Poster 125: Determining Biological Age and Prolonging the Healthspan by Targeting Senescence

Abstract

Objectives:

Although age-related chronic conditions are major drivers of morbidity and healthcare costs, most have proven difficult to prevent or treat. Fundamental aging processes may be root cause contributors to all these disorders, and among these mechanisms is cellular senescence. These cells can release pro-inflammatory cytokines/chemokines and other factors, otherwise known as the senescence-associated secretory phenotype (SASP). Specifically, senescent CD3+ T-Cells have been linked to conditions such as frailty, arthritis, and bone loss. Recently, several senolytics have been proven effective at eliminating senescent cell burden in vitro and in preliminary clinical trials. The senolytic fisetin selectively targets and eliminates senescent cells without affecting healthy cells. This clinical study further supports the body of evidence illustrating an increase in systemic senescent cell burden, SASP biomarkers, and mRNA transcripts in peripheral blood mononuclear cells (PBMCs) from blood with age as well as differences between genders. Importantly, we also found that fisetin reduces a variety of systemic senescence indices when compared to baseline. This body of work presents quantifiable measurements in a large group of participants that could be leveraged to create a composite score of healthy aging, as well as demonstrates the beneficial effects of fisetin in a small subgroup.

Methods:

Study participants were accrued between December 2019 and August 2021 (n=225, n=443 visits) at the Steadman Clinic in Vail, Colorado (IRB#2019-58). Subjects had an average age of 61.6±15.0 between 20 and 100, 48% female and 52% male (Fig. 4). For each of 3 visits per patient, 30mL blood and 3mL serum were collected. Samples were divided with one part enriched for CD3+ T-Cells and the other for total PBMCs. Both PBMCs and T-Cell samples were processed according to a recently optimized a detection strategy for senescent cells using C₁₂FDG staining and flow cytometry. RNA was isolated from the CD3+ T-Cells. qRT-PCR was performed using SYBR Green. For biomarker analysis, both multiplex and singleplex assays were run. After establishing these methods, we isolated and analyzed a subgroup (n=9) of patients that reported taking fisetin (100mg/day) after their first visit.

Results:

In 225 patients across 367 clinic visits, highly senescent (or “bright”) T-Cells did not significantly change with age; however, bright highly senescent PBMCs were found to significantly increase by 0.04% per year of age (p=0.0454) (Fig 1A-B). Both senescent T-Cells (p=0.0395) and PBMCs (p=0.0142) were more abundant in males when compared to healthy, aged-matched females (Fig 1C-D). No biomarkers showed significant differences between genders. Positive age (GDF-15, SOST, FSH) controls, a variety of SASP (MMP3, MMP9, MMP12, IL-6, IL-15, FGF23, PAI, CHD), OA biomarkers (CRP, Eotaxin, PTH), and inflammatory cytokines (TNFα, TGFβ1) had significant, positive correlations with PBMC senescence (Fig. 2) and age. Expression of p16 and p21 in RNA isolated from T-Cells was also not significantly affected by age or gender. Within our fisetin-taking subgroup, many serum SASP factors trended downward in individual patients after taking fisetin (Fig. 3A) with MMP-3, TGFβ-1, and MMP-12 significantly decreasing (p<0.05). Fisetin also decreased the percent of bright PBMCs (p=0.0039) (Fig. 3B) but did not impact bright T-Cell burden (Fig. 3C).

Conclusions:

With the expanding body of research in the field of aging, the complexity of senescence and senolytics has become ever more apparent. Preliminary data suggests that no individual markers of senescent cells appear to be fully sensitive and specific and that senescence and SASP expression vary drastically between cell types. This is supportive of the lack of significant correlations with age and changes in senescent T-Cell populations in patients taking fisetin – it is possible that assaying T-Cells as an entire population is not sufficiently sensitive to track the effects of senolytics or relation to age. Thus, we believe specific T-Cell subsets (CD4, CD8) may be more reflective of the healthspan and warrant further investigation. This study illustrated the correlation between age and senescent PBMCs as well as increasing circulating SASP factors with increases in senescent PBMC burden. Furthermore, fisetin was shown to significantly reduce these hallmarks of aging with no adverse side-effects. Our findings support the potential that fisetin could alleviate multiple age-related morbidities common in elderly patients. Follow-up analysis is needed to determine how the benefits of fisetin adapt over time. Randomized, controlled trials are needed to determine optimum senolytic regimens dependent on patient’s gender, specific cells’ senescent burden, SASP expression, and health condition. Aging is associated with severe musculoskeletal decline including OA and OP for which there are few treatment options. Several recent reports suggest that targeting senescent cells may be a viable therapeutic modality. However, with the wide variety of senescent cell populations and effects via SASP, it is paramount that we understand which outputs of senescence in which cell populations are most determinative of a negative outcome, as well as being able to track senolytics effects on specific outcomes. This study begins to pool results required to answer these bigdata questions. Ultimately, we believe these (among other) factors can be leveraged into a composite score for biological age as well as an early-detection clinical diagnostic tool to more specifically diagnose/monitor age-related diseases and serve as a novel platform to track senolytic efficacy and/improve autologous orthobiologic therapies.

Fig 1.

Senescent PBMCs Increase ivitb Age; Males Have Higher Senescent PBMC and T-Cell Burden than Females. (A-B) Senescent PBMC percent—quantified via C_UFDG flow cytometry—significantly increases with age (P=0.0454). Simple linear regression (black) is shown with 95% confidence intervals. (C-D) Males exhibit increased T-Cetl (£=0.0395) and PBMC (P=0.0142) burden when compared to females.

Fig 3.

Fiserin Decreases serum SASP markers and Seiiescent PBMCs. (A) Serum SASP factors were assayed at baseline (no fisetin) and after treatment with fisetin. Colors indicate each individual’s and the overall average (right column) percent change from baseline after treatment: "X" indicates data was not available. (B) Fisetin treatment significantly decreased bright PBMCs (P=0.0039) in all patients, with an average decrease of 35.6% (dottedred line). (C) Fisetin had no significant effect on senescent T-Cell burden.

Fig 2.

Bright, Senescent PBMCs Positively Correlate with SASP and Inflammatory Biomarkers. All correlations were determined through non-parametric, Spearman tests. Error bars show 95% confidence intervals.

Fig 4.

Study Demographics by Gender, Age, and Fisetin.