Khosla, Sundeep

Abstract The liver is organized into tightly regulated zones with distinct metabolic functions but zonation erodes with age. Cellular senescence contributes to aging and liver diseases, however, its impact on aging biology is ill-defined. As part of The Cellular Senescence Network Consortium, we used multiple spatial transcriptomics approaches (GeoMx, Visium, CosMx) with snRNA-seq to profile senescence signatures, zonation markers, and metabolic pathways in livers from wild-type (WT) mice of multiple ages. We observed a loss of canonical zone signatures in aged mouse livers characterized by “expansion” of midlobular (zone 2) marker gene expression, accompanied by diminished expression of zone 3 marker genes by middle-age (18 months), indicative of loss of cell identity. Multiple analytic approaches identified distinct age-, zone- and sex-specific senescence signatures, which were significantly associated with zonation markers changes. This was recapitulated in Ercc1 mutant models of accelerated senescence, supporting a causal role of senescent cells in liver aging. A “no-zone” hepatocyte-like cluster expanded with age and with the strongest Senescence-Associated Secretory Phenotype (SASP) profile. Gene expression profiles from senescent hepatocytes implicate decreased WNT signaling and increased BMP as contributing to age-related loss of zonation. Together, these data elucidate the role of senescent cells in driving aging biology in non-diseased liver through disruption of cell:cell signaling and the loss of metabolic and cell identity gene expression necessary for hepatocyte function.

Abstract Cellular senescence has been implicated in the pathophysiology of radiotherapy-associated bone loss. Based on our previous work, clearance of senescent cells using genetic and pharmacological tools alleviates the anomalies associated with radiation-associated bone deterioration. The pro-inflammatory senescence associated secretome referred to as senescence associated secretory phenotype (SASP), is a hallmark of cellular senescence. The modulation of SASP by senomorphic drugs, potentially can suppress the pro-inflammatory secretome of senescent cells, irrespective of the underlying senescence mechanism. In this study we tested a senomorphic drug, ruxolitinib, a Janus kinase inhibitor (JAKi), during acute and chronic radiotherapy related effects on the bone. Our clinical data indicate an early increase in several pro-inflammatory SASP proteins following radiotherapy of spinal metastasis in prostate cancer patients. Longitudinal assessment of SASP-related genes confirmed this acute elevation in several SASP markers in systemic circulation following irradiation of male mouse femurs. In studies done in male mice, following three preclinical radiotherapy regimens of 30Gy (5 x 6Gy), 60Gy (5 x 12Gy) and a single dose of 24Gy, suppression of SASP by JAKi was efficacious in suppressing radiation-induced bone damage. In comparison and as shown before, the senolytic combination of D+Q was also able to alleviate radiation-associated bone loss. Early and intermittent suppression of SASP using JAK inhibitors in male mice alleviated chronic bone deterioration, diminished telomere dysfunction, lowered senescence and SASP marker expression and reduced bone-marrow adiposity. Overall, our study shows that early targeting of SASP proteins could be a potential therapeutic to prevent radiotherapy-related chronic bone loss and risk of fractures.

Abstract Senescent cells accumulate with age and contribute to tissue dysfunction, yet their identification in tissues is challenging due to low abundance, heterogeneous phenotypes, and the lack of specific markers. Senescence-associated features span multiple subcellular compartments, including nuclear DNA damage foci, cytosolic protein changes, and perinuclear alterations, each requiring tailored detection strategies. To overcome these challenges, we developed SenoQuant ( https://github.com/HaamsRee/senoquant ), a versatile software designed for comprehensive, accurate, and unbiased spatial quantification and prediction of senescence markers across diverse tissue contexts. Utilizing AI models, SenoQuant enables precise nuclear and cytoplasmic segmentation and detection of senescence markers across low- and high-plex imaging modalities, applicable to cultured cells and tissue sections from mice and humans. The platform also supports custom AI models; for example, we built SenCeption, a proof-of-concept predictor of single-cell p21 status from DAPI-stained nuclei in human skin. Available as a free napari plugin, SenoQuant is widely accessible to researchers. By providing a unified approach to senescence analysis and prediction, SenoQuant opens new opportunities for exploring the complex biology of senescence and its impacts on aging and disease.

