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1.
Nature ; 594(7861): 100-105, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33981041

RESUMO

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.


Assuntos
Envelhecimento/imunologia , Envelhecimento/fisiologia , Sistema Imunitário/imunologia , Sistema Imunitário/fisiologia , Imunossenescência/imunologia , Imunossenescência/fisiologia , Especificidade de Órgãos/imunologia , Especificidade de Órgãos/fisiologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Dano ao DNA/imunologia , Dano ao DNA/fisiologia , Reparo do DNA/imunologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Feminino , Envelhecimento Saudável/imunologia , Envelhecimento Saudável/fisiologia , Homeostase/imunologia , Homeostase/fisiologia , Sistema Imunitário/efeitos dos fármacos , Imunossenescência/efeitos dos fármacos , Masculino , Camundongos , Especificidade de Órgãos/efeitos dos fármacos , Rejuvenescimento , Sirolimo/farmacologia , Baço/citologia , Baço/transplante
2.
Eur Spine J ; 33(3): 892-899, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37046075

RESUMO

PURPOSE: Lumbar spinal stenosis (LSS) is the most common reason for spinal surgery in patients over the age of 65, and there are few effective non-surgical treatments. Therefore, the development of novel treatment or preventative modalities to decrease overall cost and morbidity associated with LSS is an urgent matter. The cause of LSS is multifactorial; however, a significant contributor is ligamentum flavum hypertrophy (LFH) which causes mechanical compression of the cauda equina or nerve roots. We assessed the role of a novel target, microRNA-29a (miR-29a), in LFH and investigated the potential for using miR-29a as a therapeutic means to combat LSS. METHODS: Ligamentum flavum (LF) tissue was collected from patients undergoing decompressive surgery for LSS and assessed for levels of miR-29a and pro-fibrotic protein expression. LF cell cultures were then transfected with either miR-29a over-expressor (agonist) or inhibitor (antagonist). The effects of over-expression and under-expression of miR-29a on expression of pro-fibrotic proteins was assessed. RESULTS: We demonstrated that LF at stenotic levels had a loss of miR-29a expression. This was associated with greater LF tissue thickness and higher mRNA levels of collagen I and III. We also demonstrated that miR29-a plays a direct role in the regulation of collagen gene expression in ligamentum flavum. Specifically, agents that increase miR-29a may attenuate LFH, while those that decrease miR-29a promote fibrosis and LFH. CONCLUSION: This study demonstrates that miR-29a may potentially be used to treat LFH and provides groundwork to initiate the development of a therapeutic product for LSS.


Assuntos
Cauda Equina , MicroRNAs , Estenose Espinal , Humanos , Colágeno Tipo I , Hipertrofia , MicroRNAs/genética , Procedimentos Neurocirúrgicos , Estenose Espinal/terapia
3.
Pain Med ; 24(Suppl 1): S71-S80, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-36525387

RESUMO

The Biospecimen Collection and Processing Working Group of the National Institutes of Health (NIH) HEAL Initiative BACPAC Research Program was charged with identifying molecular biomarkers of interest to chronic low back pain (cLBP). Having identified biomarkers of interest, the Working Group worked with the New York University Grossman School of Medicine, Center for Biospecimen Research and Development-funded by the Early Phase Pain Investigation Clinical Network Data Coordinating Center-to harmonize consortium-wide and site-specific efforts for biospecimen collection and analysis. Biospecimen collected are saliva, blood (whole, plasma, serum), urine, stool, and spine tissue (paraspinal muscle, ligamentum flavum, vertebral bone, facet cartilage, disc endplate, annulus fibrosus, or nucleus pulposus). The omics data acquisition and analyses derived from the biospecimen include genomics and epigenetics from DNA, proteomics from protein, transcriptomics from RNA, and microbiomics from 16S rRNA. These analyses contribute to the overarching goal of BACPAC to phenotype cLBP and will guide future efforts for precision medicine treatment.


