Prevention of Senescence in Vasculature Through Quiescence

Introduction

Cellular senescence is a complex state of the cell that involves irreversible proliferative arrest. Senescence can be induced by DNA damage, telomere shortening, oncogenic mutations, metabolic and mitochondrial dysfunction, inflammation, and high levels of reactive oxygen species (ROS). The number of senescent cells increases in multiple tissues with increasing age and are often localized within pathological lesions in a variety of diseases. ¹

Some senescent cells express the proinflammatory senescence-associated secretory phenotype (SASP). Most likely these cells are attempting to participate in a repair response. SASP involves secretion of proinflammatory factors, including cytokines, growth factors, chemokines, and proteases, which contribute to local and systemic dysfunction with aging and in a number of diseases, including liver fibrosis, atherosclerosis, neurodegeneration, and idiopathic pulmonary fibrosis. ¹

Two therapeutic approaches to address senescence are the development of senolytic therapeutics, which kill senescent cells and attenuation of the senescent state itself. Two of the best studied senolytic approaches include genetically killing senescent cells expressing p16ink4a and treatment of such cells with quercetin/dasatinib. ² Examples of senomodulatory agents include Janus kinase 1 (JAK1) and JAK2 inhibitors or rapamycin, which can enhance physical function in older mice in part by reducing SASP. ^{3 –6}

Another approach to reducing the impact of cell senescence on aging animals or humans is to prevent senescence. For example, initial work suggests that agents that increase NAD+ levels may help prevent senescence by a SIRT1-dependent mechanism. ⁷

Caloric restriction (CR) has been observed to increase lifespan and healthspan in animals ranging from worms to mice to monkeys. Although increased autophagy is a key mechanism by which CR is believed to maintain stem cell (SC) function, modulate metabolism, and slow aging, ⁸ CR may act through other less well-characterized mechanisms. Ketone body levels are increased during fasting and CR. The mechanistic role of such metabolites such as ketone bodies (e.g., \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -hydroxybutyrate [ \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB]), which is considered a potential CR mimetic, ⁹ in potentially slowing aging has yet to be explored in depth.

\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB Prevents Vascular Senescence Through hnRNP A1-Mediated Upregulation of Oct4

In an article that reveals potentially important connections between antiaging pathways and cell senescence, Han et al. demonstrate the role of ketone body \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB and acetoacetate (AcAc) on vascular function, quiescence, and senescence. ¹⁰

Treatment with hydrogen peroxide mediates stress-induced senescence of cultured primary human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (hASMCs). Senescence was assessed by increasing senescence-associated beta-galactosidase (SA- \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} Gal) staining (SA- \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} Gal), and the appearance of enlarged flat multinucleated cells. Cotreatment with ketone body \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB but not AcAc reduced the number of senescent cells by SA- \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} Gal and morphology, but not to control levels. Levels of proinflammatory cytokines interleukin (IL)-6 and IL-1alpha, which are associated with and characteristic of SASP, were decreased to near normal levels by extended \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment. Moreover, polyploidy associated with cell senescence was almost completely prevented. Similar results were seen in a model of replicative senescence. HUVECs treated for 4 weeks with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB relative to controls had decreased levels of IL-6 and IL-1alpha levels, reduced cell numbers, and retained greater proliferative potential suggesting that the cells had entered a quiescent rather than a senescent state. Expression levels of senescence biomarkers \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\gamma$$ \end{document} H2AX, p21, and p16 were not elevated in peroxide-treated cells cotreated with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB, whereas by contrast quiescence biomarkers (p27, Lamin B1, and Oct4A) were increased. The two sets of reagents were verified as differential biomarkers for senescence and quiescence, respectively, in control experiments comparing the effects of peroxide-induced senescence with low serum-induced quiescence on human umbilical vein endothelial cells (HUVECS). Flow cytometry cell cycle analysis combining staining for DNA content to distinguish G1/G0 from S/G2/M with RNA staining to distinguish G0 from G1 indicated that \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment results in an increase in the number of cells in G0. ¹⁰

Han et al. hypothesized that the induction of G0 and quiescence prevented induction of senescence by peroxide stress by preventing DNA damage. Consistent with this contention, cotreatment of peroxide-treated HUVECS and hASMCs with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB resulted in reduced levels of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\gamma$$ \end{document} H2AX, which binds DNA double-strand breaks. Reduced levels of DNA damage could be due to repair or initiation of breaks. CHK1–P, BRCA1, or p53 levels remained unchanged in both cell lines, suggesting but not proving that DNA repair levels were not increased. If peroxide-induced stress does not result in significantly increased DNA damage, then master regulator of normative function, p53, should not be activated or induced. ¹⁰

If p53 is not involved, then senescence caused by activation of p53 should not be suppressed by normal quiescence or by \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB. Indeed, senescence induced by treatment with nutlin 3, which increased p53 activity by antagonizing p53 inhibitor MDM2, was not inhibited by preinducing a quiescent state either by low-serum or by \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment. ¹⁰ In fact, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment actually exacerbated the induction of senescence, suggesting that the effects of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB on senescence are indirect and probably through induction of quiescence as the authors hypothesized. Moreover, although not addressed in this article, this result suggests that the quiescence program does not fundamentally oppose the senescence program.

