Engineering topics

Campisi, Judith

Publications and source records attributed to Campisi, Judith.

13 records

DOE OSTI2023

Intermittent supplementation with fisetin improves arterial function in old mice by decreasing cellular senescence

Cellular senescence and the senescence-associated secretory phenotype (SASP) contribute to age-related arterial dysfunction, in part, by promoting oxidative stress and inflammation, which reduce the bioavailability of the vasodilatory molecule nitric oxide (NO). In the present study, we assessed the efficacy of fisetin, a natural compound, as a senolytic to reduce vascular cell senescence and SASP factors and improve arterial function in old mice. We found that fisetin decreased cellular senescence in human endothelial cell culture. In old mice, vascular cell senescence and SASP-related inflammation were lower 1 week after the final dose of oral intermittent (1 week on—2 weeks off—1 weeks on dosing) fisetin supplementation. Old fisetin-supplemented mice had higher endothelial function. Leveraging old p16-3MR mice, a transgenic model allowing genetic clearance of p16 INK4A -positive senescent cells, we found that ex vivo removal of senescent cells from arteries isolated from vehicle- but not fisetin-treated mice increased endothelium-dependent dilation, demonstrating that fisetin improved endothelial function through senolysis. Enhanced endothelial function with fisetin was mediated by increased NO bioavailability and reduced cellular- and mitochondrial-related oxidative stress. Arterial stiffness was lower in fisetin-treated mice. Ex vivo genetic senolysis in aorta rings from p16-3MR mice did not further reduce mechanical wall stiffness in fisetin-treated mice, demonstrating lower arterial stiffness after fisetin was due to senolysis. Lower arterial stiffness with fisetin was accompanied by favorable arterial wall remodeling. The findings from this study identify fisetin as promising therapy for clinical translation to target excess cell senescence to treat age-related arterial dysfunction.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Rutin is a potent senomorphic agent to target senescent cells and can improve chemotherapeutic efficacy

Aging is a major risk factor for most chronic disorders, for which cellular senescence is one of the central hallmarks. Senescent cells develop the pro-inflammatory senescence-associated secretory phenotype (SASP), which significantly contributes to organismal aging and age-related disorders. Development of senotherapeutics, an emerging class of therapeutic agents to target senescent cells, allows to effectively delay aging and alleviate chronic pathologies. Here we report preliminary outputs from screening of a natural medicinal agent (NMA) library for senotherapeutic candidates and validated several agents with prominent potential as senomorphics. Rutin, a phytochemical constituent found in a number of plants, showed remarkable capacity in targeting senescent cells by dampening expression of the full spectrum SASP. Further analysis indicated that rutin restrains the acute stress-associated phenotype (ASAP) by specifically interfering with the interactions of ATM with HIF1α, a master regulator of cellular and systemic homeostasis activated during senescence, and of ATM with TRAF6, part of a key signaling axis supporting the ASAP development toward the SASP. Conditioned media produced by senescent stromal cells enhanced the malignant phenotypes of prostate cancer cells, including in vitro proliferation, migration, invasion, and more importantly, chemoresistance, while rutin remarkably downregulated these gain-of-functions. Although classic chemotherapy reduced tumor progression, the treatment outcome was substantially improved upon combination of a chemotherapeutic agent with rutin. Our study provides a proof of concept for rutin as an emerging natural senomorphic agent, and presents an effective therapeutic avenue for alleviating age-related pathologies including cancer.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Senolysis induced by 25-hydroxycholesterol targets CRYAB in multiple cell types

Cellular senescence is a driver of many age-related pathologies. There is an active search for pharmaceuticals termed senolytics that can mitigate or remove senescent cells in vivo by targeting genes that promote the survival of senescent cells. We utilized single-cell RNA sequencing to identify CRYAB as a robust senescence-induced gene and potential target for senolysis. Using chemical inhibitor screening for CRYAB disruption, we identified 25-hydroxycholesterol (25HC), an endogenous metabolite of cholesterol biosynthesis, as a potent senolytic. We then validated 25HC as a senolytic in mouse and human cells in culture and in vivo in mouse skeletal muscle. Thus, 25HC represents a potential class of senolytics, which may be useful in combating diseases or physiologies in which cellular senescence is a key driver.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

The flavonoid procyanidin C1 has senotherapeutic activity and increases lifespan in mice