Assuntos
Dor Lombar , Humanos , RNA Ribossômico 16S , Biomarcadores , Dor Lombar/terapia , Fenótipo , New York
4.
Pain Med ; 24(Suppl 1): S36-S47, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-36715642

RESUMO

As a member of the Back Pain Consortium (BACPAC), the University of Pittsburgh Mechanistic Research Center's research goal is to phenotype chronic low back pain using biological, biomechanical, and behavioral domains using a prospective, observational cohort study. Data will be collected from 1,000 participants with chronic low back pain according to BACPAC-wide harmonized and study-specific protocols. Participation lasts 12 months with one required in person baseline visit, an optional second in person visit for advanced biomechanical assessment, and electronic follow ups at months 1, 2, 3, 4, 5, 6, 9, and 12 to assess low back pain status and response to prescribed treatments. Behavioral data analysis includes a battery of patient-reported outcomes, social determinants of health, quantitative sensory testing, and physical activity. Biological data analysis includes omics generated from blood, saliva, and spine tissue. Biomechanical data analysis includes a physical examination, lumbopelvic kinematics, and intervertebral kinematics. The statistical analysis includes traditional unsupervised machine learning approaches to categorize participants into groups and determine the variables that differentiate patients. Additional analysis includes the creation of a series of decision rules based on baseline measures and treatment pathways as inputs to predict clinical outcomes. The characteristics identified will contribute to future studies to assist clinicians in designing a personalized, optimal treatment approach for each patient.


Assuntos
Dor Lombar , Humanos , Dor Lombar/diagnóstico , Dor Lombar/terapia , Estudos de Coortes , Estudos Prospectivos , Dor nas Costas , Fenótipo , Estudos Observacionais como Assunto
5.
Pain Med ; 24(Suppl 1): S95-S104, 2023 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-36721327

RESUMO

OBJECTIVE: One aim of the Back Pain Consortium (BACPAC) Research Program is to develop an integrated model of chronic low back pain that is informed by combined data from translational research and clinical trials. We describe efforts to maximize data harmonization and accessibility to facilitate Consortium-wide analyses. METHODS: Consortium-wide working groups established harmonized data elements to be collected in all studies and developed standards for tabular and nontabular data (eg, imaging and omics). The BACPAC Data Portal was developed to facilitate research collaboration across the Consortium. RESULTS: Clinical experts developed the BACPAC Minimum Dataset with required domains and outcome measures to be collected by use of questionnaires across projects. Other nonrequired domain-specific measures are collected by multiple studies. To optimize cross-study analyses, a modified data standard was developed on the basis of the Clinical Data Interchange Standards Consortium Study Data Tabulation Model to harmonize data structures and facilitate integration of baseline characteristics, participant-reported outcomes, chronic low back pain treatments, clinical exam, functional performance, psychosocial characteristics, quantitative sensory testing, imaging, and biomechanical data. Standards to accommodate the unique features of chronic low back pain data were adopted. Research units submit standardized study data to the BACPAC Data Portal, developed as a secure cloud-based central data repository and computing infrastructure for researchers to access and conduct analyses on data collected by or acquired for BACPAC. CONCLUSIONS: BACPAC harmonization efforts and data standards serve as an innovative model for data integration that could be used as a framework for other consortia with multiple, decentralized research programs.


Assuntos
Dor Lombar , Humanos , Dor Lombar/terapia , Avaliação de Resultados em Cuidados de Saúde , Projetos de Pesquisa
6.
Eur Spine J ; 31(5): 1088-1098, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35524071