The authors tested several chemical derivatives of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB and found that the S- \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB enantiomer is the bioactive form. Using \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB-conjugated beads (SP- \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB), Han et al. pulled down and identified \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB interacting proteins from extracts of HUVECs using matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis. The four most prominent proteins identified were (1) SFPQ (splicing factor, proline- and glutamine-rich), (2) heat shock protein 70, (3) FUS (RNA-binding protein FUS), and (4) hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1). RNAi knockdown of SFPQ and FUS did not induce senescence. HSP70 a key cell protective protein was not further explored. Most interestingly, silencing of hnRNP A1 caused HUVECs to become senescent, especially after treatment with hydrogen peroxide. ¹⁰

Han et al. then focused on the possibility that Oct4 mRNA interacts with and is stabilized by hnRNP A1. Although no mention was made, they may have investigated the interaction of Lamin B, p27 and NPM1 (a protein that proteome analysis indicates has increased levels after \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment) without finding any changes. However, Oct4 mRNA differentially interacted with hnRNP A1 in the presence of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB and its half life in HUVECs increased from 1.5 to 5 hours. Interestingly, treatment with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB causes hnRNP A1 to translocate from the nucleus to the cytoplasm where it localizes to stress granules—presumably interact with key mRNA targets such as Oct4. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB-induced interaction with Oct4 mRNA is presumed to be the explanation for increased Oct4 levels observed. ¹⁰

Ectopic expression of Oct4 increased the expression of p21, p27, and Lamin B1 with reduced levels of DNA-damage \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\gamma$$ \end{document} H2AX. As expected, Oct4 slowed cell growth and inhibited H₂O₂- and aphidicolin (APC)-induced senescence, but not p53-mediated senescence induced by Nutlin 3a. APC can trigger DNA damage and senescence in cells through inhibition of DNA polymerase alpha in S phase of the cell cycle. Moreover, CRISPR/Cas9 knockout (KO) of Oct4 interferes with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB-induced cell cycle arrest in G0/G1, and instead promotes the G1/S transition and increased induction of senescence. These data suggest that Oct4 expression is necessary for \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB-induced quiescence. Moreover, Oct4 KO HUVECS were more subject to peroxide-induced damage and also increased apoptosis as well as senescence. Oct4 KO also significantly reduced expression of quiescence-associated proteins Lamin B1, p27, and p-eIF2a. ¹⁰

Oct4 expression did not alter the expression of reprogramming pluripotency factors SOX2, KLF4, c-Myc, and Nanog, suggesting these factors are not involved and that a major change in cell differentiation state or stemness does not occur at least in the endothelial cells under study. ¹⁰

To study the in vivo effects of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB, mice were injected intraperitoneally with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB or \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$S - \beta$$ \end{document} -HB. Only in the heart, brain, and aorta were Oct4 protein levels increased, which was accompanied by augmentation of quiescence biomarkers Lamin B1, p27, and phospho-eIF2a in the thoracic aorta. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$S - \beta$$ \end{document} -HB was more bioactive than \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB consistent with the results on human cells. Since, \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$S - \beta$$ \end{document} -HB is not utilized in the ketolysis pathway (unlike \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB), these results suggest that energy homeostasis is not involved in \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB increasing quiescence in endothelial cells. These effects also hold for fasting. Fasting for 3 days, which increases \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB, strongly increased Oct4 in the thoracic aorta, endothelial, and smooth muscle cells. ¹⁰

In middle-aged mice, blood levels of proinflammatory IL-1 alpha are elevated, whereas in older mice (2-year-old) levels of proinflammatory Il-6 are increased. Inverse correlations of IL-1alpha and Il-6 levels with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB levels, which varied over a fourfold range, were observed. The expression of Oct4 and Lamin B1 was positively correlated with higher levels of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB in the blood in both middle-aged and old mice. SA- \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} Gal staining for senescent cells was decreased in association with higher \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB levels. In old mice, close examination of areas of aortic roots, which develop atherosclerosis and are prone to senescence, showed that senescent positive areas of the tissue did not colocalize with Oct 4 expressing areas. ¹⁰

The authors conclude that chronic increased levels of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB reduce or delay cellular senescence through increasing Oct4 expression, likely through stabilization of Oct4 mRNA through hnRNP A1. ¹⁰

These results are consistent with previous work showing that Oct4 is induced in smooth muscle cells in blood vessels during atherosclerosis, where it plays a protective role given that reducing its expression exacerbates atherosclerosis. ¹¹

Confluence of \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB Senopreventive Pathways with Sirtuins?