Abstract In 2024, the National Academies of Sciences, Engineering, and Medicine (NASEM) published a report entitled Advancing Research on Chronic Conditions in Women. A major purpose of this report was to summarize the current state of research into a series of 21 chronic conditions that affect women differently or that are more common in women. The American Society for Bone and Mineral Research (ASBMR) established a working group to evaluate the NASEM report’s implications for women’s musculoskeletal (MSK) health. This perspective summarizes key research gaps identified in the NASEM report, including the lack of sex-specific data, underrepresentation of women in clinical studies, and minimal integration of MSK health with other chronic conditions. The NASEM report underscores the need for more comprehensive study designs that consider hormonal, genetic, and social determinants of health across the lifespan. In addition to osteoporosis, in this perspective, the ASBMR working group highlights emerging but poorly understood evidence of detrimental effects on musculoskeletal biology in non-MSK, chronic diseases that were identified in the NASEM report to disproportionately affect women. To advance the field, we advocate for better diagnostics, increased investment, and stronger representation of women in research studies. Addressing these gaps is critical for improving prevention, care, and outcomes for chronic conditions that uniquely or disproportionately affect women.

SUMMARY Neurodegenerative diseases are often accompanied by systemic comorbidities, including changes in bone health, but the molecular relationship between neurodegeneration, skeletal decline, and cellular senescence remains poorly understood. In this study, we investigated sex-specific changes in circulating bone- and senescence-related proteins across the spectrum of Alzheimer’s disease(AD), Lewbody dementia(LB) and Parkinson’s disease(PD). Plasma proteomic profiling was performed on samples from 408 participants deeply phenotyped for neurodegenerative diseases, followed by differential protein and pathway analyses. This study reveals sex-dependent alterations in bone and senescence-related circulating proteins in AD-related, PD and LB-related neurodegenerative diseases, providing insights into the complex relationship between neurodegeneration and bone health. Several candidate proteins were also associated with established plasma neurodegeneration biomarkers, particularly pTau181. Pathway analyses revealed shared mitochondrial and metabolic dysfunction across neurodegenerative diseases, with disease-specific features including vesicle trafficking disruption in AD and inflammatory-senescence pathways in LB, plus sex-divergent patterns in inflammatory signaling and bone-related pathways.