RESUMO

BACKGROUND: The intervertebral disc degenerates with age and has a poor propensity for regeneration. Small molecule transport plays a key role in long-term degradation and repair. Convection (bulk flow), induced by low rate cyclic loading of the intervertebral disc, has been shown to increase transport of small molecules. However, the potential therapeutic benefit of low rate cyclic loading on degenerated discs has not been described. The purpose of this study was to determine if a sustained (daily) low rate cyclic loading regimen could slow, arrest, or reverse intervertebral disc degeneration in the rabbit lumbar spine. METHODS: Fifty-six New Zealand white rabbits (>12 months old) were designated as either Control (no disc puncture), 8D (disc puncture followed by 8 weeks of degeneration), 16D (disc puncture followed by 16 weeks of degeneration), or Therapy (disc puncture followed by 8 weeks of degeneration and then 8 weeks of daily low rate cyclic loading). Specimens were evaluated by T2 mapping, Pfirrmann scale grading, nucleus volume, disc height index, disc morphology and structure, and proteoglycan content. RESULTS: In every metric, mean values for the Therapy group fell between Controls and 8D animals. These results suggest that sustained low rate cyclic loading had a therapeutic effect on the already degenerated disc and the regimen promoted signs of regeneration. If these results translate clinically, this approach could fulfil a significant clinical need by providing a means of non-invasively treating intervertebral disc degeneration.


Assuntos
Distinções e Prêmios , Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Bioengenharia , Modelos Animais de Doenças , Humanos , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/terapia , Coelhos , Regeneração
7.
Int J Mol Sci ; 23(7)2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35409374

RESUMO

Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10-15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-ßgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health.


Assuntos
Anel Fibroso , Degeneração do Disco Intervertebral , Disco Intervertebral , Animais , Anel Fibroso/metabolismo , Senescência Celular , Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Radiação Ionizante
8.
Eur Spine J ; 28(5): 993-1004, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30847707

RESUMO

PURPOSE: The loss of nutrient supply is a suspected contributor of intervertebral disc degeneration. However, the extent to which low nutrition affects disc annulus fibrosus (AF) cells is unknown as nutrient deprivation has mainly been investigated in disc nucleus pulposus cells. Hence, an experimental study was designed to clarify the effects of limited nutrients on disc AF cell fate, including autophagy, the process by which cells recycle their own damaged components. METHODS: Rabbit disc AF cells were cultured in different media with varying serum concentrations under 5% oxygen. Cellular responses to changes in serum and nutrient concentrations were determined by measuring proliferation and metabolic activity. Autophagic flux in AF cells was longitudinally monitored using imaging cytometry and Western blotting for LC3, HMGB1, and p62/SQSTM1. Apoptosis (TUNEL staining and cleaved caspase-3 immunodetection) and cellular senescence (senescence-associated ß-galactosidase assay and p16/INK4A immunodetection) were measured. RESULTS: Markers of apoptosis and senescence increased, while cell proliferation and metabolic activity decreased under the withdrawal of serum and of nutrients other than oxygen, confirming cellular stress. Time-dependent increases in autophagy markers, including LC3 puncta number per cell, LC3-II expression, and cytoplasmic HMGB1, were observed under conditions of reduced nutrition, while an autophagy substrate, p62/SQSTM1, decreased over time. Collectively, these findings suggest increased autophagic flux in disc AF cells under serum and nutrient deprivation. CONCLUSION: Disc AF cells exhibit distinct responses to serum and nutrient deprivation. Cellular responses include cell death and quiescence in addition to reduced proliferation and metabolic activity, as well as activation of autophagy under conditions of nutritional stress. These slides can be retrieved under Electronic Supplementary Material.


Assuntos
Anel Fibroso , Autofagia/fisiologia , Animais , Anel Fibroso/citologia , Anel Fibroso/metabolismo , Apoptose/fisiologia , Células Cultivadas , Senescência Celular , Meios de Cultura , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Oxigênio/metabolismo , Coelhos
9.
Biomolecules ; 14(10)2024 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-39456209

