Fig 2: Evidence for EC efferocytosis and PCSK9 regulation of MerTK expression. (A) Uptake of normal or apoptotic Jurkat cells by human aortic endothelial cells (HAECs, P3) after 1 hour of co-incubation. (B) Efferocytosis of apoptotic Jurkat cells by P3 HAECs after 1 hour of co-incubation. (C) Confocal microscopy at 63x magnification verifies efferocytosis by P3 HAECs. (D) Efferocytosis in P3 and P15 HAECs. (A-D) Jurkat cells were labeled with green PKH67-GL (2 μM; Sigma-Aldrich) and HAECs were labeled with red PKH67-GL (2 μM; Sigma-Aldrich) per the manufacturer's instructions. Green cells are apoptotic Jurkat cells that were not engulfed by HAECs. Green/red small round cells are apoptotic Jurkat cells that were engulfed by HAECs. Large red cells are HAECs. (E-F) RNA-seq or western blot for MerTK expression in HAECs. (G) Expression of PCSK9 and MerTK in young (P3) and aged (P15) HAECs. (H) Treatment with PCSK9 or control PBS for 24 hours at indicated concentrations and MerTK expression in young P3 HAECs. (I) MerTK expression in young and aged HAECs treated with PBS (control) or PCSK9 at 1 µg/mL for 24 hours. (J) MerTK expression in P3 and P15 primary mouse aortic endothelial cells (MAECs) isolated from WT and PCSK9-/- mice. Statistical analyses were performed with GraphPad Prism 9.0 using a two-tailed unpaired t-test. Data represent mean ± SD (n=4-6). *P< 0.05, **P<0.01, ****P< 0.0001.

Fig 3: Pep2-8 treatment postpones the aging process. (A) Bodyweight of Pep2-8 or saline treated WT mice. (B) More detailed bodyweight from Pep2-8 treated group based on (A) in aged WT mice. (C) β-Galactosidase staining as a senescence marker in the whole aorta from WT and PCSK9-/- 24-month-old mice. (D) β-Galactosidase staining in P3 (left six panels) and P15 (right six panels) MAECs isolated from WT and PCSK9-/- mice. WT MAECs were treated with mouse recombinant PCSK9 at 0.5 µg/mL for 1 week. (E) Volcano plot illustrating differentially expressed proteins in the aortic arch from Pep2-8 or saline treated aged WT mice. Relative protein abundance (log2) plotted against significance level (- log10 P-value), showing significantly (p < 0.05) downregulated (blue), upregulated (red) or non-differentially expressed proteins (gray). (F) Pep2-8 regulates LDL and HDL-cholesterol (HDL-C), but not VLDL, based on activation z-score. (G-H) IPA prediction of HDL-C and LDL networks. Upregulated and downregulated proteins are highlighted in red and green, respectively, and the color depth is correlated to the fold change. Orange and blue dashed lines with arrows indicate indirect activation and inhibition, respectively. Yellow and gray dashed lines with arrows depict inconsistent effects and no prediction, respectively.

Fig 4: Effect of PCSK9 and Pep2-8 treatment on aging markers. (A) Western blotting for expression of eNOS, NLRP3, pro-IL-1β, NF-κB, TNF-α and p22phox in HAECs. PCSK9 at 1 µg/mL or PBS control were treated with P15 HAECs for 24 hours. (B) ELISA analysis for PCSK9 concentration in serum from young (3-month-old) and aged (24-month-old) WT mice. (C) Immunofluorescent staining for eNOS, pro-IL-1β, NF-κB and p22phox in the aortic arch from the aged mice treated with saline or Pep2-8. Statistical analyses were performed with GraphPad Prism 9.4.1 using a two-tailed unpaired t-test. Data represent mean ± SD (n=3 in vitro and n=10 in vivo). ***P<0.001, ****P< 0.0001.

Fig 5: Pep2-8 treatment and protein changes in the aortic arch of WT 24-month-old mice injected with Pep2-8 vs saline every two weeks for 10 weeks. (A-B) 60 representative proteins downregulated or upregulated by Pep2-8 in aged WT mice based on activation z-score. (C) Pep2-8 treatment regulates inflammatory cytokines or chemokines based on activation score in the aortic arch of 24-month-old mice. (D) Canonical pathways based on a -log(p-value) greater than 1.5 and an absolute value z-score greater than 2.0. Color by z-score. Blue signifies negative value; orange signifies positive value; and grey signifies no activity pattern. Size is proportional to the number of genes that overlap the pathway. (E) Graphical summary for the signaling pathways. Orange and blue indicate activation and inhibition, respectively. Gray dashed lines depict no prediction. (F) Schematic cartoon for the relationship between PCSK9-mediated defective efferocytosis and vascular aging. This figure was created with Biorender.com.