Background: Vascular endothelial dysfunction is a key antecedent to cardiovascular diseases (CVD) in older adults. Reduced nitric oxide (NO) bioavailability secondary to excess mitochondria-related reactive oxygen species (mitoROS) bioactivity are key mechanisms of endothelial dysfunction with aging. Cellular senescence is a state of largely irreversible cell cycle arrest that drives endothelial dysfunction. This occurs in part through secretion of bioactive factors into serum, collectively referred to as the circulating senescence-associated secretory phenotype (SASP), that promote excess mitoROS and reduce NO. We have shown fisetin improves endothelium-dependent dilation in old mice by reducing cellular senescence and mitoROS. Here, we sought to translate our preclinical findings to older adults. Hypotheses: We tested the primary hypothesis that intermittent fisetin treatment would improve endothelial function in older adults and that this would be mediated by reductions in mitoROS and associated with lower cell senescence. We also determined if fisetin altered the serum to improve endothelial cell (EC) NO and reduce whole-cell ROS and mitoROS ex vivo. Exploratory analyses assessed if fisetin changed circulating SASP factors. Methods: In a randomized, placebo-controlled, parallel-group design pilot trial, 41 older men and postmenopausal women were randomized to fisetin (2 mg/kg/day) (age: 71±1 yrs [mean±SEM], n=22 [13 W]) or placebo (age: 71±1 yrs, n=19 [12 W]) administered in an intermittent manner (2, 3-day dosing periods separated by 14 days). Outcomes were measured at baseline and 1-3 weeks after the final dose. Endothelial function was assessed as brachial artery flow-mediated dilation (FMDBA). The change in FMDBA with an acute supratherapeutic dose (160mg) of the mitochondrial antioxidant MitoQ was used to assess mitoROS-related inhibition of endothelial function. Gene expression of cellular senescence markers Cdkn1a (encodes p21) and Lmnb1 (encodes Lamin B1) were assessed in peripheral blood mononuclear cells (PBMCs) by Q-PCR. Human aortic ECs (HAECs) in culture were exposed to 10% participant serum for 2 hrs; acetylcholine (ACh)-stimulated NO production (DAR-4M-AM) and basal whole-cell ROS (CellROX) and mitoROS (MitoBright Deep Red) bioactivity were assessed via immunofluorescence. Luminex analyses (Biotechne) measured circulating levels of SASP factors in serum. Results: FMDBA was improved by 41% with fisetin (pre: 3.9±0.4%, post: 5.5±0.5%, P< 0.0001), but unchanged with placebo (pre: 4.4±0.5%, post: 4.5±0.4%) (group x time interaction effect: P< 0.01). Similar efficacy was observed in men and women. Acute MitoQ enhanced FMDBA at baseline and after placebo (all P< 0.05) but led to no further improvements in FMDBA after fisetin treatment. Consistent with lower cell senescence, expression of Cdkn1a was reduced (P< 0.05) by 12% and expression of Lmnb1 increased (P=0.06) by 16% in PBMCs collected after fisetin but not placebo. Compared with pre-intervention, serum collected after fisetin, but not placebo, increased HAEC ACh-stimulated NO by 6% (P< 0.01) and lowered whole-cell and mitoROS bioactivity by ~10% (both P< 0.05). Protein levels of CXCL12 in serum were 5% lower (P< 0.05) after fisetin compared with pre-intervention and after placebo. Traditional cardiometabolic risk factors were unchanged. Conclusions: Intermittent fisetin treatment improves vascular endothelial function in older adults in part by suppressing mitoROS. These effects are linked to reduced cell senescence and select changes to the circulating SASP. Funding: AHA 23CDA1056582; NIH F31AG087709; NIH/NCATS CTSA UL1TR002535; Life Extension® provided fisetin (BioFisetin) and placebo. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Context Patients with mild autonomous cortisol secretion (MACS) have higher rates of vertebral fractures compared to patients with non-functioning adrenal nodules, yet bone density is not always reduced. Objective To characterize the effect of MACS on bone density, metabolism, and microstructure as measured by high-resolution peripheral quantitative computed tomography (HR-pQCT). Design Cross-sectional study, 2019-2022 Setting Tertiary referral center Participants Seventy-five patients with MACS and 75 age-, sex-, and gonadal status–matched referent subjects. Measurements Bone turnover markers, dual-energy x-ray absorptiometry (DXA) scan, and HR-pQCT. Outcomes Areal and volumetric bone mineral density (aBMD, vBMD), trabecular bone score (TBS), microstructural parameters including trabecular bone volume to total volume (BV/TV) ratio. Results While no differences in aBMD were observed at any skeletal site, patients with MACS had decreased TBS (1.389 vs 1.475, P &lt; .001) and lower osteocalcin concentration (17.1 vs 19.5 ng/mL, P = .030) than referent subjects. On HR-pQCT at the tibia, patients with MACS had reduced trabecular BV/TV (0.220 vs 0.233, P = .015) compared to referent subjects. On multivariable analysis, MACS was associated with lower TBS (odds ratio [OR] per SD decrease 2.87 [1.65-5.00]), lower trabecular BV/TV at the radius (OR per SD decrease 2.58 [1.33-5.00]), and lower trabecular BV/TV at the tibia (OR per SD decrease 4.06 [2.10-7.86]). Conclusion Patients with MACS have microstructural deterioration that may not be evident on routine DXA aBMD testing.