RESUMO

Hypertrophy of the ligamentum flavum (HLF) is a common contributor to lumbar spinal stenosis (LSS). Fibrosis is a core pathological factor of HLF resulting in degenerative LSS and associated low back pain. Although progress has been made in HLF research, the specific molecular mechanisms that promote HLF remain to be defined. The molecular factors involved in the onset of HLF include increases in inflammatory cytokines such as transforming growth factor (TGF)-ß, matrix metalloproteinases, and pro-fibrotic growth factors. In this review, we discuss the current understanding of the mechanisms involved in HLF with a particular emphasis on aging and mechanical stress. We also discuss in detail how several pathomechanisms such as fibrosis, proliferation and apoptosis, macrophage infiltration, and autophagy, in addition to several molecular pathways involving TGF-ß1, mitogen-activated protein kinase (MAPKs), and nuclear factor-κB (NF-κB) signaling, PI3K/AKT signaling, Wnt signaling, micro-RNAs, extracellular matrix proteins, reactive oxygen species (ROS), etc. are involved in fibrosis leading to HLF. We also present a summary of the current advancements in preclinical animal models for HLF research. In addition, we update the current and potential therapeutic targets/agents against HLF. An improved understanding of the molecular processes behind HLF and a novel animal model are key to developing effective LSS prevention and treatment strategies.


Assuntos
Hipertrofia , Ligamento Amarelo , Ligamento Amarelo/metabolismo , Ligamento Amarelo/patologia , Humanos , Animais , Hipertrofia/metabolismo , Fibrose/metabolismo , Transdução de Sinais
10.
JOR Spine ; 7(1): e1303, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38222800

RESUMO

Background: Low back pain is a global health problem that originated mainly from intervertebral disc degeneration (IDD). Autophagy, negatively regulated by the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway, prevents metabolic and degenerative diseases by removing and recycling damaged cellular components. Despite growing evidence that autophagy occurs in the intervertebral disc, the regulation of disc cellular autophagy is still poorly understood. Methods: Annulus fibrosus (rAF) cell cultures derived from healthy female rabbit discs were used to test the effect of autophagy inhibition or activation on disc cell fate and matrix homeostasis. Specifically, different chemical inhibitors including rapamycin, 3-methyladenine, MK-2206, and PP242 were used to modulate activities of different proteins in the PI3K/Akt/mTOR signaling pathway to assess IL-1ß-induced cellular senescence, apoptosis, and matrix homeostasis in rAF cells grown under nutrient-poor culture condition. Results: Rapamycin, an inhibitor of mTOR complex 1 (mTORC1), reduced the phosphorylation of mTOR and its effector p70/S6K in rAF cell cultures. Rapamycin also induced autophagic flux as measured by increased expression of key autophagy markers, including LC3 puncta number, LC3-II expression, and cytoplasmic HMGB1 intensity and decreased p62/SQSTM1 expression. As expected, IL-1ß stimulation promoted rAF cellular senescence, apoptosis, and matrix homeostatic imbalance with enhanced aggrecanolysis and MMP-3 and MMP-13 expression. Rapamycin treatment effectively mitigated IL-1ß-mediated inflammatory stress changes, but these alleviating effects of rapamycin were abrogated by chemical inhibition of Akt and mTOR complex 2 (mTORC2). Conclusions: These findings suggest that rapamycin blunts adverse effects of inflammation on disc cells by inhibiting mTORC1 to induce autophagy through the PI3K/Akt/mTOR pathway that is dependent on Akt and mTORC2 activities. Hence, our findings identify autophagy, rapamycin, and PI3K/Akt/mTOR signaling as potential therapeutic targets for IDD treatment.

11.
Front Immunol ; 14: 1155746, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37122738

RESUMO

Intervertebral disc degeneration (IDD) is a primary contributor to low back pain. Immune cells play an extremely important role in modulating the progression of IDD by interacting with disc nucleus pulposus (NP) cells and extracellular matrix (ECM). Encased within the annulus fibrosus, healthy NP is an avascular and immune-privileged tissue that does not normally interact with macrophages. However, under pathological conditions in which neovascularization is established in the damaged disc, NP establishes extensive crosstalk with macrophages, leading to different outcomes depending on the different microenvironmental stimuli. M1 macrophages are a class of immune cells that are predominantly pro-inflammatory and promote inflammation and ECM degradation in the NP, creating a vicious cycle of matrix catabolism that drives IDD. In contrast, NP cells interacting with M2 macrophages promote disc tissue ECM remodeling and repair as M2 macrophages are primarily involved in anti-inflammatory cellular responses. Hence, depending on the crosstalk between NP and the type of immune cells (M1 vs. M2), the overall effects on IDD could be detrimental or regenerative. Drug or surgical treatment of IDD can modulate this crosstalk and hence the different treatment outcomes. This review comprehensively summarizes the interaction between macrophages and NP, aiming to highlight the important role of immunology in disc degeneration.