Ageing-associated functional decline of organs and increased risk for age-related chronic pathologies is driven in part by the accumulation of senescent cells, which develop the senescence-associated secretory phenotype (SASP). Here we show that procyanidin C1 (PCC1), a polyphenolic component of grape seed extract (GSE), increases the healthspan and lifespan of mice through its action on senescent cells. By screening a library of natural products, we find that GSE, and PCC1 as one of its active components, have specific effects on senescent cells. At low concentrations, PCC1 appears to inhibit SASP formation, whereas it selectively kills senescent cells at higher concentrations, possibly by promoting production of reactive oxygen species and mitochondrial dysfunction. In rodent models, PCC1 depletes senescent cells in a treatment-damaged tumour microenvironment and enhances therapeutic efficacy when co-administered with chemotherapy. Intermittent administration of PCC1 to either irradiated, senescent cell-implanted or naturally aged old mice alleviates physical dysfunction and prolongs survival. We identify PCC1 as a natural senotherapeutic agent with in vivo activity and high potential for further development as a clinical intervention to delay, alleviate or prevent age-related pathologies.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Cdkn1a transcript variant 2 is a marker of aging and cellular senescence

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16 Ink4a and p21 Cip1/Waf1 . In mice, the p21 Cip1/Waf1 encoding locus, Cdkn1a , is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo , variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21 Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Age-related telomere attrition causes aberrant gene expression in sub-telomeric regions

Telomere attrition has been proposed as a biomarker and causal factor in aging. In addition to causing cellular senescence and apoptosis, telomere shortening has been found to affect gene expression in subtelomeric regions. Here, we analyzed the distribution of age-related differentially expressed genes from the GTEx RNA sequencing database of 54 tissue types from 979 human subjects and found significantly more upregulated than downregulated genes in subtelomeric regions as compared to the genome-wide average. Our data demonstrate spatial relationships between telomeres and gene expression in aging.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Age-associated expression of p21and p53 during human wound healing

In mice, cellular senescence and senescence-associated secretory phenotype (SASP) positively contribute to cutaneous wound healing. In this proof-of-concept study, we investigated the expressions of p16, p21, and other senescence-associated biomarkers during human wound healing in 24 healthy subjects using a double-biopsy experimental design. The first punch biopsy created the wound and established the baseline. The second biopsy, concentric to the first and taken several days after wounding, was used to probe for expression of biomarkers by immunohistochemistry and RNA FISH. To assess the effects of age, we recruited 12 sex-matched younger (30.2 ± 1.3 years) and 12 sex-matched older (75.6 ± 1.8 years) subjects. We found that p21 and p53, but not p16, were induced during healing in younger, but not older subjects. A role for Notch signaling in p21 expression was inferred from the inducible activation of HES1. Further, other SASP biomarkers such as dipeptidyl peptidase-4 (DPP4) were significantly induced upon wounding in both younger and older groups, whereas matrix metallopeptidase 9 (MMP9) was induced only in the younger group. Senescence-associated β-galactosidase (SA-β-gal) was not detectable before or after wounding. This pilot study suggests the possibility that human cutaneous wound healing is characterized by differential expression of p21 and p53 between younger and older subjects.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

The progeria research foundation 10 th international scientific workshop; researching possibilities, ExTENding lives – webinar version scientific summary

Progeria is an ultra-rare (prevalence 1 in 20 million), fatal, pediatric autosomal dominant premature aging disease caused by a mutation in the LMNA gene. This mutation results in accumulation of a high level of an aberrant form of the nuclear membrane protein, Lamin A. This aberrant protein, termed progerin, accumulates in many tissues and is responsible for the diverse array of disease phenotypes. Children die predominantly from premature atherosclerotic cardiovascular disease. The Progeria Research Foundation’s 10th International Scientific Workshop took place via webinar on November 2 and 3, 2020. Participants from 30 countries joined in this new, virtual meeting format. Patient family presentations led the program, followed by updates on Progeria’s first-ever application for FDA drug approval as well as initial results from the only current Progeria clinical trial. This was followed by presentations of unpublished preclinical data on drugs in development targeting the disease-causing DNA mutation, the aberrant mRNA, progerin protein, and its downstream effector proteins. Tying bench to bedside, clinicians presented new discoveries on the natural history of disease to inform future clinical trial development and new Progeria aortic valve replacement procedures. The program engaged the Progeria research community as a single unit with a common goal – to treat and cure children with Progeria worldwide.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2021

Quantitative Proteomic Analysis of the Senescence-Associated Secretory Phenotype by Data-Independent Acquisition

