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1.
Nat Commun ; 12(1): 5213, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34480023

RESUMO

Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts show lower incidences of degeneration, and the treatment results in a significant decrease in senescence markers p16INK4a, p19ARF, and SASP molecules IL-6 and MMP13. Treatment also preserves cell viability, phenotype, and matrix content. Although transcriptomic analysis shows disc compartment-specific effects of the treatment, cell death and cytokine response pathways are commonly modulated across tissue types. Results suggest that senolytics may provide an attractive strategy to mitigating age-dependent disc degeneration.


Assuntos
Envelhecimento/efeitos dos fármacos , Dasatinibe/uso terapêutico , Degeneração do Disco Intervertebral/tratamento farmacológico , Quercetina/uso terapêutico , Agrecanas/metabolismo , Envelhecimento/metabolismo , Animais , Anel Fibroso/efeitos dos fármacos , Anel Fibroso/metabolismo , Anel Fibroso/patologia , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibrose , Inflamação , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Fenótipo , Transcriptoma/efeitos dos fármacos
2.
FASEB J ; 33(11): 12364-12373, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31408372

RESUMO

Cellular senescence is a phenotypic state that contributes to age-related diseases through the secretion of matrix-degrading and inflammatory molecules. An emerging therapeutic strategy for osteoarthritis (OA) is to selectively eliminate senescent cells by initiating apoptosis. This study establishes a cartilage explant model of senescence induction and senolytic clearance using p16Ink4a expression as a biomarker of senescence. Growth-factor stimulation of explants increased the expression of p16Ink4a at both the mRNA and protein levels. Applying this culture system to cartilage from p16tdTom reporter mice (a knockin allele with tdTomato fluorescent protein regulated by the endogenous p16Ink4a promoter) demonstrated the emergence of a p16-high population that was quantified using flow cytometry for tdTomato. Cell sorting was used to separate chondrocytes based on tdTomato fluorescence and p16-high cells showed higher senescence-associated ß-galactosidase activity and increased gene expression of the senescence-associated secretory phenotype as compared with p16-low cells. The potential for effective senolysis within the cartilage extracellular matrix was assessed using navitoclax (ABT-263). Navitoclax treatment reduced the percentage of p16-high cells from 17.9 to 6.1% (mean of 13 matched pairs; P < 0.001) and increased cleaved caspase-3 confirmed apoptotic activity. Together, these findings establish a physiologically relevant cartilage explant model for testing the induction and elimination of senescent chondrocytes, which will support investigations of senolytic therapy for OA.-Sessions, G. A., Copp, M. E., Liu, J.-Y., Sinkler, M. A., D'Costa, S., Diekman, B. O. Controlled induction and targeted elimination of p16INK4a-expressing chondrocytes in cartilage explant culture.


Assuntos
Cartilagem Articular/citologia , Separação Celular , Senescência Celular , Condrócitos/citologia , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Compostos de Anilina/farmacologia , Animais , Apoptose , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/terapia , Sulfonamidas/farmacologia , Técnicas de Cultura de Tecidos
3.
Proc Natl Acad Sci U S A ; 116(7): 2603-2611, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30683717

RESUMO

The activation of cellular senescence throughout the lifespan promotes tumor suppression, whereas the persistence of senescent cells contributes to aspects of aging. This theory has been limited, however, by an inability to identify and isolate individual senescent cells within an intact organism. Toward that end, we generated a murine reporter strain by "knocking-in" a fluorochrome, tandem-dimer Tomato (tdTom), into exon 1α of the p16INK4a locus. We used this allele (p16tdTom ) for the enumeration, isolation, and characterization of individual p16INK4a -expressing cells (tdTom+). The half-life of the knocked-in transcript was shorter than that of the endogenous p16INK4a mRNA, and therefore reporter expression better correlated with p16INK4a promoter activation than p16INK4a transcript abundance. The frequency of tdTom+ cells increased with serial passage in cultured murine embryo fibroblasts from p16tdTom/+ mice. In adult mice, tdTom+ cells could be readily detected at low frequency in many tissues, and the frequency of these cells increased with aging. Using an in vivo model of peritoneal inflammation, we compared the phenotype of cells with or without activation of p16INK4a and found that tdTom+ macrophages exhibited some features of senescence, including reduced proliferation, senescence-associated ß-galactosidase (SA-ß-gal) activation, and increased mRNA expression of a subset of transcripts encoding factors involved in SA-secretory phenotype (SASP). These results indicate that cells harboring activation of the p16INK4a promoter accumulate with aging and inflammation in vivo, and display characteristics of senescence.


Assuntos
Senescência Celular/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Regiões Promotoras Genéticas , Animais , Proliferação de Células , Ativação Enzimática , Fibroblastos/metabolismo , Meia-Vida , Humanos , Camundongos , Fenótipo , RNA Mensageiro/genética , beta-Galactosidase/metabolismo
4.
Aging Cell ; 17(4): e12771, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29744983

RESUMO

Cellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro-inflammatory molecules known as the senescence-associated secretory phenotype (SASP). The senescence biomarker p16INK4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16INK4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16INK4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16INK4a in murine and human models of chondrocyte aging. We observed that p16INK4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50-fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r2  = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin-dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16INK4a expression. Moreover, increased chondrocyte p16INK4a expression correlated with several SASP transcripts. Despite the relationship between p16INK4a expression and these features of senescence, somatic inactivation of p16INK4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16INK4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.


Assuntos
Senescência Celular/genética , Condrócitos/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/genética , Idoso , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Óxidos N-Cíclicos , Inibidor p16 de Quinase Dependente de Ciclina/antagonistas & inibidores , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Humanos , Indolizinas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Osteoartrite/genética , Osteoartrite/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Compostos de Piridínio/farmacologia , RNA Interferente Pequeno/farmacologia , Adulto Jovem
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