Assuntos
Degeneração do Disco Intervertebral , Núcleo Pulposo , Humanos , Núcleo Pulposo/metabolismo , Degeneração do Disco Intervertebral/patologia , Macrófagos/metabolismo , Matriz Extracelular/metabolismo , Inflamação/metabolismo
12.
Biomolecules ; 13(4)2023 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-37189433

RESUMO

Closely associated with aging and age-related disorders, cellular senescence (CS) is the inability of cells to proliferate due to accumulated unrepaired cellular damage and irreversible cell cycle arrest. Senescent cells are characterized by their senescence-associated secretory phenotype that overproduces inflammatory and catabolic factors that hamper normal tissue homeostasis. Chronic accumulation of senescent cells is thought to be associated with intervertebral disc degeneration (IDD) in an aging population. This IDD is one of the largest age-dependent chronic disorders, often associated with neurological dysfunctions such as, low back pain, radiculopathy, and myelopathy. Senescent cells (SnCs) increase in number in the aged, degenerated discs, and have a causative role in driving age-related IDD. This review summarizes current evidence supporting the role of CS on onset and progression of age-related IDD. The discussion includes molecular pathways involved in CS such as p53-p21CIP1, p16INK4a, NF-κB, and MAPK, and the potential therapeutic value of targeting these pathways. We propose several mechanisms of CS in IDD including mechanical stress, oxidative stress, genotoxic stress, nutritional deprivation, and inflammatory stress. There are still large knowledge gaps in disc CS research, an understanding of which will provide opportunities to develop therapeutic interventions to treat age-related IDD.


Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Humanos , Senescência Celular/genética , Degeneração do Disco Intervertebral/terapia , Degeneração do Disco Intervertebral/genética , Estresse Oxidativo
13.
JOR Spine ; 5(2): e1202, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35783914

RESUMO

Background: Previous animal models of intervertebral disc degeneration (IDD) rely on open surgical approaches, which confound the degenerative response and pain behaviors due to injury to surrounding tissues during the surgical approach. To overcome these challenges, we developed a minimally invasive percutaneous puncture procedure to induce IDD in a rat model. Methods: Ten Fischer 344 male rats underwent percutaneous annular puncture of lumbar intervertebral discs (IVDs) at L2-3, L3-4, and L4-5. Ten unpunctured rats were used as controls. Magnetic resonance imagings (MRIs), serum biomarkers, and behavioral tests were performed at baseline and 6, 12, and 18 weeks post puncture. Rats were sacrificed at 18 weeks and disc histology, immunohistochemistry, and glycosaminoglycan (GAG) assays were performed. Results: Punctured IVDs exhibited significant reductions in MRI signal intensity and disc volume. Disc histology, immunohistochemistry, and GAG assay results were consistent with features of IDD. IVD-punctured rats demonstrated significant changes in pain-related behaviors, including total distance moved, twitching frequency, and rearing duration. Conclusions: This is the first reported study of the successful establishment of a reproducible rodent model of a percutaneous lumbar annular puncture resulting in discogenic pain. This model will be useful to test therapeutics and elucidate the basic mechanisms of IDD and discogenic pain.