Cellular senescence is a complex stress response that induces an essentially permanent cell cycle arrest and a complex secretory phenotype termed the senescence-associated secretory phenotype (SASP), which drives numerous aging pathologies. Characterization of the SASP can provide insights into aging and disease mechanisms, aging biomarker candidates, and targets for counteracting the deleterious effects of senescent cells. Here we describe a mass spectrometry (MS)-compatible protocol to (1) generate senescent cells using different stimuli, (2) collect conditioned medium containing proteins secreted by senescent cells (i.e., SASP), and (3) prepare the SASP for quantitative proteomic analysis using data-independent acquisition (DIA) MS. Basic Protocol 1: Generating ionizing radiation-induced senescent and control cells Alternate Protocol 1: Generating doxorubicin-induced senescent and control cells Alternate Protocol 2: Generating oncogenic RAS-induced senescent and control cells Alternate Protocol 3: Generating mitochondrial dysfunction-induced senescent and control cells Alternate Protocol 4: Generating atazanavir/ritonavir-induced senescent and control cells Support Protocol: A multiple-assay approach to confirm the phenotype of senescent cells Basic Protocol 2: Generating conditioned medium from senescent cells cultured in low serum and quiescent control cells Alternate Protocol 5: Generating conditioned medium from senescent cells cultured in complete medium and quiescent control cells Basic Protocol 3: Quantitative proteomic analysis of the SASP.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

ARDD 2020: from aging mechanisms to interventions

Aging is emerging as a druggable target with growing interest from academia, industry and investors. New technologies such as artificial intelligence and advanced screening techniques, as well as a strong influence from the industry sector may lead to novel discoveries to treat age-related diseases. The present review summarizes presentations from the 7 th Annual Aging Research and Drug Discovery (ARDD) meeting, held online on the 1 st to 4 th of September 2020. The meeting covered topics related to new methodologies to study aging, knowledge about basic mechanisms of longevity, latest interventional strategies to target the aging process as well as discussions about the impact of aging research on society and economy. More than 2000 participants and 65 speakers joined the meeting and we already look forward to an even larger meeting next year. Please mark your calendars for the 8 th ARDD meeting that is scheduled for the 31 st of August to 3 rd of September, 2021, at Columbia University, USA.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

FOXO3 targets are reprogrammed as Huntington's disease neural cells and striatal neurons face senescence with p16 INK4a increase

Neurodegenerative diseases (ND) have been linked to the critical process in aging-cellular senescence. However, the temporal dynamics of cellular senescence in ND conditions is unresolved. Here, we show senescence features develop in human Huntington's disease (HD) neural stem cells (NSCs) and medium spiny neurons (MSNs), including the increase of p16 INK4a , a key inducer of cellular senescence. We found that HD NSCs reprogram the transcriptional targets of FOXO3, a major cell survival factor able to repress cell senescence, antagonizing p16 INK4a expression via the FOXO3 repression of the transcriptional modulator ETS2. Additionally, p16 INK4a promotes cellular senescence features in human HD NSCs and MSNs. These findings suggest that cellular senescence may develop during neuronal differentiation in HD and that the FOXO3-ETS2-p16 INK4a axis may be part of molecular responses aimed at mitigating this phenomenon. Our studies identify neuronal differentiation with accelerated aging of neural progenitors and neurons as an alteration that could be linked to NDs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Role of immune cells in the removal of deleterious senescent cells

Cellular senescence is an essentially irreversible arrest of cell proliferation coupled to a complex senescence-associated secretory phenotype (SASP). The senescence arrest prevents the development of cancer, and the SASP can promote tissue repair. Recent data suggest that the prolonged presence of senescent cells, and especially the SASP, could be deleterious, and their beneficial effects early in life can become maladaptive such that they drive aging phenotypes and pathologies late in life. It is therefore important to develop strategies to eliminate senescent cells. There are currently under development or approved several immune cell-based therapies for cancer, which could be redesigned to target senescent cells. This review focuses on this possible use of immune cells and discusses how current cell-based therapies could be used for senescent cell removal.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Inhibition of USP7 activity selectively eliminates senescent cells in part via restoration of p53 activity

The accumulation of senescent cells (SnCs) is a causal factor of various age-related diseases as well as some of the side effects of chemotherapy. Pharmacological elimination of SnCs (senolysis) has the potential to be developed into novel therapeutic strategies to treat these diseases and pathological conditions. Here we show that ubiquitin-specific peptidase 7 (USP7) is a novel target for senolysis because inhibition of USP7 with an inhibitor or genetic depletion of USP7 by RNA interference induces apoptosis selectively in SnCs. The senolytic activity of USP7 inhibitors is likely attributable in part to the promotion of the human homolog of mouse double minute 2 (MDM2) ubiquitination and degradation by the ubiquitin–proteasome system. This degradation increases the levels of p53, which in turn induces the pro-apoptotic proteins PUMA, NOXA, and FAS and inhibits the interaction of BCL-XL and BAK to selectively induce apoptosis in SnCs. Further, we show that treatment with a USP7 inhibitor can effectively eliminate SnCs and suppress the senescence-associated secretory phenotype (SASP) induced by doxorubicin in mice. These findings suggest that small molecule USP7 inhibitors are novel senolytics that can be exploited to reduce chemotherapy-induced toxicities and treat age-related diseases.

59 BASIC BIOLOGICAL SCIENCES↗