A key question that arises is whether \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB affects senescence solely through the described hnRNP A1/Oct4 mechanism. What about \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB receptors such as the hydroxycarboxylic acid receptor 2 (HCAR2), which may play a role in stimulating mitochondrial biogenesis through the PGC1α-SIRT3-UCP2 Axis ¹² ? It turns out that HCAR2 is not expressed in HUVECs or endothelial cells (The Human Protein Atlas), which explains why it was not found in Han et al.'s mass spectrometry/affinity screen. But \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB may play a beneficial role in other cell types by other mechanisms as well as by preventing or slowing DNA damage-induced senescence.

Furthermore, hnRNP A1 is widely expressed and plays a key role in RNA homeostasis. For example, mutations in hnRNP A1 can cause amyotrophic lateral sclerosis (ALS). ¹³ Directly relevant to senescence is the finding that hnRNP A1 is reported to bind to and stabilize sirtuin SIRT1 mRNA, increasing its expression. Ectopic expression of hnRNP A1 delays replicative senescence and prevents oncogenic Ras from inducing SASP through upregulating SIRT1 expression. SIRT1 deacetylates and inactivates proinflammatory master regulator nuclear factor (NF)-κB, attenuating induction of IL-6/IL-8, key components of SASP. ¹⁴ Interestingly, it has been observed that hnRNP A1 mRNA levels decrease significantly with age in the liver, heart, and adipose tissue. Given that experimentally manipulated low hnRNP A1 expression directly leads to senescence, ¹⁴ it follows that agents that safely stimulate hnRNP A1 expression may help prevent cell senescence. In any case, SIRT1 represents a second independent way in which hnRNP A1 may prevent senescence. It would be of interest to determine if \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB also enhances SIRT1 binding to hnRNP A1 as well or increases its stabilization as would be predicted by the translocation of hnRNP A1 to stress granules.

Stimulation of SIRT1 activity has also been reported to directly rejuvenate the vasculature of old mice by transducing proangiogenic signals secreted by skeletal muscle cells. Treatment of mice with nicotinamide mononucleotide (NMN), which increases NAD+ levels, improves blood flow and endurance in old mice through SIRT1-dependent stimulation of blood vessel formation and capillary density. Exercise and/or increasing the levels of hydrogen sulfide (H₂S), which itself is a CR mimetic and NAD+ regulator, cooperate with NMN to further increase the benefit of NMN. ¹⁵ Presumably rejuvenating vasculature through NMN and H₂S stimulation of SIRT1 is effective because senescence associated with old animals is repressed or reduced. Potential cooperation of NMN/H₂S SIRT1 stimulation with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB and hnRNP A1 levels is likely.

Adult SCs are critical for tissue maintenance and regeneration. However, SCs can senesce during aging. Reduced NAD+ levels act as a switch to modulate muscle SC (MuSC) senescence through altered mitochondrial activity. Treatment with the NAD+ precursor nicotinamide riboside (NR) raises NAD+ levels, induces the mitochondrial unfolded protein response, restores SIRT1 activity, and increases the synthesis of prohibitins. NR rejuvenates MuSCs functionally in old mice. NR also prevents MuSC senescence in Mdx mice, which models muscular dystrophy and delays senescence of neural SCs (NSCs) and melanocyte SCs. NR treatment showed modestly increased mouse lifespan in one report ⁷ but not in another report. ¹⁶ Thus, agents that replenish NAD+, such as NR and NMN, are also potential senopreventive agents that may synergize with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB.

A Cautionary Tale in the Senescence Story

The senescent phenotype remains ill-defined. The senescent state is characterized by a set of changes that often include some of the following: expression of SA-βgal, p16ink4a, SASP cytokines such as IL-1alpha and IL-6, and nuclear phenotypes such as senescence-associated heterochromatin foci and DNA segments with chromatin alterations reinforcing senescence. ¹⁷ However, each of these features is observed in some nonsenescent cells as well. For example, macrophages can reversibly express SA-βgal and p16ink4a. ¹⁸ Perhaps the key feature that separates senescence from quiescence is that cell cycle arrest is irreversible, similar to some terminally differentiated cells such as neurons. Establishing irreversible withdrawal from the cell cycle is experimentally difficult, because it is always possible that some particular condition for cell cycle reentry is not known. Han et al. did not definitively establish that the cells they considered senescent are truly irreversibly cell cycle arrested. Even expression of the SASP phenotype can be observed in quiescent rather than just senescent fibroblasts. ¹⁹ Therefore, destruction of cells expressing SASP or p16ink4a may also destroy innocent normal bystanders expressing a phenotype associated with senescence, diminishing tissue function. Thus, senolytic therapies may produce a net positive effect, but at a price that eventually may lead to later dysfunction.

