Showing papers by "James L. Kirkland published in 2023"

Local senolysis in aged mice only partially replicates the benefits of systemic senolysis.

Abstract: Clearance of senescent cells (SnCs) can prevent several age-related pathologies, including bone loss. However, the local versus systemic roles of SnCs in mediating tissue dysfunction remain unclear. Thus, we developed a mouse model (p16-LOX-ATTAC) that allows for inducible SnC elimination (senolysis) in a cell-specific manner and compared the effects of local versus systemic senolysis during aging using bone as a prototype tissue. Specific removal of Sn osteocytes prevented age-related bone loss at the spine, but not the femur, by improving bone formation without affecting osteoclasts or marrow adipocytes. By contrast, systemic senolysis prevented bone loss at the spine and femur and not only improved bone formation, but also reduced osteoclasts and marrow adipocytes. Transplantation of SnCs into the peritoneal cavity of young mice caused bone loss and also induced senescence in distant host osteocytes. Collectively, our findings provide the first proof-of-concept evidence that local senolysis has health benefits in the context of aging, but importantly, local senolysis only partially replicates the benefits of systemic senolysis. Further, we establish that SnCs, through their SASP, lead to senescence in distant cells. Therefore, our study indicates that optimizing senolytic drugs may require systemic instead of local SnC targeting to extend healthy aging.

Cellular senescence in skin‐related research: Targeted signaling pathways and naturally occurring therapeutic agents

Abstract: Despite the growing interest by researchers into cellular senescence, a hallmark of cellular aging, its role in human skin remains equivocal. The skin is the largest and most accessible human organ, reacting to the external and internal environment. Hence, it is an organ of choice to investigate cellular senescence and to target root‐cause aging processes using senolytic and senomorphic agents, including naturally occurring plant‐based derivatives. This review presents different aspects of skin cellular senescence, from physiology to pathology and signaling pathways. Cellular senescence can have both beneficial and detrimental effects on the skin, indicating that both prosenescent and antisenescent therapies may be desirable, based on the context. Knowledge of molecular mechanisms involved in skin cellular senescence may provide meaningful insights for developing effective therapeutics for senescence‐related skin disorders, such as wound healing and cosmetic skin aging changes.

In vitro and in vivo effects of zoledronate on senescence and senescence-associated secretory phenotype markers

Abstract: In addition to reducing fracture risk, zoledronate has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronate could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronate killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronate or vehicle for 8 weeks, zoledronate significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronate demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronate, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronate significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronate has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronate and/or other bisphosphonate derivatives for senotherapeutic efficacy.

A chronic wound model to investigate skin cellular senescence

Abstract: Wound healing is an essential physiological process for restoring normal skin structure and function post-injury. The role of cellular senescence, an essentially irreversible cell cycle state in response to damaging stimuli, has emerged as a critical mechanism in wound remodeling. Transiently-induced senescence during tissue remodeling has been shown to be beneficial in the acute wound healing phase. In contrast, persistent senescence, as observed in chronic wounds, contributes to delayed closure. Herein we describe a chronic wound murine model and its cellular senescence profile, including the senescence-associated secretory phenotype.

Senescent Cells: A Therapeutic Target in Cardiovascular Diseases

Abstract: Senescent cell accumulation has been observed in age-associated diseases including cardiovascular diseases. Senescent cells lack proliferative capacity and secrete senescence-associated secretory phenotype (SASP) factors that may cause or worsen many cardiovascular diseases. Therapies targeting senescent cells, especially senolytic drugs that selectively induce senescent cell removal, have been shown to delay, prevent, alleviate, or treat multiple age-associated diseases in preclinical models. Some senolytic clinical trials have already been completed or are underway for a number of diseases and geriatric syndromes. Understanding how cellular senescence affects the various cell types in the cardiovascular system, such as endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells, progenitor cells, and cardiomyocytes, is important to facilitate translation of senotherapeutics into clinical interventions. This review highlights: (1) the characteristics of senescent cells and their involvement in cardiovascular diseases, focusing on the aforementioned cardiovascular cell types, (2) evidence about senolytic drugs and other senotherapeutics, and (3) the future path and clinical potential of senotherapeutics for cardiovascular diseases.

