Extended Data Fig. 1: Aged BMT promotes polyclonal SMC progenitor expansion.
a–d, Young and aged Myh11-CreERT2, ROSA26R(Rb/+) mice were injected with AAV-Pcsk9 and fed a WD for 16 weeks. Brachiocephalic artery sections were imaged for Rb colors (a). Percent of DAPI+ plaque cells marked by any Rb color was quantified (b). For marked plaque (c) or underlying media (d) cells, the percent of cells of each color was quantified for each age group. In a given plaque, color 1 has greatest number of cells, color 2 is second most common color and color 3 is least frequent. In c, n = 5 (young) and 7 (aged) mice and 10 plaques per age group, 5 sections with a total of ~1100–1350 cells and spanning 200 μm per plaque. In d, n = 5 (young) and 7 (aged) mice and media underlying 14 plaques analyzed per group. e-i, Young Ldlr(−/−), Myh11-CreERT2, ROSA26R(Rb/+) mice underwent young or aged BMT. In e, after 4 weeks CD feeding, peripheral leukocyte counts were analyzed. n = 6 mice. In f,g, following 16 weeks WD feeding, transverse aortic root sections were stained with SMA or Sirius red, with dashed lines demarcating cap. Percent of cap cells that are SMA+ and percent of plaque area stained with Sirius red (h,i). n = 6 mice. j–q, Young Apoe(−/−), Myh11-CreERT2, ROSA26R(Rb/+) mice underwent young or aged BMT, AAV-Pcsk9 injection and 16 weeks WD feeding. Aortic sections were stained with H&E and Oil Red O (j,k), with quantification of lesion area (l; demarcated by dashed lines in j), lipid content (m), necrotic core area (n). Sections were imaged for Rb colors (o), and of the marked plaque (p) or medial cells (q), the percent of cells of each color was quantified. n = 5 mice, 12 plaques per group, 5–6 sections with a total of ~1000–1150 cells and spanning 200 μm per plaque. r,s, Murine aortic SMCs were cultured for 8 h with EdU in medium pre-conditioned by BM-derived macrophages of young vs. aged mice. In r, SMCs were stained for EdU and DAPI, and in s, percent of DAPI+ nuclei expressing EdU was quantified. n = 6 mice. Lu, lumen; Med, tunica media; Pl; plaque. All data are means ± SD. Two-tailed Student’s t-test. Scale bars, 100 µm (j,k), 50 µm (a,f,g,o), 25 µm (r).