A Possible Nexus of Senescence and Reprogramming

The induction of Oct4 by \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB, which is one of the Yamanaka reprogramming factors, places \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB at a nexus between SC maintenance, regeneration, and cell senescence. Although Oct4 apparently is not widely expressed postdevelopmentally and, in general, is insufficient by itself to induce pluripotency or stemness, there is at least one exception. In NSCs, ectopic expression of only Oct4 is sufficient to reprogram cells to induced pluripotent stem cells (IPSc). ²⁰ Given that \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB induces Oct4 in the brain, it is possible that \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB helps maintain the stemness of NSC, and may even increase the multipotency of NSC, or rejuvenate them, a hypothesis that should be tested. Such function could be quite beneficial, especially in the elderly, with the caveat that too much or too prolonged Oct4 induction through \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB might be oncogenic. It is known that \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB can help fuel the growth of some preexisting cancers, whereas suppressing the growth of others through inhibition of histone deacetylases, which would also make it a potential epigenetic modifier in its own right. ^21,22 Because \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB reduces ROS, ²³ and optimal moderate to high ROS levels are needed for reprogramming, ²⁴ there is a built-in safety mechanism that probably would prevent \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB from fully reprogramming NSC.

The Unreasonable Pathogenicity of Senescent Cells and Unreasonable Effectiveness of Senolytics

How efficiently must induction of cell senescence be repressed to achieve effective prevention of associated pathologies? Because elimination of senescent cells appears to be beneficial in so many diseases and pathologies, especially those in which inflammation is thought to play a role, the key question is how pathological are senescent cells? In a recent report, varying numbers of gamma-irradiated senescent luminescent preadipocytes were injected intraperitoneally into young mice. They determined that mice with 0.28% senescent cells or greater in localized adipose tissue near the site of injection suffered dysfunctional consequences such as reduced exercise capability, whereas mice with 0.11% or fewer senescent cells (0.20 × 10 ⁶ injected cells) were unaffected. Moreover, even though most of the original injected senescent cells are cleared after 40 days, nonluminescent senescent cells were still present and appear to spread systemically suggesting that inflammatory signals from the original senescent cells induced more senescent cells. ²⁵ The ability of senescent cells to act as new sources of inflammatory signals in trans and maintain a proinflammatory state are the most likely explanations for the not so unreasonable pathogenicity that senescent cells display. Although the 0.11% minimum threshold is only a ballpark number and likely to vary with tissue type, species, and age, it is a good starting point for determining how efficacious a senolytic, senomodulatory, or senopreventive strategy must be, which for the reported numbers suggests at least a 60% reduction. The senoprevention rate for \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB reported by Han et al. over a 4-week period in vitro was about 60% over control (peroxide induces only a 4 × rate of increase of SA-βgal+ senescent cells when combined with \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment versus 11 × rate of increase of senescent cells with no \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB treatment for 4 weeks), which suggests that this modality of senoprevention is in the right ballpark as well. In other words, increasing \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB indeed may be reasonably effective in preventing senescence.

Medical Implications

\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB may very well act as a senopreventive agent in humans. Of course, this needs to be investigated and established by clinical studies. That said, increasing \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB is achievable today by fasting, chronic CR, exercise, and by ketogenic diets or supplements such as ketone salts, ketone esters, and medium chain triglycerides, which indirectly raise ketone body levels. The problem is that, as with any preventive regimen, it will be difficult to assess benefit.

Combining a regimen that increases \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB with other potentially senopreventive therapies such as NR supplementation is attractive, especially for middle-aged or older people, but again efficacy needs to be established.

Why bother with senoprevention when the prospect of senolytic or senomodulation therapies should solve the senescence problem?

Senolytic approaches have the consequence that they address the problem after senescence and concomitant loss of cells that might have served a useful function. Eventually, the pool of dividing cells, especially SCs, which are nonimmortal, will be depleted. That said, senolytic approaches have proven useful in mouse models of atherosclerosis, ^26,27 among many other pathologies. ² Potential senopreventive therapeutics, such as \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\beta$$ \end{document} -HB that act by inducing quiescence may help preserve cell function, inducing SC function by slowing the approach to replicative senescence. Moreover, combining senopreventive therapeutics with senolytic therapeutics may reduce loss of functional bystander cells that express characteristics of the senescent phenotype but are not senescent, such as some macrophages. Ultimately, a combination of senolytic, senomodulatory, and senopreventive therapeutics may achieve the best results.