14.
Neurospine ; 17(1): 69-76, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32252156

RESUMO

OBJECTIVE: Rabbit annulus fibrosus (AF) cells were exposed to isolated or combined mechanical and inflammatory stress to examine the expression of neuropeptide Y (NPY). This study aims to explore the ability of AF cells to produce NPY in response to mechanical and inflammatory stress. METHODS: Lumbar AF cells of 6- to 8-month-old female New Zealand white rabbits were harvested and exposed to combinations of inflammatory (interleukin-1ß) and mechanical (6% or 18%) tensile stress using the Flexcell System. NPY concentrations were measured in the media via enzyme-linked immunosorbent assay. The presence of NPY receptor-type 1 (NPY-1R) in AF cells of rabbit intervertebral discs was also analyzed via immunohistochemistry and immunofluorescence. RESULTS: Exposure to inflammatory stimuli showed a significant increase in the amount of NPY expression compared to control AF cells. Mechanical strain alone did not result in a significant difference in NPY expression. While combined inflammatory and mechanical stress did not demonstrate an increase in NPY expression at low (6%) levels of strain, at 18% strain, there was a large-though not statistically significant-increase in NPY expression under conditions of inflammatory stress. Lastly, immunofluorescence and immunohistochemistry of AF cells and tissue, respectively, demonstrated the presence of NPY-1R. CONCLUSION: These findings demonstrate that rabbit AF cells are capable of expressing NPY, and expression is enhanced in response to inflammatory and mechanical stress. Because both inflammatory and mechanical stress contribute to intervertebral disc degeneration (IDD), this observation raises the potential of a mechanistic link between low back pain and IDD.

15.
Mech Ageing Dev ; 180: 97-106, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31002926

RESUMO

Cellular senescence is a phenotype characterized by irreversible growth arrest, chronic elevated secretion of proinflammatory cytokines and matrix proteases, a phenomenon known as senescence-associated secretory phenotype (SASP). Biomarkers of cellular senescence have been shown to increase with age and degeneration of human disc tissue. Senescent disc cells in culture recapitulate features associated with age-related disc degeneration, including increased secretion of proinflammatory cytokines, matrix proteases, and fragmentation of matrix proteins. However, little is known of the metabolic changes that underlie the senescent phenotype of disc cells. To assess the metabolic changes, we performed a bioenergetic analysis of in vitro oxidative stress-induced senescent (SIS) human disc cells. SIS disc cells acquire SASP and exhibit significantly elevated mitochondrial content and mitochondrial ATP-linked respiration. The metabolic changes appear to be driven by the upregulated protein secretion in SIS cells as abrogation of protein synthesis using cycloheximide decreased mitochondrial ATP-linked respiration. Taken together, the results of the study suggest that the increased energy generation state supports the secretion of senescent associated proteins in SIS disc cells.


Assuntos
Senescência Celular , Metabolismo Energético , Disco Intervertebral/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Consumo de Oxigênio , Adulto , Feminino , Humanos , Disco Intervertebral/patologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/patologia
16.
Redox Biol ; 17: 259-273, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29747066

RESUMO

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.


Assuntos
Envelhecimento/genética , Senescência Celular/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Mitocôndrias/genética , Animais , Antioxidantes/metabolismo , Senescência Celular/fisiologia , Óxidos N-Cíclicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio/metabolismo
17.
Spine (Phila Pa 1976) ; 42(20): 1521-1528, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28570296

RESUMO

STUDY DESIGN: ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. SUMMARY OF BACKGROUND DATA: We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. METHODS: Three-month-old WT (C57BL/6) and ADAMTS5 mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-κB, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. RESULTS: Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-κB, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. CONCLUSION: The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. LEVEL OF EVIDENCE: N/A.


Assuntos
Proteína ADAMTS5/deficiência , Degeneração do Disco Intervertebral/metabolismo , Disco Intervertebral/metabolismo , Fumar Tabaco/efeitos adversos , Fumar Tabaco/metabolismo , Proteína ADAMTS5/biossíntese , Adulto , Animais , Células Cultivadas , Feminino , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Fumar Tabaco/patologia
18.
J Orthop Res ; 34(8): 1289-306, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26890203

