Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence

Abstract

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 p16(INK4a) and senescence-associated beta-galactosidase activity are detected primarily in the old tissues. VSMC from p16(INK4a) 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.

Fig. 1

Polyploid vascular smooth muscle cells (VSMC) express p16^INK4a and lose replication potential. (A) Aortic tissue sections derived from young and old Brown Norway rats were fixed with paraformaldehyde and subjected to immunostaining as detailed under the Supplementary material. The orange arrows point to p16^INK4a-positive cells (brown staining localized to the nucleus) and the blue ones to p16^INK4a-negative cells. Staining with anti-IgG was used as control. Shown are phase-contrast images at ×600 magnification, representative of three rats in each group. Staining was also carried out in cryosections, showing similar results (not shown). (B) The filled bars depict the percentage of polyploid cells identified based on nuclear size (for tissue sections), or 4′,6-diamindino-2-phenylindole (DAPI) staining (for isolated VSMC), the empty bars represent the percentage of polyploid cells that are also p16^INK4a-positive, and the striped bars represent the percentage of p16^INK4a-positive cells that are polyploid. Five slides per rat were analyzed, and a total of two or three rats were examined in each age group. Shown are average percentages ± standard deviations. (C) VSMC were isolated from 34-month-old aortas, and sorted by flow cytometer to obtain diploid (2N) and ≥ 4N populations, as described in Jones & Ravid (2004). Equal numbers of cells were cultured and monitored as in Nagata et al. (2005). Shown are phase-contrast microscopy images of the cells at 2 (lower panels) and 7 days after culturing. A small fraction of the polyploid population proliferated, but this also corresponds to the number of contaminating diploid cells in this pool (up to 10%).

Fig. 2

Increased senescence-associated β-galactosidase (SA-β-gal) activity is noted in aortic vascular smooth muscle cells (VSMC) of old rats, and predominantly in the polyploid cells. (A) The SA-β-gal staining assay (blue color; upper panel) was performed on 5-µm cryosectioned aortas and counterstained with 4′,6-diamindino-2-phenylindole (DAPI) to display nuclei (lower panel) (see Supplementary material). The upper panels show aortic sections viewed with phase-contrast microscopy and the bottom panels display respective cell nuclei stained with DAPI (examined with Olympus microscope; ×100 objective). Arrows point to SA-β-gal–positive cells (upper panels) and to corresponding large nuclei (lower panels). (B) Tissue sections or isolated VSMC prepared from 24- or 34-month-old Brown Norway rats as described in the Supplementary material were quantitated. The empty bars depict the percentage of polyploid cells identified based on nuclear size (for tissue sections) or DAPI staining (for isolated VSMC), and the filled bars represent the percentage of polyploid cells that are also SA-β-gal-positive. Three to five slides per rat were analyzed, and a total of two to three rats were examined in each age group. Shown are average percentages ± standard deviations.