Fig 1: Vascular attrition precedes and dictates cellular aging.(A) Bar graphs of the kidney, muscle, spleen, and thymus for vessel density (top, n = 5) and PDGFRß+ cells (bottom, n = 4 or 5). (B) Bar graphs show concentration of CDKN2A measured by ELISA in tissues from p16LUC mice aged 10, 30, 55, and 90 weeks (n = 5). (C) Representative FACS plots of side scatter area (SSC-A) versus PE-Texas red-A (representing DHE+ cells) in the thymus at 10 and 30 weeks and the positive control (Luperox treated). Bar graphs showing the quantification of DHE+ cells in tissues at different ages (kidney and spleen, n = 5; others, n = 4). (D) Representative FACS plots of MSPCs of thymus at 10, 30, and 55 weeks. Bar graphs showing quantification of MSPCs (%) in tissues at different ages (n = 5). (E) 3D images of thymus from Vegfr2i?EC (M) and littermate control (C) mice immunostained as indicated. Bar graphs showing the quantifications of CDKN2A concentration, MSPCs (%), and fibroblast numbers (n = 5). Data represent means ± SD. The P value derived from two-tailed unpaired t tests is given for two groups and one-way ANOVA test with Tukey’s multiple comparisons test for more than two groups. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Scale bars, (E) 50 µm.
Fig 2: Diminished IGF1 and Akt mRNA in human COPD lungs and loss of p16 increases Akt signaling in CS-exposed lungs. a, b Gene microarray analysis of whole lung lysates from 9 normal and 94 COPD donors **p = .85e−6. c–e Protein profile of phosphor-Akt Thr308 (*p = 0.0359), total Akt (*p = 0.0396, **p = 0.0416), and Cyclin D (**p = 0.0249). f Representative western blotting of whole lung lysates exposed to RA and CS (n = 4–6). g Representative images of fibroblasts isolated from p16+/+ and p16−/− lungs then treated with DMSO or Akt I (10 μM, scale bar = 400 µm). h QPCR was performed to determine Cyclin D gene expression. Data are expressed as the average ± SEM of at least three independent experiments, *p = 0.0471, **p = 0.0230, ***p < 0.0001, #p = 0.001
Fig 3: p16−/− lungs maintain function and structure when challenged with CS. a Lung compliance (**p < 0.0177), pressure/volume, and area (**p < 0.0001) between the inflation and deflation limb of the PV loop measured using Flexi-Vent. b Representative Masson’s trichrome staining of lungs exposed to RA or CS (scale bar = 40 µm), mean linear intercept was determined c from these images and quantified (*p < 0.0001 and **p < 0.0001 vs. p16−/− RA). d Body weight increases over 4 months, values are the percentage increase from day 0 (*p = 0.0031, **p < 0.0001). Quantitative RT-PCR analysis e of MMP-12 (*p = 0.029 vs. p16+/+ RA, **p = 0.0077 vs. p16+/+ CS), IL-33 (*p = 0.012 vs. p16+/+ RA, **p = 0.045 vs. p16+/+ CS), and TGFβ1 (*p = 0.0021 vs. p16+/+ RA, **p = 0.0036). After 4 months of CS, all samples were normalized to mouse GAPDH. f MMP-12 (*p = 0.0004 vs. p16+/+ RA, **p = 0.0088 vs. p16+/+ CS), IL-33 (*p = 0.004 vs. p16+/+ RA, **p = 0.023 vs. p16+/+ CS, ***p = 0.0452 vs. p16−/− RA), and TGFβ1 (*p = 0.0005 vs. p16+/+ RA, **p = 0.0097, ***p = 0.0036 vs. p16+/+ RA), protein levels determined by ELISA. All protein levels are normalized to total protein in the lysate. All data is expressed as mean ± SEM, n = 4–14 mice per group
Fig 4: SASPs and Inflammatory cytokines upregulated with CS ameliorated in p16−/− lungs. a Cytokines associated with senescent-associated secretory phenotype (SASP) and b inflammation on p16+/+ and p16−/− lungs exposed to RA or CS. IL-6 *p = 0.0499, **p = 0.0043 vs. p16+/+ CS, CXCL-1 *p = 0.0084, IL-13 *p = 0.05, **p = 0.0260, CCL-2 *p = 0.0006 vs. p16+/+ RA **p = 0.0009 vs. p16+/+ CS, RANTES *p = 0.0346, **p = 0.0267 vs. p16+/+ CS, Eotaxin *p = 0.0221 vs. p16+/+ RA, **p = 0.0010 vs. p16+/+ CS, IP-10 *p = 0.0372 vs. p16+/+ RA, **p = 0.0108, IL-5 *p = 0.0308, **p = 0.0377, IL-9 *p = 0.0425, IL-17a *p = 0.0234, **p = 0.05 vs. p16+/+ CS. N = 4–12 lungs per group
Fig 5: Proposed mechanism of protection. In the presence of chronic CS, p16 levels increase and cells become senescent. CS induces pro-inflammatory cytokines throughout the lung, and in the senescent cells SASPs are secreted, which perpetuate senescence and alveolar destruction (right side). When the cell cycle inhibitor p16 is not present (left side), basal levels of IGF1 are upregulated, resulting in stimulated insulin receptors and activation of Akt. Activated Akt stimulates cell proliferation evidenced by increases in Cyclin D expression and the maintenance of healthy alveoli
Supplier Page from Abcam for Mouse CDKN2A/p16INK4a ELISA Kit