RESUMO

Advanced age is the greatest risk factor for the majority of human ailments, including spine-related chronic disability and back pain, which stem from age-associated intervertebral disc degeneration (IDD). Given the rapid global rise in the aging population, understanding the biology of intervertebral disc aging in order to develop effective therapeutic interventions to combat the adverse effects of aging on disc health is now imperative. Fortunately, recent advances in aging research have begun to shed light on the basic biological process of aging. Here we review some of these insights and organize the complex process of disc aging into three different phases to guide research efforts to understand the biology of disc aging. The objective of this review is to provide an overview of the current knowledge and the recent progress made to elucidate specific molecular mechanisms underlying disc aging. In particular, studies over the last few years have uncovered cellular senescence and genomic instability as important drivers of disc aging. Supporting evidence comes from DNA repair-deficient animal models that show increased disc cellular senescence and accelerated disc aging. Additionally, stress-induced senescent cells have now been well documented to secrete catabolic factors, which can negatively impact the physiology of neighboring cells and ECM. These along with other molecular drivers of aging are reviewed in depth to shed crucial insights into the underlying mechanisms of age-related disc degeneration. We also highlight molecular targets for novel therapies and emerging candidate therapeutics that may mitigate age-associated IDD. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1289-1306, 2016.


Assuntos
Envelhecimento/metabolismo , Degeneração do Disco Intervertebral/etiologia , Disco Intervertebral/metabolismo , Envelhecimento/patologia , Animais , Humanos , Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/patologia , Degeneração do Disco Intervertebral/prevenção & controle
19.
J Orthop Res ; 34(8): 1466-74, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27246627

RESUMO

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC-I. Live-cell imaging revealed uptake of EMPs by AF cells. RT-PCR analysis demonstrated increased mRNA expression of MMP-1 (50.3-fold), MMP-3 (4.5-fold) and MMP-13 (5.5-fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP-treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo-angiogenesis of degenerating disc. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1466-1474, 2016.


Assuntos
Anel Fibroso/metabolismo , Micropartículas Derivadas de Células , Células Endoteliais/fisiologia , Degeneração do Disco Intervertebral/etiologia , Neovascularização Patológica , Linhagem Celular , Feminino , Humanos , Degeneração do Disco Intervertebral/metabolismo , Masculino , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade
20.
Spine J ; 15(6): 1339-46, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-24361347

RESUMO

BACKGROUND CONTEXT: Glucosamine has gained widespread use among patients, despite inconclusive efficacy data. Inconsistency in the clinical literature may be related to lack of understanding of the effects of glucosamine on the intervertebral disc, and therefore, improper patient selection. PURPOSE: The goal of our study was to investigate the effects of glucosamine on intervertebral disc cells in vitro under the physiological conditions of inflammation and mechanical loading. STUDY DESIGN: Controlled in vitro laboratory setting. METHODS: Intervertebral disc cells isolated from the rabbit annulus fibrosus were exposed to glucosamine sulfate in the presence and absence of interleukin-1ß and tensile strain. Outcome measures included gene expression, measurement of total glycosaminoglycans, new proteoglycan synthesis, prostaglandin E2 production, and matrix metalloproteinase activity. The study was funded by NIH/NCCAM, and the authors have no conflicts of interest. RESULTS: Under conditions of inflammatory stimulation alone, glucosamine demonstrated a dose-dependent effect in decreasing inflammatory and catabolic mediators and increasing anabolic genes. However, under conditions of mechanical stimulation, although inflammatory gene expression was decreased, PGE2 was not. In addition, matrix metalloproteinase-3 gene expression was increased and aggrecan expression decreased, both of which would have a detrimental effect on matrix homeostasis. Consistent with this, measurement of total glycosaminoglycans and new proteoglycan synthesis demonstrated detrimental effects of glucosamine under all conditions tested. CONCLUSIONS: These results may in part help to explain the conflicting reports of efficacy, as there is biological plausibility for a therapeutic effect under conditions of predominate inflammation but not under conditions where mechanical loading is present or in which matrix synthesis is needed.


Assuntos
Expressão Gênica/efeitos dos fármacos , Glucosamina/farmacologia , Disco Intervertebral/efeitos dos fármacos , Agrecanas/genética , Agrecanas/metabolismo , Animais , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Feminino , Glicosaminoglicanos/metabolismo , Interleucina-1beta/farmacologia , Disco Intervertebral/citologia , Disco Intervertebral/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Coelhos , Estresse Mecânico
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