Abstract Cellular senescence has been implicated in the pathophysiology of radiotherapy-related bone loss. Based on our previous work, clearance of senescent cells using genetic and pharmacological tools alleviates the anomalies associated with radiation-associated bone deterioration. The pro-inflammatory senescence associated secretome referred to as senescence associated secretory phenotype (SASP), is a hallmark of cellular senescence. The modulation of SASP by senomorphic drugs, potentially can suppress the pro-inflammatory secretome of senescent cells, irrespective of the underlying senescence mechanism. In this study we tested a senomorphic drug, ruxolitinib, a Janus kinase inhibitor (JAKi), during acute and chronic radiotherapy related effects on the bone. Our clinical data indicate an early increase in several pro-inflammatory SASP proteins following radiotherapy of spinal metastasis in prostate cancer patients. Longitudinal assessment of SASP-related genes confirmed this acute elevation in several SASP markers in systemic circulation following irradiation of mouse femurs. In a proof-of-concept study, following two preclinical radiotherapy regimens of cumulative doses of 30Gy (5 x 6Gy) and 60Gy (5 x 12Gy), a senomorphic approach of JAKi treatment was more effective in alleviating radiation-related bone loss compared to the senolytic cocktail of D+Q. Early and intermittent suppression of SASP using JAK inhibitors alleviated chronic bone deterioration, diminished telomere dysfunction, lowered senescence and SASP marker expression, reduced bone-marrow adiposity, and mitigated radiation related lymphatic impairment. Overall, our study shows that early targeting of SASP proteins could be a potential therapeutic to prevent radiotherapy-related chronic bone loss and risk of fractures. Lay Summary Radiotherapy as part of cancer treatment is correlated with an acute increase in senescent cells. Here we show that the pro-inflammatory senescence associated secretory phenotype (SASP) becomes much more prominent soon after radiotherapy in both patients and mice. Suppression of the SASP using a Janus kinase inhibitor, ruxolitinib, reduced the overall senescent cell burden, improved bone architecture by promoting bone formation, reduced bone marrow adiposity, and mitigated radiation-induced lymphatic impairment. Overall, these findings suggest that early inhibition of the SASP may help mitigate several bone anomalies and prevent long-term bone loss and fractures after radiotherapy. Graphical Abstract

Cellular senescence is a heterogeneous phenotype characterized primarily in mesenchymal cells, but the extent to which immune cells differ in their senescence phenotype, or “senotype,” is unclear. Here, we applied single-cell approaches alongside both global and cell-specific genetic senolytic mouse models to evaluate the senotype of immune cells in the bone marrow of aging mice. We found that myeloid-lineage cells exhibited the highest expression of p16 and senescence-associated secretory phenotype markers among all immune cell types. In contrast with clearance of p16 + senescent mesenchymal cells, targeted clearance of p16 + myeloid cells in aged mice had only minor effects on age-related bone loss in male mice, with no effects in females. In more detailed analyses, p16 + myeloid cells were only acutely cleared, being repopulated back to basal levels within a short time. This led to a lack of long-lasting reduction in senescent cell burden, unlike when targeting bone mesenchymal cells. In vitro, myeloid-lineage cells differed markedly from mesenchymal cells in the development of a senescent phenotype. Collectively, our findings indicate that aged bone marrow myeloid cells do not achieve the fully developed senescent phenotype originally described in mesenchymal cells, justifying further characterization of senotypes of immune cells across tissues.

Abstract Cellular senescence is a key mechanism of skeletal aging in both physiological and accelerated conditions, such as radiotherapy. This study aimed to identify common differentially regulated microRNAs (miRs) across these contexts. We performed miR sequencing on three models: femurs from young (5-month-old) versus old (24-month-old) mice; focally radiated versus non-radiated femurs; and osteocytes from young versus old mice. Osteocytes were included in the comparison, as they have the longest lifespan in the mineralized bone matrix and they form 90-95% of all mesenchymal bone cell types. Among the three groups, miR-135a-5p and miR-671-5p were the common (i.e., shared) miRs that were downregulated, and miR-183-5p, a miR that regulates the WNT pathway, was the only shared upregulated miR, while miR-155-5p, a miR that regulates the Senescence-Associated Secretory Phenotype (SASP), was elevated in two conditions. The WNT-pathway has been positively associated with bone health and Sclerostin, a WNT-pathway inhibitor produced and secreted by osteocytes, has been implicated in accelerated skeletal deterioration following radiation. Thus, we used a neutralizing antibody to Sclerostin (Scl-Ab), to assess genes related to the WNT pathway and senescence, which are regulated by miR-183-5p and miR-155-5p, respectively. We further performed miR sequencing in radiated bones from mice treated with Scl-Ab and identified miR-133a-3p, a key miR that inhibits bone metabolism and function, which is upregulated in accelerated skeletal aging (i.e., focal radiation) downregulated by Scl-Ab. Overall, our study identifies potential regulatory gene pathways that modulate skeletal aging in the presence and absence of a WNT activator, Scl-Ab.