White, Thomas A.

We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.

Produced by senescent cells, the senescence-associated secretory phenotype (SASP) is a potential driver of age-related dysfunction. We tested whether circulating concentrations of SASP proteins reflect age and medical risk in humans. We first screened senescent endothelial cells, fibroblasts, preadipocytes, epithelial cells, and myoblasts to identify candidates for human profiling. We then tested associations between circulating SASP proteins and clinical data from individuals throughout the life span and older adults undergoing surgery for prevalent but distinct age-related diseases. A community-based sample of people aged 20–90 years (retrospective cross-sectional) was studied to test associations between circulating SASP factors and chronological age. A subset of this cohort aged 60–90 years and separate cohorts of older adults undergoing surgery for severe aortic stenosis (prospective longitudinal) or ovarian cancer (prospective case-control) were studied to assess relationships between circulating concentrations of SASP proteins and biological age (determined by the accumulation of age-related health deficits) and/or postsurgical outcomes. We showed that SASP proteins were positively associated with age, frailty, and adverse postsurgery outcomes. A panel of 7 SASP factors composed of growth differentiation factor 15 (GDF15), TNF receptor superfamily member 6 (FAS), osteopontin (OPN), TNF receptor 1 (TNFR1), ACTIVIN A, chemokine (C-C motif) ligand 3 (CCL3), and IL-15 predicted adverse events markedly better than a single SASP protein or age. Our findings suggest that the circulating SASP may serve as a clinically useful candidate biomarker of age-related health and a powerful tool for interventional human studies.

Abstract TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

Background Accumulation of senescent cells and the release of proteins from those cells contribute to aging‐related disorders. Estrogen may reduce physiological changes associated with aging through inhibition of cell senescence. This study aimed to determine the effects of menopausal hormone treatments on circulating levels of cell senescence markers. Methods GDF 15, TNF R1, FAS, and MIP1α were measured in serum using bead‐based assays and compared among women who were within 3 years of natural menopause (mean age 52 years) participating in the Kronos Early Estrogen Prevention Study (KEEPS). Women were randomized to either placebo (n=38), oral conjugated equine estrogen (oCEE, n=37), or transdermal 17β‐estradiol (tE2, n=34). Serum levels of the senescent markers for each treatment group were compared to placebo 36 months after randomization using the Wilcoxon rank‐sum test. Results Serum levels of GDF 15, TNF R1, and FAS but not MIP1α were lower in both the oCEE and tE2 groups compared to placebo. However, the difference in levels between treatment and placebo were greater for oCEE [−108 pg/mL (p=0.008), −234 pg/mL (p=0.0006), and −1374 pg/mL (p<.0001), respectively] than for tE2 [−76 pg/mL (p=0.072), −105 pg/mL (p=0.076), and −695 pg/mL (p=0.036), respectively]. Conclusions In recently menopausal women, oCEE and tE2 reduced circulating levels of some markers of cell senescence. Differences in the magnitude of effect may reflect the differences in circulating levels of 17β estradiol, estrone, and sulfonated estrogens between the two active treatments. Support or Funding Information The research is supported by the Kronos Early Estrogen Prevention Study, NIA U54‐AG44170, and the Mayo Fnd.

Abstract Cellular senescence is a state of stable growth arrest in response to stress, which is a fundamental process of biological aging. They secrete products, the Senescence-Associated Secretory Phenotype (SASP), which consists of inflammatory cytokines, chemokines, growth factors and matrix remodeling proteins. Senescent cells accumulate with advancing age and partial elimination of senescent cells can reverse age-related dysfunction and increase mean lifespan in mice. However, it is not clear whether components of the SASP can be measured in human plasma and serve as aging biomarkers. Here we generated a candidate panel of senescence markers based on a multiplexed bead-based assay of proteins secreted by senescent preadipocytes, endothelial cells, fibroblasts, myoblasts, preadipocytes, and epithelial cells compared to non-senescent controls. The SASP undoubtedly varies by cell type; however, we observed that multiple components of the SASP are conserved. We then assessed circulating SASP components in human plasma samples from Mayo Clinic Biobank participants (n=280, 20 male and 20 female per decade, age 20-90) using the same method. We confirmed that components of the SASP can be quantified in human plasma with the multiplexed bead-based assay and observed several SASP components robustly increase with chronological age in humans. Our study illustrates that senescence-associated secretome components are detectable in human plasma and could potentially serve as biomarkers of systemic aging and senescent cell burden.

Abstract Senescent cells drive age-related tissue dysfunction through their potent secretome, termed the senescence associated secretory phenotype (SASP). Circulating concentrations of SASP factors may reflect biological age and serve as clinically useful biomarkers of surgical risk and ultimately, surrogate endpoints in clinical trials. However, they remain largely uncharacterized. We tested associations between circulating concentrations of SASP proteins and biological age, as determined by the accumulation of age-related health deficits, and/or postoperative outcomes in a sample of residents in Olmstead County, MN, age 60-90 years (n = 115) and cohorts of older adults undergoing surgery for severe aortic stenosis (prospective; n = 97) or ovarian cancer (case-control; n = 36). Circulating concentrations of SASP factors were associated with biological age and adverse postoperative outcomes, including risk of any adverse event or rehospitalization within the year following surgery (aortic stenosis group) or admission to an intensive care unit within 30 days of hospital discharge (ovarian cancer group). Gradient boosting machine modeling revealed a panel of SASP factors that predicted adverse outcomes across both surgical groups better than biological age or chronological age and sex. This suggests that the circulating SASP is a robust indicator of age-related health status and may help guide clinical decision making. Furthermore, circulating SASP factors may be harnessed as a readily quantifiable biomarkers in senescence-targeting interventional human studies.

AbstractAging and obesity increase multimorbidity and disability risk, and determining interventions for reversing healthspan decline is a critical public health priority. Exercise and time‐restricted feeding (TRF) benefit multiple health parameters when initiated in early life, but their efficacy and safety when initiated at older ages are uncertain. Here, we tested the effects of exercise versus TRF in diet‐induced obese, aged mice from 20 to 24 months of age. We characterized healthspan across key domains: body composition, physical, metabolic, and cardiovascular function, activity of daily living (ADL) behavior, and pathology. We demonstrate that both exercise and TRF improved aspects of body composition. Exercise uniquely benefited physical function, and TRF uniquely benefited metabolism, ADL behavior, and circulating indicators of liver pathology. No adverse outcomes were observed in exercised mice, but in contrast, lean mass and cardiovascular maladaptations were observed following TRF. Through a composite index of benefits and risks, we conclude the net healthspan benefits afforded by exercise are more favorable than those of TRF. Extrapolating to obese older adults, exercise is a safe and effective option for healthspan improvement, but additional comprehensive studies are warranted before recommending TRF.

Abstract Adipose tissue inflammation and dysfunction are associated with obesity‐related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug‐inducible “suicide” genes driven by the p16 Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra‐abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity‐related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity‐related metabolic dysfunction and its complications.