Volume 21, Issue 6, Pages 1471-1480 (November 2017) Downregulation of the Apelinergic Axis Accelerates Aging, whereas Its Systemic Restoration Improves the Mammalian Healthspan  Rahul Rai, Asish K. Ghosh, Mesut Eren, Alexander R. Mackie, Daniel C. Levine, So-Youn Kim, Jonathan Cedernaes, Veronica Ramirez, Daniele Procissi, Layton H. Smith, Teresa K. Woodruff, Joseph Bass, Douglas E. Vaughan  Cell Reports  Volume 21, Issue 6, Pages 1471-1480 (November 2017) DOI: 10.1016/j.celrep.2017.10.057 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 21, 1471-1480DOI: (10.1016/j.celrep.2017.10.057) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Aging- and Stress-Induced Senescence Downregulate the Endogenous Apelinergic Axis (A) Effect of aging on Aplnr transcript levels (n = 4). (B) Immunoblots showing the effect of aging on renal and hepatic aplnr. (C) Quantification of (B). (D) Effect of aging on cardiac and renal apln protein (n = 4). See also Figure S1A. (E) Effect of aging on Apln transcript levels (n = 4). (F) Apln transcript levels in the heart and aorta of placebo- or L-NAME- and saline- or Ang II-treated mice. n = 4 (L-NAME heart), n = 8 (L-NAME aorta), n = 3 (placebo/ saline/Ang II). See also Figure S1B. (G) HCAECs stained with senescence-associated-β-gal (SA-β-gal) stain after 4 days of TGF-β treatment. Apln and Aplnr transcript levels were assessed after 4 days (n = 3). All data are shown as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Reports 2017 21, 1471-1480DOI: (10.1016/j.celrep.2017.10.057) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Aged aplnr−/− and apln−/− Mice Exhibit Accelerated Multi-organ Aging (A) ACh-mediated dilatory responses of WT, aplnr−/−, and apln−/− mesenteric arteries ex vivo. See also Figures S2A and S2B. (B) Age-dependent changes in systolic blood pressure (SBP) in WT, aplnr−/−, and apln−/− mice (n = 3–5). See also Figure S2C. (C) i: representational periodic acid-Schiff (PAS)-stained cardiac sections, showing cardiomyocyte morphology and size. ii and iii: representational images of PAS-stained renal sections, showing glomerular morphology (ii) and tubular lumens (iii). Protein casts are noticeable in aplnr−/− renal sections (black arrow). (D) Quantification of cardiomyocytic surface area in WT, aplnr−/−, and apln−/− hearts at 15 months. (E) Comparison of heart weight to tibial length (HW/TL) ratios in WT, aplnr−/−, and apln−/− mice at 9 and 15 months of age (n = 4–6). (F) Quantification of glomerular diameters in 15-month-old WT, aplnr−/−, and apln−/− kidneys. (G) Comparison of renal weight (RW)-to-TL ratios in WT, aplnr−/−, and apln−/− mice at 9 and 15 months of age (n = 4–6). (H) Comparison of urinary protein-to-creatinine ratios at 15 months in WT, aplnr−/−, and apln−/− mice (n = 3). (I) Blood glucose levels at 9 and 15 months in WT, aplnr−/−, and apln−/− mice (n = 3–5). All data are shown as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. Cell Reports 2017 21, 1471-1480DOI: (10.1016/j.celrep.2017.10.057) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Genetic or Pharmacological Repression of Apelinergic Signaling Induces Cellular Senescence (A) i: apln immunostaining in control (ctrl) and aplnKd HCAECs. ii: SA-β-gal staining of ctrl and aplnKd HCAECs. (B) 50,000 ctrl and aplnKd cells were plated on day 0 and counted every 24 hr for 4 days. (C) Quantification of SA-β-gal-positive ctrl and aplnKd HCAECs from (A), ii (600 cells were counted). (D) Apln, p21, p53, and pai1 transcript levels in ctrl and aplnKd HCAECs. (E) Immunoblots showing levels of p53, IGFBP3, p21, p16, and β-actin in ctrl and aplnKd HCAECs. (F) Immunoblots showing levels of p16 and β-actin in HCAECs treated with DMSO (0.01%) or aplnr antagonist (5 μM ML221). (G) p21, p53, and TGF-β transcript levels in HCAECs treated with DMSO or aplnr antagonist. (H) Quantification of SA-β-gal-positive HCAECs and HCFs after 4 days of incubation with DMSO or aplnr antagonist, (approximately 400 cells counted). See also Figure S2I. (I) Apln, aplnr, and p21 transcript levels in HCFs treated with DMSO or aplnr antagonist. (A–I) n = 3 (biological replicates). (J) DNA transcription factor (TF) array showing TFs affected by aplnKd in HCAECs. An increase or decrease of ≥ 2-fold was considered significant. n = 2 (biological replicates). All data are shown as the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Cell Reports 2017 21, 1471-1480DOI: (10.1016/j.celrep.2017.10.057) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Systemic Restoration of apln Ameliorates Age-Associated Pathologies and Extends the Murine Healthspan (A) Changes in LV posterior wall (LVPW) thickness after 4 weeks of apln infusion in 15-month-old mice. (B) Changes in heart weight-to-TL ratio. (C) Post-exercise elevation in SBP and diastolic blood pressure (DBP). (A–C), n = 4 (saline), n = 6 (apln-13). (D) Total distance covered by mice during the week of behavioral assessment. (E) Total movement during the active and inactive phases. (F) Rhythmicity of food consumption. The feeding curve of saline-infused mice is out of sync because food consumption during the inactive phase is also seen (denoted by arrows). Apln rejuvenates the feeding patterns. The curve was generated during the third to fifth day. (G) Energy expenditure (EE) of apln- or saline-infused mice. No differences are seen during the active phase, whereas apln lowers EE during the inactive phase, suggesting an improved, less fragmented resting profile. Values were normalized by body weight. Shown is a mean analysis of 4-day measurements. (D–G) n = 4. (H) Co-treatment with apln decreases TGF-β-induced endothelial senescence. HCAECs were treated with either PBS, TGF-β (10 ng/mL), apln-13 (1 μM), or apln-13 + TGF-β for 4 days and then stained with SA-β-gal. All data are shown as the mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001. Cell Reports 2017 21, 1471-1480DOI: (10.1016/j.celrep.2017.10.057) Copyright © 2017 The Authors Terms and Conditions