, senescent cells acquire gene expression/phenotypic changes that may contribute to aging and detrimental pathologies (Patil et al., 2005).
In the current study, we examined the contention that polyploid aortic VSMC develop a senescence phenotype in vivo. Increased incidence of p16INK4a expression and in some cases of p21CIP1/WAF1 (p21) are considered to be biomarkers of senescence (Herbig et al., 2004, 2006). Aortas derived from old Brown Norway rats are marked by a large fraction of polyploid aortic VSMC, as also indicated by a high frequency of cells with large nuclei (Fig. 1A) (Jones & Ravid, 2004). Immunohistochemistry indicated that p16INK4a is virtually not detected in aortic VSMC of young Brown Norway rats, while frequently noted in tissues of old rats (Fig. 1A and Supplementary Fig. S1). Similar results were obtained with individual VSMC dispersed from whole aortas (Supplementary Fig. S2). About 50–60% of the polyploid cells are positive for p16INK4a in aortas derived from old rats, and approximately 30% of the p16INK4a-positive cells are diploid (Fig. 1B), suggesting that p16INK4a up-regulation alone is not a sufficient inducer of polyploidy. Of interest, p16INK4a overexpression in smooth muscle cells does not inhibit cell proliferation (Tanner et al., 2000). In contrast to p16INK4a, p21 is detected at low levels in VSMC from both young and old aortas, with no particular distribution among diploid and polyploid cells (Supplementary Fig. S3A). Analysis of commercially available p21 knockout mice (Deng et al., 1995) or p16INK4a knockout mice (Sharpless et al., 2001) indicated no change in aortic VSMC ploidy compared to control (Supplementary Fig. S3B,C).
Another hallmark of cellular senescence is the inability of cells to re-enter the cell cycle. Diploid aortic VSMC isolated by flow cytometry, as in Nagata et al. (2005), readily attach to the tissue-culture flasks and proliferate. In contrast, the majority of polyploid aortic VSMC remain permanently growth-arrested (Fig. 1C).
A traditionally accepted marker for this phenotype is senescence-associated β-galactosidase (SA-β-gal) activity (Dimri & Campisi, 1994; Dimri et al., 1995). A recent study identified this activity to be contributed to in part by lysosomal β-galactosidase, and although it is a marker of aging cells, it is not required for senescence (Lee et al., 2006). Analysis of aortic sections and of freshly isolated aortic VSMC showed that SA-β-gal-positive cells are polyploid, but not all polyploid VSMC are SA-β-gal-positive (Fig. 2 and Supplementary Fig S4). In comparing aortic sections to isolated cells, lower percentages of SA-β-gal-positive cells were found, perhaps due to weak SA-β-gal staining, which is likely more challenging to detect in tissues. Interestingly, about 70% of the p16INK4a-positive VSMC in 34-month-old samples are also positive for SA-β-gal (Supplementary Fig. S5).
In summary, our study is the first to show that senescence markers in aging aortas are primarily detected in polyploid cells, but not in all of them. We propose that the ploidy state along with other signals gradually trigger a complete senescent phenotype and potential vascular pathology.
This work was supported by grant AG022623 to K.R. K.R. is an established investigator with the American Heart Association. R.A.D. is an American Cancer Society Research Professor and Ellison Medical Foundation Senior Scholar.
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Supplementary Figure 1 Validation of the immunohistochemistry method used to stain aortic tissue sections with anti-p16,INK4a. Supplementary Figure 2 Immunohistochemistry of isolated aortic vascular smooth muscle cells (VSMC) stained with anti-p16INK4a. Supplementary Figure 3 A. Immunohistochemistry of aortic sections with anti-p21. B. Ploidy analysis of VSMC derived from p21 knockout mice. C. Ploidy analysis of VSMC derived from p16INK4a knockout mice. Supplementary Figure 4 SA-β-gal activity assay in isolated aortic vascular smooth muscle cells. Supplementary Figure 5 Isolated aortic VSMC subjected to immunohistochemistry with anti-p16INK4a and to SA-β-gal activity assay.
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