Senolytic therapy to modulate the progression of Alzheimer’s Disease (SToMP-AD) – Outcomes from the first clinical trial of senolytic therapy for Alzheimer’s disease

Abstract: Cellular senescence has been identified as a pathological mechanism linked to tau and amyloid beta (Aβ) accumulation in mouse models of Alzheimer’s disease (AD). Clearance of senescent cells using the senolytic compounds dasatinib (D) and quercetin (Q) reduced neuropathological burden and improved clinically relevant outcomes in the mice. Herein, we conducted a vanguard open-label clinical trial of senolytic therapy for AD with the primary aim of evaluating central nervous system (CNS) penetrance, as well as exploratory data collection relevant to safety, feasibility, and efficacy. Participants with early-stage symptomatic AD were enrolled in an open-label, 12-week pilot study of intermittent orally-delivered D+Q. CNS penetrance was assessed by evaluating drug levels in cerebrospinal fluid (CSF) using high performance liquid chromatography with tandem mass spectrometry. Safety was continuously monitored with adverse event reporting, vitals, and laboratory work. Cognition, neuroimaging, and plasma and CSF biomarkers were assessed at baseline and post-treatment. Five participants (mean age: 76±5 years; 40% female) completed the trial. The treatment increased D and Q levels in the blood of all participants ranging from 12.7 to 73.5 ng/ml for D and 3.29–26.30 ng/ml for Q. D levels were detected in the CSF of four participants ranging from 0.281 to 0.536 ng/ml (t(4)=3.123, p=0.035); Q was not detected. Treatment was well-tolerated with no early discontinuation and six mild to moderate adverse events occurring across the study. Cognitive and neuroimaging endpoints did not significantly differ from baseline to post-treatment. CNS levels of IL-6 and GFAP increased from baseline to post-treatment (t(4)=3.913, p=008 and t(4)=3.354, p=0.028, respectively) concomitant with decreased levels of several cytokines and chemokines associated with senescence, and a trend toward higher levels of Aβ42 (t(4)=−2.338, p=0.079). Collectively the data indicate the CNS penetrance of D and provide preliminary support for the safety, tolerability, and feasibility of the intervention and suggest that astrocytes and Aβ may be particularly responsive to the treatment. While early results are promising, fully powered, placebo-controlled studies are needed to evaluate the potential of AD modification with the novel approach of targeting cellular senescence.

Abstract B020: Pre-malignant plasma cells exhibit a senescence-like phenotype and accumulation of transposable elements

Abstract: This abstract is being presented as a short talk in the scientific program. A full abstract is available in the Short Talks from Proffered Abstracts section (PR012) of the Conference Proceedings. Citation Format: Gabriel Alvares Borges, Angelo Jose Guilatco, Christine M. Hachfeld, Ming Ruan, Sonya Royzenblat, Ming Xu, Claire M. Edwards, Marta Diaz-delCastillo, Thomas L. Andersen, Taxiarchis Kourelis, Tamar Tchkonia, James L. Kirkland, Matthew T. Drake, Megan Weivoda. Pre-malignant plasma cells exhibit a senescence-like phenotype and accumulation of transposable elements [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B020.

Abstract B023: Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells

Abstract: Multiple myeloma (MM) is an incurable plasma cell cancer. Therapeutic advances have greatly improved patient survival; however, all patients are expected to relapse. New treatments are critically needed to overcome relapse and provide durable response. There is evidence that dormant MM cells exist in the bone marrow, where they exhibit reduced proliferative capacity and resistance to treatment. These cells are present over extended periods of time and may regain proliferative capacity to drive relapse. Tumor dormancy shares features with senescence, a state of stress-induced growth arrest. Chemotherapies, including those used in the treatment of MM, have been shown to cause therapy-induced senescence in a variety of tissues. Dormant MM cells have been shown to be maintained in their non-proliferative state by interactions with bone lining cells. Interestingly, matrix signaling has also been shown to protect against chemotherapy-induced apoptosis in an in vitro ovarian cancer organoid model. Taken together, this suggests that interaction of MM cells with the bone niche maintains growth arrest and survival. We hypothesize that therapy-induced senescence drives dormancy in MM cells that depends on interactions with the mesenchymal bone niche. GFP-expressing 5TGM1 mouse MM cells were treated with vehicle or melphalan for 48 hours and then cultured in the presence or absence of ex vivo mouse bone marrow stromal cells (BMSCs). Cells were maintained for 12 additional days to allow for the development of senescence after the initial stress response. Cells were imaged and quantified during media replacements to assess adherence/survival. Cells were then fixed and stained for nuclei, phospho-γH2AX, and telomeres by immunofluorescence/fluorescence in-situ hybridization (IF/FISH). Melphalan-treated 5TGM1 cells cultured with BMSCs were significantly growth arrested over 2 weeks compared to vehicle-treated cells. Co-culture with BMSCs significantly enhanced melphalan-treated 5TGM1 adherence compared to culture without BMSCs during media replacement. Of note, melphalan-treated 5TGM1 did not survive in the absence of BMSCs after a week in culture. Melphalan-treated 5TGM1 also exhibited significantly greater adherence with BMSCs than vehicle-treated cells. Melphalan-treated 5TGM1 were significantly larger than vehicle-treated cells and exhibited both heterochromatin foci and persistent DNA damage foci associated with telomeres, which are all markers of cellular senescence. Our findings suggest that interactions with BMSCs promote the survival of melphalan-treated MM tumor cells. The enhanced adherence of these cells also suggests that current methods for minimal residual disease assessment may be inadequate, as rare populations of MM cells adhered to the marrow stroma may not be captured by routine bone marrow aspiration. These cells exhibit features of therapy-induced senescence which may be key to MM dormancy. Thus, targeting senescence survival pathways via senolytic therapy may be a novel approach to eliminate dormant MM cells and prevent disease relapse. Citation Format: Angelo J. Guilatco, Gabriel Alvares Borges, Tamar Tchkonia, James L. Kirkland, Taxiarchis Kourelis, Matthew T. Drake, Megan Weivoda. Enhanced survival and adherence of melphalan-induced senescent-like dormant multiple myeloma cells co-cultured with bone marrow stromal cells [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B023.

Senescence markers in subcutaneous preadipocytes differ in childhood‐ versus adult‐onset obesity before and after weight loss

Abstract: The aim of this study was to determine the effect of age of obesity onset on senescence‐related markers in abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) before and after moderate (~10%) weight loss.

In vitro and in vivo effects of zoledronic acid on senescence and senescence-associated secretory phenotype markers.

Abstract: In addition to reducing fracture risk, zoledronic acid has been found in some studies to decrease mortality in humans and extend lifespan and healthspan in animals. Because senescent cells accumulate with aging and contribute to multiple co-morbidities, the non-skeletal actions of zoledronic acid could be due to senolytic (killing of senescent cells) or senomorphic (inhibition of the secretion of the senescence-associated secretory phenotype [SASP]) actions. To test this, we first performed in vitro senescence assays using human lung fibroblasts and DNA repair-deficient mouse embryonic fibroblasts, which demonstrated that zoledronic acid killed senescent cells with minimal effects on non-senescent cells. Next, in aged mice treated with zoledronic acid or vehicle for 8 weeks, zoledronic acid significantly reduced circulating SASP factors, including CCL7, IL-1β, TNFRSF1A, and TGFβ1 and improved grip strength. Analysis of publicly available RNAseq data from CD115+ (CSF1R/c-fms+) pre-osteoclastic cells isolated from mice treated with zoledronic acid demonstrated a significant downregulation of senescence/SASP genes (SenMayo). To establish that these cells are potential senolytic/senomorphic targets of zoledronic acid, we used single cell proteomic analysis (cytometry by time of flight [CyTOF]) and demonstrated that zoledronic acid significantly reduced the number of pre-osteoclastic (CD115+/CD3e-/Ly6G-/CD45R-) cells and decreased protein levels of p16, p21, and SASP markers in these cells without affecting other immune cell populations. Collectively, our findings demonstrate that zoledronic acid has senolytic effects in vitro and modulates senescence/SASP biomarkers in vivo. These data point to the need for additional studies testing zoledronic acid and/or other bisphosphonate derivatives for senotherapeutic efficacy.

Ex Vivo Perfusion of Discarded Donor Liver Segments for Comparative Therapeutic Investigations

Abstract: Mohamed, Mohamed I MBBS; Kang, Lu MD; Katta, Lavanya MD; Song, ZuoRui MD; Brown, Thomas CCP; Orlando, Christopher MD; Nakhleh, Raouf MD; Tchkonia, Tamar PhD, MS; Kirkland, James MD, PhD; Nguyen, Justin Hung MD, FACS Author Information

Key elements of cellular senescence involve transcriptional repression of mitotic and DNA repair genes through the p53-p16/RB-E2F-DREAM complex

Abstract: Cellular senescence is a dynamic stress response process that contributes to aging. From initiation to maintenance, senescent cells continuously undergo complex molecular changes and develop an altered transcriptome. Understanding how the molecular architecture of these cells evolve to sustain their non-proliferative state will open new therapeutic avenues to alleviate or delay the consequences of aging. Seeking to understand these molecular changes, we studied the transcriptomic profiles of endothelial replication-induced senescence and senescence induced by the inflammatory cytokine, TNF-α. We previously reported gene expressional pattern, pathways, and the mechanisms associated with upregulated genes during TNF-α induced senescence. Here, we extend our work and find downregulated gene signatures of both replicative and TNF-α senescence were highly overlapped, involving the decreased expression of several genes associated with cell cycle regulation, DNA replication, recombination, repair, chromatin structure, cellular assembly, and organization. We identified multiple targets of p53/p16-RB-E2F-DREAM that are essential for proliferation, mitotic progression, resolving DNA damage, maintaining chromatin integrity, and DNA synthesis that were repressed in senescent cells. We show that repression of multiple target genes in the p53/p16-RB-E2F-DREAM pathway collectively contributes to the stability of the senescent arrest. Our findings show that the regulatory connection between DREAM and cellular senescence may play a potential role in the aging process.